








key: cord-296253-bxyzhsfs
authors: Elham, Elzat; Wumaier, Reziya; Wang, Chengji; Luo, Xiangying; Chen, Tao; Zhong, Nanshan
title: Anatomic evidence shows that lymphatic drainage exists in the pituitary to loop the cerebral lymphatic circulation
date: 2020-05-30
journal: Med Hypotheses
DOI: 10.1016/j.mehy.2020.109898
sha: 
doc_id: 296253
cord_uid: bxyzhsfs

Respiratory infections can result in intracranial infections and unknown neurological symptoms. The central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (CNS) remain unknown. To determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing CNS infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of Evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. Surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. Taken together, our study may be the first to anatomically demonstrate the existence of novel lymphatic vessel structures in the pituitary, as well as their communication with the perinasal (lymphatic) tissue. Our findings suggest the existence of an ultimate loop for “classical” meningeal lymphatic drainage and are relevant to cerebral infection and immune defence.

Respiratory infections can result in intracranial infections and unknown neurological symptoms. The central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (CNS) remain unknown.

To determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing CNS infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of Evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. Surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner.

Respiratory infections (e.g., fungi, bacteria, and coronavirus) can result in unknown intracranial infections and consequent neurological symptoms (1) (2) (3) . For example, in the current COVID-19 epidemic in China, 78 (36.4%) of 214 patients with COVID-19

were admitted with neurological symptoms to Wuhan Union Hospital (2) , and we observed 2 cases of diabetes insipidus (DI) related to pituitary disorder in patients with severe COVID-19 (in the First Affiliated Hospital of Guangzhou Medical University). It is generally believed that pathogens cause intracranial infection by entering the subarachnoid space via nasopharyngeal or middle ear passages, blood flow, blood-brain, and cerebrospinal fluid (CSF) barriers, although we still cannot explain the existence of pathogens in the CSF, as the blood-brain barrier (BBB) can prevent the transmission of pathogens to the meninges(1).

CSF originates from the choroid plexus of the intracranial lateral ventricle (4) . The reflux of CSF to the lymphatic system plays an important role in cerebral immunity (4, 5) . CSF is drained through meningeal lymphatic vessels, which allow immune cells to enter draining lymph nodes (DLNs) and play an important role in cerebral immune defence. However, the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (CNS) remain unknown (4, 6) . The perinasal lymphatic system is the first-line barrier of respiratory immunity against pathogen invasion of the respiratory tract and body (7) . Respiratory infections can lead to CNS infections, but it is unclear whether the perinasal lymphatic system and lymphatic vessels are involved in cerebral immune defence and play a role in CNS infections caused by respiratory pathogens (1, 7) .

To elucidate the roles of the perinasal lymphatic system during cerebral infection (especially respiratory-related infections) and cerebral immune defence, we carried out an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems. Under an anatomic (20× magnification) microscope, we dissected the mouse intracranial nervous system after injection of Evans blue (perinasal lymphatic reflux assay) and found that lymphatic vessels that exist in the pituitary and loop the cerebral lymphatic circulation are responsible for the perinasal-pituitary lymphatic drainage.

The Lyve1-Alexa 488 antibody was purchased from eBioscience (catalogue # 53-0443-80) and used at a 1:250 dilution. Anti-CD31 was purchased from Abcam (catalogue # ab222783) at a 1:100 dilution. Anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody was purchased from Invitrogen Co., Ltd., (catalogue # A32740) and used at a 1:1000 dilution. Evans blue and other reagents were purchased from Sangon Biotech (Shanghai) and were of high analytical grade.

Mice (BALB/c, 7 weeks old) were divided into different groups (5 for each group).

For the treated groups, mice were anaesthetized with pentobarbital sodium (70 mg/kg) by intraperitoneal injections and then subcutaneously injected with 0.1 mL of Evans blue (5%) by microsyringe via either or both limbs, the tail and the perinasal area (e.g., bilateral the hindlimbs, the second toe of the dorsal feet, both flanks, the dorsal sides of the bilateral forelimbs, the bilateral retroauricular regions, the parietal midpoint between both ears, the tip of the nose, and the bilateral ventral mucosae of the tongue). The control groups received saline instead.

After injection, the mice were placed on a heating pad at a stable (25°C) temperature for 4 hours and then euthanized (350 mg/kg) for anatomic analysis. In brief, with the abdomen facing down, the dorsal fur of the mouse was moistened with saline, after which the dorsal skin was cut transversely and then longitudinally to the mouse nose to fully expose the skull with scissors. Then, the cervical muscles were cut off the skull from the foramen magnum to expose the brain. The brain was then removed with tweezers; nerves connecting the brain were also cut off to expose the pituitary for observation under a Zeiss operating microscope (Zeiss Opmi Primo ceiling-mount microscope, 20× magnification). Photos were taken with a Canon 5DSR camera (Micro Lens: Canon EF 100 mm f/2.8L IS USM; Micro Flash: Canon mr-14ex II).

The pituitary was removed from the mouse, immersed in 4% PFA for fixation, and embedded in paraffin. The pituitary was transversely sectioned (Leica CM 1950) and adhered to a glass slide. The sections were then deparaffinized, after which they underwent antigen retrieval, 3% hydrogen peroxide solution blocking for endogenous peroxidase and 3% BSA blocking. The blocking buffer was discarded, and primary antibody in PBS was added to the section and incubated in a wet box at 4°C overnight.

The glass slides were washed with PBS three times (5 min each) after incubation.

Then, the slides were incubated with secondary antibody (HRP-labelled) for 50 min at room temperature. The sections then underwent DAB and Harris haematoxylin staining after three washes in PBS. Then, the section was mounted using neutral balsam, and the blue staining was reversed in ammonia water. Photos were taken under a fluorescence microscope (Leica DMI4000B).

The sections and slide preparation, antigen retrieval, BSA blocking, and primary and secondary antibody incubation processes were the same as the aforementioned conditions used for immunohistochemistry.

After staining with secondary antibodies, the glass slide was washed in PBS three times (5 min each). After the section dried slightly, DAPI was added to the sample, followed by 10 min of room temperature incubation in the dark. The glass slide was then washed again three times and mounted using an antifluorescence quenching mounting agent. Photos were taken under a confocal microscope (LSM710 laser confocal microscope, Zeiss).

Adobe Photoshop, Fiji Image Analysis, and GraphPad Prism software were chosen for image processing.

Two to four hours after the subcutaneous injection of Evans blue into the mouse limbs, tail, and perinasal area, we unexpectedly observed a light-blue, well-discriminated, regular "birds eye" region in the central pituitary (8) (Fig. 1A; We then dissected other untreated mice and found that the "birds eye" region exists in the central pituitary, which was identifiable but can be carefully discriminated from peripheral white matter ( Fig. 1C ; see green arrow and circle). We further performed pathological staining and confirmed that the "birds eye" region was actually the posterior pituitary or adenohypophysis(8) (Fig. 1D) . Moreover, lymphatic endothelial growth factor was found to be highly expressed in the "birds eye" region ( Fig. 1E and 

We acknowledge that the brain lacks classic lymph circulation according to our current understanding. However, it remains unclear how respiratory pathogens cause intracranial infection, how immune cells enter the brain for immune defence and whether the peripheral lymphatic system communicates with the central nervous system in addition to the meningeal lymphatic draining system. A, Colocalization analysis of Lyve-1 and CD31 under the scope in Figure 2D (upper right, the most colocalized area in Figure 2D was selected and marked with a yellow line; scale bar: 100 μm). B, Immunofluorescence analysis showed no colocalization of Lyve-1 and CD31 (Fiji Image analysis).

Funding: The study was financially supported by grants from the National Natural Science We declare that none of the authors has any potential conflicts of interest with regard to this manuscript. A, Colocalization analysis of Lyve-1 and CD31 under the scope in Figure 2D (upper right, the most colocalized area in Figure 2D was selected and marked with a yellow line; scale bar: 100 μm). B, Immunofluorescence analysis showed no colocalization of Lyve-1 and CD31 (Fiji Image analysis).

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None of the authors has any potential conflicts of interest.

