Layout 1 Our understanding of spasticity has evolved in recent decades. The numerous definitions of this complex phenomenon often focus on neural structures and processes,1-4 but the transformation occurring in spastic muscles and soft tissues adds further complexity and is even less understood. Histological and imaging methods have shown spasticity and chronic disuse to be associated with greater variability in muscle fiber size and type, as well as a proliferation of disorganized extracellular matrix material, increased fat content, muscle shortening, atrophy, and sarcopenia, po- tentially leading to muscle stiffness and contractures.5-8 The umbrella term muscle fibrosis is often used to refer to these changes, but controversy remains as to what consti- tutes fibrosis.7,8 With the increasing use of US guidance for chemodener- vation,10-11 the US may also serve as a reliable tool to as- sess spastic muscle and its evolution over time.12 In neuromuscular disease, fibrofatty infiltrations correspond to increased EI.13-14 In spasticity, muscle fibrosis is often considered a potential cause of treatment failure.15 Muscle and soft tissues are often targeted by different spasticity treatments.16 Thus, further elucidating the relationship be- tween muscle changes and spasticity manifestation may better inform treatment. This paper aims to provide a narrative review of muscle Abstract Botulinum Neurotoxin Type A (BoNT-A) injections using Ultrasound (US) guidance have led to research evaluating changes in muscle architecture. Controversy remains as to what constitutes increased Echo-Intensity (EI) in spastic muscles and whether this may affect outcomes. We aim to provide a narrative review of US muscle architecture changes following Central Nervous Sys- tem (CNS) lesions and explore their relationship to spasticity. Medline, CINAHL, and Embase databases were searched with keywords: ultrasonography, hypertonia, spasticity, fibrosis, and Heckmatt. Three physicians reviewed the results of the search to select relevant papers. Reviews identified in the search were used as a resource to identify additional studies. A total of 68 papers were included. Four themes were identified, including histopathological changes in spastic muscle, effects of BoNT-A on the muscle structure, available US modalities to assess the muscle, and util- ity of US assessment in clinical spasticity. Histopathological studies revealed atrophic and fibro- fatty changes after CNS lesions. Several papers described BoNT-A injections contributing to those modifications. These changes translated to increased EI. The exact significance of increased mus- cle EI remains unclear. The Modified Heckmatt Scale (MHS) is a validated tool for grading muscle EI in spasticity. The use of the US may be an important tool to assess muscle architecture changes in spasticity and improve spasticity management. Treatment algorithms may be developed based on the degree of EI. Further research is needed to determine the incidence and impact of these EI changes in spastic muscles. Key Words: botulinum neurotoxin; echo-intensity; Modified Heckmatt Scale; muscle spasticity; muscle fibrosis; ultrasound. Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 - 134 - Thematic Section: Advances in Musculoskeletal and Neuromuscular Rehabilitation | Maccarone & Masiero Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 Assessing muscle architecture with ultrasound: implications for spasticity Ève Boissonnault,1,2 April Jeon,2,3 Michael C. Munin,2,3 Mirko Filippetti,2,4 Alessandro Picelli,2,4 Chloe Haldane,2,5 Rajiv Reebye2,5 1Faculty of Medicine, Université de Montréal, Montreal, Canada; 2Canadian Advances in Neuro- Orthopedics for Spasticity Consortium (CANOSC), Kingston, Canada; 3Physical Medicine and Rehabilitation School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, United States; 4Physical and Rehabilitation Medicine section, Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy; 5Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, Canada. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 architecture changes following CNS lesions, describe their relationship to spasticity, and explore the potential role of the US in muscle evaluation to suggest areas of future re- search. This is the first review to synthesize evidence of spastic muscle changes and to highlight the utility of the US (beyond targeting) for investigating often overlooked peripheral phenomena in spasticity research and clinics. Materials and Methods A literature search was performed by the College of Physi- cians and Surgeons of British Columbia librarians for re- lated articles published between 1986 and March 2022, using Medline, CINAHL, and Embase databases. The five- stage methodological framework of Arksey and OʼMalley was followed. The research question was the following: “What muscle architectural changes are seen in spasticity and how can these changes be evaluated using ultrasound?”. Keywords used were: ultrasonography, muscle tonus, hy- pertonia, spasticity, fibrosis, and Heckmatt. Three physi- cians independently reviewed the results of the search at title, abstract, and full text. Reviews identified in the search were used as a resource to identify additional individual studies. Following this, reference lists of full text and a hand search of Google Scholar was performed which also in- cluded relevant French and Italian literature. Inclusion crit- eria were: i) any level of evidence, ii) studies conducted on animals and/or humans, iii) studies reporting muscle archi- tecture/histology changes post-CNS lesions, and/or effects of BoNT-A on the muscle structure, and/or use of US in spasticity evaluation, and iv) full-text available for review. One reviewer independently extracted relevant data from included articles and recorded data in a spreadsheet. De- mographic information included author, year of publica- tion, sample size, study design, level of evidence, patient diagnoses, muscle architectural changes, and US changes. The level of evidence was recorded as provided by the study (Supplementary Material, Table 1). Results Sixty-eight papers were included in this review (Supple- mentary Material, Table 1). Studies were published from 1986 to 2021. Most studies were of Level III Evidence (N= 41), followed by Level V (N=9), Level IV (N=7), Level II (N=2), and Animal Studies (N=7). Four themes were identified, including histopathological changes in spastic muscle, effects of BoNT-A on the muscle structure, available US modalities to assess the muscle, and utility of US assessment in clinical spasticity. Histological changes in spastic muscle In peripheral nervous system disease, a strong correlation between the extent of nerve damage and the degree of muscle pathology was found in horses with laryngeal hemiplegia (Table 1).17 Affected laryngeal muscles also showed extensive atrophy and fiber-type grouping, indi- cative of denervation and reinnervation.18 In horses af- fected by Australian Stringhalt, abnormally wide distribution in fiber size and a reduction in type II fibers were observed as a consequence of distal axonopathy.19 Following transection of the recurrent laryngeal nerve, echogenicity of affected laryngeal muscles increased sig- nificantly over time compared to the side with no neurec- tomy.20 However, Pillen et al. showed that although the calculation of mean muscle EI strongly correlates with structural muscle changes and the severity of muscle pathology, it is impossible to know whether an increased EI has resulted from fibrosis, fatty infiltration, or both without a complementary muscle biopsy.21 Other vari- ables, such as overlying skin and superficial fascia super- position, can also interfere with muscle EI.21 Structural and functional changes also occur following CNS lesions. Lieber et al.’s work on rats with spinal cord transection demonstrated long-term changes in muscle contractile properties affecting slow muscles to a greater extent compared to predominately fast muscle, with slow- to-fast fiber type conversion associated with a decrease in cross-sectional area and an increase in contractile speed and specific tension.22,23 The morphometric properties of muscles are also altered, with fiber atrophy and an in- creased proportion of endomysial and perimysial connec- tive tissue in both slow and fast muscle.23,24 In human subjects, previous studies revealed a significant positive correlation between collagen type I accumulation in thickened endomysium, decrease in fiber cross-sec- tional area, and more severe muscle stiffness in children with spastic cerebral palsy.25,26 Though these muscles are stiffer,26,27 the bundles are more mechanically fragile with more disorganized, low-quality extracellular material.28 Further structural changes include loss of sarcomeres, in- crease in the ratio of collagen to muscle fiber, increase in fat content within muscles and tendons, and shortening of muscle fibers with decreased passive extension capacity.5- 7 Concerning sarcomere length and fiber type conversion, results are more heterogeneous and hardly predictable, emphasizing the complex character of spastic muscle in human subjects.26-29 Other crucial unanswered questions remain regarding the role of extracellular matrix adapta- tion30 and hyaluronan accumulation31 in reduced range of motion and stiffness, the intrinsic and extrinsic factors in- fluencing those changes, the influence of genetics and epigenetics, and the limitations of current methods to quantify and understand muscle rheological properties and architectural transformation.29,32 Effects of BoNT-A on the muscle structure Concerning extrinsic factors potentially affecting muscle structure and biomechanical properties, BoNT-A injec- tions are suspected to have lasting effects that must be considered (Table 2).33 Mathevon et al. conducted a sys- tematic review of the effects of BoNT-A injections in ani- mals and humans. They found that in animals, a single injection of BoNT-A induced muscle atrophy that was still incompletely recovered at one year. After one injection, the percentage of fast type IIb fibers decreased in favor of intermediate type IIa fibers and slow type I fibers. With monthly injections, the number of myosin-heavy chains associated with faster phenotypes decreased after the third - 135 - Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 month.33 In humans, neurogenic atrophy compensated by fibrosis was also noted, but in only one study.34 The lack of standardized measurement procedures for assessing the architectural consequences of BoNT-A injections on mus- cles with 2D US was outlined.33 A prospective histopathologic study by Valentine et al. on ten ambulatory children with cerebral palsy also con- firmed a positive association between BoNT-A injections and neurogenic atrophy between four months and three years after the last treatment.35 Regarding the BoNT-A ef- fect on the different muscle fiber types, they showed a slow type I fiber loss and a fast type II fiber predominance significantly related to the number of treatments of BoNT- A. It is however worth mentioning that the distinction be- tween type IIa and IIb fibers was not made by Valentine’s group. It remains that the histopathological outcome of post-BoNT-A treated human muscle is variable. In 2020, Picelli et al. investigated the clinical and US ar- chitectural changes induced by BoNT-A in 21 post-stroke patients with spastic equinus.36 At four weeks post-injec- tion, they did not observe any significant effect of BoNT- A treatment on ultrasonographic characteristics (EI, muscle thickness, and pennation angle). In 2021, Battaglia et al. investigated the effects of BoNT- A by conducting a cross-sectional observational study on 53 spastic hemiparetic stroke survivors.37 They concluded that BoNT-A does not seem to influence muscle degener- ation and that EI increase appears to be primarily related to spastic muscle evolution and functional impairment. In- terestingly, in subjects with preserved walking capability and lower spasticity grade, an increase in EI of the medial gastrocnemius was observed in the paretic limb alone, but in patients with impaired gait and more severe spasticity, similar US changes were observed in both calves. - 136 - Table 1. Changes in muscle histology. First Author, Date Population Change in Muscle Histology Booth, 2001 Pediatric (26, mean age 10.6 years) Increased collagen accumulation in spastic Diplegic or quadriplegic cerebral palsy muscle endomysium Cahill, 1986 Animal - Equine (15 horses) Muscle damage reflects nerve damage present Laryngeal hemiplegia Chalmers, 2015 Animal - Equine (28 horses) Increased echo intensity of muscle on Recurrent laryngeal nerve transection ultrasound Friden, 2003 Adult (41 control without neuromuscular Muscle fibers developed passive tension at condition) shorter sarcomere length Pediatric (15 experimental) Cerebral palsy Harrison, 1991 Equine (18 foals) Muscle fiber grouping Equine recurrent laryngeal neuropathy Neurogenic atrophy Lieber, 1986 Animal - Rats (24, 10 experimental, Slow to fast fiber transformation 14 control) Slow muscle less able to generate prolonged Thoracic spinal cord transection contractionIncreased type 1 fiber atrophy Lieber, 2003 Adult (21 control without neuromuscular Muscle cells stiffer but extra-cellular matrix condition) of inferior mechanical strength Pediatric (9 experimental) Cerebral palsy Pillen, 2009 Animal (14 golden retrievers) Increased intersitial fibrous tissue correlated Muscular dystrophy with increase echo intensity on ultrasound Slocombe, 1981 Animal - Equine (9 horses) Increased type I fibers Australian Stringhalt Loss of type II fibers Smith, 2011 Pediatric (33 experimental, 19 control) Muscle bundles including extra-cellular Cerebral palsy matrix stiffer Non -co mmerc ial us e o nly - 137 - Table 2. Change in muscle structure with botulinum neurotoxin A (BoNT-A). First Author, Date Population Intervention Assessment Change in Muscle Structure with BoNT-A Battaglia, 2021 53 patients with spastic Patients had received Structural • No relevant influence of hemiparesis following toxin (dosing not ultrasonographic BoNT-A in contributing stroke reported), mean differences to tissue degeneration in treatment cycles 6 between medial spastic muscles (range 4-8). gastrocnemius and Time of stroke to soleus in affected first treatment mean and unaffected limb. 1.1 years Assessed: cross sectional area, muscle thickness, pennation angle and mean gray value Mathevon, 2015 Systematic review of 21 articles Muscle measures • Muscle atrophy (involving humans and animals) (N=9) – balance, • Reduction in muscle optical microscopy, thickness histochemistry; • Reduced pennation Imaging (N=10) – angle B-mode ultrasound, • Decreased fast type MRI, elastography; IIb fibers in favour of Biomechanical type IIa/slow type I measurements (N=3) • Myosin heavy chains – passive torque reduced Picelli, 2020 21 chronic stroke BoNT-A injection into Ultrasonographic • No significant effect of patients affected gastrocnemiuscharacteristics at BoNT-A injection on medialis and lateralis one-month ultrasound characteristics (dose not reported) post-injection (i.e. Muscle echo, thickness, pennation angle, achilles tendon thickness and hardness) Schroeder, 2009 Two healthy adult male Single dose 74 units MRI imaging at 3, 6, • Denervation of volunteers (47 and to lateral 9 and 12 months neuromuscular junction 31 years) gastrocnemius after injection; on electron microscopy (3 sites/muscle), Signal intensity • High signal intensity Saline of 2 m: alternations, pattern on STIR sequence into contralateral cross-sectional area; in injected muscles limb lateral Histopathology; persistent at 12 months gastrocenmius Electron microscopy post-injection. • Neurogenic fiber atrophy with some compensatory fiber hypertrophy Valentine, 2015 10 patients with Onabotulinum toxin Open muscle biopsy • Neurogenic atrophy in cerebral palsy in 2-4 sites per from medial medial gastroscnemius (mean age gastrocnemius muscle gastrocnemius and between 4 months to 11.6 years) vastus lateralis 3 years post BoNT-A (control) • Type I fiber loss with type II predominance Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 Available ultrasound modalities for muscle assessment The US has become a popular tool that has risen to fulfill the need for a more reliable and accessible method to as- sess spastic muscle and its evolution over time (Table 3).12 Three main US modalities have been used to study spastic muscles: morphological changes,36-52 sonoelastogra- phy,36,38,43,45,47,51,53-65 and echogenicity.36,37,44,49,52,66-71 Morphological changes Morphological changes observable with the 2D US in- clude muscle thickness, pennation angle, and muscle depth. Assessing morphological changes is a more con- ventional approach and can provide useful information about the properties of the muscle fascicle.46 Given the high variability of each parameter, there are no established reference values, and the collected information requires manipulations to be interpreted.36,38-52,72 Sonoelastography Sonoelastography provides information about tissue stiff- ness.73,74 It may be used to estimate muscle strain qual- itatively47,51,53-57 or quantitatively with shear wave elastography.36,38,43,45,54,58-63 A systematic review and meta- analysis reported that US elastography has moderate reli- ability when used in neurological populations.74 Moreover, its usage requires expensive software, and highly qualified technicians, and does not assess muscle echotexture when we know there is growing interest regarding differentia- tion of EI.67,68 Echogenicity Interestingly, a strong correlation was found between elas- tography and EI in stroke-impaired muscles.75 Echogenicity can also be assessed qualitatively36,44,49,67-71,76 or quanti- tatively with software-generated gray-scale score and pixel analysis.37,41,52,61,69,75 Brightness or B-mode US can be used to visualize tissues with varying sonoacoustic properties; these properties in turn determine the number of echoes re- turning to the transducer. Structures will appear bright or hyperechoic when they are highly reflective of sound waves, whereas they will appear dark or hypoechoic when they reflect few sound waves to the transducer. In the US, skeletal muscle appears as a mix of hypoechoic contractile fascicles and hyperechoic intramuscular connective tissue. By altering the histopathological properties of muscle, spas- ticity also alters its solo acoustic features, presumably due to atrophy and fibro-fatty hyperechoic replacement of hy- poechoic contractile elements.10 However, coexisting vari- ables such as aging, muscle strength, and sarcopenia can also influence EI.77-80 Like all medical imaging modalities, US images exhibit - 138 - Table 3. Available US modalities for muscle assessment. Morphological Changes Sonoelastrophy Echogenity • Muscle thickness • Muscle strain • Gray scale score • Pennation angle • Shear wave elastrography • Pixel analysis • Muscle depth • Brightness Battaglia, 2021 Askin, 2017 Battaglia, 2021 Calvo-Lobo, 2018 Cosenza, 2020 Filippetti, 2022 Calvo-Lobo, 2018 Eby, 2016 Hara, 2018 Cosenza, 2020 Eby, 2017 Kenis-Coskun, 2020 Dias, 2017 Gao, 2018 Kim, 2021 Fröhlich-Zwahlen, 2014 Gao, 2018 Moreta, 2020 Hadi, 2018Hong, 2018 Gao, 2019 Picelli, 2012 Jakubowski, 2017 Hong, 2018 Picelli, 2014 Kesikburun, 2015 Jakubowski, 2017 Picelli, 2017 Kim, 2021 Kesikburun, 2015 Picelli, 2020 Lee, 2019 Lee, 2015 Santamato, 2014 Mathevon, 2018 Lee, 2019 Picelli, 2014 Leng, 2019 Picelli, 2017 Liu, 2020 Picelli. 2020 Mathevon, 2018 Thielman, 2019 Picelli, 2020 Yang, 2014 Rasool, 2016 Wu, 2017 Yoldas, 2021 Yasar, 2016 Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 various image artifacts. In particular, the US is subject to a locally correlated multiplicative noise called speckle, which degrades image quality and compromises diagnos- tic confidence.81,82 Speckle noise is an inherent property of medical US imaging that tends to reduce the image res- olution and contrast, thereby reducing the diagnostic value of this imaging modality. As a result, speckle noise reduc- tion is essential whenever US imaging is used for tissue characterization.83 US visual evaluation of muscle can be complex because muscles have an inhomogeneous, speck- led appearance on US images. In addition, fascicles can give a strong or weak reflection, depending on size, direc- tion, and rheological properties, making it difficult to de- tect slight differences in reflections. Different degrees of experience and visual judgment are also known to con- tribute to the relatively high rates of inter-and intra-ob- server variations of visual evaluation.84 Quantitative assessment of muscle EI is more objective and allows for statistical analysis, which is very suitable for research purposes. However, in addition to being more time-consuming, it requires normal values of each muscle EI for each US device.85 Therefore, while one study was conducted on the reliability of the normative data between US devices,84 this makes the quantitative analysis more difficult to apply in everyday clinical practice, particularly in an outpatient setting. In addition, system settings will strongly influence the value in both types of assessment (visual and quantitative), so it is crucial to keep all settings that can affect the yield of grey [such as the ambient light, gain, compression, focus, depth and time gain compensa- tion (TGC), and the set frequency of the probe] constant throughout the measurements.86 The Heckmatt scale is commonly used for semi-quanti- tative assessment of EI.84,87,88 It was originally developed by Heckmatt and Dubowitz, who proposed it as a radio- logic tool to visually evaluate muscles in Duchenne mus- cular dystrophy.89 One of the main advantages of semi-quantitative echogenicity assessment is that it can be performed visually by anyone with a standard US device, without the need to purchase expensive software (unlike sonoelastography).69 There has been an increasing trend of utilization of the Heckmatt scale in patients with spas- ticity after stroke,36,44,49,67-71 although it has never been ex- plicitly validated for this population.66 In 2020, Moreta et al. modified the Heckmatt scale to obtain greater speci- ficity for the spastic muscle evaluation. Their MHS dem- onstrated good reliability and validity in using EI to assess pathologic muscle changes that occur in patients with spasticity.66 Utility of ultrasound assessment in clinical spasticity An observational study conducted by Picelli et al. ex- plored the relationship between ultrasonographic, electro- myographic, and clinical parameters in 43 stroke patients with spastic equinus.49 They showed that spastic gastroc- nemius muscle EI was directly associated with the Mod- ified Ashworth Scale (MAS) score and inversely correlated with muscle thickness, posterior pennation angle, compound muscle action potential amplitude, and ankle passive range of motion. In the medial gastrocne- mius, 37.2% were Heckmatt grade 2, 39.5% grade 3, and 23.2% grade 4. In the lateral gastrocnemius, 41.9% were Heckmatt grade 2, 39.5% grade 3, and 18.6% grade 4.49 Since the study was conducted on patients with equino- varus, it is not surprising that the proportion of Heckmatt grade 4 was higher in the medial gastrocnemius. The au- thors then compared the features of spastic equinus foot in 38 patients with chronic stroke and 38 patients with multiple sclerosis.44 They found a significant difference in muscle EI between the two groups, with a mean Heckmatt score of 3.00 in stroke patients, compared to 1.00 and 2.00 for the lateral and medial gastrocnemius respectively in patients with multiple sclerosis. Interestingly, the MAS was also higher in the stroke group.44 Another observa- tional study reported a mean Heckmatt score of 3.00 in the tibialis posterior of 46 stroke survivors with spastic equinovarus foot.90 Echogenicity has been less studied in the upper extrem- ities, but one observational study focused on the Flexor Digitorum Superficialis (FDS) and Flexor Digitorum Pro- fundus (FDP) muscles of 48 post-stroke patients using quantitative analysis. They showed significant differences between the cross-sectional area and EI values between affected and unaffected sites, as well as a strong correla- tion between mean EI (grey scale values) and Heckmatt scores. In FDS, 43.2% were Heckmatt 1 or 2, while 56.8% were Heckmatt 3 or 4. In FDP, 29.5% were Heckmatt 1 or 2, while 70.5% were Heckmatt 3 or 4.69 Kim et al. found increased EI in hemiparetic limbs com- pared to the normal side in stroke patients who were less than 1 month out from a first stroke.52 The authors deter- mined muscle EI using quantitative grey scale analysis. In addition, they measured pennation angle, fascicle length, and muscle thickness. They found reduced mean muscle thickness in the brachialis and medial gastrocnemius. Ho- wever, the differences in EI between the hemiparetic and normal muscles were greater than the differences in mus- cle thickness, which suggests that EI is a more sensitive measure of structural change in hemiparetic muscle.52 US guidance is not only useful for muscle assessment, but it also has the potential to predict spasticity treatment outcomes. A prospective study by Santamato et al. aimed to assess the effects of Extracorporeal Shock Wave Ther- apy (ESWT) for the treatment of post-stroke plantar flexor muscle spasticity. They found a significant posi- tive correlation between time since stroke onset and grade on the Heckmatt scale. In addition, they found that the reduction in spastic plantar flexor tone in re- sponse to ESWT persisted at 30 days in patients with EI graded 1, 2, and 3 on the Heckmatt scale, but not in those graded 4.70 Two studies specifically looked at the influence of muscle EI on the response to BoNT-A injections. The first one is a cohort study of 56 patients with spastic equinus foot re- sulting from stroke, followed for 4 weeks. One-third of patients had received less than 3 treatments with BoNT- A, while two-thirds had received BoNT-A at least 3 times before being enrolled in the study. Regarding their Heck- matt score, 28.6% were Heckmatt grade 2, 39.3% were grade 3, and 32.1% were grade 4. The authors observed - 139 - Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 that patients with EI of the spastic gastrocnemius graded 2 on the Heckmatt scale showed greater improvement in spasticity than those with higher scores after injection of the same dose of BoNT-A.68 The second study investigating this question was a retro- spective study of 102 post-stroke patients with spasticity due to lower limb paralysis, who were treated with a com- bination of BoNT-A injection and a 2-week inpatient mul- tidisciplinary rehabilitation program. At baseline, 17 patients were Heckmatt grade 1, 55 were grade 2, 24 were grade 3, and 6 were grade 4. Patients were on average 63 years old, and the mean time between the onset of spas- ticity and BoNT-A injection was 6.3 years. They observed significant improvement in MAS scores after the combi- nation of BoNT-A and rehabilitation. However, subjects with Heckmatt grade 4 showed less improvement in motor function compared to those whose muscle EI was classi- fied as grades 1-3, suggesting that BoNT-A and multidis- ciplinary rehabilitation may not be indicated for patients with high spastic muscle EI.67 A publication by Filippetti et al. also supports the inverse correlation between response to spasticity treatment and muscle echogenicity. They observed a significant inverse association of the spastic calf muscles EI with the affected ankle dorsiflexion passive range of motion after a lido- caine diagnostic tibial nerve block, suggesting that in- creased EI also correlates negatively with response to treatment.71 A shared limitation of those three papers is that they used a non-validated tool, the original Heckmatt scale, to assess patients with spasticity. As spastic muscle may not show uniform pathologic changes throughout the entire muscle, the MHS may be a more valuable visual semi-quantitative scale that could be used in future research assessing mus- cle EI post-spasticity treatment.66 Discussion This narrative review of sixty-eight papers assessing the use of US in the assessment of the spastic muscle revealed four main themes: i) histological changes in the spastic muscle; ii) effects of BoNT-A on the muscle structure; iii) US modalities for muscle assessment; iv) utility of US as- sessment in clinical spasticity. Histopathological studies revealed muscle atrophy as well as an increase in fatty tissue and extracellular matrix adap- tation after CNS lesions in animals23,24 and humans,25-28 correlating with reduced range of motion and stiff- ness.30,32,33 Reviews have also underlined the challenges and limitations of current methods to quantify and under- stand the adaptability of muscle architectural composition and stiffness.29,30,32 Several papers described BoNT-A injections contributing to muscle atrophy, increased collagen, and shifts in myo- sin-heavy chains on histological slides of animal subjects.33 Although these changes can be translated into a decrease in muscle thickness and an increase in EI on US images, its effects on long-term functional outcomes are unknown and researchers have yet to consistently show a deleterious ef- fect of BoNT-A injections on the treated spastic muscle with this modality.33,36,37 Histopathological studies are less con- sistent in humans, and outcomes of post-BoNT-A treated muscle biopsies are less predictable.33,35,37 There is a need for more robust research in this area. Regarding US modalities for the assessment of spastic muscle, semi-quantitative echogenicity assessment with the Heckmatt scale has become more common.36,44,49,67- 71 It can be performed by anyone with access to a stan- dard US device, without the need to purchase expensive software.69 The limitation is that the Heckmatt scale has never been explicitly validated for the population with a CNS lesion,66 unlike the MHS, which was developed to improve specificity in the spastic muscle evaluation. In a clinical setting, US assessment of spastic muscle has multiple utilities. It is an easily accessible and useful tool to improve our understanding of the changes in muscle composition following a CNS lesion. Concerning EI spe- cifically, studies have shown a direct correlation between muscle EI and clinical spasticity parameters like MAS, passive range of motion, and Tardieu scale.44,49,69 It is also suggested that EI is a more sensitive measure of structural change in hemiparetic muscle compared to pennation angle, fascicle length, and muscle thickness at only one month after a stroke.52 US guidance also has the potential to optimize spasticity treatment. Not only is there level 1 evidence that instrumented guidance (using US, electro- myography, or electrical stimulation) is superior to injec- tions done solely with manual guidance,10 but the reduced efficacy of treatment in muscles with increased EI sug- gests that EI assessment should be part of our spasticity management algorithm.67,68,70,71,73 Our understanding of the histopathological phenomena occurring in the spastic muscle and contributing to in- creased passive stiffness needs to be deepened. The exact significance of increased muscle EI in human subjects with spasticity in vivo remains unclear.32 Increased col- lagen, fat content, and hyaluronan,31,91 skin changes, fas- cia,21 extracellular matrix, sarcomere length, neural control, muscle atrophy, disuse,32 muscle strength, aging, sarcopenia,78-80,92 and genetics29 each potentially play a role in the resulting EI, development of contractures, and re- sponse to spasticity treatment. Despite many years of fundamental and clinical research, spasticity management remains challenging, and the out- come of our treatments is unfortunately often suboptimal. There is without a doubt a need to develop more accurate and accessible tools to assess the spastic muscle. The spas- ticity-validated MHS could be used in future spasticity studies involving multimodal treatment and assessment of muscle EI evolution with time. International US spasticity courses93,94 should include an assessment of muscle and surrounding structures in their curriculum design. With the increased accessibility and affordability of the US, it will likely be an important tool in clinical spasticity prac- tice. In addition to its key role in muscle identification and localization for chemodenervation,10,11 US can also en- hance our understanding of the consequences of CNS le- sions on muscle content and may help in selecting the most appropriate treatment combination to reach our pa- tients’ goals. - 140 - Non -co mmerc ial us e o nly Assessing muscle architecture with ultrasound Eur J Transl Myol 34 (2) 12397, 2024 doi: 10.4081/ejtm.2024.12397 In the future, large-scale multicentric prospective obser- vational studies could be conducted to determine which factors have the most impact on muscle EI evolution and what treatment modality should be prioritized concerning the pairing of clinical and EI assessments. For example, we might opt for a more aggressive and surgical approach to manage a patient with an MAS score of 2 or more and a grade of 3 or 4 on the MHS. We can also hypothesize that by precisely delivering BoNT-A to more hypoechoic and healthy intramuscular pockets, we could optimize the outcome of our injections. Finally, US muscle assessment could aid in investigations of the pathophysiological mechanisms involved in innovative spasticity treatments such as collagenase95 and hyaluronidase injections,96,97 as well as cryo neurolysis,98 and to define their place in the future algorithm of spasticity management. Conclusions Animal and human studies describe muscle architectural changes after upper motor neuron injury and after BoNT- A injection. Human studies have revealed that muscle-in- creased EI may affect spasticity treatment outcomes. Sonoelastography and quantitative analysis of muscle EI have been shown to detect changes in muscle US archi- tecture in spasticity, but its practical utility in clinical prac- tice may be challenging to incorporate. The semi-quantitative validated tool MHS demonstrated good reliability and validity in assessing pathologic muscle changes in patients with spasticity and is easy to use in clinical practice. The use of US may be an important tool to assess architectural muscle changes in spasticity and improve spasticity management. We encountered some limitations while writing this paper. As this is a narrative review, including every histologic paper on post-CNS lesion muscle changes was impossible. However, thanks to the method applied for the literature search, we are confident that we included the most rel- evant papers to support our main hypothesis that US is an accessible tool that could change the way we assess pe- ripheral muscle changes in research and clinical settings. These changes remain only partially understood and re- quire more investigation. In the future, treatment algorithms may be developed based on the level of muscle stiffness coupled with the de- gree of EI to achieve patient goals. Further epidemiologi- cal studies are needed to determine the incidence of these EI changes in spastic muscles and their effect on function and treatment outcomes. List of abbreviations BoNT-A: Botulinum Neurotoxin Type A, US: Ultrasound, EI: Echo-Intensity, CNS: Central Nervous System, MHS: Modified Heckmatt Scale, TGC: Time Gain Compensation, MAS: Modified Ashworth Scale, FDS: Flexor Digitorum Superficialis, FDP: Flexor Digitorum Profundus, ESWT: Extracorporeal Shock Wave Therapy Acknowledgments We would like to thank the librarian service of the College of Physicians and Surgeons of British Columbia for their help with the literature review. Funding This research received no external funding. Conflicts of interest There are no conflicts of interest associated with this pub- lication to report. Corresponding Author Rajiv Reebye, GF Strong Rehabilitation Centre, 4255 Laurel Street, Vancouver BC, V5Z 2G9, Canada. ORCID ID: 0000-0002-0415-9401 E-mail: rajiv.reebye@vch.ca Ève Boissonnault ORCID ID: 0000-0002-5994-2903 E-mail: eve.boissonnault@umontreal.ca April Jeon ORCID ID: 0009-0008-2986-2620 E-mail: anhyon16@gmail.com Michael C. Munin ORCID ID: 0000-0002-4140-3004 E-mail: muninmc@upmc.edu Mirko Filippetti ORDCID ID: 0000-0001-5930-5974 E-mail: mirko.filippetti@univr.it Alessandro Picelli ORCID ID: 0000-0002-3558-8276 E-mail: alessandro.picelli@univr.it Chloe Haldane ORCID ID: 0000-0003-1569-8416 E-mail: chloe.haldane@gmail.com References 1. Lance JW. The control of muscle tone, reflexes, and movement: Robert Wartenberg Lecture. Neurology 1980;30:1303-13. 2. Li S, Francisco GE, Rymer WZ. 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Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 19 February 2024. Accepted: 21 April 2024. Early access: 30 May 2024. - 144 - Online supplementary materials Table 1. Characteristics of the studies included. Non -co mmerc ial us e o nly https://www.aapmr.org/education/step-certificate-programs/step-interventional-spasticity-certificate-program https://www.aapmr.org/education/step-certificate-programs/step-interventional-spasticity-certificate-program