Neuromuscular Research Research Articles

Isolated semitendinosus involvement in the initial stages of limb-girdle muscular dystrophy 2L

Unit of Neuromuscular Disorders, Laboratory of Molecular Medicine, Bambino Gesu’ Children’s Research Hospital, Rome, Italy Folkhalsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland Pediatric Neurology Unit, Catholic University School of Medicine, Rome, Italy d Institute of Neurology, Catholic University School of Medicine, Rome, Italy Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland Neurology Department, Vaasa Central Hospital, Vaasa, Finland

Muscle utilization patterns vary by skill levels of the practitioners across specific yoga poses (asanas)

To compare muscle activation patterns in 14 dominant side muscles during different yoga poses across three skill levels. Mixed repeated-measures descriptive study. University neuromuscular research laboratory, Miami, US. A group of 36 yoga practitioners (9 M/27 F; mean ± SD, 31.6 ± 12.6 years) with at least 3 months yoga practice experience. Each of the 11 surya namaskar poses A and B was performed separately for 15s and the surface electromyography for 14 muscles were recorded. Normalized root mean square of the electromyographic signal (NrmsEMG) for 14 muscles (5 upper body, 4 trunk, 5 lower body). There were significant main effects of pose for all fourteen muscles except middle trapezius (p<.02) and of skill level for the vastus medialis; p=.027). A significant skill level × pose interaction existed for five muscles (pectoralis major sternal head, anterior deltoid, medial deltoid, upper rectus abdominis and gastrocnemius lateralis; p<.05). Post hoc analyses using Bonferroni comparisons indicated that different poses activated specific muscle groups; however, this varied by skill level. Our results indicate that different poses can produce specific muscle activation patterns which may vary due to practitioners' skill levels. This information can be used in designing rehabilitation and training programs and for cuing during yoga training.

Cooperative International Neuromuscular Research Group Duchenne Natural History Study demonstrates insufficient diagnosis and treatment of cardiomyopathy in Duchenne muscular dystrophy.

Cardiomyopathy is a common cause of morbidity and death in patients with Duchenne muscular dystrophy (DMD). This investigation was a cross-sectional cross-sectional analysis of clinical data from the multi-institutional Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study of 340 DMD patients aged 2-28 years. Cardiomyopathy was defined as shortening fraction (SF) <28% or ejection fraction (EF) <55%. Two hundred thirty-one participants reported a prior clinical echocardiogram study, and 174 had data for SF or EF. The prevalence of cardiomyopathy was 27% (47 of 174), and it was associated significantly with age and clinical stage. The association of cardiomyopathy with age and clinical stage was not changed by glucocorticoid use as a covariate (P > 0.68). In patients with cardiomyopathy, 57% (27 of 47) reported not taking any cardiac medications. Cardiac medications were used in 12% (15 of 127) of patients without cardiomyopathy. We found that echocardiograms were underutilized, and cardiomyopathy was undertreated in this DMD natural history cohort.

The CNDR: Collaborating to Translate New Therapies for Canadians

Patient registries represent an important method of organizing "real world" patient information for clinical and research purposes. Registries can facilitate clinical trial planning and recruitment and are particularly useful in this regard for uncommon and rare diseases. Neuromuscular diseases (NMDs) are individually rare but in aggregate have a significant prevalence. In Canada, information on NMDs is lacking. Barriers to performing Canadian multicentre NMD research exist which can be overcome by a comprehensive and collaborative NMD registry. We describe the objectives, design, feasibility and initial recruitment results for the Canadian Neuromuscular Disease Registry (CNDR). The CNDR is a clinic-based registry which launched nationally in June 2011, incorporates paediatric and adult neuromuscular clinics in British Columbia, Alberta, Ontario, Quebec, New Brunswick and Nova Scotia and, as of December 2012, has recruited 1161 patients from 12 provinces and territories. Complete medical datasets have been captured on 460 "index disease" patients. Another 618 "non-index" patients have been recruited with capture of physician-confirmed diagnosis and contact information. We have demonstrated the feasibility of blended clinic and central office-based recruitment. "Index disease" patients recruited at the time of writing include 253 with Duchenne and Becker muscular dystrophy, 161 with myotonic dystrophy, and 71 with ALS. The CNDR is a new nationwide registry of patients with NMDs that represents an important advance in Canadian neuromuscular disease research capacity. It provides an innovative platform for organizing patient information to facilitate clinical research and to expedite translation of recent laboratory findings into human studies.

Leg Amyotrophic Diplegia

To identify the frequency of leg amyotrophic diplegia (LAD) at a US academic center, describe the pattern of weakness, and provide comparative data from 8 additional major US academic institutions. LAD is a leg onset variant of progressive muscular atrophy (PMA). LAD weakness is confined to the legs for at least 2 years, and there are no upper motor neuron signs. We present a retrospective chart review of 24 patients with the LAD presentation from the University of Kansas Medical Center ( n = 8 cases) and from 8 US academic institutions (n = 16 cases). Of the 318 subjects identified in the University of Kansas Medical Center Neuromuscular Research Database, 82% (260 subjects) had amyotrophic lateral sclerosis (ALS), 1.9% (6) had familial ALS, 6.6% (21) had primary lateral sclerosis, and 9.2% (29) had lower motor neuron (LMN) disease. Of these 29 cases, 16 had PMA, 5 had brachial amyotrophic diplegia, while 8 had LAD. The mean LAD age of onset was 58 years with a male/female ratio of 3/1. Onset was asymmetric in 7/8. We identified a pelviperoneal pattern of weakness (sparing of knee extension and/or ankle plantar flexion) in 4 cases and distal predominant weakness in 3 cases. All patients had electrodiagnostic findings consistent with motor neuron disease confined to the lower extremities. We present LAD disease duration and survival data from 8 major academic neuromuscular centers. At last follow-up, weakness progressed to involve the arms in 6/24 LAD cases and of these 6 cases, 2 patients died from progression to typical ALS. From onset of symptoms, mean survival in LAD is 87 months, with 92% of cases being alive. The natural history of LAD differs from typical forms of ALS and PMA. LAD is a slowly progressive disorder that accounts for a fourth of LMN disease cases. An asymmetric pelviperoneal pattern of weakness should heighten the suspicion for LAD.

A functional system for high-content screening of neuromuscular junctions in vitro.

High-content phenotypic screening systems are the logical extension of the current efficient, yet low information content, pre-clinical screens for drug discovery. A physiologically accurate in vitro neuromuscular junction (NMJ) screening system would therefore be of tremendous benefit to the study of peripheral neuropathies as well as for basic and applied neuromuscular research. To date, no fully-defined, selective assay system has been developed which would allow investigators to determine the functional output of cultured muscle fibers (myotubes) when stimulated via the NMJ in real time for both acute and chronic applications. Here we present the development of such a phenotypic screening model, along with evidence of NMJ formation and motoneuron initiated neuromuscular transmission in an automated system. Myotubes assembled on silicon cantilevers allowed for measurement of substrate deflection in response to contraction and provided the basis for monitoring the effect of controlled motoneuron stimulation on the contractile behavior. The effect was blocked by treatment with D-tubocurarine, confirming NMJ functionality in this highly multiplexed assay system.

P.2.11 CINRG Duchenne Natural History Study: Relationship of longitudinal measures of ambulatory timed function tests and loss of clinical milestones

The Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study enrolled 340 individuals with DMD aged 2–28 years in a longitudinal observational study. Clinical assessments were obtained every 3 months for the first year and annually, thereafter. The aim of this sub analysis was to evaluate the predictive capabilities of timed tests in assessing loss of clinically meaningful milestones. We created a 6-level composite of individual “milestone” tasks combining the results from the ability to perform the timed function tests and the Brooke and Vignos functional scales. Three GC groups (naive, current user, past user) were compared on timed function velocities at ages Timed function velocities increased with age in children 10 s. predicted loss of ability to stand (log rank p p 8 s. predicted loss of stair climbing (log rank p p 12 s. predicts loss of ambulation over 12 months (log rank p 10% over a year predicted the likelihood of loss of ambulation over the following 4 years (log rank p Longitudinal studies of timed function tests show predictable maturational changes and improved timed function velocities in those currently treated with GC. Timed function tests are clinically meaningful in terms of predictive ability for loss of function.

P.2.16 Cooperative International Neuromuscular Research Group (CINRG) study of echocardiographic outcome measures for use in clinical trials in muscular dystrophy

Cardiomyopathy is a significant clinical outcome for muscular dystrophy. Echocardiography (echo) is a versatile measure of cardiac function that is available in most muscular dystrophy centers and, therefore, has high utility for clinical care and multicenter clinical trials. Echo outcomes for treatment trials in muscular dystrophy have traditionally measured ejection fraction (EF). However, this measure can be challenging to obtain with more advanced disease due to chest wall deformities and difficulty with positioning for testing. This study was performed to pilot prospective measures of echo outcomes at 5 centers of the Cooperative International Neuromuscular Research Group (CINRG) using a central sonography protocol and 2 central readers. Forty-eight participants 8–18 years old with genetically confirmed Duchenne muscular dystrophy, Becker muscular dystrophy or limb girdle muscular dystrophy underwent electrocardiogram, echo and B-type natriuretic peptide testing. Notably, the echo quality was insufficient to read EF for 23 studies by one reader and for 5 studies by the other reader. In contrast, myocardial performance index (MPI) measured by Doppler could be read by both readers in 46 studies. Furthermore, the agreement of cardiac measures between the 2 independent readers and between one reader performing 2 different reads was greater for MPI than for EF. In 35% of studies, an abnormal MPI while EF remained normal suggested early cardiac dysfunction. This finding suggests that MPI may demonstrate increased sensitivity to detect cardiomyopathy when compared to EF. In subsequent analyses to explore echo measures of even greater sensitivity to detect cardiac dysfunction, speckle tracking was performed to measure longitudinal and circumferential strain. The results suggest that MPI and strain measures are sensitive and amenable to central reading for muscular dystrophy clinical trials. Cardiomyopathy is a significant clinical outcome for muscular dystrophy. Echocardiography (echo) is a versatile measure of cardiac function that is available in most muscular dystrophy centers and, therefore, has high utility for clinical care and multicenter clinical trials. Echo outcomes for treatment trials in muscular dystrophy have traditionally measured ejection fraction (EF). However, this measure can be challenging to obtain with more advanced disease due to chest wall deformities and difficulty with positioning for testing. This study was performed to pilot prospective measures of echo outcomes at 5 centers of the Cooperative International Neuromuscular Research Group (CINRG) using a central sonography protocol and 2 central readers. Forty-eight participants 8–18 years old with genetically confirmed Duchenne muscular dystrophy, Becker muscular dystrophy or limb girdle muscular dystrophy underwent electrocardiogram, echo and B-type natriuretic peptide testing. Notably, the echo quality was insufficient to read EF for 23 studies by one reader and for 5 studies by the other reader. In contrast, myocardial performance index (MPI) measured by Doppler could be read by both readers in 46 studies. Furthermore, the agreement of cardiac measures between the 2 independent readers and between one reader performing 2 different reads was greater for MPI than for EF. In 35% of studies, an abnormal MPI while EF remained normal suggested early cardiac dysfunction. This finding suggests that MPI may demonstrate increased sensitivity to detect cardiomyopathy when compared to EF. In subsequent analyses to explore echo measures of even greater sensitivity to detect cardiac dysfunction, speckle tracking was performed to measure longitudinal and circumferential strain. The results suggest that MPI and strain measures are sensitive and amenable to central reading for muscular dystrophy clinical trials.

‘SIT’ down and relax: the interpolated twitch technique is still a valid measure of central fatigue during sustained contraction tasks

‘SIT’ down and relax: the interpolated twitch technique is still a valid measure of central fatigue during sustained contraction tasks

The cooperative international neuromuscular research group Duchenne natural history study--a longitudinal investigation in the era of glucocorticoid therapy: design of protocol and the methods used.

Contemporary natural history data in Duchenne muscular dystrophy (DMD) is needed to assess care recommendations and aid in planning future trials. The Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study (DMD-NHS) enrolled 340 individuals, aged 2-28 years, with DMD in a longitudinal, observational study at 20 centers. Assessments obtained every 3 months for 1 year, at 18 months, and annually thereafter included: clinical history; anthropometrics; goniometry; manual muscle testing; quantitative muscle strength; timed function tests; pulmonary function; and patient-reported outcomes/health-related quality-of-life instruments. Glucocorticoid (GC) use at baseline was 62% present, 14% past, and 24% GC-naive. In those ≥6 years of age, 16% lost ambulation over the first 12 months (mean age 10.8 years). Detailed information on the study methodology of the CINRG DMD-NHS lays the groundwork for future analyses of prospective longitudinal natural history data. These data will assist investigators in designing clinical trials of novel therapeutics.

The cooperative international neuromuscular research group Duchenne natural history study: Glucocorticoid treatment preserves clinically meaningful functional milestones and reduces rate of disease progression as measured by manual muscle testing and other commonly used clinical trial outcome measures

introduction: Glucocorticoid (GC) therapy in Duchenne muscular dystrophy (DMD) has altered disease progression, necessitating contemporary natural history studies. The Cooperative Neuromuscular Research Group (CINRG) DMD Natural History Study (DMD-NHS) enrolled 340 DMD males, ages 2-28 years. A comprehensive battery of measures was obtained. A novel composite functional "milestone" scale scale showed clinically meaningful mobility and upper limb abilities were significantly preserved in GC-treated adolescents/young adults. Manual muscle test (MMT)-based calculations of global strength showed that those patients <10 years of age treated with steroids declined by 0.4 ± 0.39 MMT unit/year, compared with -0.4 ± 0.39 MMT unit/year in historical steroid-naive subjects. Pulmonary function tests (PFTs) were relatively preserved in steroid-treated adolescents. The linearity and magnitude of decline in measures were affected by maturational changes and functional status. In DMD, long-term use of GCs showed reduced strength loss and preserved functional capabilities and PFTs compared with previous natural history studies performed prior to the widespread use of GC therapy.

A Clinical Analysis of Alzheimer?s Research in Neuromuscular Physical Medicine and Rehabilitation

The purpose of this commentary is to discuss the role of clinical research as an effective method for studying the comparative effects of selected therapeutic interventions, upon the rehabilitation outcome of persons with Alzheimer’s disease. Furthermore it is an analysis which will endeavor to suggest that within reason, physical therapy and orthopedic practitioners can expect patients with neurological and cognitive losses, to demonstrate functional improvement [1]. Implementing reliable experimental, clinical research and quasiexperimental design methodologies will contribute to the establishment of scientifically based standard-of-care treatment protocols [2]. The construct of innovation in neuromuscular research, is an evident trend in the rehabilitation literature [3-5]. Consistent with this trend, it is hypothesized that by integrating physical therapy treatment with cognitive therapy treatment, patients with progressive neuromuscular decline will achieve better functional outcomes.

Additional evidence that the ryanodine receptor gene (RYR1) causes malignant hyperthermia and severe skeletal malformations

Additional Evidence That the Ryanodine Receptor Gene (RYR1) Causes Malignant Hyperthermia and Severe Skeletal Malformations Yosuke Kakisaka, Kazuhiro Haginoya,* Yuko Takahashi, Tatsuhiro Ochiai, Ikuma Fujiwara, Atsuo Kikuchi, Keisuke Wakusawa, Satoru Kobayashi, Hirosato Kikuchi, Yasuko Ichihara, Shinichiro Takahashi, and Ichizo Nishino Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan Department of Pediatric Neurology, Takuto Rehabilitation Center for Children, Sendai, Japan Department of Orthopedics, Takuto Rehabilitation Center for Children, Sendai, Japan Department of Anesthesiology, Saitama Medical University Hospital, Saitama, Japan Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan

Developing normalized strength scores for neuromuscular research

The Accurate Test of Limb Isometric Strength (ATLIS) device can reliably measure the strength of 12 muscle groups using a fixed load cell. The purpose of this study was to analyze ATLIS data from healthy adults to calculate an individual's predicted strength scores. ATLIS data were collected from 432 healthy adults. Linear regression models were developed to predict each muscle group's strength. The R-squared statistic assessed variability accounted for by the models. Simple main effects models stratified by gender were used to establish regression equations for each muscle using factors of age, weight, and height. Normalizing raw strength scores controls for biometric factors, thus enabling meaningful comparisons between subjects and allowing each muscle to contribute equally to a summary score. Normalized scores are easily interpreted for broad clinical uses, and derived summary scores establish individuals' disease progression rates using a common scale, allowing for more efficient clinical trials.

S.P.17 Standards of care: Ensuring physiotherapists are competent to treat babies and children with neuromuscular disorders

Abstract There are 16 centres in the UK offering specialist neuromuscular services; some very large, with five full time neuromuscular physiotherapists and additional research physiotherapists. Others are small having one or two clinics per month. There are no specific qualifications in the UK that actually designate a paediatric physiotherapist, although there is the qualification of a masters degree in paediatric physiotherapy. This is an academic qualification and does not confer experience or expertise in any specific area. Many newly qualified therapists however are going straight to a Masters degree in the hope of securing future employment. So which physiotherapists are qualified to assess and treat children with neuromuscular disorders? Who teaches them and who teaches the teachers? The Association of Paediatric Physiotherapists (APCP) a clinical interest group and the official voice of paediatric physiotherapy in the UK has initiated a programme of published competencies for physiotherapists; the standards for physiotherapists working in neonatology having been completed. The standards for physiotherapists working in neuromuscular disorders are now being agreed by the relevant stakeholder physiotherapists in the field, based where possible, on published evidence. Understanding the underlying pathology, knowing the presenting features and progression of individual conditions; and managing the many aspects of treatment and rehabilitation are covered. The topics include assessment, respiratory treatment, mobility, management of contractures and deformity and appropriate exercise and activity. Once finalised, these topics will form the basis for development of expert clinical practise and will enable the running of courses in advanced practise.

Quality of life and measures of quality of life in patients with neuromuscular disorders

In this review we present an overview of quality of life (QOL) and QOL measures in neuromuscular disorders. We discuss the characteristics of QOL measures used in neuromuscular research, highlighting differences between generic versus disease-specific and global versus health-related QOL instruments. The phenomenon of response shift is reviewed. Commonly used QOL instruments are reviewed for amyotrophic lateral sclerosis, muscle diseases, myasthenia gravis, and polyneuropathy. We also review some of what is known about QOL for patients with these neuromuscular disorders.

Ultrasound Is Superior to Magnetic Resonance Imaging for Detecting Peripheral Nerve Pathology in Mononeuropathies and Brachial Plexopathies (P06.149)

Objective: Compare accuracy of magnetic resonance imaging (MRI) and ultrasound (US) in detecting peripheral nerve pathology. Background It is unknown whether MRI or US is superior for detecting peripheral nerve pathology. Design/Methods: We reviewed charts of 141 cases referred for neuromuscular US between 6/2007 and 10/2011 for suspected upper and lower extremity mononeuropathy(ies) or brachial plexopathy, excluding idiopathic carpal tunnel syndrome and ulnar neuropathy at the elbow, and identified cases who underwent MRI of the same limb for the same indication as the US. Imaging results were compared to the gold standard of surgical or, if unavailable, clinical/electrodiagnostic evaluation. Results: We identified 52 cases evaluated with both MRI and US. Focal nerve pathology was established in 46 and excluded in six by surgical (39) or clinical/electrodiagnostic evaluation (13). Diagnoses included peripheral nerve sheath tumors (18), traumatic (10) or idiopathic (8) neuropathy, fibrosis (4), compression by ganglion/synovial cysts (4) or other soft-tissue structures (3), non-nerve soft tissue tumor (3), intraneural granuloma (1) and vasculitis (1). US was more sensitive than MRI (93 vs. 70%) and equally specific (both 83%). Pathology accurately identified on US but not MRI included nerve enlargement (7), peripheral nerve sheath tumor (3), compression by anomalous muscle (1), and a hemangioma identified as non-nerve pathology on US but not MRI. Of these 12 cases with inaccurate MRI but not US, eight (67%) had lesions larger than two centimeters; only two lesions were outside the field of view of the MRI. In three cases classified as accurate for a single lesion on both MRI and US, US identified 11 additional lesions (multifocal neurofibromas, nerve enlargements, and fibrous thickening) not seen on MRI. Conclusions: Ultrasound is more sensitive, has equivalent specificity, and may better identify multifocal lesions than MRI in suspected mononeuropathies and brachial plexopathies. Supported by: This publication was made possible in part by the Washington University Neuromuscular Research Fund and the Grant Number UL1RR024992 from the NIH-National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Disclosure: Dr. Zaidman has nothing to disclose. Dr. Seelig has nothing to disclose.

TDP-43 Drives Nuclear Factor- B Activation in ALS (P03.179)

Objective: Our objective was to search for proteins which might interact with TDP-43 and to investigate the physiological significance of such interaction. Background TDP-43 inclusions are a hallmark of amyotrophic lateral sclerosis (ALS) and dominant mutations in TARDBP, which codes for TDP-43, were reported by several groups as a primary cause of ALS. However, the physiological role of TDP-43 and the pathogenic pathways of TDP-43 abnormalities are not well understood. Design/Methods: Real-time RT-PCR and ELISA revealed that the levels of mRNA and protein for TDP-43 was elevated in the spinal cord of sporadic ALS cases. Mass spectrometry and co-immunoprecipitation assays were carried out to identify proteins which interact with TDP-43. Transgenic mice overexpressing human TDP-43 by 3 folds and control littermates were used as a source of primary cells and for therapeutic testing of withaferin A. Results: We have found that TDP-43 and NF-κB p65 mRNA and protein expression is higher in spinal cords if ALS patients than healthy individuals. TDP-43 interacts with and colocalizes with p65 in glial and neuronal cells from ALS patients and mice expressing wild-type and mutant TDP-43 transgenes, but not in cells from healthy individuals or nontransgenic mice. TDP-43 acted as a co-activator of p65, and glial cells expressing higher amounts of TDP-43 produced more proinflammatory cytokines and neurotoxic mediators after stimulation with lipopolysaccharide or reactive oxygen species. TDP-43 overexpression in neurons also increased their vulnerability to toxic mediators. Treatment of TDP-43 mice with withaferin A, an inhibitor of NF-κB activity, reduced denervation in the neuromuscular junction and ALS disease symptoms. Conclusions: We conclude that TDP-43 deregulation contributes to ALS pathogenesis in part by enhancing NF-κB activation, and that NF-κB may constitute a therapeutic target for the disease. Supported by: The Canadian Institutes of Health Research (CIHR) and Neuromuscular Research Partnership, the Robert Packard Center for ALS Research at Johns Hopkins and the Fondation Andre-Delambre. Disclosure: Dr. Julien has received personal compensation for activities with Biogen Idec as a consultant. Dr. Swarup has nothing to disclose. Dr. Dupre has nothing to disclose. Dr. Kriz has nothing to disclose.

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures. Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium. The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context. Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures. Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium. The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context. Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.