Volumetric muscle loss (VML) resulting from extremity trauma presents chronic and persistent functional deficits which ultimately manifest disability. Acellular biological scaffolds, or decellularized extracellular matrices (ECMs), embody an ideal treatment platform due to their current clinical use for soft tissue repair, off-the-shelf availability, and zero autogenous donor tissue burden. ECMs have been reported to promote functional skeletal muscle tissue remodeling in small and large animal models of VML injury, and this conclusion was reached in a recent clinical trial that enrolled 13 patients. However, numerous other pre-clinical reports have not observed ECM-mediated skeletal muscle regeneration. The current study was designed to reconcile these discrepancies. The capacity of ECMs to orchestrate functional muscle tissue remodeling was interrogated in a porcine VML injury model using unbiased assessments of muscle tissue regeneration and functional recovery. Here, we show that VML injury incites an overwhelming inflammatory and fibrotic response that leads to expansive fibrous tissue deposition and chronic functional deficits, which ECM repair does not augment. Encouraging results in animal models, in which implantation of dissociated native muscle elements necessary for canonical muscle fiber regeneration effectively promoted de novo muscle fiber regeneration and neuromuscular functional recovery7, highlight the potential of regenerative therapeutics. The challenge now is to delineate which of these native regenerative elements (or their surrogates) must be implanted to instruct and interact with the host in vivo.Biomaterial-based technologies that enhance host tissue regenerative responses hold significant promise for restoration of functional soft tissue. Traumatic partial ablation of skeletal muscle, or volumetric muscle loss (VML) injury is particularly in need of an effective regenerative therapy. VML injury presents a defect region in which all native elements required for canonical skeletal muscle regeneration  are removed; and, because adult mammalian skeletal muscle is not adept at 8). Implanted ECMs are hypothesized to create a regeneration-permissive environment within the defect area that chemoattracts and modulates the myriad of cell types needed to orchestrate regeneration of functional skeletal muscle tissue. The existing literature reports mixed results on the capacity of ECMs to promote this form of skeletal muscle tissue specific constructive remodeling10. The potential of constructive remodeling, however is supported by an impressive report of a physiologically relevant magnitude of muscle tissue regeneration following an ECM implantation in a large animal model11.Acellular biological scaffolds, or extracellular matrices (ECMs), are biological materials that have been widely evaluated for VML repair due to their off-the-shelf availability, and zero autogenous donor tissue burden. Various ECMs derived from different species and tissues using proprietary decellularization methods are in clinical use for several soft tissue indications, to include herniated abdominal muscle wall repair. While ECM devices are FDA approved for soft tissue reinforcement with general claims of host tissue deposition, which may potentially be fibrotic, the basic literature is comprised of numerous reports that have investigated if ECMs specifically promote functional muscle tissue regeneration  and urinary bladder matrix (UBM) derived ECM repair of VML injury to improve functional outcomes. Using muscle function as a metric of efficacy, in comparison to the injured limb pre-surgically 7 patients improved function, 3 had no change, and 3 had subsequent loss of function. ECM repair resulted in bulking of the muscle unit; however, further investigation using muscle biopsies does not conclusively present evidence of muscle fiber regeneration in the area of implantation beyond islands of muscle fibers (~10–50 muscle fibers). Moreover, these fibers are unlikely to significantly contribute to voluntary contractile force in human muscles, which are comprised of hundreds of thousands of muscle fibers14. Regardless, the stated conclusion drawn in this work is that biomaterial mediated constructive remodeling promotes de novo muscle tissue regeneration that improves limb function15.A recent clinical trialThe difficulty of powering clinical trials to determine efficacy and mechanism of action, matched with the discrepancies within the basic science literature in demonstrating ECM-mediated functional muscle tissue regeneration currently confounds clinical recommendation of ECMs for VML injury repair. Herein we performed a translational study in a porcine VML injury model to interrogate the capacity of commercially available ECMs to promote functional recovery of VML injured musculature and used unbiased analyses for functional recovery and regeneration of muscle tissue.17. Herein, VML injury was surgically created bilaterally in the porcine peroneous tertius (PT) muscle, the primary dorsiflexor muscle, by excising 5.10 ± 0.03 g muscle tissue to create a defect of ~3 × 3 × 1 cm in the middle third of the musculature  was selected because it lacks cell binding motifs and matricryptic peptides20 that are presumptively pivotal to scaffold mediated regeneration9. In a pilot study, Hya was implanted into a tibialis anterior (TA) muscle rat VML model and assessed for in vivo TA muscle strength as a function of stimulation frequency and muscle tissue regeneration 8 weeks post-injury. Non-repaired TA muscles presented a 46–61% strength deficit, which Hya did not improve at any stimulation frequency . Lastly gene expression of inflammatory cytokines  and myogenic transcription factors (myogenin and Pax7) key to skeletal muscle regeneration was analyzed in TA muscles 2 weeks post-injury to determine if Hya impacted the molecular phenotype of the healing response. Expression of all genes was similarly up-regulated in Hya and non-repaired muscles (p ≥ 0.421) when normalized to uninjured contralateral muscles. Collectively, these results supported Hya as a suitable negative control for further testing alongside selected ECMs in the porcine PT VML model.As an initial step in determining the capacity of an ECM to promote constructive remodeling of skeletal muscle in a porcine VML model, we sought to establish a biomaterial negative control incapable of orchestrating ECM-mediated constructive remodeling. In previous rodent VML models, ECM implantation promoted functional improvements via putative improvements in fibrosis-mediated force transmissionThe remaining studies were conducted to determine if ECM repair (SIS or UBM) of VML injury improves neuromuscular strength and muscle tissue regeneration compared to injuries left non-repaired or repaired with Hya . PT muscle VML injuries were repaired immediately by an orthopaedic surgeon and observed out to 12 weeks post-injury.Swelling was obvious within the initial weeks post-surgery in the non-repaired and UBM-repaired limbs compared to sham-operated limbs. Negligible swelling on par with sham-operated limbs was observed in the Hya-repaired limbs. SIS-repaired muscles presented greater swelling than all other conditions. 12 weeks post-VML injury, the majority of limbs displayed ambiguous muscle unit borders between the PT muscle and the lateral or medial synergist muscles in the anterior compartment. There was apparent tethering between the PT muscle and synergists muscle, fascia, and the skin. In all cases, the fibrosis was greatest on the lateral aspect of the limb. The fibrosis and the tethering throughout the lower limb was most pronounced in the SIS-repaired limb, in which often there were nodules or hardened aspects of the fascia outside of the muscle. During muscle harvesting, the fibrous tissue bled extensively. In contrast to the ECM-repaired limbs, discrete muscle boundaries within the anterior compartment were observed and tissue tethering was present but markedly reduced following repair with Hya.2 and involved implantation of three overlying sheets, introduced too great of a biological burden and caused wound dehiscence. Importantly, no adverse outcomes were reported using SIS or any other ECM device in a recently completed clinical trial of VML repair22.Notably, following the initial surgery and repair, unexpected and significant complications developed after SIS ECM-repair  positive muscle fibers were observed in close proximity to the remaining musculature in each group; however, muscle fibers were not present throughout the defect Fig. . Moreove9. We therefore performed immunohistological analysis of full-thickness PT muscle sections to gain insight into which cell types successfully migrated and survived in the defect area 12 weeks post-injury  and 11,506 total mRNAs were detected. In total, 3,996 genes were differentially expressed when compared to sham-operated, and the SIS-repaired displayed the highest number of differentially expressed genes compared to other repaired and non-repaired muscle . Thus, an alternative mechanism of functional recovery was proposed in rodent VML injuries, wherein ECM-related fibrous tissue deposition improved the transmission of forces produced by the remaining muscle mass 19. We therefore anticipated that ECM-repaired muscles would impart a modest functional improvement and that this mode of functional improvement could therefore explain the modest gains in isometric or isokinetic strength observed clinically12. However, the current findings do not support any mechanism of strength improvement and suggests that the putative mechanism of ECM improved force transmission is not conserved in larger VML defects. The drastic difference in fibrotic tissue deposition between porcine and rat VML models may account for this discrepancy  and therefore by-pass voluntary muscle recruitment strategies, while human strength assessments are voluntary and dependent on recruitment characteristics. It is therefore possible that the modest strength gains in the clinic may be due to neuromuscular recruitment adaptations that occur in the absence of de novo muscle tissue regeneration or even skeletal muscle hypertrophy.Previous preclinical observations in rodent models of VML reported ECM-mediated improvements in nerve-evoked muscle strength. For instance, rodent studies using the same commercial UBM37, where increases in expression of inflammatory and ECM remodeling genes temporally subsided after injury38, VML induces overexpression of these transcripts up to three months after injury. Detection of these transcripts was consistent with histological observations of macrophage infiltration into the VML defect site. Macrophages have been shown to secrete pro-fibrogenic factors such as TGF-β1 and upregulate components of the Complement system to perform phagocytosis after muscle injury. Both of these elements were significantly upregulated and enhance Wnt signaling, which has also been shown to attenuate satellite cell proliferation and enhance fibroblast production of collagen39. Thus, sustained activation of these gene expression programs from a VML injury may contribute to a poor environment for satellite cells and hinder their repair of the defect site, regardless of increased progenitor recruitment from an ECM. Since none of the ECMs directly impacted these expression programs, the underlying pathogenesis of VML was effectively unaltered and produced nearly indistinguishable transcriptomes at the tissue level. Collectively, the gene expression programs observed support a model whereby incessant inflammation from infiltrating macrophages results in over-activation of Complement, which in turn initiates Wnt signaling in mesenchymal progenitors and fibrogenesis40. This model of chronic inflammation resulting in defective satellite cell responses, enhanced proliferation of matrix-producing cells and increased ECM deposition has also been observed in muscular dystrophy41 and is uniquely consistent with other models of muscle tissue degeneration and fibrosis25.In contrast to previous muscle regeneration studies43, but remain at odds with the robust regeneration presented by Turner et al.11. While large-scale tissue regeneration is challenged by the absolute distances required for revascularization and reinnervation, the fairly consistent observation of sub-physiological muscle regeneration following ECM repair for VML in both small and large animal models challenges the broader efficacy of ECM mediated muscle tissue regeneration9. The current study unequivocally indicates that ECM repair provides no functional benefit and suggests a unique pathology of chronic inflammation and persistent connective tissue deposition which may inhibit satellite cell based repair. The sub-physiological level of muscle tissue regeneration observed herein with any of the biomaterials implanted supports the continued development of biomaterial-based therapies that utilize biological adjuncts46 to potentially achieve a functionally meaningful magnitude of muscle tissue regeneration. However, the unwavering pathobiology of sterile VML injury observed herein regardless of immediate biomaterial repair draws significant concerns regarding the prolonged health of the remaining musculature and suggests that therapeutic intervention may be better served at attenuating pathology within the remaining musculature, as opposed to focusing solely on de novo muscle tissue regeneration. Collectively, this study argues that the use of ECMs for functional muscle repair following VML injury clinically is at best premature.The current observations are in line with the low magnitude of muscle fiber regeneration presented after VML repair in a host of small and large animal modelsFemale Yorkshire Cross pigs (n = 13) were purchased form Midwest Research Swine . In pilot studies, male Lewis rats (n = 20) were purchased from Harlan Laboratories . All protocols and animal care guidelines were approved by the United States Army Institute of Surgical Research Institutional Animal Care and Use Committee (A-14-018 & A-14-017), in compliance with National Institute of Health Guidelines and the United States Department of Agriculture. All studies were conducted in compliance with the Animal Welfare Act, the Implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals.Studies were conducted to test currently clinically utilized ECMs and a biomaterial negative control. At the time of surgery bilateral VML injuries (or sham procedures) were performed and then each limb was randomized to no repair or implantation with a commercially available ECM or Hya  was surgically removed using sharp dissection from the middle third of the PT muscle  for pain management. Rats were randomized to repair with Hya or left non-repaired. Data was compared to the contralateral non-injured limb at 2 or 8 weeks post-surgery.2, 37 °C). Following the VML injury, the muscle fascia was sutured with interrupted and locking proline sutures and the Hya was placed in the fascia pocket with a 16 G syringe. For the sham operated and non-repaired limbs, sutures were placed to mark the muscle and VML defect, respectively  that was explored (Lot #LB835143) is a multi-layer acellular ECM manufactured from animal derived non-cross linked collagen . The second biologic wound matrix  that was used is a multi-layer ECM manufactured from urinary bladder matrix (UBM) with an intact epithelial basement membrane and various collagens . Prior to implantation each ECM was prepared following individual manufacture suggestions. Briefly, under sterile conditions the ECM was rehydrated in room temperature saline and trimmed to fit the VML defect site. The ECM was circumferentially anchored in place by ~12–15 interrupted and locking proline sutures. An alternative biomaterial negative control using Hya was explored. Hya is a naturally occurring component of the extracellular matrix; it is biodegradable, non-immunogenic and anti-adhesive. In this context the use of Hya as a hydrogel was to provide a barrier for cell migration within the VML defect site. A pre-polymer solution consisting of 1% weight/volume thiol-modified hyaluronan  and 1% PEGDA (MW 3.4kDA) in degassed sterile water was mixed, drawn into a 30 ml syringe, and subsequently allowed to cross-link at 37 °C for 30 minutes in a humidified incubator . In all cases, torque was measured over a range of joint angles. Body weight was assessed immediately prior to each procedure and used to normalize torque values. When available the torque was also normalized by the muscle volume of the anterior compartment as determined by CT imaging.The strength of the dorisflexor muscles of the anterior compartment in each leg of pigs was tested with the knee stabilized at 90° and activated by subdermal stimulation of the peroneal nerve using needle electrodes6. The muscle was activated via subcutaneous needle electrodes placed on both sides of the common peroneal nerve and function was assessed with the ankle at a right angle using a dual-mode muscle lever system elicited using 0.1 ms pulse width over 400 ms across a range (10–200 Hz) of frequencies (Grass S88 and 305B Aurora Scientific servomotor).To assess the function of the isolated rat TA muscle the distal tendons of the extensor digitorum longus and extensor hallicus longus muscles severed above the retinaculumin vivo testing parameters. In each pig, both the right and left limbs were tested for isometric torque across seven joint angles . At each angle tested, two or three measurements were taken, with the exception of 55° of plantarflexion when only one measurement was taken due to near end range of motion (plantarflexion 62.4 ± 1.3°). At each joint angle, isometric torque was normalized to body weight and evaluated to determine if differences existed between right and left limbs, the order of the limb tested, and minimum and maximum values obtained for all values. In the whole model  there was no difference between right and left limbs (p = 0.972) or the order of limbs tested (p = 0.999). Furthermore, there were also no differences when examined at individual joint angles  underwent scans prior to surgical procedures and at 2 weeks post-surgery (n = 4). Scans were analyzed with Vitrea Vital Medical Imaging Software to estimate volume of the anterior compartment. The volume was estimated from the coronal 2D images and then reconstructed into a 3D model by an investigator blinded to experimental group. Volume measurements were normalized to body mass, additionally anterior compartment volume was used to normalize muscle torque.Standard imaging studies of the lower limb were conducted using a Toshiba 160 slice CT at 0.5 mm slice thickness. Scans were taken prior to in vivo functional assessment, samples from the PT, tibialis and digitorum synergist muscles were collected. In all cases muscle samples were excised using a 10 mm biopsy punch through the full thickness of the muscle and then frozen in melting isopentane and stored at −80 °C until analysis. Each muscle sample was cross-sectioned at 8 µm and stained with standard histologic procedures for hematoxylin and eosin and Masson’s trichrome. Bright filed images were acquired with 20x  objective on a Zeiss Axio Scan.Z1  and stitched into a large composite image. Composite images were saved separately as a 24-bit, 96 dpi color images. Additionally, an independent investigation of the hematoxylin and eosin and Masson’s trichrome staining of the skeletal muscle was conducted by a board certified pathologist blinded to experimental group. Narrative reports were provided for all muscle sections. Histologic samples acquired from the rat TA muscle in the pilot study were processed with the same approach. Quantitative assessments of Hya presence at 2 and 8 weeks post injury were performed by manually circling a region of interest over the observed material using MATLAB .Following the terminal Immunofluorescent staining of the PT muscle was conducted using a combination of sarcomeric MyHC , laminin , Pax7 , CD163  DAPI , and Wheat Germ Agglutinin . Appropriately paired secondary antibodies at a dilution of 1:200  were subsequently used. In all cases the expected staining patterns in normal skeletal muscle were observed and the specificity of anti-labeling was confirmed by the absence of staining outside expected structures and was consistent with manufacturer’s technical information.Confocal imaging was conducted with an Olympus FluoView FV1000 laser scanning confocal microscope  mounted on an inverted Olympus IX81 microscope and equipped with Multi-line Argon , HeNeG (543 nm), diode (405 nm) and diode (635 nm) lasers using an Olympus UPLSAPO 20x/0.85 N.A. or 60x/1.35 N.A. oil immersion lenses. Images were acquired in a 512 × 512 array with pixel dimensions (0.5 × 0.5 μm). Confocal images were saved as individual channel 12-bit multi-TIFF files, and reconstructed in MetaMorph . For display purposes only images were down-converted, without introducing any changes in brightness or contrast. Laser intensity was held consistent across imaging of the same antibody probes. During all imaging and investigation, investigators were blinded to experimental group.http://www.hartfaalcentrum.nl) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the endogenous control49.Real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to quantify gene expression within the VML defect region at 2 weeks post-injury. RNA was isolated from the homogenized tissue using the TRIzol method. The RNA samples were reverse transcribed into cDNA using a QuantiTech Rev Transcription kit  according to the manufacturer’s protocol. Gene expression for target markers was determined using custom-designed primers  for 30 seconds at room temperature and total RNA was isolated from the slurry using the miRNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. RNA concentration and integrity were measured with a Nanodrop spectrophotometer (Nanodrop 2000c) and Bioanalyzer (Agilent 2100), respectively. 1 μg of isolated total RNA was used to produce cDNA libraries using the Truseq protocol (Illumina), as per the manufacturer’s instructions. Biological replicates or triplicates were produced from each condition and individual libraries were pooled and sequenced using 76 base-pair (bp) paired-end reads on a Hi-Seq. 2500 in high output mode to an average depth of 55 M reads per library.Sus scrofa (Pig) genome (build 9.2) and transcript abundances were quantified using Kallisto50 with default parameters. Differential expression (DE) of transcripts was calculated using the R/Bioconductor implementation of DESeq. 251. Analysis of expression data, such as set analysis based on treatment specific expression profiles, principal component analysis, and correlation of inter-treatment gene expression were performed with R. Count data was log2 transformed, after adding a pseudocount of 0.5 to all measurements. Differential gene expression (relative to sham controls) in gene matrixes and plots were called significant at a False Discovery Rate  of less than 0.05. Given the high number of genes exhibiting significant differential expression, additional filters were set to obtain DE genes for further analysis . Treatment-group-specific DE genes were computed and set analysis was used to determine overlapping and unique genes between groups using base R libraries and plotted using R package VennDiagram (version 1.6.17). Principal component analysis was performed on significant DE genes using base R libraries and plotted using ggplot2 (version 2.1.0).High-throughput sequencing reads were pseudoaligned to the University of California, Santa Cruz, annotated 52, REACTOME53, and BioCyc54. GO terms were limited to Biological Processes. Pathway analysis was conducted on qualified genes using the R/Bioconductor package GAGE55 and significant  pathways were ranked in order of significance. Pathways selected were within the top 50 ranked up- or down-regulated pathways and reflected general trends with those top 50 pathways. A matrix of treatment group x treatment group Pearson correlation values, generated using treatment expression profiles and base R libraries, was calculated and plotted using the R package pheatmap (version 1.0.8).Significant DE genes were also used to compute Gene Set Enrichment Analyses. Gene Sets used for enrichment analyses were obtained from the Gene Ontology ConsortiumRNA-Seq datasets are uploaded onto GEO and accession numbers are pending.All quantitative data was analyzed using JMP . Treatment group sample sizes of at least 3 were determined prior to the study to allow for the detection of a 33% increase in muscle torque production at power (1 – ß) of 0.80 and α of 0.05, with a shared standard deviation of 0.025 Nm/kg determined in a functional optimization study. Body weight was analyzed by one-way repeated measures ANOVA  over the 12 weeks study. Pre-surgical data was analyzed one-way ANOVA to determine any differences existed across groups, no differences in pre-surgical data were determined (p ≥ 0.070) and therefore pre-surgical data was collapsed across experimental groups. Data was analyzed by one-way ANOVA (group) for anterior compartment volume, torque, RT-PCR, and Hya degradation, or two-way ANOVA for peak torque over time or stimulation frequency (group x time point/frequency). Tukey’s post-hoc analyses were performed upon significant ANOVA. Statistical handling of RNAseq data are described above. Data are reported as mean ± SE and significance was determined at the α < 0.05 level.The opinions or assertions contained here are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army, the Department of Defense, nor the U.S. Government.Supplementary Data


Walking is of high priority for people with multiple sclerosis (PwMS). It remains unclear whether aerobic exercise can improve walking ability and upregulate neurotrophins. This review aims to consolidate evidence to develop optimal aerobic training parameters to enhance walking outcomes and neuroplasticity in PwMS. Clinical studies examining aerobic exercise for ≥3 weeks, having outcomes on walking with or without neurotrophic markers, were included. Studies utilizing animal models of MS were included if they employed aerobic exercise with outcomes on neurological recovery and neurotrophins. From a total of 1783 articles, 12 clinical and 5 animal studies were included. Eleven clinical studies reported improvements in walking ability. Only two clinical studies evaluated both walking and neurotrophins, and neither found an increase in neurotrophins despite improvements in walking. Patients with significant walking impairments were underrepresented. Long-term follow-up revealed mixed results. Two animal studies reported a positive change in both neurological recovery and neurotrophins. Aerobic exercise improves walking ability in PwMS. Gains are not consistently maintained at 2- to 9-month follow-up. Studies examining levels of neurotrophins are inconclusive, necessitating further research. Aerobic exercise enhances both neurological recovery and neurotrophins in animal studies when started 2 weeks before induction of MS. Multiple sclerosis (MS) is a demyelinating autoimmune disease affecting approximately 2.3 million people worldwide . ImproveWalking is of high priority for PwMS  and therAerobic exercise is one intervention that has potential to affect multiple underlying targets such as enhancing markers of neuroplasticity, attenuating neural inflammation, and improving tolerance for physical activity, and because of reciprocal limb movements, it also helps restore walking ability , 15. EviThe regulation of neurotrophic factors has been implicated in the repair of neural structures damaged by the demyelination process, resulting in functional recovery in PwMS . CurrentThe primary aim of this review was to systematically evaluate the clinical (human) studies examining the effects of aerobic exercise on walking ability in MS. The second aim was to determine the aerobic exercise training parameters (FITT) that enhance both walking ability and proneuroplastic biomarkers (neurotrophins) in PwMS. The third aim was to analyze the extent to which aerobic exercise protocols evaluated in animal research can be translated into clinical practice.Randomized clinical studies that evaluated the effects of aerobic/endurance-type exercise programs  among PwMS for a duration of at least 3 weeks were eligible for this review. Studies with outcomes on walking ability (primary study outcome) evaluating spatiotemporal parameters and/or endurance with or without serum levels of neurotrophins  were included.We also included randomized controlled studies in animal models of MS  or cuprizone). Animal studies in which aerobic-type exercise  was evaluated for its effects on gait and neurotrophins in the blood/muscle/brain/spinal cord, performed both before and after disease induction, were included.The studies that evaluated slow-paced exercise or combination training with low aerobic workload  were excluded. Only English language articles were included.https://doi.org/10.1155/2017/4815958). Two authors screened and assessed the eligibility of each article separately. Review articles and eligible articles were hand-searched for relevant references. The search strategy is presented in A systematic literature search was conducted in PubMed, EMBASE, Cochrane, Scopus, and Physiotherapy Evidence Database (PEDro), using a combination of keywords  and MESH/EMTREE terms in the respective databases  included in this systematic review were assessed for methodological quality using the Physiotherapy Evidence Database (PEDro) scale criteria , analysis , 42, 44 analysis .In total, only four of the twelve studies evaluated the retention of training effects after the conclusion of aerobic intervention  43, 46––46–48. An = 3) reported varied results, with one study having no improvements in both walking domains compared to over-ground walking training [Our results indicate that most aerobic inventions that utilize the reciprocal motion of walking  training , 48.2/kg/m) [There were conflicting findings in the studies that provided aerobic exercise without gait training such as leg/arm cycling, calisthenics, and combined endurance and resistance training. One study that evaluated leg cycling reported improvements in 6-minute walking endurance but not in the cost of walking (mL O2/kg/m) . Another2/kg/m) , and las2/kg/m)  Table 32/kg/m [T  Frequency: three times per week for at least 6–8 weeks  Intensity: 40–75% age predicted maximum heart rate or 30–60% work rate for those with low to moderate levels of disability (EDSS < 6); maximum walking speed tolerated for people with higher levels of disability (EDSS ≥ 6)  Time: at least 30 minutes per session  Type: aerobic-type training on a treadmill (EDSS < 6)/leg cycling (EDSS ≤ 6)/game based or combined aerobic and strengthening exercise (EDSS < 6)/calisthenics (EDSS < 4.5)/robot-assisted treadmill (EDSS 5–7)We summarized the findings in We identified eleven clinical studies that reported significant improvements in walking outcomes, out of which two measured both walking and serum levels of neurotrophins , 45.In the study by Schulz et al. , aerobicIn the study by Briken et al. , walking  Frequency: 2 to 3 times per week for at least 8 to 9 weeks  Intensity: light to hard , interva  Time: at least 30 minutes per session  Type: aerobic-type leg cycling (EDSS < 6)There is not enough data to extrapolate our findings and suggest optimal exercise parameters that could improve walking and upregulate neurotrophins. However, based on two clinical studies , 45, 54, before EAE induction [n = 2), spinal cord (n = 1), serum (n = 1), and muscle (n = 1) (online supplement c). All of these studies also initiated exercise before induction of EAE. In one study [We identified 5 studies that investigated the effects of aerobic exercise on neurological status and neurotrophins in animal models of MS (online supplement c). Only two studies showed significant improvements in neurological status and both instituted exercise for 2 weeks or morenduction , 51   recommen before the induction of EAE in the animal.In this review, we sought to identify the optimal type of aerobic exercise and training parameters that could lead to improvements in walking ability in PwMS and promote brain repair through the upregulation of neurotrophic factors. We report five key findings: (1) the clinical studies were of fair to good quality and consistently showed that aerobic interventions (ranging from mild to vigorous intensities) improved walking endurance and spatiotemporal parameters of gait in people with EDSS scores less than 6 ; interventions that did not employ the reciprocal motions of walking  improved walking endurance more consistently than they did for the spatiotemporal parameters; (2) very few studies examined whether effects were sustained after cessation of the intervention, and those that did showed that most outcomes return to baseline within a few months; (3) people with severe MS-related walking impairments (EDSS 6 and above) were relatively underrepresented in the studies; (4) in clinical studies, neurotrophins were not reliably changed with aerobic exercise; (5) in animal studies, both neurotrophins and neurological status were improved when aerobic exercise began more than 2 weeksOur findings from 12 clinical studies suggest that aerobic exercise targeting the reciprocal movements of gait per se is not required in order to improve walking in MS. Participants also had improved walking endurance and walking quality with nongait activities such as leg/arm cycling, swimming, and calisthenics. Physical therapists, therefore, can use multiple aerobic exercise modalities to affect gait. This is particularly important for home-based and community-based exercise which may make use of arm cycling or swimming. Our findings are similar to those in chronic neurological conditions like stroke, cerebral palsy, and Parkinson's disease –60 whichOnly four of the 12 studies examined whether improvements were sustained after cessation of the training program and most showed that outcomes return to baseline within a few months . It is nIt is important that research undertaken to improve gait include people with MS who have problems with walking. Eight of the 12 studies included participants who had EDSS scores less than 3 and even EDSS 1, suggesting very minimal impairment levels . ClearlyPeople having an MS-related disability often report higher rates of exercise-induced fatigue . Future We aimed to examine the findings in animal studies to determine their applicability to MS clinical research. Of the five studies examining aerobic exercise in an animal model of MS (EAE), exercise benefited walking and increased neurotrophins only when instituted two weeks or more before EAE induction. This suggests that aerobic exercise is likely neuroprotective but provides little benefit when employed after MS is induced in the animal. The neuroprotective effects of exercise have been reported in animal models of ischemic stroke  in whichWe also showed that, with only two clinical studies and four animal studies examining BDNF as a potential biomarker of plasticity, the results are inconclusive on whether serum levels of BDNF indicated exercise-related repair of the CNS. However, a recent meta-analysis of 13 studies on a mixed population  showed increased magnitude of BDNF responsivity and higher resting levels of BDNF after exercise training . FurtherConsolidated evidence suggests that aerobic exercise training can improve walking ability  in people having MS without severe walking impairments. In this review, we have outlined the optimal aerobic training parameters (30 min 3x week for 6–8 weeks at mild to vigorous intensity) that improved walking in people with EDSS scores less than 6.0 . Although individual studies reported that gait-specific and non-gait-specific types of aerobic exercise improved both endurance and spatiotemporal parameters of walking, the effects of the aerobic exercise were not sustained more than six months after the end of intervention. There is a need to build exercise programs for people living with MS having higher disability, especially EDSS 6.0 or above, to restore their lost ability to walk.In PwMS, the serum levels of neurotrophins measured at rest did not significantly change after completing a course of aerobic training. In contrast, the animal studies showed a significant change in both neurological recovery and neurotrophins in blood, muscle, and nervous tissue, especially when aerobic exercise begins 2 weeks before EAE induction.Search strategy and further details of included studies.


Trisomy of chromosome 21, the genetic cause of Down syndrome, has the potential to alter expression of genes on chromosome 21, as well as other locations throughout the genome. These transcriptome changes are likely to underlie the Down syndrome clinical phenotypes. We have employed RNA-seq to undertake an in-depth analysis of transcriptome changes resulting from trisomy of chromosome 21, using induced pluripotent stem cells (iPSCs) derived from a single individual with Down syndrome. These cells were originally derived by Li et al, who genetically targeted chromosome 21 in trisomic iPSCs, allowing selection of disomic sibling iPSC clones. Analyses were conducted on trisomic/disomic cell pairs maintained as iPSCs or differentiated into cortical neuronal cultures. In addition to characterization of gene expression levels, we have also investigated patterns of RNA adenosine-to-inosine editing, alternative splicing, and repetitive element expression, aspects of the transcriptome that have not been significantly characterized in the context of Down syndrome. We identified significant changes in transcript accumulation associated with chromosome 21 trisomy, as well as changes in alternative splicing and repetitive element transcripts. Unexpectedly, the trisomic iPSCs we characterized expressed higher levels of neuronal transcripts than control disomic iPSCs, and readily differentiated into cortical neurons, in contrast to another reported study. Comparison of our transcriptome data with similar studies of trisomic iPSCs suggests that trisomy of chromosome 21 may not intrinsically limit neuronal differentiation, but instead may interfere with the maintenance of pluripotency. ADARB1, encoding one of two genes responsible for adenosine-to-inosine RNA editing [Down Syndrome (DS) results from an extra copy of chromosome 21, and this change in gene dosage has been proposed to alter chromosome 21 gene expression. Chromosome 21 trisomy also has the potential to alter the global transcriptome, either by secondary effects of chromosome 21 gene over-expression, or as a byproduct of additional genetic material itself [ editing ; and DYR editing . IdentifInitial gene expression studies that sought to establish chromosome 21-dependent transcriptome changes using human material were confounded by the effects of different genetic backgrounds in the DS and control individuals who were studied . An alteRecently, the development of paired disomic/trisomic induced pluripotent stem cells (iPSCs) derived from the same trisomic individual (or from discordant monozygotic twins) has allowed the measurement of human trisomy 21-dependent gene expression independent of genetic background differences –15. ThesIn contrast to the Jiang et al study , the triTrisomic (C2 and C3) and disomic iPSC cell lines , which had been developed from an individual with Down syndrome by Li et al. , were ob3 cells/cm2 in mTeSR1 medium + 10 μM Y27632 ROCK inhibitor (Tocris). After 2–4 days, the cells formed a meshwork of dense ridges with mostly open spaces in between. The differentiation process was begun (day 0) by feeding the cells every other day for 12 days with NIM neural induction medium (Stem Cell Technologies), followed by DDM medium supplemented with 2% B27 [iPSC cells were induced to differentiate to cortical neurons by a modification of the method of Espuny-Camacho et al. . Brieflyh 2% B27  every oth 2% B27 . Every 4Successful differentiation of iPSCs into cortical neuronal cultures was confirmed by immunofluorescence staining for markers of cortical layers V  and VI (transcription factor TBR1), as well as for glutamatergic markers vGLUT1 (encoded by SLC17A7) and GRIK2 .For live cell staining, StemGent antibodies conjugated to Dylight 488: Tra1-60 or Tra1-81, were applied to iPSC cultures for 30–60 min according the manufacturer’s instructions, and the stained colonies were examined with an Evos FL microscope.For staining of fixed cells, cells were grown on chamber slides (LabTek II CC2), rinsed with Dulbecco’s PBS, fixed 15 min in 4% p-formaldehyde/4% sucrose in PBS, permeabilized 7 min in 0.25% Triton X-100 in PBS, and blocked 1–2 hrs with 5% sheep or goat serum in PBS. Primary antibodies, applied overnight at 4°C, were from Abcam: Oct4 (ab19857), Tbr1 (ab31940), Ctip2 (ab18465), GRIK2 (ab53092), vGlut1 (ab72311) and βIII-tubulin (ab7751); from Millipore: MAP2 , MAP2 . The slides were rinsed (5X PBS), secondary antibodies (Life Technologies) were applied for 2 hrs at room temperature, rinsed (4X PBS), stained with DAPI (Sigma D9542), and mounted with ProLong Gold AntiFade (Life Technologies). Images were captured using an epifluorescence microscope (Zeiss Axioskop) equipped with a CCD camera and Slidebook image analysis software (3i). All images comparing trisomic and disomic cells were captured using identical exposures and digital adjustments.Lysates of disomic and trisomic iPSCs were prepared with RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma), and the protein content of each lysate was determined (Bradford assay). Using BioRad reagents and equipment, an equal amount of protein from each lysate was denatured by boiling 5 minutes in 4x Sample Buffer containing DTT; run on a Criterion XT pre-cast gradient gel (4%-12%) in XT-MOPS running buffer at 150 volts for 1.5–2 hours; and blotted via a Trans-Blot Turbo system for 7 minutes using the Mixed Molecular Weight protocol . The blot was washed in TBS-T, , stained with Ponceau S to affirm protein transfer, washed again and blocked in 5% milk in TBS-T before being stained sequentially with MAP2 rabbit polyclonal antibody  and goat anti-rabbit HRP (1:1000). The washed blot was then treated with SuperSignal West Femto Maximum Sensitivity substrate (Life Technologies), diluted 1:10 in distilled water, for 5 minutes while covered, and images were taken using a ChemiDoc-it imaging system and VisionWorks LS software. The blot was stripped and stained for GAPDH (1:2000) as a loading control. Images were analyzed using Fiji (ImageJ) and Excel; integrated density of each band was measured and normalized to the GAPDH signal before comparing the amount of MAP2 in each protein sample.RNA for poly(A) RNA sequencing libraries was extracted from IPSC and neuronal cultures via TRIzol extraction. Genomic DNA was removed using TURBO DNase (Invitrogen). RNA integrity (RIN) was verified on an Agilent 2100 bioanalyzer and only samples with RIN ≥ 7.0 were used for sequencing. Single-end sequencing libraries were prepared by the UCCC Genomics Core  using the TruSeq Stranded mRNA Library Prep Kit (Illumina). Cluster generation and sequencing were performed on the Illumina HiSeq 2000 platform. The reads were de-multiplexed and converted to FASTQ format using CASAVA software from Illumina. Read numbers per sample ranged from 78,091,984–158,679,037, and uniquely aligned reads ranged from 56,727,581 to 100,169,823.Analysis of library complexity and per-base sequence quality (i.e. q > 30) was assessed using FastQC (v0.11.2) software . Low quaWe identified A-G RNA editing sites in the 6 (3 disomic and 3 trisomic) RNA-seq cortical neuronal samples using the SPRINT RNA editing software . First, Splicing analysis was performed using Hartley’s QoRTs / JunctionSeq pipeline . Tophat 5’ TCTCACAGCTCCGACACTAT 3’, REV 5’ GAGTCCAATAAGGCCAGCAA 3’). For the APOO loading control, primers bordering exon 6 and 7 were used . 30ng/μl of cDNA made from a mix of random hexamers, oligo dt, and Superscript IV reverse transcriptase  were used as input for all samples. PCR cycling conditions were set as follows: denaturation for 5 min at 95 °C, followed by 40 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 54 °C, and polymerization for 30s at 72 °C, and a final extension for 10 min at 72 °C. APOO exon 4 exclusion was resolved on 2% agarose gel.To capture the exclusion of APOO exon 4 in trisomic samples we designed primers bordering exon 3 and 5 . qRT-PCRs were run in an ABI 7500 Real-Time PCR System . Relative quantification was determined using the ΔΔCt method.To further confirm splicing changes in APOO we performed quantitative PCR on the remaining cDNA from IPSC disomic samples C244, C243, and IPSC trisomic sample C2B. Following standard protocols, quantitative real-time PCRs (qRT-PCR) were conducted using PerfeCTa SYBR Green SuperMix, Low ROX  for all samples in technical triplicates. Primers used to capture the trisomic exon inclusion were: FWD RepEnrich software was used to generate a count table containing uniquely aligned reads assigned to repetitive elements . An hg19The full datasets of normalized transcript accumulation values for the iPSC studies by Weick et al , Jiang eAll raw sequencing data has been deposited in GEO (GSE101942).Paired trisomic/disomic iPSCs were obtained from the Russell lab and maintained in feeder-free media. Cortical neuronal cultures were derived from the iPSCs following the procedure of Espuny-Camacho et al  with minUsing a false discovery rate (FDR) of 10%, we identified 1644 transcripts  with differential expression between trisomic and disomic iPSCs see . Gene onTo confirm the surprising observation that the trisomic iPSCs appeared to express neuronal transcripts at higher levels than the paired disomic iPSCs, we performed quantitative RT-PCR on a set of neuronal-specific transcripts that showed significant upregulation. Specifically, we chose neurotransmitter receptors , a neuronal cytoskeletal regulator , and two neuronal transmembrane proteins . As shown in To further confirm the more neuronal character of the trisomic iPSCs, we focused on MAP2, a classic neuronal-specific microtubule-binding protein whose transcript was increased an average of 6 fold in the trisomic iPSCs compared to the disomic iPSCs. As shown in Letourneau et al  claimed Using the same parameters applied in the iPSC comparison to the RNA-seq data from the neuronal cultures, we identified 1,377 transcripts  differentially expressed in the trisomic and disomic neuronal cultures See . Gene onAn important caveat of our analysis is that although transcripts for general neuronal markers  were not increased in the trisomic neuronal cultures compared to the disomic neuronal cultures, cortical neuronal differentiation leads to the production of multiple glial and neuronal cell types, and we do not have sufficient data to demonstrate that all cultures were equivalently differentiated. Thus, our RNA-seq data from the differentiated cultures can best be interpreted as demonstrating that the trisomic iPSCs were not limited in their ability to differentiate into cortical neuronal cultures.In both the iPSC and neuronal trisomic cultures, chromosome 21 transcripts were increased overall, at close to the 1.5 fold increase predicted from a simple gene dosage effect . The specific chromosome 21 genes over-expressed were dependent on whether iPSCs or cortical neuronal cultures were being analyzed .ADARB1 encodes one of the two enzymes responsible for post-transcriptional A-to-I editing of RNA. This gene is encoded on chromosome 21, leading to suggestions that excessive RNA editing may occur in trisomy 21 cells. To test this hypothesis, we used SPRINT software to globally identify RNA editing sites in transcriptome data from trisomic and disomic differentiated cortical neuronal cultures.  We identified and scored >250,000 editing sites in both the trisomic and disomic transcriptomes, and quantified fraction of editing across all these sites. Although ADARB1 transcripts appear to be increased in the trisomic neuronal cultures, we did not observe a significant increase in editing levels in the trisomic cultures . We concTo identify differences in splicing between trisomic and disomic cells, we tested multiple splicing algorithms, and chose JunctionSeq see . Using tRetrotransposons, one class of repetitive elements, appear to play an important role in the maintenance of stem cell pluripotency , 31. To The iPSCs and induced neuronal cultures that we have characterized were derived from a single individual, thereby intrinsically limiting the generalization of our results. However, three datasets have been published that have attempted a similar transcriptome characterization using trisomic/disomic iPSC pairs that have the same genetic background within each study . Weick eThe relevant data from previous studies were downloaded from online repositories, and trisomic/disomic ratios were calculated for all the annotated genes assayed. This approach enabled us to use a dimensionless measure that allowed comparison of RNA-seq and microarray data. Our study and the twin study by Letourneau et al  producedAn inherent limitation o human transcriptome studies is accounting for the differing genetic background of individuals. In the context of Down syndrome, this limitation has hindered determining the degree to which trisomy of chromosome 21 alters expression of genes on this chromosome and in the genome as a whole. However, recent technical advances have allowed the derivation of iPSCs trisomic or disomic for chromosome 21 from the same individual. We have used RNA-seq to characterize in depth the transcriptome of one such trisomic/disomic iPSC pair, originally generated by Li and colleagues . We haveAs observed in other studies, the majority of significant transcriptome changes we identified in both iPSCs and derived neuronal cultures occur in non-chromosome 21 genes. Gene ontology analysis of genes with transcript abundance changes in trisomic iPSCs unexpectedly revealed increases in transcripts from neuronal-associated genes and decreases in transcripts from genes involved in cell adhesion function and germ layer/mesoderm development. These differences did not result from visible spontaneous differentiation in the trisomic iPSCs, which were monitored daily.APP gene, and then with gancyclovir to select for loss of the chromosome 21 bearing the inserted selection cassette. It is possible that these rounds of selection resulted in epigenetic  changes that added to the transcriptional differences between the trisomic and disomic iPSCs. In fact, we were unable to efficiently differentiate into cortical neuronal cultures any of the 4 disomic clones that had gone through these selection procedures. As the differentiation protocol we used has been employed by other research groups to produce cortical neuron cultures from iPSCs with normal karyotypes, we suspect that the derivation of disomic clones may have resulted in a subtle intrinsic change in the iPSC state. Fortunately, we were able to derive a spontaneous disomic line from the C3 trisomic iPSC clone, which did readily differentiate into cortical neurons. The C3 disomic iPSCs (C3-D21) were therefore used to generate the disomic cortical neuronal cultures. Importantly, C3-D21 disomic iPSCs expressed less MAP2 than trisomic iPSCs . Global chromatin effects of chromosome 21 trisomy were also proposed by Letourneau et al , who ideWe did observe trisomy-dependent splicing changes in both the iPSCs and neuronal cultures. Our identification of splicing alterations in trisomic cells is not completely novel, as splicing differences in a selected set of genes has been observed in fetal DS tissue . RNA-seqExamination of the accumulation of repetitive element transcripts revealed a highly significant decrease in transcripts from the endogenous retrovirus HERVH in the trisomic iPSCs. HERVH transcripts have been specifically identified as a pluripotency marker , and disAs described above, consideration of the possible effects of the selection procedures used to generate the disomic derivative clones suggests a possible technical reason why we have observed transcriptional differences not reported by other investigators. However, cluster analysis of our dataset with other comparable studies suggests that our results are not anomalous, but may reflect the range of states assumed by human stem cells trisomic for chromosome 21. The dendrogram shown in An alternative interpretation of our cluster analysis is that due to an inherent instability/variability of iPSCs in general, the grouping of trisomic transcriptome profiles we have observed reflects a divergence of individual iPSC clones in culture, rather than true trisomic/disomic transcriptional differences. While we cannot exclude this possibility, this seems unlikely to explain the grouping differences in the Jiang study, where the iPSCs were only grown for 3 weeks after XIST-based knockdown of chromosome 21 gene expression.Why might trisomic iPSCs show a range of transcriptome signatures? Given our observation that the trisomic iPSCs we characterized had enhanced accumulation of transcripts associated with differentiated neurons, and reduced expression of the HERVH pluripotency marker, we speculate that trisomic iPSCs may have an inherent inability to maintain a full pluripotent state. Following this assumption, trisomic iPSCs would have a more constrained differentiation capacity than corresponding disomic iPSCs, which could explain their reported defects in neuronal differentiation . On the S1 FigLines show regions of increased or decreased gene expression in cells trisomic for chromosome 21 as calculated using the smoothing algorithm employed by Letourneau et al. The red (iPSCs) and black (fibroblast) traces are taken from the Letourneau study; the green trace is from the iPSCs used in this study. Note that there are significantly different expression domains calculated from the different studies (arrows).(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Further studies indicated this phenomenon was not simply a result of enhanced soluble signaling factors, including vascular endothelial growth factor (VEGF), and that CAFs generated significantly larger deformations in 3D gels compared to normal fibroblasts. Inhibition of the mechanotransductive pathways abrogated the ability of CAFs to deform the matrix and suppressed vascularization. Finally, utilizing magnetic microbeads to mechanically stimulate mechanically-inhibited CAFs showed partial rescue of vascularization. Our studies demonstrate enhanced mechanical activity of CAFs may play a crucial and previously unappreciated role in the formation of tumor-associated vasculature which could possibly offer potential novel targets in future anti-cancer therapies.The role of cancer-associated fibroblasts (CAFs) as regulators of tumor progression, specifically vascular growth, has only recently been described. CAFs are thought to be more mechanically active but how this trait may alter the tumor microenvironment is poorly understood. We hypothesized that enhanced mechanical activity of CAFs, as regulated by the Rho/ROCK pathway, contributes to increased blood vessel growth. Using a 3D  This may be due, in part, to our limited understanding of other factors, such as the mechanical properties of the tumor microenvironment, in controlling cancer associated vascular growth. Mechanical force can be both normal and parallel to a substrate (or cell) and are generally referred to as tensile/compressive and shear, respectively. Previous work has demonstrated that the mechanical force of shear flow can drive vascular growth7, but there is limited work describing how tensile forces may regulate vascular growth9.The formation of new blood vessels in the tumor microenvironment is essential for tumor progression and generally occurs when the metabolic demands of the tumor exceed the existing supply. Although blood vessel growth is an attractive target for anti-cancer treatments16. CAFs are a heterogeneous population of stromal cells present in the tumor microenvironment that can be derived from several tissue sources18. There are no significant somatic differences between CAFs and their normal counterparts, indicating that oncogenic mutations do not contribute to the CAF phenotype19. CAFs have a myofibroblast-like phenotype, characterized by enhanced secretory functions and mechanical activity compared to resident fibroblasts. For example, CAFs secrete elevated levels of stromal derived factor 1 (SDF1) and VEGF22, as well as demonstrate increased tensile forces potentially mediated through the YAP-pathway24. While CAF signaling pathways, including VEGF and hypoxia inducible factor 1-alpha (HIF-1α) have been extensively studied, the role of mechanics in tumor progression specifically CAF-generated mechanical forces is poorly understood26. We hypothesized that CAF-mediated mechanical forces may contribute to enhanced vascular growth in the tumor microenvironment in a way that is complementary to, but distinct from, VEGF signaling.Recently, cancer-associated fibroblasts (CAFs) have been described as potent regulators of tumor progression, including angiogenesis and metastasisin vitro model of blood vessel formation, we demonstrate that CAFs, derived from human breast cancer27, support the self-assembly of vascular networks when co-cultured with endothelial cells in fibrin hydrogels. Similar vascular growth is not seen in samples containing normal breast fibroblasts (NBFs) and endothelial cells. Further studies demonstrate that CAF-supported vessel growth is not solely dependent on soluble factors, including VEGF, but that inhibition of mechanotransductive pathways including ROCK, YAP, and Snail1 (SN1) in the CAF attenuates vascular growth. Finally, inducing mechanical perturbations with magnetic beads in the absence of CAF-generated extracellular matrix (ECM) deformations and upregulating the mechanical activity of NBFs both stimulate blood vessel growth. Additionally, we examined the mechanical activity of normal human lung fibroblasts (NHLFs), which have been shown by our lab to be effective stromal cells for vascularization studies32. Studies showed similar results to CAF vascularization potential and biomechanical behaviors, indicating that mechanobiology may regulate vascular growth in noncancerous environments as well. Taken together, our data demonstrate that CAF-mediated biomechanical activity can regulate vascular growth and might contribute to blood vessel formation in the tumor microenvironment.Using a 3D When co-cultured with endothelial cells (ECs), CAFs support the formation of interconnected vascular networks in 3D fibrin and fibrin-collagen gels Fig. . In contMedia from CAF/EC and NHLF/EC cultures exhibit approximately twice the steady state level of VEGF compared to NBF/EC Fig. . A smallThe ability for CAFs to promote blood vessel growth was significantly enhanced by increasing fibrin concentration from 2.5 mg/L to 5.0 mg/mL Fig. . Above t33. Increasing the fibrin concentration, or effective stiffness of the gels, signficantly decreases the magnitude of displacements for all cell types. Average deformation values and more complete statistical data can be found in Supplemental Table Using our custom bead-tracking algorithm, we measured displacements in 3D fibrin gels in the presence of NHLFs, NBFs or CAFs. At all concentrations of fibrin gels tested, CAFs produce significantly higher mean deformations in the ECM compared to NBFs Fig. . Previou24. Additionally, our deformations measurements highlight the heterogeneity of biomechanical properties of CAF behaviors. These results indicate that the biomechanical activity of CAFs may play a role in the development of blood vessel networks and promoted further investigations.Based on our experiments, we observed some changes in vascularization potential and mechanical behavior of CAFs in response to changes in mechanical properties of the ECM. This aligns with previous results in the literature, showing CAFs have a more myofibroblast-like phenotype including enhanced contractility in 2DPrevious work has shown that ROCK, YAP, and SN1 are all parts of the mechanotransductive pathways of CAFs, regulating how the cells interact with the matrix surrounding them. Utilizing shRNA to inhibit YAP and SN1 pathway inhibited the ability of CAFs to support blood vessel formation in 3D compared to CAF empty vector (EV) controls Fig. . InteresWe measured the VEGF concentration in 3D fibroblast only cultures to determine how the presence of ECs might affect overall steady state VEGF levels. Based on this comparison, we hypothesize that mechanical activity of CAF-EVs may promote ECs present in co-cultures to consume or utilize more VEGF, resulting in lower measured steady state levels when compared to modified CAF/EC cultures Fig. . Since oThese results demonstrate that biomechanical activity induced in the ECM by CAF mechanotransduction regulated by ROCK2, SN1, and YAP are important in the regulation of CAF promoted blood vessel growth. Furthermore, enhancing mechanical perturbations to the microenvironment by constitutively activating Rho in NBFs generated greater vascularization in these samples, representing nearly a 2-fold increase in vascularization potential. Moving forward, we chose to utilize a magnet-based system to physically induce mechanical deformations in the microenvironment regardless of the presence of stromal-secreted factors.Magnetic microbeads coated with thrombin were embedded in fibrin gels to provide a direct means of mechanically perturbing cells in the absence of factors secreted by CAFs. Thrombin-coated beads generate ECM deformations in the presence of a magnetic field Fig.  and thesin vitro tumor microenvironment model. This biomechanical regulation occurs in concert with soluble signaling cues, including VEGF, to promote vascularization associated with tumor progression.These studies support our hypothesis that mechanical deformations or perturbations of the ECM generated by cells regulate the growth of blood vessels as promoted by CAFs in the tumor microenvironment. In the absence of any stromal cells, EC only groups demonstrated ~3-fold increases in blood vessel growth stimulated by dynamic magnetic bead movements. In mechanically inhibited CAF samples, we were able to rescue the vascularization potential with dynamic magnetic stimulation. Overall, we believe this study demonstrates the role of biomechanical activity of CAFs as a potent regulator of vascular growth in an in vitro 3D microtissue model. Our results demonstrate that CAFs generate larger deformations within the ECM, compared to NBFs, which stimulate self-assembly of ECs into a vascular network. This process is mediated via mechanotransductive pathways in the CAF including ROCK2, YAP, and SN1. While VEGF signaling is important, the inhibition of VEGF receptors only partially abrogates the CAF-promoted vascular growth. Furthermore, CAF conditioned media only moderately rescues blood vessel growth in NBF samples, demonstrating that soluble factors alone are not sufficient to explain the enhanced blood vessel growth in the presence of CAFs. These conclusions are further supported by NHLF studies, showing that the mechanical activity of these cells correlates with blood vessel growth in fibrin gels and that VEGFR inhibition does not completely abrogate vessel growth; this work suggests that fibroblast mechanobiology may regulate vascularization in contexts other than cancer biology. On the other hand, our data also show that NHLFs generate smaller deformations in 2.5 mg/mL fibrin gels compared to NBFs, despite also showing approximately 10-fold higher level of vascularization potential. We believe this indicates that large mechanical deformations alone are insufficient to drive blood vessel growth supported by stromal cells and that it is an important interaction between biomechanics and soluble signaling factors that ultimately directs vasculogenic processes. Taken together, this enhanced understanding of CAF-mediated vascular network formation could potentially provide alternative therapeutic strategies that focus on modulating the mechanobiology of CAFs.Our study demonstrates a role for CAF mechanics in the development of vasculature using an 35. Additionally, enhanced ROCK signaling is associated with stabilization of SN136. Other groups have demonstrated that an inhibitor against both isoforms of ROCK abrogates YAP signaling and limits cell contraction39. Our key results indicate that CAFs induce vascularization in a 3D in vitro microtissue model via active mechanical deformations induced in the ECM, and that this process is not solely dependent on VEGF signaling. Inhibition of the mechanotransductive pathway, specifically ROCK2 and SN1, decreases vascularization potential of CAFs compared to control lines while still showing more vessel growth than NBF samples. This might indicate compensatory mechanisms downstream of these factors that still promote blood vessel growth. In CAFs with depleted YAP, limited vessel growth is observed (similar to NBF groups), demonstrating a role as a mechanoregulator of CAF-associated vascular growth.To understand the mechanism of mechanically driven vascular growth, we chose to inhibit SN1, ROCK, and YAP to test different points along the canonical signaling pathways of actin-myosin contractility in CAFs; this also presented a parallel opportunity to induce expression of a contractility regulator, Rho, in NBFs to promote a “CAF-like” phenotype. We opted to use shRNA inhibition instead of small molecule inhibitors because the majority of our experiments are co-cultures where off target effects would confound interpretation of results. Recent work has indicated that SN1 is required for transforming growth factor beta induced expression of alpha-smooth muscle actin, a necessary protein for contractile machinery and that SN1 inhibition reduces activity of Rho41. Taken in context with our data, this suggests that cells need stable actin stress fibers, but not necessarily enhanced actin polymerization associated with focal adhesions at the cell membrane, to induce deformations in the ECM and ultimately drive blood vessel growth. Furthermore, the results demonstrate that ROCK2 may be the more potent regulator of biomechanically controlled blood vessel growth as regulated by CAFs compared to ROCK1. Western blot analysis of CAF-shROCK2 cells showed only ~50% decrease in protein expression compared to CAF-EV  over the others as key regulators of CAF biomechanics. Rather, we view our work as a compellation, showing that inhibiting mechanotransduction at 4 different locations along a pathway affects the ability of CAFs to deform the ECM and thus abrogates vascularization potential of these cells.42), or interstitial fluid flow. Tensile forces, on the other hand, can be generated when neighboring cells contract. As differences in cell concentrations were not a factor in regulating blood vessel in our model and interstitial flow was absent, our in vitro system represents an environment to isolate and investigate predominantly tensile forces generated by CAFs and their subsequent role in the formation of blood vessels. Although our system does not include interstitial flow, there is strong evidence supporting interstitial flow-derived forces and their role in vessel growth, as well44.It has become increasingly apparent that cell and ECM mechanics impacts tumor progression, and should be considered in the development of novel treatment strategies. Mechanical force can be described as either shear  or normal (perpendicular). While shear forces exist in a viscoelastic material such as tissue, they are due predominantly from fluid flowing over surfaces or through a material . Normal forces can be either compressive (reducing volume) or tensile (expanding volume). Compression can occur when the activated tumor stroma produces excessive ECM proteins , excessive cell proliferation  and longer term (ECM remodeling) events that may apply additional forces to the surface of ECs to impact vascular growth.The remodeling of the peritumoral microenvironment occurs through multiple time-scales, ranging from rapid (on the order of seconds to minutes) fluctuations of the cellular edges via focal adhesion protrusionsThe study presented here illustrates how deformations in the ECM induced by CAFs regulate the growth of vasculature in a manner not solely dependent on soluble signaling factors, including VEGF. Further, the studies utilizing magnetic beads demonstrate that ECs respond to direct mechanical stimulation by initiating a vasculogenic process, in the absence of secreted factors produced by CAFs. Based on our magnetic bead studies, we demonstrate that the movements induced in the ECM by mechanical perturbations partially recapitulate vascular growth driven by CAF biomechanics. Furthermore, our magnetic bead studies recapitulate ECM deformations on a similar length scale to those induced by CAFs (4–6 μm), or roughly 25% of the diameter of the average cell. For our static bead displacement experiments, these deformation scales do not represent the dynamic or time-varying features of strains in the ECM, rather they capture a mean tensile strain induced over the observed time period. In contrast, our moving magnetic bead experiments attempted to capture the dynamic behavior of mechanotranductive signaling.in vitro assay of vasculogenesis. While vasculogenic vessel growth was previously believed to occur only during embryogenesis60, we now understand that multiple cell types, including endothelial progenitor cells, bone marrow stromal cells, tumor cells, and CAFs can promote vasculogenesis in association with the progression of cancer. Our work supports the premise that CAFs can enhance vascular growth through a vasculogenic process that is mediated, in part, by biomechanical forces and that the Rho/ROCK pathway and YAP signaling are involved. Although all of our studies were conducted in an in vitro assay, we believe this work correlates with previous in vivo work describing the mechanical activity of CAFs as it relates to SN1 signaling in tumor progression61. Ultimately, we believe this information will help elucidate potential novel targets for anti-cancer therapeutic strategies designed to modulate the biomechanical behaviors of CAFs that contribute to tumor progression.Our studies demonstrate the role of active mechanical forces, generated by CAFs as a regulator of blood vessel growth in a 3D 63. NHLFs were obtained from Lonza (#CC-2512); multiple donors were utilized throughout the studies between passages 4–8. Fibroblasts were cultured in DMEM  with 10% FBS (ThermoFisher #26140079), 1% L-glutamine , 1% non-essential amino acids , 1% sodium pyruvate (ThermoFisher #11360070), and 1% penicillin/streptomycin . Characterization of NBFs and CAFs has been completed previously in our lab64. ECs were isolated and derived as described previously from umbilical cord blood samples65, cultured in EGM-2 , and utilized between passages 3–6. Microtissues were created by dissolving fibrinogen  in DPBS, and then mixing with cell suspensions to create final concentrations of 5 × 104 of each cell type per gel. Thrombin was added to a final concentration of 3 U/mL before the suspension was plated into a 1 cm diameter poly(dimethyl siloxane) ring on a glass coverslip. Gels were allowed to solidify for a minimum of 20 min before the addition of EGM-2. To test gel composition, fibrinogen concentration was varied and in some studies collagen I  was added to the solution prior to the addition of thrombin. Media was changed every 2 days and experiments were halted via fixation at the end of 7 days.To form vascular networks in 3D gels, a 1:1 ratio of endothelial cell colony forming cell-derived endothelial cells (ECFC-ECs or just ECs) derived from umbilical cord blood and fibroblasts were embedded in fibrin gels . NBFs and CAFs were previously isolated and immortalized utilizing insertion of hTERT into patient-derived fibroblasts66. Soluble VEGF produced during the ring assay was measured using VEGF Human ELISA Kit ; a second series of VEGF measurements were taken from samples containing only fibroblasts at the same concentration used in the ring assay. In some experiments, VEGF signaling was inhibited by adding a small molecule inhibitor of VEGFR, SU-5402,  to the media after 3 days of culture. To analyze conditioned media, 100% conditioned media from ring assays containing both CAFs/ECs or NHLFs/ECs was harvested, spun down at 1000 rpm for 5 min to remove cell debris, and then fed directly to ring assays containing NBFs and ECs.Vessel growth was analyzed by staining the hydrogels for the endothelial marker CD31 . Briefly, gels were rinsed with PBS, fixed at −20 °C for 20 min with a 1:1 acetone-methanol mixture, rinsed, blocked for 1 h at room temperature with 2% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20. Antibodies were diluted in the same solution and incubated overnight at 4 °C . Images were acquired on an Olympus IX83 inverted microscope and analyzed using AngioTool software to quantify vessel length4 fibroblasts only. Bead movement was tracked in 3D over the course of 1 h, after gels had been incubated overnight. This algorithm thus captured long-term ECM deformations introduced by cell behavior.To analyze the mechanical behavior of fibroblasts, we developed a bead displacement algorithm in MATLAB™. Bead displacement events were interpreted as ECM deformations induced by cell contractility events. Using the vasculogenic ring assay setup, 1 μm diameter blue fluorescent polystyrene microbeads  were included as fiducial markers in gels containing 5 × 1068 and YAP, which has been shown to be involved with CAF mechanosensitive behavior38. We also chose to inhibit SN1, a transcription factor shown to be upregulated in CAFs compared to normal fibroblasts69, that has recently been shown to be involved in CAF mechanotransduction61. In a parallel study, we were able to promote enhanced contractility of NBFs using a constitutively active form of Rho inserted into the cells via lentiviral transduction (referred to as NBF-caRho cells). shRNAis were from the Broad collection (Washington University Genome Center). There were 5–7 shRNAis initially selected for each target. These were all screened and 2 shRNAis were chosen. All shRNAis and constitutively active RhoA(Q63L)-Flag tagged cDNA were subcloned into the pLVX-hygro vector. Lentiviruses were produced in HEK 293T cells, and human CAFs were transduced and selected in 200 μg/mL hygromycin. To check knockdown efficiency, cells were lysed in 1X RIPA buffer supplemented with protease inhibitors. Protein lysates were processed following standard procedures. shRNAi target sequences as well as antibody description and working dilutions used can be found in Supplemental Tables 61; for these studies, the targeting sequence listed as letter A in Table To interrogate the mechanotransductive pathways involved in mechanically regulated vessel growth, we utilized shRNA to inhibit CAF contractility. Specifically, we focused on the Rho/ROCK pathway, which are potent regulators of the actin cytoskeletal structure70. Beads were added to the vasculogenic ring assay. To stimulate movement, rare earth magnets  were placed directly adjacent to the well plate containing rings for static magnetic studies. For dynamic stimulation studies, the magnets were located on an orbital shaker 3 cm from the bottom of the well plate. Speed of rotation was varied to adjust magnetic stimulation.To eliminate signaling of soluble factors that promote vessel growth, a magnetic bead assay was developed as a means to mechanically stimulate endothelial cells. Iron oxide microbeads  with a tosyl-coated surface were mixed with 50 U/mL thrombin in 0.1% BSA in PBS to create thrombin-coated magnetic beadsVasculogenic assays represent a minimum of 3 biological replicates. Bead displacement results represent average magnitudes of displacement based on a minimum of 40 beads analyzed per gel over a minimum of 2 technical replicates. Average displacement vector magnitudes were compared among sample groups. All data was compared via ANOVA, followed by post-hoc Holm-Sidak tests as appropriate with α = 0.05. Unless noted, all data is presented as average ± standard error of the mean, except for deformation data which is presented as average deformation magnitude ± standard deviation.The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.Supplemental Information


Force control deficits have been repeatedly documented in autism spectrum disorder (ASD). They are associated with worse social and daily living skill impairments in patients suggesting that developing a more mechanistic understanding of the central and peripheral processes that cause them may help guide the development of treatments that improve multiple outcomes in ASD. The neuromuscular mechanisms underlying force control deficits are not yet understood. Seventeen individuals with ASD and 14 matched healthy controls completed an isometric index finger abduction test at 60% of their maximum voluntary contraction (MVC) during recording of the first dorsal interosseous (FDI) muscle to determine the neuromuscular processes associated with sustained force variability. Central modulation of the motorneuron pool activation of the FDI muscle was evaluated at delta (0–4 Hz), alpha (4–10 Hz), beta (10–35 Hz) and gamma (35–60 Hz) frequency bands. ASD patients showed greater force variability than controls when attempting to maintain a constant force. Relative to controls, patients also showed increased central modulation of the motorneuron pool at beta and gamma bands. For controls, reduced force variability was associated with reduced delta frequency modulation of the motorneuron pool activity of the FDI muscle and increased modulation at beta and gamma bands. In contrast, delta, beta, and gamma frequency oscillations were not associated with force variability in ASD. These findings suggest that alterations of central mechanisms that control motorneuron pool firing may underlie the common and often impairing symptoms of ASD. Sensorimotor impairments are common in autism spectrum disorder (ASD) ,2. DisruMultiple types of sensorimotor abnormalities have been identified in individuals with ASD, including reduced eye movement accuracy ,8, postuForce control involves excitatory cortical commands relayed from primary motor cortex (M1) to spinal motor neurons which innervate skeletal muscle fibers . Motor nMotorneuron pool firing is coordinated by descending central commands generated at multiple frequencies to dynamically ensure precise force production . SimultaBeta band 10–35 Hz) frequency modulation of the motorneuron pool coincides with a default mode of cortical innervation from contralateral M1 during tasks involving low to moderate force contractions ,31,32,33 Hz frequDefining alterations in the central modulation of the motorneuron pool activity associated with the increased force variability previously documented in ASD ,39,40,41Three study aims were pursued in the present study. First, whereas deficits in controlling grip force may reflect impairments in coordinating force production across multiple effectors, we tested whether individuals with ASD show deficits controlling sustained force produced by one finger in isolation. Second, we examined motorneuron pool firing properties and central modulation of the motorneuron pool at different frequency bands  during isometric index finger abduction in order to characterize neuromuscular properties underlying increased force variability in ASD . Third, 1,30 = 0.007, p = 0.935; CNT = 22.500 N, SE = 2.198 N; ASD = 22.747 N, SE = 2.055 N) and mean sustained force   and the interactive effect of frequency band and group  were significant (p = 0.018), while patients showed greater normalized power than controls at beta  and gamma  frequencies. No differences between groups were found for alpha band power.Individuals with ASD and healthy controls showed similar FDI muscle motorneuron pool mean discharge rate  scores for healthy controls or individuals with ASD . For heaThe present study adds to the growing literature documenting deficits of voluntary hand movements and force production in ASD. Our findings also provide new evidence that motorneuron pool activity power during isometric index finger force production is abnormal in ASD at delta (0–4 Hz), beta (10–35 Hz), and gamma (35–60 Hz) frequency bands. Results further indicate that neuromuscular oscillations at these frequency bands are tightly linked to force control in healthy individuals, but that the central-peripheral communication processes that support the attenuation of force output variability in ASD are distinct and less organized. Taken together, alterations of central modulation of the motorneuron pool activity of the FDI muscle during constant force production may represent a key neurophysiological deficit related to both motor impairment and ASD symptoms.Our findings were consistent with prior studies showing increased variability of manual motor output in ASD during precision gripping ,17,39, wRelative to controls, individuals with ASD showed similar levels of FDI muscle motorneuron pool discharge rate A and disMotorneuron pool oscillations within the delta band (0–4 Hz) represent a default mode of neural processing with the reductions seen as individuals engage in skilled actions, and associated with greater sustained motor precision ,24. At rThe nature of these compensatory processes remains unclear. Consistent with prior studies, we found that increased modulation of the motorneuron pool at beta (10–35 Hz) and gamma (35–60 Hz) as well as decreased modulation at delta (0–4 Hz) were highly associated with force variability reduction in healthy individuals ,38,46,47Unlike with controls, we did not find any relationship in ASD between modulation of motorneuron pool at beta and gamma bands and sustained force variability suggesting that excessive central oscillations were needed to support patients maintaining the target force level, but that these central processes had no effect on the precision of motor output. These findings suggest that central modulations may not be organized in ASD in the same manner as they are in health. For example, it is possible that, for individuals with ASD, fast-twitch and fatigable motor units are not recruited  or are rMVC production was more strongly associated with age in ASD compared to controls suggesting that increases in strength during development likely are delayed in ASD. This finding may help explain inconsistencies of prior studies showing both reductions in manual strength ,14,15 anIncreased age was associated with greater force variability reduction B in healWhile the present study documents several novel findings useful for understanding neuromuscular processes underpinning force control deficits in ASD, our results should be considered in the context of multiple limitations. First, our sample spans a relatively broad age range. The small sample size may have contributed to insufficient power for characterizing the developmental trajectory of hand MVC and force variability increases in ASD. Further, the high functioning individuals may show less force variability as opposed to patients with lower IQ scores. Second, some of the participants with ASD in our sample may have shown comorbid conditions that are common in this disorder  and depression). Systematic studies of contributions of these comorbid conditions to force variability increase as well as atypical central modulation of the motorneuron pool at different frequency bands in ASD are needed. Third, interpretations of our current findings, particularly the central origins of delta, alpha, beta and gamma frequency bands will need to be tested by integrating simultaneous measures of central oscillations using electroencephalogram (EEG) or magnetoencephalography (MEG). Such studies, in combination with EMG recording, will allow us to better understand altered central-to-peripheral mechanisms related to both motor and the defining symptoms of ASD. Lastly, antihypertensive and antidepressant medications have unclear effects on psychomotor functioning, with studies documenting both improvement and decline ,59,60,61Seventeen individuals with ASD and 14 healthy controls matched on age, gender, IQ and handedness  performeParticipants with ASD were excluded if they had a known genetic or metabolic disorder associated with ASD  or history of non-febrile seizures. Healthy controls were recruited from the community and were required to have a score of 8 or lower on the Social Communication Questionnaire (SCQ) . ControlNo participants were taking medications known to affect sensorimotor control at the time of testing, including antipsychotics, stimulants, or anticonvulsants . Seven iParticipants were seated in a darkened room facing a 27-inch Dell  LCD monitor  located 60 cm in front of them. They sat with their shoulder abducted at 45°, elbow flexed and forearm resting on a customized arm brace A. The arPrior to testing, each individual’s index finger MVC was measured for their dominant hand. Participants completed three separate 5-s trials in which they were instructed to press against the load cell with as much force as possible by abducting their index finger. The amount of force they generated was displayed on the monitor as a red bar moving upwards with increased force. Participants rested for 1-min between consecutive trials to minimize the effect of muscle fatigue. The average maximum force across trials was calculated as the estimate of each participant’s index finger MVC ,17,39.TM, Delsys Inc.) was placed on the back of participants’ hand in alignment with their FDI muscle. The sEMG electrode consists of five non-invasive probes (0.5 mm diameter of each) with four arranged in a square and the fifth probe located in the center of the square at a fixed distance of 3.6 mm from each of the surrounding four probes. Pairwise signal subtraction of these probes results in four differential sEMG channels from the FDI muscle. The sEMG time series was then amplified at 1 K, sampled at 20 KHz, and band-pass filtered at 20–450 Hz. Four-channel sEMG time series were decomposed offline into distinct motor unit action potential trains using Delsys decomposition algorithms (v42) [Prior to sEMG sensor attachment, individuals’ skin over the FDI muscle was shaved and cleansed with rubbing alcohol to remove oil, debris and dead skin cells. A reference electrode was taped over the lateral epicondyle of participants’ humerus. Subsequently, a specialized sEMG sensor  was selected from each trial to represent motorneuron pool activation of the FDI muscle and included in analyses of the mean discharge rate of the motorneuron pool. For those trials with a total number of retained motor units greater than eight, we selected eight motor units based on the following procedures: (1) the smallest and largest motor units were identified as the first (MU1) and the last (MU8) recruited motor units; (2) assigning motor units in the middle of the scale as the fourth (MU4) and fifth (MU5) recruited motor units; (3) assigning motor units in the middle of MU1 and MU4 as the second (MU2) and third (MU3) recruited motor units; and (4) assigning motor units in the middle of MU5 and MU8 as the sixth (MU6) and seventh (MU7) recruited motor units  of the inter-spike intervals calculated using the formula below:Modulation of the motorneuron pool activity was quantified using power spectrum analyses in the frequency domain. Inter-spike intervals of the motorneuron pool were initially transformed into a continuous time series by interpolating each trial . Then, fEach specific frequency bin refers to each of the four frequency bands; thus, normalized power was derived for delta (0–4 Hz), alpha (4–10 Hz), beta (10–35 Hz) and gamma (35–60 Hz) frequency bands.The ADOS-2 was used to confirm participants’ diagnosis and rate ASD symptoms based on the observation of each participant’s behavior . The ADOt-test was used to compare index finger MVCs between individuals with ASD and healthy controls. A series of one-way ANOVAs were conducted to examine between-group (ASD vs. Control) differences on mean sustained force, standard deviation of force, mean discharge rate and discharge rate variability of the FDI muscle motorneuron pool. A 2 (group) × 4 (frequency band: delta (0–4 Hz), alpha (4–10 Hz), beta (10–35 Hz) and gamma (35–60 Hz)) fixed effect repeated measure ANOVA was applied to identify between-group differences in motorneuron pool activations of the FDI muscle across the power spectrum. For all analyses, results were interpreted as significant if p < 0.05. Where Mauchly’s test indicated a violation of sphericity, the Greenhouse–Geisser estimate was used to provide a conservative test of ANOVA main and interaction effects.A student r|) was greater than 0.5.To determine the relationships between FDI muscle motorneuron pool activity at each frequency band and force variability, Pearson correlations were conducted separately for individuals with ASD and healthy controls. Pearson correlation coefficients also were used to examine the relationships between individuals’ muscle strength , force and dEMG measurements found to be different between groups and age, IQ and clinical ratings of ASD severity from the ADOS-2. Results were interpreted as significant if alpha was less than 0.05 and the absolute value of the correlation coefficient  and gamma 35–60 Hz) frequency bands as well as delta 0–4 Hz) band reduction were all associated with lower force variability in healthy participants, although these relationships were all attenuated in patients. These findings suggest a lack of central communication to skeletal muscles in ASD as well as less organized motor unit recruitment at the periphery. An emerging literature has indicated that early motor developmental abnormalities are among the earliest signs of ASD [5 Hz and  Hz band 0 Hz freq