Muscle Injury Model Research Articles

Regulatory T cells and skeletal muscle regeneration.

Skeletal muscle regeneration results from the activation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Inflammatory and immune cells have a crucial role in the regeneration process. Acute muscle injury causes an immediate transient wave of neutrophils followed by a more persistent infiltration of M1 (proinflammatory) and M2 (anti-inflammatory/proregenerative) macrophages. New studies show that injured muscle is also infiltrated by a specialized population of regulatory T (Treg) cells, which control both the inflammatory response, by promoting the M1-to-M2 switch, and the activation of satellite cells. Treg cells accumulate in injured muscle in response to specific cytokines, such as IL-33, and promote muscle growth by releasing growth factors, such as amphiregulin. Muscle repair during aging is impaired due to reduced number of Treg cells and can be enhanced by IL-33 supplementation. Migration of Treg cells could also contribute to explain the effect of heterochronic parabiosis, whereby muscle regeneration of aged mice can be improved by a parabiotically linked young partners. In mdx dystrophin-deficient mice, a model of human Duchenne muscular dystrophy, muscle injury, and inflammation is mitigated by expansion of the Treg-cell population but exacerbated by Treg-cell depletion. These findings support the notion that immunological mechanisms are not only essential in the response to pathogenic microbes and tumor cells but also have a wider homeostatic role in tissue repair, and open new perspectives for boosting muscle growth in chronic muscle disease and during aging.

Muscle injury in rats induces upregulation of inflammatory cytokines in injured muscle and calcitonin gene-related peptide in dorsal root ganglia innervating the injured muscle.

ABSTRACT Introduction: In this study we evaluated the relationships among the behavioral changes after muscle injury, histological changes, changes in inflammatory cytokines in the injured muscle, and changes in the sensory nervous system innervating the muscle in rats. Methods: We established a model of muscle injury in rats using a dropped weight. Behavior was assessed using the CatWalk system. Subsequently, bilateral gastrocnemius muscles and dorsal root ganglia (DRGs) were resected. Muscles were stained with hematoxylin and eosin, and inflammatory cytokines in injured muscles were assayed. DRGs were immunostained for calcitonin gene–related peptide (CGRP). Results: Changes of behavior and upregulation of inflammatory cytokines in injured muscles subsided within 2 days of injury. Repaired tissue was observed 3 weeks after injury. However, upregulation of CGRP in DRG neurons continued for 2 weeks after injury. Conclusion: These findings may explain in part the pathological mechanism of persistent muscle pain. Muscle Nerve 54: 776–782, 2016

Perivascular Stem Cells Diminish Muscle Atrophy and Retain Viability in a Rotator Cuff Tear Model

Objectives:Rotator cuff tears (RCTs) are a common cause of shoulder pain and often necessitate surgical repair. Muscle changes including atrophy, fibrosis, and fatty degeneration can develop after RCTs, which may compromise surgical repair and clinical outcomes. Lipoaspirate-derived human perivascular stem cells (PSCs) have demonstrated myogenic and angiogenic potential in other small animal models of muscle injury. We hypothesized that the administration of PSCs following massive RCTs may help to diminish these muscle changes in a small animal model.Methods:A total of 90 immunodeficient mice were used (15 groups, N=6). Each was assigned to one of three surgical groups: i) sham, ii) supraspinatus and infraspinatus tendon transection (TT), or iii) TT and suprascapular nerve denervation (TT+DN). PSCs were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into small blood vessel residing pericytes (CD146+ CD34- CD45- CD31-) and large blood perivascular adventitial cells (CD146- CD34+ CD45- CD31-). Mice received either a) no injection, b) saline injection, c) pericyte injection, or d) adventitial cell injection at the time of the index procedure or at two weeks following index surgery. The supraspinatus muscles were harvested six weeks after the index procedure. Muscle atrophy was assessed by measuring percent wet muscle weight change for each sample. Muscle fiber cross-sectional area (CSA), fibrosis, and fatty degeneration were analyzed using Image J™. Additionally, pericytes and adventitial cells were transduced with a luciferase-containing construct. Animals were given injections of luciferin and imaged using IVIS to track in vivo bioluminescence following injections to assess cell viability.Results:Treatment with PSC injection after TT resulted in less wet weight loss and greater muscle fiber CSA than control groups (P<0.05). The TT+DN groups treated with PSC injections two weeks post-op also had less muscle weight loss and greater muscle fiber CSA than their respective control groups. The TT+DN groups treated with PSC injections at the time of surgery demonstrated no differences in weight loss, but had greater muscle fiber CSA than their respective controls. There was no difference in fibrosis between the TT groups. However, TT+DN groups treated with pericyte injections at both time points and adventitial cell injections two weeks post-op had less fibrosis than TT+DN controls. There was less fatty degeneration in the TT groups treated with pericyte injections at both time points and adventitial cells at the time of surgery compared to matched controls. There were no differences in the amount of fatty degeneration between the TT+DN groups. Bioluminescence imaging demonstrated viability of the injected cells at three weeks following injections (Figure 1).Conclusion:Our findings demonstrate significantly less muscle atrophy in the groups treated with PSC injections compared to respective controls for both TT and TT+DN procedures. These results suggest that the use of PSCs may have a role in the prevention of muscle atrophy by aiding in the maintenance of muscle bulk without leading to increased fibrosis or fatty infiltration. Additionally, bioluminescence data suggests that these cells maintain viability and engraft within the native muscle to improve muscle bulk. Improved muscle quality in the setting of rotator cuff tears may increase the success rates of rotator cuff repair and lead to superior clinical outcomes.

Dendritic Cell-Like Cells Accumulate in Regenerating Murine Skeletal Muscle after Injury and Boost Adaptive Immune Responses Only upon a Microbial Challenge.

Skeletal muscle injury causes a local sterile inflammatory response. In parallel, a state of immunosuppression develops distal to the site of tissue damage. Granulocytes and monocytes that are rapidly recruited to the site of injury contribute to tissue regeneration. In this study we used a mouse model of traumatic skeletal muscle injury to investigate the previously unknown role of dendritic cells (DCs) that accumulate in injured tissue. We injected the model antigen ovalbumin (OVA) into the skeletal muscle of injured or sham-treated mice to address the ability of these DCs in antigen uptake, migration, and specific T cell activation in the draining popliteal lymph node (pLN). Immature DC-like cells appeared in the skeletal muscle by 4 days after injury and subsequently acquired a mature phenotype, as indicated by increased expression of the costimulatory molecules CD40 and CD86. After the injection of OVA into the muscle, OVA-loaded DCs migrated into the pLN. The migration of DC-like cells from the injured muscle was enhanced in the presence of the microbial stimulus lipopolysaccharide at the site of antigen uptake and triggered an increased OVA-specific T helper cell type 1 (Th1) response in the pLN. Naïve OVA-loaded DCs were superior in Th1-like priming in the pLN when adoptively transferred into the skeletal muscle of injured mice, a finding indicating the relevance of the microenvironment in the regenerating skeletal muscle for increased Th1-like priming. These findings suggest that DC-like cells that accumulate in the regenerating muscle initiate a protective immune response upon microbial challenge and thereby overcome injury-induced immunosuppression.

Establishing a Functional Model of Mouse Muscle Injury and Recovery

PURPOSE: To establish an in vivo mouse model of muscle injury and recovery. METHODS: Sixteen genetically identical female mice were randomized to two 8-mouse injury cohorts (either Phosphate Buffered Saline (PBS) (control) or cardiotoxin (injured)). Injections were performed unilaterally to the right tibialis anterior. One protocol (nocturnal wheel running) was evaluated longitudinally throughout the 17 day study period, while the remaining 5 protocols (dynamic weight bearing, treadmill gait analysis, Rota-Rod performance, open field testing, and grip strength) were evaluated as one-time assays over post-injury days 5-6. All experiments were conducted at Jackson Laboratory, Bar Harbor, Maine. RESULTS: Of the six protocols examined, only the Rota-Rod assay demonstrated a statistically significant difference between injured and control mice (p<0.0018). The Rota-Rod is a rotating rod that increases velocity until the mouse is no longer able to stay on and falls to a soft surface for a maximum trial duration of 300 seconds. The mean latency (seconds until falling) was 298 seconds for control mice and 186 seconds for cardiotoxin-injured mice, with a significant difference of 111 seconds (95% CI 17.0-205.5). There was no significant difference in the two groups in terms of wheel running, weight bearing, gait, open field, or grip strength. CONCLUSION: The Rota-Rod consistently and significantly delineated between cardiotoxin-injured mice and placebo mice at day 5 post-injury. It is likely that even greater statistical significance would result from Rota-Rod trials lasting greater than 300 seconds. It is unknown how Rota-Rod would perform at other time points. Unilateral cardiotoxin injection to the tibialis anterior and subsequent Rota-Rod trial provides a low cost easy to perform in vivo murine model of muscle injury functional assessment within which novel therapies can be assessed.

Effects of Tart Cherry Extract and L-Arginine on Regeneration of Myotube Cultures after Mechanical Strain

Eccentrically-biased muscle loading produces temporary damage to muscles leading to repair and adaptation. The muscle adaptation process after exercise induced muscle damage results in temporary de-habilitation and compromised resiliency of the muscle. Both tart cherry extract (TCE) and L-arginine (L-Arg) have been shown to reduce strength loss due to muscle loading, but the mechanisms by which they operate are currently unknown. PURPOSE: To determine: a) the activation threshold of mechanical loading that induces muscle cell damage and regeneration, and b) to determine the influence of TCE and L-Arg on markers of damage, regeneration, and metabolism in cultured muscle cells. METHODS: A FlexCell™ FX5000 system was used to apply mechanical strain to C2C12 myotube cultures. The strain protocol consisted of a fixed magnitude of strain for 0, 2, 4, 6, 12, 18, or 24h. Cultures were then assayed at all time points for lactate dehydrogenase (LDH) release (damage), satellite cell proliferation (regeneration) using a Click-iT® EdU Imaging Kit and mitochondrial metabolism via XTT assay, in the presence or absence of TCE (5 ng/ml) or L-Arg (150 μm). RESULTS: Mechanical strain induced significant (p<0.05) LDH release from myotube cultures after 12h (64.4±31.5%), and 24h (60.5±20.3%) of strain, relative to controls. Satellite cell proliferation increased by (39.5±5.4%) at 6h, (87.5±6.1%) at 12, peaked at 18h (135 ±7.6%), and remained elevated at 24h (99.5 ±11%) post-strain, relative to controls (p<0.05). Mitochondrial metabolism was elevated in strained myotube cultures (vs. controls) at 18h (25.1 ±4.3%) and 24h (24 ±10.8%) post-injury (p<0.05). Changes in satellite cell activation (r=0.77, p=0.01) and metabolism (r=0.78, p=0.01) were highly correlated to LDH release. TCE (5 ng/ml) and L-Arg (150 μm); however, had no effect on damage, satellite cell activation, or metabolism, compared to untreated cultures. CONCLUSION: These findings demonstrate in vitro that the magnitude of the regenerative response to muscle damage is associated with the degree of the initial mechanical insult. Moreover, TCE and L-Arg do not lessen the initial mechanical insult or accelerate regeneration in this model of strain-induced muscle injury. Funding source: Combat Feeding Research & Engineering Program

Quantitative evaluation of striated muscle injury by multiscale blob features method.

This study aimed to use the multiscale blob feature (MBF) method to quantitatively evaluate porcine striated muscle injuries. A porcine striated muscle injury model was induced by microwave ablation and anhydrous acetic acid injection, respectively. Then, both 2D sonographic and histological features of the lesions were recorded and compared. Later, MBF was used to quantitatively evaluate the porcine striated muscle injuries by extracting the texture features from the 2D ultrasonogram via measuring eight textural parameters (Mean, SDev, NOB, [Formula: see text], [Formula: see text], HOD, DOD, and POD). Microwave ablation produced oval or round-like lesions, which had a pale gray color, with an echo attenuation detected at lesion center due to carbonization; anhydrous acetic acid injection produced long, stripped lesions, which had a slate-gray color, with a gas-like intense echo at lesion center. There were significant differences in Mean, [Formula: see text] and POD between the muscle samples treated by microwave ablation and the control samples, as well as significant differences in NOB, [Formula: see text] and POD between the muscle samples treated by anhydrous acetic acid injection and the control. There were significant differences in Mean, [Formula: see text], NOB, and [Formula: see text] between the muscle samples treated by microwave ablation and those treated by anhydrous acetic acid injection. Quantitative evaluation of striated muscle injuries using the MBF method was able to differentiate the muscle injuries caused by microwave ablation and anhydrous acetic acid injection, suggesting that this method may be a potential and reliable tool for quantitative evaluation of muscle injuries.

Customized platelet-rich plasma with transforming growth factor β1 neutralization antibody to reduce fibrosis in skeletal muscle

The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-β1, that can cause fibrosis and inhibit optimal muscle healing. ObjectiveTo test if neutralizing TGF-β1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. MethodsPRP was isolated from in-bred Fisher rats. TGF-β1 neutralization antibody (Ab) was used to block the TGF-β1 within the PRP prior to injection. The effects of customized PRP (TGF-β1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. ResultsA significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. ConclusionNeutralizing TGF-β1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.

Discovery of Novel Small Molecules that Activate Satellite Cell Proliferation and Enhance Repair of Damaged Muscle.

Skeletal muscle progenitor stem cells (referred to as satellite cells) represent the primary pool of stem cells in adult skeletal muscle responsible for the generation of new skeletal muscle in response to injury. Satellite cells derived from aged muscle display a significant reduction in regenerative capacity to form functional muscle. This decrease in functional recovery has been attributed to a decrease in proliferative capacity of satellite cells. Hence, agents that enhance the proliferative abilities of satellite cells may hold promise as therapies for a variety of pathological settings, including repair of injured muscle and age- or disease-associated muscle wasting. Through phenotypic screening of isolated murine satellite cells, we identified a series of 2,4-diaminopyrimidines (e.g., 2) that increased satellite cell proliferation. Importantly, compound 2 was effective in accelerating repair of damaged skeletal muscle in an in vivo mouse model of skeletal muscle injury. While these compounds were originally prepared as c-Jun N-terminal kinase 1 (JNK-1) inhibitors, structure-activity analyses indicated JNK-1 inhibition does not correlate with satellite cell activity. Screening against a broad panel of kinases did not result in identification of an obvious molecular target, so we conducted cell-based proteomics experiments in an attempt to identify the molecular target(s) responsible for the potentiation of the satellite cell proliferation. These data provide the foundation for future efforts to design improved small molecules as potential therapeutics for muscle repair and regeneration.

Chronic Paraspinal Muscle Injury Model in Rat.

ObjectiveThe objective of this study is to establish an animal model of chronic paraspinal muscle injury in rat.MethodsFifty four Sprague-Dawley male rats were divided into experimental group (n=30), sham (n=15), and normal group (n=9). Incision was done from T7 to L2 and paraspinal muscles were detached from spine and tied at each level. The paraspinal muscles were exposed and untied at 2 weeks after surgery. Sham operation was done by paraspinal muscles dissection at the same levels and wound closure was done without tying. Kyphotic index and thoracolumbar Cobb's angle were measured at preoperative, 2, 4, 8, and 12 weeks after the first surgery for all groups. The rats were sacrificed at 4, 8, and 12 weeks after the first surgery, and performed histological examinations.Results At 4 weeks after surgery, the kyphotic index decreased, but, Cobb's angle increased significantly in the experimental group (p<0.05), and then that were maintained until the end of the experiment. However, there were no significant differences of the kyphotic index and Cobb's angle between sham and normal groups. In histological examinations, necrosis and fibrosis were observed definitely and persisted until 12 weeks after surgery. There were also presences of regenerated muscle cells which nucleus is at the center of cytoplasm, centronucleated myofibers.ConclusionOur chronic injury model of paraspinal muscles in rats shows necrosis and fibrosis in the muscles for 12 weeks after surgery, which might be useful to study the pathophysiology of the degenerative thoracolumbar kyphosis or degeneration of paraspinal muscles.

Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride.

Models of skeletal muscle injury in animal models are invaluable tools to assess muscle stem cell (MuSC)-mediated tissue repair. The optimization and comprehensive evaluation of these approaches have greatly improved our ability to assess MuSC regenerative potential. Here we describe the procedures for skeletal muscle injury with notexin and BaCl2 and assessment of the dynamics of tissue regeneration.

A New Surgical Model of Skeletal Muscle Injuries in Rats Reproduces Human Sports Lesions.

Skeletal muscle injuries are the most common sports-related injuries in sports medicine. In this work, we have generated a new surgically-induced skeletal muscle injury in rats, by using a biopsy needle, which could be easily reproduced and highly mimics skeletal muscle lesions detected in human athletes. By means of histology, immunofluorescence and MRI imaging, we corroborated that our model reproduced the necrosis, inflammation and regeneration processes observed in dystrophic mdx-mice, a model of spontaneous muscle injury, and realistically mimicked the muscle lesions observed in professional athletes. Surgically-injured rat skeletal muscles demonstrated the longitudinal process of muscle regeneration and fibrogenesis as stated by Myosin Heavy Chain developmental (MHCd) and collagen-I protein expression. MRI imaging analysis demonstrated that our muscle injury model reproduces the grade I-II type lesions detected in professional soccer players, including edema around the central tendon and the typically high signal feather shape along muscle fibers. A significant reduction of 30% in maximum tetanus force was also registered after 2 weeks of muscle injury. This new model represents an excellent approach to the study of the mechanisms of muscle injury and repair, and could open new avenues for developing innovative therapeutic approaches to skeletal muscle regeneration in sports medicine.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury

Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. Statement of significanceCOX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model.The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Effect of administration of antibodies against nerve growth factor in a rat model of muscle injury

IntroductionAlthough muscle injury is a common source of pain, the mechanism causing such pain is not completely known. We have previously reported nerve growth factor (NGF) as a proinflammatory mediator involved in acute pain, and clinical trials have shown the effectiveness of anti-NGF antibodies for management of low back pain. Here, we aim to examine the effects of anti-NGF antibodies on muscle-derived pain by studying their effects on sensory innervation in a rat muscle injury model. MethodsA nervous system tracer, Fluoro-Gold, was applied to both gastrocnemius muscles of 24 male Sprague Dawley rats to stain the sensory nerves. Then, the drop-mass method was used to damage the right gastrocnemius muscle of the posterior limb. Anti-NGF antibodies (50μL) were injected into the injured muscles in 12 rats. Tissues were evaluated 1, 3, and 7 days post-injury by performing haematoxylin-and-eosin (HE) staining. The percentage of the total number of FG-positive cells that were also positive for a pain-related neuropeptide, calcitonin gene-related peptide (CGRP), was determined for the bilateral dorsal root ganglia from L1 to L6 7 days post-injury. ResultsHE staining showed active inflammation, indicated by increased basophil and eosinophil accumulation, at the injury site 1 and 3 days post-injury, as well as scar tissue formation 7 days post-injury. Injection of anti-NGF reduced muscle necrosis 1 and 3 days post-injury, and resulted in replacement of granulation tissue and muscle fibre regeneration 7 days post-injury. Anti-NGF also significantly inhibited CGRP among FG-positive cells (treatment group 38.2%, control group 49.6%; P<0.05). DiscussionThis study found active inflammation induced by NGF, which may contribute to pain after muscle injury. Anti-NGF antibodies successfully suppressed the pain mediator NGF and inhibited inflammation, suggesting NGF as a target for control in pain management.

Choosing the appropriate model for studying muscle regeneration in mice: A comparative study of classical protocols

Skeletal muscle is a very relevant model to study tissue regeneration because of its striking capacities to structurally and functionally regenerate, after one or many rounds of degeneration. Almost all research teams working on muscle regeneration exploit numerous experimental models of muscle injury, displaying a differential effect on the fate of resident or circulating cells in the muscle bed after injury. The main focus of these studies is generally the stem cell and little is known about the relative role of the different other muscle components (myofibers, stroma, vascularisation and immune cells) during tissue reconstruction after an injury. Our project, based on the comparison between different injury models (chemicals, snake venoms or mechanical/physical injury), is in fine to understand the involvement of the different muscle tissue compartments (stem cells, inflammatory cells, vascular system and connective tissue) as well as their interactions in the tissue destruction/regeneration processes. Using morphological approaches, we first characterised the kinetics of muscle regeneration in different injury models, and demonstrated significant differences, in the early stages, in the tissue effects of these models, in contrast to an ad integrum restoration of the tissue properties in the late stages for all models. Collectively, these data allows us to decipher the fine interactions between muscle compartments and their importance in the different stages of the tissue regeneration process, a fundamental knowledge for the set up of new innovative therapeutic strategies.

Diabetic mice exhibited a peculiar alteration in body composition with exaggerated ectopic fat deposition after muscle injury due to anomalous cell differentiation.

BackgroundSarcopenic obesity, age‐related muscle loss, which is compensated by an increase in fat mass, impairs quality of life in elderly people. Although the increase in intramuscular fat is associated with decreased insulin sensitivity and increased metabolic risk factors, the origin of diabetes‐associated intramuscular fat has not been elucidated. Here, we investigated intramuscular fat deposition using a muscle injury model in type 2 diabetic mice.MethodsMale 8‐week‐old C57BL/6 and 8‐week‐old and 26‐week‐old KKAy underwent intramuscular injection of cardiotoxin (Ctx) (100 μL/10 μM) into the tibialis anterior (TA) muscles. After 2 weeks, the muscles were removed and evaluated.ResultsKKAy exhibited impaired muscle regeneration and ectopic fat deposition. Such impairment was more marked in older KKAy. These changes were also observed in another diabetic mouse model, db/db and diet‐induced obese mice but not in streptozocin‐induced diabetic mice. Deposited fat was platelet‐derived growth factor (PDGF) receptor alpha positive and its cytoskeleton was stained with Masson's trichrome, indicating it to be of fibro‐adipocyte progenitor cell origin. Expression of a myogenic marker, myoD, was lower and that of PDGF receptor alpha and CCAAT/enhancer binding protein (CEBP) alpha was higher in Ctx‐injured TA of KKAy compared with that of C57BL/6. Peroxisome proliferator‐activated receptor γ (PPARγ) was highly expressed in fat‐forming lesions in older KKAy. Treatment with all‐trans retinoic acid prevented the formation of intramuscular fat; however, treatment with GW9662, a PPARγ antagonist, increased the fibrotic change in muscle.ConclusionsDiabetic mice showed impaired muscle regeneration with fat deposition, suggesting that diabetes may enhance sarcopenic obesity through a mechanism involving anomalous fibro‐adipocyte progenitor cell differentiation.

P21 deficiency delays regeneration of skeletal muscular tissue.

The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

Longitudinal evaluation of local muscle conditions in a rat model of gastrocnemius muscle injury using an in vivo imaging system.

This study aimed to evaluate the time course of local changes during the acute phase of gastrocnemius muscle strain, in a rat model, using an in vivo imaging system. Thirty-eight, 8-week-old Sprague-Dawley male rats were used in our study. Experimental injury of the right gastrocnemius muscle was achieved using the drop-mass method. After inducing muscle injury, a liposomally formulated indocyanine green derivative (LP-iDOPE, 7 mg/kg) was injected intraperitoneally. We evaluated the muscle injuries using in vivo imaging, histological examinations, and enzyme-linked immunosorbent assays. The fluorescence peaked approximately 18 h after the injury, and decreased thereafter. Histological examinations revealed that repair of the injured tissue occurred between 18 and 24 h after injury. Quantitative analyses for various cytokines demonstrated significant elevations of interleukin-6 and tumor necrosis factor-α at 3 and 18 h post-injury, respectively. The time course of fluorescence intensity, measured using in vivo imaging, demonstrated that the changes in cytokine levels and histopathologic characteristics were consistent. Specifically, these changes reached peaked 18 h post-injury, followed by trends toward recovery.

Mesenchymal-stem-cell-derived exosomes accelerate skeletal muscle regeneration

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle-repair-related cytokines, a number of repair-related miRNAs were identified. This study suggests that the MSC-derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR-494.

MiR-30 family microRNAs regulate myogenic differentiation and provide negative feedback on the microRNA pathway.

microRNAs (miRNAs) are short non-coding RNAs that can mediate changes in gene expression and are required for the formation of skeletal muscle (myogenesis). With the goal of identifying novel miRNA biomarkers of muscle disease, we profiled miRNA expression using miRNA-seq in the gastrocnemius muscles of dystrophic mdx4cv mice. After identifying a down-regulation of the miR-30 family (miR-30a-5p, -30b, -30c, -30d and -30e) when compared to C57Bl/6 (WT) mice, we found that overexpression of miR-30 family miRNAs promotes differentiation, while inhibition restricts differentiation of myoblasts in vitro. Additionally, miR-30 family miRNAs are coordinately down-regulated during in vivo models of muscle injury (barium chloride injection) and muscle disuse atrophy (hindlimb suspension). Using bioinformatics tools and in vitro studies, we identified and validated Smarcd2, Snai2 and Tnrc6a as miR-30 family targets. Interestingly, we show that by targeting Tnrc6a, miR-30 family miRNAs negatively regulate the miRNA pathway and modulate both the activity of muscle-specific miR-206 and the levels of protein synthesis. These findings indicate that the miR-30 family may be an interesting biomarker of perturbed muscle homeostasis and muscle disease.