Muscle atrophy and neuromuscular impairment are consequences of spinal cord injury (SCI) that impede quality of life and functional recovery. PURPOSE: To examine time course changes in muscle fiber type distribution, fiber cross-sectional area (fCSA), and other histologic characteristics of muscle pathology occurring in rats in response to moderate-severe contusion SCI. METHODS: Twenty-four 4-month old male Sprague-Dawley rats received SHAM surgery or T9 laminectomy plus moderate-severe (250 kilodyne) contusion SCI by a computer-guided impactor. Body weight and Basso-Beattie-Bresnahan (BBB) hindlimb locomotor rating scores were measured weekly. Animals were euthanized and soleus were harvested at 2-weeks, 1-month, 2-months, or 3-months post-surgery. Soleus fiber-type distribution, fCSA, and muscle-nerve bundle morphology were assessed by immunofluorescent staining, imaged using an epifluorescent microscope, and quantified with semi-automatic muscle analysis using segmentation of histology (SMASH). RESULTS: At 1-week post-surgery, SCI animals exhibited near-complete hindlimb paralysis (indicated by BBB scores <3), with minimal improvement in voluntary hindlimb locomotor function thereafter. Body weight, soleus mass, and median fCSA were significantly lower in SCI vs SHAM animals (p<0.01 at all timepoints). A slow-to-fast fiber-type shift was observed in SCI animals, with a progressive ~20% decrease in the number of type I fibers, ~8% increase in type IIa fibers, and ~5% increase in hybrid type I/IIa fibers at each consecutive timepoint, along with the emergence of unstained type IIx/b muscle fibers (~30% of total) at 3-months. In addition, muscle fiber splitting was present in SCI animals at 2-months, as well as reduced neurofilament staining in SCI muscle-nerve bundles. Furthermore, evidence of growth related remodeling occurred in SHAM muscles from increased centrally nucleated fibers across timepoints but not in SCI. CONCLUSION: Deterioration in motor ability accompanying SCI produced muscle atrophy and progressive impairments in muscle oxidative capacity that may have resulted from repetitive denervation-reinnervation cycles. These factors may have contributed to muscle pathology resulting in limited capacity for muscle growth and remodeling.
Skeletal muscle atrophy is a hallmark of severe spinal cord injury (SCI) that is precipitated by the neural insult and paralysis. Additionally, other factors may influence muscle loss, including systemic inflammation, low testosterone, low insulin-like growth factor (IGF)-1, and high-dose glucocorticoid treatment. The signaling cascades that drive SCI-induced muscle loss are common among most forms of disuse atrophy and include ubiquitin-proteasome signaling and others. However, differing magnitudes and patterns of atrophic signals exist after SCI versus other disuse conditions and are accompanied by endogenous inhibition of IGF-1/PI3K/Akt signaling, which combine to produce exceedingly rapid atrophy. Several well-established anabolic agents, including androgens and myostatin inhibitors, display diminished ability to prevent SCI-induced atrophy, while ursolic acid and β2-agonists more effectively attenuate muscle loss. Strategies combining physical rehabilitation regimens to reload the paralyzed limbs with drugs targeting the underlying molecular pathways hold the greatest potential to improve muscle recovery after severe SCI.
Abstract Background The rate and magnitude of skeletal muscle wasting after severe spinal cord injury (SCI) exceeds most other disuse conditions. Assessing the time course of molecular changes can provide insight into the progression of muscle wasting post‐SCI. The goals of this study were (1) to identify potential targets that may prevent the pathologic features of SCI in soleus muscles and (2) to establish therapeutic windows for treating these pathologic changes. Methods Four‐month‐old Sprague–Dawley male rats received T9 laminectomy (SHAM surgery) or severe contusion SCI. Hindlimb locomotor function was assessed weekly, with soleus muscles obtained 1 week, 2 weeks, 1 month and 3 months post‐surgery ( n = 6–7 per group per timepoint). RNA was extracted from muscles for bulk RNA‐sequencing analysis ( n = 3–5 per group per timepoint). Differentially expressed genes (DEGs) were evaluated between age‐matched SHAM and SCI animals. Myofiber size, muscle fibre type and fibrosis were assessed on contralateral muscles. Results SCI produced immediate and persistent hindlimb paralysis, with Basso–Beattie–Bresnahan locomotor scores remaining below 7 throughout the study, contributing to a progressive 25–50% lower soleus mass and myofiber atrophy versus SHAM ( P < 0.05 at all timepoints). Transcriptional comparisons of SCI versus SHAM resulted in 184 DEGs (1 week), 436 DEGs (2 weeks), 133 DEGs (1 month) and 1200 DEGs (3 months). Upregulated atrophy‐related genes included those associated with cell senescence, nuclear factor kappa B, ubiquitin proteasome and unfolded protein response pathways, along with upregulated genes that negatively influence muscle growth through the transforming growth factor beta pathway and inhibition of insulin‐like growth factor‐I/Akt/mechanistic target of rapamycin and p38/mitogen‐activated protein kinase signalling. Genes associated with extracellular matrix (ECM), including collagens, collagen crosslinkers, proteoglycans and those regulating ECM integrity, were enriched within upregulated DEGs at 1 week but subsequently downregulated at 2 weeks and 3 months and were accompanied by >50% higher ECM areas and hydroxyproline levels in SCI muscles ( P < 0.05). Myofiber remodelling genes were enriched in upregulated DEGs at 2 weeks and 1 month and were downregulated at 3 months. Genes that regulate neuromuscular junction remodelling were evident in muscles post‐SCI, along with slow‐to‐fast fibre‐type shifts: 1 week and 2 weeks SCI muscles were composed of 90% myosin heavy chain (MHC) type I fibres, which decreased to only 16% at 3 months and were accompanied by 50% fibres containing MHC IIX ( P < 0.05). Metabolism genes were enriched in upregulated DEGs at 1 month and were further enriched at 3 months. Conclusions Our results substantiate many known pathologic features of SCI‐induced wasting in rat skeletal muscle and identify a progressive and dynamic transcriptional landscape within the post‐SCI soleus. Future studies are warranted to consider these therapeutic treatment windows when countering SCI muscle pathology.
Insulin-like growth factor I (IGF-I) is essential for muscle and bone development and a primary mediator of growth hormone (GH) actions. While studies have elucidated the importance of IGF-I specifically in muscle or bone development, few studies to date have evaluated the relationship between muscle and bone modulated by IGF-I in vivo, during post-natal growth. Mice with muscle-specific IGF-I overexpression (mIgf1+/+) were utilised to determine IGF-I- and muscle-mass-dependent effects on craniofacial skeleton development during post-natal growth. mIgf1+/+ mice displayed accelerated craniofacial bone growth when compared to wild-type animals. Virus-mediated expression of IGF-I targeting the masseter was performed to determine if post-natal modulation of IGF-I altered mandibular structures. Increased IGF-I in the masseter affected the mandibular base plane angle in a lateral manner, increasing the width of the mandible. At the cellular level, increased muscle IGF-I also accelerated cartilage thickness in the mandibular condyle. Importantly, mandibular length changes associated with increased IGF-I were not present in mice with genetic inhibition of muscle IGF-I receptor activity. These results demonstrated that muscle IGF-I could indirectly affect craniofacial growth through IGF-I-dependent increases in muscle hypertrophy. These findings have clinical implications when considering IGF-I as a therapeutic strategy for craniofacial disorders.
Background/Aims: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion.Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration.A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s.The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown.The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase.Methods: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice.We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13.We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals.Outcome measures included muscle morphology and function.
Loading and testosterone may influence musculoskeletal recovery after spinal cord injury (SCI). Our objectives were to determine (a) the acute effects of bodyweight-supported treadmill training (TM) on hindlimb cancellous bone microstructure and muscle mass in adult rats after severe contusion SCI and (b) whether longer-term TM with adjuvant testosterone enanthate (TE) delivers musculoskeletal benefit. In Study 1, TM (40 min/day, 5 days/week, beginning 1 week postsurgery) did not prevent SCI-induced hindlimb cancellous bone loss after 3 weeks. In Study 2, TM did not attenuate SCI-induced plantar flexor muscles atrophy nor improve locomotor recovery after 4 weeks. In our main study, SCI produced extensive distal femur and proximal tibia cancellous bone deficits, a deleterious slow-to-fast fiber-type transition in soleus, lower muscle fiber cross-sectional area (fCSA), impaired muscle force production, and levator ani/bulbocavernosus (LABC) muscle atrophy after 8 weeks. TE alone (7.0 mg/week) suppressed bone resorption, attenuated cancellous bone loss, constrained the soleus fiber-type transition, and prevented LABC atrophy. In comparison, TE+TM concomitantly suppressed bone resorption and stimulated bone formation after SCI, produced near-complete cancellous bone preservation, prevented the soleus fiber-type transition, attenuated soleus fCSA atrophy, maintained soleus force production, and increased LABC mass. 75% of SCI+TE+TM animals recovered voluntary over-ground hindlimb stepping, while no SCI and only 20% of SCI+TE animals regained stepping ability. Positive associations between testosterone and locomotor function suggest that TE influenced locomotor recovery. In conclusion, short-term TM alone did not improve bone, muscle, or locomotor recovery in adult rats after severe SCI, while longer-term TE+TM provided more comprehensive musculoskeletal benefit than TE alone.
