Muscle Cryoinjury and Quantification of Regenerating Myofibers in Mice (original) (raw)
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Scientific Reports, 2016
The application of cryotherapy is widely used in sports medicine today. Cooling could minimize secondary hypoxic injury through the reduction of cellular metabolism and injury area. Conflicting results have also suggested cryotherapy could delay and impair the regeneration process. There are no definitive findings about the effects of cryotherapy on the process of muscle regeneration. The aim of the present study was to evaluate the effects of a clinical-like cryotherapy on inflammation, regeneration and extracellular matrix (ECM) remodeling on the Tibialis anterior (TA) muscle of rats 3, 7 and 14 days post-injury. It was observed that the intermittent application of cryotherapy (three 30-minute sessions, every 2 h) in the first 48 h post-injury decreased inflammatory processes (mRNA levels of TNF-α, NF-κB, TGF-β and MMP-9 and macrophage percentage). Cryotherapy did not alter regeneration markers such as injury area, desmin and Myod expression. Despite regulating Collagen I and III and their growth factors, cryotherapy did not alter collagen deposition. In summary, clinical-like cryotherapy reduces the inflammatory process through the decrease of macrophage infiltration and the accumulation of the inflammatory key markers without influencing muscle injury area and ECM remodeling. Skeletal muscle lesions are responsible for the majority of the functional limitations observed in sports and occupational medicine 1. After primary injury, muscle regeneration occurs in a highly orchestrated process that involves the activation of muscle satellite cells to proliferate and differentiate into a new muscle fiber 2 with a constant pattern irrespective of the cause (contusion, strain, or laceration). After muscle injury it is possible to observe four independent phases, despite their etiology: degeneration, inflammation, regeneration, and fibrosis 2-4. The activation and differentiation of satellite cells is characterized by the rapid upregulation of myogenic differentiation 1 (MyoD) and insulin-like growth factor 1 (IGF-1) 5,6. In addition, in vitro and in vivo studies indicate that anti-inflammatories such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and nuclear factor-κ B (NF-κ B) produced by macrophages could activate satellite cells, stimulating myoblast proliferation and differentiation into myotube formation 7,8. The fibrosis and remodeling phases of muscle regeneration involve the deposition of Collagen I and III fibers and reorganization of the tissue, which could be induced by TGF-β 9 , IGF-I 10 , and connective tissue growth factor (CTGF) 11. In addition, matrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM) 12. MMP-2 (or gelatinase A) activity is concurrent with the regeneration of new myofibers probably due to degradation of type IV collagen of the basement membrane during myoblast proliferation, migration, and fusion. MMP-9 (or gelatinase B) activation is related to the early inflammatory phase and to the activation of satellite cells 13,14 .
Abstract Background: Accelerated muscle regeneration is highly desirable after direct injury in trauma patients. Though the advantage of extracellular matrix extracted from porcine urine bladder (UBM) on tissue regeneration is feasible, the mechanisms by which skeletal myogenesis is improved are not clear. The current study aim was to determine whether UBM affects skeletal satellite cells during muscle repair after cryoinjury. Methods: RAG2-/-, γc-/- mice underwent bilateral gluteus muscle cryoinjury under general anesthesia. 200μg UBM suspended in 20 μl of Matrigel solution was implanted on one side of the wound intramuscularly, and 20μl of vehicle was applied on the contralateral side as a sham treatment. Five animals without any operation served as baseline. Mice were sacrificed from days 1 to 28 after injury for muscle tissue collection. Tissue morphology was estimated via H&E staining. Satellite cell proliferation was examined by immunofluorescence staining and western blot. Myogenesis markers were assessed by qPCR. Results: H&E staining results showed that muscle regeneration area increased in both sham and UBM treated muscles following injury. The number of regenerating myotubes was significantly higher in UBM treated tissue at 28 days (p<0.05, vs. sham group). Both Pax7+ and Ki-67+ satellite cell number significantly increased in muscle with UBM treatment compared to sham treated muscles (p<0.05). Protein levels of proliferating cell nuclear antigen (PCNA) were greater in muscle with UBM treatment at day 14 and 28 (p<0.05). MyoD1 and myogenin mRNA levels were also significantly higher in UBM treated animal muscle at day 28 (p<0.05). Conclusion: UBM treatment increased skeletal satellite cell proliferation and myogenic differentiation at 28 days after local muscle injury. Keywords: Skeletal muscle injury, muscle regeneration, satellite cell, proliferation, differentiation © 2014 Song et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Skeletal muscle represents the largest tissue mass in the body, consisting of 40% to 45% of total body weight and is responsible for supportive and locomotive functions [1]. This tissue is at risk for injury whether purposeful (exercise) [2] or in traumatic incidents. In the case of exercise, regeneration of the injured tissue is the desired response, and for direct injury from trauma this is also true. However, after direct injury the muscle mass is lost with minimal regeneration affecting long term recovery and function. Current clinical management options include functional free muscle transfer and the use of advanced bracing [3], which have limited efficiency on muscle recovery. Improvement of muscle regrowth is then of major concern in the treatment of those with severe and direct muscle injury. Muscle regeneration involves both resident and non-resident cells with myogenic properties. The major participants in adult muscle regeneration are muscle satellite cells which reside underneath the basal lamina of mature muscle fibers. In response to skeletal muscle
Influence of icing on muscle regeneration after crush injury to skeletal muscles in rats
Journal of Applied Physiology, 2011
The influence of icing on muscle regeneration after crush injury was examined in the rat extensor digitorum longus. After the injury, animals were randomly divided into nonicing and icing groups. In the latter, ice packs were applied for 20 min. Due to the icing, degeneration of the necrotic muscle fibers and differentiation of satellite cells at early stages of regeneration were retarded by ∼1 day. In the icing group, the ratio of regenerating fibers showing central nucleus at 14 days after the injury was higher, and cross-sectional area of the muscle fibers at 28 days was evidently smaller than in the nonicing group. Besides, the ratio of collagen fibers area at 14 and 28 days after the injury in the icing group was higher than in the nonicing group. These findings suggest that icing applied soon after the injury not only considerably retarded muscle regeneration but also induced impairment of muscle regeneration along with excessive collagen deposition. Macrophages were immunohis...
Scandinavian Journal of Medicine & Science in Sports, 2020
Cryotherapy is a therapeutic modality widely used for the treatment of muscle injuries to control pain and inflammatory processes. This study aimed to investigate the effects of cryotherapy on the inflammatory and oxidative stress parameters and mechanical properties of, and pain in, the skeletal muscles of rats with lacerative muscle injury. The rats were anesthetized with 4% isoflurane and subjected to gastrocnemius muscle laceration injury. After injury, all animals in the intervention groups received cryotherapy treatment for 20 minutes using plastic bags containing crushed ice. The protocol comprised three daily applications at 3‐hour intervals on the day of injury, with reapplication 24 hours later. Seventy‐two male Wistar rats were divided into three groups: sham, muscle injury (MI), and MI + cryotherapy (MI + cryo). Muscle mechanical properties were analyzed by mechanical tensile testing on day 7 after injury. The MI + cryo group showed reduced TNF‐α, IFN‐γ, and IL1β levels;...
Absence of exogenous satellite cell contribution to regeneration of frozen skeletal muscle
Journal of Muscle Research and Cell Motility, 1986
Rat extensor digitorum longus (EDL) muscles were repeatedly frozen and thawed to kill completely all cellular constituents. Within four days, blood vessels and phagocytic cells invaded the muscles. Migrating or circulating myoblasts were not among the invading cells, and could be induced to invade the frozen muscle only when a physical bridge was created with an adjacent intact muscle. A further requirement for migration was that the connective tissue investments of both the frozen EDL and adjacent muscle had to be disrupted. This study demonstrates that regeneration of a muscle is primarily dependent upon the intrinsic satellite cell population, although under some circumstances recruitment of extrinsic cells is possible.
Neuropathology and Applied Neurobiology, 1993
In regenerating skeletal muscle, sarcoplasmic extensions containing variable numbers of nuclei, widely referred to as 'buds' or 'stumps', are formed at the ends of damaged myofibres. In this paper we investigated whether the nuclei seen in the buds results from fusion of myogenic cells or from migration of myonuclei to the sealed ends of damaged myofibres in murine muscle regenerating after crush injury. The fusion of mononuclear and multinucleate myogenic cells to the buds was demonstrated by transmission electron microscopy. In order to elucidate the frequency and kinetics of cytoplasmic continuity between myotubes and sealed myofibres, we labelled the damaged myofibres with carbocyanine dye DiI (which inserts into the lipid bilayer and travels down continuous membranes) and the samples were then examined by confocal scanning microscopy. This technique showed that there was little fusion between myotubes and myofibres during the first 6 days after crush injury, but significant fusion had occurred by the tenth day especially at the newly sealed region of the damaged myofibre. A scheme for the repair of damaged skeletal muscle is presented.
Journal of Cell Science, 1993
Using muscle as an in vivo model system, we have tested the hypothesis that basic fibroblast growth factor is released from a cytoplasmic storage site into the extracellular environment via diffusion through survivable, mechanically-induced plasma membrane disruptions. Normal and dystrophic (mdx) mouse muscle were studied. Strong immunostaining for bFGF was detected in the cytoplasm of myofibers of uninjured muscle fixed in situ by perfusion. By contrast, myofibers did not stain cytoplasmically for bFGF after suffering lethal disruptions of their plasma membranes caused by freezing and thawing followed by sectioning. Sub-lethal, transient disruptions of myofiber plasma membranes - termed plasma membrane ‘wounds’ - were shown to be induced by needle puncture or exercise of muscle. Quantitative image analysis revealed that these wounded fibers contained significantly reduced levels of bFGF. Dystrophic exercised and unexercised muscle was found to possess an ∼6-fold higher proportion o...
Tissue triage and freezing for models of skeletal muscle disease
2014
Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.
Brazilian Archives of Biology and Technology, 2013
This study examined the effect of three sessions of cryotherapy (three sessions of 30 minutes applied each 2 h) and muscle compression in the regenerating skeletal muscle of the rats. The middle belly of tibialis anterior muscle was injured by a frozen iron bar and received one of the following intervention: injury + cryotherapy (treated with cryotherapy); injury + placebo (sand pack), and injury (I).The enzymatic activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the presence of 1mM or 10mM pyruvate. The ANOVA and Tukey's test (p≤0.05) were performed for the statistical analysis. In summary, the intermittent sessions of cryotherapy, associated to muscle compression and applied immediately after the primary muscle injury minimized the CS and LDH activity at 4h30 and 24h periods post-lesion, which could be related to the reduction in the secondary muscle injury inherent to cryotherapy treatment.