Molecular and Metabolic Mechanism of Low-Intensity Pulsed Ultrasound Improving Muscle Atrophy in Hindlimb Unloading Rats.

Low-intensity pulsed ultrasound (LIPUS) has been proved to promote the proliferation of myoblast C2C12. However, whether LIPUS can effectively prevent muscle atrophy has not been clarified, and if so, what is the possible mechanism. The aim of this study is to evaluate the effects of LIPUS on muscle atrophy in hindlimb unloading rats, and explore the mechanisms. The rats were randomly divided into four groups: normal control group (NC), hindlimb unloading group (UL), hindlimb unloading plus 30 mW/cm2 LIPUS irradiation group (UL + 30 mW/cm2), hindlimb unloading plus 80 mW/cm2 LIPUS irradiation group (UL + 80 mW/cm2). The tails of rats in hindlimb unloading group were suspended for 28 days. The rats in the LIPUS treated group were simultaneously irradiated with LIPUS on gastrocnemius muscle in both lower legs at the sound intensity of 30 mW/cm2 or 80 mW/cm2 for 20 min/d for 28 days. C2C12 cells were exposed to LIPUS at 30 or 80 mW/cm2 for 5 days. The results showed that LIPUS significantly promoted the proliferation and differentiation of myoblast C2C12, and prevented the decrease of cross-sectional area of muscle fiber and gastrocnemius mass in hindlimb unloading rats. LIPUS also significantly down regulated the expression of MSTN and its receptors ActRIIB, and up-regulated the expression of Akt and mTOR in gastrocnemius muscle of hindlimb unloading rats. In addition, three metabolic pathways (phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism) were selected as important metabolic pathways for hindlimb unloading effect. However, LIPUS promoted the stability of alanine, aspartate and glutamate metabolism pathway. These results suggest that the key mechanism of LIPUS in preventing muscle atrophy induced by hindlimb unloading may be related to promoting protein synthesis through MSTN/Akt/mTOR signaling pathway and stabilizing alanine, aspartate and glutamate metabolism.

Cerium Oxide Nanoparticle Administration to Skeletal Muscle Cells under Different Gravity and Radiation Conditions.

For their remarkable biomimetic properties implying strong modulation of the intracellular and extracellular redox state, cerium oxide nanoparticles (also termed "nanoceria") were hypothesized to exert a protective role against oxidative stress associated with the harsh environmental conditions of spaceflight, characterized by microgravity and highly energetic radiations. Nanoparticles were supplied to proliferating C2C12 mouse skeletal muscle cells under different gravity and radiation levels. Biological responses were thus investigated at a transcriptional level by RNA next-generation sequencing. Lists of differentially expressed genes (DEGs) were generated and intersected by taking into consideration relevant comparisons, which led to the observation of prevailing effects of the space environment over those induced by nanoceria. In space, upregulation of transcription was slightly preponderant over downregulation, implying involvement of intracellular compartments, with the majority of DEGs consistently over- or under-expressed whenever present. Cosmic radiations regulated a higher number of DEGs than microgravity and seemed to promote increased cellular catabolism. By taking into consideration space physical stressors alone, microgravity and cosmic radiations appeared to have opposite effects at transcriptional levels despite partial sharing of molecular pathways. Interestingly, gene ontology denoted some enrichment in terms related to vision, when only effects of radiations were assessed. The transcriptional regulation of mitochondrial uncoupling protein 2 in space-relevant samples suggests perturbation of the intracellular redox homeostasis, and leaves open opportunities for antioxidant treatment for oxidative stress reduction in harsh environments.

Low-Frequency Electrical Stimulation Promotes Satellite Cell Activities to Facilitate Muscle Regeneration at an Early Phase in a Rat Model of Muscle Strain.

The capability of regeneration for skeletal muscle after injury depends on the differentiation and proliferation ability of the resident stem cells called satellite cells. It has been reported that electrical stimulation was widely used in clinical conditions to facilitate muscle regeneration after injury, but the characterization of satellite cell responses to the context of low-frequency electrical stimulation in early-phase muscle strain conditions has not been fully clarified. In this study, we aim to investigate the effects of low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily) on satellite cell activities in a rat model for the early phase of muscle strain. Firstly, we adopted our previously developed rat model to mimic the early phase of muscle strain in human. After then, we examined the effects of low-frequency electrical stimulation on histopathological changes of the muscle fiber by hematoxylin and eosin (H&E) staining. Finally, we investigated the effects of low-frequency electrical stimulation on satellite cell proliferation and differentiation by quantification of the expression level of the specific proteins using western blot analyses. The muscle strain in biceps femoris muscles of rats can be induced by high-speed rotation from knee flexion 50° to full knee extension at 960°·s-1 angular velocity during its tetany by activating the sciatic nerve, as evidenced by a widening of the interstitial space between fibers, and more edema or necrosis fibers were detected in the model rats without treatment than in control rats. After treatment with low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily), the acute strained biceps femoris muscles of rats showed obvious improvement of histomorphology as indicated by more mature muscle fibers with well-ordered formation with clear boundaries. Consistently, the expression levels of the MyoD and myogenin were marked higher than those in the rats in the animal model group, indicating increased satellite cell proliferating and differentiating activities by low-frequency electrical stimulation. This study shows that low-frequency electrical stimulation provides an effective stimulus to upregulate the protein expression of MyoD/myogenin and accelerate the restoration of structure during the early phase of muscle strain. This may have significance for clinical practice. Optimization of low-frequency electrical stimulation parameters may enhance the therapeutic outcome in patients.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model.

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

Effectiveness of electrical stimulation on nerve regeneration after crush injury: Comparison between invasive and non-invasive stimulation.

Several studies have investigated the use of invasive and non-invasive stimulation methods to enhance nerve regeneration, and varying degrees of effectiveness have been reported. However, due to the use of different parameters in these studies, a fair comparison between the effectiveness of invasive and non-invasive stimulation methods is not possible. The present study compared the effectiveness of invasive and non-invasive stimulation using similar parameters. Eighteen Sprague Dawley rats were classified into three groups: the iES group stimulated with fully implantable device, the tES group stimulated with transcutaneous electrical nerve stimulation (TENS), and the injury group (no stimulation). The iES and tES groups received stimulation for 6 weeks starting immediately after the injury. Motor function was evaluated using the sciatic functional index (SFI) every week. The SFI values increased over time in all groups; faster and superior functional recovery was observed in the iES group than in the tES group. Histological evaluation of the nerve sections and gastrocnemius muscle sections were performed every other week. The axon diameter and muscle fiber area in the iES group were larger, and the g-ratio in the iES group was closer to 0.6 than those in the tES group. To assess the cause of the difference in efficiency, a 3D rat anatomical model was used to simulate the induced electric fields in each group. A significantly higher concentration and intensity around the sciatic nerve was observed in the iES group than in the tES group. Vector field distribution showed that the field was orthogonal to the sciatic nerve spread in the tES group, whereas it was parallel in the iES group; this suggested that the tES group was less effective in nerve stimulation. The results indicated that even though rats in the TENS group showed better recovery than those in the injury group, it cannot replace direct stimulation yet because rats stimulated with the invasive method showed faster recovery and superior outcomes. This was likely attributable to the greater concentration and parallel distribution of electric field with respect to target nerve.

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration.

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

Alterations in pectoralis muscle cell characteristics after radiation of the human breast in situ.

The life-time risk of being diagnosed with breast cancer is ~12%, hence breast cancer is by far the most common cancer among women. The multimodal treatment concept of breast cancer often intends radiation. The utilized ionizing radiation leads changes in the tissue resulting in tissue damage due to an alteration of molecular factors. The goal of this study was to identify the role of muscle-catabolic proteins after radiation of human pectoralis major muscles in situ. Tissue of the pectoralis major muscle was collected in 12 breast cancer patients after radiation (maximum 3 years after radiation) undergoing a deep inferior epigastric perforator free-flap breast reconstruction. At the same time, an intraindividual comparison to rectus abdominis muscle was carried out upon free-flap elevation. Immunological properties, cell proliferation, differentiation as well as the expression profile of the muscle tissue were investigated through immunohistological reactions, a DNA-microarray and histology. We found significantly increased neutrophil immigration in the radiated muscle tissue. At the same time, proteins responsible for muscular atrophy and apoptosis were significantly elevated in immunohistochemistry. A DNA microarray detected immunological upregulation and myo-differentiative disorders in radiated muscle tissue. This novel study investigating catabolism in radiated muscle in situ can serve as a basis for the treatment of radiation-accompanied muscle disorders.

Low-level laser irradiation induces a transcriptional myotube-like profile in C2C12 myoblasts.

Low-level laser irradiation (LLLI) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLI exerts these effects remain largely unknown. Here, we described global gene expression profiling analysis in C2C12 myoblasts after LLLI that identified 514 differentially expressed genes (DEG). Gene ontology and pathway analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLI with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLI. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLI myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLI myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLI, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLI regulates a set of mRNAs that control myoblast proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLI myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLI on skeletal muscle cells.

A Low-Cost Pulse Generator for Exacerbating Muscle Fiber Detachment Phenotypes in Zebrafish.

Muscle fiber detachment from myoseptal boundaries is a common finding in zebrafish models of muscular dystrophies. In some instances, there is a weakening of the interaction between muscle fiber and myosepta, which is yet to manifest as a fiber detachment phenotype. Therefore, to push the fiber detachment of muscle, mutant fish but not their wild-type siblings, beyond their binding threshold, a series of small electrical pulses can be applied to the larvae to create a maximal force contraction and ultimately fiber detachment. To do this, we built a digital pulse generator which delivers four 8 ms 30 V pulses in quick succession, and it has the advantage over older analog approaches to pulse generation because it improves accuracy and is appreciably less expensive. Our pulse generator significantly increases fiber detachment in the laminin-α2 deficient, congenital muscular dystrophy type 1a (MDC1a) model lama2-/- fish when compared with controls.

Promotion of Myogenic Maturation by Timely Application of Electric Field Along the Topographical Alignment.

Engineered muscular substitutes can restore the impaired muscle functions when integrated properly into the host tissue. To generate functional muscles with sufficient contractility at the site of transplant, the in vitro construction of fully differentiated muscle fibers would be desired. Many previous reports have identified either topographical alignment or electrical stimulation as an effective tool to promote myogenic differentiation. However, optimization of spatial and temporal arrangement of these two physical cues for better differentiation and maturation of skeletal muscles has not been investigated. In this article, we introduce a novel cell culture system that allows simultaneous application of these two independent directional cues at both orthogonal and parallel arrangements. We then show that the parallel arrangement of the aligned topography and the electric field synergistically facilitates better differentiation and maturation of C2C12, generating myotubes with more fused nuclei. Addition of the electric stimulation at the late stage of myogenic differentiation is found to further improve cell fusion to form multinucleate myotubes through a phosphatidylinositol-3-OH-kinase-dependent pathway. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance both myogenic differentiation and maturation in a temporal and orientation-dependent manner, providing the basis for therapeutic strategies for regenerative tissue engineering.

Muscle fiber conduction velocity and EMG amplitude of the upper trapezius muscle in healthy subjects after low-level laser irradiation: a randomized, double-blind, placebo-controlled, crossover study.

Although low-level laser therapy (LLLT) is an important resource for the treatment of non-specific neck pain patients, the dose which presents the greatest therapeutic potential for the treatment of this pathology is still unclear. The present study aimed to evaluate the immediate effect of LLLT on the muscle fiber conduction velocity (MFCV) and electromyographic activity (EMG) of the upper trapezius (UT) muscle in healthy individuals. A total of 20 healthy subjects were enrolled in a randomized, double-blind, crossover study. Active LLLT (820 nm wavelength, 30 mW, energy total 18 J) or placebo LLLT (pLLLT) was delivered on the UT muscle. Each subject was subjected to a single session of active LLLT and pLLLT. Surface electromyography (sEMG) signal of the UT muscle was recorded during five different step contractions of shoulder elevation force (10-30% maximal voluntary contraction) pre- and post-LLLT irradiation. The values of MFCV and sEMG global amplitude (RMSG) were used to calculate the effects of LLLT. The results showed no difference in the MFCV comparing the LLLT and pLLLT groups (F = 0.72 p = 0.39, η p2 = 0.004). However, a significant difference was observed in the RMSG between the LLLT and pLLLT (F 1,2 = 16.66; P < 0.0001, η p2 = 0.09). Individuals who received active LLLT presented a significant decrease in RMSG after laser application (F = 61.28; p < 0.0001, η p2 = 0.43). In conclusion, the 820 nm LLLT, with energy total of 18 J, did not alter the MFCV but significantly reduced the sEMG signal amplitude of the upper trapezius muscle in healthy subjects to a level of up to 30% of maximal voluntary contraction.

Optogenetic approach for targeted activation of global calcium transients in differentiated C2C12 myotubes.

Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.

Influence of Nitric Oxide generated through microwave plasma on L6 skeletal muscle cell myogenesis via oxidative signaling pathways.

Myogenic precursors are myoblasts that have a potency to differentiate into muscle fibers on injury and maintain the regenerative power of skeletal muscle. However, the roles of exogenous nitric oxide (NO) in muscle development and myoblast differentiation are largely unknown. Therefore, in this study, we examined the effects of exogenous NO generated by a microwave plasma torch on rat myoblastic L6 cell proliferation and differentiation. We observed that the differentiation of L6 myogenic precursor cells into myotubes was significantly enhanced after NO treatment. The expression of the myogenesis marker proteins and mRNA level, such as myoD, myogenin, and myosin heavy chain (MHC), as well as the cyclic guanosine monophosphate (cGMP) level, were significantly increased after the NO treatment, without creating toxicity. Moreover, we observed that the oxidative stress signaling [extracellular-signal-regulated kinase (Erks), and Adenosine monophosphate-activated protein kinase (AMPK)] phosphorylation was higher in NO treated cells than in the control cells [without NO treatment]. Therefore, these results reveal the exogenous NO role in regulating myoblast differentiation through the oxidative stress signaling pathway. Through this work, we can suggest that exogenous NO can help in cell differentiation and tissue regeneration, which provides new possibilities for plasma medicine.

[WEAK COMBINED MAGNETIC FIELDS ADJUSTED TO THE PARAMETRIC RESONANCE FOR Ca2+ INTENSIFY DYSTROPHIN SYNTHESIS IN MDX MICE SKELETAL MUSCLES AFTER CELL THERAPY].

The mdx mice are an X-linked myopathic mutants, an animal model for human Duchenne muscular dystrophy (DMD). Mdx mice muscles are characterized by high level of striated muscle fibers (SMF) death followed by regeneration. As a result most SMFs of mdx mice have centrally located nuclei. The possibility of using stem cells therapy for the correction of DMD is actively being studied. One of the approaches to the usage of bone marrow stem cells for cellular therapy of DMD is the replacement of bone marrow after irradiation by X-rays. This method however does not give significant increase of dystrophin synthesis in mdx mice muscles fibers. We have tried to affect the mice after bone marrow transplantation by weak combined magnetic fields adjusted to the parametric resonance for Ca2+(Ca(2+)-MF) based on the data that the weak combined magnetic fields influence on tissues regeneration. We observed a significant increase in the proportion of dystrophin-positive SMFs in group of mdx mice radiation chimera 5 Gy and 3 Gy which was additionally exposed in Ca(2+)-MF in comparison with the control mdx mice and the group of mdx mice radiation chimera 5 Gy and 3 Gy which was kept in terrestrial magnetic field 2 months after chimera preparation--up to 15.8 and 18.3%, respectively. Also, there was an accumulation of SMFs without central nuclei. These data indicate a significanly increased efficacy of cell therapy in the case of additional exposition in Ca(2+)-MF. Thus, the efficiency of bone marrow transplantation mdx mice after both in doses 3 and 5 Gy was considerably enhanced by additional exposition to Ca(2+)-MF. Apparently, such magnetic field can intensify functioning of donor's nuclei which had been incorporated into muscle fibers.

Direct optical activation of skeletal muscle fibres efficiently controls muscle contraction and attenuates denervation atrophy.

Neural prostheses can restore meaningful function to paralysed muscles by electrically stimulating innervating motor axons, but fail when muscles are completely denervated, as seen in amyotrophic lateral sclerosis, or after a peripheral nerve or spinal cord injury. Here we show that channelrhodopsin-2 is expressed within the sarcolemma and T-tubules of skeletal muscle fibres in transgenic mice. This expression pattern allows for optical control of muscle contraction with comparable forces to nerve stimulation. Force can be controlled by varying light pulse intensity, duration or frequency. Light-stimulated muscle fibres depolarize proportionally to light intensity and duration. Denervated triceps surae muscles transcutaneously stimulated optically on a daily basis for 10 days show a significant attenuation in atrophy resulting in significantly greater contractile forces compared with chronically denervated muscles. Together, this study shows that channelrhodopsin-2/H134R can be used to restore function to permanently denervated muscles and reduce pathophysiological changes associated with denervation pathologies.

Hyperspectral deep ultraviolet autofluorescence of muscle fibers is affected by postmortem changes.

After slaughter, muscle cells undergo biochemical and physicochemical changes that may affect their autofluorescence characteristics. The autofluorescent response of different rat extensor digitorum longus (EDL) and soleus muscle fiber types was investigated by deep ultraviolet (UV) synchrotron microspectroscopy immediately after animal sacrifice and after 24 h of storage in a moist chamber at 20 °C. The glycogen content decreased from 23 to 18 µmol/g of fresh muscle in 24 h postmortem. Following a 275 nm excitation wavelength, the spectral muscle fiber autofluorescence response showed discrimination depending upon postmortem time (t0 versus t24 h) on both muscles at 346 and 302 nm and, to a lesser extent, at 408 and 325 nm. Taken individually, all fiber types were discriminated but with variable accuracy, with type IIA showing better separation of t0/t24 h than other fiber types. These results suggest the usefulness of the autofluorescent response of muscle cells for rapid meat-aging characterization.

Deep UV excited muscle cell autofluorescence varies with the fibre type.

The rat skeletal muscle consists of four pure types of muscle cells called type I, type IIA, type IIX and type IIB, and their hybrids in different proportions. They differ in their contraction speeds and metabolic pathways. The intracellular composition is adapted to the fibre function and therefore to fibre types. Given that small differences in composition are likely to alter the optical properties of the cells, we studied the impact of the cell type on the fluorescence response following excitation in the deep UV region. Rat soleus and extensor digitorum longus (EDL) muscle fibres, previously identified based on their cell types by immunohistofluorescence analysis, were analyzed by synchrotron fluorescence microspectroscopy on stain-free serial muscle cross-sections. Muscle fibres excited at 275 nm showed differences in the fluorescence emission intensity among fibre types at 302, 325, 346 and 410 nm. The 410/325 ratio decreased significantly with contractile and metabolic features in EDL muscle, in the order of I > IIA > IIX > IIB fibres (p < 0.01). Compared to type I fibres, the 346/302 ratio of IIA fibres decreased significantly in both EDL and soleus muscles (p < 0.01). This study highlights the usefulness of autofluorescence spectral signals to characterize histological cross-sections of muscle fibres with no staining chemicals.

Low-level laser therapy modulates musculoskeletal loss in a skin burn model in rats.

PURPOSE: To investigate the effectiveness of low-level laser therapy (LLLT) on gastrocnemius muscle morphology and Myod immunoexpression in a model of dorsal burn in rats. METHODS: Sixteen male Wistar rats were distributed into two groups: control group (CG): rats submitted to scald burn injury without treatment and laser treated group (LG): rats submitted to scald burn injury and treated with laser therapy. Fourteen days post-surgery, gastrocnemius muscle was evaluated being the specimens stained with HE and morphometric data was evaluated. MyoD expression was assessed by immunohistochemistry. RESULTS: The results showed that laser treated animals presented more organized tissue morphology compared to the non-treated animals, with a higher number of nucleus in the fibers. Also, the cross sectional area of the fibers and the MyoD immunoexpression in the laser treated groups was higher. CONCLUSION: Low-level laser therapy had positive effects on gastrocnemius muscle, improving tissue muscle morphology, increasing cross sectional area and MyoD immunoexpression.

Low-level laser therapy modulates musculoskeletal loss in a skin burn model in rats

PURPOSE: To investigate the effectiveness of low-level laser therapy (LLLT) on gastrocnemius muscle morphology and Myod imunoexpression in a model of dorsal burn in rats. METHODS: Sixteen male Wistar rats were distributed into two groups: control group (CG): rats submitted to scald burn injury without treatment and laser treated group (LG): rats submitted to scald burn injury and treated with laser therapy. Fourteen days post-surgery, gastrocnemius muscle was evaluated being the specimens stained with HE and morphometric data was evaluated. MyoD expression was assessed by immunohistochemistry. RESULTS: The results showed that laser treated animals presented more organized tissue morphology compared to the non-treated animals, with a higher number of nucleus in the fibers. Also, the cross sectional area of the fibers and the MyoD immunoexpression in the laser treated groups was higher. CONCLUSION: Low-level laser therapy had positive effects on gastrocnemius muscle, improving tissue muscle morphology, increasing cross sectional area and MyoD immunoexpression. .

Skeletal Muscle Cell Behavior After Physical Agent Treatments.

Apoptosis is essential for skeletal muscle development and homeostasis. It has been frequently involved in several muscle myopathies and sarcopenia, as well as in denervation, in disuse and acute strenuous or eccentric physical exercise. In this work skeletal muscle cell death, induced in vitro by a variety of physical triggers, has been investigated. C2C12 myoblasts and myotubes were exposed to UVB for 30 min, hyperthermia for 1 h at 43 °C, low pH for 3 h, hypothermia for 4h at 0 - 6°C, all followed by 2 - 4 h recovery. Their effects have been analysed by means of morpho- functional and molecular approaches. After UVB radiation, hyperthermia and acidosis, morphological apoptotic features and in situ DNA fragmentation appeared, more evident in myoblasts. Interestingly, apoptotic, non apoptotic and necrotic nuclei could be occasionally observed within the same myotube. Low pH induced apoptosis and necrosis, both characterized by swollen nuclei. In all these experimental conditions, the molecular investigations revealed a caspase pathway involvement in inducing cell death. Differently, hypothermia showed a scant and initial chromatin margination, in the presence of a diffused autophagic component. In this case, in situ DNA fragmentation and caspase activation have not been detected. Myoblasts and myotubes appeared sensitive to physical agents, some of which, induced apoptotic cell death. Moreover, hypothermia exposure seemed to enhance autophagic response, thus representing a way to delay trauma-correlated muscle inflammation. This study permits to highlight skeletal muscle cell behavior in response to physical agents, by adding important information to muscle cell death knowledge. UVB radiation and hyperthermia, usually used in clinical therapy, have also adverse effects on skeletal muscle such as myonuclei loss and cell death, contributing to muscle mass decrease. Acidosis occurs physiologically in muscular fatigue, reducing not only the athlete performance, but causing muscle cell damage or death too. Finally, hypothermia, stimulating the autophagic response, could have a key role in muscle injury prevention.