Rat Gastrocnemius Research Articles

Acute high-intensity muscle contraction moderates AChR gene expression independent of rapamycin-sensitive mTORC1 pathway in rat skeletal muscle.

The relationship between mechanistic target of rapamycin complex 1 (mTORC1) activation after resistance exercise and acetylcholine receptor (AChR) subunit gene expression remains largely unknown. Therefore, we aimed to investigate the effect of electrical stimulation-induced intense muscle contraction, which mimics acute resistance exercise, on the mRNA expression of AChR genes and the signalling pathways involved in neuromuscular junction (NMJ) maintenance, such as mTORC1 and muscle-specific kinase (MuSK). The gastrocnemius muscle of male adult Sprague-Dawley rats was isometrically exercised. Upon completion of muscle contraction, the rats were euthanized in the early (after 0, 1, 3, 6 or 24h) and late (after 48 or 72h) recovery phases and the gastrocnemius muscles were removed. Non-exercised control animals were euthanized in the basal state (control group). In the early recovery phase, Agrn gene expression increased whereas LRP4 decreased without any change in the protein and gene expression of AChR gene subunits. In the late recovery phase, Agrn, Musk, Chrnb1, Chrnd and Chrne gene expression were altered and agrin and MuSK protein expression increased. Moreover, mTORC1 and protein kinase B/Akt-histone deacetylase 4 (HDAC) were activated in the early phase but not in the late recovery phase. Furthermore, rapamycin, an inhibitor of mTORC1, did not disturb changes in AChR subunit gene expression after muscle contraction. However, rapamycin addition slightly increased AChR gene expression, while insulin did not impact it in rat L6 myotube. These results suggest that changes in the AChR subunits after muscle contraction are independent of the rapamycin-sensitive mTORC1 pathway.

Normobaric hypoxia accelerates high-intensity intermittent training-induced mitochondrial biogenesis (PGC-1α)- and dynamics (OPA1)-related protein expressions in rat gastrocnemius muscle.

High-intensity intermittent training (HIIT) in a normobaric hypoxic environment enhances exercise capacity, possibly by increasing the mitochondrial content in skeletal muscle; however, the molecular mechanisms underlying these adaptations are not well understood. Therefore, we investigated whether HIIT under normobaric hypoxia can enhance the expression of proteins involved in mitochondrial biogenesis and dynamics in rat gastrocnemius muscle. Five-week-old male Wistar rats (n = 24) were randomly assigned to the following four groups: (1) sedentary under normoxia (20.9% O2) (NS), (2) training under normoxia (NT), (3) sedentary under normobaric hypoxia (14.5% O2) (HS), and (4) training under normobaric hypoxia (HT). The training groups in both conditions were engaged in HIIT on a treadmill five to six days per week for nine weeks. From the fourth week of the training period, the group assigned to hypoxic conditions was exposed to normobaric hypoxia. Forty-eight hours after completing the final training session, gastrocnemius muscles were surgically removed, and mitochondrial enzyme activity and mitochondrial biogenesis and dynamics regulatory protein levels were determined. Citrate synthase (CS) activity and mitochondrial oxygen phosphorylation (OXPHOS) subunits in the gastrocnemius muscle in the HT significantly exceeded those in the other three groups. Moreover, the levels of a master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and a mitochondrial fusion-related protein, optic atrophy 1 (OPA1), were significantly increased by HIIT under normobaric hypoxia. Our data indicates that HIIT and normobaric hypoxia increase the expression of mitochondrial biogenesis- and dynamics-related proteins in skeletal muscles.

Effects of intermittent exposure to hypobaric hypoxia and cold on skeletal muscle regeneration: Mitochondrial dynamics, protein oxidation and turnover

Muscle injuries and the subsequent regeneration events compromise muscle homeostasis at morphological, functional and molecular levels. Among the molecular alterations, those derived from the mitochondrial function are especially relevant. We analysed the mitochondrial dynamics, the redox balance, the protein oxidation and the main protein repairing mechanisms after 9 days of injury in the rat gastrocnemius muscle. During the recovery rats were exposed to intermittent cold exposure (ICE), intermittent hypobaric hypoxia (IHH), and both simultaneous combined stimuli. Non-injured contralateral legs were also analysed to evaluate the specific effects of the three environmental exposures. Our results showed that ICE enhanced mitochondrial adaptation by improving the electron transport chain efficiency during muscle recovery, decreased the expression of regulatory subunit of proteasome and accumulated oxidized proteins. Exposure to IHH did not show mitochondrial compensation or increased protein turnover mechanisms; however, no accumulation of oxidized proteins was observed. Both ICE and IHH, when applied separately, elicited an increased expression of eNOS, which could have played an important role in accelerating muscle recovery. The combined effect of ICE and IHH led to a complex response that could potentially impede optimal mitochondrial function and enhanced the accumulation of protein oxidation. These findings underscore the nuanced role of environmental stressors in the muscle healing process and their implications for optimizing recovery strategies.

BMSCs-Derived Extracellular VesiclemiR-29a-3p Improved the Stability of Rat Myasthenia Gravis by Regulating Treg/Th17 Cells

ABSTRACT Introduction Myasthenia gravis (MG) is an autoimmune disorder. Microvesicle-derived miRNAs have been implicated in autoimmune diseases. However, the role of microvesicle-derived miR-29a-3p in MG remains poorly understood. This study aimed to investigate the therapeutic effect and mechanism of miR-29a-3p derived from stem cell microvesicles (MVs) on experimental autoimmune myasthenia gravis (EAMG) rats. Methods EAMG was induced in rats by injection of the subunit of the rat nicotinic anti-acetylcholine receptor (AChR) R97–116 peptide.Besides the control group, EAMG rats were randomly allocated into the EAMG model group, MV group, MV-NC-agomir group, and MV- miR-29a-3p-agomir group. Results Our results found that BMSCs-MV promoted miR-29a-3p expression in gastrocnemius of EAMG rats. Bone marrow mesenchymal stem cells (BMSCs) derived microvesicle miR-29a-3p improved the hanging ability and swimming time of EMGA rats and weakened the degree of muscle fiber atrophy. Furthermore, microvesicles from miR-29a-3p overexpressing BMSCs reduced the content of AchR-Ab in the serum of EAMG rats. BMSC-derived microvesicle miR-29a-3p further suppressed the expression of IFN-γ and enhanced the IL-4 and IL-10 in the serum of EAMG rats by restoring the Th17/Treg cells balance. Discussion BMSCs-derived microvesicle miR-29a-3p improved the stability of rat myasthenia gravis by regulating Treg/Th17 cells. It may be an effective treatment for MG.

Preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model

We previously demonstrated that transcutaneous CO2 application promotes muscle fiber-type switching, fracture healing, and osteogenesis by increasing blood flow and angiogenesis. Here, we aimed to investigate the preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model. Eleven-week-old male Sprague-Dawley rats were divided into hindlimb suspension (HS), HS with transcutaneous CO2 application (HSCO2), and control groups. HSCO2 rats were administered transcutaneous 100 % CO2 gas in their bilateral hindlimbs, five times a week for 20 min. After 3 weeks, we harvested the gastrocnemius, femur, and tibia for assessment. Histological analysis revealed a significant decrease in the gastrocnemius myofiber cross-sectional area in HS rats compared to the control rats, whereas HSCO2 rats exhibited a significant increase compared to HS rats. Micro-computed tomography showed significant bone atrophy in the trabecular and cortical bones of the femur in HS rats compared to those of the control rats, whereas significant improvement was noted in HSCO2 rats. Histological analysis of the proximal tibia revealed more marrow adipose tissue in the HS rats than in the control rats. However, in the HSCO2 rats, fewer marrow adipose tissue and osteoclasts were observed. Moreover, HSCO2 rats had more osteoblasts and higher expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) than the HS rats. The gastrocnemius and distal femur of HSCO2 rats also exhibited elevated PGC-1α and VEGF expression and upregulation of the myogenesis markers and osteogenesis markers compared to those of HS rats. This treatment effectively prevented disuse osteoporosis and muscle atrophy by promoting local angiogenesis and blood flow. PGC-1α is crucial for promoting this angiogenic pathway. Transcutaneous CO2 application may be a novel preventive procedure for disuse osteoporosis and muscle atrophy, complementing medication and rehabilitation.

Endurance exercise-induced histone methylation modification involved in skeletal muscle fiber type transition and mitochondrial biogenesis

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.

The Impact of Sciatic Nerve Injury on Extracellular Matrix of Lower Limb Muscle and Thoracolumbar Fascia: An Observational Study.

Peripheral nerve injury (PNI) is a complex clinical challenge resulting in functional disability. Neurological recovery does not always ensure functional recovery, as extracellular matrix (ECM) alterations affect muscle function. This study evaluates hyaluronan (HA) and collagen concentration in the gastrocnemius muscle and thoracolumbar fascia (TLF) in unilateral lower limb PNI rats to explore systemic ECM alterations following PNI and their impacts on functional recovery. Eighteen 8-week-old male Sprague-Dawley rats were divided into experimental (n = 12 left sciatic nerve injury) and control (n = 6) groups. After six weeks, motor function was evaluated. Muscle and TLF samples were analysed for HA and collagen distribution and concentrations. SFI and gait analysis confirmed a functional deficit in PNI rats 6 weeks after surgery. HA concentration in both sides of the muscles decreased by approximately one-third; both sides showed significantly higher collagen concentration than healthy rats (12.74 ± 4.83 µg/g), with the left (32.92 ± 11.34 µg/g) significantly higher than the right (20.15 ± 7.03 µg/g). PNI rats also showed significantly lower HA (left: 66.95 ± 20.08 µg/g; right: 112.66 ± 30.53 µg/g) and higher collagen (left: 115.89 ± 28.18 µg/g; right: 90.43 ± 20.83 µg/g) concentrations in both TLF samples compared to healthy rats (HA: 167.18 ± 31.13 µg/g; collagen: 47.51 ± 7.82 µg/g), with the left TLF more affected. Unilateral lower limb PNI induced HA reduction and collagen accumulation in both the lower limb muscles and the TLF, potentially exacerbating motor function impairment and increasing the risk of low back dysfunctions.

Ameliorative effect of nano-pregabalin in gastrocnemius muscle of gamma irradiated rats with an experimental model of fibromyalgia: Crosstalk of Sirt3, IL-1β and PARP1 pathways

BackgroundFibromyalgia (FM) is a complex syndrome with somatic symptoms connected to the operational state of muscles. Although radiotherapy is a cornerstone in cancer treatment, it is implicated in the aggravation of FM. Lately, formulation of medicines in nano-forms become of great prominence due to their prospective applications in medicine. So, this study aimed to assess possible therapeutic benefits of formulating pregabalin in a nono-form (N-PG) for managing FM during exposure to gamma radiation. MethodsGamma rays administered in fractionated doses (2 Gy/day) to male rats after one hour of s.c. injection of reserpine (1 mL/kg per day) to induce FM, then treated with single daily dose of (30 mg/kg, p.o.) PG or N-PG for ten successive days. Rats were subjected to behavioral tests, then sacrificed to obtain serum and gastrocnemius muscles. ResultsN-PG significantly antagonized reserpine-induced FM as proved by; the immobility and performance times in forced swim and rotarod performance tests, respectively were restored near to the normal time, serum IL-8 and MCP-1 chemokines were nearby the normal levels, mitigated oxidative stress through increasing total thiol, Sirt3, CAT enzyme and decreasing COX-1, inhibition of inflammation via IL-1β and MIF significant reduction, it possessed anti-apoptotic effect verified by decreasing PARP-1 and increasing Bcl-XL, gastrocnemius muscles had minimal fibrosis levels as seen after Masson trichrome staining. Histopathological results were coincidence with biochemical inspection. ConclusionThis study identifies N-PG as a novel drug that could be of a value in the management of FM particularly in cancer patients undergoing radiotherapy.

Cannabigerol-A useful agent restoring the muscular phospholipids milieu in obese and insulin-resistant Wistar rats?

Numerous strategies have been proposed to minimize obesity-associated health effects, among which phytocannabinoids appear to be effective and safe compounds. In particular, cannabigerol (CBG) emerges as a potent modulator of the composition of membrane phospholipids (PLs), which plays a critical role in the development of insulin resistance. Therefore, here we consider the role of CBG treatment on the composition of PLs fraction with particular emphasis on phospholipid subclasses (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)) in the red gastrocnemius muscle of Wistar rats fed the standard or high-fat, high-sucrose (HFHS) diet. The intramuscular PLs content was determined by gas-liquid chromatography and based on the composition of individual FAs, we assessed the stearoyl-CoA desaturase 1 (SCD1) index as well as the activity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) pathways. Expression of various proteins engaged in the inflammatory pathway, FAs elongation, and desaturation processes was measured using Western blotting. Our research has demonstrated the important association of obesity with alterations in the composition of muscular PLs, which was significantly improved by CBG supplementation, enriching the lipid pools in n-3 PUFAs and decreasing the content of arachidonic acid (AA), which in turn influenced the activity of PUFAs pathways in various PLs subclasses. CBG also inhibited the local inflammation development and profoundly reduced the SCD1 activity. Collectively, restoring the PLs homeostasis of the myocyte membrane by CBG indicates its new potential medical application in the treatment of obesity-related metabolic disorders.

Transcriptome sequencing and bioinformatics analysis of gastrocnemius muscle in type 2 diabetes mellitus rats

ObjectiveType 2 diabetes mellitus (T2DM) is one of the high risk factors for sarcopenia. However, the pathogenesis of diabetic sarcopenia has not been fully elucidated. This study obtained transcriptome profiles of gastrocnemius muscle in normal and T2DM rats based on high-throughput sequencing technology, which may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.MethodsTwelve adult male Sprague-Dawley rats were randomly divided into Control group and T2DM group, and gastrocnemius muscle tissue was retained for transcriptome sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) 6 months later. Screening differentially expressed genes (DEGs), Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Gnomes (KEGG) functional annotation and enrichment analysis were performed for DEGs. Six DEGs related to apoptosis were selected for qTR-PCR verification.ResultsTranscriptomic analysis showed that there were 1016 DEGs between the gastrocnemius muscle of T2DM and normal rats, among which 665 DEGs were up-regulated and 351 DEGs were down-regulated. GO analysis showed that the extracellular matrix organization was the most enriched in biological processes, with 26 DEGs. The extracellular matrix with 35 DEGs was the most abundant cellular component. The extracellular matrix structural constituent, with 26 DEGs, was the most enriched in molecular functions. The highest number of DEGs enriched in biological processes, cellular components and molecular functions were positive regulation of transcription by RNA polymerase II, nucleus and metal ion binding, respectively. There were 78, 230 and 89 DEGs respectively. KEGG pathway enrichment analysis showed that ECM-receptor interaction, PI3K-Akt signaling pathway and TGF-β signaling pathway(p < 0.001) had higher enrichment degree and number of DEGs. qRT-PCR results showed that the fold change of Map3k14, Atf4, Pik3r1, Il3ra, Gadd45b and Bid were 1.95, 3.25, 2.97, 2.38, 0.43 and 3.6, respectively. The fold change of transcriptome sequencing were 3.45, 2.21, 2.59, 5.39, 0.49 and 2.78, respectively. The transcriptional trends obtained by qRT-PCR were consistent with those obtained by transcriptome sequencing.ConclusionsTranscriptomic analysis was used to obtain the “gene profiles” of gastrocnemius muscle of T2DM and normal rats. qRT-PCR verification showed that the genes related to apoptosis were differentially expressed. These DEGs and enrichment pathways may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.

Astragaloside IV protects against autoimmune myasthenia gravis in rats via regulation of mitophagy and apoptosis.

Astragaloside IV (AS‑IV) has various pharmacological effects, including antioxidant and immunoregulatory properties, which can improve myasthenia gravis (MG) symptoms. However, the potential mechanism underlying the effects of AS‑IV on MG remains to be elucidated. The present study aimed to investigate whether AS‑IV has a therapeutic effect on MG and its potential mechanism of action. By subcutaneously immunizing rats with R97‑116 peptide, an experimental autoimmune (EA) MG rat model was established. AS‑IV (40 or 80 mg/kg/day) treatment was then applied for 28 days after modeling. The results demonstrated that AS‑IV significantly ameliorated the weight loss, Lennon score and pathological changes in the gastrocnemius muscle of EAMG rats compared with the model group. Additionally, the levels of acetylcholine receptor antibody (AChR‑Ab) were significantly decreased, whereas mitochondrial function [ATPase and cytochrome c (Cyt‑C) oxidase activities] and ultrastructure were improved in the AS‑IV treated rats. Moreover, the mRNA and protein expression levels of phosphatase and tensin homolog‑induced putative kinase 1, Parkin, LC3II and Bcl‑2, key signaling molecules for mitophagy and apoptosis, were upregulated, whereas the mRNA and protein expression levels of p62, Cyt‑C, Bax, caspase 3 and caspase 9 were downregulated following AS‑IV intervention. In conclusion, AS‑IV may protect against EAMG in a rat model by modulating mitophagy and apoptosis. These findings indicated the potential mechanism underlying the effects of AS‑IV on MG and provided novel insights into treatment strategies for MG.

Role of GPR81 in regulating intramuscular triglyceride storage during aerobic exercise in rats.

Lactate, a metabolite of exercise, plays a crucial role in the body. In these studies, we aimed to investigate the role of G protein-coupled receptor 81 (GPR81), a specific receptor for lactate, in regulating lipid storage in the gastrocnemius muscle of rats. To achieve this, we measured the impact of sodium 3-hydroxybutyrate (3-OBA) concentration and time on the cAMP-PKA signaling pathway in the gastrocnemius muscles ofrats. Our investigation involved determining the effects of administering 3-OBA at a concentration of 3 mmol L-1 just 15 min before exercise. As expected, exercise led to a notable increase in intramuscular lactate concentration in rats. However, injecting 3-OBA prior to exercise yielded intriguing results. It not only further augmented the cAMP-PKA signaling pathway but also boosted the expression of lipolysis-related proteins such as hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). Simultaneously, it decreased the expression of fat-synthesizing proteins, including acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS), while increasing the protein expression of cytochrome c oxidase subunit Ⅳ(COX Ⅳ) and the activity of citrate synthetase (CS). Unfortunately, there was no significant change observed in intramuscular triglyceride (IMTG) content. In summary, our findings shed light on the role of lactate in partially regulating intramuscular triglycerides during exercise.

Circulating and Tissues Concentrations of Selected Trace Elements in Response to Moderate Physical Exercise: Protective Effects of Cucurbita pepo Seeds Oil Extraction

Background: Physical exercise and the subsequent recovery processes have been proposed to affect the essential hemostatic balance of trace elements, either by increasing degradation rates or by enhancing loss from the body. This might disturb the function of vital metabolic organs, such as muscles and liver, leading to unsatisfactory performance. It appears that taking advantage of mineral-rich herbal supplements could replenish the body's mineral sources. Objectives: The present study aimed to investigate the effects of Cucurbita pepo supplementation on exercise-induced dynamics of micronutrients in serum, liver, and skeletal muscle of Wistar rats. Methods: Forty male Wistar rats (aged 4 - 6 weeks old, weighing 130 - 150 g) were randomly divided into four groups of ten rats each: (1) Saline control group (SC), (2) training at 25 m/min group (ST), (3) pumpkin seed oil supplementation group (PC), and (4) pumpkin seed oil supplementation + Training at 25m/min (PT). Moderate-intensity aerobic exercise (25 m/min, eliciting approximately 65% VO2Peak) and consumption of pumpkin seed oil (4 ml/kg/day) were conducted for 6 weeks. The ICP-OES technique was used to measure plasma, liver, and skeletal muscle levels of zinc (Zn), iron (Fe), manganese (Mn), magnesium (Mg), and copper (Cu) approximately 48 to 72 hours after the final session of the training protocol. Data were analyzed using one-way ANOVA and post-hoc Bonferroni tests (P &lt; 0.05). Results: No significant changes in serum Fe (P = 0.728) and Mn (P = 0.52) levels, as well as liver tissue levels of Zn (P = 0.633), Fe (P = 0.512), Mn (P = 0.194), Mg (P = 0.156), and Cu (P = 0.711), were observed among the intervention groups. However, all trace mineral element (TME) concentrations, except for Cu (P = 0.907) in the gastrocnemius tissue, significantly changed among the study groups (P &lt; 0.05). Additionally, the results of the Bonferroni test showed that six weeks of moderate physical activity decreased the serum, liver, and gastrocnemius levels of TMEs, and subsequent supplementation with pumpkin seed oil played a replenishing role in preserving the resources. Conclusions: Pumpkin seed oil supplementation was demonstrated to be an effective ergogenic aid for the up-regulation and replenishment of exercise-induced TME excretion from the liver, gastrocnemius, and body fluids in healthy rats.

Both acute glyphosate and the aminomethylphosphonic acid intoxication decreased the acetylcholinesterase activity in rat hippocampus, prefrontal cortex and gastrocnemius muscle

It has been reported that glyphosate, one of the most common herbicides used in agriculture, impairs locomotion and cognition. Glyphosate has a variable half-life in soil up to biotic and/or abiotic factors transform the molecule in metabolites such as the aminomethylphosphonic acid (AMPA) that has a longer half-life. In this study, female Sprague Dawley rats were acutely exposed to different doses of glyphosate or AMPA (i.e. 10, 56 or 100 mg/kg) and, subsequently, the acetylcholinesterase (AChE) activity was measured in the hippocampus, prefrontal cortex (PFC) and the gastrocnemius muscle. Both glyphosate and AMPA produced a similar decrease in the AChE activity in all the tissues tested. These results suggest that interference with normal cholinergic neurotransmission may be one of the mechanisms involved in glyphosate-induced motor alterations in rats. Moreover, our results highlight the biological importance of AMPA as a molecule with anticholinesterase action in brain and skeletal muscle. To our knowledge, this is the first report showing in vivo that AMPA, the major metabolite of glyphosate, behaves as an organophosphate.

Inhibitory effect of acupoint electrostimulation with different layers and intensities on muscular inflammatory pain and spinal WDR neuron activity.

To observe the analgesic effects of different levels and intensities of electrical stimulation on the local acupoints in the pain source area and their impact on wide dynamic range (WDR) neurons in the spinal dorsal horn, in order to provide a basis for selecting appropriate parameters for electroacupuncture (EA) stimulation. Wistar rats were used in 3 parts of the experiment. Complete Freund's adjuvant was used to establish a model of inflammation-induced pain in the gastrocnemius muscle. After modeling, 6 rats were randomly selected for multi-channel extracellular electrophysiological recording of the electrical activity of WDR neurons, to determine the threshold for activating the A-component (Ta) and the C-component (Tc), which were used as the intervention intensities for skin transcutaneous electrical acupoint stimulation (TEAS) or EA. Thirty-six rats were randomly divided into normal , model , TEAS-Ta , TEAS-Tc, EA-Ta , and EA-Tc groups, with 6 rats in each group. In the pain source area , Ta or Tc intensity of TEAS or EA intervention at"Chengshan"(BL57) was performed for 30 min each time, once a day, for 3 consecutive days. A small animal pressure pain measurement instrument was used to measure the mechanical pressure pain threshold of the gastrocnemius muscle in rats, and the Von Frey filament was used to measure the mechanical pain threshold of the footpad. Thirteen rats were randomly selected to observe the immediate responsiveness of WDR neurons to Ta/Tc intensity of EA or TEAS in BL57. The thresholds of TEAS to activate WDR neuron A-component or C-component were (2.43±0.57) mA and (7.00±1.34) mA, respectively, while the thresholds for EA to activate muscle WDR neuron A-component or C-component were (0.72±0.34) mA and (1.58±0.35) mA, respectively. After injection of CFA into the gastrocnemius muscle, compared with the normal group both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad of rats in the model group were significantly decreased (P<0.001). After TEAS-Ta, TEAS-Tc or EA-Ta intervention in the BL57, both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad were significantly higher than those in the model group (P<0.05, P<0.001). Compared with the normal group, the electrical threshold for evoking WDR neuron C-component discharge was significantly decreased (P<0.001) in the model group, while increased after TEAS-Ta, TEAS-Tc, or EA-Ta intervention (P<0.01) compared with the model group. The evoked discharge frequency of muscle WDR neurons decreased significantly after immediate intervention with TEAS-Ta, TEAS-Tc, or EA-Ta (P<0.01, P<0.05). EA-Tc had no significant improvement on the evoked electrical activity of WDR neurons or pain behavior. TEAS-Ta, TEAS-Tc, or EA-Ta can all alleviate the local and footpad mechanical pain in rats with muscle inflammation and inhibit the responsiveness of WDR neurons, indicating that different intensities are required for analgesic effects at different levels of acupoints in the pain source area.

HucMSCs Delay Muscle Atrophy After Peripheral Nerve Injury Through Exosomes by Repressing Muscle-Specific Ubiquitin Ligases.

Cell therapy based on mesenchymal stem cells (MSCs) alleviate muscle atrophy caused by diabetes and aging; however, the impact of human umbilical cord mesenchymal stem cells on muscle atrophy following nerve injury and the underlying mechanisms remain unclear. In this study, we evaluated the therapeutic efficacy of human umbilical cord MSCs (hucMSCs) and hucMSC-derived exosomes (hucMSC-EXOs) for muscle atrophy following nerve injury and identified the underlying molecular mechanisms. Sciatic nerve crush injury in rats and the induction of myotubes in L6 cells were used to determine the ameliorating effect of hucMSCs and hucMSC-EXOs on muscle atrophy. Q-PCR and Western blot analyses were used to measure the expression of muscle-specific ubiquitin ligases Fbxo32 (Atrogin1, MAFbx) and Trim63 (MuRF-1). Dual-luciferase reporter gene experiments were conducted to validate the direct binding of miRNAs to their target genes. Local injection of hucMSCs and hucMSC-EXOs mitigated atrophy in the rat gastrocnemius muscle following sciatic nerve crush injury. In vitro, hucMSC-EXOs alleviated atrophy in L6 myotubes. Mechanistic analysis indicated the upregulation of miR-23b-3p levels in L6 myotubes following hucMSC-EXOs treatment. MiR-23b-3p significantly inhibited the expression of its target genes, Fbxo32 and Trim63, and suppressed myotube atrophy. Notably, an miR-23b-3p inhibitor reversed the inhibitory effect of miR-23b-3p on myotube atrophy in vitro. These results suggest that hucMSCs and their exosomes alleviate muscle atrophy following nerve injury. MiR-23b-3p in exosomes secreted by hucMSCs contributes to this mechanism by inhibiting the muscle-specific ubiquitination ligases Fbxo32 and Trim63.

Effect of Infrared and Green Photomodulation Exposure on the Number of Active Myosatellite Cells in Regenerating Muscles.

Reparative properties of infrared laser exposure are well known, but the effects of green laser light are little studied. We analyzed the effects of short (60 sec) and longer (180 sec) exposure to infrared (980 nm) and green (520 nm) laser on the number of activated myosatellite cells in the regenerating m. gastrocnemius of Wistar rats after infliction of an incision wound. Histological preparations were used for morphometric evaluation of myosatellite cells with MyoD+ nuclei. Increased numbers of MyoD+ nuclei were observed on days 3 and 7 after 60-sec exposure to infrared and green laser.

Pre-heating stress associated with acute oral leucine supplementation effects in rat gastrocnemius muscle: Implications for protein synthesis signaling pathways

Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7–8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle.

Effects of preconditioning with heat stress on acute exercise-induced intracellular signaling in male rat gastrocnemius muscle.

Heat stress (HS) induces Akt/mTOR phosphorylation and FoxO3a signaling; however, whether a prior increase in heat shock protein 72 (HSP72) expression affects intracellular signaling following eccentric exercise remains unclear. We analyzed the effects of HS pretreatment on intramuscular signaling in response to acute exercise in 10-week-old male Wistar rats (n = 24). One leg of each rat was exposed to HS and the other served as an internal control (CT). Post-HS, rats were either rested or subjected to downhill treadmill running. Intramuscular signaling responses in the red and white regions of the gastrocnemius muscle were analyzed before, immediately after, or 1 h after exercise (n = 8/group). HS significantly increased HSP72 levels in both deep red and superficial white regions. Although HS did not affect exercise-induced mTOR signaling (S6K1/ERK) responses in the red region, mTOR phosphorylation in the white region was significantly higher in CT legs than in HS legs after exercise. Thr308 phosphorylation of Akt showed region-specific alteration with a decrease in the red region and an increase in the white region immediately after downhill running. Overall, a prior increase in HSP72 expression elicits fiber type-specific changes in exercise-induced Akt and mTOR phosphorylation in rat gastrocnemius muscle.

Swimming alters some proteins of skeletal muscle tissue in rats with Alzheimer-like phenotype

ObjectivesExercise training plays a significant role in preventing the destruction of central nerve neurons and muscle atrophy. The purpose of the present study was to investigate the effect of a period of swimming training on the expression of Neural cell adhesion molecule (NCAM), Semaphorin 3A (SEMA3A), and Profilin-1 (PFN1) proteins in the gastrocnemius muscle of Alzheimer-like phenotype rats. Methods & materials32 Wistar males were (6 weeks of age) divided into four groups: Healthy Control (HC), Alzheimer-like phenotype's Control (AC), Healthy Training (HT), and Alzheimer-like phenotype's Training (AT). Alzheimer-like phenotypes were induced by beta-amyloid injection in the hippocampus. The training program consisted of 20 swimming sessions. Gastrocnemius muscle was removed after the intervention, and NCAM, SEMA3A, and PFN1 proteins were measured by the immunohistoflorescent method. ResultsThe results showed that SEMA3A was increased (p = 0.001), and NCAM (p = 0.001), and PFN1 (p = 0.001) were decreased in AC compared to the HC group. Also, the results showed that NCAM (p = 0.001) and Pfn1 (p = 0.002) increased in the HT group compared to HC, and the NCAM (p = 0.001) and Pfn1 (p = 0.002) in AT group compared to AC (p = 0.001) increased significantly, while SEMA3A was reduced in the HT group compared to HC (p = 0.001) and AT group compared to AC (p = 0.001) ConclusionSwimming effectively improves axon regeneration and neuronal formation in motor neurons and, therefore, can be an effective intervention to prevent and control the complications of Alzheimer-like phenotype.