Rat Muscle Research Articles

Hypoxia treatment and resistance training alters microRNA profiling in rats skeletal muscle

MicroRNAs (miRNAs) may play a crucial regulatory role in the process of muscle atrophy induced by high-altitude hypoxia and its amelioration through resistance training. However, research in this aspect is still lacking. Therefore, this study aimed to employ miRNA microarray analysis to investigate the expression profile of miRNAs in skeletal muscle from an animal model of hypoxia-induced muscle atrophy and resistance training aimed at mitigating muscle atrophy. The study utilized a simulated hypoxic environment (oxygen concentration at 11.2%) to induce muscle atrophy and established a rat model of resistance training using ladder climbing, with a total intervention period of 4 weeks. The miRNA expression profile revealed 9 differentially expressed miRNAs influenced by hypoxia (e.g., miR-341, miR-32-5p, miR-465-5p) and 14 differentially expressed miRNAs influenced by resistance training under hypoxic conditions (e.g., miR-338-5p, miR-203a-3p, miR-92b-3p) (∣log2(FC)∣ ≥ 1.5, p < 0.05). The differentially expressed miRNAs were found to target genes involved in muscle protein synthesis and degradation (such as Utrn, mdm2, eIF4E), biological processes (such as negative regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-dependent), and signaling pathways (such as Wnt signaling pathway, MAPK signaling pathway, ubiquitin-mediated proteolysis, mTOR signaling pathway). This study provides a foundation for understanding and further exploring the molecular mechanisms underlying hypoxia-induced rats muscle atrophy and the mitigation of atrophy through resistance training.

Curcumin regulates autophagy through SIRT3-SOD2-ROS signaling pathway to improve quadriceps femoris muscle atrophy in KOA rat model

Knee osteoarthritis (KOA) usually leads to quadriceps femoris atrophy, which in turn can further aggravate the progression of KOA. Curcumin (CUR) has anti-inflammatory and antioxidant effects and has been shown to be a protective agent for skeletal muscle. CUR has been shown to have a protective effect on skeletal muscle. However, there are no studies related to whether CUR improves KOA-induced quadriceps femoris muscle atrophy. We established a model of KOA in rats. Rats in the experimental group were fed CUR for 5 weeks. Changes in autophagy levels, reactive oxygen species (ROS) levels, and changes in the expression of the Sirutin3 (SIRT3)-superoxide dismutase 2 (SOD2) pathway were detected in the quadriceps femoris muscle of rats. KOA led to quadriceps femoris muscle atrophy, in which autophagy was induced and ROS levels were increased. CUR increased SIRT3 expression, decreased SOD2 acetylation and ROS levels, inhibited the over-activation of autophagy, thereby alleviating quadriceps femoris muscle atrophy and improving KOA. CUR has a protective effect against quadriceps femoris muscle atrophy, and KOA is alleviated after improvement of quadriceps femoris muscle atrophy, with the possible mechanism being the reduction of ROS-induced autophagy via the SIRT3-SOD2 pathway.

The Effect of Cold-Water Swimming on Energy Metabolism, Dynamics, and Mitochondrial Biogenesis in the Muscles of Aging Rats.

Our study aimed to explore the potential positive effects of cold water exercise on mitochondrial biogenesis and muscle energy metabolism in aging rats. The study involved 32 male and 32 female rats aged 15 months, randomly assigned to control sedentary animals, animals training in cold water at 5 ± 2 °C, or animals training in water at thermal comfort temperature (36 ± 2 °C). The rats underwent swimming training for nine weeks, gradually increasing the duration of the sessions from 2 min to 4 min per day, five days a week. The results demonstrated that swimming in thermally comfortable water improved the energy metabolism of aging rat muscles (increased metabolic rates expressed as increased ATP, ADP concentration, TAN (total adenine nucleotide) and AEC (adenylate energy charge value)) and increased mRNA and protein expression of fusion regulatory proteins. Similarly, cold-water swimming improved muscle energy metabolism in aging rats, as shown by an increase in muscle energy metabolites and enhanced mitochondrial biogenesis and dynamics. It can be concluded that the additive effect of daily activity in cold water influenced both an increase in the rate of energy metabolism in the muscles of the studied animals and an intensification of mitochondrial biogenesis and dynamics (related to fusion and fragmentation processes). Daily activity in warm water also resulted in an increase in the rate of energy metabolism in muscles, but at the same time did not cause significant changes in mitochondrial dynamics.

Differences in neuromuscular junctions between intrinsic muscles of the forepaw and biceps muscles in rats.

Motor endplates of the interossei muscles become destabilized, whereas those of the biceps muscles remain stable in a rat model of obstetric brachial plexus palsy. However, it is unclear whether the morphology of the motor endplates of the interossei muscles is different from that of the biceps muscles in normal rat. We hypothesized that the motor endplates in the interossei muscles have specific characteristics different from those in the biceps muscles. The motor endplates were labeled with α-bungarotoxin and synaptophysin. The cross-sectional areas of the muscle fibers, the morphologies of the motor endplates, and the absolute and normalized areas (corrected by muscle fiber diameter) of the motor endplates of the interossei muscles and the biceps muscles were compared in rats at 1, 3, and 5 weeks after birth. The cross-sectional area of the interossei muscles and biceps muscle fibers were found to have increased gradually at 1, 3, and 5 weeks, but that of the biceps muscles was larger than that of the interossei muscles. The motor endplates of the interossei muscles and the biceps muscles gradually develop from crescent to pretzel shape after birth, and those of the interossei muscles have a smaller area. At 1, 3, and 5 weeks postnatally, the area of postnatal normalized motor endplates of the interossei muscles was much smaller than that of the biceps muscles. A better understanding of the morphological differences of the motor endplates between the interossei muscles and the biceps muscles may help to understand their physiological and pathological changes.

Mechanism of muscle atrophy in a normal-weight rat model of type 2 diabetes established by using a soft-pellet diet

Dietary factors such as food texture affect feeding behavior and energy metabolism, potentially causing obesity and type 2 diabetes. We previously found that rats fed soft pellets (SPs) were neither hyperphagic nor overweight but demonstrated glucose intolerance, insulin resistance, and hyperplasia of pancreatic β-cells. In the present study, we investigated the mechanism of muscle atrophy in rats that had been fed SPs on a 3-h time-restricted feeding schedule for 24 weeks. As expected, the SP rats were normal weight; however, they developed insulin resistance, glucose intolerance, and fat accumulation. In addition, skeletal muscles of SP rats were histologically atrophic and demonstrated disrupted insulin signaling. Furthermore, we learned that the muscle atrophy of the SP rats developed via the IL-6–STAT3–SOCS3 and ubiquitin–proteasome pathways. Our data show that the dietary habit of consuming soft foods can lead to not only glucose intolerance or insulin resistance but also muscle atrophy.

Active constituents of essential oil from Curcuma phaeocaulis for treatment of dysmenorrhea

This study aims to identify the active constituents of essential oil from the rhizomes of Curcuma phaeocaulis for the treatment of dysmenorrhea. The compounds were separated and purified by molecular distillation, silica gel and Sephadex LH-20 column chromatography, preparative thin layer chromatography, and semi-preparative high performance liquid chromatography. Their structures were identified by mass spectrometry and nuclear magnetic resonance spectroscopy. The animal model of primary dysmenorrhea and the contraction model of isolated uterine smooth muscle of rats were established to examine the active constituents in the essential oil for treating dysmenorrhea. Six sesquiterpenes were isolated and identified as dehydrocommiterpene A(1), comosone Ⅱ(2), 5α(H)-eudesma-3(4),7(11)-dien-9β-ol-6-one(3), guaia-6(7)-en-11-ol(4), curcumenol(5), and isocurcumenol(6), among which compound 1 was a novel compound. The animal experiments showed that the essential oil from C. phaeocaulis significantly lowered the level of PGF_(2α) in uterine tissue compared with the model group. The experiment with the contraction model of isolated uterine smooth muscle demonstrated that the components with high boiling points outperformed those with low boiling points in relaxing the uterine smooth muscle, and compounds 1, 2, 5, and 6 isolated from the fraction with a high boiling point had the effect of relaxing the uterine smooth muscle. Among them, compounds 5 and 6 inhibited the extracellular Ca~(2+) influx and intracellular Ca~(2+) release to relax the uterine smooth muscle. In conclusion, the components with high boiling points and sesquiterpenes are the active components in the essential oil of C. phaeocaulis for treating dysmenorrhea.

Early Movement Restriction Affects FNDC5/Irisin and BDNF Levels in Rat Muscle and Brain.

Interaction with the environment appears necessary for the maturation of sensorimotor and cognitive functions in early life. In rats, a model of sensorimotor restriction (SMR) from postnatal day 1 (P1) to P28 has shown that low and atypical sensorimotor activities induced the perturbation of motor behavior due to muscle weakness and the functional disorganization of the primary somatosensory and motor cortices. In the present study, our objective was to understand how SMR affects the muscle-brain dialogue. We focused on irisin, a myokine secreted by skeletal muscles in response to exercise. FNDC5/irisin expression was determined in hindlimb muscles and brain structures by Western blotting, and irisin expression in blood and cerebrospinal fluid was determined using an ELISA assay at P8, P15, P21 and P28. Since irisin is known to regulate its expression, Brain-Derived Neurotrophic Factor (BDNF) levels were also measured in the same brain structures. We demonstrated that SMR increases FNDC5/irisin levels specifically in the soleus muscle (from P21) and also affects this protein expression in several brain structures (as early as P15). The BDNF level was increased in the hippocampus at P8. To conclude, SMR affects FNDC5/irisin levels in a postural muscle and in several brain regions and has limited effects on BDNF expression in the brain.

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.

EFFECT OF AMMONIUM PYRROLIDINEDITHIOCARBAMATE ON THE DEVELOPMENT OF OXIDATIVE STRESS IN BICEPS FEMORIS MUSCLES OF RATS DURING MODELLED METABOLIC SYNDROME

Excessive consumption of high-calorie food, a sedentary lifestyle and increased psycho-emotional stress are risk factors for the development of metabolic syndrome. These factors are especially relevant for residents of countries with highly developed economies. Metabolic syndrome is accompanied not only by metabolic disorders, but also leads to the development of a systemic inflammatory response, which is associated with excessive production and circulation of cytokines in the blood. As a rule, the activation of the transcription factor NF-κB leads to an increase in the production of pro-inflammatory cytokines.&#x0D; The aim of this work is to determine the effect of the NF-κB transcription factor activation inhibitor on the activity of antioxidant enzymes, the production of superoxide anion radical, the content of oxidatively modified proteins, and the concentration of malondialdehyde in the biceps femoris muscle of rats who underwent experimental metabolic syndrome.&#x0D; The study included 24 sexually mature male Wistar rats weighing 200-260 g. The animals were divided into 4 groups of 6 animals each. The first group was the control group; the second group involved the rats exposed to metabolic syndrome simulated by adding a 20% fructose solution as the only source of drinking water to the standard vivarium diet for 60 days; the third group involved the animals received ammonium pyrrolidine dithiocarbamate in a dose of 76 mg/kg intraperitoneally 3 times a week for 60 days; the fourth group was exposed to the combined effect of the ammonium pyrrolidinedithiocarbamate administration during modeled metabolic syndrome. In this study we investigated a 10% homogenate of the biceps femoris muscle with the research focus on several parameters: superoxide anion radical production, superoxide dismutase and catalase activity, malondialdehyde concentration, and the presence of oxidatively modified proteins.&#x0D; Simulation of the metabolic syndrome led to the development of oxidative stress in the biceps muscle of rats that was accompanied by an increase in the production of the superoxide anion radical and a decrease in the activity of antioxidant enzymes. The administration of ammonium pyrrolidinedithiocarbamate during modeled metabolic syndrome caused a decrease in the basic production of superoxide anion radical, production of superoxide anion radical by the microsomal electron transport chain and production of superoxide anion radical by the mitochondrial electron transport chain by 30.28%, 26.21 %, and 27.00%, respectively, compared to the rats in metabolic syndrome group. Superoxide dismutase activity increased by 78.81% and catalase activity grew by 144.74% compared to the metabolic syndrome group. The concentration of free malondialdehyde under the administration of ammonium pyrrolidinedithiocarbamate during metabolic syndrome reduced by 51.80%, and the content of oxidatively modified proteins decreased by 31.74% compared to the metabolic syndrome group.&#x0D; The administration of ammonium pyrrolidinedithiocarbamate, an NF-κB transcription factor activation inhibitor, effectively prevents the development of oxidative stress in the biceps femoris muscle of rats with modeled metabolic syndrome.&#x0D; This study is a part of the initiative research project No. 0124U000092 "High- and low-intensity phenotypes of systemic inflammatory response: molecular mechanisms and new medical technologies for their prevention and correction".

Differential impact of unilateral stroke on the bihemispheric motor cortex representation of the jaw and tongue muscles in young and aged rats.

Dysphagia commonly occurs after stroke, yet the mechanisms of post-stroke corticobulbar plasticity are not well understood. While cortical activity associated with swallowing actions is bihemispheric, prior research has suggested that plasticity of the intact cortex may drive recovery of swallowing after unilateral stroke. Age may be an important factor as it is an independent predictor of dysphagia after stroke and neuroplasticity may be reduced with age. Based on previous clinical studies, we hypothesized that cranial muscle activating volumes may be expanded in the intact hemisphere and would contribute to swallowing function. We also hypothesized that older age would be associated with limited map expansion and reduced function. As such, our goal was to determine the impact of stroke and age on corticobulbar plasticity by examining the jaw and tongue muscle activating volumes within the bilateral sensorimotor cortices. Using the middle cerebral artery occlusion rat stroke model, intracortical microstimulation (ICMS) was used to map regions of sensorimotor cortex that activate tongue and jaw muscles in both hemispheres. Young adult (7 months) and aged (30 months) male F344 × BN rats underwent a stroke or sham-control surgery, followed by ICMS mapping 8 weeks later. Videofluoroscopy was used to assess oral-motor functions. Increased activating volume of the sensorimotor cortex within the intact hemisphere was found only for jaw muscles, whereas significant stroke-related differences in tongue activating cortical volume were limited to the infarcted hemisphere. These stroke-related differences were correlated with infarct size, such that larger infarcts were associated with increased jaw representation in the intact hemisphere and decreased tongue representation in the infarcted hemisphere. We found that both age and stroke were independently associated with swallowing differences, weight loss, and increased corticomotor thresholds. Laterality of tongue and jaw representations in the sham-control group revealed variability between individuals and between muscles within individuals. Our findings suggest the role of the intact and infarcted hemispheres in the recovery of oral motor function may differ between the tongue and jaw muscles, which may have important implications for rehabilitation, especially hemisphere-specific neuromodulatory approaches. This study addressed the natural course of recovery after stroke; future work should expand to focus on rehabilitation.

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.

Laser-assisted synthesis of nano-hydroxyapatite and functionalization with bone active molecules for bone regeneration

The main goal of bone tissue engineering research is to replace the allogenic and autologous bone graft substitutes that can promote bone repair. Owing to excellent biocompatibility and osteoconductivity, hydroxyapatite is in extensive research and high demand for both medical and non-medical applications. Although various methods have been developed for the synthesis of hydroxyapatite, in the present study we have shown the use of nanosecond laser energy in the wet precipitation method of nano-hydroxyapatite (nHAP) synthesis without using ammonium solution or any other chemicals for pH maintenance. Here, the present study aimed to fabricate the nanohydroxyapatite using a nanosecond laser. The X-ray diffraction and Fourier transform infrared spectroscopy have confirmed the hydroxyapatite formation under laser irradiation in less time without aging. A transmission electron microscopy confirmed the nano size of synthesized nHAP, which is comparable to conventional nHAP. The length and width of the laser-assisted nHAP were found to be in the range of 50–200 nm and 15–20 nm, respectively, at various laser parameters. The crystallite size obtained by Debye Scherrer formulae was found to be in the range of ∼ 16–36 nm. In addition, laser-assisted nHAP based composite cryogel (nanohydroxyapatite/gelatin/collagen I) was synthesized and impregnated with bioactive molecules (bone morphogenic protein and zoledronic acid) that demonstrated significant osteogenic potential both in vitro in cell experiment and in vivo rat muscle pouch model (abdomen and tibia muscles). Dual-energy X-ray analysis, micro-CT, and histological analysis confirmed ectopic bone regeneration. Micro-CT based histomorphometry showed a higher amount (more than 10-fold) of mineralization for animal groups implanted with composite cryogels loaded with bioactive molecules compared to only composite cryogels groups. Our findings thus demonstrate a controlled and rapid synthetic method for the synthesis of nHAP with various physical, chemical, and biological properties exhibited as comparable to conventionally synthesized nHAP.

Novel Bioresorbable Drug-Eluting Mesh Scaffold for Therapy of Muscle Injury.

A novel bioresorbable drug-eluting polycaprolactone (PCL) mesh scaffold was developed, utilizing a solvent-cast additive manufacturing technique, to promote therapy of muscle injury. The degradation rate and mechanical properties strength of the PCL mesh were characterized after immersion in a buffer solution for different times. The in vitro release characteristics of vancomycin, ceftazidime, and lidocaine from the prepared mesh were evaluated using a high-performance liquid chromatography (HPLC) assay. In addition, the in vivo efficacy of PCL meshes for the repair of muscle injury was investigated on a rat model with histological examinations. It was found that the additively manufactured PCL meshes degraded by 13% after submission in buffered solution for four months. All PCL meshes with different pore sizes exhibited greater strength than rat muscle and survived through 10,000 cyclic loadings. Furthermore, the meshes could offer a sustained release of antibiotics and analgesics for more than 3 days in vitro. The results of this study suggest that drug-loaded PCL mesh exhibits superior ability to pure PCL mesh in terms of effectively promoting muscle repair in rat models. The histological assay also showed adequate biocompatibility of the resorbable meshes. The additively manufactured biodegradable drug-eluting meshes may be adopted in the future in humans for the therapy of muscle injuries.

Effects of age and diet consistency on the expression of myosin heavy-chain isoforms on jaw-closing and jaw-opening muscles in a rat model.

Skeletal craniofacial morphology can be influenced by changes in masticatory muscle function, which may also change the functional profile of the muscles. To investigate the effects of age and functional demands on the expression of Myosin Heavy-Chain (MyHC) isoforms in representative jaw-closing and jaw-opening muscles, namely the masseter and digastric muscles respectively. Eighty-four male Wistar rats were divided into four age groups, namely an immature (n = 12; 4-week-old), early adult (n = 24; 16-week-old), adult (n = 24; 26-week-old) and mature adult (n = 24; 38-week-old) group. The three adult groups were divided into two subgroups each based on diet consistency; a control group fed a standard (hard) diet, and an experimental group fed a soft diet. Rats were sacrificed, and masseter and digastric muscles dissected. Real-time quantitative polymerase chain reaction was used to compare the mRNA transcripts of the MyHC isoforms-Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx)-of deep masseter and digastric muscles. In the masseter muscle, hypofunction increases Myh1 (26, 38 weeks; p < .0001) but decreases Myh4 (26 weeks; p = .046) and Myh2 (26 weeks; p < .0001) expression in adult rats. In the digastric muscle, hypofunction increases Myh1 expression in the mature adult rats (38 weeks; p < .0001), while Myh2 expression decreases in adult rats (26 weeks; p = .021) as does Myh4 (26 weeks; p = .001). Myh7 expression is increased in the digastric muscle of mature adult rats subjected to hypofunction (38 weeks; p = <.0001), while it is very weakly expressed in the masseter. In jaw-opening and jaw-closing muscles, differences in myosin expression between hard- and soft-diet-fed rats become evident in adulthood, suggesting that long-term alteration of jaw function is associated with changes in the expression of MyHC isoforms and potential fibre remodelling. This may give insight into the role of function on masticatory muscles and the resultant craniofacial morphology.

Celecoxib attenuates hindlimb unloading-induced muscle atrophy via suppressing inflammation, oxidative stress and ER stress by inhibiting STAT3.

Improving inflammation may serve as useful therapeutic interventions for the hindlimb unloading-induced disuse muscle atrophy. Celecoxib is a selective non-steroidal anti-inflammatory drug. We aimed to determine the role and mechanism of celecoxib in hindlimb unloading-induced disuse muscle atrophy. Celecoxib significantly attenuated the decrease in soleus muscle mass, hindlimb muscle function and the shift from slow- to fast-twitch muscle fibers caused by hindlimb unloading in rats. Importantly, celecoxib inhibited the increased expression of inflammatory factors, macrophage infiltration in damaged soleus muscle. Mechanistically, Celecoxib could significantly reduce oxidative stress and endoplasmic reticulum stress in soleus muscle of unloaded rats. Furthermore, celecoxib inhibited muscle proteolysis by reducing the levels of MAFbx, MuRF1, and autophagy related proteins maybe by inhibiting the activation of pro-inflammatory STAT3 pathway in vivo and in vitro. This study is the first to demonstrate that celecoxib can attenuate disuse muscle atrophy caused by hindlimb unloading via suppressing inflammation, oxidative stress and endoplasmic reticulum stress probably, improving target muscle function and reversing the shift of muscle fiber types by inhibiting STAT3 pathways-mediated inflammatory cascade. This study not only enriches the potential molecular regulatory mechanisms, but also provides new potential therapeutic targets for disuse muscle atrophy.

Simultaneous Effect of Eight Weeks High-fat Diet, Continuous Moderate-intensity Training, and High-intensity Intermittent Training on the Gene Expression Levels of PLIN2 and ATGL in the Twin Muscle of Male Wistar Rats

Background: A high-fat diet can cause obesity and endanger health. Therefore, the present study aimed to investigate the effect of eight weeks of high-fat diet, continuous moderate-intensity training, and high-intensity intermittent training on the gene expression levels of PLIN2 and ATGL in the twin muscles of male Wistar rats. Materials and Methods: Thirty-two male Wistar rats were divided into four groups (n=8): 1- control group with a standard diet (ND) 2- control group with a high-fat diet (HFD) 3- high-fat diet and continuous aerobic training with Moderate intensity (HFD+MICT) 4- high-fat diet and intermittent aerobic training (HFD+HIIT). For one training group, eight weeks of moderate-intensity continuous aerobic training (MICT), and for the other group, eight weeks of high-intensity intermittent aerobic training (HIIT) were performed on the treadmill. Then, 48 hours after the last training session, the twin muscle tissue was isolated to check the expression of PLIN2 and ATGL genes. Gene expression analysis was done by Real-time PCR technique. One-way analysis of variance (ANOVA) and Tukey's post-hoc test were used to analyze the collected data. Results: After eight weeks of training, the level of PLIN2 gene expression in the two training groups was significantly lower than in the HDF group, and the level of ATGL gene expression was significantly higher in both groups than in the HDF group (P&lt;0.05). Conclusion: It was shown that two exercises had a modulating effect on the expression of genes that are effective in the process of autophagy and the formation of fat cells.

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

Ultrasound-based assessment of the expression of inflammatory markers in the rectus femoris muscle of rats.

Ultrasonographic characteristics of skeletal muscles are related to their health status and functional capacity, but they still provide limited information on muscle composition during the inflammatory process. It has been demonstrated that an alteration in muscle composition or structure can have disparate effects on different ranges of ultrasonogram pixel intensities. Therefore, monitoring specific clusters or bands of pixel intensity values could help detect echotextural changes in skeletal muscles associated with neurogenic inflammation. Here we compare two methods of ultrasonographic image analysis, namely, the echointensity (EI) segmentation approach (EI banding method) and detection of selective pixel intensity ranges correlated with the expression of inflammatory regulators using an in-house developed computer algorithm (r-Algo). This study utilized an experimental model of neurogenic inflammation in segmentally linked myotomes (i.e., rectus femoris (RF) muscle) of rats subjected to lumbar facet injury. Our results show that there were no significant differences in RF echotextural variables for different EI bands (with 50- or 25-pixel intervals) between surgery and sham-operated rats, and no significant correlations among individual EI band pixel characteristics and protein expression of inflammatory regulators studied. However, mean numerical pixel values for the pixel intensity ranges identified with the proprietary r-Algo computer program correlated with protein expression of ERK1/2 and substance P (both 86-101-pixel ranges) and CaMKII (86-103-pixel range) in RF, and were greater (p < 0.05) in surgery rats compared with their sham-operated counterparts. Our findings indicate that computer-aided identification of specific pixel intensity ranges was critical for ultrasonographic detection of changes in the expression of inflammatory mediators in neurosegmentally-linked skeletal muscles of rats after facet injury.

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.

Effect of robotic gait training on muscle and bone characteristics in spinal cord transected rats.

Osteoporosis and loss of muscle mass are secondary issues with spinal cord injury. Robotic gait training has provided evidence of increasing bone density and muscle mass, but its effect on bone strength is undetermined. The purpose of this study was to determine the effect of a 6-week robotic locomotion training program on skeletal muscle mass and bone characteristics. Twelve female Sprague-Dawley rats received a mid-thoracic spinal cord transection at 5 days old and at 3 weeks old were assigned to a Control or Trained Group. The Trained Group performed 5-min sessions on the Rat Stepper 5 days a week for 6 weeks with 90% of body weight supported. At the end of the 6 weeks, body mass was obtained and right femurs and four lower extremity muscles were harvested. Femur bone mineral density was measured with DXA and mechanical characteristics of the femur were determined via 3-point bending testing. Independent t-tests, effects sizes and percent differences were computed between the two groups (p < 0.05). The Trained Group had significantly larger normalized femur mass (p = 0.007) and normalized soleus muscle mass (p = 0.033) when compared to the Control Group. There was a medium or large effect size with the Trained Groups' femurs having larger mass, bone mineral density, rupture loads, cortical wall thickness, shaft cross sectional area, soleus mass, normalized gastrocnemius mass, and smaller shaft inner diameters compared to the Control Group. These changes may contribute to decreasing osteoporosis and fracture risk in those with spinal cord injuries.