Eccentric Exercise Leads to Glial Activation but not Apoptosis in Mice Spinal Cords.

The aim of this investigation was to evaluate the effects of 3 overtraining (OT) protocols on the glial activation and apoptosis in the spinal cords of mice. Rodents were divided into control (C; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). The incremental load test, ambulation test, exhaustive test and functional behavioural assessment were used as performance evaluation parameters. 36 h after the exhaustive test, the dorsal and ventral parts of the lumbar spinal cord (L4-L6) were dissected for subsequent protein analysis by immunoblotting. The OT protocols led to similar responses of some performance parameters. The ventral glial fibrillary acidic protein (GFAP) protein levels were diminished in the OTR/up and OTR compared to CT and OTR/down groups. The ventral ionized calcium binding adaptor molecule 1 (Iba-1), and the dorsal GFAP and Iba-1 protein levels were increased in the OTR/down compared to the other groups. The ratio between the cleaved capase-3/caspase-3 and cleaved caspase-9/caspase-9 measured in the spinal cord were not sensitive to the OT protocols. In summary, the OTR/down activated the glial cells in the motor (i. e. Iba-1) and sensory (i. e. GFAP and Iba-1) neurons without leading to apoptosis.

Effects of different physical exercises on leptin concentration in obese adolescents.

In order to compare the effects of leisure physical activity (LPA), aerobic training (AT) and aerobic plus resistance training (AT + RT) as part of a 6-month interdisciplinary therapy in body composition, insulin resistance and leptin concentrations in obese adolescents, 72 volunteers (n = 24 in each group) ages 15-19 years were evaluated. Delta (Δ) body mass (kg) and Δ BMI (kg/m(2)) was different between AT and LPA groups and in AT + RT group compared to both LPA and AT groups; Δ body fat mass (kg and %) was different only in AT + RT group compared to both LPA and AT; Δ body lean mass (%) was different only in AT + RT group; Δ body lean mass (kg) was negative only in AT and positive and different from AT in AT + RT group; ΔHOMA-IR did not differ among groups; Δ leptin (ng/ml) was negative and different from LPA for both AT and AT + RT groups. In conclusion, both AT and AT + RT promoted a reduction on leptin levels, however, the adolescents subjected to AT + RT presented better results in body composition than the AT group. These results highlight the importance of associating aerobic and resistance training with nutritional and psychological approaches in the treatment of obese adolescents.

Human adipose-derived mesenchymal stromal cells injected systemically into GRMD dogs without immunosuppression are able to reach the host muscle and express human dystrophin.

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting 1 in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

Lack of ß2-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice.

ß(2)-adrenergic receptor (ß(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of ß(2)-AR activation are highly recognized, less is known about the impact of ß(2)-AR in endurance capacity. We presently used mice lacking ß(2)-AR [ß(2)-knockout (ß(2) KO)] to investigate the role of ß(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. ß(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, ß(2) KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, ß(2) KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in ß(2) KO skeletal muscle. Altogether, these data provide evidence that disruption of ß(2)-AR improves oxidative metabolism in skeletal muscle of ß(2) KO mice and this is associated with increased exercise capacity.

Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice.

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Aerobic exercise training improves skeletal muscle function and Ca2+ handling-related protein expression in sympathetic hyperactivity-induced heart failure.

The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca2+ abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha2A/alpha2C adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca2+ handling-related protein expression by Western blot. alpha2A/alpha2CARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca2+ release-related proteins [alpha1-, alpha2-, and beta1-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca2+ reuptake-related proteins [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1/2 and Na+/Ca2+ exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca2+ handling toward WT levels. We provide evidence that Ca2+ handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca2+ handling proteins.

Exercise training attenuates lipectomy-induced impaired glucose tolerance in rats.

OBJECTIVE: Since visceral adipose tissue (VAT) may account for impaired peripheral and hepatic insulin sensitivity (IS), it has been hypothesized that the partial removal of VAT could result in improved insulin action, while the re-growth of the excised tissue and/or compensatory growth of non-excised depots seems to occur. Thus, it was aimed to investigate whether or not VAT removal and exercise affect IS. METHODS: Male Wistar rats were fed a high-fat diet and subsequently assigned randomly to one of four groups: 1. exercised plus lipectomized (EL), 2. exercised plus sham-lipectomized (ES), 3. sedentary plus lipectomized (CL), 4. sedentary plus sham-lipectomized (CS). After lipectomy, EL and ES animals underwent a 7-consecutive-day training period. Body weight, food intake, basal metabolic rate, fasting glucose, and glucose tolerance were assessed before and after the interventions. Fasting insulin and the HOMA index, body fat mass, and the expression of pro-inflammatory genes were assessed after the interventions. RESULTS: EL group showed greater insulin sensitivity compared to all other groups. EL and ES groups showed lower fasting insulin levels when compared to CL and CS groups, respectively. The EL group showed improved IS when compared to the remaining groups. The CL group showed impaired glucose tolerance and increased TNF-alpha gene expression. Body weight and fat mass did not differ among the groups. PPAR gamma gene expression was increased in the EL and ES groups. CONCLUSIONS: These results showed that short-term swimming training improved insulin sensitivity, but failed to prevent fat regain in lipectomized animals. Lipectomy induced impaired glucose tolerance, which is probably related to increased TNF-alpha gene expression. It is possible that a high-fat diet might be implicated in faster regain of adipose tissue after lipectomy. Our results also show that short-term exercise associated with lipectomy could improve insulin sensitivity.

Exercise training reduces cardiac angiotensin II levels and prevents cardiac dysfunction in a genetic model of sympathetic hyperactivity-induced heart failure in mice.

The role of exercise training (ET) on cardiac renin-angiotensin system (RAS) was investigated in 3-5 month-old mice lacking alpha(2A-) and alpha(2C-)adrenoceptors (alpha(2A)/alpha(2C)ARKO) that present heart failure (HF) and wild type control (WT). ET consisted of 8-week running sessions of 60 min, 5 days/week. In addition, exercise tolerance, cardiac structural and function analysis were made. At 3 months, fractional shortening and exercise tolerance were similar between groups. At 5 months, alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction and fibrosis associated with increased cardiac angiotensin (Ang) II levels (2.9-fold) and increased local angiotensin-converting enzyme activity (ACE 18%). ET decreased alpha(2A)/alpha(2C)ARKO cardiac Ang II levels and ACE activity to age-matched untrained WT mice levels while increased ACE2 expression and prevented exercise intolerance and ventricular dysfunction with little impact on cardiac remodeling. Altogether, these data provide evidence that reduced cardiac RAS explains, at least in part, the beneficial effects of ET on cardiac function in a genetic model of HF.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice.

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom.

Detection and characterization of rabies virus in Southern Brazil by PCR amplification and sequencing of the nucleoprotein gene.

Due to the medical and socio-economical importance of both human and animal rabies infection, several studies have suggested the use of molecular techniques such as RT-PCR and DNA sequencing for diagnosis and phylogenetic studies of the rabies virus. Considering the conservancy of the nucleoprotein (N) gene of the virus, we herein describe a RT-PCR assay for rabies diagnosis and characterization. A total of 75 samples obtained from a variety of animal species in the state of Santa Catarina (SC), Southern Brazil, were comparatively studied by fluorescence antibody test (FAT), mouse inoculation test (MIT), cell infection assay and RT-PCR, which revealed itself to be as sensitive as FAT and MIT and less time-consuming than MIT. Direct sequencing of the 5' end of the N gene allowed the clustering of the SC samples with samples from the vampire bat-related or sylvatic cycle through comparative sequence analysis.