Determinação eletroquímica da capacidade antioxidante para avaliação do exercício físico

Physical training can adapt or cause injury to skeletal muscles implicating metabolic alterations, which can be detected by biochemical analysis. Apparently the increase in the production of reactive oxygen species (ROS) is involved in both processes. Enzymatic and low molecular weight antioxidants (LMWA) minimize ROS's deleterious action through redox reactions. Cyclic voltammetry (CV) has been suggested as a tool to quantify the antioxidant capacity conferred by LMWA. The use of CV to evaluate the modulation of the antioxidant capacity conferred by LMWA in response to physical exercise is discussed here.

Experimental intoxication by the leaves of Melia azedarach (Meliaceae) in cattle

Green leaves of Melia azedarach were administered at single doses ranging from 5 to 30 g/kg bw to 11 calves. Clinical signs were depression, ruminal stasis, dry feces with blood, ataxia, muscle tremors, sternal recumbency, hypothermia and abdominal pain. Serum AST and CPK were increased. Signs appeared from 8 to 24 hours after dosing, and the clinical course lasted from 2 to 72 hours. Three calves dosed with 30g/kg bw died. The macroscopic findings included intestinal congestion, yellow discoloration of the liver, brain congestion and dry feces with blood in the rectum. The liver showed swollen and vacuolated hepatocytes. Necrotic hepatocytes were scattered throughout the parenchyma or concentrated in the periacinar zone. Degenerative and necrotic changes were observed in the epithelium of the forestomachs. There was also necrosis of the lymphoid tissue. Skeletal muscles showed hyaline degeneration and fiber necrosis. The necrotic fragments contained floccular or granular debris with infiltration by macrophages and satellite cells.

Accidental and experimental salinomycin poisoning in rabbits

An outbreak of salinomycin poisoning in rabbits is described. At least 27 out of 2,000 rabbits reared on a farm died after the coccidiostatic drug sulfaquinoxaline was substituted by salinomycin in the feed. An average of 26.9ppm salinomycin was detected in the ration given to the rabbits. Clinical signs included anorexia, apathy and bradykinesia, which progressed to incoordination and recumbency. Gross lesions consisted of pale areas in the skeletal muscles. The histopathological findings showed severe necrotic degenerative myopathy in association with infiltration of neutrophils and macrophages. One rabbit exhibited similar alterations in the myocardium. Mineralization was observed in the affected skeletal muscles in some cases. In order to verify if the poisoning was due to salinomycin, 20 rabbits were divided into five groups and a ration containing the drug at doses of 10, 25, 50, 75 and 100ppm was given. The administration of doses higher than 50ppm resulted in manifestation of the clinical signs seen in the outbreak of poisoning. It was concluded, that probably an error related to the mixture of salinomycin in the feed was the cause of deaths in the spontaneous outbreak of poisoning on the rabbit farm.

Spontaneous coffee senna poisoning in cattle: report on 16 outbreaks

Sixteen outbreaks of Senna occidentalis (coffee senna) that occurred in cattle in the state of Rio Grande do Sul, Brazil, were reviewed. The great majority (75%) of the outbreaks occurred in adult cattle at pasture during the autumn and winter months with 50% in May, evidencing a striking seasonality. Mortality rates varied from 4.2% to 55.2% and cattle died 2 days up to 2 weeks after showing clinical signs that included dry feces (occasionally diarrhea), muscle weakness, reluctance to move, tachypnea, instability of the hind limbs with dragging of the toes, tremors in muscles of the thighs, neck, and head, ear dropping, sternal recumbency, lateral recumbency and death. Myoglobinuria characterized by a dark red or black discolored urine was a consistent finding in cattle affected at pasture but not in those poisoned by ration contaminated with coffee senna beans. Creatine phosphokinase serum activity was marked ly elevated. Main gross changes observed in 23 necropsies involved skeletal muscles of the hind limbs. These changes consisted of varying degrees of paleness of muscle groups. Subepicardial and subendocardial hemorrhages were present in the hearts of all affected cattle. Histologically a segmental degenerative myopathy of striated muscles was present in every case and had a multifocal polyphasic or monophasic character. Myocardial (3/23), hepatic (3/13), renal (3/10), and splenic (1/6) microscopic lesions were observed occasionally. Myocardial lesions were mild and consisted of vacuolation of cardiomyocytes or focal fibrosis. Hepatic changes consisted of diffuse hepatocelular vacuolation, cytosegrosomes within hepatocytes, and individual hepatocellular necrosis. Kidneys had vacuolar degeneration of tubular epithelium associated with acidophilic casts (proteinosis) within tubular lumina. In the spleen there was marked necrosis of lymphocytes of the white pulp. No histological changes were found in the brains of 13 affected cattle. The data of this study suggest that coffee senna poisoning is an important cause of death in cattle in southern Brazil.

Two research paths for probing the roles of oxygen in metabolic regulation

Tissues such as skeletal and cardiac muscles must sustain very large-scale changes in ATP turnover rate during equally large changes in work. In many skeletal muscles these changes can exceed 100-fold. Examination of a number of cell and whole-organism level systems identifies ATP concentration as a key parameter of the interior milieu that is nearly universally 'homeostatic'; it is common to observe no change in ATP concentration even while change in its turnover rate can increase or decrease by two orders of magnitude or more. A large number of other intermediates of cellular metabolism are also regulated within narrow concentration ranges, but none seemingly as precisely as is [ATP]. In fact, the only other metabolite in aerobic energy metabolism that is seemingly as 'homeostatic' is oxygen - at least in working muscles where myoglobin serves to buffer oxygen concentrations at stable and constant values at work rates up to the aerobic maximum. In contrast to intracellular oxygen concentration, a 1:1 relationship between oxygen delivery and metabolic rate is observed over biologically realistic and large-magnitude changes in work. The central regulatory question is how the oxygen delivery signal is transmitted to the intracellular metabolic machinery. Traditional explanations assume diffusion as the dominant mechanism, while proponents of an ultrastructurally dominated view of the cell assume an intracellular perfusion system to account for the data which have been most perplexing to metabolic biochemistry so far: the striking lack of correlation between changes in pathway reaction rates and changes in concentrations of pathway substrates, including oxygen and pathway intermediates.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

Ginseng administration protects skeletal muscle from oxidative stress induced by acute exercise in rats

Enzymatic activity was analyzed in the soleus, gastrocnemius (red and white) and plantaris muscles of acutely exercised rats after long-term administration of Panax ginseng extract in order to evaluate the protective role of ginseng against skeletal muscle oxidation. Ginseng extract (3, 10, 100, or 500 mg/kg) was administered orally for three months to male Wistar rats weighing 200 ± 50 g before exercise and to non-exercised rats (N = 8/group). The results showed a membrane stabilizing capacity of the extract since mitochondrial function measured on the basis of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities was reduced, on average, by 20% (P < 0.05) after exercise but the activities remained unchanged in animals treated with a ginseng dose of 100 mg/kg. Glutathione status did not show significant changes after exercise or treatment. Lipid peroxidation, measured on the basis of malondialdehyde levels, was significantly higher in all muscles after exercise, and again was reduced by about 74% (P < 0.05) by the use of ginseng extract. The administration of ginseng extract was able to protect muscle from exercise-induced oxidative stress irrespective of fiber type.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Cyclosporin A preferentially attenuates skeletal slow-twitch muscle regeneration

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, is associated with muscle regeneration via NFATc1/GATA2-dependent pathways. However, it is not clear whether calcineurin preferentially affects the regeneration of slow- or fast-twitch muscles. We investigated the effect of a calcineurin inhibitor, cyclosporin A (CsA), on the morphology and fiber diameter of regenerating slow- and fast-twitch muscles. Adult Wistar rats (259.5 ± 9 g) maintained under standard conditions were treated with CsA (20 mg/kg body weight, ip) for 5 days, submitted to cryolesion of soleus and tibialis anterior (TA) muscles on the 6th day, and then treated with CsA for an additional 21 days. The muscles were removed, weighed, frozen, and stored in liquid nitrogen. Cryolesion did not alter the body weight gain of the animals after 21 days of regeneration (P = 0.001) and CsA significantly reduced the body weight gain (15.5%; P = 0.01) during the same period. All treated TA and soleus muscles showed decreased weights (17 and 29%, respectively, P < 0.05). CsA treatment decreased the cross-sectional area of both soleus and TA muscles of cryoinjured animals (TA: 2108 ± 930 vs 792 ± 640 µm²; soleus: 2209 ± 322 vs 764 ± 439 m²; P < 0.001). Histological sections of both muscles stained with Toluidine blue revealed similar regenerative responses after cryolesion. In addition, CsA was able to minimize these responses, i.e., centralized nuclei and split fibers, more efficiently so in TA muscle. These results indicate that calcineurin preferentially plays a role in regeneration of slow-twitch muscle.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.