Regeneration of new fibers in muscles of old rats reduces contraction-induced injury.

Skeletal muscles are injured by their own contractions. Compared with muscles in young animals, those in old animals are injured more easily and more severely and regenerate less well afterward. Injection of a myotoxin (bupivacaine) causes complete degeneration of fibers in extensor digitorum longus (EDL) muscles of rats, followed by full regeneration within 60 days. We tested the specific hypothesis that, 3 days after a protocol of pliometric (lengthening) contractions, the newly regenerated muscle fibers in bupivacaine-treated EDL muscles in both young and old rats would show a lesser deficit in maximum force and fewer damaged fibers than muscles in nontreated EDL muscles. The treated and nontreated EDL muscles of young and old male Wistar rats were administered a protocol of 225 pliometric contractions and were evaluated 3 days afterward, when morphological damage to muscle fibers is most severe. In treated compared with nontreated EDL muscles of both young and old rats, the force deficit and the number of damaged fibers were each reduced by approximately 75%. We conclude that newly regenerated fibers in muscles of young and old animals are resistant to injury and that maintenance of newly regenerated fibers by conditioning may prevent inadvertent damage, particularly in muscles of elderly people.     

Contraction-induced injury to the extensor digitorum longus muscles of rats: the role of vitamin E

Van der Meulen, Jack H., Anne McArdle, Malcolm J. Jackson,and John A. Faulkner. Contraction-induced injury to the extensordigitorum longus muscles of rats: the role of vitamin E. J. Appl. Physiol. 83(3): 817-823, 1997.Three days after a protocol of 225 pliometric (lengthening)contractions was administered to in situ extensor digitorum longusmuscles of rats, the force deficit was 64 ± 7% and the percentageof damaged muscle fibers was 38 ± 5% of the control values. Wethen tested the hypothesis that at 3 h and 3 days after the protocol anelevation in the muscle vitamin E content would decrease the forcedeficit, the percentage of damaged muscle fibers, and the serumactivities of creatine kinase and pyruvate kinase. The 5-8 days ofintravenous injections of -tocopherol increased muscle vitamin Econtent threefold compared with vehicle (ethanol)-treated rats. Despite the difference in vitamin E content, the force deficit and number ofdamaged fibers were not different. After the contractionprotocol, the serum creatine kinase and pyruvate kinase activities ofthe vehicle-treated rats increased fourfold at 3 h and twofold at 3 days, whereas the vitamin E-treated rats showed no change. We concludethat vitamin E treatment did not ameliorate either the induction of theinjury or the more severe secondary injury at 3 days.Despite the absence of evidence for an antioxidant function, the lackof any increase in serum enzyme activities for vitamin E-treated ratsat 3 h and 3 days supported a role for vitamin E in the prevention ofenzyme loss after muscle damage.

     

Occurrence of oxidative stress during myocardial reperfusion

Reperfusion, without doubt, is the most effective way to treat the ischaemic myocardium. Late reperfusion may however cause further damage. Myocardial production of oxygen free radicals above the neutralizing capacity of the myocytes is an important cause of this reperfusion damage. There is evidence that prolonged ischaemia reduces the naturally occurring defence mechanisms of the heart against oxygen free radicals, particularly mitochondrial manganese superoxide dismutase, and intracellular pool of reduced glutathione. Consequently, reperfusion results in a severe oxidative damage, as evidenced by tissue accumulation and release of oxidized glutathione.An oxygen free radical-mediated impairment of mechanical function also occurs during reperfusion of human heart. In fact we observed during surgical reperfusion of coronary artery disease (CAD) patients, a prolonged and sustained release of oxidized glutathione;the degree of oxidative stress was inversely correlated with recovery of mechanical and haemodynamic function. These findings represent the rationale for therapeutic interventions which increase the cellular antioxidant capacities and improve the efficacy of myocardial reperfusion.     

Muscle inflammatory cells after passive stretches, isometric contractions, and lengthening contractions.

We tested the hypotheses that lengthening contractions, isometric contractions, and passive stretches increase muscle inflammatory cells (neutrophils and macrophages) and that prior conditioning with lengthening contractions, isometric contractions, or passive stretches reduces neutrophils and macrophages after subsequent lengthening contractions. Extensor digitorum longus muscles in anesthetized mice were subjected in situ to lengthening contractions, isometric contractions, or passive stretches. Six hours or 3 days after a protocol of contractions or passive stretches, neutrophils and macrophages were quantified in muscle cross sections. Three days after isometric contractions or passive stretches, neutrophils were elevated (P < 0.05) 3.7- and 5.5-fold, respectively, relative to controls. Both macrophages and neutrophils were increased 51.2- and 7.9-fold, respectively, after lengthening contractions. Prior lengthening contractions, isometric contractions, or passive stretches reduced inflammatory cells after lengthening contractions performed 2 wk later. The major finding of this study was that passive stretches and isometric contractions elevated neutrophils without causing overt signs of injury. Because both passive stretches and isometric contractions elevated neutrophils and afforded some protection from contraction-induced muscle injury, neutrophils and/or the related inflammatory events may contribute to the induction of a protective mechanism.     

Changes in free radicals,trace elements,and neurophysiological function in rats with liver damage induced byD-galactosamine

The changes in trace elements, free radicals, and neurophysiological function were investigated in rats with liver damage induced byd-galactosamine (GalN). The elevated results showed that all the parameters related to free radical metabolism changed after administration of GalN. Relative free radical concentration, malonaldehyde (MDA), and oxidized glutathione (GSSG) elevated, but reduced glutathione (GSH) decreased. Concurrently, zinc, copper, manganese, and selenium contents in liver were significantly reduced, whereas iron was elevated. In rats with hepatic encephalopathy (HE) owing to fulminant hepatic failure (FHF) induced by a high dosage of GalN, the latencies of VEPs were delayed. Moreover, there is a correlation between Zn content of brain and the latencies of VEPs. The results of this study suggested that lipid peroxidation by free radicals might be responsible for GalN-induced liver damage in which trace elements were involved, and that change in brain Zn might play a role in the neural inhibition of HE owing to FHF.     

Free radical scavengers can modulate the DNA-damaging action of alloxan

Alloxan can generate diabetes in experimental animals and its action can be associated with the production of free radicals. It is therefore important to check how different substances often referred to as free radical scavengers may interact with alloxan, especially that some of these substance may show both pro- and antioxidant activities. Using the alkaline comet assay we showed that alloxan at concentrations 0.01-50 microM induced DNA damage in normal human lymphocytes in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 microM diminished the extent of DNA damage induced by 50 microM alloxan. Pre-treatment of the lymphocytes with a nitrone spin trap, alpha-(4-pyridil-1-oxide)- N-t-butylnitrone (POBN) or ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which mimics glutathione peroxides, reduced the alloxan-evoked DNA damage. The cells exposed to alloxan and treated with formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results confirmed that free radicals are involved in the formation of DNA lesions induced by alloxan. The results also suggest that alloxan can generate oxidized DNA bases with a preference for purines and contribute to their alkylation.     

Hyperbaric Oxygen Treatment Attenuates Glutathione Depletion and Improves Metabolic Restitution in Postischemic Skeletal Muscle

Glutathione serves as an important intracellular defence against reactive oxygen metabolites and has been shown to be depleted from a number of tissues upon oxidative stress. In the present study we have investigated the levels of total glutathione (reduced + oxidized) in skeletal muscle of the rat after prolonged ischema and reperfusion with and without treatment with hyperbaric oxygen (HBO) for the initial 45 minutes immediately following reperfusion. A tourniquet model for temporary, total ischemia was used, in which one hind leg was made ischemic for 3 or 4 hours. Muscle biopsies were taken after 5 hours of reperfusion. In postischemic muscle there was a significant decrease of total glutathione compared to control muscle, but in the 3-hour-ischema-groups the loss of total glutathione was less in HBO treated animals than in untreated. HBO treatment also preserved ATP and PCr and decreased edema formation in the postischemic muscle following 3 hours of ischemia and reperfusion when compared to untreated animals. However, after 4 hours of ischemia, HBO treatment failed to improve any of these parameters in the postischemic muscle. Thus, our results demonstrate that HBO treatment lessens the metabolic, ischemic derangements and improves recovery in postischemic muscle after 3 hours of ischemia followed by reperfusion.     

2,4,6－三氯苯酚诱导鲫鱼肝脏自由基的产生及其氧化应激

采用电子顺磁共振的方法,研究了鲫鱼腹腔注射2,4,6-三氯苯酚(2,4,6-TCP)不同时间(4、8、12、24、72 h)后其肝脏自由基强度的变化、氧化应激反应及其损伤机理.结果表明：2,4,6-TCP极显著促进了鲫鱼肝脏自由基的产生;鲫鱼肝脏内超氧化物歧化酶 (SOD) 与谷胱甘肽硫转移酶 (GST) 的活性受到显著诱导 ,过氧化氢酶 (CAT) 活性受到抑制,还原型谷胱甘肽 (GSH) 含量与对照组无明显差异,氧化型谷胱甘肽 (GSSG) 含量显著升高 ,丙二醛(MDA) 含量极显著增加.     

Neutrophil accumulation following passive stretches contributes to adaptations that reduce contraction-induced skeletal muscle injury in mice.

Skeletal muscles can be injured by their own contractions, especially when the muscle is stretched during a lengthening contraction. Exposing a muscle to a conditioning protocol of stretches without activation (passive stretches) before lengthening contractions reduces contraction-induced injury. Although passive stretching does not damage muscle fibers, neutrophils are elevated in the muscle after passive stretches. Our purpose was to investigate the relationship between neutrophil accumulation following passive stretches and the protection from subsequent contraction-induced injury provided by the passive stretches. Our hypothesis was that passive stretch conditioning would not provide protection from subsequent lengthening contraction-induced injury under circumstances when the increase in muscle neutrophils in response to the conditioning was prevented. Extensor digitorum longus muscles of mice were conditioned with passive stretches 14 days before exposure to a protocol of damaging lengthening contractions. Mice were either untreated or treated with an antibody (RB6-8C5) that reduced the level of circulating neutrophils by over 95% before administration of passive stretches. Neutrophil levels recovered in treated mice by the time lengthening contractions were performed. Lengthening contractions were also administered to muscles with no prior exposure to passive stretches. Maximum isometric force, number of damaged fibers, and muscle neutrophil concentration were measured 3 days after lengthening contractions. Compared with nonconditioned control muscles, the severity of contraction-induced injury was not reduced by prior passive stretch conditioning when mice were treated with RB6-8C5 before conditioning. We conclude that neutrophils contribute to adaptations that protect muscles from injury.     

Lengthening contractions are not required to induce protection from contraction-induced muscle injury

We tested the hypothesis that lengthening contractions and subsequent muscle fiber degeneration and/or regeneration are required to induce exercise-associated protection from lengthening contraction-induced muscle injury. Extensor digitorum longus muscles in anesthetized mice were exposed in situ to repeated lengthening contractions, isometric contractions, or passive stretches. Three days after lengthening contractions, maximum isometric force production was decreased by 55%, and muscle cross sections contained a significant percentage (18%) of injured fibers. Neither isometric contractions nor passive stretches induced a deficit in maximum isometric force or a significant number of injured fibers at 3 days. Two weeks after an initial bout of lengthening contractions, a second identical bout produced a force deficit (19%) and a percentage of injured fibers (5%) that was smaller than those for the initial bout. Isometric contractions and passive stretches also provided protection from lengthening contraction-induced injury 2 wk later (force deficits = 35 and 36%, percentage of injured fibers = 12 and 10%, respectively), although the protection was less than that provided by lengthening contractions. These data indicate that lengthening contractions and fiber degeneration and/or regeneration are not required to induce protection from lengthening contraction-induced injury.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Indices of free-radical-mediated damage following maximum voluntary eccentric and concentric muscular work

This study monitored plasma and skeletal muscle markers of free-radical-mediated damage following maximum eccentric and concentric exercise, to examine the potential role of free radicals in exercise-induced muscle damage. Fourteen male volunteers performed either (1) a bout of 70 maximum eccentric and a bout of 70 maximum concentric muscle actions of the forearm flexors (the bouts being separated by 4 weeks; n = 8) or (2) a bout of 80 maximum eccentric and a bout of 80 maximum concentric muscle actions of the knee extensors (the bouts being separated by 1 week; n=6). Plasma markers of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and diene-conjugated compounds (DCC) were monitored in the arm protocol and skeletal muscle markers of oxidative lipid and protein damage, malondialdehyde (MDA) and protein carbonyl derivatives (PCD) respectively, were monitored in the leg protocol. In both protocols, the contralateral limb was used for the second bout and the order of the bouts was randomised between limbs. Repeated measures ANOVA indicated significant changes from baseline following eccentric arm work on the measures of serum creatine kinase activity (P < 0.05), maximum voluntary torque production (P < 0.01) and relaxed arm angle (P < 0.01). Subjective muscle soreness peaked 2 days after eccentric arm work (P < 0.05, Wilcoxon test). However, there were no changes in the plasma levels of TBARS or DCC following the eccentric or concentric arm exercise. Immediately after concentric leg exercise, skeletal muscle PCD concentrations was significantly higher than that observed immediately after eccentric work (P < 0.05). However, no significant difference between the eccentric and concentric knee extensor bouts was observed on the measure of skeletal muscle MDA concentration. The results of this study offer no support for the involvement of oxygen free radicals in exercise-induced muscle damage.     

Residual force enhancement following eccentric induced muscle damage

During lengthening of an activated skeletal muscle, the force maintained following the stretch is greater than the isometric force at the same muscle length. This is termed residual force enhancement (RFE), but it is unknown how muscle damage following repeated eccentric contractions affects RFE. Using the dorsiflexors, we hypothesised muscle damage will impair the force generating sarcomeric structures leading to a reduction in RFE. Following reference maximal voluntary isometric contractions (MVC) in 8 young men (26.5±2.8y) a stretch was performed at 30°/s over a 30° ankle excursion ending at the same muscle length as the reference MVCs (30° plantar flexion). Surface electromyography (EMG) of the tibialis anterior and soleus muscles was recorded during all tasks. The damage protocol involved 4 sets of 25 isokinetic (30°/s) lengthening contractions. The same measures were collected at baseline and immediately post lengthening contractions, and for up to 10min recovery. Following the lengthening contraction task, there was a 30.3±6.4% decrease in eccentric torque (P<0.05) and 36.2±9.7% decrease in MVC (P<0.05) compared to baseline. Voluntary activation using twitch interpolation and RMS EMG amplitude of the tibialis anterior remained near maximal without increased coactivation for MVC. Contrary to our hypothesis, RFE increased (～100-250%) following muscle damage (P<0.05). It appears stretch provided a mechanical strategy for enhanced muscle function compared to isometric actions succeeding damage. Thus, active force of cross-bridges is decreased because of impaired excitation-contraction coupling but force generated during stretch remains intact because force contribution from stretched sarcomeric structures is less impaired.     

Free radicals in exhaustive physical exercise: mechanism of production, and protection by antioxidants

Moderate exercise is a healthy practice. However, exhaustive exercise generates free radicals. This can be evidenced by increases in lipid peroxidation, glutathione oxidation, and oxidative protein damage. It is well known that activity of cytosolic enzymes in blood plasma is increased after exhaustive exercise. This may be taken as a sign of damage to muscle cells. The degree of oxidative stress and of muscle damage does not depend on the absolute intensity of exercise but on the degree of exhaustion of the person who performs exercise. Training partially prevents free radical-formation in exhaustive exercise. Treatment with antioxidants such as vitamins C or E protects in part against free radical-mediated damage in exercise. Xanthine oxidase is involved in free-radical formation in exercise in humans and inhibition of this enzyme with allopurinol decreases oxidative stress and muscle damage associated with exhaustive exercise. Knowledge of the mechanism of free-radical formation in exercise is important because it will be useful to prevent oxidative stress and damage associated with exhaustive physical activity.     

Spin trapping in biological systems. Oxidation of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide by a hydroperoxide-hematin-system.

We have used the spin trap 5,5-dimethyl-1-pyrroline-1-oxide to determine if primary free radicals are involved in the hematin-cumene hydroperoxide system which has been shown to oxidize N-hydroxy-2-acetylaminofluorene into the nitroxyl free radical form of this carcinogen. We have found that the spin trap was oxidized itself rather than trapping either primary free radicals or carcinogen free radicals.     

Production, detection, and adaptive responses to free radicals in exercise

Free radicals (particularly oxygen- and nitrogen-centered radicals), and related reactive oxygen and nitrogen species, are generated in cells and tissues during exercise. Mitochondria (actually, 'leakage' of electrons from ubisemiquinone and other electron transport chain components), xanthine oxidase, and phagocytes such as neutrophils may all contribute to free radical production. In this article we review mechanisms of free radical production during exercise and methods for detecting free radicals and related reactive species, during, or immediately following exercise. The evidence presented strongly suggests that free radicals generated during mild to moderate endurance-type exercise actually form part of the mechanism of exercise adaptation that includes extensive biogenesis of muscle mitochondria, increased muscle blood supply, and altered fuel consumption patterns. We suggest, as originally proposed [1], that (at moderately increased levels) free radicals actually act as intracellular signaling molecules to initiate exercise adaptation. In contrast, endurance exercise of extreme duration and extreme intensity appears to generate much higher levels of free radicals that overwhelm cellular antioxidant defenses, and cause tissue damage. Such free radical damage requires effective protein, lipid, and DNA repair systems, and sufficient recuperation, before exercise adaptation can recommence.     

Mitochondrial Ageing and the Beneficial Role of α-Lipoic Acid

Oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the ageing process. Mitochondria are known to be a rich source for the production of free radicals and, consequently, mitochondrial components are susceptible to lipid peroxidation (LPO) that decreases respiratory activity. In the present investigation, we have evaluated mitochondrial LPO, 8-oxo-dG, oxidized glutathione, reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and electron transport chain (ETC) complex activities in the brain of young versus aged rats. In aged rats, the contents of LPO, oxidized glutathione and 8-oxo-dG were high whereas reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities were found to be low. Lipoic acid administration to aged rats reduced the levels of mitochondrial LPO, 8-oxo-dG and oxidized glutathione and enhanced reduced glutathione, ATP, lipoic acid and ETC complex activities. In young rats lipoic acid administration showed only minimal lowering the levels of LPO, 8-oxo-dG and oxidized glutathione and slight increase in the levels of reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities. These findings suggest that the dithiol, lipoic acid, provides protection against age-related oxidative damage in the mitochondria of aged rats.     

大鼠急性运动对血清睾酮、睾丸组织丙二醛和超氧化物歧化酶的影响

本实验分别在大鼠衰竭跑后即刻、0.5、1、3及24h同时检测了血清睾酮(T)、黄体生成素、睾丸组织脂质过氧化物丙二醛和超氧化物歧化酶。结果表明,大鼠衰竭跑后恢复30min时,伴随着睾丸组织丙二醛含量的明显升高及超氧化物歧化酶活性的显著降低,血清T水平也明显降低。提示,长时间衰竭跑后血清T浓度的降低很可能与缺血-再灌引起睾丸组织氧自由基剧增及脂质过氧化损伤,从而降低了酶的活性和抑制了T的合成有关。氧自由基清除剂能减轻和预防该损伤,并能有效地防止运动后血清T的降低。     

Enzymatic and non-enzymatic protective mechanisms in recalcitrant seeds of Araucaria bidwillii subjected to desiccation.

The changes in several antioxidants as well as in the level of C-centered free radicals and thiobarbituric acid reactive substances (TBARS) were studied in seeds of Araucaria bidwillii Hook desiccated to 37%, 28% and 21% moisture content. The lowest-safe moisture content for the seedling establishment was 37%. The embryo, besides double amounts of free radicals, showed higher levels of both enzymatic and non-enzymatic antioxidants than endosperm. Lutein decreased in both organs whereas alpha-tocopherol values were not affected by desiccation. In the embryo at 37% seed moisture content the antioxidant defense system increased giving rise to a decrease in free radicals. Beyond this point, free radicals and TBARS increased in agreement with the umpiring of the ascorbate/glutathione cycle by the decrease in reduced glutathione and glutathione reductase activity (GR, EC 1.6.4.2). At 21% moisture GR decreased. In the endosperm during desiccation, the consumption of ascorbate, total glutathione and lutein prevented the rise in free radicals and TBARS till 28% moisture, at which an increase in oxidized glutathione was also observed.