No effect of short-term 17beta-estradiol supplementation in healthy men on systemic inflammatory responses to exercise

Sex-based differences in inflammatory responses to exercise may be mediated by estrogen through increased muscle membrane stability and/or inhibited cytokine production. In this study, in vivo effects of estrogen on systemic inflammation-related responses to exercise were assessed in healthy men. In a double-blind, placebo-controlled, crossover design, 11 men cycled for 90 min at 65% Vo2 max after 8 days of 17beta-estradiol supplementation (ES; 2 mg/day) or placebo (PL; glucose polymer). After a 2-wk washout, exercise was repeated after 8 days on the alternate treatment. Blood was collected pre- and postexercise to determine IL-6, soluble intercellular adhesion molecule-1 (sICAM-1), neutrophil counts, and cortisol. Preexercise serum was assayed for sex hormones. ES increased estradiol (133+/-71 to 840+/-633 pmol/l, P=0.005) and reduced testosterone (19.9+/-3.7 to 16.1+/-3.9 nmol/l, P=0.007). Exercise increased cortisol (P=0.02), IL-6 (P<0.001) and neutrophil counts (P<0.001) with no influence on sICAM-1 (P=0.34) and no effect of ES on these changes. Postexercise IL-6 and neutrophil counts were correlated (r=0.58, P=0.005); postexercise IL-6 and cortisol (r=0.18, P=0.43) and postexercise cortisol and neutrophil counts (r=0.06, P=0.78) were not. Postexercise sICAM-1 was not correlated with the above variables (P>or=0.79). In conclusion, 8 days of ES in healthy men did not influence systemic inflammation-related responses to acute exercise. Future studies should investigate 17beta-estradiol effects on IL-6 production and neutrophil infiltration within skeletal muscle during and after exercise.     

Accelerated skeletal muscle recovery after in vivo polyphenol administration

Acute skeletal muscle damage results in fiber disruption, oxidative stress and inflammation. We investigated cell-specific contributions to the regeneration process after contusion-induced damage (rat gastrocnemius muscle) with or without chronic grape seed-derived proanthocyanidolic oligomer (PCO) administration. In this placebo-controlled study, male Wistar rats were subjected to PCO administration for 2 weeks, after which they were subjected to a standardised contusion injury. Supplementation was continued after injury. Immune and satellite cell responses were assessed, as well as oxygen radical absorption capacity and muscle regeneration. PCO administration resulted in a rapid satellite cell response with an earlier peak in activation (Pax7(+), CD56(+), at 4 h post-contusion) vs. placebo groups (PLA) (P<.001: CD56(+) on Day 5 and Pax7(+) on Day 7). Specific immune-cell responses in PLA followed expected time courses (neutrophil elevation on Day 1; sustained macrophage elevation from Days 3 to 5). PCO dramatically decreased neutrophil elevation to nonsignificant, while macrophage responses were normal in extent, but significantly earlier (peak between Days 1 and 3) and completely resolved by Day 5. Anti-inflammatory cytokine, IL-10, increased significantly only in PCO (Day 3). Muscle fiber regeneration (MHC(f) content and central nuclei) started earlier and was complete by Day 14 in PCO, but not in PLA. Thus, responses by three crucial cell types involved in muscle recovery were affected by in vivo administration of a specific purified polyphenol in magnitude (neutrophil), time course (macrophages), or time course and activation state (satellite cell), explaining faster effective regeneration in the presence of proanthocyanidolic oligomers.     

Antioxidant supplementation influences the neutrophil tocopherol associated protein expression, but not the inflammatory response to exercise

Intense exercise induces inflammatory-like changes and oxidative stress in immune cells. Our aim was to study the effects of antioxidant diet supplementation on the neutrophil inflammatory response and on the tocopherol associated protein (TAP) expression after exhaustive exercise. Fourteen male-trained amateur runners were randomly divided in two placebo and supplemented groups. Vitamins C (152 mg/d) and E (50 mg/d) supplementation were administrated to the athletes for a month, using an almond based isotonic and energetic beverage. Non-enriched beverage was given to the placebo group. After one month, the subjects participated in a half-marathon race (21 km-run). Neutrophil TAP mRNA expression and markers of the inflammatory response were determined before, immediately after, and 3 h after finishing the half-marathon race. TAP expression increased after exercise mainly in the neutrophils of the placebo group. Exercise induced an inflammatory response in both placebo and supplemented groups, manifested with neutrophilia, increased creatine kinase and lactate dehydrogenase serum activities, neutrophil luminol chemiluminescence and myeloperoxidase release. Plasma malondialdehyde only increased in the placebo group after exercise. Diet supplementation with moderate levels of antioxidant vitamins avoids plasma damage in response to exhaustive exercise without the effects on the inflammatory process. Neutrophil degranulation and increased tocopherol associated protein could contribute to the neutrophil protection from the oxidative stress.     

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.     

Exercise training increases oxidative capacity and attenuates exercise-induced ultrastructural damage in skeletal muscle of aged horses.

Exercise training improves functional capacity in aged individuals. Whether such training reduces the severity of exercise-induced muscle damage is unknown. The purpose of the present study was to determine the effect of 10 wk of treadmill exercise training on skeletal muscle oxidative capacity and exercise-induced ultrastructural damage in six aged female Quarter horses (>23 yr of age). The magnitude of ultrastructural muscle damage induced by an incremental exercise test before and after training was determined by electron microscopic examination of samples of triceps, semimembranosus, and masseter (control) muscles. Maximal aerobic capacity increased 22% after 10 wk of exercise training. The percentage of type IIa myosin heavy chain increased in semimembranosus muscle, whereas the percentage of type IIx myosin heavy chain decreased in triceps muscle. After training, triceps muscle showed significant increases in activities of both citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase. Attenuation of exercise-induced ultrastructural muscle damage occurred in the semimembranosus muscle at both the same absolute and the same relative workloads after the 10-wk conditioning period. We conclude that aged horses adapt readily to intense aerobic exercise training with improvements in endurance, whole body aerobic capacity, and muscle oxidative capacity, and heightened resistance to exercise-induced ultrastructural muscle cell damage. However, adaptations may be muscle-group specific.     

Body temperature modulates the antioxidant and acute immune responses to exercise

The aim of this study was to determine the effects of whole body heat in combination with exercise on the oxidative stress and acute phase immune response. Nine male endurance-trained athletes voluntarily performed two running bouts of 45 minutes at 75-80% of VO(2max) in a climatic chamber in two conditions: cold and hot humid environment. Leukocyte, neutrophil and basophil counts significantly rose after exercise in both environments; it was significantly greater in the hot environment. Lymphocyte and neutrophil antioxidant enzyme activities and carbonyl index significantly increased or decreased after exercise only in the hot environment, respectively. The lymphocytes expression of catalase, Hsp72 and CuZn-superoxide dismutase was increased in the hot environment and Sirt3 in the cold environment, mainly during recovery. In conclusion, the increased core body temperature results in the acute phase immune response associated to intense exercise and in the immune cell adaptations to counteract the oxidative stress situation.     

Influence of one bout of vigorous exercise on ascorbic acid in plasma and oxidative damage to DNA in blood cells and muscle in untrained rats

We investigated the influence of a single exhaustive bout of downhill running on oxidative damage to DNA and changes of antioxidant vitamin concentrations in rats. Plasma vitamin E levels were unchanged up to 48 hr postexercise. However, plasma ascorbic acid (AA) levels increased after the exercise, then decreased thereafter. This increase corresponded to a marked decrease in AA concentration in the adrenal glands. The activity of hepatic l-gulono-gamma-lactone oxidase, which catalyzes AA synthesis, was unaltered after the exercise. The weight of the adrenal glands was significantly increased 24 hr postexercise. These results indicate that the change in the plasma AA concentration after vigorous exercise was due mainly to the release of AA from the adrenal glands. The plasma creatine phosphokinase (CPK) activity and white blood cell (WBC) count increased 3 to 6 hr postexercise. Over this same period, a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine in DNA, increased in the WBC, but not in the foreleg muscle. Lipid peroxide and vitamin E levels were also unchanged in the foreleg muscle. There was a positive correlation between CPK activity in the plasma and DNA damage in the WBC, suggesting that the DNA damage in the WBC was closely related with muscle damage due to exercise.     

Body temperature modulates the antioxidant and acute immune responses to exercise

Abstract

The aim of this study was to determine the effects of whole body heat in combination with exercise on the oxidative stress and acute phase immune response. Nine male endurance-trained athletes voluntarily performed two running bouts of 45 minutes at 75–80% of VO_2max in a climatic chamber in two conditions: cold and hot humid environment. Leukocyte, neutrophil and basophil counts significantly rose after exercise in both environments; it was significantly greater in the hot environment. Lymphocyte and neutrophil antioxidant enzyme activities and carbonyl index significantly increased or decreased after exercise only in the hot environment, respectively. The lymphocytes expression of catalase, Hsp72 and CuZn-superoxide dismutase was increased in the hot environment and Sirt3 in the cold environment, mainly during recovery. In conclusion, the increased core body temperature results in the acute phase immune response associated to intense exercise and in the immune cell adaptations to counteract the oxidative stress situation.     

Muscle and blood redox status after exercise training in severe COPD patients

Beneficial effects of exercise training in patients with chronic obstructive pulmonary disease (COPD) are acknowledged. However, high-intensity exercise may enhance muscle oxidative stress in severe COPD patients. We hypothesized that high-intensity exercise training of long duration does not deteriorate muscle redox status. In the vastus lateralis and blood of 18 severe COPD patients and 12 controls, before and after an 8-week training program, protein oxidation and nitration, antioxidant systems, and inflammatory cytokines were examined. At baseline, COPD patients showed greater muscle oxidative stress and superoxide dismutase activity and circulating inflammatory cytokines than controls. Among COPD patients, muscle and blood protein carbonylation levels were correlated. Both groups showed training-induced increase in VO(2) peak and decreased blood lactate levels. After training, among the COPD patients, blood protein nitration levels were significantly reduced and muscle protein oxidation and nitration levels did not cause impairment. Muscle and blood levels of inflammatory cytokines were not modified by training in either patients or controls. We conclude that in severe COPD patients, high-intensity exercise training of long duration improves exercise capacity while preventing the enhancement of systemic and muscle oxidative stress. In addition, in these patients, resting protein oxidation levels correlate between skeletal muscle and blood compartments.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Do invading leucocytes contribute to the decrease in glutathione concentrations indicating oxidative stress in exercised muscle, or are they important for its recovery?

Mice were subjected to one session of strenuous running exercise and their soleus muscles were examined in respect of changes in ultrastructure and to their concentration of reduced glutathione [GSH] which are indicators of oxidative stress. It was hypothesized that invading leucocytes contributed to oxidative stress and they were functionally inhibited in one experimental group by the administration of colchicine. Exercise led to an immediate decrease in [GSH] of about 60%, which slowly recovered during 96 h after exercise. With the administration of colchicine after exercise, [GSH] was higher than in the untreated exercise group 48 h after exercise, indicating an inhibition of the ability of leucocytes to produce oxidative stress. However, at 96 h after exercise, [GSH] was lower in the treated exercise group than in the untreated group. The morphological evaluation of the percentage of affected fibres showed that the invasion of leucocytes increased muscle fibre damage. The results suggested that invading leucocytes enhanced production of reactive species of oxygen that may have participated in inducing muscle damage. However, inhibition of leucocyte invasion did not permit their scavenger action of removing cell debris, which appeared to produce even more oxidative stress in the muscle.     

Inflammatory markers CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase in eccentric exercised human skeletal muscles

The aim of the present study was to investigate leucocyte markers, CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase on skeletal muscle biopsies from biceps brachii after unaccustomed eccentric exercise followed by the second bout of exercise 3 weeks later. The subjects (10 subjects received COX-2 inhibitor (Celecoxib) and 13 subjects received placebo) were divided into three categories: mild, moderate and severe effect of eccentric exercise, according to the reduction and recovery of muscle force-generating capacity after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer. The results showed that the CD66b antibody was applicable for localization of neutrophils in human skeletal muscle, whereas the other studied neutrophil markers recognized also other leucocytes than neutrophils. The number of CD66b positive cells in skeletal muscle was very low and was not affected by the exercise. The macrophage marker CD68 showed reactivity also against satellite cells and fibroblast-like cells in skeletal muscle and therefore cannot be applied as a quantitative value for inflammatory cells. Skeletal muscle fibre injury, shown as dystrophin negative fibres, was observed approximately in half of the biopsies at 4 and 7 days after the first exercise bout in the categories moderate and severe effect of eccentric exercise. These subjects represent the most prominent loss in muscle force-generating capacity both at the category and the individual levels. Furthermore, deformed skeletal muscle fibres were observed in five subjects in these categories after the second bout of exercise. The present results suggest that neutrophils are not involved in skeletal muscle fibre injury and the reduction in muscle force-generating capacity after a single bout of eccentric exercise is a good indirect indicator of muscle damage in humans. Furthermore, prolonged regeneration process could be one of the reasons for impaired peripheral muscle function after high-force eccentric exercise.     

Human blood neutrophil responses to prolonged exercise with and without a thermal clamp.

The purpose of this study was to investigate the effects of prolonged exercise with and without a thermal clamp on neutrophil trafficking, bacterial-stimulated neutrophil degranulation, stress hormones, and cytokine responses. Thirteen healthy male volunteers (means +/- SE: age 21 +/- 1 yr; mass 74.9 +/- 2.1 kg; maximal oxygen uptake 58 +/- 1 ml x kg(-1) x min(-1)) completed four randomly assigned, 2-h water-immersion trials separated by 7 days. Trials were exercise-induced heating (EX-H: water temperature 36 degrees C), exercise with a thermal clamp (EX-C: 24 degrees C), passive heating (PA-H: 38.5 degrees C), and control (CON: 35 degrees C). EX-H and EX-C was comprised of 2 h of deep water running at 58% maximal oxygen uptake. Blood samples were collected at pre-, post-, and 1 h postimmersion. Core body temperature was unaltered on CON, clamped on EX-C (-0.02 degrees C), and rose by 2.23 degrees C and 2.31 degrees C on EX-H and PA-H, respectively. Exercising with a thermal clamp did not blunt the neutrophilia postexercise (EX-C postexercise: 9.6 +/- 1.1 and EX-H postexercise: 9.8 +/- 1.0 x 10(9)/liter). Neutrophil degranulation decreased (P < 0.01) similarly immediately after PA-H (-21%), EX-C, and EX-H (-28%). EX-C blunted the circulating norepinephrine, cortisol, granulocyte-colony stimulating factor, and IL-6 response (P < 0.01) but not the plasma epinephrine and serum growth hormone response. These results show a similar neutrophilia and decrease in neutrophil degranulation after prolonged exercise with and without a thermal clamp. As such, the rise in core body temperature does not appear to mediate neutrophil trafficking and degranulation responses to prolonged exercise. In addition, these results suggest a limited role for cortisol, granulocyte-colony stimulating factor, and IL-6 in the observed neutrophil responses to prolonged exercise.     

Sympathoadrenal response to repetitive exercise in normal and asthmatic subjects

To explore the role of catecholamine release in the pathogenesis of exercise-induced asthma, we had seven asthmatic and seven normal subjects undergo three hourly exercise challenges that were matched for inspired air temperature, minute ventilation, and relative work loads. Pulmonary mechanics and plasma epinephrine and norepinephrine were measured before, at end exercise, and serially after each challenge. There were no differences in the pattern of sympathoadrenal response of asthmatic and normal subjects, and both groups released sufficient quantities of epinephrine and norepinephrine into the peripheral circulation to allow these compounds to function as circulating hormones. As the catecholamines rose with repetitive exercise, progressive bronchodilation occurred in the asthmatics at the end of the work load, thus decreasing the apparent magnitude of the obstructive response. In addition to their effects on airway smooth muscle, the alpha-adrenergic actions of both catecholamines may have reduced airway wall hyperemia and edema. These data demonstrate that asthmatics do not have a defect in catecholamine release during exercise and that the physiological expression of exercise-induced asthma can be modulated by the sympathoadrenal epiphenomena that are associated with physical exertion.     

Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function

Uncoupling protein-3 (UCP3) expression has been shown to increase dramatically in response to muscular contraction, but the physiological significance of UCP3 upregulation is still elusive. In this study, UCP3 mRNA and protein expression were investigated along with mitochondrial respiratory function, reactive oxygen species (ROS) generation, and antioxidant defense in rat skeletal muscle during and after an acute bout of prolonged exercise. UCP3 mRNA expression was elevated sharply at 45 min of exercise, reaching 7- to 8-fold above resting level at 150 min. The increase in UCP3 protein content showed a latent response but was elevated approximately 1.9-fold at 120 min of exercise. Both UCP3 mRNA and UCP3 protein gradually returned to resting levels 24 h postexercise. Mitochondrial ROS production was progressively increased during exercise. However, ROS showed a dramatic drop at 150 min although their levels remained severalfold higher during the recovery. Mitochondrial State 4 respiration rate was increased by 46 and 58% (p < 0.05) at 90 and 120 min, respectively, but returned to resting rate at 150 min, when State 3 respiration and respiratory control index (RCI) were suppressed. ADP-to-oxygen consumption (P/O) ratio and ATP synthase activity were lowered at 3 h postexercise, whereas proton motive force and mitochondrial malondialdehyde content were unchanged. Manganese superoxide dismutase gene expression was not affected by exercise except for an increase in mRNA abundance at 3 h postexercise. These data demonstrate that UCP3 expression in rat skeletal muscle can be rapidly upregulated during prolonged exercise, possibly owing to increased ROS generation. Increased UCP3 may partially alleviate the proton gradient across the inner membrane, thereby reducing further ROS production by the electron transport chain. However, prolonged exercise caused a decrease in energy coupling efficiency in muscle mitochondria revealed by an increased respiration rate due to proton leak (State 4/State 3 ratio) and decreased RCI. We thus propose that the compromise of the oxidative phosphorylation efficiency due to UCP3 upregulation may serve an antioxidant function to protect the muscle mitochondria from exercise-induced oxidative stress     

Imbalance in SOD/CAT activities in rat skeletal muscles submitted to treadmill training exercise

The association between physical exercise and oxidative damage in the skeletal musculature has been the focus of many studies in literature, but the balance between superoxide dismutase and catalase activities and its relation to oxidative damage is not well established. Thus, the aim of the present study was to investigate the association between regular treadmill physical exercise, oxidative damage and antioxidant defenses in skeletal muscle of rats. Fifteen male Wistar rats (8-12 months) were randomly separated into two groups (trained n=9 and untrained n=6). Trained rats were treadmill-trained for 12 weeks in progressive exercise (velocity, time, and inclination). Training program consisted in a progressive exercise (10 m/min without inclination for 10 min/day). After 1 week the speed, time and inclination were gradually increased until 17 m/min at 10% for 50 min/day. After the training period animals were killed, and gastrocnemius and quadriceps were surgically removed to the determination of biochemical parameters. Lipid peroxidation, protein oxidative damage, catalase, superoxide dismutase and citrate synthase activities, and muscular glycogen content were measured in the isolated muscles. We demonstrated that there is a different modulation of CAT and SOD in skeletal muscle in trained rats when compared to untrained rats (increased SOD/CAT ratio). TBARS levels were significantly decreased and, in contrast, a significant increase in protein carbonylation was observed. These results suggest a non-described adaptation of skeletal muscle against exercise-induced oxidative stress.     

Cellular adaptation to repeated eccentric exercise-induced muscle damage.

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 and bout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.     

Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91^phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.