DIVERSITY OF ENDURANCE TRAINING EFFECTS ON ANTIOXIDANT DEFENSES AND OXIDATIVE DAMAGE IN DIFFERENT BRAIN REGIONS OF ADOLESCENT MALE RATS

At rest, the brain uses about 1/5 of the total oxygen consumed by man to cover energy expenditure (about 240 kcal/kg organ mass/day as compared to about 30 kcal/kg whole body mass/day (1, 2). During physical exercise of moderate intensity, global and regional cerebral blood flow increase by 40 –70% to satisfy the increased metabolic demand for oxygen. The affected brain regions include motor-related structures and central command network (3). Increased oxygen consumption results in increased production of a variety of reactive oxygen species (ROS), e.g. superoxide anion, hydroxyl radicals, nitric oxide (NO) and singlet oxygen, due to oxidative phosphorylation-related electron leakage from mitochondrial transport chain (4-8). Being considered unavoidable by products of aerobic metabolism, ROS may cause oxidative damage to the cell components by impairing cellular energetics and modulating signaling pathways ("redox signaling") that lead to diverse acute and chronic changes in cellular environment dependent on the affected tissue (9).

According to the most recent studies, about 1% of total oxygen consumed by the brain will form superoxide anion radical, with mitochondria (mainly complex 1), NADPH oxidase and cytosolic xanthine oxidase being most important contributors (10-12). The superoxide radical can be readily dismutated by superoxide dismutase (SOD; E.C. 1.15.1.1) to H2O2 and singlet oxygen. H2O2 is next converted by either catalase (CAT; E.C. 1.11.1.6) or glutathione peroxidase (GPx; E.C. 1.11.1.9) to H2O and O2 (10, 11). The antioxidant enzymes CAT, GPx and SOD, and the ratio of reduced to oxidized glutathione (GSH/GSSG) are critical for protection against oxyradicals toxicity. Glutathione consumed (oxidized) in the GPx-mediated detoxification reaction is recycled back to GSH by glutathione reductase (GR; E.C. 1.6.4.2). The levels of glutathione and these antioxidant enzymes are much lower in the CNS as compared to erythrocytes and peripheral tissues (15, 16). Because of this characteristic and high consumption of inspired oxygen as well as high abundance of polyunsaturated fatty acids, the brain is also particularly prone to oxidative damage (17).

The imbalance between ROS generation and their scavenging by enzymatic and nonenzymatic antioxidants-based systems in the host is termed oxidative stress. This type of stress is gaining particular attention for its presumed causative role in physiological aging and many pathological processes (18, 19). However, regular exercise can cause an adaptation of the respective cellular antioxidant systems, resulting in increased resistance to oxidative stress and reduced cellular damage (20-23).

Various investigators have determined the effect of exercise on oxidative damage and/or the free radicals scavenging enzymes in different tissues: liver (24), skeletal muscle (25), cardiac muscle, blood (26) and brain (27-29). A great majority of studies on the effect of physical activity on CNS oxidative stress were performed in adult or aged rats, with particular focus on aging-related phenomena. However, it is adolescence that is the period of the highest sensitivity to a variety of endogenous and exogenous stimuli (30-32). Moreover, it was shown that moderate exercise from young ages may counteract the decline of some aging-associated detoxication processes in the brain; interestingly, at least some of these processes show considerable differences between various brain regions (33, 34).

Previous studies have shown that physical activity affects oxidative stress in peripheral tissues as well as in the entire brain differently in adolescent as compared with adult rats, and that endurance training can increase antioxidant defenses in these localizations (35-38). The purpose of this study was to determine the effect of endurance (moderate intensity aerobic) training on the major enzymatic components of antioxidant barrier in the brain of adolescent rats, with consideration of different involvement of various brain regions in the initiation, control and execution of locomotion. Aerobic exercise is known to elevate the activity of two constitutively expressed nitric oxide synthases (NOSs) in the brain that are postulated to exert a beneficial effect on brain function (39, 40). Both these enzymes can also contribute to brain lipid peroxidation due to NO reaction with the superoxide radical production and the resulting production of the peroxynitrite radical (41). Hence, we decided also to assess total NOS activity as well as the contents of glutathione that acts as a key protector of brain proteins under the conditions of enhanced NO production (42).

MATERIAL AND METHODS

Animals and experimental protocol

All animal use procedures were in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and with the current laws of Poland. The study protocol was approved by the First Warsaw Ethical Committee for Animal Experiments of the Polish Academy of Sciences, Warsaw, Poland (Certificate of Approval No. 251 of February 8, 2006).

Adolescent (5 - 6 weeks old) male Wistar rats of 112 – 135 g body weight from the animal breeding facility of Mossakowski Medical Research Centre (Warsaw, Poland) were used for the study. The rats were housed 4 - 5 per cage, at 12 h light/12 h dark cycle (lights on at 7:00 a.m.), 22 - 24°C ambient temperature and 45 - 65% relative humidity, and were given free access to standard pellet rat chow and tap water. All efforts were made to keep at minimum the number of rats used for the study and to minimize animal discomfort.

Before the start of training, all rats were pretested on a motorized rodent treadmill (3 × 5 min, with 15-min breaks, beginning at 10 m/min and gradually increasing the speed up to 20 m/min at the end of each 5 min episode, at 0° inclination) for three successive days to habituate them to the training environment and to identify and eliminate rats unwilling to run. The preselected rats were randomly divided between the sedentary control group (Ctrl, N = 18) and the endurance-trained group (EndTr, N = 18). The rats scheduled for EndTr were exercised on the treadmill (at 0° inclination) for 6 weeks, 5 days a week, beginning at treadmill speed of 16 m/min during the first week. The speed was increased by 4 m/min weekly over the next 3 weeks, and then was kept at 28 m/min for the remainder of the training period. The session duration began each week at 40 min/day and was increased by 5 min daily during the first 4 weeks; during the final two weeks, the rats ran for 1 h daily (38). The EndTr program employed has been shown previously by a variety of measures to induce a number of health-promoting changes and to greatly improve endurance in rats (43-46), similar to the effects of analogous training reported in young men (47).

All rats were sacrificed by simple decapitation 48 h after the last training session to determine chronic effects of the training. Trunk blood was collected into heparinized tubes. After taking an aliquot for the assessment of blood GSH, the remainder of each blood sample was immediately spun for 10 min at 1000 × g and +4°C to separate plasma (for the assessment of thiobarbituric acid-reactive substances, TBARS) and erythrocytes (for the assessment of antioxidant enzymes). The erythrocytes were washed three times with cold (4°C) sterile 0.9% NaCl solution and then both the plasma and the washed erythrocytes were aliquoted and kept frozen at –80°C until analyzed. The brains were removed and chilled on ice, and the brain cortex, cerebellum, hippocampus, midbrain and striatum were dissected, snap-frozen in liquid nitrogen and stored at –80°C until analyzed.

Preparation of brain homogenates

Brain samples were homogenized with all-glass Dounce homogenizers using 14 strokes of the looser-fitted pestle A and tissue to buffer ratio of 1:9 (w/v). The buffers used were as follows: 1) 10 mM Tris-HCl buffer pH 7.4 containing 0.25 M sucrose, 1 mM EDTA and one mini tablet/10 ml of complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) for homogenates for the assessment of total NOS activity and TBARS; and 2) 0.1 M phosphate buffer pH 7.0 containing 1% Triton X-100 for homogenates for the assessment of GSH and of GPx, GR and CAT activities. Homogenate protein content was determined by the method of Lowry.

Determination of total nitric oxide synthase activity

Total NOS activity in brain samples was assayed as described elsewhere (48). Briefly, homogenate aliquots (0.2 mg protein) were incubated at 37°C for 20 min in 50 mM Tris-HCl buffer pH 7.4 containing 100 µM [U–14C]L-arginine (0.2 µCi), 2 mM CaCl2, 15 µM FAD, 1 µM calmodulin, 10 µM tetrahydrobiopterin, 1 mM EDTA, 1 mM dithiothreitol and 1 mM NADPH, in the final volume of 300 µl. The reaction was stopped by adding 1 ml of 100 mM Tris-HCl buffer pH 5.5 containing 10 mM EDTA. Gross cellular debris was spun down at 3000 × g for 10 min. The resulting supernatant was passed through 1 ml Na+ form DowexTM 50W × 8 columns, and [14C]L-citrulline was eluted with 2 × 1 ml H2O; the eluate was mixed with 10 ml of Bray’s scintillation liquid and the radioactivity was measured in a β-counter.

Determination of thiobarbituric acid-reactive substances

TBARS content in brain tissue samples was determined according to Asakawa and Matsushita (49). Briefly, brain regional homogenates were diluted with 10 mM Tris buffer pH 7.4 to give protein concentration of about 1 mg/ml. One-ml aliquots of the diluted homogenates were mixed with 1 ml of 30% TCA and left on ice for 5 min. Next, 0.1 ml of 5 N HCl was added to each sample and the mixtures were centrifuged for 10 min at 4000 × g. The supernatants were collected, mixed with 1 ml of 0.75% thiobarbituric acid (TBA), capped and heated for 15 min in boiling water bath. The absorbance of the reaction mixtures was read at 535 nm using Shimadzu UV 1202 spectrophotometer. TBARS level in plasma was assayed using the TBA method (50) with some modifications, including the addition of 0.01% butylated hydroxytoluene to lower the auto-oxidation of lipids during heating with TBA, the extraction of the colored reaction product with n-butanol, and reading the absorbance of the organic layer at 532 nm (51). TBARS concentration was calculated based on a standard curve generated from 1,1,3,3-tetraethoxypropane solutions and was expressed as nmol of malondialdehyde per mg protein.

Determination of glutathione

Brain tissue total glutathione content was assessed by using the method of Tietze (52) as modified by Bhat et al. (53). Briefly, diluted brain tissue homogenate aliquots (100 µg protein/100 µl) were mixed with 2 volumes of 9% TCA and after 5 min incubation on ice were centrifuged for 10 min at 9000 × g and 4°C to remove precipitated protein. To determine the total glutathione level, 0.1 ml aliquots of supernatant samples were mixed with 10 µl of GR solution in 0.2 M phosphate buffer pH 8.0 (49 U/ml) and 0.2 ml of the same phosphate buffer containing 0.01 mM EDTA, 0.6 mM NADPH and 6 mM 5,5'-dithiobis(2-nitrobenzoic) acid. After 10 min incubation at room temperature the absorbance of all the samples was measured at 412 nm with Bio-Rad (Wien, Austria) ELISA Reader 3500. Glutathione content was calculated on the basis of standard curve prepared with a series of GSSG solutions according to the same procedure. Blood GSH concentration was determined using fresh whole blood as described elsewhere (54).

Determination of superoxide dismutase activity

SOD activity in brain tissue samples was measured using a modification of the method for the xanthine/xanthine oxidase/nitro blue tetrazolium (NBT) assay (55). Briefly, diluted brain sample homogenate aliquots (50 µg of protein/50 µl) were mixed with 245 µl aliquots of 0.1 M phosphate buffer pH 8.0 containing 0.4 mM xanthine and 0.24 mM NBT, and the reaction mixtures were brought to 37°C in the BioRad ELISA Reader 3500. The reaction was started by adding 5 µl of xanthine oxidase solution in the phosphate buffer (2.94 U/ml) and immediate mixing. After instantly taking the first absorbance measurement (at 560 nm), the incubation was continued for 20 min and was next stopped by adding 2 µl of 69 mM sodium dodecyl sulfate; then the absorbance was read again at 560 nm. One unit of SOD activity corresponds to 50% inhibition of the xantine oxidase-catalyzed reaction per mg protein. Blood SOD activity was measured in erythrocyte lysates using the commercially available Ransod SD125 kit (Randox Laboratories, Antrim, UK) according to manufacturer's instructions.

Determination of glutathione peroxidase activity

Brain regional GPx activities were determined using the BioxyTech GPx-340 kit (Oxis Health Products Inc., Portland, OR, USA) according to manufacturer's instructions. Blood GPx activity was measured in erythrocyte lysates using the commercially available Ransel RS505 kit (Randox Laboratories, Antrim, UK) according to manufacturer's instructions.

Determination of glutathione reductase activity

Brain regional GR activities were determined using the BioxyTech GR-340 kit (Oxis Health Products Inc., Portland, OR, USA) according to manufacturer's instructions. Blood GR activity was determined in erythrocyte lysates as described elsewhere (56).

Determination of catalase activity

Catalase activity was assayed according to the method of Aebi (57). Blood CAT activity was determined in erythrocyte lysates. Brain sample homogenates were centrifuged at 9000 × g for 10 min to obtain supernatant. Twenty-five µl aliquots of the supernatant where mixed with 975 µl of 0.1 M phosphate buffer pH 8.0 and 500 µl of 10 mM H2O2 and the decomposition of H2O2 was directly estimated by the net decrease in absorbance at 240 nm.

Determination of hemoglobin

Lysate hemoglobin concentration was determined spectrophotometrically by the cyanmethemoglobin method using a Randox total hemoglobin assay kit HG980 (Randox Laboratories, Antrim, UK).

Statistical analysis

All results are expressed as the mean ± S.E.M. Between-group differences in blood antioxidant barrier indices and TBARS levels were tested using the Student t-test for independent variables, with significance set at P < 0.05. Data on brain regional antioxidant enzymes and total NOS activities, brain regional total glutathione level and brain regional TBARS levels were first analyzed using repeated measures 2-way ANOVA with training (group) as the main factor and brain region as the repeated measures factor. Since the analyses showed significant training × brain region interactions, the data were next analyzed for each brain region separately using Student's t-tests for dependent and independent variables as appropriate. To keep the family-wise probability of type I error < 0.05, the sequential Bonferroni-Holm correction was applied to the results of the post-hoc t-tests. All statistical analyses were performed using the Statistica v. 7.1 software package (StatSoft Inc., Tulsa, OK, USA), except for the Bonferroni-Holm correction that was applied 'manually'.

RESULTS

The effect of endurance training on enzymatic antioxidant system in different brain regions

A two-way ANOVA yielded significant effects of training and region, and of training × region interaction on GR activity (F1,6 = 19.4, P = 0.005; F4,24 = 163.7, P < 10–3; and F4,24 = 5.37, P = 0.0031, respectively). Post-hoc analysis showed dramatically higher GR activity in the neocortex of EndTr rats compared to Ctrl rats (+760%); no such effect was found in the other regions studied (from –4 to +17%, not significant), see Fig. 1A.

A two-way ANOVA revealed significant effects of region and training × region interaction (F4,24 = 13.0, P < 10–3; and F4,24 = 3.34, P = 0.026, respectively), but not of training (F1,6 = 0.34, P = 0.58) on GPx activity. Brain regional differences in average GPx activity between the EndTr rats and their sedentary counterparts ranged from -40% in the neocortex to +27% in the hippocampus, but did not reach significance in any region studied, see Fig. 1B.

A two-way ANOVA demonstrated significant effects of training and region, and of training × region interaction (F1,6 = 44.0, P = 0.0006; F4,24 = 15.3, P < 10-3; and F4,24 = 4.05, P = 0.012, respectively) on CAT activity. Post-hoc test showed that these effects translated into significantly higher average CAT activity in the neocortex (+226%) and the cerebellum (+115%) of EndTr rats compared to Ctrl rats, see Fig. 1C.

A two-way ANOVA yielded significant effects of training and region, and of training × region interaction on SOD activity (F1,6 = 21.7, P = 0.003; F4,24 = 99.6, P < 10–3; and F4,24 = 3.13, P = 0.033, respectively). Post-hoc analysis showed significantly higher SOD activity in the neocortex of EndTr vs. Ctrl rats (+173%), whereas the differences in the other regions studied (ranging from –12% in the cerebellum to +46% in the midbrain) did not reach significance, see Fig. 1D.

Fig. 1. The effects of endurance training (EndTr) on glutathione reductase (A), glutathione peroxidase (B), catalase (C) and superoxide dismutase (D) activity in selected brain regions of EndTr vs. sedentary (Ctrl) adolescent male rats (4 rats/group) 48 hours after the accomplishment of the last training session. * P < 0.05, ** P < 0.01 vs. the respective Ctrl group value.

The effect of endurance training on glutathione content in different brain regions

A two-way ANOVA yielded significant effects of training, region and training × region interaction on total tissue GSH content (F1,6 = 34.1, P = 0.0011; F4,24 = 246.2, P < 10–3; and F4,24 = 4.78, P = 0.006, respectively). As revealed by post-hoc analysis, the EndTr rats compared to their sedentary counterparts had significantly higher GSH contents in the cortex, cerebellum and striatum (+41, +71 and +34%, respectively), but not in the hippocampus and midbrain (+20 and +44%, respectively), see Fig. 2.

Fig. 2. The effect of endurance training (EndTr) on total glutathione (GSH + GSSG) contents in different brain regions of EndTr vs. sedentary (Ctrl) adolescent male rats (4 rats/group) 48 hours after the accomplishment of the last training session. * P < 0.05, *** P < 0.001 vs. the respective Ctrl group value.

The effect of endurance training on brain regional total nitric oxide synthase activity

A two-way ANOVA yielded highly significant effects of training, region and training × region interaction on total NOS activity (F1,6 = 160.0, P < 10–3; F4,24 = 299.7, P < 10–3; and F4,24 = 26.9, P < 10–3, respectively). Post-hoc analysis showed significant while moderately higher total NOS activity in the cerebellum, striatum and midbrain (+19, +57 and +28%, respectively), but not in the cortex and hippocampus (+2 and –5%, respectively) of EndTr vs. Ctrl rats, see Fig. 3.

Fig. 3. The effect of endurance training (EndTr) on total NOS activity in homogenates of selected brain regions of EndTr vs. sedentary (Ctrl) adolescent male rats (4 rats/group) 48 hours after the accomplishment of the last training session. ** P < 0.01, *** P < 0.001 vs. the respective Ctrl group value.

The effect of endurance training on brain regional thiobarbituric acid-reactive substances levels

A two-way ANOVA yielded significant effects of training, region and training × region interaction on TBARS content (F1,8 = 8.38, P = 0.020; F4,32 = 67.6, P < 10–3; and F4,32 = 10.8, P < 10–3; respectively). Post-hoc analysis showed significantly higher TBARS content in the EndTr compared to Ctrl rats in the cerebellum and striatum only (+57 and +48%, respectively), see Fig. 4.

Fig. 4. The effect of endurance training (EndTr) on TBARS content in homogenates of selected brain regions of EndTr vs. sedentary (Ctrl) adolescent male rats (5 rats/group) 48 hours after the accomplishment of the last training session. *** P < 0.001 vs. the respective Ctrl group value.

The effect of endurance training on antioxidant systems and thiobarbituric acid-reactive substances level in peripheral blood

Forty-eight hours after the last training session, EndTr rats showed significantly higher contents of total glutathione (+46%) and TBARS (+34%) in circulating blood than their sedentary counterparts. Simultaneously, the EndTr compared to the untrained rats showed only a negligible tendency for increase (+23%, P = 0.12) in CAT activity and significantly lower SOD, GR and GPx activities in their blood (–48, –47 and –55%, respectively), see Table 1.

Table 1. Effects of moderate intensity endurance training (EndTr) on blood antioxidant barrier and concentration of lipid peroxidation products in adolescent male rats.

aStatistically significant between-group differences are marked in boldtype.

The effect of endurance training on rat body weight gain

The rats were weighed at the study entry and just before sacrifice. Two-way ANOVA of the body weight data revealed significant effects of time, training status and time × training status interaction on body weight. There was no difference between the study groups at the entry, whereas the EndTr rats weighed markedly less than their sedentary counterparts 48 h after completion of the training, see Table 2.

Table 2. Effects of moderate intensity endurance training (EndTr) on body weight gain in adolescent male rats.

***P < 0.001 vs. the respective starting body weight, ### vs. the respective value for the Ctrl group.

DISCUSSION

Effects of physical exercise on brain antioxidant defenses may greatly differ depending on the exercise type, mode and intensity, as well as the training protocol employed. Some data indicate that non-exhaustive aerobic exercise/training promotes a protective antioxidant function on the brain in adults (58). However, as claimed by those authors, "The wide range of exercise protocols at different intensities and volumes does not allow us to provide reliable conclusions. This lack of homogeneity in the protocols could be due to the difficulty to establish the intensity of the effort when using animal models“. Effects of various factors on the brain may also greatly differ depending on its maturity (59). This study was aimed at testing the effect of moderate-intensity training on brain antioxidative 'barrier' in adolescent rats. The training started at about the 7th week of their life, which corresponds roughly to 12.5 human years, and the effects of the training were assessed at 13 weeks, which is well below full (including social aspects) maturity (60, 61). The development of different systems of mammals follows different trajectories (61). Importantly, imaging studies using a variety of techniques demonstrate that CNS development extends deep into adolescence, and that the development follows different time course in various brain structures in humans (62). There is no ground to suppose that this is not the case also in other placentalia.

This study demonstrates that endurance training-induced changes in enzymatic defenses in the brain and blood follow diverse patterns. Moreover, there were major differences in the pattern of the changes between brain regions directly involved with the execution and coordination of locomotion-related muscular activity (the cerebellum, midbrain and striatum) and those engaged in locomotion planning and initiation (the cortex), or those that respond to enhanced physical activity, but are not directly appointed for any of the said actions (the hippocampus). Furthermore, even the changes in the striatum, midbrain and cerebellum showed substantial dissimilarity. Interestingly, no changes in either the antioxidant enzymes' activities (CAT, GR, GPx and SOD) or in oxidative stress (TBARS content) were observed in these brain regions in male rats subjected to a single bout of graded exercise (up to 100% VO2max) (63). This suggests that the effects observed in our study were due to the endurance type of exercise and/or the training intensity and volume. No increase in prefrontal cortical, striatal and hippocampal SOD and GPx activities was also observed in 22-week old rats subjected to 8-week chronic running exercise of similar volume (1 h, 5 days a week), but lower intensity (20 m/min) (64). We cannot tell for sure whether the disagreement between our results and that previous report is related to the age-related difference between the subject rats and/or the difference in the respective exercise/training programs. However, the fact that the chronic exercise program employed in that study did not significantly modify brain regional TBARS levels suggest an important role of the latter.

In contrast to changes in enzymatic antioxidant defenses, changes in glutathione levels showed considerable similarity between the different brain regions studied and circulation (Fig. 2 and Table 1). Whereas brain glutathione level is predominantly maintained by recycling its constituents within the brain, there is experimental evidence for active GSH transport from the periphery. However, the relative contribution of extra-CNS glutathione source(s) to brain glutathione content is not known (65, 66). Notably, the most evident EndTr-related increases in total glutathione were found in the brain regions directly involved in the initiation and execution of locomotion. However, GSH may also be engaged in a variety of oxidative stress-unrelated regulatory mechanisms in the brain (65, 66).

The only oxidative stress measure employed, namely TBARS content, while showing less heterogeneous changes than those in antioxidant defenses, demonstrated an enhancement of lipid peroxidation only in some brain regions directly involved with locomotor activity (the cerebellum and striatum) and in peripheral blood. The latter most likely reflected continuing, endurance-training-related elevated TBARS content in skeletal muscles, mainly in aerobic (red) muscle fibers (37), consequential to the excess of post-exercise oxygen consumption and increased resting metabolic rate (67). Since only the cerebellum and striatum showed endurance training-related elevations in TBARS, the differences in the studied brain regional TBARS levels were clearly of local origin. Since exercise enhances brain synthesis of dopamine (68) that may significantly contribute to TBARS formation due to its autooxidation or metabolism by monoamine oxidase (69), one might expect a particularly high elevation of TBARS in dopamine-rich brain regions, i.e. the striatum and midbrain. Surprisingly, we found the highest training-related increase in TBARS content in the cerebellum that shows extremely low total dopamine content (70) and dopamine usage (71) in the rat. The differences in the brain regional effects of EndTr on TBARS contents may explain the lack of significant effect of regular exercise on TBARS content measured in homogenates of entire brain in earlier studies (72, 73). However, that failure could also be related to the fact that the exercise scheme employed was not a true training program, because it involved no increase in either exercise volume or exercise intensity.

TBARS level was also significantly higher in the plasma of EndTr compared with Ctrl rats, which finding is in apparent conflict with earlier studies that showed no change in this index 24 or 48 h after the last bout of exercise. The disagreement may be attributed to differences in training schemes. The scheme used in our study included manipulations of both running volume and intensity, whereas the other researchers either used a different exercise mode (swimming, (74)) or exercise sessions of much lower and constant volume and only changed the intensity of exercise that also included a higher strength component (75). Importantly, their training schemes also resulted in different changes in TBARS contents in the heart and skeletal muscles than those obtained with our training schedule, (37, 38, 74).

On the other hand, some researchers reported no significant effect of exercise training on SOD and GPx activities in the cortex, striatum and hippocampus and on glutathione levels in the cortex and striatum (28). While their findings appear to be in a conflict with our data shown above, this discrepancy could be consequential to a major strength training-oriented component in that training program, whereas our training scheme was strictly of endurance type.

In line with the present findings, we have previously reported that EndTr lastingly increases the expression and activities of both endothelial and neuronal NOS isoforms, but not of the inducible NOS, and elevates the activity of the NO/soluble guanylyl cyclase/cGMP pathway in the striatum, midbrain and cerebellum, but not in the cortex or hippocampus. Those changes were associated with markedly increased spontaneous locomotor activity (76). Altogether, these observations may be related to the role of NO in synaptic plasticity and locomotor learning (77-79). The activity of the various NOS isoforms is an important source of free radicals in the CNS. Interestingly, whereas EndTr significantly elevated total NOS activity in all studied brain regions that are directly involved in the execution and coordination of locomotion-related body movements (the striatum, cerebellum and midbrain), but not in those engaged in locomotion planning and initiation (the cortex) or in those that react to enhanced physical activity but are not directly appointed for any of the said functions (the hippocampus). Since the midbrain compared to the other studied brain regions showed no superiority in any studied component of antioxidant defenses, this observation suggests that NOS is not the main source of oxidative free radicals in this region.

The brain region that showed increases in most antioxidant enzymes studied was the neocortex. Notably, this region showed the highest EndTr-induced relative increase in CAT activity and no training-induced increases in TBARS content, suggesting a successful prevention of exercise-related increase in oxidative stress. On the other hand, the absence of a rise in cortical TBARS content could also be explained by the absence of an EndTr-induced increase in NOS activity. Interestingly, the striatum that was the only region to show a significant endurance training-related elevation of GSH, but no change in antioxidant enzymes studied, revealed also the highest increase of the oxidative stress biomarker TBARS.

Another brain region studied that showed a major EndTr-related change in antioxidant defenses was the cerebellum. Because of its indispensability for movement coordination and balance, the cerebellum is heavily engaged in all locomotion-related muscular work. Notably, the cerebellum showed the highest CAT activity among the studied brain regions in the sedentary rats, and this difference was even more prominent in their EndTr counterparts. It has been shown that a single bout of either volitional physical exercise or pharmacologically-induced muscle contractions (e.g., in pilocarpine-induced status epilepticus), does not significantly modify cerebellar CAT activity (63, 80). Whereas EndTr caused considerable elevations in some elements of cerebellar antioxidant barrier (CAT activity and glutathione levels), these changes did not prevent the EndTr-related increase in cerebellar TBARS content. This may be related either to the insufficient activity of CAT, or to the concomitant increase in NOS activity in this region, or both.

In summary, the moderate intensity EndTr program employed was found to significantly enhance a number of components of antioxidant barrier in the adolescent rat brain, mostly in the neocortex and - to a lesser extent - in the cerebellum and striatum, whereas no change in this respect was found in the brain regions that are not directly involved in the initiation or execution and control of locomotion. However, as evidenced by marked elevations in striatal and cerebellar TBARS, the observed changes were insufficient to fully prevent the training-related increases in oxidative damage.

Acknowledgments: This work was supported by the grant No. 2011/01/B/NZ5/01397 from the National Science Centre of Poland and by the statutory funds from the Jerzy Kukuczka Academy of Physical Education in Katowice.

Conflict of interests: None declared.

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