THE EFFECTS OF STRENGTH, AEROBIC, AND CONCURRENTEXERCISE ON SKELETAL MUSCLE DAMAGE IN RATSANDERSON RECH, MS,1 REGIS RADAELLI, MS,1 ADRIANO M. DE ASSIS, PhD,2 JO~AO R. FERNANDES, MS,1

ALINE LONGONI, PhD,2 MAGDOLNA M. VOZARI-HAMPE, PhD,2 RONEI S. PINTO, PhD,1 and CRISTIANE MATT�E, PhD2

1 Exercise Research Laboratory, School of Physical Education, Federal University of Rio Grande do Sul, St. Felizardo,750, LAPEX Building, Porto Alegre, RS, 90690-200, Brazil

2 Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

Accepted 4 October 2013

ABSTRACT: Introduction: In this study we examined oxidativestress and skeletal muscle damage resulting from acutestrength, aerobic, or concurrent exercise in rats. Methods: Theanimals were divided into control (C), strength (SE), aerobic(AE), and combined (CE) exercise groups. They were eutha-nized at 3 different time-points (6, 24, and 48 h) after acuteexercise. Results: SE exercise rats had increased dichlorofluor-escein oxidation at 6 h post-exercise and decreased superoxidedismutase activity at all time-points. Glutathione peroxidaseactivity and sulfhydryl levels were increased in the AE group at48 h post-exercise. Serum lactate dehydrogenase activity wasincreased in the SE and CE groups at 24 h and in the AEgroup at 48 h. Echo intensity was elevated at 24 h for allgroups. Conclusions: Forty-eight hours was sufficient for com-plete recovery from oxidative stress and muscle damage in theSE and CE groups, but not in the AE group.

Muscle Nerve 000:000–000, 2014

The combination of strength and aerobic stimuliduring the same training session, known as concur-rent training, has been demonstrated to be anexcellent option for athletic training aimed at per-formance, as well as for people who wish toenhance their quality of life.1,2 The main long-term benefits of concurrent training are gains inmuscle strength and power, enhancements in max-imal oxygen uptake (VO2max), increases in leanbody mass, improved body composition, and areduction of metabolic risk factors, such as hyper-tension and high serum glucose.2,3 Despite thelarge number of studies detailing the effects ofconcurrent training, the time course of skeletalmuscle damage after a concurrent training sessionhas been less well explored.

Muscle damage is associated with disorganiza-tion of sarcomeres, Z-line dissolution, A-band dis-ruption, and t-tubule alteration, leading toincreased blood levels of muscle enzymes [lactatedehydrogenase (LDH) and creatine kinase] andecho intensity, as measured by ultrasound.3,6 Fur-thermore, muscular performance is impaired bymuscle damage, resulting in reductions in maximalstrength. Performing subsequent training sessionswithout optimal muscle recovery may impairchronic neuromuscular adaptations, because thesubject may not be able to complete the estimatedtotal volume in the subsequent training session.

Oxidative stress may play a major role in theprocess of muscle damage due to an increase information of free radicals during exercise.7 Thesefree radicals may promote damage to muscle struc-tures and lead to protein, lipid, or DNA damagewithin muscle cells.8 There are few studies thathave evaluated the relationship between oxidativestress and muscle damage after exercise. Nikolaidiset al.9 reported that muscle-damaging exercise mayincrease free radical levels and oxidize lipids, pro-teins, glutathione, and possibly DNA. According toBailey et al.,10 oxidative stress is present in muscleafter muscle damaging exercise. Thus, it is impor-tant to enhance our knowledge about the relation-ship between oxidative stress and muscle damageafter different types of exercise, as well as therecovery time that allows for muscle repair.

The implementation of an animal model thatsimulates concurrent training is important forenhancing our knowledge of this type of training.Identifying the appropriate time interval for mus-cular recovery will permit application of long-termconcurrent training in rats. Furthermore, this typeof model will benefit future studies involving theeffect of concurrent training under different con-ditions in animals, such as diabetic, obese, or olderrats.

Based on the lack of studies on the time courseof recovery and the muscle damage induced by anacute concurrent training session, the purpose of thisstudy was to compare indirect markers of muscledamage and oxidative stress in skeletal muscle afteran acute session of strength, aerobic, or concurrenttraining in adult Wistar rats. We hypothesized that

Abbreviations: AE, aerobic exercise group; ANOVA, analysis of variance;C, control group; CAT, catalase; CE, combined strength and aerobic exer-cise group; DCF, dichlorofluorescein; EI, echo intensity; GSH, glutathione;GSH-Px, glutathione peroxidase; LDH, lactate dehydrogenase; NADPH,nicotinamide adenine dinucleotide phosphate; RNS, reactive nitrogen spe-cies; ROS, reactive oxygen species; SE, strength exercise group; SH, sulf-hydryl; SOD, superoxide dismutase; VO2max, maximal oxygen uptakeKey words: concurrent exercise; exercise recovery; gastrocnemius; mus-cle damage; oxidative stressThis project was supported by the Brazilian agencies CAPES, CNPq (Con-selho Nacional de Desenvolvimento Cient�ıfico e Tecnol�ogico), PROPESQ/UFRGS (Pr�o-reitoria de Pesquisa), and FAPERGS (Fundac~ao de Apoio aPesquisa do Estado do Rio Grande do Sul)

Correspondence to: A. Rech; e-mail: ufrgs.anderson@gmail.com

VC 2013 Wiley Periodicals, Inc.Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24091

Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014 1

48 h would be sufficient time for complete recoveryfrom exercise-induced muscle damage.

METHODS

Ethics Statement. The NIH’s “Guide for the Careand Use of Laboratory Animals” (NIH PublicationNo. 80-23, revised 1996) was followed in all experi-ments. All procedures and experiments wereapproved by the local ethics committee for animaluse (CEUA, No. 21206).

Animals and Reagents. Considering an a 5 0.05and a power estimated at 0.80, we utilizedG*Power (version 3.1.1) software to designate thenumber of animals necessary for study. The esti-mate was based on the mean values of antioxidantenzymes (superoxide dismutase, catalase, and glu-tathione peroxidase) found in previous studies,21

and it was determined that 8 animals per groupwere necessary for an analysis of variance(ANOVA). The experiments were performed on80 male Wistar adult rats (70–90 days old, 315.3 6

32.3 g body weight) from the central animal houseof the Department of Biochemistry. The animalswere maintained under a 12-h/12-h dark/lightcycle with temperature ranging between 22� and24�C and housed in a propylene box (4 rats perbox). The rats had free access to a 20% (w/w) pro-tein commercial chow (Rodent Show; Nutrilab,Colombo) and water. All chemical reagents wereobtained from Sigma Chemical Co. (St. Louis,Missouri).

Exercise Protocols. The rats were divided into4 groups: control (C, n 5 8); strength exercise(SE, n 5 24); aerobic exercise (AE, n 5 24); andcombined strength and aerobic exercise (CE, n 5

24). Each exercise treatment group contained24 animals divided into 3 different time-points foreuthanasia (6 h, n 5 8; 24 h, n 5 8; 48 h, n 5 8).The animals in group C were only manipulated tosimulate stressor conditions similar to the othergroups. The rats were placed in a cage that was dif-ferent from the one they were maintained inthroughout the day to mimic manipulation and achange of environment for the same total time asthe exercised rats.

The rats in the SE group performed a protocoladapted from previous studies.11 During the exer-cise protocol, the animals were not exposed to anytraumatic stimulation (such as electric shock orheat). Briefly, the strength training apparatus wascomposed of wood and was lined with rubber. Theramp was 1 m in length and inclined at 30� (Fig.1). The borders of the ramp were opaque andpainted white so that the rats could not maintaineye contact with other exercising rats. The animalswere stimulated to climb the ramp toward the top

where there was a dark box where they rested untilthe interval period was completed. Because theseanimals felt safe in the ambient dark after the3 days of adaptation, they did not require anyform of stimulus to climb the ramp. The rats wereadapted to the ramp with a progressive number ofclimbs on 3 different days before the exercise pro-tocol, which were always at the same hour of theday (between 9:00 a.m. and 12:00 p.m.). On thefirst day, the rats were familiarized with the exer-cise protocol and climbed the ramp 4 or 5 timeswithout carrying any weight. On the second day ofthe adaptation process, the animals then climbedthe ramp 4 or 5 times, carrying 30% of their bodyweight. During the last day of adaptation, the ani-mals had to climb the ramp 10–12 times, carryingthe equivalent of 30% of their body weight. Atleast 24 h after the last familiarization session, onthe acute exercise day, the rats performed 10–12sets of ramp-climbing movements, carrying a loadequivalent to 85–95% of their corporal weight with2 min of rest between each climb. The load wasdragged over a plastic board that was attached to asteel cable connected to a plastic belt surroundingthe rat’s body.

The AE group performed the protocol appliedby Cechetti et al.,12 which was performed in anadapted motorized rodent treadmill (Imbramed;Porto Alegre, Brazil). No painful stimulation wasused. The rats went through the same 3 days ofadaptation as the SE group with daily increases inthe intensity and volume of exercise until thefourth day when they executed the acute aerobictraining session. Some animals were unable to per-form the treadmill exercise; they were removed onthe first day of adaptation and did not participatein the study. The acute session lasted for 30 minand began with an initial warm up of 5 min, 20 min

FIGURE 1. Strength training apparatus.

2 Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014

of exercise at 60% VO2max, followed by 5 min ofcool-down. The velocity target of 60% VO2max wasbased on previous studies from our laboratory,which measured the VO2max using the same tread-mill equipment.12 The animals that refused to runwere gently encouraged with small taps on theback. In the concurrent training protocol, weaimed to analyze the recovery phase from a differ-ent exercise stimulus (neuromuscular andcardiovascular).

It was stipulated that the CE group wouldaccomplish half of each protocol (aerobic andstrength training), avoiding excessive muscle dam-age due to a higher volume of exercise. Thus, theconcurrent training group performed 5 or 6 setsof ramp climbing, carrying a load equivalent to85–95% of body weight, followed by 15 min ofexercise on the treadmill at 60% VO2max. The ses-sion total time was 30 min for each exercise proto-col with a variation of 62 min for each rat.Therefore, the total exercise time was not differentbetween the training groups.

Tissue Preparation. After the particular acute exer-cise session for each rat and after the specificrecovery time (6, 24, or 48 h), the animals hadultrasound images taken from the posterior part ofthe right leg by the same evaluator who had takenthe control image before the exercise session. Theimages were saved for further analysis. Subse-quently, the animals were euthanized by decapita-tion, and their blood was collected (5 ml) forcentrifugation and further analysis of serum LDH.The rats did not receive any type of anesthesia,because it can interfere with the biochemical tech-niques used in this study.13 Immediately after theblood collection, the gastrocnemius muscle was dis-sected, weighed, and homogenized in 10 volumes(1:10, w/v) of 20 mM sodium phosphate and 140mM potassium chloride buffer. The homogenatewas centrifuged at 1500 g (4�C) for 10 min. Themuscle supernatant was then stored at 280�C forfurther biochemical analysis of antioxidant enzymeactivities, dichlorofluorescein (DCF) oxidation,and sulfhydryl group content. Each analysis wasperformed in duplicate to minimize methodologi-cal issues.

Dichlorofluorescein Oxidation. Reactive oxygen(ROS) and nitrogen species (RNS) productionwas assessed using the DCF oxidation method.14

Briefly, 60 ll of muscle supernatant was incubatedat 37�C in the dark for 30 min with the additionof 240 ll of 20,70-dichlorofluorescein diacetate(H2DCFDA; Sigma-Aldrich, St. Louis, Missouri) ina 96-well plate. H2DCFDA is cleaved by cellularesterases and forms H2DCF, which is oxidized bythe reactive species present in the samples. This

reaction produces a fluorescent compound thatcan be measured fluorimetrically using a 488-nmexcitation and 525-nm emission wavelength. Astandard curve using standard DCF (0, 25–10 mM)is pipetted in parallel with the samples. Theresults were calculated as nanomoles DCF permilligram protein and are presented as a percentof control.

Sulfhydryl Group Content Measurement. The sulfhy-dryl (SH) group content of reduced groups wasverified spectrophotometically through the reactionbetween the free SH groups from the samples and5,50-dithiobis-(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich) in the dark for 30 min. This reactionforms thionitrobenzoate (TNB), a compound thatabsorbs light at 412 nm.15 The results are expressedas nanomoles TNB per milligram protein.

Catalase Assay. Catalase (CAT) activity was meas-ured as described previously.16 Muscle homoge-nates (40 ll) from all groups were taken with 150ll of phosphate buffer in different wells (125 mM,pH 7.4). The reaction was initiated by adding 10ll of H2O2 (0.5 mM). A blank was prepared with190 ll of phosphate buffer and 10 ll of H2O2 (0.5mM). The rate of decrease in optical density dueto degradation of H2O2 (Sigma-Aldrich) was meas-ured at the end of 1 min against the blank at 240nm. One CAT unit was defined as the amount ofenzyme that decomposed 1 M H2O2 per minute(37�C). The specific activity is expressed in unitsper milligram of protein.

Superoxide Dismutase Assay. Superoxide dismu-tase (SOD) activity was evaluated by quantifyingthe inhibition of superoxide-dependent autoxida-tion of epinephrine, verifying the absorbance ofsamples at 480 nm.17 In a 96-well plate, we addedtissue homogenates (30 ll), 150 ll of 50 mM gly-cine buffer (pH 10.2), and 10 ll of 10 lM catalase(Sigma-Aldrich). In the auto-oxidation wells, 180ll of 50 mM glycine buffer (pH 10.2) and 10 ll of10 lM catalase were added. The reaction was initi-ated by adding 10 ll of 60 mM epinephrine. Theabsorbance was measured at 480 nm and recordedfor 10 min at 32�C. One unit of SOD activity wasdefined as the amount of enzyme required todecrease the oxidation of epinephrine by superox-ide by 50%. The specific activity is expressed inunits per milligram of protein.

Glutathione Peroxidase Assay. Glutathione peroxi-dase (GSH-Px) activity was measured according tothe method described by Wendel18 using tert-butylhydroperoxide as a substrate. The disappearanceof nicotinamide adenine dinucleotide phosphate(NADPH; Sigma-Aldrich) was monitored spectro-photometrically at 340 nm in a medium containing

Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014 3

2 mM glutathione (Sigma-Aldrich), 0.15 U/ml glu-tathione reductase, 0.4 mM azide, 0.5 mM tert-butyl hydroperoxide, and 0.1 mM NADPH. Oneunit of GSH-Px was defined as 1 lmol of NADPHconsumed per minute, and the specific activity isrepresented as units per milligram of protein.

Serum Lactate Dehydrogenase Activity. Afterexercise-induced skeletal muscle damage, the con-centration of muscle enzymes, such as LDH, isenhanced in the bloodstream. The activity ofserum LDH was determined spectrophotometri-cally using a commercial kit (Labtest DiagnosticsSA, Lagoa Santa, Brazil). LDH activity is expressedin units per liter.

Echo Intensity. The echo intensity (EI) of gastro-cnemius muscle was determined according to aprevious study.19 After anesthetic induction by iso-flurane, B-mode ultrasound images were obtained(Ultravision Flip Plus; Amsterdam, Philips) using a7.5-MHZ linear probe (38 mm) placed perpendicu-larly at the middle point of the gastrocnemius mus-cle. For image acquisition, we used a water-basedgel to promote acoustic contact without causingexcessive probe pressure on the skin. In eachimage, the gastrocnemius muscle was identified,

and a 1-cm2 region of interest was determined andused to quantify the EI based on a gray-scale histo-gram (0: black, 255: white) using a standard func-tion of ImageJ software, version 1.37 (NationalInstitutes of Health, Silver Spring, Maryland). Themean EI value was calculated using values from 3images. The same investigator obtained all imagesand analyzed the EI.

Statistical Analyses. All data are expressed as themean 6 SD. Data normality was tested using theShapiro–Wilk test. After the data presented a nor-mal distribution (P > 0.05), a 1-way analysis of var-iance (ANOVA) test was applied to comparedifferent exercises at the same recovery time. Whenthe ANOVA indicated a main effect for group, Bon-ferroni post hoc tests were used to detect differencesbetween exercise types. The significance level wasset as a < 0.05. All statistical analyses were per-formed using GraphPad Prism 5.0 (GraphPad, Inc.,San Diego, California) for Windows.

RESULTS

Strength Exercises Alter Reactive Species Production

and Sulfhydryl Groups in Gastrocnemius. The ani-mals subjected to strength exercise had a signifi-cant increase in reactive species production,indicated by increased DCF oxidation (Fig. 2A) at6 h post-exercise when compared with groups C (P< 0.01) and CE (P < 0.01). DCF oxidation wasnormalized at 24 h and decreased at 48 h post-exercise. These results reached statistical signifi-cance when compared with all other groups (C: P< 0.05; AE: P < 0.05; CE: P < 0.001). AE and CEdid not alter DCF oxidation in the muscle at thetime-points evaluated. We also measured total SHgroup content, which could include tissue glutathi-one (GSH) levels or SH groups provided by pro-teins, reflecting protein damage. AE (P < 0.01)and CE (P < 0.001) increased total SH group con-tent 24 h after acute exercise when compared withthe SE group (Fig. 2B). The increased values wereonly maintained in the AE group 48 h post-exercise (P < 0.001).

Antioxidant Enzyme Activities Were Modulated by

Exercise. Figure 2 shows the effect of differenttypes of exercise on SOD, CAT, and GPx activities.SOD activity was decreased significantly in the SEgroup 6 h (P < 0.01), 24 h (P < 0.01), and 48 h(P < 0.05) post-exercise compared with controls(Fig. 3A). The SE group also displayed a signifi-cant decrease in SOD activity at 6 h compared withthe AE group (P < 0.05) and at 48 h (P < 0.05)compared with the CE group (Fig. 3A).

CAT activity was increased in the SE group 6 hpost-exercise when compared with the AE (P <0.05) and CE (P < 0.01) groups, but was not

FIGURE 2. Oxidative stress analysis (*P < 0.05; **P < 0.01;***P < 0.001). To facilitate understanding of the results, group C

is always included, although these are the same animals. (A)

DCF oxidation. Significant differences exist between groups SE

and C (P < 0.01) and group CE (P < 0.01), between groups

SE and CE (P < 0.001) at 48 h post-exercise, and between

groups SE with C and group E (P < 0.05) at the same time-

point. (B) Thiol group content. Significant differences exist

between groups SE with AE (P < 0.01) and group CE (P <

0.001) at 24 h post-exercise and between group AE and all

other groups (P < 0.001). The results are expressed as the

mean 6 SD (n 5 8 per group).

4 Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014

altered when compared with control rats (P >0.05) (Fig. 3B). Twenty-four hours after an exercisesession, CAT activity was normalized in the SEgroup. Nevertheless, CAT activity was decreased inthe SE group 48 h post-exercise (P < 0.05) whencompared with the AE group (Fig. 3B).

GPx activity was augmented in the AE group at48 h when compared with groups C (P < 0.05)and CE (P < 0.001) (Fig. 3C). In contrast, the CEgroup displayed a decrease at 48 h when comparedwith the control group (P < 0.05) (Fig. 3C).

Exercise Increases Serum Lactate Dehydrogenase

Activity and EI. The data from the LDH activityand EI measurements are shown in figure 4. Bothgroups SE (P < 0.05) and CE (P < 0.01) had sig-nificant increases in LDH activity when comparedwith group C at 24 h post-exercise (Fig. 4A). LDHactivity was increased significantly in the AE groupat 48 h post-exercise when compared with group C(P < 0.05) (Fig. 4A).

A significant increase was observed in EI in theAE (P < 0.01) and CE groups (P < 0.001) com-pared with group C at 6 h post-exercise (Fig. 4B).All groups differed significantly from group C at24 h post-exercise (P < 0.05), due to increased

FIGURE 4. Serum LDH activity and EI (*P < 0.05; **P < 0.01;***P < 0.001). To facilitate understanding of the results, group C

is always included, although these are the same animals. (A)

Serum LDH activity. Significant differences exist at 6 h post-

exercise between groups AE and CE (P < 0.05) and at 24 h

post-exercise between groups C and SE (P < 0.05) and group

AE (P < 0.01). At 48 h post-exercise, a significant difference

exists between groups C and AE (P < 0.05). (B) EI. A signifi-

cant difference exists between groups C and AE (P < 0.01)

and group CE (P < 0.001) at 6 h post-exercise and between all

groups compared with group C at 24 h post-exercise (P <

0.05). However, no difference was observed at 48 h post-

exercise. The results are expressed as the mean 6 SD (n 5 6

per group).

FIGURE 3. Antioxidant enzyme activities (*P < 0.05; **P <

0.01; ***P < 0.001). To facilitate understanding of the results,

group C is always included, although these are the same ani-

mals. (A) SOD activity. Significant differences exist between

group SE at 6 h and group C (P < 0.01) and group AE (P <

0.05), group SE at 24 h and group C (P < 0.01), groups SE

and C (P < 0.05), and group CE (P < 0.05). (B) CAT activity.

Significant differences exist between groups SE and AE (P <

0.05) and group CE (P < 0.01) at 6 h post-exercise. Moreover,

a significant difference exists between groups AE and SE at 48

h post-exercise (P < 0.05). (C) GPx activity. Significant differen-

ces exist between groups AE and C (P < 0.05) and group CE

(P < 0.001) at 48 h post-exercise and between groups CE and

C (P < 0.05) at the same time-point. The results are expressed

as the mean 6 SD (n 5 8 per group).

Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014 5

gastrocnemius EI. No significant difference (P >0.05) was observed among the groups at 48 h post-exercise (Fig. 4B).

DISCUSSION

The main finding of this study was that 48 hwas sufficient for significant recovery from muscledamage and oxidative stress in the SE and CEgroups. However, in contrast to our hypothesis, 48h was not sufficient for significant recovery in theAE group. These data indicate that skeletal muscleappears to be more responsive in terms of oxida-tive stress parameters when the animal is submittedto a strength exercise protocol. In contrast, ratssubjected to aerobic exercise (groups AE and CE)appear to suffer more muscle damage, as evaluatedby serum LDH activity and muscle EI.

The oxidative stress generated within the mus-cle cell may be responsible for several types ofdamage to the integrity and function of the neuro-muscular system. The increase in reactive speciesproduction may result in damage to the t-tubules,impairments in sarcolemma function, and myofila-ment contraction, in addition to altered mitochon-drial metabolism. These injuries to muscle cellstructure may impair muscle contraction, thecapacity for force generation, and performance ofany activity that requires force production.8

Intense physical exercise promotes an increase inproduction of ROS and RNS within muscle fibers.This augmented generation can have positiveeffects, such as cellular signaling, or negativeeffects, such as reactive species-induced apoptosisor altered cellular function.8 Measuring DCF oxi-dation is a parameter of reactive species produc-tion with no specificity for ROS or RNS.14 Wefound an increase in DCF oxidation in skeletalmuscle in the SE group 6 h after acute exercise.However, this same group had a significantdecrease 48 h post-exercise. Surprisingly, thegroups that had an aerobic component in theirexercise session, the AE and CE groups, had noalterations in their DCF oxidation values whencompared with control animals at any time-pointduring the recovery phase.

Despite augmented ROS generation duringmuscle contraction, oxidative stress may still bepresent several days after the acute exercise sessionas a result of inflammation and immunological cellinfiltration into skeletal muscle tissue.9 Some stud-ies have demonstrated that antioxidant enzymesact as a response to an acute exercise session.20,21

Uchiyama et al.21 evaluated weight lifting–inducedmuscle damage (soleus and plantaris muscles)after an acute exhaustive exercise session usingmale Wistar rats. They found enhancement inSOD, CAT, and GPx activities during the first 3–6

h post-exercise, followed by a return to basal levelsat 12 h and a new increase after 48–72 h. First, theincrease in oxidative stress may be evident becauseof the repeated ischemia–reperfusion state afterresistance exercise, whereas the second increasewas due to infiltration of phagocytic cells into theinjured tissue.21 Kayatekin et al.20 investigated amouse model of sprint exercise and the responseof antioxidant enzymes in skeletal muscle after anacute high-intensity exercise session. Although theydid not determine any influence of this exercisemodality on the SOD or GPx activities in the gas-trocnemius muscle until 24 h post-exercise, previ-ous studies have shown the importance ofevaluation periods of longer than 24 h post-exercise.7,21

The SE model utilized in our study showed adecrease in SOD activity at all time-points afterexercise and only an increase in CAT activity at 6h post-exercise. Considering that we found anincrease in reactive species at 6 h post-exercise, wesuggest that SOD inhibition could result fromincreased ROS.22 Salo et al.22 demonstrated thatthe SOD protein structure could be affected by theproduct of its reaction (H2O2), and this structuralmodification may lead to a decrease in SOD activ-ity. The results from our investigation demonstratethat, in the gastrocnemius muscle, the antioxidantenzyme activities are almost unaffected by the aero-bic protocol applied in this exercise model. Theexception is GPx, which exhibited an increase at48 h in the group subjected to treadmill training,decreasing its activity in the CE group. The pre-dominant fiber type in the gastrocnemius musclemay have influenced these results, because gastro-cnemius is composed primarily of type II non-oxidative muscle fibers.23 The SH content couldindicate protein oxidation or GSH levels.15 SHgroup oxidation may modify the functional state ofa protein, impairing its functioning.15 Silva et al.24

evaluated the effect of an acute session of 90 minof downhill treadmill running on oxidative stressin the quadriceps muscles of male rats. Asexpected, the exercise acted as a potent oxidativestress inducer, and they observed a decrease in thereduced SH, which may indicate a state of redoximbalance or protein damage.24 Surprisingly, wefound an increase in reduced SH content at 24 hpost-exercise in the AE and CE groups comparedwith the SE group. In addition, the AE group hadan increase in SH content 48 h post-exercise, sig-nificantly different from all the other groups.Recently, one study suggested that muscle mayexport reduced GSH when a stress situation suchas exercise and/or dehydration occurs and thatthis release would stimulate the synthesis of GSHwithin the muscle.25 However, that study was

6 Different Exercises Alter Muscle Damage MUSCLE & NERVE Month 2014

conducted in humans, which does not allow fordirect comparisons with our results.

Some studies have shown an increase in EI andLDH activity in serum after exercise-induced mus-cle damage.12,27 Fujikake et al.19 evaluated muscledamage induced by bupivacaine (BPVC), a chemi-cal compound that causes myonecrosis. In thatstudy, muscle EI was elevated for up to 7 days aftermuscle BPVC damage.19 In our study, EI wasincreased significantly at 6 h and 24 h post-exercise in the AE and CE groups. The SE groupdisplayed an increase during the first 6 h; however,a significant difference was observed only at 24 hpost-exercise. After 48 h, there was no significantincrease in any group, suggesting that a 2-dayperiod is sufficient for tissue repair. We also meas-ured serum LDH, which, although representingcell disruption without tissue specificity, appearsincreased in the blood after a large amount of cel-lular damage, such as what occurs in exercise. Theresults of this study indicate significant increases inserum LDH in the SE and CE groups comparedwith the control group at 24 h post-exercise. How-ever, after 48 h, the values were not significantlydifferent compared with the control group. Therewas an increase in serum LDH activity in the AEgroup at 48 h post-exercise. Our results agree withhuman studies that have also found an elevation inEI and LDH after an eccentric exercise protocol.27

Milias et al.7 demonstrated an increase in serumLDH 24 h after eccentric maximal exercise, andthe values remained elevated until 96 h post-exercise. However, that study utilized a maximaleccentric protocol, making a comparison with ourresults difficult.7 The LDH overflow and increasein EI of humans or rats may indicate necrosis ortissue damage generated by the intense exercise.29

The LDH and EI values may indicate that the aero-bic component can lead to more muscle damagecompared with the SE protocol used in our investi-gation. This result may be due to the presence ofthe eccentric component in the AE protocol com-pared with the SE, which had less intensity andduration of an eccentric stimulus due to climbingupwards.

The resistance exercise model has some limita-tions with regard to the eccentric phase of themovement. Because the rats had to climb theramp with a load attached to a steel cable con-nected to a plastic belt surrounding the animal’sbody, the eccentric component during thestrength exercise was minimal, which may have ledto a minor impact on muscle damage comparedwith the aerobic exercise protocol. Nevertheless,there are other resistance training protocols forrats that have the same limitation of the eccentricphase of the movement.29,30 We only assessed the

gastrocnemius muscle. Thus, the results may havelimited applicability to other muscles because ofthe predominance of type II muscle fibers.9 There-fore, assessment of other muscles to distinguishbetween the responses is suggested.

In conclusion, these data indicate that a 48-hperiod was sufficient for complete recovery frommuscle damage and oxidative stress in the SE andCE groups, but it was not sufficient in the AEgroup. Our results further demonstrate how aero-bic and strength training or combined training(strength and aerobic) can induce differentresponses during muscle oxidative stress and dam-age. This suggests that aerobic training damagesthe structure of muscle cells in a more significantmanner compared with strength training or com-bined exercises in male Wistar rats. Thus, based onthe protocols used in this study, aerobic trainingmay require more time for recovery between work-outs. The choice of an adequate time intervalbetween workouts should take muscle recovery as awhole into account to prevent muscle damagebeyond the capacity of tissue regeneration.

The authors are grateful to Fernanda Cechetti, PhD, for technicalsupport and laboratory assistance, and to C�ıntia Botton and EuricoNestor Willhelm for technical support.

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