ORIGINAL ARTICLE

Phototherapy and resistance training prevent sarcopeniain ovariectomized rats

Adalberto Vieira Corazza & Fernanda Rossi Paolillo &

Francisco Carlos Groppo & Vanderlei Salvador Bagnato &

Paulo Henrique Ferreira Caria

Received: 7 August 2012 /Accepted: 10 December 2012# Springer-Verlag London 2013

Abstract The aim of this study was to histologically andbiochemically analyze the effects of light-emitting diodetherapy (LEDT) associated with resistance training to pre-vent sarcopenia in ovariectomized rats. Forty female Wistarrats (12 months old, 295–330 g) were bilaterally ovariecto-mized and divided into four groups (n=10 per group):control–sedentary (C), resistance training (T), LEDT–sed-entary (L), and LEDT plus resistance training (LT). Trainedrats performed a 12-week water-jumping program (3 daysper week) carrying a load equivalent to 50–80 % of theirbody mass strapped to their back. Depending on the groupprotocol, the LED device (850 nm, 100 mW, 120 J/cm2, spotsize 0.5 cm2) was used either as the only method or after theresistance training had been performed. The device wasused in the single point contact mode (for 10 min). Theirradiated region was the center of the greater trochanter ofthe right femur and the middle third of the rectus femoris

muscle was subsequently analyzed histomorphometrically.Significant increases (p<0.05) were noted for the musclevolume of the T (68.1±19.7 %), the L (74.1±5.1 %), and theLT (68.2±11.5 %) groups compared to the C group (60.4±5.5 %). There were also significant increases in the concen-trations of IGF-1, IL-1, and TNF-α in the muscles of thetreated groups (p<0.05). Animals in the LT group showed asignificant increase in IL-6 compared to T, L, and C groups(p<0.05). These findings suggest that resistance trainingand LEDT can prevent sarcopenia in ovariectomized rats.

Keywords LEDT .Ovariectomy .Phototherapy .Resistancetraining . Sarcopenia

Introduction

For women, menopause causes metabolic changes, whichare related to a reduction of the estrogen production. Estro-gen decreases the inflammatory response and acceleratesmuscle healing, through a proliferation and activation ofthe muscle fiber satellite cells [1] and expression of theinsulin-like growth factor 1 (IGF-1) gene [2].

IGF-1 plays a vital role in regulating somatic growth andcellular proliferation, thereby contributing to human longev-ity. In aging, somatopause is associated with reduced activ-ity of the hypothalamic–pituitary (growth hormone (GH)–IGF) system that decreases GH production by ∼14 % perdecade after middle age. This hormone deficit decreases thedensity of muscle fibers and leads to sarcopenia, a conditioncharacterized by the loss of muscle mass and strength [3].

Resistance training (RT) is recognized as a means ofpreventing and treating sarcopenia in postmenopausal wom-en by inflammatory cytokines modulation [4] and IGF-1

A. V. Corazza (*) : F. C. Groppo : P. H. F. CariaDepartment of Morphology, Piracicaba Dental School (FOP),University of Campinas (UNICAMP), Av. Limeira, 901,13414-903 Piracicaba, SP, Brazile-mail: avcorazza@gmail.com

F. C. Groppoe-mail: fcgroppo@fop.unicamp.br

P. H. F. Cariae-mail: phcaria@fop.unicamp.br

F. R. Paolillo :V. S. BagnatoOptics Group from Physics Institute of São Carlos (IFSC),University of São Paulo (USP), Av. Trabalhador Sãocarlense,400–Centro,13560-970 São Carlos, SP, Brazil

F. R. Paolilloe-mail: fer.nanda.rp@hotmail.com

V. S. Bagnatoe-mail: vander@ifsc.usp.br

Lasers Med SciDOI 10.1007/s10103-012-1251-8

stimulation [5] with increases in number and activity ofsatellite cells and consequent muscle hypertrophy [6]. De-spite the benefits of RT, the high muscle tension achievedwith unusual exercise loads and/or detrimental effects onimmune function promote a disturbed skeletal muscle result-ing in muscle damage and low-grade inflammatory reaction[7]. These symptoms are accompanied by local glycogendepletion, excessive lactate production, and lymphocytesproliferation which decreases blood pH and causes celldeath (apoptosis) [8]. Phenomenon apoptosis and sarcope-nia in elderly are associated with an increased generation ofreactive oxygen species (ROS) during sustained hyperemiafollowing high-intensity exercise by biochemical and struc-tural changes in the mitochondria [9]. ROS accumulationcan promote oxidative stress by the addition of a singleelectron to the oxygen molecule and are usually generatedby an altered function of the mitochondrial respiratory chainand an insufficient functioning of the antioxidant cellulardefense mechanisms [10].

In order to regulate the deficiencies in cell activity inyoung and elderly subjects after physical training, aminoacid, myostatin inhibitors, testosterone treatment, calorierestriction [11], and non-invasive physical procedures likeultrasound therapy, electrical current modalities [12], andphototherapy have been used.[13].

Phototherapy is justified in biochemical theory by theincreased activity of the mitochondria and changes to theredox state. By the conversion of electromagnetic to bio-chemical energy with an increase in the oxygen binding,cellular respiration rate and production of adenosine triphos-phate [14], avoiding release ROS high levels [15].

Low-level laser therapy (LLLT) is a form of light mono-chromatic, coherence, and colimation with proprieties tomodulate pain and can promote healing in different biolog-ical systems [16]. Phototherapy showed excellent therapeu-tic response in muscle injury, especially in a metabolicallyweakened environment [17]. The inflammatory phase ofmuscle microlesion repair can be regulated by LLLT witha reduction of inflammatory cells and their mediators likecytokines as tumor necrosis factor α (TNF-α) [18] as wellas photobiomodulation of oxygen-free radicals by modulateof antioxidants [15, 17, 19]. Consequently in the prolifera-tive phase, LLLT stimulates growth factors such as IGF-1[19], growth of blood vessels (angiogenesis) by signalingvascular endothelial growth factor and stimulated satellitecells of muscle fibers to prevent muscle atrophy [20].

An alternative to LLLT is the light-emitting diode therapy(LEDT), which yields similar results while offering an excel-lent cost–benefit ratio and short time of application [21] byusing probes with large numbers of LEDs [22]. Device-basedLED that is not a monochromatic and coherent source never-theless shows a narrower emission band compared to conven-tional lamps. Concerning LLLT, LED energy density is

distributed in a broader spectrum, possibly interacting with ahigher number of specific photoreceptors and wide absorptionwindow in biological tissues [23]. LEDT has been investigat-ed for use in the repair process of tissues [24] and optimize thephysical performance of athletes [13] and elderly people [22].

Depending on the fluence delivered on the surface of thetissue, different responses may be observed during the healingprocess [21] and prevention of muscle atrophy by photother-apy [25]. In this way, scientific research for correct dosimetricdetermination of wavelength, fluence, and irradiance, as wellas each clinical condition and specific cellular modulation isof great relevance to promote the photobiomodulation inmuscle tissue.

Given the lack of information regarding the action ofLEDT in association with resistance training, and their effectson aging muscles, the aim of this study was to assess theeffects of LEDT associated with resistance training protocolto prevent sarcopenia in ovariectomized rats. The hypothesesof this study is that LEDT plus RT can improve skeletalmuscle metabolism after physical exercise for a long period,as evaluated by muscle volume fraction, muscle IGF-1, andinflammatory mediators (IL-1, IL-6, and TNF-α).

Materials and methods

Animals

Forty female Wistar rats (12 months old, 295–330 g) werehoused in plastic cages (five rats per cage) in a temperature-controlled room (22±2 °C), with lights switched on from 6AM to 6PM. The experiments were approved by the EthicsCommittee for Animal Experimentation (CEUA/UNI-CAMP, protocol number 2115–1), and were performed inaccordance with the ethical guidelines of the Brazilian So-ciety of Laboratory Animal Science.

Ovariectomy

Bilateral ovariectomy was performed via translumbar inci-sions under ketamine/xylazine anesthesia (47.5 and 12 mg/kg, respectively, i.m.). The uterine tubes were ligated (catgut4.0) and the ovaries were removed. Intact control groups weresham operated. The efficiency of the surgical procedure wasverified by postmortem dissection to confirm the uterine tubeatrophy. The rats were randomly divided into four groups (n=10 per group): control–sedentary (C), resistance training (T),LEDT–sedentary (L), and LEDT plus resistance training (LT).

Training protocol

For high-intensity resistance training, the rats underwentweightlifting sessions every 48 h over a 12-week period in

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a container (75 cm high, 30 cm diameter) with water at 30±2 °C. For training, a small backpack filled with lead ballscorresponding to 50–80 % of the body mass was strapped tothe back of each animal. Prior to the exercise sessions, therats underwent an adaptation period (in training groups)during which they were subjected to an increasing numberof exercise sets (2–4) and repetitions (5–10; protocol adap-ted from Cunha et al. [26]) in the water every other day for1 week (a total of 3 days). The animals were allowed to restfor 30 s between each set of exercises.

The body mass of the animals was measured pre-operatively (12 days before the RT), after the adaptationperiod (1 week) and finally on the last day of each of the12 weeks of the resistance training. The modified protocolof Hornberger and Farrar [27] was used with load weightsthat were appropriate for the training cycle. The load weightwas proportional to each rat’s body mass (Fig. 1) andincreased from 50 % of the body mass (weeks1 and 2) to60 % (weeks3–6), 70 % (weeks 7–10), and 80 % (weeks11and 12). The water column height started at 30 cm (week1)and was increased to 35 cm (week2) and 40 cm (week3onwards). The rats were killed 24 h after the end of theprotocol and the rectus femoris muscle was removed forbiochemical and histological analyses.

LED therapy

The prototype LED device used was developed in the PhysicsInstitute of the University of São Paulo, São Carlos, SP, Brazil.The device was operated at 100 mW using a continuouswavelength of 850 nm with a 200 mW/cm2 irradiation overan illuminated area of 0.5 cm2. Phototherapy was startedpostoperatively on day12. The fluence of 120 J/cm2 [22, 25]was performed punctually, through a single point contactmode in the center of the greater trochanter of the right femurfor 10min every 48 h over a 12-week period. The center of thegreater trochanter was located by palpation and was relabeledevery week. Infrared LED shows a nonlocal energy absorp-tion during this procedure by photography measurement

(Fig. 2) using an infra-red digital camera (Seco EletronicsInc, CCD B/W Ontário, Canada). For the LT group, photo-therapy was applied immediately after the training.

Parameters evaluated

Histomorphometry

The middle third of the rectus femoris muscle was removedand placed in 10 % formaldehyde solution for 24 h. Thetissue samples were embedded in paraffin blocks and cutinto 5 μm sections, stained with hematoxylin and eosin andexamined by light microscopy (Axioskop 2 Plus, Zeiss,Jena, Germany) coupled to a digital camera (DFC280,Leica, Germany). The images (×20 magnification) wereprocessed using a IM50 software (Leica). The muscle vol-ume fractions were determined according to the stereologi-cal principles of Weibel et al. [28] using an image analysissystem (Image-Pro, Media Cybernetics, Silver Spring, MD,USA) and a digital grid with 667 intersections, with onecentral and two marginal fields in relation to the transverseaxis of the middle third of the muscle.

Cytokines and IGF-1

Muscle samples were collected from the right thigh(middle third of the rectus femoris muscle) 24 h afterthe final training session. The muscles were frozen andstored at −80 °C. Just before performing the analysis, thesamples were defrosted and cut into cubes (±1.52 g),with one cube from each animal being macerated inliquid nitrogen and then resuspended in 5 mL of phos-phate buffer solution. The samples were assayed togetherin triplicate in the tests described below.

TNF-α, IL-1, and IL-6

Muscle cytokine concentrations were assayed using ELISAkits (Peprotech Inc.) with the absorbance being measured at

jump

load

backpack

/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\water level

Time (weeks)

Ove

rlo

ad(%

bo

dy

mas

s)

a b

Fig. 1 Schematic presentationof training protocol. a Increasein exercise overload (expressedas a percentage of the bodymass) during resistancetraining. The training protocollasted 12 weeks and waspreceded by a period ofadaptation (a). *Water level(in centimeters) in the containerused to train the rats. b Trainingcontainer

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450 nm in a microplate reader (ELX 800, Bio-Tek Instru-ments, Winooski, Vermont, USA). The concentration rangesfor the sandwich ELISAs were 63–3,000, 16–1,000, 62–8,000 pg/mL and for TNF-α, IL-1, and IL-6, respectively.

IGF-1

The IGF-1 concentration in rectus femoris muscle was mea-sured using a quantitative, high sensitivity (range, 62–6,000 pg/mL) ELISA kit (Peprotech Inc., Rock Hill, NJ,USA) according to the manufacturer’s instructions.

Statistical analysis

The Levene and Shapiro–Wilk tests were used to analyzethe data variance and distribution, respectively. The Krus-kal–Wallis test was used to compare the muscle volumefraction, IGF-1 and cytokine, and the data of the fourgroups. BioEstat 5.0 software (Fundação Mamirauá, Bélem,PA, Brazil) was used for all statistical analyses, with thelevel of significance set at 5 %.

Results

Muscle volume fraction

The treated groups showed a significant increase (p<0.05)in the volume fraction of the rectus femoris muscle com-pared to the control group (Fig. 3) as illustrated in musclehistology (Fig. 4).

Cytokines

The muscle TNF-α concentrations for the LT, T, and L groupswere also significantly higher than for C group, although theconcentration for the LT group was significantly lower than

for Tand L groups (Fig. 5). The muscle IL-1 concentrations ofthe LT, T, and L groups were significantly higher (p<0.05)than for C group (Fig. 6). The IL-6 concentration inmuscles ofthe LT group was significantly greater than in T, L, and Cgroups (Fig. 7).

IGF-1

There was a significant increase (p<0.05) in the muscleIGF-1 concentration for the LT, T, and L groups comparedto C group (Fig. 8).

Fig. 2 LEDT illuminated muscle volume during the procedure. Thecentral point contact irradiation was 0.5 cm2 and showed light scatter-ing in the rat’s thigh after transmitted to the skin

Fig. 3 Total muscle volume fraction for the four groups of rats studied.C Control–sedentary, L LEDT sedentary, LT LEDT and resistancetraining, T resistance training. Columns with different letters (a–c)show significant differences (p<0.05)

Fig. 4 Photomicrograph (HE) of rectus femoris muscle after 14 weeksof rats ovariectomy. LT LED therapy plus resistance training groupapplied in rats showing increase diameter of muscle fibers transversesection. T resistance training performed in rats showing increase mus-cle fibers transverse section. L LED therapy irradiated in rats showingincrease diameter of muscle fibers transverse section. C control–sed-entary group with absence treatment in rat’s presenting decrease andirregular fiber sizes

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Discussion

The association of resistance training to LEDT showed toprevent muscle fiber atrophy of ovariectomized rats, but didnot supplemented muscle volume compared to T group.However, the LT group demonstrated low TNF-α concen-tration in high-intensity exercise protocol. Scientific studiesindicate that high intensity training stimulates TNF-α andleads to an activation of caspase-3 and caspase-8 (cysteine–aspartic acid proteases) which induces muscle apoptosis andsarcopenia [29]. Thus, photobiomodulation inflammatorymediator TNF-α can collaborate in preventive action ofsarcopenia for RT long periods.

RTovertraining in hormonal deficient subjects can promotebiochemical alterations of mitochondrial structures with anincreased unbalance in ROS production and depletion of thebody’s antioxidant reserve, leading to increased fragility ofmuscle that accompanies mechanical injury and subsequentinflammation. Therefore, these factors can contribute to oxi-dative stress stem and reduced regenerative potential of mus-cle fibers due to a reduction of satellite cells [8].

When we used protocol training of a long period of RT onrats with an absent of estrogen associated with advanced age

(15 months old in protocol completed) was with objective ofcollaborate in metabolic deficits. In this regard, LEDT wasapplied immediately after RT demonstrating the reduction ofinflammatory mediator TNF-α in longitudinal trainingovariectomized rats. Similarly, phototherapy applied during[22] and after exercise [30] showed optimized physicalperformance in longitudinal training program. On the otherhand, other studies showed results in muscle performancewith phototherapy applied before exercise to reducingmarkers of fatigue, oxidative stress, and muscle damagebut only in athletes and transversal exercise protocols [13,25]. In this sense, the photobiomodulation can improveinflammatory modulation after RT because RT overload inthe elderly accentuates muscle fragility [8] and phototherapyshowed better biomodulation when applied in metabolicdeficit [15, 17, 19].

The decrease in the TNF-α concentration in the LT groupcompared to L and T groups was possibly influenced by theincrease in the IL-6. Exercise stimulates an IL-6 secretion bythe muscles and this mediator attenuates the production ofboth IL-1β and TNF-α while also increasing the levels of theanti-inflammatory cytokines such as IL-10 [4]. Despite theconflicting behavior of IL-6 as both an anti-inflammatory and

Fig. 5 Muscle TNF-α concentrations for rats after 12 weeks of resis-tance training. C control–sedentary, L LEDT–sedentary, LT LEDT andresistance training, T resistance training. Columns with different letters(a–c) show significant differences (p<0.05)

Fig. 6 Muscle IL-1 concentrations for rats after 12 weeks of resistancetraining. C control–sedentary, L LEDT–sedentary, LT LEDT and resis-tance training, T resistance training. Columns with different letters (a–c) show significant differences (p<0.05)

Fig. 7 Muscle IL-6 concentrations for rats after 12 weeks of resistancetraining. C control–sedentary, L LEDT–sedentary, LT LEDT and resis-tance training, T resistance training. Columns with different letters (a–c) show significant differences (p<0.05)

Fig. 8 Muscle IGF-1 concentrations for rats after 12 weeks of resis-tance training. C control–sedentary, L LEDT–sedentary, LT LEDT andresistance training, T resistance training. Columns with different letters(a–c) show significant differences (p<0.05)

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a pro-inflammatory marker [31], we suggest that the LEDTincreased in the muscle IL-6 formation facilitated and en-hanced the physical recovery after RT, as reported in anotherstudy regarding IL-6 and post-training recovery [4]. The pos-itive result observed in the LT group was the IL-6 signaling toIGF-1 synthesis [3], which can be stimulated to avoid atrophymuscle volume in middle-age rats ovariectomized.

When irradiance of 120 J/cm2 was used in the rats therewas photobiomodulator effects on TNF-α and IL-6 concen-tration post-training, suggesting the prevention in the lossmuscle due to an increase of IGF-1 concentration. Similarly,Sussai et al. [25] when using LLLT (1, 660 nm) with fluenceof 133.3 J/cm2 before high intensity exercise in the ratsgastrocnemius muscle prevented apoptosis of muscle cells.On the other hand, Wu et al. [32] irradiated with a He–Nelaser for 10 min under a fluence of 120 J/cm2 human lungadenocarcinoma cells (ASTC-a-1) and in an African greenmonkey SV40-transformed kidney fibroblast cells (COS-7)showed an increase of ROS and apoptose cellular evolution.In this context, when fluence is applied in cell culture,experimental, and clinical studies show variations in theabsorption of energy, which answers phototherapeutics dis-tinct need for adjustments in the therapeutic window asphototherapeutic goals differ in each type of body tissue.

LEDT (1, 850 nm) with fluence of 120 J/cm2 applied inmuscle tissue was regarded as high as related by Huangh etal. [33]. However, the light interacted with several interfacesof skin, adipose, muscle, and bone volume around of irra-diance central point, allowing higher number of specificphotoreceptors and wide absorption window in tissues[24]. Tunner and Hode [34] related that the usage of highfluences in the skin promotes light attenuation throughoutthe deep tissue volume, resulting in a dose within the ther-apeutic window. Other author comment that initial fluenceof 100–300 J/cm2 on surface tissues is attenuated to 20 J/cm2 to the depth of 5–10 cm [35]. Nakano et al. [20] suggestthat LLLT irradiation (1, 830 nm) with power output of60 mW can promote recovery of muscle atrophy, fromdisuse, by proliferation of satellite cells and angiogenesis,but the power was reduced to 20 and 5 mW by in rat skinpenetration and rat skin plus gastrocnemius muscle penetra-tion, respectively. Despite difficulties involved in complyingenergy dose simulation to be used in the present study, thefluence was distributed to the rat’s thigh muscle, whichsuggests that light attenuation in the tissue layer promotesfluence within range of the therapeutic window. Therefore,it demonstrated a preventive action in the rectus femorismuscle volume loss in 14 weeks after rat’s ovariectomy.

The higher IGF-1 concentration for the L group comparedto C group indicated the prevention of sarcopenia, possiblythrough the stimulation of pathways in the hormonal IGF-1/GH axis, same without influencing estrogen in the productionof IGF-1 [1]. As reported by other authors, phototherapy can

help prevent muscle atrophy by angiogenesis stimulation [36],increase IGF-I [37] with signalizing to protein synthesis sim-ilar to intracellular concentrations of cytoprotective heat shockproteins (HSP-70i), and consequent satellite cell proliferation[38]. Different to our results, in an in vivo study, LLLT (at830 nm) did not stimulate IGF-1 production in the gastrocne-mius muscle of rats irradiated with a fluence of 0.6 J/cm2 (perpoint) on a total of 60 points (3.6 J total energy) [20]. Thisfinding suggests that LEDT with an energy density of 120 J/cm2 applied at a single point, as used in our study, was highlyeffective in stimulating the IGF-1 production and to maintainmuscle volume.

Beside the fluence, the wavelengths and light type influen-ces in the energy distribution. Our study used infrared LED (1,830 nm) due to scatter of light with elliptic shape [34], tocompensate the scattering of LED light after skin transmitted(Fig. 2), due low coherence in relation to laser low level [13],thus ensure more light penetration efficiency [15].

In the T group, the exercise frequency (48 h intervals) mayhave contributed to the benefits observed in the anti-atrophicaction. An interval of 48 h shows a positive effect on theprotein synthesis during recovery from inflammation follow-ing an overload resistance training [39]. Another study, how-ever, found that resistance training for five times per week ledto atrophy in rat plantaris muscles [40]. A functional overloadof physical exercise could decrease the protein synthesis [41],with increased rates of catabolism/anabolism and a reductionin muscle volume. Furthermore, an RTwith adequate intervalsbetween exercises can increase mitochondrial content anddecrease oxidative stress in older adults [42].

Physical exercise promoting an increase in cytokines maybe pro-inflammatory (IL-1β, TNF-α, and IL-6), anti-inflammatory (IL-6, IL-10, IL-4, IL-5, IL-13, and IL-1rα),or may contribute to a biomodulation of the inflammation[31]. In this study, it was observed that despite an increasingIL-1 and TNF-α for the T and L groups there was no impair-ment in the IGF-1 synthesis when compared to the sedentarygroup. These results are in agreement with previous work inwhich the optimization of the IGF-1 production during a high-intensity resistance training was associated with an enhancedsignaling of the inflammatory cytokines [43].

Another response to the positive effects of the sarcopeniaprevention by means of RT arises from the increase in IGF-1, and consequently this may be added to the effect ofestrogen deficiency developed by the ovariectomy done onthe rats. Enhanced IGF-1 production in response to resis-tance exercises [5] inhibits protein degradation in myotubesby stimulating the phosphatidylinositol-3 kinase/protein ki-nase B pathway [44] and improves muscle hypertrophy [7].It is therefore possible that the RT for 12 weeks providedsufficient anabolic signaling to avoid sarcopenia for the ratsin this study. Suetta et al. [45] showed that RT in elderlypostoperative patients helps regulate muscle hypertrophy by

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IGF-1 regarding electrical stimulation and functional train-ing. When transposing this experiment to humans, we mayexpect to find that the gradual increase of the load during thetraining promotes the prevention of sarcopenia and developsmuscle hypertrophy as has been reported by other authors[5]. Clinical studies are needed to establish the mechanismsby which LEDT may prevent sarcopenia in elderly womensubjected to resistance training.

Conclusion

Our results indicate that both the RT and the LEDT canenhance the anabolic activity by stimulating the IGF-1 pro-duction, thereby increasing the muscle volume of middle-aged ovariectomized rats. Despite the increased muscle vol-ume being similar for all treated groups we can report thatthe phototherapy may also regulate the TNF-α and IL-6concentrations and can optimize the metabolic recovery ofthe rats after high-intensity resistance training.

Acknowledgments The authors would like to thank Felippe Furlan,Júlio César Domingues, Luis Vaz de Lima Júnior, and Mariana SabinoBortolozzo for valuable technical assistance. We would also like tothank the Conselho Nacional de Desenvolvimento Científico e Tecno-lógico (CNPq) for their financial support.

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