metabolic and functional changes that derive from the natural aging process increase the 
 number of diseases , fractures , and falls experienced by individuals , thereby causing 
 patients to experience brief periods of inactivity , which culminate in joint immobilization 
 and may directly influence the development of age - related sarcopenia1 .
studies show that after 48 hours of joint immobilization , 
 skeletal muscle develops atrophy2 , 3 , and that six hours after immobilization , a 
 37% reduction of protein synthesis occurs3 , 4 . upon joint immobilization , type i fibers ( slow ) are more vulnerable to hypotrophy due to 
 their oxidative metabolic profile and to the anti - gravitational characteristics of postural 
 or tonic muscles5 , 6 .
prior strength training induces hypertrophy and protects 
 the structural and mechanical properties of skeletal muscle during a disuse period , through 
 the preservation of new cell nuclei that are developed in the muscle tissue during the 
 training period9 .
therefore , the previous 
 physical training load may influence muscle recovery after a period of inactivity10 .
aerobic training performed regularly 
 favors the skeletal muscle cellular response through an improvement in capillary 
 density11 .
high - intensity resistance training is an effective method for the treatment of atrophic 
 muscles12 .
however , the effect of 
 regular exercise without load on atrophy also needs to be investigated .
based on the negative effects of joint immobilization on the aging process and on the 
 cellular alterations derived from regular aerobic training , the hypothesis tested in the 
 present study was that , training performed during a long period prior to immobilization 
 might minimize the effects of muscle disuse and favor post - immobilization muscle recovery . 

thus , the objective of this study was to evaluate the efficacy of pre - exercise on 
 immobilization and subsequent recovery of the white gastrocnemius ( wg ) and soleus ( sol ) 
 muscles of female rats .
thirty , 8-month - old , female wistar rats were maintained under controlled temperature 
 conditions , with 12-hour light / dark cycles , and with ad libitum access to food and water . 

this study was approved by the ethics commission for animal use ( comisso de tica sobre o 
 uso de animais ceua ) of the methodist university of piracicaba under the protocol 
 09/13 .
female wistar rats were randomly and evenly allocated to six groups : sedentary ( s ) ; 
 immobilized sedentary ( is ) ; immobilized / rehabilitated sedentary ( irs ) ; trained ( t ) ; 
 immobilized trained ( it ) ; and immobilized / rehabilitated trained ( irt ) . for four months ,
the t , it and irt group animals performed swimming exercise ( water 
 temperature 30  c  2  c ) that lasted for 60 minutes per session , at a frequency of three 
 sessions per week on alternate days , for a total of 48 training sessions . during this time , 
 the s , is and irs group animals were housed in cages and could move freely . after four months of training
, the left hindlimbs of the animals in the is , irs , it and irt 
 groups were immobilized for five days using an acrylic resin orthosis that kept the ankle at 
 90 and the left knee and hip free , following the method described by silva et al13 .
female rats from the irs and irt groups 
 were physically rehabilitated in swimming sessions starting 24 hours after orthosis removal . 

remobilization was performed five times per week ( 60-minute sessions without a load 
 increase ) for two weeks , a total of 10 sessions , according to the protocol described by 
 nascimento et al10 .
all of the animals 
 that performed swimming exercise had previously been adapted to this training .
animals in 
 the t group continued the pre - immobilization exercise protocol , as follows : three sessions 
 per week , 60 minutes per session , for an additional six sessions .
the choice of females was based on the studies of nascimento et al.10 and milani et al.14 , which conducted swimming after training immobilization of female 
 wistar rats .
the animals were euthanized using 1.16  g/10  ml ketamine hydrochloride and 2  g/100  ml 
 xylazine hydrochloride at a dose of 0.09  ml/100  g body weight .
the left wg and sol muscles 
 were collected , weighed , and divided into two equal parts : one part was used to perform 
 light microscopy , and the other part was used to perform immunoblotting .
samples were fixed on wooden supports with tragacanth gum and were then frozen in 80  c 
 isopentane and 196  c liquid nitrogen .
the muscles were cross - sectioned on a cryostat 
 ( microm - hs505e ) to a thickness of 8 m and mounted on slides .
the sections were then stained 
 with hematoxylin and eosin ( he ) . to analyze the muscle cross - sectional area ( csa ) ,
the samples were viewed under a 
 light microscope ( 20 magnification ) coupled to a camera and analyzed using image 
 pro - plus 6.0 software ( media cybernetics ) . the transforming growth factor ( tgf)-1 protein level ( tgf-1 ; mouse monoclonal antibody , 
 sigma - aldrich , t7039 , st .
louis , mo , usa ) was quantified in all of the experimental groups . 

muscle extracts were centrifuged for 20 minutes at 4  c ; the supernatant was 
 treated with laemmli buffer ( 10% sds , 50% glycerol , 0.1% bromophenol blue , 100  mm tris - hcl , 
 ph 7.4 ) , and 50  mg of protein was loaded onto a 12% sds - polyacrylamide gel .
the proteins 
 were transferred from the gel to a nitrocellulose membrane using electrophoretic transfer 
 equipment from bio - rad .
the membranes were blocked for one hour at room temperature ( rt ) in 
 5% skim milk diluted in basal solution ( 10  mm trisma base , 150  mm nacl , and 0.02% tween-20 ) . 

then , the membranes were incubated in the primary antibody overnight at 4  c , washed in 
 basal solution , and incubated for 2 hours at rt in basal solution containing a 
 peroxidase - conjugated secondary antibody ( goat anti - mouse igg - hrp , santa cruz : sc-2005 , 
 dallas , tx , usa ) .
to detect immunoreactive bands , the membranes were exposed using a 
 chemiluminescence kit ( super signal west pico chemiluminescence , pierce , therm scientific , 
 waltham , ma , usa ) for 5 minutes , and the signal was detected using g - box equipment ( gene 
 sys ) .
the membranes were then washed in basal solution and incubated for one hour at 60  c in 
 10  ml of stripping buffer ( 10  mm tris - hcl ph 7.5 , 0.1  m -mercaptoethanol , 8  m urea ) .
then , 
 the membranes were incubated in 1  m tris - hcl ( ph 7.5 ) for 30 minutes , to neutralize the 
 stripping buffer , washed in basal solution , and processed as previously described to detect 
 glyceraldehyde-3-phosphate dehydrogenase ( gadph mouse monoclonal , santa cruz , sc-59540 , 
 dallas , tx , usa ) , which is an internal control protein , as its expression is not altered by 
 changes in physiologic conditions .
optical densitometry quantification was performed using 
 image j software ( the national institute of health , usa ) .
the data of three groups , one - way 
 anova followed by tukey s post - test was performed on the data with a parametric 
 distribution , while the kruskal - wallis test was performed on the data with a non - parametric 
 distribution . to perform a comparative analysis of two groups ,
student s t - test for 
 independent samples was used for parametric data , and the wilcoxon test was used for 
 non - parametric data . in all cases ,
training favored the gain of sol muscle weight in t group animals compared with s group 
 animals ( p<0.01 , table 1table 1.white gastrocnemius ( wg ) muscle and soleus ( sol ) muscle mean weights in grams ( g ) 
 and muscle fiber mean cross - section area ( m ) with the standard deviation 
 for all groupsgroupsmuscle weight ( g)csa ( m)wbsolwbsols0.417  0.070.135  0.023,884  5103,446  356is0.412  0.010.132  0.032,720  3442,234  91irs0.363  0.030.121  0.022,773  6062,636  315t0.459  0.280.172  0.015,038  700 3,926  310it0.426  0.110.157  0.033,515  5603,064  302irt0.381  0.060.148  0.033,043  505 3,441  300 s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : 
 trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained . *
different 
 from group t , p<0.01 ; * * different from groups is and irs , p<0.05 ; 
 different from groups it and irt , p<0.05 ; different from 
 group irs , p<0.05 . ) .
immobilization significantly reduced the wg csa in the animals from the is , 
 irs , it , and irs groups compared with the animals from the control groups ( s and t , 
 p<0.05 ) .
immobilization also significantly reduced the sol csa in the animals from the is 
 and irs groups compared with the animals from the s group ( p<0.05 ) ; however , no 
 significant difference was found among the trained groups , and the irt group showed a 
 significant increase in csa compared with the irs group ( p<0.05 , table 1 ) .
s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : 
 trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained . *
different 
 from group t , p<0.01 ; * * different from groups is and irs , p<0.05 ; 
 different from groups it and irt , p<0.05 ; different from 
 group irs , p<0.05 .
1.cross sections of white gastrocnemius ( a  f ) and soleus ( g  l ) muscle stained with he . 

a and g : sedentary ( s ) ; b and h : immobilized sedentary ( is ) ; c and i : 
 immobilized / rehabilitated sedentary ( irs ) ; d and j : trained ( t ) ; e and k : immobilized 
 trained ( it ) ; f and l : immobilized / rehabilitated trained ( irt ) .
observe reduction in 
 cross - sectional area of the muscle fibers of immobilized groups ( b , c , h , i ) .
1b , c , h , i ) and increased csa of the soleus 
 muscle fibers in the irt group ( fig .
cross sections of white gastrocnemius ( a  f ) and soleus ( g  l ) muscle stained with he . 

a and g : sedentary ( s ) ; b and h : immobilized sedentary ( is ) ; c and i : 
 immobilized / rehabilitated sedentary ( irs ) ; d and j : trained ( t ) ; e and k : immobilized 
 trained ( it ) ; f and l : immobilized / rehabilitated trained ( irt ) . observe reduction in 
 cross - sectional area of the muscle fibers of immobilized groups ( b , c , h , i ) .
bar = 
 100 micron the level of tgf-1 protein in the wg muscle was higher in the s group than in the is , irs , 
 and t groups ( p<0.05 ) , but no significant difference was found among animals from the 
 trained groups .
immobilization induced an increase in sol tgf-1 in the is group compared 
 with that in the other sedentary groups ( p<0.05 ) .
sol tgf-1 was similar among the 
 trained groups , with tgf-1 in the irt group being significantly higher than that of the irs 
 group ( p<0.02 , fig .
2.white gastrocnemius ( wg ) muscle and soleus ( sol ) muscle tgf-1 mean protein levels in 
 arbitrary units , with the standard deviation .
s : sedentary ; is : immobilized sedentary ; 
 irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : 
 immobilized / rehabilitated trained . * different from group t ; * * different from group 
 s ; # different from group is ;  different from group irs ; p0.02 ) .
white gastrocnemius ( wg ) muscle and soleus ( sol ) muscle tgf-1 mean protein levels in 
 arbitrary units , with the standard deviation .
s : sedentary ; is : immobilized sedentary ; 
 irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : 
 immobilized / rehabilitated trained . * different from group t ; * * different from group 
 s ; # different from group is ;  different from group irs ; p0.02
pre - immobilization training was able to prevent hypotrophy of the sol muscle ( mainly 
 composed of type i fibers ) , as immobilization did not reduce the csa in the it group , and 
 during the rehabilitation period , the csa was higher in the irt group than in the irs group . 

the wg muscle ( mainly composed of type ii fibers ) exhibited csa reduction after 
 immobilization in both the is and it groups compared with their respective controls , the s 
 and t groups , respectively .
a short immobilization period , ranging from four to ten days , is sufficient to cause 
 metabolic and structural changes to muscle tissue2 , 
 13 , 15 , 
 16 .
the results of the present study 
 show that five days of inactivity , even with an orthosis model that allowed for unloading of 
 the animal , were sufficient to induce changes in the muscles analyzed , particularly in the 
 sol .
changes in the joint , such as adhesions of fibrous hyperplasia tissue in the synovial 
 membrane and fibrosis in the articular cartilage surface , are also observed in the early 
 hours of immobilization17 .
these data underline the clinical importance of having a rehabilitation program for 
 patients who undergo joint immobilization , even for short periods of time .
in addition , it 
 is noteworthy that according to wall , dirks , and van loon18 , the repetitive exposure of individuals to short periods of 
 inactivity might culminate in the development of sarcopenia .
pitanga et al.19 emphasize that reduced levels of physical 
 activity also contribute to the development of sarcopenia and that regular physical exercise 
 is a protective factor against sarcopenia in women in the postmenopausal period .
the pre - immobilization , long - term training protocol chosen for use in the present study was 
 an attempt to approximate the protocols used in humans and was performed three times per 
 week , without a load , for a total of 180 minutes every week .
the remobilization protocol considered that striated skeletal 
 muscles require more time to recover than to undergo atrophy8 , 14 . although no significant differences were observed between the csa of sedentary and trained 
 female rats , the sol muscle weight was significantly higher in the trained female rats than 
 in the sedentary animals .
the results show that swimming remobilization did not contribute to the recovery of wg 
 muscle csa , which can be justified by the physical training performed .
milani et al.14 compared the biomechanical effects of 
 immobilization for seven days and remobilization in the skeletal muscle of trained and 
 sedentary female rats .
training was conducted with swimming ( 60-minute sessions , five times 
 per week ) with a progressive load increase ( up to 8% of body mass ) for six weeks .
milani et 
 al.14 observed that compared with the 
 sedentary group , the group that underwent pre - immobilization training presented a positive 
 effect on the mechanical behavior of the gastrocnemius muscle after a two - week 
 remobilization swimming plan , without a load increase .
notably , coupling an external weight equivalent to 6% of the body mass in experimental 
 models during swimming training is considered high - intensity physical activity10 , 21 .
tanaka et al.22 observed that the soleus 
 ( predominantly type i fibers ) and plantaris muscles ( predominantly type ii fibers ) are able 
 to recover after 14 days of tail suspension and three days of reloading , however , the 
 process leading to recovery seems to differ .
the production of hepatocyte growth factor 
 ( hgf ) , a mitogen for satellite cells , in the soleus muscle was approximately 3-fold greater 
 than that of controls .
the tgf-1 cytokine family is known to regulate several cellular processes , such as 
 angiogenesis , differentiation , and apoptosis , in addition to stimulating fibroblast 
 proliferation and extracellular matrix production23 . following a muscle lesion , tgf-1 increases , which may inhibit 
 regeneration and promote fibrosis24 .
czarkowska - paczek et al.25 investigated 
 the effects of training on the formation of tgf-1 in the sol muscle and observed increased 
 mrna expression but reduced protein levels after six weeks of treadmill training . because 
 tgf-1 is a strong stimulator of collagen expression , the increase of tgf-1 in muscle might 
 be harmful , and on the basis of the cellular responses to training , czarkowska - paczek et 
 al.25 suggested that this reduction 
 might be due to a regulatory mechanism of transcription
the results of the present study show there was a similar level of tgf-1 among the trained 
 groups , in both muscles , which suggests that a trained muscle has the capacity to adapt to 
 different stimuli , such as immobilization and remobilization . in groups of sedentary 
 animals , however , immobilization increased the level of tgf-1 in the wg and sol muscles 
 only in the is group .
future studies are necessary to compare aerobic and anaerobic training with a similar 
 weekly frequency , and to perform additional molecular analyses , such as the quantification 
 of tnf- , metalloproteinases , free radical markers , collagen and satellite cells , as well as 
 functional tests . in conclusion ,
the tgf-1 protein level highlights the muscle s adaptive capacity , indicating that regular 
 exercise , even without loading and performed three times per week , can have positive effects 
 on the skeletal muscle of female rats .
 [ purpose ] to evaluate the efficacy of pre - exercise on immobilization and subsequent 
 recovery of white gastrocnemius ( wg ) and soleus ( sol ) muscles of female rats . 
 [ subjects 
 and methods ] thirty , 8-month - old , female wistar rats were randomly and evenly allocated to 
 six groups : sedentary ( s ) ; immobilized sedentary ( is ) ; immobilized / rehabilitated sedentary 
 ( irs ) ; trained ( t ) ; immobilized trained ( it ) ; and immobilized / rehabilitated trained ( irt ) . 
 for four months , t , it and irt group animals performed swimming exercise ( three sessions 
 per week , 60 minutes per session ) , while s , is and irs groups animals remained housed in 
 cages . 
 after this period , the left hindlimb of the animals from the is , irs , it and irt 
 groups was immobilized for five days , with the ankle at 90. after removal of the 
 orthosis , animals from the irs and irt groups followed a rehabilitation program based on 
 swimming ( five sessions per week , 60 minutes per session ) for two weeks . 
 [ results ] 
 immobilization significantly reduced the cross - sectional area of the white gastrocnemius 
 muscle ; no changes were observed in the soleus muscles of the trained animals . 
 transforming growth 
 factor-1 protein levels were similar among the trained groups . 
 
 [ conclusion ] prior swimming prevents hypotrophy of the soleus muscle after immobilization , 
 and protein levels reflected the adaptive capacity of the skeletal muscle . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION