studies have shown that physical exercise can have profound effects on cardiovascular , pulmonary , and the musculoskeletal system , as well as the central nervous system ( cns ) [ 1 , 2 ] .
moderate physical activity improves memory and learning [ 38 ] and is associated with a lower risk for alzheimer 's dementia , parkinson 's disease   and other types of neurodegenerative diseases .
one of the regions of cns more affected by exercise is the hippocampus . a putative mechanism , through which the exercise exerts its effects on the hippocampus , is the induction of brain - derived neurotrophic factor ( bdnf ) .
astrocytes , the most abundant glial cells , particularly those of the glutamatergic type , are very important elements in neurotransmission [ 12 , 13 ] and antioxidant defense , and this action involves the synthesis and secretion of glutathione ( gsh ) .
these cells are responsible for glutamate removal from the synaptic cleft and its conversion , through glutamine synthetase ( gs ) catalysis , into glutamine for replacement in the neurons .
moreover , increments in energy demand and functional activity during exercise may require functional and structural alterations in astrocytes [ 15 , 16 ] , due to their involvement in the blood brain barrier .
the involvement of astrocytes in brain plasticity is commonly related to changes in specific proteins , such as glial fibrillary acidic protein ( gfap ) , s100b , glutamate transporters and glutamine synthetase . with regard to bdnf
, it is important to mention that this neurotrophin , which is de novo synthesized in neurons , is released as mature bdnf and pro - bdnf , and that pro - bdnf is uptaken and released by astrocytes .
therefore , recycling of bdnf by astrocytes seems to contribute to the regulation of synaptic plasticity .
physical exercise induces biochemical alterations in astrocytes [ 2023 ] and enhances the behavioral performance of animals in spatial memory tasks [ 7 , 20 , 24 ] .
the contribution of astrocytes to the brain plasticity induced by physical exercise needs further characterization .
this study aimed to investigate the effect of moderate exercise for 4 weeks on some parameters of astrocytic activity in the rat hippocampus .
forty - eight male wistar rats ( 90 days old , weighing 250320  g ) were obtained from our breeding colony ( at the department of biochemistry , federal university of rio grande do sul ) and were maintained under controlled light and environmental conditions ( 12  h light/12  h dark cycle at a constant temperature of 22    1c ) with free access to food and water .
all animal experiments were carried out in accordance with the directive 2010/63/eu of the european parliament and following the regulations of the local animal house authorities .
rats were habituated with the treadmill apparatus to minimize novelty stress and submitted to maximal exercise test .
after the test , rats that refused to run or who had the worst performance were assigned to the control / sedentary group ( sed ) ( n = 24 ) .
the other animals were assigned to the exercise group ( exe ) ( n = 24 ) .
three sets of experiments were carried out separately ( table 1 ) . to perform the training ,
all animals were adapted to walk on a treadmill for three consecutive days ( days 1 to 3 ) .
this adaptation consisted of walking on a treadmill for 10  min at 5  m / min . on the fourth day
the test consisted of a graded exercise on the treadmill , with speed increments of 5  m / min every 3  min , starting at 5  m / min and continuing up to the mec of each rat .
the exercise training consisted of running sessions on an adapted motorized rodent treadmill ( inbramed tk 01 , porto alegre , brazil ) at 60% of mec ( maximal velocity ) .
treadmill training was performed between 8:00 and 12:00  h. the animals ran for 20 minutes , 5 days a week , for 4 weeks .
the control / sedentary group was transported to the experimental room and handled exactly as the experimental animals were and maintained in the turned off treadmill for 5 min without forcing them to run .
two hours after the last exercise session ( 20  min / day , 5 days / week during four weeks ) animals ( n = 5 - 6 ) were anesthetized with ketamine and xylazine ( 75 and 10  mg / kg , resp . , i.p . ) , and then whole blood was obtained through an intracardiac puncture using a 0.37  mm diameter needle that was inserted into the intercostal space above the sternum .
twenty - four hours after the last exercise session ( 20  min / day , 5 days / week during four weeks ) the animals were killed by decapitation , brains were removed and placed in cold saline medium with the following composition ( in mm ) : 120 nacl ; 2 kcl ; 1 cacl2 ; 1 mgso4 ; 25 hepes ; 1 kh2po4 and 10 glucose , adjusted to ph 7.4 , and previously aerated with o2 . the hippocampi were dissected out , and transverse slices of 0.3  mm were obtained using a mcilwain tissue chopper .
slices were transferred immediately to 24-well culture plates , each well contains 0.3  ml of the solution described previously for measuring s100b secretion and 0.3  ml of hbss ( hank 's balanced salt solution ) for measuring glutamate uptake and glucose uptake .
slice samples ( hippocampus ) were then frozen ( 80c ) for biochemical measurements , described as follows .
briefly , 50  l of sample plus 50  l of tris buffer were incubated for 2  h on a microtiter plate previously coated with monoclonal anti - s100b ( sh - b1 ) .
polyclonal anti - s100b was incubated for 30  min and then peroxidase - conjugated anti - rabbit antibody was added for a further 30  min .
elisa for gfap   was carried out by coating the microtiter plate with 100  l samples containing 30  g of protein for overnight at 4c .
incubation with a rabbit polyclonal anti - gfap for 2  h was followed by incubation with a secondary antibody conjugated with peroxidase for 1  h , at room temperature ; the standard gfap curve ranged from 0.1 to 10  ng / ml .
rats were anesthetized using ketamine / xylazine ( 75 and 10  mg / kg , resp . , i.p . ) and were perfused through the left cardiac ventricle with 200  ml of saline solution , followed by 200  ml of 4% paraformaldehyde in 0.1  m phosphate buffer saline ( pbs ) , ph 7.4 .
after perfusion , the brains were removed , postfixed in the same fixative solution for 4  h at room temperature , and cryoprotected by immersion in 15% and 30% sucrose solution in pbs at 4c .
the brains were then frozen by immersion in isopentane cooled with co2 and stored in a freezer ( 80c ) for later analyses .
coronal slices ( 45  m ) were obtained at 20c using a cryostat ( leitz ) .
the free - floating sections were incubated with polyclonal anti - gfap from rabbit , diluted 1  :  3000 in pbs - triton x-100 0.4% and 2% bovine serum albumin ( bsa ) , for 48  h at 4c . after washing several times in pbs
, tissue sections were incubated with anti - rabbit alexa 488 ( molecular probes ) diluted 1  :  500 in pbs - tx 0.1% and 2% bsa for 1  h at room temperature .
afterwards , the sections were washed several times in pbs , mounted on slides with fluor save , and covered with coverslips .
briefly , hippocampal slices were transferred to 24-well plates and incubated for 23  min at 37c in a hank 's balanced salt solution ( hbss ) containing ( in mm ) : 137 nacl , 5.36 kcl , 1.26 cacl2 , 0.41 mgso4 , 0.49 mgcl2 , 0.63 na2hpo47h2o , 0.44 kh2po4 , 4.17 nahco3 , and 5.6 glucose , adjusted to ph 7.4 .
the assay was started by the addition of 0.1  mm l - glutamate and 0.33  ci / ml l-[2 , 3-h ] glutamate .
incubation was stopped after 5  min by removal of the medium and rinsing the slices twice with ice - cold hbss .
sodium - independent uptake was determined using n - methyl - d - glucamine instead of nacl .
sodium - dependent glutamate uptake was obtained by subtracting the nonspecific uptake of the total uptake to obtain the specific uptake .
results ( nmol / mg protein / min ) were expressed as a percentage of the control .
briefly , homogenized tissue samples were added to a reaction mixture containing ( in mm ) : 10 mgcl2 ; 50 l - glutamate ; 100 imidazole - hcl buffer ( ph 7.4 ) ; 10 2-mercaptoethanol ; 50 hydroxylamine - hcl ; 10  atp and incubated for 15  min at 37c .
the reaction was stopped by the addition of 0.4  ml of a solution containing : 370  mm ferric chloride ; 670  mm hcl ; 200  mm trichloroacetic acid .
after centrifugation , the supernatant was measured at 530  nm and compared to the absorbance generated by standard quantities of -glutamylhydroxamate treated with ferric chloride reagent .
this assay detects only the reduced glutathione content . briefly , homogenized slices were assayed in sodium phosphate buffer ( 0.1  m , ph 8.0 ) , and protein was precipitated with 1.7% meta - phosphoric acid .
supernatant was assayed with o - phthaldialdehyde ( 1  mg / ml methanol ) at room temperature for 15  min .
fluorescence was measured using excitation and emission wave lengths of 350 and 420  nm , respectively .
briefly , homogenates from hippocampal were mixed with 25% trichloroacetic and centrifuged at 1800  g for 10  min .
the total no2 in the incubation was measured by a colorimetric assay at 540  nm , based on the griess reaction .
intracellular ros production was detected using the nonfluorescent cell permeating compound , 27-dichlorofluorescein diacetate ( dcf - da ) .
samples homogenized in sodium phosphate buffer , ph 7.4 with 140  mm kcl were treated with dcf - da ( 10  m ) for 30  min at 37c .
the fluorescence was measured in a plate reader ( spectra max gemini xps , molecular devices , usa ) with excitation at 485  nm and emission at 520  nm , as described previously .
values are obtained as a unit of fluorescence / mg protein and are expressed as percentage of control .
briefly , slices were transferred to 24-well plates and incubated for 30  min at 37c in a hank 's balanced salt solution ( hbss ) containing ( in mm ) : 137 nacl , 5.36 kcl , 1.26 cacl2 , 0.41 mgso4 , 0.49 mgcl2 , 0.63 na2hpo47h2o , 0.44 kh2po4 , 4.17 nahco3 , and 5.6 glucose , adjusted to ph 7.4 .
the assay was started by addition of 0.1  ci / ml [ 2,3-h]-d - glucose .
the incubation was stopped after 30  min by removal of the medium and rinsing the slices twice with ice - cold hbss .
nonspecific uptake was determined by using 25  m cytochalasin b. final glucose uptake was obtained by subtracting the nonspecific uptake of the total one to obtain the specific uptake .
corticosterone concentrations in serum were measured using a commercial eia kit conforming the manufacturer 's protocol ( cayman chemicals , mi , usa ) .
briefly , 50  l aliquots were added to 96-well plates precoated with rabbit antisheep igg antibody .
we added corticosterone - specific acetylcholinesterase tracer and specific corticosterone antiserum and then placed plates on an orbital shaker for two hours .
the concentration of corticosterone was calculated by comparing samples to the standard curve generated with the kit .  
bdnf protein was assessed using the chemikine brain - derived neurotrophic factor ( bdnf ) sandwich elisa kit ( millipore , usa ) , according to the manufacturer 's recommendations .
briefly , the slices of hippocampus were individually homogenized in buffer containing 100  mm tris - hcl ( ph 7.0 ) , containing 2% bovine serum albumin ( bsa ) , 1  m nacl , 4  mm edtana2 , 2% triton x-100 , 0.1% sodium azide , and the cocktail protease inhibitors ( sigma ) .
the supernatant was incubated on a 96-well microplate previously coated with anti - bdnf monoclonal antibody . after blocking
, plates were incubated with biotinylated mouse anti - bdnf monoclonal antibody for 2  h and streptavidin - hrp conjugate solution for 1  h. then color reaction with 3,3,5,5-tetramethylbenzidine substrate was quantified in a plate reader at 450  nm .
an increase in lactate level ( reflecting an improved muscle metabolism ) in the blood is commonly used as a marker of the effect of exercise training .
blood samples were collected from a cut at the tip of the tail at the end of the last exercise bout .
the lactate concentrations were determined with a lactate analyzer ( accutrend plus , roche diagnostic , germany ) .
parametric data from the experiments are presented as means    standard error and statistically evaluated by student 's t - test , assuming p < 0.05 .
in order to characterize whether the protocol for the treadmill exercise was aerobic , we analyzed the blood lactate levels in animals .
the concentration of blood lactate in both groups , exercise ( exe ) and control / sedentary ( sed ) , remained below the anaerobic threshold of 7.17  0.16  mm .
moreover , a significant decrease in blood lactate levels was observed in the exercise group , as compared to the control / sedentary group ( p = 0.04 ) ( figure 1(a ) ) .
a small but significant increase was observed in serum corticosterone levels ( p = 0.04 , figure 1(b ) ) .
the bdnf content in hippocampus was not changed by this exercise protocol ( p = 0.17 , figure 2(a ) ) . however , the mean gfap content was decreased in exercised rats ( p = 0.02 , figure 2(b ) ) .
the immunohistochemistry of the stratum radiatum of ca1 hippocampal region ( figure 3(a ) and 3(b ) ) suggests a decrease in gfap immunoreactivity .
no significant differences in s100b content , as evaluated by elisa , were observed in the hippocampus of rats submitted to treadmill exercise ( p = 0.92 , figure 4(a ) ) .
hippocampal slice preparations from the two groups were used to evaluate in vitro s100b secretion .
no significant changes in basal s100b secretion were found at 1  h ( figure 4(b ) ) .
this exercise protocol did not change the glutamate uptake ( p = 0.56 , figure 5(a ) ) in hippocampal tissue .
in contrast , we observed an increase in gs activity in exercised rats ( p = 0.003 , figure 5(b ) ) .
two parameters were investigated to evaluate oxidative stress in the hippocampus : gsh ( reduced form of glutathione ) and no ( nitric oxide ) contents .
no significant differences were observed in gsh content ( p = 0.92 , figure 6(a ) ) .
conversely , a decrease in no content ( based on nitrite measurement ) was found in the hippocampal tissue of exercised rats ( p = 0.049 , figure 6(b ) ) .
. however , relatively little is known about mechanisms underlying these benefits [ 2 , 38 ] .
synaptic remodeling in hippocampus induced by physical exercise has been described in animal models ( e.g. , ) .
astrocytes have been shown to play an important role in synaptic remodeling [ 4042 ] and protecting the cns against various pathologies .
the aim of this study was to evaluate the effect of moderate treadmill running on astroglial parameters in the rat hippocampus .  
our results show that 4 weeks of moderate exercise decrease the gfap content in the rat hippocampus .
this is in agreement with a decrease in the number and immunoreactivity of gfap cells in the rat cerebral cortex after running .
however , there are some controversies in the literature regarding the effect of physical exercise on gfap expression in astrocytes .
some studies have shown that physical exercise seems to increase gfap expression in different brain regions , such as the cerebral cortex , striatum , and hippocampus .
on the other hand , some studies have not found alterations in this parameter in the rat hippocampus [ 21 , 23 ] .
the discrepancy in results probably arises from methodological differences , including brain regions , exercise protocol ( frequency , intensity ) , and duration of exercise training .
moreover , we can not rule out that the hippocampal changes observed ( on gfap and other parameters ) reflect differences in rats , based on inherent differences ; that is , rats that eagerly exercised versus rats that refused to run during the initial exercise test .
however , blood lactate levels measured previously to this test were not altered ( data not shown ) .  
lactate is an important energetic substrate for the brain , and it has been proposed that an increment of blood lactate , during physical activity , could induce brain changes .
interestingly , we found a decrease in serum lactate with our protocol of physical activity .
this apparent paradoxical decrease has also been described in mice submitted to 6 weeks of moderate - intensity treadmill exercise .
this decrease could be interpreted as an adaptation to exercise , in particular with an improvement of the cori cycle ( between the muscle and liver ) . in support of this idea
, authors found an increase in lactate transporter in the liver , during this study .
however , we can not exclude an increment of blood lactate at the beginning of the exercise protocol , leading to hippocampal changes .
gfap is a highly regulated protein , whose expression is induced by brain development and injury .
we observed a decrease in hippocampal gfap , induced by physical exercise . at this moment , our work hypothesis is that corticosterone , which we found increased in our protocol of physical exercise , can downregulate gfap expression .
in fact , gfap levels are negatively regulated by glucocorticoids in the rat hippocampus ( see , for a review ) .
moreover , we can speculate that this downregulation of gfap ( induced by physical exercise ) could be associated with other neuronal changes that accompany gfap downregulated conditions , such as neurite outgrowth [ 49 , 50 ] .
therefore , we may suggest that gfap decreases as in indirect signal of neuronal plasticity in the rat hippocampus in response to treadmill exercise .
it has been suggested that gfap expression has an important role in astrocyte - neural interactions ; however , the consequence of this change in the cognitive process remains unclear .
in contrast to gfap , s100b was not changed by physical exercise in the rat hippocampus , confirming previous reports [ 21 , 23 ] .
this protein is secreted by astrocytes and has a trophic extracellular effect on neuron and glial cells [ 52 , 53 ] . moreover ,
no changes in basal in vitro secretion of this protein in the hippocampal slices were observed , in agreement with unaltered levels of cerebrospinal fluid s100b in rats submitted to treadmill exercise .
bdnf is a neurotrophin , associated with brain plasticity that is induced by physical exercise [ 2 , 9 , 54 ] .
this neurotrophin had no effect on gfap expression in the retina   but was able to reduce gfap expression in the cerebral cortex after ischemia .
on the other hand , bdnf was able to restore hippocampal gfap levels reduced by chronic unpredictable mild stress in rats . under these conditions
, we did not observe any significant physical exercise - induced changes in bdnf that could be related to decreased gfap levels .
corticosterone is able to transiently downregulate bdnf mrna in the hippocampus , possibly contributing , in this study , to maintain the levels of this neurotrophin unaltered .
moreover , other studies using moderate acute physical exercise ( for 1  h )   ( or for 2 weeks ) [ 60 , 61 ] did not find changes in hippocampal bdnf either .  
in addition to the decrease in gfap , we found an increment in gs activity . whether this effect also depends on corticosterone remains unclear , but it has been reported that expression of gfap and gs in vivo shows opposite responses to corticosterone in the hippocampus . however , there is not enough information about changes in these proteins induced by physical exercise .
recently , we found that gs activity was increased by caloric restriction but not by physical exercise ( treadmill running , 20  min sessions , for 12 weeks ) .
this could suggest that the gs increment induced by the 4-week protocol of treadmill running could be transitory ( as well as gfap decrease ) .
the increased gs activity induced by physical exercise could be beneficial in the brain ; this enzyme is involved in the modulation of the turnover of glutamate through the glutamate - glutamine cycle and detoxification of ammonia , and its decrease has been observed in neurodegenerative disorders .
the enzyme activity is negatively modulated by nitration on the tyrosine residue , through a nmda receptor - mediated cgmp - no signal transduction pathway ( see butterworth   for a review ) .  
note that while no works as a physiological mediator , excessive no release due to the high expression of inducible no synthase ( inos ) mediates inflammatory / degenerative brain diseases . herein , we observed a decrease in no content ( measured by nitrite content ) , which in turn could be connected to the increased gs activity . in agreement with these findings ,
physical exercise was able to prevent the increase in hippocampal no induced by intracerebroventricular streptozotocin administration .
the mechanism for this is unclear , but it may also be mediated by corticosterone . in support of this hypothesis ,
swimming exercise induced a decrease in the rat hippocampal inos , possibly dependent on corticosterone .
other experimental evidence reinforces the importance of treadmill exercise for decreasing inos in murine models of neurodegenerative diseases , such as parkinson 's   and alzheimer 's diseases .
considering the close metabolic relationship between glutamine and glutamate , we also investigated glutamate uptake in hippocampal slices of exercised rats .
no changes in glutamate uptake were observed , in agreement with our previous study using a longer protocol of physical exercise .
hippocampal gsh content and ros ( data not shown ) were not altered by physical exercise under our conditions .
a glutathione decrease could suggest oxidative stress , and an increment induced by physical exercise has been interpreted as signal of improvement in antioxidant defense [ 20 , 23 ] .
an elegant study has shown that moderate treadmill running did not change the basal content of glutathione , but it was able to reverse the decrease in glutathione induced by intraperitoneal buthionine - sulfoximine administration .
another important aspect for brain activity , during physical exercise , is the increment cerebral blood flow that is commonly related to energetic substrate uptake .
for example , diabetic type 2 patients may benefit from physical exercise , as muscles dramatically increase glucose uptake
. however , brain alterations induced by physical alterations have not been evaluated , and potential changes in the hypometabolism that precedes neurodegenerative diseases ( see cunnane , et al . ) could be very useful to improve life quality .
herein , no changes were observed in the glucose uptake of hippocampal slices of healthy exercised animals ( data not shown ) .
however , this parameter should be investigated in brain disease models , where physical exercise has been proposed as a neuroprotective strategy ( e.g. , rodrigues et al . ) .
our data show that moderate treadmill exercise was able to induce a decrease in gfap content ( evaluated by elisa and immunohistochemistry ) and to increase gs activity .
bdnf , another putative regulator of the expression of these astroglial proteins , was not altered in hippocampal tissue .
we did not observe changes in other astroglial parameters such as glutamate uptake , gsh content , and s100b protein .
taken together , these data indicate specific changes in astrocyte markers induced by physical exercise , the importance of astrocytes in brain plasticity , as well as reinforce the relevance of physical exercise as a strategy for neuroprotection .
 physical exercise effects on brain health and cognitive performance have been described . 
 synaptic remodeling in hippocampus induced by physical exercise has been described in animal models , but the underlying mechanisms remain poorly understood . 
 changes in astrocytes , the glial cells involved in synaptic remodeling , need more characterization . 
 we investigated the effect of moderate treadmill exercise ( 20  min / day ) for 4 weeks on some parameters of astrocytic activity in rat hippocampal slices , namely , glial fibrillary acidic protein ( gfap ) , glutamate uptake and glutamine synthetase ( gs ) activities , glutathione content , and s100b protein content and secretion , as well as brain - derived neurotrophic factor ( bdnf ) levels and glucose uptake activity in this tissue . 
 results show that moderate treadmill exercise was able to induce a decrease in gfap content ( evaluated by elisa and immunohistochemistry ) and an increase in gs activity . 
 these changes could be mediated by corticosterone , whose levels were elevated in serum . 
 bdnf , another putative mediator , was not altered in hippocampal tissue . 
 moreover , treadmill exercise caused a decrease in no content . 
 our data indicate specific changes in astrocyte markers induced by physical exercise , the importance of studying astrocytes for understanding brain plasticity , as well as reinforce the relevance of physical exercise as a neuroprotective strategy . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions