stroke is an acute and progressive neurodegenerative disorder that has become one of the 
 leading causes of mortality and various disabilities1 .
ischemic stroke is the result of a transient or permanent reduction 
 in cerebral arterial blood flow caused by residual tissue infarction via an embolus or local 
 thrombosis2
. the major pathobiological 
 mechanisms of cerebral ischemic injury include intracellular excitotoxicity , oxidative 
 stress , apoptosis , and inflammation3 . 

cerebral ischemia triggers a complex series of biochemical and molecular mechanisms that 
 impairs the neurological functions through breakdown of cellular integrity .
these 
 alterations are by mediated by excitotoxic mechanisms , glutamatergic signalling , 
 free - radical reactions , ionic imbalance , and other processes3 .
physical exercise within a relatively short period of time ( weeks to months ) is able to 
 increases the expression of a number of neurotrophic genes involved in any enhancement of a 
 physical performance4 . among several 
 exercise paradigms ,
voluntary wheel running , forced treadmill running , and involuntary 
 muscle contraction caused by electrical stimulation are the commonly adopted exercise 
 models .
apart from their physical benefits , these exercises have been separately 
 demonstrated to improve cognitive function and facilitate neural rehabilitation after brain 
 injury5,6,7 .
it is important to know 
 which rehabilitation intervention is more effective in facilitating motor function recovery 
 and brain - derived neurotrophic factor ( bdnf ) upregulation , which is a leading factor 
 responsible for motor learning and memory following brain ischemia8 .
it is widely expressed in the rodent brain , and is particularly 
 abundant and widely distributed in the hippocampus , cerebral cortex , cerebellum , striatum , 
 and amygdala11 , 12 .
bdnf expression was also reported in various parts of the human 
 brain , including the hippocampus , claustrum , amygdala , bed nucleus of the stria terminalis , 
 septum and the nucleus of the solitary tract13 .
bdnf is involved in memory formation , including learning and 
 behavior , synaptic plasticity , synaptic efficacy and neuronal connectivity .
it also promotes 
 the development of enhances the survival of adult neurons9 , 14 , 15 .
although extensive studies have been performed to investigate the role of exercise in 
 promoting stroke rehabilitation .
it remains unclear whether the endogenous production of 
 neurotrophins induced by physical exercise have neuroprotective effects after stroke . in the 
 present study
, we examined rats after transient middle cerebral artery occlusion(mcao ) to 
 determine whether regular motor exercise on a treadmill immediately after ischaemia .
thus , 
 we hypothesized that antecedent exercise treadmill training promotes motor function and 
 changes the expressions of bdnf in focal cerebral ischemic injury in rats .
forty 8-week - old male sprague - dawley rats , weighing 250260 g were used in this study 
 following a 1-week acclimatization period .
the rats were housed at a temperature of 25.0  c 
  1.0  c and a humidity level of 50  5% under a 12-h light - dark cycle ; they had free access 
 to food and water .
group i included untreated 
 normal rats ( n=10 ) ; group ii included untreated focal cerebral ischemia rats ( n=10 ) ; group 
 iii included rats that performed treadmill exercise ( 20 m / min ) training after focal cerebral 
 ischemia ( n=10 ) ; and group iv included rats that performed antecedent treadmill exercise ( 20 
 m / min , 14 days ) training before focal cerebral ischemia ( n=10 ) as well as treadmill exercise 
 after ischemia .
all animal experimental protocols were performed in accordance with the 
 guidelines of the dongshin university animal care and use committee .
focal cerebral ischemia 
 was induced using a modified intraluminal suture method as described previously16 .
briefly , the left common internal and 
 external carotid arteries were exposed through a midline incision in the neck and then 
 carefully dissected from the surrounding tissues under an operating microscope .
after 
 electro - coagulation of the external and common carotid arteries , a 30 silicon rubber - coated 
 monofilament was inserted through the common carotid artery into the internal carotid artery 
 to 1820  mm beyond the carotid bifurcation at the base of the middle cerebral artery .
an 
 atraumatic aneurysm clip was placed on the internal carotid artery to prevent bleeding .
the 
 clip and the monofilament were removed 1  h later for transient ischemia and left in place 
 for 24  h for permanent ischemia .
the treadmill velocity was set at 20 
 m / min , and 20  min / day antecedent treadmill exercise was performed during the 14 days period 
 at a 0 degree incline . during this time , the rats in group iii were allowed to move freely 
 in their cages , but no additional treadmill running was performed .
the rats in both group 
 iii and group iv performed treadmill exercise for 21 days , which began 24  h after the 
 surgery . after 3 weeks of treadmill exercise , the animals were sacrificed by decapitation 
 the morning following the last treatment day and their hippocampi were removed immediately 
 placed on dry ice , and stored at 70  c until used for protein measurements .
the motor 
 behavior index of the rats was measured blindly using garcia s index at 10 a.m. on 
 postoperative days 1 , 7 , 14 , and 21 to account for diurnal variation .
six items were 
 measured and the total score ranged from 3 to 18 ; the higher the score , the better the motor 
 performance .
items 14 ( spontaneous activity , symmetry of movements , symmetry of the 
 forelimbs , and climbing the wall of wire cage ) measure motor performance , and items 56 
 ( reaction to touch on and response to vibrissae touch ) measure sensory function18 .
the hippocampus was homogenized in lysis 
 buffer b ( 137  mm nacl , 20  mm tris , 1% np40 , 10% glycerol , 1  mm phenylmethylsulfonyl fluoride 
 [ pmsf ] , 10 g / ml aprotinin , 1 g / ml leupeptin , 0.5  mm sodium vanadate , ph8.0 ) for western 
 blot analysis .
tissue was homogenized in freshly prepared lysis buffer ( 1:10 w / v ) and 
 centrifuged at 12,000 g for 30  min .
the supernatants were collected , 
 divided into aliquots and stored at 70  c .
the total protein concentration of the 
 hippocampal homogenates were determined with a microbca kit , using bovine serum albumin as 
 the standard .
aliquots containing an equal amount of hippocampal protein extracts ( 20 g ) 
 were mixed with gel loading buffer and separated on 10% sodium dodecyl sulfate 
 ( sds)-polyacrylamide gels .
after electrophoresis , the proteins were transferred onto 
 polyvinylidene fluoride ( pvdf ) membranes and nonspecific binding was blocked with 5% nonfat 
 dry milk in tris - buffered saline and tween 20 .
after incubation with the anti - bdnf primary antibody ( sc-546 , santa 
 cruz , usa ) , monoclonal anti- actin ( a-5316 , 1:5,000 , sigma , usa ) antibodies membranes were 
 washed with tbst and incubated with the appropriate horse radish peroxidase - conjugated 
 secondary antibody ( 1:4,000 dilution ) .
immuno- complexes were visualized by 
 chemiluminescence using the ecl kit according to the manufacturer s instructions .
the film 
 signals were digitally scanned and then quantified using nih image j software .
differences between groups 
 were tested by one - way anova , followed by the student 
a : p<0.05 , compared to group i ; b : p<0.05 , 
 compared to group ii ; c : p<0.05 , as compared to group iii .
a : p<0.05 , compared to group i ; b : p<0.05 , 
 compared to group ii ; c : p<0.05 , as compared to group iii .
the garcia s motor behavior scores were 18 for all of the rats in group i ( normal ) at 1 day 
 postoperatively .
these scores for group ii ( mcao ) at 1 day postoperatively ( 9.70.9 ) were 
 significantly different from those of the normal group ( 18.00.0 ) . on day 21 , there were 
 apparently significant differences in the gracia scores between the ischemia group and the 
 exercise groups .
after antecedent treadmill exercise , the garcia s scores increased from 9.3 
 to 14.5 in the antecedent treadmill exercise group ( group iii ) , while the garcia s scores 
 increased from 9.1 to 12.5 in the exercise group ( table 
 1 ) .
there was a 
 trends toward a higher expression of bdnf protein in the exercise groups ( group iii and 
 group iv ) compared to the control group ( group i ) .
the antecedent treadmill exercise group 
 showed higher expression of bdnf than the treadmill exercise ( group iii ) group ( table 2 ) .
stroke patients display various symptoms and disabilities such as motor and sensory 
 weakness , cognitive impairment , perceptual disorders and limitations in daily 
 activities19 .
several physical 
 therapeutic interventions such as exercise , exposure to an enriched environment and 
 rehabilitation enhance functional recovery after stroke20 .
therefore , it is necessary to improve the abilities of motor 
 function , cognition , and perception for successful rehabilitation21 , 22 .
beneficial 
 effects of exercise therapies include improved learning and memory , improved motor function , 
 and enhancement of proteins involved in brain plasticity , such as bdnf and transforming 
 tyrosine receptor kinase ( trk)a23,24,25,26 .
the purpose of this study was to compare 
 the effect of antecedent treadmill exercise training with that of treadmill exercise 
 training after mcao on the motor function and changes the expressions of bdnf in focal 
 cerebral ischemia . in stroke studies ,
the rat is the most commonly used animal model due to its size , which 
 allows easy monitoring of the physiologic variables and handling of vascular structures . 
 nonetheless since the rodent is the best - characterized animal in genetics and molecular 
 biology , an increasing number of stroke studies
many animal studies have demonstrated the beneficial effects of 
 treadmill exercise , such as a smaller brain infarct volume or improvement in neurological 
 function , either before or after stroke when compared with spontaneous recovery7 , 29 . 

the action of exercise on the regulation of bdnf , a molecule that plays an important role in 
 rat hippocampal motor learning , might involve epigenetic regulatory mechanisms20 . based on information that bdnf is a 
 critical mediator of the effects of antecedent treadmill exercise on synaptic plasticity and 
 motor function , and our results showed that the change in bdnf is crucial to accomplish this 
 process .
our results revealed significant motor function recovery by garcia s scores improvement in 
 the treadmill exercise groups .
stroke patients had lower energy expenditure and improved 
 gross motor efficiency and gained improved ambulatory ability after treadmill exercise30 .
this is consistent with the findings of 
 another study which reported that improved motor performance after exercise was associated 
 with elevated expression of bdnf and trkb31 .
therefore , it appears that the brain plasticity mediated by bdnf 
 and trkb might play a trivial role in motor recovery after cerebral ischemia in mild 
 cerebral ischemic rats compared to moderate - to - severe ischemic rats .
according to the study 
 of cechetti et al.32 , no significant 
 changes in the bdnf expression were detected in the hippocampi of rats that performed daily 
 moderate intensity exercise ( 2 weeks of 20  min / day of treadmill training ) .
this result 
 differs from previous studies demonstrating that physical activity , running , and swimming 
 increases the expression of bdnf in the rodent brain20 , 33,34,35 . in the present study
, there was a significant effect of the antecedent treadmill exercise 
 groups on bdnf expression , and this group of rats showed greater improvement of motor 
 function than the treadmill exercise group .
the change in bdnf expression also showed 
 characteristics of neuroprotective characteristics that promoted survival of hippocampal , 
 striatal , and septal neurons in animal experiments against insults such as focal brain 
 ischemia .
previous investigations have found that increasing bdnf levels or 
 activating bdnf - associated signaling pathways leads to improved recovery following 
 stroke24 , 38 .
however , the present results provide the first direct 
 demonstration that antecedent exercise is crucial for mediating the motor recovery that 
 takes place as a result of post - stroke rehabilitation .
our data clearly show that antecedent 
 treadmill exercise appears to act as a major homeostatic regulator of motor function and 
 bdnf expression , with important implications for brain plasticity .
it has been suggested 
 that the antecedent treadmill exercise are important in improving motor function .
thus , 
 antecedent treadmill exercise rather than post - stroke exercises may provide the beneficial 
 effect for the recovery of stroke patients .
   [ purpose ] in the present study , we investigated the effect of antecedent exercise on 
 functional recovery and brain - derived neurotrophic factor ( bdnf ) expression following 
 focal cerebral ischemia injury . 
 [ subjects ] the rat middle cerebral artery occlusion ( mcao ) 
 model was employed . 
 adult male sprague - dawley rats were randomly divided into 4 groups . 
 
 group i included untreated normal rats ( n=10 ) ; group ii included untreated rats with focal 
 cerebral ischemia ( n=10 ) ; group iii included rats that performed treadmill exercise ( 20 
 m / min ) training after focal cerebral ischemia ( n=10 ) ; and group iv included rats that 
 performed antecedent treadmill exercise ( 20 m / min ) training before focal cerebral ischemia 
 ( n=10 ) as well as treadmill exercise after ischemia . at different time points ( 1 , 7 , 14 , 
 and 21 days ) 
 garcia s score , and the hippocampal expressions level of bdnf were examined . 
 
 [ results ] in the antecedent exercise group , improvements in the motor behavior index 
 ( garcia s score ) were observed and hippocampal bdnf protein expression levels increased . 
 
 [ conclusion ] these results indicate that antecedent treadmill exercise , before permanent 
 brain ischemia exerts a neuroprotective effect against ischemia brain injury by improving 
 motor performance and increasing the level of bdnf expression . 
 furthermore , the antecedent 
 treadmill exercise of appropriate intensity is critical for post - stroke 
 rehabilitation . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION