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 , and it is a leading cause of long - term motor disabilities2 . about 85% of stroke patients have had 
 ischemic strokes , which are mainly caused by acute thromboembolic occlusion or local 
 thrombosis of the intracranial arteries3
. 
 the major pathological mechanisms of cerebral ischemic injury include protease activation , 
 oxidative stress , disruption of ca homeostasis disruption , inflammation , and 
 intracellular excitotoxity4,5,6 .
exercise that is performed within a relatively short period of time results in effects on 
 serum lipids , inflammation , oxidative stress markers and blood pressure as well as reducing 
 the risk of stroke7 . among several 
 exercise paradigms ,
voluntary wheel running , forced treadmill running , and involuntary 
 muscle contraction from neuromuscular electrical stimulation ( nmes ) are commonly adopted 
 exercise models8 , 9 .
it is important to know which rehabilitation intervention is most 
 effective in facilitating motor function recovery .
in addition , upregulating the levels of 
 proteins in the caspase and calpain families has been shown to be a leading factor that is 
 responsible for motor learning and neuronal plasticity following brain degenerative 
 processes10 .
calpain is one of a family of natural 
 cysteine proteases that have been implicated in cell death following brain damage12 .
the main calpain isoforms are -calpain 
 and m - calpain , and their activation has been linked to synaptic plasticity , as well as to 
 neurodegeneration , in the central nervous system13 .
these proteases have been implicated in numerous cell functions , 
 including cell survival , division , proliferation , maturation , migration , and apoptosis14 , 15 .
many of the substrates of calpains have been localized to the 
 presynaptic and postsynaptic compartments of neurons in the hippocampus and cortex16 , 17 .
calpain substrates include cytoskeletal and associated proteins , 
 kinases and phosphatases , membrane receptors and transporters , and steroid receptors18 .
calpain inhibition has been shown to be 
 neuroprotective in most models of neurodegeneration19,20,21 , but it remains unclear whether the endogenous inhibition of 
 calpain that is induced by physical exercise has neuroprotective effects after stroke .
thus , 
 we hypothesized that antecedent exercise treadmill training would promote motor function and 
 change the levels of expression of calpain in focal cerebral ischemic injury in rats .
forty male 8-week - old sprague - dawley rats , weighing 250260  g were used following a 1-week 
 acclimatization period .
the rats were housed at a temperature of 25.0  1.0  c and a 
 humidity level of 50  5% a 12-h light - dark cycle .
group i comprised untreated normal rats 
 ( n=10 ) ; group ii comprised untreated focal cerebral ischemia rats ( n=10 ) ; group iii 
 comprised rats that performed treadmill exercise ( 20 m / min ) training after focal cerebral 
 ischemia ( n=10 ) ; and group iv comprised rats that performed antecedent treadmill exercise 
 ( 20 m / min , 14 days ) training before focal cerebral ischemia ( n=10 ) .
all animal experimental 
 protocols were performed in accordance with the guidelines of the institution 's animal care 
 and use committee .
focal cerebral ischemia was induced by a modified intraluminal suture , as 
 described previously22 .
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 3 - 0 
 silicon rubber - coated monofilament was inserted through the common carotid artery into the 
 internal carotid artery to a depth of 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 in order to prevent bleeding .
the clip and the monofilament were removed 1  h later 
 for transient ischemia , and they were left in place for 24  h for permanent ischemia .
the treadmill velocity was set at 20 
 m / min on a 0 degree incline , and antecedent treadmill exercise was performed for 20  min / day 
 over a the 14-day period . at that same time , the rats in group iii were allowed to move 
 freely in their cages , but no additional treadmill running was employed . the rats in the 
 groups iii and iv performed treadmill exercise over a 21-day period , which began 24  h after 
 the surgery . at the end of experiment ,
the animals were sacrificed by decapitation the 
 morning following the last exercise day , and their hippocampi were removed immediately , 
 placed on dry ice , and stored at 70  c for protein measurements . in the limb - placement test , 
 rats were graded from 0 to 2 in each of the 8 subtests as follows : score 0 , unable to place 
 limb ; score 1 , partial or delayed over 2-s placement ; and score 2 , immediate placement24 .
the hippocampus was homogenized in lysis 
 buffer b ( 137  mm nacl , 20  mm tris , 1% np40 , 10% glycerol , 1  mm phenylmethylsulfonyl 
 fluoride , 10 g / ml aprotinin , 1 g / ml leupeptin , 0.5  mm sodium vanadate , ph 8.0 ) for western 
 blot analysis .
the tissue was homogenized in freshly prepared lysis buffer ( 1:10 w / v ) and 
 centrifuged at 12,000  g for 30  min .
the total protein concentrations of the hippocampal homogenates were determined 
 with a microbca kit with the use of bovine serum albumin as a 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 -polyacrylamide gels .
after electrophoresis , the 
 proteins were transferred onto polyvinylidene fluoride membranes and nonspecific binding was 
 blocked with 5% nonfat dry milk in tris - buffered saline and tween 20 .
after incubation with the rabbit polyclonal 
 anti - m - calpain antibody ( 1:1,000 triple point biologics ; forest grove , or , usa ) and the 
 monoclonal anti- actin ( a-5316 , 1:5,000 , sigma , usa ) antibody , the membranes were washed 
 with tbst and incubated with appropriate horse radish peroxidase - conjugated secondary 
 antibody ( 1:4,000 dilution ) .
the immunocomplexes were visualized by chemiluminescence with 
 an ecl kit according to the manufacturer 's instructions .
the film signals were digitally 
 scanned and then quantified with nih image j software .
data analysis was performed with spss for windows , version 18.0 ( spss inc . ,
all of the data are expressed as mean  standard deviation ( sd ) of 3 replications .
the 
 differences between 2 groups were tested by one - way anova , which were followed by the 
 student  newman  keuls multiple comparison tests when differences were detected .
p - values less 
 than 0.05 at a 95% confidence level were considered significant .
data were presented as mean  sd , a : p<0.05 as compared to group i , b : p<0.05 as 
 compared to group ii , c : p<0.05 as compared to group iii .
data are presented as meansd , a : p<0.05 as compared to group i , b : p<0.05 as 
 compared to group ii , c : p<0.05 as compared to group iii .
the limb placement test behavior scores were 18 for all of the rats in group i on 
 postoperative day 1 .
the scores for group ii on postoperative day 1 were 4.5  0.8 points , 
 which were significantly different from those of the normal group ( 16.0  0.0 points ) .
on 
 day 21 , there were apparently significant differences in these scores between the ischemia 
 group ( group ii ) and the exercise groups ( group iii and gooup iv ) .
after antecedent 
 treadmill exercise , the limb placement test behavior score increased from 4.6  0.5 points 
 to 8.4  0.5 points in the treadmill exercise group , while the limb placement behavior test 
 significantly increased from 4.5  0.5 point to 11.5  0.5 points in the exercise group 
 ( p<0.05 ) ( table 1 ) .
there 
 was a trend toward lower expression levels of calpain protein in the exercise groups 
 compared to the control group .
the antecedent exercise group ( group iv ) showed lower levels 
 of expression of calpain than the treadmill exercise group ( table 2 ) .
stroke patients display various symptoms and disabilities , such as motor impairment , 
 depression , cognitive impairments , physical disabilities , sensory weakness , and limited 
 activities of daily activities25,26,27,28 .
these are many of the factors that have 
 been shown to influence the quality of life of stroke patients .
physiotherapists have tried 
 to aid in return of upper limb function for patients , by conducting repetitive practice 
 through several therapeutic interventions , paying special attention to strength , endurance , 
 coordination , speed , and ways to integrate motor function into the patient 's everyday 
 activities29 , 30 .
a number of previous studies have investigated the role of 
 exercise in promoting stroke rehabilitation .
the pathological mechanisms underlying ischemic 
 stroke , at least in part , converge on impaired intracellular calcium homeostasis , leading to 
 the activation of calpain . owing to their dependence on calcium ,
calpains have attracted 
 much attention as modulators implicated in a wide variety of biological phenomena including 
 cell migration , proliferation , cell signalling , and protein homoeostasis31,32,33,34 . 

thus , we hypothesized that antecedent exercise treadmill training would promote motor 
 function and change the levels of expression of calpain in focal cerebral ischemic injury in 
 rats .
physical exercise training has been a well - established way to protect neuronal cells 
 against ischemia - induced brain injury35 , 36 .
exercise increases capillary density by 
 inducing angiogenesis factors , and it is protective against ischemic damage37 .
our results revealed significant motor 
 function recovery , as shown - by the limb placement test score improvements in the treadmill 
 exercise groups .
neuronal synaptic remodeling in the brain hippocampus that is induced by 
 physical exercise has been described in a number of animal models38 .
an important mechanism of neuronal cell death following brain ischemia is the disruption of 
 ca homeostasis39 .
the 
 activation of calpain could be just one of the various pathways that lead to neuronal injury 
 and cell death .
calpains are abundantly present in the nervous tissue as well as in the 
 hippocampus , cortex , and cellebellum40 ) .
the best 
 known members of the calpain family are the - and m - calpains , which are referred to occur 
 in practically all mammalian tissues and cell types17 .
an increase of cytosolic ca in neurons that are 
 exposed to glutamate may cause the activation of calpain , which is a cytosolic 
 ca - dependent protease30 , 41 .
the main characteristics of the different 
 members of the calpain large family of proteases , traditionally divided into classical and 
 non - classical calpains according to their domain structure .
this includes the binding of calpain to its 
 specific endogenous inhibitor calpastatin in a substrate - competitive manner , a differential 
 intracellular localization , and the cellular control of ca homoeostasis42 .
our results indicate that physical 
 exercise inhibited mitochondrial release of apoptotic cell death in ischemic rat brain 
 injury .
while the deleterious effects of calpain activation in neuronal ischemia have been well 
 documented , other factors , such as energy deficits in the cell , the activation of poly 
 polymerase-1 and ca dependent phospholipase a2 , and the degradation 
 of membrane phospholipids , are also thought to be important43,44,45,46 .
however , in our study , there were significant inhibitory effects antecedent treadmill 
 exercise groups on the levels of calpain expression , and improvement limb motor function 
 compared to the effects in the treadmill exercise group .
the changes in calpain expression 
 might reflect neuroprotective characteristics that promote the survival of hippocampal 
 neurons , as has been shown in animal experiments of insults , such as focal brain ischemia . 

based on the findings that calpain is a critical mediator of the effects of antecedent 
 treadmill exercise on synaptic plasticity and motor function , our results showed that 
 changes in calpain are crucial for accomplishing this process .
our data clearly show that antecedent treadmill exercise acted as a major homeostatic 
 regulator of motor function and calpain expression inhibition , with important implications 
 for neuronal plasticity .
these findings suggest that the antecedent treadmill exercise is 
 important for improving limb motor function .
thus , antecedent treadmill exercise rather than 
 other post - stroke exercises may provide beneficial effects for stroke patients .
   [ purpose ] in the present study , we investigated the effects of antecedent exercise on 
 functional recovery and calpain protein expression following focal cerebral ischemia 
 injury . 
 [ subjects and methods ] the rat middle cerebral artery occlusion model was 
 employed . 
 adult male sprague - dawley rats were randomly divided into 4 groups . 
 group i 
 comprised untreated normal rats ( n=10 ) ; group ii comprised untreated rats with focal 
 cerebral ischemia ( n=10 ) ; group iii comprised rats that performed treadmill exercise ( 20 
 m / min ) training after focal cerebral ischemia ( n=10 ) ; and group iv comprised rats that 
 performed antecedent treadmill exercise ( 20 m / min ) training before focal cerebral ischemia 
 ( n=10 ) . at different time points ( 1 , 7 , 14 , and 21 days ) , limb placement test score and 
 the levels of calpain protein in the hippocampus 
 were examined . 
 [ results ] in the 
 antecedent exercise group , improvements in the motor behavior index ( limb placement test ) 
 were observed and hippocampal calpain protein levels were decreased . 
 [ conclusion ] these 
 results indicated that antecedent treadmill exercise prior to focal cerebral ischemia 
 exerted neuroprotective effects against ischemic brain injury by improving motor 
 performance and decreasing the levels of calpain expression . 
 furthermore , these results 
 suggest that antecedent treadmill exercise of an appropriate intensity is critical for 
 post - stroke rehabilitation . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION