stroke is the most common progressive neurodegenerative disorder and is one of the leading 
 causes of dysfunction in upper extremity movements and walking1 .
ischemic stroke occurs when cerebral arterial blood flow is either 
 transiently or permanently reduced , and includes both embolic and thrombotic subtypes2 .
stroke is a major cause of morbidity and 
 mortality worldwide3 , and is the third 
 leading cause of death in the united states .
approximately 85% of patients with stroke experience ischemic strokes , which are mainly 
 caused primarily by local thrombosis or acute thromboembolic occlusion of intracranial 
 arteries5 .
the major pathological 
 mechanisms that follow cerebral ischemic injury include inflammation , protease activation , 
 oxidative stress , excessive production of reactive oxygen species , and intracellular 
 excitotoxicity6 , 7 ) . in the human brain , about 25% of the energy
is 
 expended on synaptic transmission , protein synthesis , and intracellular transport8 , 9 . 
 typically , the first consequence of cerebral ischemia is attenuated synaptic activity and 
 transmission10 .
changes in synaptic 
 function are generally considered reversible , but persistent neuronal damage can cause 
 membrane failure and cell death11 .
thus , 
 an improved understanding of the pathological mechanisms that underlie neuronal cell death 
 in ischemic stroke may help improve neuroprotective strategies after stroke .
traditional stroke treatments may include neurodevelopmental training ( ndt ) , proprioceptive 
 neuromuscular facilitation ( pnf ) , a motor relearning program , the rood approach , and 
 exercise12,13,14,15,16 .
the most commonly used 
 exercise programs include wheel running , voluntary exercise , involuntary exercise , and 
 forced treadmill running17,18,19 .
considering the 
 many exercise programs available , it is important to know which of these rehabilitation 
 strategies is most effective in facilitating motor function recovery .
recent studies 
 indicate that exercise training protects neural cells from inflammation , apoptosis , and 
 oxidative stress and can enhance choline acetyltransferase activity and inhibit 
 acetylcholinesterase ( ache)20,21,22,23 . acetylcholine ( ach ) is considered to be the most 
 important neurotransmitter for cognition and motor function24 , 25 .
nevertheless , it 
 remains unclear whether the endogenous inhibition of ache that occurs with physical exercise 
 can provide neuroprotection after stroke .
we therefore hypothesized that , in rats with focal 
 cerebral ischemic injury , treadmill training would improve motor function and increase ache 
 activity .
thirty - six male sprague - dawley rats ( age = 8 weeks ; weight = 250260 g ) were used in the 
 study .
rat were housed at a temperature of 25.0  c  1.0  c and 50  5% humidity , with a 
 12-h light - dark cycle , and had free access to food and water .
the rats were introduced to 
 the study after a 1-week acclimatization period , and were divided randomly into 3 groups . 

group i included untreated normal rats ( n=12 ) , group ii included untreated focal cerebral 
 ischemia rats ( n=12 ) , and group iii included rats that performed treadmill exercise training 
 ( 20 m / min ) training after focal cerebral ischemia ( n=12 ) .
all animal experimental protocols 
 were performed in accordance with the guidelines of the institutional animal care and use 
 committee of dongshin university .
focal cerebral ischemia was induced with a modified intraluminal suture method , which was 
 previously described in detail26 . 

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 electrocoagulation 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 after 1  h to model transient ischemia and after 24  h to model 
 permanent ischemia .
the treadmill exercise group ( group iii ) 
 trained for 20  min / day with a 0 degree incline for the entire 21-day period .
group ii rats 
 were allowed to move freely in their cages during the same 21-day period , and did not 
 participate in treadmill training .
the 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 placement or placement delayed over 2-s ; and score 2 , immediate 
 placement28 .
ache activity was determined by the colorimetric assay of ellman et al.29 , as previously described .
after rats were 
 euthanized , whole ischemic brains were mixed in 0.1  m phosphate buffer ( ph 8.0 ) and 
 homogenized .
briefly , in the 96 well plates , 25  l of 15  mm acetylthiocholine iodide , 75  l 
 of 3  mm dtnb(dithiobisnitrobenzoic acid ) and 75  l of 50  mm tris  hcl , ph 8.0 , containing 
 0.1% bsa , were combined , and the absorbance was read at 405  nm after a 5-min incubation 
 period at room temperature .
any increase in absorbance due to spontaneous substrate 
 hydrolysis was corrected by subtracting the reaction rate of the reaction before adding the 
 enzyme .
next , 25  l of sample ( brain homogenates ) was added , and the absorbance was read 
 again after a second 5-min incubation period at room temperature .
the ache activity was 
 expressed as  mol / min / g of tissue protein .
data analysis was performed with spss for windows , version 18.0 ( spss inc . ,
differences in limb 
 placement test scores were examined with a parametric one - way anova and scheffe 's post hoc 
 procedure at 95% significance levels . for
test - based on scoring systems ( ordinal measures ) , 
 the nonparametric kruskal 
wallis test was used with the post hoc multiple - comparison test to 
 determine the number and relation of the group differences at a 95% significance level .
data were analyzed by anova and scheffe 's test . 
 within a given characteristic ,
data were analyzed by anova and scheffe 's test . 
 within a given characteristic ,
all group i rats scored 18 points in the limb placement test on postoperative day 1 .
the 
 group ii mean score was 4.4  0.7 points on postoperative day 1 , which differed 
 significantly from the group i mean ( 17.00.0 points ) .
significant differences in limb 
 placement test scores were observed on day 21 between the focal cerebral ischemia group and 
 the exercise group .
after treadmill training , the limb placement behavior score increased 
 significantly in the exercise group from 4.4  0.6 points on day 1 to 8.2  0.6 points on 
 day 21 ( p < 0.05 ) ( table 1 ) .
we analyzed each brain fraction extract at the end of the experiment to determine ache 
 activity levels . compared with the control group ,
the treadmill training group showed a 
 trend toward decreased ache activity ( table 2 ) . 

after treadmill training , the ache activity significantly decreased by 1.450.5  nmole / mg 
 protein / min in group ii and 0.850.17 
nmole / mg protein / min in the treadmill training group 
 ( p < 0.05 ) ( table 1 ) .
patients with stroke experience various symptoms or disabilities that limit their daily 
 activities , which includes physical disability , motor impairment , cognitive impairment , or 
 sensory weakness30,31,32 .
these factors have been 
 shown to influence the quality of life of patients with stroke .
physiotherapists attempt to 
 rehabilitate upper limb function through repetitive practice and other therapeutic 
 interventions that pay special attention to strength , endurance , coordination , speed , and 
 reintegration of specific motor functions into a patient 's everyday routine33 , 34 .
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 an impaired cholinergic system , which leads to decreased 
 synaptic transmission .
thus , we hypothesized that treadmill training would promote motor 
 function and change the levels of activation of ache levels in rats with focal cerebral 
 ischemia rats .
physical exercise training is a well - established method for protecting neural cells from 
 ischemia - induced brain injury35 , 36 .
exercise shows a tendency to 
 simultaneously promote cell survival mechanisms , while inhibiting neuronal apoptotic 
 pathways35 .
in addition , it is 
 increases capillary density by inducing angiogenic factors and protects against ischemic 
 damage37 .
our results revealed 
 significant motor function recovery , as indicated by improvements in the limb placement test 
 score .
maintaining a balanced cholinergic system homeostasis is crucial for central and peripheral 
 nervous system function , as well as for a healthy neuromuscular junction38 .
a common mechanism for neuronal cell 
 death following brain ischemia is disruption of the cholinergic system38,39,40 .
neuronal cell death , like ischemia , triggers a 
 series of events , including neuroinflammation , which can disrupt the cholinergic system41 .
central cholinergic neurotransmission 
 through muscarinic receptor activation contributes to cognition and memory formation and 
 influences ltp42 .
therefore it is likely that cholinergic 
 factors , such as ach concentration and ache inhibition , are important for plasticity in the 
 human motor system38 . in the present study ,
there were significant inhibitory effects on ache activity and 
 improvements in limb motor function in the treadmill training group .
changes in ache 
 activity might reflect neuroprotective characteristics that promote the survival of 
 hippocampal neurons , as has been shown in animal experiments modeling focal cerebral 
 ischemia44 .
based on the findings that 
 ache is a critical mediator for the effects that treadmill training imposes on synaptic 
 plasticity and motor function , our results showed that changes in ache are crucial for 
 accomplishing this process .
the present results suggest that treadmill training after stroke 
 can enhance cholinergic system activity and thereby improve limb motor function45 .
thus , our data clearly show that treadmill training appears to act as a major homeostatic 
 regulator of limb motor function and ache inhibition , which has important implications for 
 cholinergic system recovery .
thus , compared with other poststroke exercise routines , 
 treadmill training may provide further beneficial effects for patients with stroke .
   [ purpose ] in the present study , we investigated the effects of treadmill training on 
 limb motor function and acetylcholinesterase activity following focal cerebral ischemia 
 injury . 
 [ methods ] focal cerebral ischemia was examined in adult male sprague - dawley rats 
 by using a middle cerebral artery occlusion model . 
 rats were randomly divided into 3 
 groups . 
 group i included untreated normal rats ( n=12 ) , group ii included untreated rats 
 with focal cerebral ischemia ( n=12 ) , and group iii included rats that performed treadmill 
 exercise ( 20 m / min ) training after focal cerebral ischemia ( n=12 ) . 
 we determined the limb 
 placement test score for each rat on days 1,7 , 14 , and 21 ; acetylcholinesterase activity 
 in the hippocampus was examined at the end of the experiment . 
 [ results ] we observed that 
 the motor behavior index improved in the treadmill group , and hippocampal 
 acetylcholinesterase activity was decreased . 
 [ conclusion ] these results indicated that 
 treadmill training after focal cerebral ischemia exerts a neuroprotective effects against 
 ischemic brain injury by improving motor performance and decreasing the levels of 
 acetylcholinesterase activity . 
 furthermore , these results suggest that treadmill training 
 at an appropriate intensity is critical for post - stroke rehabilitation . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION