the majority of deaths and disabilities from trauma occur with traumatic brain injury 
 ( tbi)1 .
tbi is an insult to the brain 
 that results in impairments of cognitive and physical functioning as well as disturbances in 
 behavioral or emotional functioning2 . 

previous studies have attempted to establish laboratory models of tbi to assess the damage 
 phase and treatment in a variety of primates and conditions3 . among them , controlled cortical impact ( cci ) , occasionally referred 
 to as the rigid percussion model , is a model of traumatic brain injury in ferrets , rats , and 
 mice that may potentially prove useful in elucidating the mechanisms underlying 
 neurodegeneration using genetically altered animals4 , 
 5 .
injury results in a considerable hematoma under the injury 
 site that is immediately visible , and the impact induces primary necrosis at the center of 
 the contusion .
spontaneous exercise may be therapeutic in the management of cns injury , by reducing the 
 degree of initiatory damage , limiting the degree of secondary neuronal death , and promoting 
 neural repair and behavioral rehabilitation7 .
the effects of exercise on genes encoding for neurotrophins and 
 other proteins suggest that exercise could regulate anatomical changes that support brain 
 plasticity .
it has been previously demonstrated that exercise increases the number of new 
 neurons in the dentate gyrus8 .
epileptic , 
 ischemic , and traumatic insults to the brain induce marked changes in the expression of 
 genes encoding for neurotrophins , nerve growth factor ( ngf ) , brain - derived neurotrophic 
 factor ( bdnf ) , and neurotrophin-3 ( nt-3 ) in cortical and hippocampal neurons9 .
the purpose of the present study was to investigate the expression of nt-3 , which provides 
 a neuroprotective effect that promote the maintenance and survival of neurons in rats 
 undertaking spontaneous wheel running exercise after experimental tbi using a cci model in 
 rats .
thirty male sprague - dawley rats weighing between 250  g and 300  g were used and maintained 
 on a 12 h on/12 h off light / dark cycle with ad libitum access to food and 
 water .
all the experiments were performed in accordance with protocols approved by the 
 university of daegu animal experiment committee , based on the nih guidelines for the care 
 and use of laboratory animals ( nih publication , 1996 ) .
animals were divided into 3 groups 
 randomly : namely , the normal ( nrm ) , tbi , and swr groups .
the tbi and swr groups were 
 anesthetized with 2  ml / kg 50% zoletil and 50% xylazine hydrochloride mixture and secured in 
 a stereotaxic frame .
animals were subjected to a 3-mm diameter craniotomy centered on the 
 left side , 3  mm lateral to the sagittal suture and 1  mm posterior to the bregma .
injury was 
 induced with a weight 3  mm in diameter and weighing 45  g that was dropped through the guide 
 tube from a height of 20  cm .
the exercise groups were 
 put on a wheel running exercise regimen for 15 min per day , at the same time every day . the 
 wheel ( 21  cm diameter and 8  cm width ) ( jd - a-06 type , jeungdo bio & plant co. , ltd . , 

seoul , south korea ) was fixed on one side , and the rat was permitted to run spontaneously 
 within the wheel .
the swr groups were also made to exercise . when sacrificed , the animals were anesthetized with a mixture of 2  ml / kg 50% zoletil and 
 50% xylazine hydrochloride and perfused through the heart with 200  ml of 0.9% nacl solution 
 followed by 4% paraformaldehyde solution .
the brains were removed , maintained in 
 post - fixative overnight , and then sectioned to a thickness of 30  l for 
 immunohistochemistry . in brief , the sections were washed ( 310  min ) in 0.01  m phosphate - buffered saline solution 
 ( pbs ; ph 7.2 ) and incubated for 12  h at room temperature with mouse monoclonal anti - nt-3 
 ( chemicon , temecula , ca , usa ) .
the antibody was diluted to 1:200 with a solution of triton 
 x-100 and normal donkey serum .
after incubation in primary antibody , the sections were 
 rinsed ( 310  min ) in pbs , incubated for 90  min at room temperature with anti - mouse igg 
 ( vector laboratories inc , burlingame , ca , usa ) , and diluted to 1:25 with a solution of 
 triton x-100 and normal donkey serum .
after incubation in secondary antibody , the sections 
 were rinsed ( 310  min ) in pbs , and then incubated for 1  h at room temperature with a 
 vectastain elite abc - kit ( vector laboratories inc . ,
the sections were 
 rinsed with pbs and incubated for 10  min in 0.04  mg of 3,3-diaminobenzidine ( dab , in 200  ml 
 distilled water ) .
the sections were then incubated for 1  min in dab solution with 35% 
 h2o2 .
the dab sections were rinsed with pbs ( 310  min ) to halt the 
 chromogen reaction , wet - mounted onto gelatin / chromium - coated slides , and allowed to air - dry 
 overnight .
the sections were then dehydrated through a series of alcohols , soaked in xylene , 
 and cover - slipped with clarion ( biomedia , usa ) .
after the animals were anesthetized and sacrificed , their brains were extracted , immersed 
 for 10  min in cold pbs , and sliced into 2.0-mm - thick sections .
the sliced brain samples were 
 incubated in 2% 2,3,5-triphenyltetrazolium chloride ( ttc , sigma - aldrich , st .
louis , mo , usa ) 
 dissolved in pbs for 20  min at 37  c and subsequently transferred to 4% paraformaldehyde 
 solution for fixation .
brain damage was revealed by a lack of ttc staining , which indicated 
 that the tissues were dehydrogenase deficient .
the volume of brain damage was measured in 
 each slice , and the injured area was determined in terms of a percentage of total brain area 
 pixels .
the brains of each group were collected , washed twice in pbs , and then homogenized and 
 lysed with buffer ( 137  mm nacl , 8.1  mm na2hpo4 , 2.7  mm kcl , 1.5  mm 
 kh2po4 , 2.5  mm edta , 1  mm dithiothreitol , 0.1  mm pmsf , 10  g / ml 
 leupeptin [ ph 7.5 ] ) for 30  min on ice . the lysates were centrifuged for 10  min at 15,000  rpm 
 at 4  c , and the protein concentration was determined as described previously12 .
equal amounts of protein ( 40  g ) were 
 resolved via 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and 
 transferred to nitrocellulose membranes .
the blots were washed with tbst ( 10  mm tris - hcl [ ph 
 7.6 ] , 150  mm nacl , 0.05% tween 20 ) , blocked with 5% skim milk for 1 h , and then , incubated 
 with the appropriate primary antibodies at the dilutions recommended by the suppliers .
the 
 membranes were washed , and the primary antibodies were detected using horseradish 
 peroxidase - conjugated goat anti - rabbit igg or goat - anti mouse igg .
the bands were then 
 visualized via enhanced chemiluminescence ( amersham pharmacia biotech , piscataway , nj , 
 usa ) .
the ladder walk device consisted of a 1  m long wooden - rung walkway with a varying distance 
 of 1.5  cm between rungs .
the 
 animals could traverse the walkway freely and without reinforcement . during the third 
 session ,
each group was tested at week 1 
 after surgery , and then once per week for the following 2 weeks . in a single testing 
 session
, the animals crossed the walkway three times and received a score for the number of 
 fore and hind limb feet faults per each 10 steps .
only the lesion - affected limbs were 
 analyzed , and a foot fault was characterized as a total miss or slip from the rung , or a 
 misplacement of the paw on the rung .
the results were expressed as the means  standard error ( se ) or deviation ( sd ) .
all 
 experiments were analyzed via analysis of variance , and some experiments were analyzed via 
 comparisons of the treatment mean with the controls using the bonferroni - dunn test . 

all analyses were 
 performed using spss for windows ( v. 12.0  k , spss inc . , chicago , il , usa ) .
skilled ladder rung walking test was conducted for the with forelimbs ( a ) and hind 
 limbs ( b ) .
the error ratio 
 ( % ) is the mean  sd of the values to measure during the test .
* p < 0.05 versus before exercise between periods ; 
  p < 0.05 versus 1 week after exercise between periods ;  p < 0.05 versus 2 weeks 
 after exercise between periods ;  p < 0.05 versus con between the group at 3 weeks 
 after exercise   the effect of spontaneous wheel running after tbi on nt-3 expression in 
 immunohistochemistry . to confirm nt-3 expression , immunohistochemistry ( a ) was conducted 
 in the nrm ( a ) , tbi ( b ) , and swr ( c ) groups as described in the materials and methods 
 section . each example shown is representative of three experiments . scale bar = 200 
 m . to confirm nt-3 expression
, western blotting analysis was conducted as described in the 
 materials and methods section .
the optical density values denote the mean  se of three experiments for 
 each condition determined from densitometry relative to -actin , respectively . 

statistical analysis was performed by using one - way anova . * p < 0.05 versus nrm .  p 
 < 0.05 versus tbi .
skilled ladder rung walking tests were conducted to evaluate the effects of spontaneous 
 wheel running after tbi , which is sensitive to alterations of motor function after sensory 
 motor cortex damage ( table 1 ) .
the error ratio 
 was measured in the forelimbs ( a ) and hind limbs ( b ) .
the results for both affected limbs in 
 all three groups showed a significant decrease in the error ratio compared with the before 
 exercise period as time passed significantly ( p<0.05 ) .
comparison with the nrm group 
 showed that there was the more increase in the error ratio in the tbi group .
moreover , the 
 increase tended to be less profound in the swr group than in the tbi group ( p<0.05 ) . to confirm the ischemic volume of the brain - injured area ,
ttc staining was conducted in 
 the nrm , tbi , and swr groups as described in the materials and methods section .
the 
 results provide the percentage of injured area in total brain area pixels and represent 
 the mean  se .
p < 0.05 versus tbi . immunohistochemistry and immunoblotting for nt-3 expression were performed in each group 
 ( fig . 1 and table 2 ) .
it was observed that the tbi - induced reduction in nt-3 expression 
 increased wheel running for 3 weeks . a more profound significant increase in nt-3 expression 

was noted in the swr group than in the nrm group ( p<0.05 ) . to confirm the area of the brain injured by tbi ,
no ischemic areas were detected in the nrm 
 group . in the swr group ,
the ischemic area was statistically significant decrease than in 
 the tbi group ( p<0.05 ) .
tbi may induce scalp laceration , skull fracture , contusion , intracranial hemorrhage , 
 blood - brain barrier disruption , and diffuse axonal injury according to the features , 
 direction , and degree of force applied to the head14 , 
 15 .
brain and whole body physiological 
 changes engendered by primary mechanical injuries are associated with a series of 
 morphological and cellular changes as secondary injuries1 , 16 .
pathological pathways that may 
 potentially harm brain cells include excitotoxicity , free radical formation , inflammation , 
 and apoptosis , among others .
auto - protective mechanisms include the formation of heat shock 
 proteins ( hsps ) , anti - inflammatory cytokines , growth factors ( gfs ) , and endogenous 
 antioxidants17 . among endogenous protective mechanisms ,
growth factors are special endogenous signaling 
 proteins that promote survival , division , growth , and the differentiation and morphological 
 plasticity of neural cells .
moreover , some of these growth factors are required for certain 
 trophic or all of these functions in selected neural populations of the nervous system18 .
growth factors can generally be divided 
 into four major groups according to their receptor systems and their downstream signal 
 transduction pathways .
many types of growth factors are induced very early after brain 
 lesions , including ngf , bdnf , glial cell line - derived growth factor ( gdnf ) , basic and acidic 
 fibroblastic growth factors ( fgf ) , and members of the transforming growth factor super 
 family ( tgf ) .
nt-3 was detected in the corpus callosum , the hippocampus , cortex ( layer v ) , the primary 
 olfactory cortex , the amygdala , the purkinje cells of the cerebellum and spinal cord , and so 
 on19 .
it has been suggested that this 
 neurotrophin may serve a neuroprotective function by playing a role in the maintenance and 
 survival of neurons after tbi .
thus , it is important to characterize the spatial and 
 temporal patterns and levels of nt-3 expression following experimental brain injury20 . in the present study ,
it was reported that physical exercise , such as treadmill and wheel running exercises , 
 induced increases in the levels of neurotrophic factor , and this fact is consistent with our 
 findings21 . additionally , these 
 neurotrophic factors prevent programmed cell death and promote cell survival in the 
 progression of brain injury22 .
these 
 imply that increased nt-3 expression as a result of wheel running exercise reduced 
 programmed cell death and tissue ischemic injury and induced brain recovery via the 
 regulation of cellular signaling .
the aim of motor training , such as running exercise , is to diminish functional disability 
 and promote brain recovery .
this can be achieved by proper intervention for rehabilitation , 
 which is useful in stimulating therapy - induced recovery in brain - injured patients23 .
exercise , as a simple and widely 
 practiced behavior , can enhance the activation of molecular and cellular cascades and 
 promote brain vascularization , neurogenesis , and functional changes in neuronal structure , 
 as well as resistance to injury8 .
in conclusion , spontaneous running exercise promotes brain recovery and motor function as 
 the result of nt-3 expression .
these facts imply that spontaneous exercise would be needed 
 for rehabilitation of brain - injured patients when properly directed by physical therapist 
 properly .
moreover , it the present study contributes evidence showing that spontaneous 
 rehabilitation training is helpful to biological and functional recovery in brain 
 injured - patients by stimulating the metabolic activation in the brain .
   [ purpose ] the aim of the present study was to investigate the expression of 
 neurotrophin-3 ( nt-3 ) after applying spontaneous wheel running exercises ( swr ) after 
 experimental traumatic brain injury ( tbi ) . 
 [ subjects and methods ] thirty male 
 sprague - dawley rats were divided into 3 groups ; 20 rats were subjected to controlled 
 cortical impact for tbi , and then , animals were randomly collected from the swr group and 
 subjected to wheel running exercise for 3 weeks . 
 ten rats were not subjected to any injury 
 or running exercise to compare with the effect of tbi and swr . 
 immunohistochemistry , 
 western blotting , skilled ladder rung walking test , and 2,3,5-triphenyltetrazolium 
 chloride staining analysis for the evaluation of nt-3 expression were used to assess brain 
 damage and recovery . 
 [ results ] the tbi - induced decrease in nt-3 expression was recovered 
 by wheel running exercise . moreover , decreased ischemic volume and progressive 
 neurobehavioral outcome were observed in the swr group . 
 [ conclusion ] spontaneous running 
 exercise promotes brain recovery and motor function through an increase in expression of 
 nt-3 . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION