stroke is defined as a sudden disturbance in the blood supply to the brain caused by 
 hemorrhage or ischemia that provokes an acute inflammatory response and secondary cascade of 
 neurodegenerative processes leading to cell death in the weeks and months following the 
 injury1 .
the symptoms , which vary 
 according to the site and extent of brain damage , can be severely disabling and may result 
 in impaired motor and sensorimotor domains and cognitive task performance .
physical 
 rehabilitation therapy remains the first - line intervention strategy for attenuating chronic 
 impairments in sensory - motor function2 . during skilled reaching ,
an individual reaches for a food item that has been placed in the 
 mouth for eating .
this natural human behavior the first complex behavior displayed by human 
 infants is engaged in daily by adults and requires no special training3 . among the many motor impairments that follow neurological 
 damage in humans , the loss of manual dexterity , i.e. , skilled use of the hands , is one of 
 the most debilitating .
the adult brain responds to physical exercise and environmental stimulation with increased 
 neurogenesis in the dentate gyrus6 .
these 
 new cells mature locally into granule neurons , form dendritic arbors and axonal projections , 
 and functionally integrate into the existing hippocampal network7 .
several studies have demonstrated an association between 
 increased dentate neurogenesis and improved performance in hippocampal learning tasks8 .
gap-43 , a growth- and plasticity - associated protein , is developmentally regulated , 
 concentrated in growth cones , excluded from dendrites , and selectively transported to axons 
 and their terminals .
protein kinase c ( pkc ) regulates gap-43 function via phosphorylation at 
 ser-41 in rats , mice , and humans and at ser-42 in chicks9 .
phosphorylation of the pkc site is highly correlated with memory 
 formation and the synaptic plasticity of long - term potentiation ( ltp)10 .
for example , enriched 
 rehabilitation ( er ) , which is the coupling of environmental enrichment and daily reach 
 training , improves forelimb use following middle cerebral artery occlusion ( mcao ) in 
 rats12 .
therefore , the principal 
 objective of the present study was to assess the effects of a skilled reaching task on 
 cognition , as indexed by the pattern of gap-43 expression in the hippocampus , following 
 intracerebral hemorrhage ( ich ) in rats .
all procedures were performed according to the protocols established by the animal 
 experiment committee of daegu university , based on the nih guidelines for the care and use 
 of laboratory animals ( nih , 1996 ) .
sixty male sprague - dawley rats ( aged 12 weeks ) , weighing 
 250270 g , were maintained on a 12-hour on/12-hour off light / dark cycle with ad 
 libitum access to food and water .
rats were anesthetized with 2  ml / kg of a 50% zoletil/50% xylazine hydrochloride mixture and 
 placed in a stereotactic frame . a needle ( hamilton instrument syringe , 75 n
, 5 ul ; hamilton 
 company usa , reno , nv , usa ) was implanted into the left striatum ( 3.5  mm lateral and 7  mm 
 deep relative to the bregma ) , according to the atlas of paxinos and watson13 .
five minutes after insertion of the 
 needle , collagenase ( type vii ; sigma - aldrich coporation , st .
louis , mo , usa ; 0.12 u in 1  l 
 nacl , 0.9% ) was injected over the course of the following 5  min using a pump 
 ( precidor , infors ht , basel , switzerland ) .
the needle was left in place for 
 another 5  min and then removed slowly .
body temperature was monitored 
 throughout surgery using a rectal probe and maintained at 37.5  0.5  c using a 
 homoeothermic blanket control unit ( harvard apparatus , edenbridge , uk ) .
animals were 
 randomly assigned to either the control ( n = 30 ; con ) or skilled reaching training ( n = 30 ; 
 srt ) group . following surgery , animals were returned to their home cage , which was maintained at 
 2628  c to allow recovery from the anesthesia .
the animals in the srt group were trained on a single pellet - reaching task modified from 
 that of whishaw and pellis14 .
boxes were 
 made of clear plexiglas ( 45  14   35  cm ) .
a 1-cm - wide vertical slit was placed in the 
 center of the front wall . on the exterior of the wall in front of the slit ,
two indentations on the surface of the shelf , 
 located 1  cm from the inside of the wall and aligned with the edges of the slit ( 1  cm 
 apart ) , ensured that the rats could reach the pellet with either paw .
a pellet was placed in 
 the indentation contralateral to the limb with which the rat preferred to use for reaching . 

the lateral placement of the food pellet prevented the rats from using their tongue to 
 obtain the pellet and also prevented use of the non - preferred paw to grasp the food14 .
a metal bar was positioned in front of 
 the slit and approximately 1  cm from the horizontal shelf , thereby preventing the animal 
 from dragging the food after reaching and preventing performance of a full and correct 
 grasping movement .
the first 5 days comprised a shaping phase during which a number of food pellets were 
 placed on the shelf to attract the animals . at the end of this period , rats could reliably 
 retrieve the food from the shelf using the preferred limb in each trial .
subsequently , a 
 single pellet was placed in the food indentation contralateral to the rat s preferred paw , 
 and the training phase , consisting of 25 trials , was repeated three times during each daily 
 session on the 7 days prior to surgery15 .
animals were sacrificed 1 , 2 , and 4 weeks after ich via anesthesia using 2  ml / kg of a 50% 
 zoletil/50% xylazine hydrochloride mixture .
comparisons between treatment and control means 
 were performed via the bonferroni - dunn test .
a pattern of gap-43 expression in the hippocampus was observed for each group following 
 extraction of the brains .
optical density values determined at 1 , 2 , and 4 weeks post 
 intracranial hemorrhage are presented in table 
 1table 1.gap-43 expression in the hippocampusgrouprelative optical density ( percentage of 
 control)1 week2 weeks4 weekscon10014.058.88.549.77.6*srt10025.8156.923.1197.723.5*the amounts of gap-43 were detected via western blotting with anti - gap-43 as 
 described in the materials and methods section .
the values represent the meansse of three independent 
 experiments conducted in triplicate dishes . * p<0.05 vs. 1 week groups .
# p<0.05 
 vs. 2 weeks and 4 weeks groups , respectively , in con .. gap-43 expression was shown to increase in the srt group in accordance with 
 greater time elapsed ; however , it decreased in the con group ( p < 0.05 ) .
statistical 
 significance was only reached after 4 weeks ; additionally , a significant difference was 
 observed between the 1- and 4-week groups ( table 
 1 ) . at 1 week
post injury , there was no significant difference between the con and 
 srt groups ( p > 0.05 ) , but at 2 and 4 weeks , significant differences were observed ( p 
 > 0.05 ) ( table 1 ) . the amounts of gap-43 were detected via western blotting with anti - gap-43 as 
 described in the materials and methods section .
the values represent the meansse of three independent 
 experiments conducted in triplicate dishes . * p<0.05 vs. 1 week groups .
# p<0.05 
 vs. 2 weeks and 4 weeks groups , respectively , in con .
this study demonstrated that specific motor training via srt and following experimental ich 
 promotes a histological change in the hippocampus associated with the time course of gap-43 
 expression .
the model of ich used in the present study consisted of an intrastriatal injection of 
 collagenase , a proteolytic enzyme that interrupts the basal lamina of cerebral blood 
 vessels , thus causing intraparenchymal bleeding16 . in addition to its quick and easy execution , another feature of 
 this model is that hemorrhage is spontaneous in nature , in contrast to the alternative model 
 of ich , which involves autologous blood injection .
furthermore , a recent study comparing 
 collagenase to blood infusion models of ich in rats demonstrated that neurological deficits 
 resolve more rapidly and completely in the blood . despite similar initial hematoma volumes , 
 rats infused with collagenase
did indeed exhibit greater neurological impairment at every 
 time point evaluated following striatal injection and remained significantly impaired at 28 
 days ( the endpoint of the study ) .
consequently , the collagenase model should be preferred in 
 studies assessing long - term functional outcomes17 .
several recent studies in mice and rats clearly demonstrated that dentate neurogenesis is 
 strongly stimulated by voluntary exercise in a running wheel18 , 19 and that the new neurons 
 are functionally integrated into the hippocampal circuitry7 .
another study showed that this training increased the physical 
 fitness of the animals , stimulated the release of local hippocampal and circulating 
 neurogenic growth factors , and enhanced angiogenesis in the hippocampal vasculature8 .
wurm et al.20 observed that daily 
 training of a single forelimb greatly improved dentate neurogenesis in the healthy brain . 

reaching training significantly increased the survival rate of newly developed neurons , 
 whereas environmental enrichment did not influence dentate neurogenesis .
in addition , 
 following cortical infarcts in the forelimb sensorimotor cortex , both rehabilitative 
 training of the impaired forelimb and environmental stimulation increased dentate 
 neurogenesis .
the present study indicated that gap-43 expression in the hippocampus was significantly 
 reduced by ich at 4 weeks in the con group . in contrast , the srt group exhibited a robust 
 increase in gap-43 expression , but the difference in expression between the two groups was 
 statistically significant only at 2 and 4 weeks .
mirescu et al.21 reported that animals with cortical infarcts in the 
 forelimb sensorimotor cortex exhibited reduced reaching success during the first 510 days 
 following formation of the lesion , which might serve to stress the animals and thereby 
 impair the neurogenic response .
moreover , wurm et al.20 observed that skilled forelimb training effectively stimulated 
 dentate neurogenesis and spatial learning in both infarcted and healthy brains ; however , 
 this training - induced increase in neurogenesis was reduced following cortical infarcts .
no 
 significant difference was noted between the con and srt groups at 1 week in this study .
auriat et al.22 reported that 
 rehabilitation comprising an enriched environment and skilled reach training improved 
 recovery after ich in rats via a plasticity response ( e.g. , increased dendritic growth ) that 
 did not involve neurogenesis .
the growth - associated protein gap-43 is developmentally 
 regulated , concentrated in growth cones , and highly correlated with memory formation and the 
 synaptic plasticity of ltp9 , 10 .
the present study indicates that short daily sessions of skilled reaching training 
 significantly increase gap-43 expression in the hippocampus at 2 and 4 weeks post ich .
the 
 present findings suggest that increases in gap-43 expression in the hippocampus following 
 skilled reaching training might result in enhanced cognition and neural plasticity post ich . 

although further study of therapeutic interventions is necessary to confirm their 
 neurological mechanisms , the results of this study may nevertheless help to establish novel 
 rehabilitative strategies .
 [ purpose ] the primary objective of this study was to assess the effects of a skilled 
 reaching task on cognition , as indexed by the pattern of gap-43 expression in the 
 hippocampus , following intracerebral hemorrhage ( ich ) in rats ( when the hippocampus plays 
 a critical role in spatial memory and learning ) . [ subjects and methods ] the model of ich 
 used in the present study involved intrastriatal injection of collagenase . 
 sixty male 
 sprague - dawley rats ( aged 12 weeks ) were randomly assigned to either a control ( n = 30 ; 
 con ) or skilled reaching training group ( n = 30 ; srt ) . the srt group were trained 5 days 
 per week for 4 weeks following ich . 
 animals were sacrificed 1 , 2 , or 4 weeks after ich . 
 
 western blot analysis was used to evaluate gap-43 expression . 
 [ results ] gap-43 expression 
 was increased in the srt group , in accordance with greater elapsed time , but decreased in 
 the con group . at 1 week 
 post injury , there were no significant differences between the 
 con and srt groups . 
 however , there were significant differences at both 2 and 4 weeks . 
 
 [ conclusion ] the present findings suggest that increased gap-43 expression in the 
 hippocampus following skilled reaching training may result in enhanced cognition and 
 neural plasticity following ich . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION