recently , there have been numerous reports published concerning the effect of exercise on central nervous disorders in experimental models .
for instance , treadmill exercise decreased lesion size and expression of caspase-3 in an intracerebral hemorrhage model ( lee et al . , 2003 ) .
some previous studies suggested that functional recovery after stroke could be improved by complex motor training rather than simple repetitive exercise ( jones et al .
physical activity in an enriched environment substantially improves sensorimotor function outcome after brain injury ( johansson , 1996 ; johansson and ohlsson , 1996 ) .
in addition , previous studies suggested that brain - derived neurotrophic factor ( bdnf ) has a role in plasticity following cerebral infarction .
post - ischemic intravenous bdnf treatment improves functional motor recovery after photo thrombotic stroke and induces widespread neuronal remodeling ( schbitz et al . , 2004 ) .
improvements in motor behavior index were found with an increasing level of bdnf and the level of trkb proteins in contralateral hemisphere was increased by treadmill exercise ( kim et al . , 2005 ) .
glial cell line - derived neurotrophic factor ( gdnf ) is another potent neuroprotective factor produced chiefly by astrocytes .
intracerebroventricular and intraparenchymal administrations of gdnf reduced infarct sizes induced by transient middle cerebral artery ( mca ) occlusion ( wang et al . , 1997 ) .
however , few reports have described the relationship between functional recovery and gdnf after cerebral infarction .
we have reported on the expression of gdnf in an accelerated phase of functional recovery in a rat model of cerebral infarction ( horinouchi et al . , 2007 ) .
however , the relationships among exercise , functional recovery , and the expression of gdnf protein have to be investigated .
the present study investigates the expression of gdnf protein with functional recovery upon exercise after photochemically induced cerebral infarction .
animals were maintained at a constant temperature under a 12:12 h light : dark cycle and allowed free access to food and water until the experiments began .
this study was carried out in accordance with the guidelines for animal experimentation of the graduate school of medical and dental sciences , kagoshima university , japan .
initially , to investigate the effects of exercise on functional recovery after photochemically induced cerebral infarction , 22 adult male wistar rats ( 231  12 g ) were used .  
these animals were housed in plastic cages ( 60 x 40 x 25 cm ) with an environmentally controlled room with a 12/12 hour light - dark cycle , standard rat food and water were provided ad libitum .
these animals were divided into two groups : non - exercise ( n = 11 ) and beam - walking exercise ( n = 11 ) .
rats were deeply anesthetized with sodium pentobarbital ( 40 mg / kg ) . to induce a cerebral infarction , photochemical dye rose bengal ( 20 mg / kg ) was injected into the tail vein and illuminated with green light ( 560 nm ) through the intact cranium for 20 min ( watson et al .
, 1985 ; dietrich et al . , 1987 ; schroeter et al . , 1999 ) .
a green light beam of 10 mm in diameter was irradiated using an mhf - g150lr halogen lamp ( moritex , japan ) through optical fiber to the exposed skull localized to an area 6 mm lateral to the midline and 4 mm posterior to the bregma in order to induce infarction in the right sensorimotor area of the cerebral cortex .  
the rats of the beam - walking group were trained for 20 min every day to walk on a narrow beam ( 122 cm long  2.5 cm wide ) from one day after infarction and those of the non - exercise group were left to follow a natural course .
the locomotion of postoperative rats was evaluated using a beam - walking task with an elevated narrow beam ( 122 cm long x 2.5 cm wide ) ( feeney et al . , 1982 ; abo et al .
the lowest score ( a score of  1  ) was given if the animal was unable to traverse the beam and could not place the affected hind limb onto the horizontal surface .
a score of  2  was given if the animal was unable to traverse the beam but placed the affected hind limb on the horizontal surface of the beam and maintained balance .
a score of  3  was given if the animal traversed the beam while dragging the affected hind limb .
a score of  4  was given if the animal traversed the beam and at least once placed the affected hind paw on the horizontal surface of the beam .
a score of  5  was given if the animal used the affected limbs in fewer than half of its steps along the beam .
the highest score ( a score of  7  ) was given if the animal traversed the beam normally with no more than two foot slips .
the day prior to the photochemical procedure , animals in all groups were given a single beam - walking test to confirm that their performance merited a score of 7 .
the performance of each animal was evaluated by two different observers . in the post - infarct portion of the study , rats that achieved a score of "
days on which a score of  7  was achieved by the non - exercise group were compared with those of beam - walking group using the unpaired - sample student 's t test .
statistically significant differences were considered when the p value was less than 0.05 . to investigate the expression of gdnf protein , 42 adult wistar rats ( 232  9.5 g ) were used . at various days ( non - exercise group ; 1 , 3 , 5 , and 7 days ; n = 6 per day , beam - walking group ; 3 , 5 , and 7 days ; 6 animals for each point ) after photochemical infarction , the brains were removed under deep anesthesia .  
animals were perfused transcardially with 300 ml of 4 % paraformaldehyde in 0.1 m phosphate buffer at ph 7.4 under deep anesthesia .
the brains were removed and post - fixed in perfusion fixative overnight and then left in 20 % buffered sucrose at 4c .
the sections prepared as described earlier were stained using the peroxidase - antiperoxidase ( pap ) technique .
the sections were incubated for 15 min in 0.1 % hydrogen peroxide ( santoku chemical industry , japan ) in ethanol in order to inactivate the endogenous peroxidase activity , and rinsed three times with phosphate - buffered saline ( pbs ) at a ph of 7.4 for 5 min .
the sections were then incubated for 1 h in 10 % normal swine serum ( dako a / s , denmark ) in pbs to block the nonspecific binding of gdnf antiserum to the tissue .
sections were incubated for 24 h with the primary gdnf antibody ( final antibody dilution 1:200 , santa cruz biotechnology , usa ) , rinsed for 5 min with pbs three times , and incubated for 1 h with the swine anti - rabbit secondary antibody ( dilution 1:100 , dako a / s , denmark ) .
the sections were then incubated for 1 h with pap ( 1:100 , dako a / s , denmark ) and rinsed three times with pbs for 5 min .
sections were washed twice for 5 min in 0.05 m tris - hcl buffer at ph 7.6 .
this was followed by a dark - violet to black color reaction with 0.02 % 3,3'-diaminobenzidine ( dab : wako pure chemical industries , osaka , japan ) and 0.6 % nickel ammonium sulfate ( nacalai tesque , kyoto , japan ) in 0.05 m tris - hcl buffer at ph 7.6 containing 7 l of h2o2 ( nickel - enhanced dab reaction ) for 9 min at room temperature .
the sections were rinsed twice for 5 min in 0.05 m tris - hcl buffer at ph 7.6 , dehydrated through a series of ethanols ( 90 % , 95 % , 99 % ) , cleared in xylene , and mounted with coverslips .
the number of gdnf - like immunoreactive cells was counted with a computer - assisted image analyzer using the public domain software scion image program ( scion corp . ,
one - way analysis of variance ( anova ) was used to compare the numbers of gdnf immunoreactive cells in non - exercise and beam - walking exercise groups over time .
post hoc analyses were performed with the least significant difference ( lsd ) test when the f ratio for the anova was significant .
tokyo , japan ) . a p value < 0.05 was considered to be statistically significant .
paralysis after infarction was evaluated using the beam - walking task . in this study ,
rats that achieved a score of  1  24 h after the procedure were used .
functional recoveries of non - exercise and beam - walking groups evaluated with the beam - walking task are shown ( figure 1(fig .
days on which a score of  7  was achieved by the non - exercise and beam - walking groups are shown ( figure 1a(fig .
1 ) ) . functional recovery of the beam - walking exercise group was significantly earlier than that of the non - exercise group ( p < 0.01 ) .
recoveries of the beam - walking score in non - exercise and beam - walking exercise groups after the photochemical procedure are shown ( figure 1b(fig .
subsequently , in the beam - walking exercise group , recovery was remarkable , and the average score became  6.7  6 days after infarction . in the beam - walking exercise group in particular ,
rats recovered remarkably at 2 - 4 days after the operation . however , in the non - exercise group , recovery was gradual and the average score became  6.7  14 days after infarction .
localizations of gdnf - positive cells were almost equal within cortical layers ii to vi , with limited expression of gdnf in layer i. in the beam - walking exercise group , the number of gdnf - like immunoreactive cells reached a maximum on day 3 and then decreased over time ( figure 2(fig .
the number of gdnf - like immunoreactive cells reached a maximum at day 1 and then decreased over time . at 3 days after infarction , the number of gdnf - positive cells in the temporal cortex surrounding the infarction ( 446  30 / mm ) of the beam - walking exercise group was significantly higher than that of the non - exercise group ( 384  7.5 / mm ) ( p < 0.05 ) ( figure 3(fig .
, there was no statistically significant difference between the non - exercise group and the beam - walking exercise group .
in previous studies , animals exhibited significantly improved motor performance after performing various kinds of exercise .   in many studies , treadmill training was used for exercise in various contexts ( yang et al . , 2003 ; wang et al . ,
there have been studies comparing the quality of training with the amount of training . in other studies ,
experimental exercise was performed with wheel running for voluntary exercise ( kleim et al . , 2002 ; marin et al . , 2003 ) and rotarod for complex motor skills training ( ding et al . , 2002 , 2004 ) .
an enriched environment ( in a large cage with ladders , rope , a chain , and so on ) promoted functional recovery after ischemic injury in rat ( biernaskie and corbett , 2001 ; risedal et al . , 2002 ) .
the present results showed that the functional recovery of the beam - walking exercise group was observed significantly earlier than that of the non - exercise group .
our results indicated that the period of functional recovery was reduced by about half by the beam - walking exercise .
additionally , gdnf expression was increased in the exercise group compared with that in the control group on day 3 after infarction .
there are few studies that have examined gdnf levels and functional recovery after cerebral infarction .
for example , focal ischemia after transient mca occlusion induces cell proliferation , and exogenous gdnf increases the cell proliferation following focal ischemia in adult rats ( dempsey et al . , 2003 ) .
cerebral ischemia triggers an increase in gene expression of not only gdnf but also its receptors , c - ret and gfr-1 ( arvidsson et al . , 2001 ) . in this study
, the present results showed that the number of gdnf - positive cells in the temporal cortex surrounding the infarction of the beam - walking exercise group was significantly increased compared with that in the non - exercise group .
these findings suggested that the beam - walking task enhanced the expression of gdnf protein at an early stage after cerebral infarction .
interestingly , in the beam - walking exercise group , motor function was recovered remarkably and the number of gdnf - like immunoreactive cells reached a maximum at day 3 .
these results suggested that the expression of gdnf protein was induced by training and that functional recovery was promoted . over the last decade
a number of molecular mechanisms by which brain cells die , or survive , after focal cerebral ischemia have been unraveled . in the core of ischemic lesions ,
brain tissue undergoes necrosis ; however , in penumbra , biochemical and molecular processes determine the survival of tissue . within minutes after focal ischemia
, excitotoxic mechanisms lead neurons and glia to cell death . in the following days after the onset of infarction , excitotoxic triggers contribute to the death of tissue .
such events include peri - infarct depolarization and the more delayed mechanisms of inflammation and apoptosis ( dirnagl et al . , 1999 , 2003 ) .
previous studies suggested that administration of gdnf reduced infarct sizes induced by transient mca occlusion ( wang et al .
therefore , it is possible that gdnf has a role of anti - excitotoxicity or anti - inflammation and anti - apoptosis in areas of penumbra .
however , in this study , cerebral ischemia was induced by irradiation with green light .
most of the blood vessels exposed to the green light became occluded , thus abolishing all collateral circulation .
therefore , rats of photochemical infarction model have few areas of penumbra ( abo et al . , 2001 ) .
in this study , the role of gdnf protein after photochemical infarction might be related to the plasticity of the brain rather than nerve protection .
recently , experimental study indicated that gdnf and its receptor gfr-1 have a function in synapse formation in hippocampal neurons ( ledda et al . , 2007 ) .   in conclusion
, this study suggested that exercise enhances functional recovery from cerebral infarction and induces increased expression of neurotorophic factor gdnf .
rehabilitation soon after cerebral infarction could enhance cortical repair and regeneration after infarction in clinical practice .  
this study was supported in part by a grant - in - aid for scientific research ( no .
18500410 , 2006 ) from the ministry of education , science , sports and culture , japan .
 exercise has been considered to affect the functional recovery from central nervous damage . 
 neurotrophic factors have various effects on brain damage 
 . however , the effects of exercise for expression of gdnf on functional recovery with brain damage are not well known . 
 we investigated the difference in functional recovery between non - exercise and beam - walking exercise groups , and the expression of gdnf in both groups after photochemical infarction . 
 adult male wistar rats ( n = 64 ) were used . 
 animals were divided into two groups : non - exercise ( n = 35 ) , and beam - walking exercise ( n = 29 ) . 
 all rats underwent surgical photochemical infarction . 
 the rats of the beam - walking group were trained every day to walk on a narrow beam after a one - day recovery period and those of the non - exercise group were left to follow a natural course . 
 animals were evaluated for hind limb function every day using a beam - walking task with an elevated narrow beam . 
 the number of gdnf - like immunoreactive cells in the temporal cortex surrounding the lesion was counted 1 , 3 , 5 , and 7 days after the infarction . 
 functional recovery of the beam - walking exercise group was significantly earlier than that of the non - exercise group . at 3 days after infarction 
 , the number of gdnf - positive cells in the temporal cortex surrounding the infarction was significantly increased in the beam - walking exercise group compared with that in the non - exercise group . in the exercise group , motor function 
 was remarkably recovered with the increased expression of gdnf - like immunoreactive cells . 
 our results suggested that a rehabilitative approach increased the expression of gdnf and facilitated functional recovery from cerebral infarction . 
Introduction
Materials and Methods
Results
Discussion
Note