The Role And Mechanism Of Preconditioning Lesion On The Regeneration Of Injured Spinal Cord

PartⅠThe role of preconditioning lesion in the regeneration of injured spinal cordObjective:To observe the effect of preconditioning lesion(sciatic nerve transection or L5 ventral root transection(L5VRT)one week prior to dorsal column injury)on the regeneration of fasciculus gracilis and fasciculus cuneams in the injured spinal cord.Methods:Adult female Sprague-Dawley(SD)rats were randomly divided into normal group,spinal cord injury group and combined injury (preconditioning lesion combined with spinal cord injury)group.The rats survived for 2 weeks after spinal cord injury.FB was injected immediately after spinal cord injury into 5mm rostra1 to the spinal cord lesion site to retrogradely label sensory neurons in the dorsal root ganglia (DRG).CTB was injected into the sciatic nerve to anterogradely label ascending axons 3 days before sacrifice.Results:In the normal spinal cord,CTB-ir was observed in the gracile and cuneatus nucleus.After spinal cord injury,no CTB labeled nerve terminals were observed in the gracile and cuneatus nucleus,axons were stopped before the cavity and no axons were found in the glia scar of injured spinal cord.In the combined injury group,the CTB-ir axons were found caudal to,within and rostral to the cavity of injured spinal cord.Moreover,the number of CTB-labeled axons in the glia scar in the combined injury group was greater than those in the spinal cord injury group.In the ipsilateral DRG,the number of FB-labeled sensory neurons was significantly increased in the combined injury group compared to the spinal cord injury group.Conclusion:Preconditioning lesion promotes regeneration of ascending sensory tract in the injured spinal cord. PartⅡThe mechanism of preconditioning lesion on the enhanced regeneration of injured spinal cordObjective:To investigate the mechanism of preconditioning lesion on the enhanced regeneration of injured spinal cord,the expression of brain-derived neurotrophic factor(BDNF)was observed in spinal cord and DRG and the regeneration of the injured spinal cord was evaluated after preconditioning lesion with BDNF antiserum treatment.Methods:Adult female Sprague-Dawley(SD)rats were randomly divided into normal group,preconditioning lesion group,spinal cord injury group and combined injury group.Rats in the combined injury group were intraperitoneally injected with BDNF antiserum or normal sheep serum(NSS).Animals were allowed to survive for 1,7 and 14 days. Immunohistochemistry,in situ hybridization and ELISA were used to detect the expression of BDNF in the injured spinal cord,lumbar spinal cord and bilateral lumbar 4,5 DRG.And also,BDNF expression was detected in FB labeled neurons of the DRG.The regeneration was evaluated with FB and CTB labeling in the combined injury group after BDNF neutralization.βⅢ-tubulin immunohistochemistry was carried out to evaluate the neurite growth of the cultured DRG neurons and explants after BDNF neutralization.Results:It was found that the expression of BDNF protein and mRNA in the DRG,lumbar spinal cord and injured spinal cord were up-regulated in the preconditioning lesion group.Compared with the spinal cord injury group,more BDNF positive fibers with a club-like shape in the white matter were seen in the spinal cord segment caudal to lesion site ipsilateral to preconditioning lesion side in the combined injury group.Besides,in the ipsilateral DRG,there were more FB+ neurons, most of which were immunoreactive for BDNF,in the combined injury group than in the spinal cord injury group.After treated with BDNF antiserum,axons were stopped before the cavity and only very few axons grew short distance into the lesion site in the combined injury group.The length of axons from the caudal boundary of the lesion site towards the lesion site in the NSS group was longer than that of BDNF antiserum treated rats.In the ipsilateral DRG,the number of FB-labeled regenerating neurons was significantly reduced in the BDNF antiserum treated group,compared to those in the NSS group.Neurite length of cultured DRG neurons or explants in the Matrigel was significantly shorter in the BDNF antiserum treated group than that in the NSS group.Conclusion:The up-regulation of BDNF maybe involved in the enhanced regeneration of injured spinal cord induced by preconditioning lesion. PartⅢThe mechanism of endogenous BDNF in the enhanced regeneration of injured spinal cord after preconditioning lesionObjective:To study the mechanism of endogenous BDNF in the enhanced regeneration of injured spinal cord after preconditioning lesion by observing the expression of GAP-43,p-Erk and RhoA with or without neutralizing endogenous BDNF.Methods:Adult female Sprague-Dawley(SD)rats were randomly divided into preconditioning lesion group,spinal cord injury group and combined injury group.Rats in the preconditioning lesion group and combined injury group were intraperitoneally injected with BDNF antiserum or normal sheep serum(NSS).Immunohistochemistry, immunoblotting or RhoA pull-down assays were performed to study the expression of GAP-43 and p-Erk or the activation of RhoA in the different groups with or without neutralizing endogenous BDNF.Results:One week after preconditioning lesion in the NSS treated animals,the number of GAP-43+ and p-Erk+ neurons was significantly increased in the ipsilateral DRG.The number of GAP-43+ and p-Erk+ neurons in the ipsilateral DRG was reduced after BDNF antiserum treatment.The expression of GAP-43 and RhoA was upregulated at the third days after spinal cord injury and maintained until the 14^thday.The highest level of GAP-43 and RhoA expression presented at the 7^thdays after spinal cord injury.After spinal cord injury,the level of GAP-43 was higher but RhoA was lower in the combined injury group than that of the spinal cord injury group.After treatment with the BDNF antiserum,the expression of GAP-43 was reduced and RhoA was upregulated after BDNF antiserum treatment in the combined injury group.Conclusion:The upregulation of GAP-43,activation of p-Erk signal pathway and downregulation of RhoA by increased BDNF maybe involved in the enhanced regeneration of injured spinal cord after preconditioning lesion.