Effects Of Netrin-1 On The Axonal Plasticity And Functional Reconstruction In Rat Brain After Cerebral Infarction

Objects In this study,we use AAV-mediated Netrin-1 over-expression and AAV-mediated Netrin-1 RNAi to regulate the expression of netrin-1 in rat brain after ischemic injury.Aiming at exploring the role and relevant molecular mechanism of Netrin-1 on nerve remodeling and functional reconstructionMethods 1.The establishment,Selection and packing of the AAV-mediated Netrin-1 over-expression and AAV-mediated Netrin-1 RNAi vector. 2.The SPF 8 weeks male SD rats were divided into 5 groups randomly:sham group(n=12),Mcao,group(n=12),mcao+AAV-empty-viral(n=12),mcao+nt-1overexpression(M+NT-1-AAv,n=12),maco +Nt-1 RNAi(M+NT-1-AAV-RNAi,n=12) 3.The establishment of Middle cerebral artery occlusion models,(MCAO),based on the modified Zea-longas method. 4.AAV-netrin-1 over-expression vectors,AAV-empty vectors,AAV-NT-1-RNAi vectors were respectively injected 2 weeks before Mcao in brain regions medial and posterior to the ischemic lesion in the ipsilateral striatum at the respective stereotaxic coordinates. We use fluorescence microscope to observe the virus transfection,western-blot to observe the regulation of NT-1. 5.The NDS,modified neurological severity score(m NSS) and modified adhesive removal test were performed 1 day?3 days?7 days?10days and 14 days after MCAO to understand the changes of nerve function in each group rats. 6.The brains are removed after 14 days after mcao,Protein expression level of NT-1,NF-200, syn,PSD-95 were tested by Western-Bolt. 7.The expression level of NF-200 in the tissue corresponding to the peri-infract cortex was detected by immunofluorescent stainingResults 1.Western-blot shows that AAV-mediated Netrin-1 over-expression vector can significantly increased the level of Netrin-1 around the ischemic brain tissue while AAV-mediated Netrin-1 RNAi can decrease the level of Netrin-1. The results demonstrated that vectors were transfection successfully.2.Rats in netrin-1 over-expression group performed better than MCAO group and netrin-1 RNAi group in neurological scores. 3.Western-blot shows that compared with MCAO group and netrin-1 RNAi group,the expression of NF-200,Synaptophysin and PSD-95 in netrin-1 over-expression group were higher than MCAO group and netrin-1 RNAi group(P<0.05).Compared with MCAO group,Those proteins' expression except PSD-95 were lower in netrin-1 RNAi group(P<0.05). 4.Immunofluorescent staining showed that in netrin-1 over-expression group,NF-200 had a higher expression and better structure than MCAO group and netrin-1 RNAi group.Conclusions We demonstrates that the regulation of netrin-1 after cerebral ischemic injury may be beneficial to the axonal plasticity and functional reconstruction. The expression of NF-200,Synaptophysin and PSD-95 may contribute to those functions...