Experimental Research On Acute Spinal Cord Injury In Rats By GDNF Mediated By RAAV2in Combination With The Early Rehabilitative Treatment

Objective: Choose male SD rats as the experimental subject, use GDNF mediatedby rAAV2joint early early rehabilitation training on acute spinal cord injury in rats afterthe injury, adopt the kinematics scores, histomorphology and immunohistochemicaltechniques, compared the effects on SCI of early rehabilitative treatment, gene therapyand combined therapy through analysising the changes of the rats’ movement functionatat different time, the expression of GDNF in the injured region, the changes of motorneurons’ form and amount, etc.Methods: This experiment adopted GDNF mediated by rAAV2gene therapy, earlyrehabilitative treatment and combined therapy for SCI:90male SD rats, divided intosham-operated group(n=18), the natural healing group (n=18), gene therapygroup(n=18), rehabilitation training group(n=18), combined therapy group (n=18),randomly. Each group based in accordance with different time points were randomlydivided into7d group(n=6),14d group(n=6),21d group(n=6). InjectrAAV2-GDNF-GFP after24hours for the gene therapy group and the combined therapygroup. For rehabilitation training group and combined therapy group，after the successof the third day began to early rehabilitative training. The methods and indicators,(i)Evaluating the SCI rats’ movement function through the BBB score and inclined plateexperiment.(ii)Observating the changes of spinal motoneuron’and neurogliocyte’ formand amount by HE dyeing.(iii)Observating the efficiency of rAAV2-GDNF-GFPinfected the injured spinal cord by fluorescence microscope.(iv)Detecting GDNFantigen distribution in injured spinal cord tissue and the expression of GDNF by SABCkit.Results:(i)Kinematics scores display that the effect of rehabilitation training on SCI rats’ movement function recovery is better than gene therapy. The scores hassignificant difference on the7th day (P <0.05)and very significant difference on the14th and the21th day (P <0.01).(ii)Through HE dyeing sections we can see that theprotection of motor neurons of gene therapy is better than rehabilitation training,thescores have significant difference on the7th day (P <0.05).(iii)Immunohistochemicalresults show that the GDNF’ AOD of rehabilitation training group is higher than thenatural healing group, the scores have significant difference on the7th day(P <0.05)andvery significant difference on the14th,21th day(P <0.01).(iv)Compared with everyother group, the effects of the combination group are the best in the kinematics scoreafter injury, the number of survival motoneurons, the efficiency of rAAV2-GDNF-GFP’infection and the expression of GDNF. The scores are all have significant differences onthe7th and14th day(P <0.05).Conclusion:(i)At the cellular level, rAAV2-GDNF-GFP has the ability to infect293T cell and express GDNF protein; At the tissular level, GDNF is expressed at boththe transcriptional and translational level in the damaged spinal tissue.The AOD is thehighest on14d.(ii)Early rehabilitation training can evidently improve the motorfunction of the rats’ hind legs (recover the fastest from7d to14d) and promote GDNFexpression in damaged rats’ spine cord(most evident on14d).(iii)rAAV2mediatedGDNF gene therapy can protect damaged spinal motoneurons and it significantlyimprove the expression of GDNF mRNA and protein, which accelerates therehabilitation process of motor function.(iv)The combined therapy is better than singletherapy in the rehabilitation of motor function, the protection of damaged spinalmotoneurons and the expression of GDNF, which indicates that the combined therapy isan important tendency of SCI treatment.