| ObjectiveDetect RhoA expression in different phase after Wistar rat's spinal cord injury to discuss the correlation between neurofiber axonal regeneration and spinal cord function and by interfering RhoA expression,discuss the healing effect for spinal cord injury in order to supply new strategy for the repair of spinal cord injury.Method1.Observe RhoA expression in different phase after spinal cord injury on the double level of gene expression and protein translation80 Wistar rats,divided into 2 groups randomly:40 rats in control group,40 rats in experimental group,T10 acute spinal cord injury model was made by IMPACT-Ⅱ,4 rats in each group was taken out respectively after 8h,24h,3h,7d and 2w after successfully modeling,then extract the gross RNA which exist in the spinal cord injury area,assay RhoA mRNA expression by real-time quantitative PCR and gel electrophoresis.Another 4 rats'injuried spinal cord tissue was carried on freeze tissue slice,immunohistochemistry to observe the RhoA protein expression.2.By interfering RhoA expression,observe recovery of rat's spinal cord injury36 Wistar rats were made into T10 acute spinal cord injury model by IMPACT-Ⅱmethod,after successfully modeling,the rats were divided into 3 groups,12 rats in each group:control group;local multipoint injection C3 group after spinal cord injury (1.75mg/kg,3 injection point);caudal vein C3 injection group(250μg/kg/d,7days in succession after modeling).7 days after modeling,take out 4 rats in each group to carry on real-time quantitative PCR assay and tissue slice immunohistochemistry stain to assay RhoA activity;BBB score was evaluated in each group every week after modeling to check motor functional recovery after spinal cord injury;After 12 weeks, carry on 10%BDA nerve antrograde tracing labeling,2 weeks after labeling,take out injuried spinal cord and make 5μm fast frozen section to carry on fluorescent staining and HE staining.Results8 hours after spinal cord injury,RhoA mRNA expression start□euritis□g,3 days after injury,RhoA mRNA expression reach the peak,and keep the high level for 7 days,2 weeks after injury,the expression dramaticly decrease and there are no significant difference comparing with control group;immunohistochemistry stain shows RhoA protein expression increase evidently 3 days after injury,and keep the high expression for 2 weeks.7 days after spinal cord injury,real-time quantitative PCR and tissue slice immunofluorescence results show that RhoA expression in two experimental groups (local multipoint injection C3 group after spinal cord injury;caudal vein C3 injection group)is lower than control group,the difference has statistic meaning(P＜0.05), however no significant difference between two experimental groups;BBB score has statistic meaning(P＜0.05)comparing between groups 4 weeks after injury,among which caudal vein C3 injection group is obviously higher than control group (P＜0.05);12 weeks later,HE staining and light microscope observation demonstrate that part nerve fiber get through injuried area in C3 application group,while control group hardly see regenerated nerve fiber get through injuried area,however spinal cord cavity as well as gial scar formation can been seen in both groups.12 weeks after injury,BDA(biotinylated dextran amine)nerve tracing and microscope observation show that axonal regeneration extension is more obvious in experimental group, corticospinal tract regenerates and extends,part of which can get through the injuried area and reach distal end.ConclusionAfter rat's spinal cord injury,RhoA mRNA expression obviously increase,and has evident time curve,RhoA protein expression increase is slightly later than mRNA;At the early stage after spinal cord injury,C3 can get through blood-spinal cord barrier and reach injuried area to interfere RhoA expression,via inhibiting RhoA activity,ameliorating local injuried microenvironment and promote nerve fiber regeneration and functional recovery of spinal cord.
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