Experimental Research Of Naomaiyihao Combined With Bone Mesenchymal Stem Cells Transplantation Treating Ischemic Stroke

ObjectiveTo observe the dynamic state changes of neurological impairment,water content of brain tissue,pathological change of brain tissue,neuronal marker NSE(neuron specific enolase,NSE),vascular endothelial cell marker CD31 at different intervals after cerebral ischemic reperfusion in operated rats.To exam their injury feature of brain tissue after cerebral ischemic-reperfusion and reveal its changeable regularity.To study the influence of these change by using BMSCs(bone mesenchymal stem cells,BMSCs) transplantation group, Naomaiyihao group and Combined treated group,discuss the protective effect and therapeutic mechanism of Naomaiyihao combined BMSCs transplantation to rats after ischemic-reperfusion.MethodsSD(Sprague Dawley,SD)(n=155,male,weight:250—320g)rats were randomly divided into sham-operated group(n=8) and operated group which involved model group(n=24),BMSCs transplantation group(n=24),Naomaiyihao group(n=24),and Combined treated group(n=24),then operated groups were randomly divided into 3d,7d and 14d.Each group had 8 rats.Bone mesenchymal stem cells were isolated and cultured from bone marrow by their abilities to proliferate in culture with an attached well-spread morphology.And the shapes and structures of the cells were observed by phase contrast microscope.Naomaiyihao group and Combined treated group were syringed through mouth into stomach 3d before animal experiment with Naomaiyihao,one time each day,sham-operated group, model group and BMSCs transplantation group with saline of equal volume. Iscliemic-perfusion animal model was duplicated in accordance with improved Longa's focal middle cerebral artery occlusion(MCAO) model in rats,After 2 hours of ischemia,the fishing line was picked out 3-4mm to make the brain reperfused,and 10μl suspension(1×10⁴/ml) containing 5×10⁵ BMSCs were respectively injected by stereotactic instrument into striatum in rats of BMSCs group and Combined treated group 24 hours after reperfusion.Combined treated groups were syringed through mouth into stomach with Naomaiyihao and BMSCs groups with saline of equal volume after transplantation.The rats were tested neural score on 3d,7d and 14d,and then part of the brain tissue was taken out for observing the water content of the brain tissue,Pathological changes in ischemic area was observed from light microscope. Immunohistochemical method was performed to investigate the expression of CD31 positive cell and NSE positive cell.Results1,Brain pathological injury in different degree could be seen in operated group.As time went by,the pathological injury became lower.The nerve function score,water content of brain tissue,athological changes of cerebral tissue of naomaiyihao group,BMSCs group and combined treated group was lighter than that of model group,the weight growth rate increased more significantly than model group.2,Changs of Neuronal marker NSE expression:Compared with the model group, its expression in BMSCs group and Naomaiyihao group were more obvious separately.BMSCs group and Naomaiyihao can cause the combined effect,and the combined effect was more increased than other group.3,Changs of CD31 positive cell expression in the brain tissue of each group rats:Compared with the model group,its expression in BMSCs group and Naomaiyihao group were more obvious separately.BMSCs group and Naomaiyihao can cause the combined effect,however the combined effect was not more increased than Naomaiyihao group.Conclusion1,Cerebral ischemia reperfusion could result in serious pathological injury of brain tissue in rats,including the changes of neural morphology,increment of brain water ratio,damage of microvessel structure.The most severe pathological injury was in the 3^th day and remaind a more higher lever of injury in the 14^th day.2,Naomaiyihao could promote BMSCs survived in the brain tissue and differentiated into vascular endothelial cell,so the number of angiogenesis increased obviously in Combined treated groups than that other groups.Promote angiogenesis may be one of the mechanisms of brain protection.3,Multiple factor variance analysis showed that Naomaiyihao could lighter the brain water content obviously as time went by,Naomaiyihao could promote BMSCs angiogenesis after ischemic-reperfusion in rats,protect the neural cells,however,neural function was not significantly improved,the possible reason is the observation time was too short.