Experimental Study On Role Of C-Jun N-terminal Kinase And Its Mechanism On Symptomatic Cerebral Vasospasm After Subarachnoid Hemorrhage

Part one:The pathological changes of the basilar artery walls in the models of cerebral vasospasm(CVS) after subarachnoid hemorrhage in rabbits.Objective The model of symptomatic cerebral vasospasm(CVS) after subarachnoid hemorrhage(SAH) was set up by injecting autologous blood twice via cistern magna after occlusion of right carotid in the experimental animals. Neurobehavioral and histopathological changes of basilar arteries were observed in experimental animal.Methods Thirty rabbits were divided randomly into Normal,Control(normal saline was injected into the cisterna magna), SAH groups(SAH3d,SAH7d,SAH10d and SAH14d) five groups.The model of SAH was set up by injecting autologous blood twice via cistern magna after occlusion of right carotid twice at 48-hour intervals in rabbits.The neurological behavior changes and general specimen in experimental animals were observed after SAH.Light and transmission electron microscopy and TUNEL staining after experimental SAH,were used to test the endothelial apoptosis in basilar arteries,and the expression of phosphorylated JNK(P-JNK) and Caspase-3 protein in the vascular endothelial cells(VEC) was examined with an immunohistochemical technique.Results No neurobehavioral changes happened between before and after injecting normal saline.There were neurobehavioral changes in SAH groups and it went on for a long time.Light and transmission electron microscopy investigation indicated that the histopathological changes including arterial norrowing,thickness of arterial wall,degeneration of endothelial cells and smooth muscle cells started on day 3,such changes progressed with time and obviously on day 7,the changes began to relieve on day 10 after SAH.The upregulation of P-JNK was detected in the vascular wall on day 3,became stronger on day 7,and decre- ased on day 10 after SAH.Apoptotic-like changes such as condensation of cytoplasm and increased condensation,positive staining of TUNEL and upregulation of Caspase-3 protein were observed in endothelial cells in SAH group.The control group had no significant performance results compared with SAH group suggest that symptomatic CVS models were set up as a reliable model,consistent with literature reports.Conclusion 1.The symptomatic model of CVS appearances neurobehavioral changes and go gradually back to normal after 2 days.Vasospasm start after SAH 3d,7d arrived peak and 10d release gradually.These results suggest that the model is more satisfied which to simulate the clinical CVS after SAH on the pathophysiological and physiological changes.2.The vascular endothelial cells increase expression of P-JNK,cytoplasmic positive for Caspase-3 expression in basilar artery of the vascular endothelial after SAH,TUNEL staining positive cells markedly increase,and the activation level at time of change and on top of the basilar artery is associated with pathophysiological changes,it suggests that JNK-mediated apoptosis probably play a certain role in the formation and development of CVS.Part two:Effect of the JNK inhibitor on the development of cerebral vasospasm after symptomatic subarachnoid hemorrhage in rabbits.Objective To evaluate the effect of JNK specific inhibitor—SP600125 on the development of symptomatic CVS after SAH,in order to explore the CVS cellular mechanism of signal transduction pathway after SAH.Methods Twenty-four rabbits were divided randomly into Control,SAH,DMSO(SAH +carrier(dimethyl sulfoxide)),and SP600125(SAH treated with SP600125) four groups in a two-hemorrhage rabbit model after occlusion of right carotid in rabbits.The morphological changes of basilar arteries were studied with light and electron transmission microscopy.TUNEL staining was used to test the VECs apoptosis in basilar arteries,and the expression of Caspase-3 protein was examined with an immunohistochemical technique.Results Basilar arteries in SAH group and DMSO group were narrower compared with control group.The upregulation of P-JNK was detected and lots of positive cells which was in brown on the vascular wall.Apoptotic-like changes such as condensation of cytoplasm,positive staining of TUNEL and upregulation of Caspase-3 protein were observed in VECs in SAH group compared with Control group.The SAH group had diffirences when it compared with control group.There was no diffirence between the SAH group and DMSO group.In the treatment group,SP600125 significantly ameliorated the pathological changes including thickness of arterial wall,degeneration and necrosis of endothelial cells(P＜0.05)when compared with SAH or DMSO group.Conclusion 1.JNK inhibitor-SP600125 could reduce the symptoms of neurobehavior. It alleviate the degree of cerebral vasospasm clearly after SAH,so that P-JNK,Caspase-3 and TUNEL positive endothelial cells in the percentage of positive cells arre decreased and apoptosis-like changes are reduced.2. It is further confirmed that JNK-mediated apoptosis may be the formation and development of symptomatic CVS and it has played an important role in inhibiting JNK signal transduction and apoptosis after the spasm of these changes,so this experiment probably provides a novel therapeutic way for symptomatic CVS.