| Part one: Establishment of the middle cerebralartery occlusion modelObjective:To establish a simple, reliable and minor trauma model of focal cerebral ischemia in Wistar rats. Methods: The focal cerebral ischemia model in Wistar rats were made by the modified suture-occuluded method of Zealonga and Koizum. Then the reliability of this model was evaluated with the examination of neurological method. Results: 2 hours after MCAO, rats showed neurologincal functional obstacle, this clearly indicated the brain infarct. ConclusionThe improved model is simple, reliable, minor trauma and stable. It is advisable to use this experimental animal model to study focal cerebral ischemia.Part two:Changes of expression of NGB and p-ERK1/2 after cerebral ischemical reperfusionObjectiveTo investigate the dynamic changes of neuroglobin and diphos- phorylated-ERK expression in Cortex of frontallobe and field CA 1 of hippocampus,and relation of changes of neuroglobin expression in serum and time of following ischemic一reperfusion injury. Through antagon PD98059 of extracellular signal-regulated kinase intervention,To investigate rudiment reation of NGB and ERK1/2 ,and regulation mechanism potential of NGB at it's upstream. MethodsHere to select sixty-five Wistar male rats were divided randomly in thirty groups,each group have five rats. A group is sham operated group,no filament insert, only separation vertebral artety and common carotid artery; B group is ischemia/re- perfusion( I/R) group, were divided again in six subgroup by 4h, 8h, 16h, 32h, 64h and 128h of reperfusion;C group is PD98059 intervention group, were divided alse in six subgroup by 4h, 8h, 16h, 32h, 64h and 128h of reperfusion, peritoneal injection PD98059 after ischemia 30min(65mg/kg). An model of focal cerebral ischemia/reperfusion was established. Observe ischemic changes in cerebral tissue by LM before and after the experiment. The expression of NGB and p-ERK were detected with immunohistochemistry.Same to select twenty-fore Wistar male rats were divided randomly in seven groups, each group have five rats.Sham operated rats not subjected to filament insert; ischemia/re- perfusion( I/R) group, were divided in six subgroup by 4h, 8h, 16h, 32h, 64h and 128h of reperfusion. The expression of NGB color saturation in serum was examined by double antibody sandwich enzyme linked immunnosorbent assay method. The data was analyzed by SPSS13.0.Results:1. The expression of NGB in Cortex of frontallobe and field CA 1 of hippocampus.Immunohistochemistry staining showed the expression of NGB was lowest after the sham operation in the coxtex of frontallobe. There was a tendency in the I/R group show that first to rise then to down, The expression of NGB began to increase at 4h(P<0.01 vs sham group),keep on increase at 8-16h, reached peak at 32h,then to down at 64h and returned to sham level at 128h. Change of expression of the positive cells in the CA1 hippocampus showed the similar result to in the coxtex of frontallobe.but the number of positive cells in the CA1 hippocampus less that corresponding time point in the coxtex of frontallobe(P<0.01).Change tendency of expression of the positive cells in the PD98059 intervention group showed the similar result to in the I/R group in the coxtex of frontallobe and CA1 hippocampus.but the number of positive cell in the PD98059 intervention group less that in the I/R group in corresponding time point. there was significant difference among the coxtex of frontallobe and the CA1 hippocampus(P<0.01).then there was no significant difference among at 128h in the coxtex of frontallobe and at 128h in the CA1 hippoca- mpus(P>0.05).according to mean of each group,we think that number of positive cell was rise following time of reperfusion to last at same group,and that number of positive cell less than in the PD98059 intervention group less thanin the I/R group at same time point(P<0.01).2. The expression of p-ERK1/2 in Cortex of frontallobe and field CA 1 of hippocampus.The expression of p-ERK1/2 was weak after the sham operation in the coxtex of frontallobe. There was a tendency in the I/R group show that first to rise then to down, The expression of p-ERK1/2 began to significant increase at 4h(P<0.01 vs sham group),keep on increase at 8h, reached peak at 16h,then to down at 32h and still higher at 128h than 4h group. Change of expression of the positive cells in the CA1 hippocampus showed the similar result to in the coxtex of frontallobe.but the number of positive cells in the CA1 hippocampus more than corresponding time point in the coxtex of frontallobe(P<0.01).Change tendency of expression of the p-ERK1/2 in the PD98059 intervention group showed the similar result to in the I/R group in the coxtex of frontallobe and CA1 hippocampus.but the number of positive cell in the PD98059 intervention group less than in the I/R group in corresponding time point. there was significant difference at the same sime piont among the PD98059 intervention group and the I/R group (P<0.01).then there was no significant difference among at 128h and at 4h in the coxtex of frontallobe (P>0.05).according to mean of each group,we think that number of positive cell was rise following time of reperfusion to last at same group,and that number of positive cell less than in the PD98059 intervention group significant less than in the I/R group at same time point(P<0.01).Conclusion1.Expressin accrescence of positive neuron of NGB is closed related to cerebral ischemia repefusion, and suggested that NGB might play an important role in adaptive protection against ischemia repefusion.in Ischemic Penumbra.2. Expressin increased of p-ERK1/2 is a protection mechanism to cerebral ischemia repefusion.3. p-ERK signal transduction pathway possible is a upstream regulatory mechanism to neuroglobin.4.Content accrescence of NGB in serum possible as a original quantizal index to evaluate nerves injury;and it has important significance in early diagnosis ,treat,prognosis assessment of cerebrovascular disease. |
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