| Dipfluzine (Dip), a new diphenylpiperazine compound was first developed by School of pharmacy, Hebei Medical University. Our previuos studies by the biochemical, physiological, and the morphological methods or techniques that Dip could markedly protect against the ischemic brain injury, and the important mechanisms of Dip protecting from ischemic brain injury might relate to its effects of selective cerebral vasodilation, increasing cerebral blood flow, improving energy metabolism, decreaseing concentration of extracellular excitatory amino acids,and blocking calcium channels in ischemic brain. Since thrombolytic therapy is frist choiced for the patients with cerebral thromboembolism and apoptosis has been thought as main form of ischemia-reperfusion brain injury, especially delayed neuron death. The present study was undertaken to observe if Dip possesses the protective effects on the brain injury from ischemia-reperfusion in model of middle cerebral artery occlusion (MCAO) of rats by measuring apoptosis and free radical formation, and to explore the molecular mechanisms of Dip protecting from ischemia-reperfusion brain injury by determining the effects of Dip on the apoptosis concerning factors, caspase-3, cytochrome c and Bcl-2 etc. The projects and results were as follow:1 Comparison of the models of focal brain ischemia-reperfusion for different time. Aim: To study the cell injury and the expression of caspase-3 after focal brain ischemia-reperfusion in different time .Methods: The model of focal cerebral ischemia-reperfusion was established with the suture-occluded. 54 rats were divided randomly to nine groups, sham operation group,I (ischemia) 1h R(reperfusion) 0.5hgroup , I 1h R 2h group, I 2h R 0.5h group, I 2h R 2h group, I 3h R 0.5h group, I 3h R 2h group, I 6h R 0.5h group, and I 6h R 2h group. Caspase-3 protein was measured by immunohistochemical method, the brain slices were stained by HE or blue Methyphenyl amine(BMPA) and morphological manifestation of apoptotic cell was examined under microscrope. Results: In HE staining, brain tissue structure was normal in sham group, and exhibited ischemic damage involving both cortex and caudate-putamen of right cerebral hemisphere in I 2h R 0.5h group, I 2h R 2h group I 3h R 0.5h group, I 3h R 2h group, I 6h R 0.5h group, and I 6h R 2h group characterized by cell swelling, broken nucleis , cell lysis, pyknotic nuclei and vacuolization. The changes of ischemic damage in I 1h R 0.5h group were milder. BMPA staining showed that the apoptotic cells after ischemia-reperfusion in all of experimental groups were remarkably increased compared to sham operation group. Immunohistochemical staining indicated that positive cells of caspase-3 protein after brain ischemia-reperfusion in all of experimental groups were remarkably increased(P(0.05) compared to sham operation group. The level of caspase-3 protein in ischemia-reperfusion 6h group was significantly higher than those of other groups. The content of MDA in all experimental groups after ischemia-reperfusion were gradually increased according to the time compared to sham group.Conclusion: The damage of neuron showed by caspase-3 protein, MDA and the brain slices stained by HE or BMPA was significantly increased with prolonged time of ischemia-reperfusion, suggested that apoptosis,MDAand caspase-3 protein are sensitive indicator representing ischemia-reperfusion brain injury. The model of ischemia for 2h and repefusion for 2h showed significant changes in the indicators above and thus was used to evaluate the drug for treating ischemia-reperfusion brain injury in our later study.2 Effect of Dip on cell apoptosis after brain ischemia-reperfusion in rats Aim : To observe the effect of Dip on cell apoptosis after brain ischemia-reperfusion in rats . Method: The model of focal cerebral ischemia-reperfusion was established with the suture-occluded. 30 rats were divided randomly to five groups(n=6 each group): a sham group(S), a vehicle gr...
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