| Dipfluzine (Dip), a novel calcium antagonist of diphenylpiperazines with similar structure to flunarizine (Flu), was synthesized by Hebei Medical University. Dip could protect against the ischemic brain injury by selective dilating cerebral vessels, increasing cerebral blood flow and improving energy metabolism. It had been tested that apoptosis was the main form of the neuronal death in ischemia reperfusion brain injury. The present study is to make model of the global cerebral ischemia reperfusion to observe if Dip possesses the antiapoptotic effect on brain injury by measuring apoptosis. Moreover, we could further explore the mechanisms of Dip protecting from ischemia reperfusion brain injury by determining the effects of Dip on the apoptosis concerning factors, caspase-3, NO, NOS,and iNOS etc.1 Relation of the neuron apoptosis and global cerebral ischemia reperfusion for different timeObjective: To select the best time for evaluating the effects of drug on the ischemia reperfusion brain injury by observing the changes of neuron injury in different ischemia-reperfusion time Methods: The rat model of reperfusion after 15 min of the global cerebral ischemia was made by using Pusinellis 4-VO metheod. Fifty SD rats (weighting 250-300g, male, healthy) were divided randomly into 5 groups: (1) sham operation control group (2) IR4h (ischemia 15min reperfusion 4h) group (3) IR8h group (4) IR24h group (5) IR72h group. The rats in test groups were anesthetized with chloral hydrate and both vertebral arteries were occluded permanently by electrocoagulation. On the next day, both carotid arteries were occluded with aneurysm clips for 15 min. Rats that lost their righting reflex and whose pupils were dilated and unresponsive to light were selected for the experiment. Rats in sham operation group only underwent an operating procedure, the common carotid arteries and vertebral arteries were not occluded. At 4h, 8h, 24h, and 72h after reperfusion or 24h after sham operation, rats were killed by decapitation. Hippocampi of four rats in each group were removed quickly and put in 70% ethanol for measuring the apoptosis rate and caspase-3 protease by FCM. The rest rats in each group were killed. Brains were removed, immersed in 4% paraformaldehude for 24-72h and processed for paraffin embedding. Coronal sections at the level of 1.2-5.2mm after optic chiasma were selected and processed for immunohistochemistry and HE staining. The expression changes of caspase-3 and morphological changes of cell and density of pyramidal cells in hippocampal CA1 sector were examined under light microscope. Results: The apoptosis rate in the sham operation group was 2.23±0.21%. It was obviously increased with prolonged time of reperfusion. The apotosis rate in IR4h group, IR8h group, IR24h group, and IR72h group were 4.97±0.49%, 8.00±0.30%, 10.17±1.31%, and 12.03±1.17% respectively, which were different from sham operation group. The changes tendency of caspase-3 expression were similar to the changes of the apoptosis rate, which was also obviously increased with the prolongation of the reperfusion. The expression of caspase-3 in sham operation group, IR4h group, IR8h group, IR24h group, and IR72h group were 5.03±0.08 channel, 5.51±0.14 channel, 5.61±0.18 channel, 6.14±0.17 channel, and 6.37±0.27 channel respectively. Morphological observation by HE staining: there were 3-4 layers pyramidal cells ranged regularly in hippocampal CA1 sector, the nucleus was big, round, and pellucid, which can be seen 1-2 nucleolus clearly. In the IR4h and IR8h group, the hippocampal structure was normal and the morphology and the number of neuron did not be obviously changed. In the IR24h group, the number of normal neuron decreased, the cell body shrank, and the nucleus became out of shape. The IR72h group characterized by hippocampal structure destroyed, cell swelling, cell lysis, pyknotic nuclei, and vacuolization.Conclusions: With prolongation of reperfusion time, the apoptosis rate and expression of caspase-3 are obviously increased and...
|
PDF Full Text Request