Effects Of Postcondition On Apoptosis Of Rats With Acute Ischemic Cerebral Infarction Followed By Reperfusion

【Objective】We investigated the neuroprotective effects of ischemic postconditioning on rats suffering from different level of acute ischemic cerebral infarction followed by reperfusion. Furthermore we studied the influence of ischemic postconditioning on neural apoptosis after acute ischemic cerebral infarction and explored the role of Bcl-2, Bax in the mechanism of neural apoptosis after ischemia/reperfusion (I/R). To make sure if ischemic postconditioning reduced acute ischemic cerebral infarction followed by reperfusion-induced neurological function and structure injury in rats by inhibiting apoptosis.Thereby ischemic postconditioning extended the therapeutic time windows of ischemic cerebrovascular disease.【Methods】There are two part of experiment. Part 1 Objective To study the effect of ischemic postconditioning on infarct size and neurologic scoring after acute ischemic cerebral infarction followed by reperfusion in rats. The present studies have shown that robust protection of ischemic postconditioning was only detected after relatively mild and short term ischemia.However. there is few study about the protective effects of postconditioning against the relatively severe and long term cerebral infarction. Some domestic studies have shown that ischemic postconditioning have neuroprotection effect even it performed at 4.5 hours after MCAO of rats then followed by reperfusion.But the foreign country have not reported it at the time being.So,we choose 4.5 hours after MCAO of rats for ischemic postcondition.And to make sure if ischemic postconditioning have neuroprotection effect when it performed at 4.5 hours after MCAO of rats.Methods Twenty-four healthy, adult, male Spraque-Dawley (SD) rats were randomly divided into sham-operated group, ischemic-reperfusion group, ischemic postconditioning group 1, ischemic postconditioning group 2 according to completely random design. Each group has six rats.We established acute Middle cerebral artery occlusion (MCAO) models.The ischemic-reperfusion group was treated with blood reperfusion after two hours of MCAO. After 2 and 4.5 hours of MCAO respectively, the rats of ischemic postconditioning group land 2 underwent postconditioning consisting of 5 cycles of 10-second/10-second reperfusion/reocclusion conducted 10 seconds after reperfusion,then followed by reperfusin. All rats were assessed neurological deficit scores at 1h and 24h after reperfusion. In all groups, these rats were sacrificed at the end of the 24-hour reperfusion.The brains of rats were removed directly and then sectioned into 2-mm coronal slices, putting the brain slices into 2% TTC solution in an incubator at 37℃for 30 minutes away from light. After that, check out the results. TTC stains viable brain tissue red. while infracted tissue remains white. TTC-stained brain sections were photographed with a computer camera. The infarct areas were calculated by an investigator blind to the study groups, using Image-Pro plus6.0.The non-infarcted areas in the left cerebral hemisphere were subtracted from the total area of the right cerebral hemisphere and multiplied by the slice thickness (2 mm) to determine the infarct volume for each section. The volumes of the five sections were added to give the total infarct volume for one rat.Using SPSS11.5 software for statistical treatment. The results were expressed as mean±standard deviation. Because the results of sham-operated group were zero, then they weren't analysed in order to avoiding heterogeneity of variance. First,to test the distribution of cerebral infarction volume of each group.If the distribution of each group is Normal we then test the heterogeneity of variance.If the test of heterogeneity of variance is homoscedasticity. we use One-Way ANOVA..And Differences were considered significant at p<0.05. Comparison of infarct volumes between each two groups of the three groups according to the S-N-K:If there is heterogeneity of variance, we use Nonparametric Tests (Kruskal-Wallis).And comparison of infarct volumes between each two groups of the three groups according to Independent-samples t test.According to the numbers of comparison,the size of test was determined.Because the results of sham-operated group weren't analysed.the numbers of comparison between each group was 3 times.So the size of test isα=0.016.Repeated measures ANOVA for the neurologic impairment scores.Differences were considered significant at p<0.05.Results 1. No infarct volumes were found in sham-operated group.There was obviously larg infract area found in the group of 4.5 hours infarction followed by postconditioning and the group of 2 hours infarction followed by reperfusion, along the left middle cerebral artery distribution area. While there was only small plaque-like ischemic focus distribute in the frontoparietal cerebral cortex and basal ganglia in the group of 2 hours infarction followed by postconditioning. There were statistical difference between the three groups at F=61.945 p=0.000 p<0.05,in terms of infarct volume 24 hours after reperfusion. Comparison of infarct volumes between each two groups of the three groups according to the Independent-samples t test showed a statistical difference atα=0.016.2. No neurological symptoms were found in sham-operated group, but were obviously in treated groups.There was considered statistically significant between each treated group and between different time (F=56.64. p=0.000;F=29.65, p =0.000).Neurological symptoms of three groups were no marked statistically significant I hour after reperfusion.but showed a statistically significant 24 hours after reperfusion. Neurological impairment scoring of the group of 4.5 hours infarction followed by postconditioning is obviously improved compared with 2 hours infarction followed by reperfusion group and postconditioning at 2 hours after infarction group, after 24 hours reperfusion. Neurological scoring of the group of postconditioning at 2 hours after infarction is minimum among the three groups.To observe from each time point of each group.2 hours infarction followed by reperfusion group and postconditioning at 2 hours after infarction group showed a statistically significant between 1 hours after reperfusion and 24 hours after reperfusion(t=4.04, p=0.002;t=7.06, p=0.000).But neurological scoring was no marked statistically significant between 1 hours after reperfusion and 24 hours after reperfusion in the group of postconditioning at 4.5 hours.The factors of time and treating are effected from each other. Neurological scoring was decreased along with time lengthened. The cross reaction at a considered statistically significant (F=8.75,p=0.001).[Conclusion]:Ischemic postcondtioning could decrease the infarct volume and relieve the neurologic impairment of rats after acute ischemic cerebral infarction followed by reperfusion.But if there was severe ischemia and the period of ischemia was long,the protective effects of ischemic postcondtioning dcreased. Part 2 Objective To observe HE dyeing and measure the number of apoptotic cells and Bcl-2, Bax protein positive cells in cerebral cortex with TUNEL or immunohistochemistry after acute ISCI injury. To make sure if ischemic postconditioning execute neuroprotection effect by inhibiting apoptosis. Methods Twenty-four healthy, adult, male Spraque-Dawley (SD) rats were randomly divided in to sham-operated group, ischemic-reperfusion group, ischemic postconditioning group 1, ischemic postconditioning group 2, according to completely random design. Each group had six rats.We established acute Middle cerebral artery occlusion (MCAO) models.The ischemic-reperfusion group was treated with blood reperfusion after two hours of MCAO. After 2 and 4.5 hours of MCAO respectively, the rats of ischemic postconditioning group 1and 2 underwent postconditioning consisting of 5 cycles of 10-second/10-second reperfusion/reocclusion conducted 10 seconds after reperfusion,then followed by reperfusin.In all groups, the rats were sacrificed at the end of the 24-hour reperfusion.Rats were anesthetized with 3.5% chloralhydrate (1ml/100g) and fixed with normal saline 250ml and 4% paraformaldehyde 300ml through apex of heart. The brains were removed from the skull.2mm before optic chiasma were paraffin embedded.4-μm-thick paraffin sections were cut and used for HE staining and TUNEL for apoptosis and immunohistochemical analysis for bcl-2, Bax protein. Using SPSS 11.5 software for statistical treatment, the results were expressed as mean±standard deviation. Because the results of sham-operated group were zero, then they weren't analysed in order to avoiding heterogeneity of variance. Comparisons among groups were made by One-Way ANOVA for the number of apoptotic cells and Bcl-2, Bax protein positive cells. Differences were considered significant at p<0.05. Results There was decreased neuronal degeneration, ischemia, cellular necrosis, interstitial edema, the count of TUNEL, bax and increased bcl-2 positive cells in the group of postconditioning at 2 hours after infarction, when compared with the group of postconditioning at 4.5 hours after infarction and the group of 2 hours infraction followed by reperfusion. Differences between the three groups were considered significant at p<0.05. The group of postconditioning at 4.5 hours after infarction with a marked difference compared with the other two groups. Conclusion ischimic postconditioning executed protection on acute ischemic cerebral infarction followed by reperfusion this neuroprotective effect was most likely achieved by antiapoptotic mechanisms through up-regulating bcl-2 and down-regulating bax.