The Relationship Between P63 And Neuronal Apoptosis After Cerebral Ischemia And Intervention Of Limb Ischemic Postconditioning In Rats

Objective:Ischemic stroke is a common cerebrovascular disease in clinical,with high morbidity,disability and mortality. Therefore, to simulate cerebrovascular disease by a variety of animal models, and the research of its pathogenesis, prevention and therapy have widely been taken serious. In this study, a rat model of asphyxial cardiac arrest global cerebral ischemia/reperfusion by endotracheal tube clamping was established. The change of neuroethology, p63 expression and the relationship between them with neuronal apoptosis after global cerebral ischemia/reperfusion in rats were investigated. The effects of limb ischemic postconditioning after global cerebral ischemia/reperfusion on p63 expression and neuronal apoptosis was investigated. And to explore neuroprotection of limb ischemic postconditioning, and p63 taking part in regulating neuronal apoptosis after cerebral ischemia/reperfusion. Eventually to provide the experimental evidence for new drug discovery to inhibit neuronal apoptosis, and for prevention and treatment of cerebral ischemia. Methods:120 male SD rats were randomly divided into three groups,40 in each group:sham group, global cerebral ischemia/ reperfusion(I/R) group and limb ischemic postconditioning (LIPC) group. All rats anesthetized with pentobarbital, catheter was inserted into excised femoral arteries and connected with biological function converter to monitor arterial blood pressure, then endotracheal tube was inserted. Rats in sham group were not received endotracheal tube clamping and occlusion of the femoral artery. Endotracheal tube were clamped to result in asphyxial cardiac arrest global cerebral ischemia/reperfusion, and received cardiopulmonary resuscitation after 4 minutes of cardiac arrest in I/R group. After global cerebral ischemia/reperfusion, rats in LIPC group were subjected to 10min of ischemia of two lower limbs by occlusion of bilateral femoral artery followed by 10min of reperfusion,3 times repeated. Rats were executed for cerebral tissues at 6h,12h,24h,48h and 72h after operation,8 in each time point. Change of neuroethology in rats was evaluated by Longa neurological scoring system, expression of p63 in hippocampus CA1 area was detected by immuno-histochemical staining, neuronal apoptosis was detected by deoxynucleotide-mediated in situ end labeling (TdT-mediated dUTP nick end labeling, TUNEL). And change of morphosis of hippocampal neurons and neuronal density(Neuronal density, ND) was observed by HE staining. Results:1. In this study,23 rats were excluded, among them,16 fail to CPR and 7 with no evedene of global cerebral ischemia, another 23 rats added to the study, a total of 120 rats entered the final analysis.2. Neurological behavior scores in group:Neurological behavioral score was 0 in Sham group.In I/R group,at 24h,48h and 72h after cerebral ischemia, neurological behavior scores were 2.2±0.7,2.6±0.5,1.4±0.5, significantly higher than those in Sham group (p<0.01). Neurological behavior scores of 48h was higher than that of 24h and 72h,there was statistical significance (p<0.05).In LIPC group, at 24h,48h and 72h after cerebral ischemia, neurological behavior scores were 1.1±0.8,1.2±1.0 and 0.4±0.5, significantly lower than I/R group (p<0.05), but significantly higher than the Sham group (p<0.05). Neurological behavioral score was 0 at 6h,12h after cerebral ischemia in I/R and LIPC group.3. p63 expression:The positive ratio of the cells expressing p63 protein less than 5% in Sham group, namely negative expression. In I/R group, the ratio was 13.0±4.9,45.6±8.2,70.6±9.2 and 61.5±7.4 at 12h,24h,48h and 72h after operation respectively, significantly higher than Sham group at the same time point(p<0.01).The ratio of 48h was higher than that of 24h and 72h, there was statistical significance (p <0.05).In LIPC group, the ratio was 12.4±2.6,34.8±5.5,47.7±5.9 and 39.7±7.3, respectively, significantly higher than Sham group (p<0.01),and significantly lower than I/R group at 48h and 72h after operation(p<0.05),but no significant difference at 6h after operation between I/R group and the LIPC group (p> 0.05).4. Neuronal apoptosis: Apoptotic nuerons was very rare in hippocampal CA1 of Sham group, and apoptosis index was 0. In I/R group, a lot of apoptotic cells were found at 24-48h after global cerebral ischemia/reperfusion, maximum at 48h. Apoptosis index at 24h,48h and 72h after operation were 14.8±3.4,17.7±1.8 and 15.1±1.2 respectively, significantly increased compared with the Sham group (p<0.05). In LIPC group, apoptotic index were 12.1±1.4,13.9±3.0 and 13.0±1.40 respectively at 24h,48h,72h after operation, compared with I/R group, significantly decreased at the same time point, (p<0.05), but higher than that in Sham group (P<0.05). There was no significant difference of apoptotic index at 6h and 12h after cerebral ischemia/reperfusion in I/R group and LIPC group, and with no significant difference between the two groups(p>0.05).5. Histology and neuronal density:HG was 0～Ⅰlevel in hippocampal CA1 of sham group. HG wasⅡ～Ⅲlevel in hippocampal CA1 of I/R group, significantly higher than sham group(p<0.05). HG wasⅠ～Ⅱlevel in hippocampal CA1 of LIPC group, significantly higher than Sham group(p<0.05),but lower than I/R group(p<0.05).Neuronal density were 221±8.7,219±6.3, 222±8.5,218±6.0 and 214±4.7 n/mm2 at 6h,12h,24h,48h and 72h in hippocampal CA1 of Sham group, respectively. Neuronal density were 153±10.8,126±12.6 and 133±10.5 n/mm2 at 24h,48h and 72h in hippocampal CA1 of I/R group respectively, significantly decreased compared with the Sham group at the same point(p<0.05). Neuronal density were 167±7.4,149±14 and 159±12n/mm2 at 24h,48h and 72h in hippocampal CA1 of LIPC group respectively, significantly decreased compared with the Sham group at the same point(p<0.05), but significantly higher than I/R group at the same point (p<0.05). There was no significant difference of neuronal density at 6h and 12h after cerebral ischemia/reperfusion in I/R group and LIPC group, and with no significant difference between the two groups(p>0.05).Conclusion:1. The expression of p63 up-regulated gradually from 12h to 48h after global cerebral ischemia/reperfusion, while decreased after 72h. 2.Neuronal apoptosis and expression of p63 first increased then decreased after cerebral ischemia/reperfusion, maximum at 48h. They were positive correlation and p63 could involved in neuronal apoptosis.3. LIPC had neuroprotection, and its mechanism could include reducing expression of p63 protein and inhibiting neuronal apoptosis, but the exact signaling pathway remains to be further studied.