| Background and objective:Studies had found that Wnt/?-catenin signaling pathway played protective role in neuronal apoptosis after cerebral ischemia,its inhibitor DKK1 could inhibit Wnt/?-catenin signaling pathway,and aggravate ischemic injury.Studies had found that over expression of p63(?Np63)could activate Wnt/?-catenin signaling pathway in cell culture and whole embryo model.Our previous studies had shown that p63 was high expression after cerebral ischemia in rats.Then,could high expression of p63 activate Wnt/(3-catenin signaling pathway after cerebral ischemia,and what should be the effect of it on cerebral ischemia?This experiment was designed to explore this question.In this study,the effect of intracerebroventricular injection of p63 antibody or Dkkl protein on p63 protein,(3-catenin protein,neuronal apoptosis in hippocampal CA1 area and the level of S100? in serum after cerebral ischemia/reperfusion were observed in rats,to explore the role of p63 on the Wnt/?-catenin signaling pathway after cerebral ischemia,and to regulate and control neuronal apoptosis after cerebral ischemia and hypoxia,and to provide experimental evidence on cerebral ischemia and infarction prevention.Methods:Eighty four male SD rats weighing 250?300g were randomly divided equally into four groups(n=21):group A,B,C and D.The rats in group A were received normal saline injection into lateral ventricle without cerebral ischemia/reperfusion;the rats in group B,C,D were respectively received normal saline,p63 antibody,DKK1 protein injection into lateral ventricle and cerebral ischemia/reperfusion.Models of cerebral ischemia/reperfusion were performed by two vessel occlusion(bilateral common carotid artery)plus hypotension(35-40mmHg)to establish cerebral ischemia/reperfusion model,ischemia time was forty minutes.All the rats neurobehavioral was respectively scored at 24h,48h,72h after operation.And double antibody sandwich enzyme-linked immunosorbent assay(ELISA)were used for detecting the level of S100?in serum.The neuronal densities were observed by HE staining.Neuronal apoptosis cells were determined by TUNEL.The expressions of p63 and?-catenin protein were detected by immuno-histochemistry(IHC)staining and semi-quantitative analyzed in hippocampal CA1 area.Results:1.Neurological behavior scores:Neurological behavior scores were normal before operation in all rats and postoperation in group A(p>0.05).Within group B,C and D,the postoperative neurological behavior scores were significantly lower than preoperative scores(p<0.01),the scores were significantly different between postoperative points,from high to low:24h,48h,72h(p<0.05).The postoperative neurobehavioral scores were significantly different at the same time points between groups,from high to low:A,C,B,D,with statistical significance.(p<0.01).2.Neuronal density:There was no statistically significant of neuronal densities at each time point after surgery in group A(p>0.05).Within group B,C and D,the postoperative neuronal densities were significantly lower at each time point,and lowest at 72h,the neuronal densities were significantly different between postoperative points,from high to low:24h,48h,72h(p<0.01).The postoperative neuronal densities were significantly different at the same time points between groups,from high to low:A,C,B,D(p<0.01).3.Neuronal apoptosis:Apoptotic neurons were very rare at each time in group A after operation,with no statistical significance(p>0.05).Within group B,C and D,the postoperative neuronal apoptosis cells were significantly higher at each time point,and highest at 48h,the neuronal apoptosis cells were significantly different between postoperative points,from high to low:48h,24h,72h(p<0.01).The postoperative neuronal apoptosis cells were significantly different at the same time points between groups,from high to low:D,B,C,A(p<0.01).4.The level of S100? in serum:The level of S100? in serum of group A was very low at each time,with no statistical significance(p>0.05).Within group B,C and D,the level of S100? in serum was significantly higher at each time point,and highest at 48h,the level of S100? was significantly different between postoperative points,from high to low:48h,24h,72h(p<0.01).The postoperative level of S100? in serum was significantly different at the same time points between groups,from high to low:D,B,C,A(p<0.01).5.The expression of p63 and ?-catenin protein:?The expression of p63 was very rare at each time in group A after operation,with no statistical significance(p>0.05).Within group B,C and D,the expression of p63 was significantly higher at each time point,and highest at 48h,the expression of p63 was significantly different between postoperative points,from high to low:48h,72h,24h(p<0.01).The postoperative expression of p63 was significantly different at the same time points between groups,from high to low:D,B,C,A(p<0.01).? The expression of ?-catenin protein was in high level in group A after operation.However,the differences between each time point were not statistically significance(p>0.05).Within group B,C and D,the expression of ?-catenin was significantly lower at each time point and lowest at 48h,and recovered slightly at 72h,the expression of ?-catenin was significantly different between postoperative points,from high to low:24h,72h,48h(p<0.05).The postoperative expression of ?-catenin was significantly different at the same time points between groups,from high to low:A,C,B,D(p<0.01).6.Correlation analysis:The expression of ?-catenin protein had negative correlation with p63 and neuronal apoptosis in hippocampal CA1 area after cerebral ischemia in rats(correlation coefficients were r=-0.941;r=-0.887).The expression of p63 and the level of S100? in serum had positive correlation with neuronal apoptosis in hippocampal CA1 area after cerebral ischemia(correlation coefficients were r= 0.956;r= 0.936).Conclusion:1.Intracerebroventricular injection of DKK1 protein could aggravate ischemic injury after cerebral ischemic.It could inhibit the Wnt/?-catenin signaling pathway after cerebral ischemia,increase the expression of p63,and then increase neuronal apoptosis.2.Intracerebroventricular injection of p63 antibody had neuroprotection after cerebral ischemia.It could reduce expression of p63 after cerebral ischemia,upregulate the Wnt/?-catenin signaling pathway,and then decrease neuronal apoptosis.3.The expression of ?-catenin protein had negative correlation with p63 and neuronal apoptosis in hippocampal CA1 area of rats after cerebral ischemia;The expression of p63 and the level of S100? in serum had positive correlation with neuronal apoptosis in hippocampal CA1 area after cerebral ischemia. |
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