Objective:Studies have shown that adapted reperfusion had neuroprotection after cerebral ischemia.But now,the effect of postconditioning with alternating of hypotensive perfusion and normal perfusion(defined as hypotensive perfusion postconditioning)on cerebral ischemia is not reported.In this study,a cerebral ischemic model in rats(2-vessel occlusion with hypotension)was established to observe the changes of neuroethology,neuronal apoptosis,TAp63 and ΔNp63 expression in hippocampal CA1,and S100β protein concentration in plasma,to explore the neuroprotection of postconditioning with alternating of hypotensive perfusion and normal perfusion on cerebral ischemia,and to provide a new clinic thoughts.Methods:Eighty male SD rats were randomly divided into four groups:sham group,cerebral ischemic/reperfusion group(I/R),hypotensive perfusion group one(HR1),hypotensive perfusion group two(HR2),20 in each group.All rats were evaluated neuroethology before anesthesia,and normal rats(16-20 points)were enrolled into study.Rats in sham group were not received cerebral ischemia.Rats in I/R group were received cerebral ischemia.The rats in HR1 and HR2 group were received postconditioning of hypotensive perfusion after cerebral ischemia.Cerebral ischemic model was established by clamping the bilateral common carotid artery(BCCA),with hypotention(MAP30-35mmHg)by drawing blood from femoral artery,maintained 20 minutes,then unclamped BCCA and injected blood back to elevate blood pressure to 80-90mmHg,maintained 30 minutes.Hypotensive perfusion postconditioning:Rats in HR1 group,continued to clamp BCCA at the beginning of reperfusion,elevated the pressure to 80-90mmHg and maintained 10 minutes,then unclamped BCCA 2min,repeatedly for 3 times.Rats in HR2 group,continued to clamp one side CCA and 1/2 of other side CCA at the beginning of reperfusion,elevated the pressure to 80-90mmHg and maintained 10 minutes,then unclamped BCCA 2min,repeatedly for 3 times.The rats in all groups were tested the neurological behavior score and then executed for cerebral tissues at 6h,1d,3d,5d after surgery.The paraffin sections were taken for HE,TUNEL staining to calculate neuron density,neuron apoptosis index and immunohistochemistry(IHC)to detect the expression of TAp63 and ΔNp63(semiquantitative analysis)in CA1 of hippocampus.The plasma S100β protein was determined by ELSA at preischemic and time to execute.Results:1.Cerebral ischemic model In this study,8 rats died:3 rats died from cardiac arrest and apnea,and other 5 rats died before the time to observe their indexs.Eight rats were supplemented.Eighty rats were enrolled in study at last.2.Neruological behavior scores Before the operation,all rats,neruological behavior scores were normal.In sham group neruological behavior scores were normal at all time points.In I/R,HR1 and HR2 group,the neruological behavior scores were significant difference between preoperation and postoperation at same time point(p<0.01),and the scores at 3d were lowest.Neruological behavior scores were no significant difference between HR1 and HR2 group(p>0.05),but both significantly higher than I/R group(p<0.05).3.Neuronal density HE staining in hippocampal CA1 was normal in sham group.The neuronal densities in I/R,HR1 and HR2 group were significantly lower than Sham group at 1d,3d,5d after operation(p<0.01),neuronal density at 3d was lowest.There were no significant difference of neuronal densities between HR1 and HR2 group(p>0.05),but both significantly higher than I/R group(p<0.05).4.Neuronal apoptosis Apoptotic neurons were very rare in hippocampal CA1 of Sham group at all time points.In I/R,HR1 and HR2 group,neuronal apoptosis indexs in hippocampal CA1 were significantly higher than Sham group at 1d,3d,5d after operation(p<0.01),and highest at 3d.Neuronal apoptosis indexs ware no significant difference between HR1 and HR2 group(p>0.05),but both significantly lower than I/R group(p<0.05).5.TAp63 and Δ Np63 protein expression TAp63 and Δ Np63 protein expression in hiappocampal CA1 of Sham group were rare at all time points.The positive expression rates of the two p63 isforms in Sham group were all less than 5%,recorded as negative expression.In I/R,HR1 and HR2 group,the rates of the two p63 isforms in hippocampal CA1 were significantly higher than Sham group at Id,3d,5d(p<0.01).TAp63 protein expression rates in I/R,HR1 and HR2 group at 3d were significantly higher than other time in same group,recorded as(+++).ΔNp63 protein expression rates in I/R,HR1 and HR2 group at 5d were significantly higher than other time in same group:I/R group recorded as(+),HR1 and HR2 group recorded as(+++).TAp63 and ANp63 ptotein expression rates were no significant difference between HR1 and HR2 group(p>0.05),but both TAp63 ptotein expression rates were significantly lower than I/R group(p<0.05),both ΔNp63 ptotein expression rates were significantly higher than I/R group(p<0.05).6.S100β concentration in plasma The S100β concentration in Sham group was normal at all time points.In I/R,HR1 and HR2 group,S100β concentrations were significantly higher than Sham group at all time points after operation(p<0.01),and highest at 3d.S100βconcentration was no significant difference between HR1 and HR2 group(p>0.05),but both significantly lower than I/R group.7.Correlation analysis TAp63 and ΔNp63 expressions in hippocampal CA1 were positive correlated(R=0.54,p<0.01).S100β concentration in plasma and TAp63 expression in hippocampal CA1 were positive correlated(R=0.38,p<0.01).But S100β concentration in plasma and ΔNp63 expression in hippocampal CA1 were no correlation.S100β concentration in plasma and neuronal apoptosis in hippocampal CA1 were positive correlated(R=0.83,p<0.01).Conclusion:1.Alternating of hypotensive perfusion and normal perfusion postconditioning had neuroprotection on cerebral ischemia in rats.Its possible mechanism was to decrease the expression of TAp63 protein and increase the expression of ΔNp63 ptotein in hippocampal CA1,and to inhibit neuronal apoptosis.2.The expression of TAp63 increased at first day,and 3d reached peak,and then decreased at 5d after cerebral ischemia.The expression ofΔNp63 was gradually increased after cerebral ischemic reperfusion.3.TAp63 expression was positive correlated with ANp63 expression in hippocampal CA1 and S100β concentration in plasma.S100β concentration in plasma and neuronal apoptosis in hippocampal CA1 were positive correlated. |