亚低温对脑缺血再灌注损伤大鼠神经细胞凋亡和caspase-3释放的影响

Effects of subhypothermia on apoptosis and caspase-3 expression in neuronal cells after cerebral ischemia / reperfusion injury in rats

目的从信号转导及细胞凋亡角度,研究亚低温对大鼠脑缺血再灌注损伤(I/R)的脑保护作用及机制。方法 72只雄性健康SD大鼠,随机分成假手术组(S组)、缺血再灌注组(IR组)、亚低温组(M组),每组24只;三组缺血10 min后分别按再灌注12 h、24 h、48 h,再分为3个亚组,各亚组动物均为8只。大鼠脑缺血再灌注损伤模型制作采用改良四血管阻滞法,免疫组化SP法动态观察各个时间点海马CA1区caspase-3蛋白的变化;光镜和电镜分别观察再灌注48 h亚组海马CA1区神经细胞形态和线粒体超微结构的改变。结果 (1)大鼠脑缺血再灌注损伤后12 h海马CA1区caspase-3即有明显表达,24 h达高峰,48 h后仍有较高表达;(2)IR组和M组各时间点caspase-3表达水平比S组明显升高(P<0.05);24 h亚组线粒体超微结构和神经细胞形态受损严重;(3)M组各个时间点caspase-3表达水平较IR组明显下降(P<0.05或P<0.01);24 h亚组线粒体超微结构和神经元形态均有不同程度的改善。结论亚低温对caspase-3依赖的线粒体凋亡通路有干预作用,通过维持线粒体膜稳定,抑制释放和激活caspase-3蛋白,保护线粒体的形态功能,从而减少神经细胞凋亡的发生,发挥脑保护作用。

Objective To explore the effects and mechanism of subhypothermia on apoptosis and caspase-3 expression in neuronal cells after cerebral ischemia/reperfusion （IR） injury in rats. Methods Seventy two healthy male SD rats were randomly divided into three groups： group S （ sham operation group） , group IR （ischemia/reperfusion） and group M （ subhypothermia treated group）, 24 rats in each group. The model of focal cerebral ischemia reperfusion injury was established by using Pulsinelli＇s method. Rats in each group were further divided into 3 subgroups （n =8 in each）, which were treated with 10 min ischemia, then 12 h, 24 h or 48 h cerebral reperfusion, respectively. The expression of caspase-3 in neuronal cells were examined by immunohistochemical SP method, the pathological changes and mitochondria ultrastructure of neuronal cells in hippocampus CA1 region were observed by light microscope and electron microscope at 24 h after reperfusion. Results The expression of caspase-3 started to increase at 12 h after reperfusion, reached the peak at 24 h and remained high level at 48 h. Compared with group S, the expression of caspase-3 in group IR and M were increased significantly （ P 〈 0.01 ）. The pathological changes of nerve cells and mitochondria ultrastructure were exacerbated at 24 h after reperfusion. Compared with IR group , the expression of caspase-3 in group M were decreased significantly （P 〈 0. 05 or P 〈 0. 01 ） ; and the pathological changes of nerve cells and mitochondria ultrastructure were attenuated at 24 h after reperfusion. Conclusion Subhypothermia protects rat neuronal cells from ischemia/ perfusion injury through stabilizing mitochondria membranes and inhibiting the caspase-3-dependent apoptosis.