亚低温对大鼠短暂性全脑缺血后皮质NOS亚型表达及神经元凋亡的影响

Effects of mild hypothermia on nitric oxide synthase subtype expression and neuron apoptosis in cerebral cortex after transient global ischemia in rats

目的观察亚低温对大鼠全脑缺血再灌注后皮质3种一氧化氮合酶亚型表达、一氧化氮产生以及神经元凋亡的影响,探讨亚低温的神经保护机制。方法成年雄性SD大鼠,采用双侧颈总动脉阻断闭塞+低血压法制备短暂性全脑缺血动物模型,随机分为常温缺血组、亚低温缺血组和常温假手术对照组;常温时的脑温为36.5℃～37.5℃,肛温为35.9℃～36.9℃;亚低温时控制在32.5℃～33.5℃,相对应肛温为32.2℃～33.1℃;分别于缺血后及亚低温治疗后30min、2h、24h和72h观察短暂缺血对脑组织一氧化氮合酶亚型表达及神经元凋亡的影响,以及亚低温对缺血性脑损伤的保护作用。行神经元尼氏体亚甲蓝染色观察神经元数目及形态学的变化;免疫组化染色检测神经元型、诱导型和内皮型一氧化氮合酶的表达水平;应用硝酸还原酶法检测硝酸盐/亚硝酸盐水平的变化;采用TUNEL染色法并结合电子显微镜观察神经元凋亡的变化。结果常温缺血组大鼠额叶皮质3种一氧化氮合酶亚型表达水平及硝酸盐/亚硝酸盐含量均明显高于常温假手术对照组(P<0.05或P<0.01),出现凋亡神经元;低温缺血组大鼠3种一氧化氮合酶亚型表达水平和硝酸盐/亚硝酸盐含量明显低于常温缺血组(P<0.05或P<0.01),未检测到凋亡神经元。结论脑缺血后一氧化氮参与了神经元的凋亡过程。

Objective To observe the effects of mild hypothermia on expressions of three nitric oxide synthase (NOS) subtype, production of nitric oxide (NO) and neuron apoptosis, for exploring the neuroprotective mechanism of mild hypothermia on cerebral cortex after reperfusion in global cerebral ischemia. Methods The transient global ischemic models of male adult SD rats were established by occlusion of bilateral common carotid arteries and artificial hypotension. Then the model rats were randomizely divided into three groups: normothermic ischemia group, hypothermic ischemia group and normothermic sham-operation control group. In normothermic group, cerebral temperature was 36.5 ℃-37.5 ℃, and rectal temperature was 35.9 ℃-36.9 ℃; while in hypothermic group, cerebral and rectal temperature was controlled at 32.5 ℃-33.5 ℃ and 32.2 ℃-33.1 ℃ respectively. The effects of transient cerebral ischemia on expression of NOS subtype and neuron apoptosis, and the effect of mild hypothermia on neuroprotection were observed at 30 min, 2 h, 24 h and 72 h after ischemia and hypothermia respectively: (1) The changes of number and morphology of neurons were detected by means of Nissl body staining with methylene blue. (2) The expression of neuron type NOS, induction type NOS and endothelial type NOS were assessed by immunohistochemical staining. (3) The change of nitrate/nitrite level was examinated by nitrate reductase method. (4) The apoptotic neurons were identified with TUNEL staining by electronic microscope. Results The expressions of three NOS subtype and nitrate/nitrite level in frontal lobe of rats from normothermic ischemia group were obviously higher than that in rats from normothermic sham-operation group (P < 0.05 or P < 0.01), and apoptotic neurons were detected in the former group. While the expression of NOS subtype and nitrate/nitrite level in hypothermic ischemia group were significantly lower than that in normothermic ischemia group (P < 0.05 or P < 0.01), and none of apoptotic neurons was detected. Conclusion The NO is involved in the process of neuron apoptosis after cerebral ischemia, and mild hypothermia treatment can down regulate the expression level of NOSs, the production of NO and neuron apoptosis after transient global ischemia in rats.