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目的本实验观察脑缺血预处理(CIP)对损伤性缺血所致大鼠海马CA1区神经元凋亡的影响,并观察抑制一氧化氮(NO)生成后这一影响的改变,进一步验证NO在脑缺血耐受(BIT)诱导中的作用及其作用机制。方法 36只永久凝闭椎动脉的Wistar大鼠,分为6组(每组n=6):①假手术组,只暴露双侧颈总动脉,不阻断血流;②损伤性缺血组,挟闭双颈总动脉10min,恢复再灌注;③缺血预处理+损伤性缺血组,预先挟闭双颈总动脉3min,再灌注3d后再挾闭10min;④一氧化氮合成酶抑制剂(L-NAME)组,于CIP前1h腹腔注射L-NAME(5mg.kg-1),其它步骤同缺血预处理+损伤性缺血组;⑤L-NAME+左旋精氨酸(L-Arg)组,于CIP前1h腹腔注射L-NAME(5mg.kg-1)和L-Arg(300mg.kg-1),其它步骤同CIP+损伤性缺血组;⑥L-NAME+损伤性缺血组,于腹腔注射L-NAME(5mg.kg-1)72h后挟闭双颈总动脉10min,恢复再灌注。各组大鼠于术后或末次缺血后3d断头取脑,分离海马CA1区脑组织,流式细胞仪检测细胞凋亡百分率。结果①全脑缺血10min使海马CA1区细胞凋亡明显增加,损伤性缺血组的细胞凋亡百分率为17.66±2.17,与假手术组(4.18±0.72)相比,明显增加(P<0.01);②缺血预处理+损伤性缺血组,细胞凋亡百分率(5.94±1.63)比损伤性缺血组明显减少(P<0.01);③L-NAME组,细胞凋亡百分率(16.53±2.31)明显增加,和缺血预处理+损伤性缺血组相比,有显著性差别(P<0.01);④L-NAME+L-Arg组细胞凋亡百分率(8.96±1.85)比L-NAME组减少(P<0.01),但仍然高于假手术组和缺血预处理+损伤性缺血组(P<0.01);⑤L-NAME+损伤性缺血组细胞凋亡百分率(17.34±2.73)与假手术组相比明显增加(P<0.01),与损伤性缺血组无显著性差别(P>0.05)。结论①减少海马CA1区细胞的凋亡是CIP诱导BIT的重要机制;②L-NAME抑制NO生成的同时,显著抑制CIP的抗凋亡作用,表明NO参与CIP的抗凋亡作用。
Objective To observe the effect of cerebral ischemic preconditioning (CIP) on the neuronal apoptosis in hippocampal CA1 area of rats induced by ischemic injury and to observe the effect of inhibiting the nitric oxide (NO) production and further verify Role of nitric oxide in the induction of cerebral ischemic tolerance (BIT) and its mechanism of action. Methods Thirty-six Wistar rats with permanent vertebral artery occlusion were divided into 6 groups (n = 6 in each group): ① sham operation group, bilateral carotid artery was exposed only, blood flow was not blocked; ② injury ischemic group , Occluding the common carotid artery 10min, recovery and reperfusion; ③ ischemic preconditioning + ischemic injury group, pre-closure occlusion of the common carotid artery 3min, reperfusion for 3min and then with 10min closure; ④ nitric oxide synthase inhibition (L-NAME) group, intraperitoneal injection of L-NAME (5mg.kg-1) one hour prior to CIP, and the other steps were the same as the ischemic preconditioning + ischemic injury group; ⑤ L-NAME + L-arginine ) Group were given intraperitoneal injection of L-NAME (5mg.kg-1) and L-Arg (300mg.kg-1) 1h before CIP. The other steps were the same as CIP + ischemic insult group. ⑥ L-NAME + After intraperitoneal injection of L-NAME (5mg.kg-1) for 72h, the two common carotid arteries were occluded for 10min and the reperfusion was resumed. The rats in each group were decapitated 3 days after the operation or the last ischemia, and the hippocampal CA1 region brain tissue was separated. The percentage of apoptosis was detected by flow cytometry. Results ① The apoptosis of hippocampal CA1 region was significantly increased 10 min after ischemia in the hippocampus. The percentage of apoptotic cells in the ischemic group was 17.66 ± 2.17, which was significantly higher than that in sham operation group (4.18 ± 0.72) (P <0.01) ); ② The percentage of apoptosis in ischemic preconditioning + ischemic injury group (5.94 ± 1.63) was significantly lower than that in ischemic injury group (P <0.01); ③The percentage of apoptosis in L-NAME group (16.53 ± 2.31 (P <0.01); ④ The percentage of apoptosis in L-NAME + L-Arg group (8.96 ± 1.85) was significantly higher than that in L-NAME group (P <0.01), but still higher than those in sham group and ischemic preconditioning + ischemic insult group (P <0.01) .⑤The percentage of apoptotic cells in L-NAME + ischemic group was 17.34 ± 2.73, There was no significant difference between the two groups (P> 0.05). Conclusion ①Important apoptosis of hippocampal CA1 region is an important mechanism of CIP-induced BIT. ②L-NAME can inhibit the generation of NO and anti-apoptotic effect of CIP, indicating that NO participates in the anti-apoptotic effect of CIP.