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 investegat the effect of cerebral ischemic preconditioning ( CIP ) on neuronal apoptosis in hippocampal CA1 subfield resulted from the ischemic insult and the influence of inhibiting nitric oxide ( NO ) production on the effect to further clarify the effect and mechanism of NO in the induction of brain ischemic tolerance( BIT ). Methods Thirty-six Wistar rats with vertebral arteries permanently occluded were randomly divided into 6 groups: 1. Sham-operated group ( n = 6 ): the bilateral common carotid arteries ( BCCA ) were separated, but without occluding blood flow; 2. Ischemic insult group ( n =6 ): the BCCA were clamped for 10 min; 3. CIP + ischemic insult group ( n = 6 ): the BCCA were clamped for 3 min as CIP and then were clamped again for 10 min at 3 d of reperfusion after CIP; 4. L-NAME group ( n = 6 ): L-NAME ( 5mg. Kg-, I. P. ) was administered 1 h prior to CIP, other procedures were the same as those in CIP + ischemic insult group. 5. L-NAME + L-Arg group ( n =6 ): L-NAME ( 5mg. Kg-, I. P. ) and L-Arg ( 300mg. Kg-, I. P. ) were administered lh prior to CIP, other procedures werethe same as those in CIP + ischemic insult group; 6. L-NAME + ischemic insult group ( n = 6 ): L-NAME ( 5 mg. Kg-, I. P. ) was administered first, 72 h later, the BCCA were clamped for 10 min. Three days after the sham-operation or the last time of ischemia, the animals were decapitated, brains were removed and the CA1 region was dissected out. The percentage of cellular apoptosis in the CA1 region was detected by means of flow cytometer. ( P <0. 01 ). Results 1. Ischemia for 10 min brought about the increase of cellular apoptosis apparently in the CA1 region, the percentage of the cellular apoptosis was 17. 66 ± 2. 17, obviously increased compared with the sham-operated group ( 4. 18 ±0. 72 ) ( P <0. 01 ); 2. The percentage of cellular apoptosis in CIP + ischemic insult group( 5. 94 ± 1. 63 ) was much lower than that in ischemic insult group ( P < 0. 01 ); 3. The percentage of cellular apoptosis increased apparently in L-NAME group ( 16. 53 ± 2. 31 ) compared with CIP + ischemic insult group ( P < 0. 01 ); 4. The percentage of cellular apoptosis was lower in L-NAME + L-Arg group ( 8. 96 ± 1. 85 ) than that in L-NAME group ( P <0.01 ), but higher than that in the sham-operated group and CIP + ischemic insult group ( P < 0. 01 ); 5. The percentage of cellular apoptosis increased apparently in L-NAME + ischemic insult group ( 17. 34 ± 2. 73 ) compared with the sham-operated group ( P<0.01 ), but was not different compared with ischemic insult group ( P >0.05 ). Conclusion 1. Protective effect of CIP against cellular apoptosis is an important mechanism in the induction of BIT in the CA1 hippocampus; 2. The inhibition of NO production is accompanied by blocking of the protection of CIP, which suggests that anti-apoptosis is a mechanism of NO participating in the induction of BIT.
展开▼
