nNOS介导小鼠局灶性脑梗死后氧化DNA损伤的机制研究

Experimental study on oxidative DNA damage after focal infarction induced by nNOS in brain

目的探讨还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)阳性神经元表达与脑梗死后DNA损伤修复过程的相关性。方法制作小鼠前脑缺血-再灌注模型(FbIR),夹闭双侧颈总动脉90 min后恢复血流,再灌注15min后处死动物制作脑组织冰冻切片。A组(n=10):FbIR90/15 min;B组(n=4):FbIR90/15 min+3BR7NI;C组(n=10):假手术对照组;D组(n=4):采用组织化学染色法观察NADPH-d阳性神经以元的分布。A、B两组分别用大肠杆菌核苷酸外切酶Ⅲ敏感位点法(escherichia coliexonucleaseⅢsensitive sites,EXOSS)检测AP位点和3-′PO4末端型两种氧化DNA损伤。用EXOSS检测AP位点和3-′PO4末端型两种氧化DNA损伤;采用组织化学染色法观察NADPH-d阳性神经元的分布。结果EXOSS法可检测到大脑不同部位的氧化DNA损伤,主要集中在大脑皮质区、下丘脑弓形核区、纹状体和海马区;特异性NOS抑制剂3BR7NI明显减弱全脑的EXOSS信号强度(P<0.000 1),差异有显著性,其作用主要表现在大脑皮质区,而对下丘脑的弓型核等区作用较弱;NADPH-d阳性神经元主要分布在大脑皮层、纹状体、丘脑和齿状核,下丘脑的弓型核区仅见少量染色,EXOSS/NADPH-d的表达对比显示,在大脑皮质区EXOSS信号主要在NADPH-d阳性神经元中。结论脑梗死后大脑不同区域氧化DNA损伤的机制不一,3BR7NI仅消除大脑皮层区的EXOSS染色,而对下丘脑弓形核区的作用不大,本研究从形态学证实了大脑皮层区nNOS可能参与了脑梗死后氧化DNA损伤修复过程,而下丘脑的弓型核区的这一过程可能有其他机制参与。

Objective To study the correlation of expressions of NADPH-d positive neurons and oxidative DNA damage after brain infarction. Methods Forebrain ischemia-reperfusion （FblR） was induced by occluding both common carotid arteries for 90 rains, followed by 15 rains of reperfusion. Forebrain sections were made after autopsy. Four groups were divided as follows： A group（ n = 10） ：FblR 90/15 min; B group（ n = 4） ： FblR 90/15min ＋ 3BR7NI; C group（ n = 10） ： a sham control; D group（ n = 4） ： normal control. In both A and B group, two types of oxidative DNA damages of AP （apyrimidinie or apurinie） sites and 3＇-PO4 terminal sites of single-strand breaks were detected by EXOSS method （Escheriehia coli exonuelease Ⅲ sensitive sites）. The morphology and distribution of NADPH-d positive neurons were examined by histochemical teehnical method. Results The oxidative DNA damage elevated unequally in different areas, mainly distributing in cortex, in areuate nuclei of hypothalamus and in hippocarnpus. In B group, miee treated with 3-bromo-7-nitroindazole （3BR7NI）, an specifically inhibiter of nNOS, showed decreased EXOSS signals in the eerebral cortical neurons, but not in areuate nuclei of hypothalamus. NAPDH-d positive neurons were mainly distributed in cortex, corpus striatum, dentatothalamus, and seldom in areuate nuclei of hypothalamus. The expression of EXOSS/NAPDH showed that the EXOSS signals were mostly in NAPDH-d positive neurons in cortex area. Conclusion The mechanisms of oxidative DNA damages after brain infaretion were different in different cortex areas. This research provided morphologie proof of nNOS involving in oxidative DNA damage in cortex, while another meehanita maybe existed in arcuate nuclei of hypothalamus.