冷诱导RNA结合蛋白对浅低温治疗心跳骤停大鼠海马神经元及线粒体损伤的影响

Effects of cold induced RNA binding protein on hippocampal neurons and mitochondrial damage after mild hypothermia in a rat model of cardiac arrest

目的浅低温治疗是心跳骤停后有效的脑复苏措施,温度降低时冷诱导RNA结合蛋白(CIRP)表达显著增强。文中旨在评估抑制CIRP对浅低温治疗心跳骤停大鼠海马神经元和线粒体损伤效果的影响并探讨其机制。方法5只雄性SD大鼠海马区注射AAV9,以0.2μL/min注入左右侧各1μL,2周后荧光显微镜观察GFP表达。60只大鼠采用随机数字表法均分为5组(n=12):假手术组、模型组、浅低温组、浅低温+CIRP抑制组、浅低温+空载对照组。浅低温+CIRP抑制组海马区注射AAV9,浅低温+空载对照组注射等量空载体,其余组注射等渗盐水。2周后采用经食道心脏起搏诱发心跳骤停+心肺复苏的方法制备心跳骤停模型。低温组降温至32~34℃,维持6 h。再灌注72 h行NDS评分、HE染色并海马CA1区锥体细胞计数;再灌注24 h电镜下观察海马锥体细胞中线粒体结构,Western blot法检测海马CIRP、线粒体动力相关蛋白1(Drp1)和细胞色素C(Cyt-C)蛋白含量。结果与假手术组NDS评分(80.0分)相比,模型组(61.5分)、浅低温组(70.0分)、浅低温+CIRP抑制组(65.0分)、浅低温+空载对照组(69.0分)评分均降低(P<0.05);与模型组相比,浅低温组、浅低温+空载对照组评分升高(P<0.05)。光镜下模型组细胞排列紊乱稀疏,空泡出现,核固缩或碎裂,大量锥体细胞死亡;浅低温组和浅低温+空载对照组组排列紊乱程度减轻,核固缩、核碎裂及死亡细胞数减少;浅低温+CIRP抑制组与模型组表现相似,较浅低温组损伤加重。模型组、浅低温组、浅低温+空载对照组存活锥体细胞计数[(25.2±3.8、51.8±3.2、50.2±4.4)个]较假手术组[(76.3±3.9)个]明显减少(P<0.05),浅低温组和浅低温+空载对照组较模型组增多(P<0.05),而浅低温+CIRP抑制组[(27.2±4.9)个]较浅低温+空载对照组少(P<0.05)。与假手术组Drp1、Cyt-C蛋白含量比较,其余4组均明显增高(P<0.05),浅低温组、浅低温+空载对照组较模型组降低(P<0.05),浅低温+CIRP抑制组较浅低温组和浅低温+空载对照组明显升高(P<0.05)。与假手术组CIRP蛋白(0.14±0.03)相比,浅低温+CIRP抑制组(0.03±0.01)含量显著降低(P<0.05);浅低温组(0.37±0.08)和浅低温+空载对照组(0.39±0.04)较模型组(0.25±0.05)显著升高(P<0.05)。结论下调海马CIRP的表达,将削弱浅低温对心跳骤停大鼠神经元的保护作用,其机制可能与线粒体分裂有关。

Objective Mild hypothermia was an effective way of cerebral resuscitation after cardiac arrest.The expression of cold-induced RNA binding protein(CIRP)was significantly enhanced when the temperature was lowered.This study was to evaluate the effects and the mechanisms of CIRP inhibition on hippocampal neurological and mitochondria function after mild hypothermia in a rat model of cardiac arrest.Methods Five male Sprague-Dawley rats were injected with AAV9 in the hippocampus,1μL on each side,speeding 0.2μL/min.The expression of GFP was observed by fluorescence microscopy after 2 w.Sixty rats were randomly divided into 5 groups(n=12 for each group):sham operation group,model group,mild hypothermia group,mild hypothermia+CIRP inhibition group and mild hypothermia+normal control group.Injection of AAV9 was performed on mild hypothermia+CIRP inhibition group,same amount of empty vector on mild hypothermia+normal control group,while normal saline on the other groups.Animal models of global cerebral IR were established by transesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation at 2 w after injection.Cooling to 32-34℃was initiated and the temperature was maintained for 6 h on mild hypothermia groups.NDS score,HE staining and pyramidal cell counting on hippocampal CA1 area were performed at 72 h after reperfusion.At 24 h after reperfusion,mitochondrial structure of pyramidal cells in hippocampal CA1 was observed under electronic microscope and the expressions of CIRP,dynamin-related protein 1(Drp1)and cytochrome C(Cyt-C)were detected by Western blot.Results The NDS score of model group was decreased,the number of pyramidal cells was reduced,and the mitochondria were severely damaged.The NDS score of mild hypothermia group was increased,and the number of pyramidal cells was increased(all P<0.05),and mitochondrial damage was reduced compared with model group.In mild hypothermia+CIRP inhibition group,the NDS score was no significant difference compared with mild hypothermia+normal control group and model group,and the number of pyramidal cells was lower than that in mild hypothermia+normal control group[(27.2±4.9)vs(50.2±4.4),P<0.05],similar to model group(25.2±3.8),the damage of mitochondria was severe.After 2 weeks of AAV9 injection,GFP was widely expressed in the hippocampus.The expression of CIRP in mild hypothermia+CIRP inhibition group was respectively small compared with sham operation group[(0.14±0.03)vs(0.03±0.01),P<0.05],which was successfully inhibited by injection of AAV9.The expression of CIRP in model group(0.25±0.05)was significantly higher than that in sham operation group.The expression of CIRP in mild hypothermia group(0.37±0.08)and mild hypothermia+normal control group(0.39±0.04)were higher than that in model group(all P<0.05).The trends of Drp1 and Cyt-C expression were the same,in model group was higher than that in sham operation group,in mild hypothermia group was lower than that in model group,in mild hypothermia+CIRP inhibition group was higher than in mild hypothermia+normal control group(all P<0.05);There were no significant differences between model group and mild hypothermia+CIRP inhibition group,and between mild hypothermia group and mild hypothermia+normal control group.Conclusion Inhibition of CIRP expression in hippocampus can weaken the protective effects of mild hypothermia on neurons in a rat model of cardiac arrest.The mechanism of those effects might be association with mitochondrial division.