氟桂利嗪对脑海马迟发性神经元死亡沙土鼠空间分辨学习能力的影响

Effect of flunarizine on spatial discrimination learning in gerbils with delayed neuronal death in hippocampus

目的:氟桂利嗪是通过血脑屏障的细胞钙超载阻滞剂。探其对迟发性神经元死亡发生后沙土鼠空间分辨学习的影响及对脑海马CA1区神经元的保护作用。方法:实验于2004-03/06在大连医科大学基础医学院生理实验室完成。将健康雄性蒙古沙土鼠56只随机分3组:假手术组(n=8),缺血再灌注组(n=24),氟桂利嗪组(n=24),后两组又分为缺血5min后再灌注2d及7d组和Y迷宫组3个亚组,每亚组8只动物。缺血再灌注组和氟桂利嗪组夹闭双侧颈总动脉制备缺血再灌注模型,假手术仅暴露双侧颈总动脉,不夹闭。氟桂利嗪组术后胃灌氟桂利嗪5mg/kg,1次/d,直至处死前1d;其他两组胃灌生理盐水0.5mL。术后第4天利用Y迷宫检测动物空间分辨学习能力,包括学习获得所需天数及条件反射次数。实验沙土鼠脑组织结构变化及细胞内显微结构变化以免疫组织化学染色法及电镜技术桧测观察。结果:经补充56只动物进入结果分析。①Y迷宫学习获得所需天数:缺血再灌注组多于假手术组和氟桂利嗪组(5.88±0.99,2.00±0.76,2.13±0.64,P<0.01),假手术组和氟桂利嗪组比较无差异(P>0.05)。②Y迷宫学习获得条件反射次数:3组间无差异(P>0.05)。③术后7d脑海马CA1区存活神经元数:缺血再灌注组少于假手术组和氟桂利嗪组犤(8.95±2.29),(65.04±8.09),(42.13±5.90)个/视野,P<0.01犦,氟桂利嗪组仍少于假手术组(q=10.94,P<0.01)。④脑组织病理变化:电镜观察可见迟发性神经元死亡早期形成大量的多泡体,并广泛分布于CA1区神经元树突,是凋亡早期的特征性表现,5mg/kg氟桂利嗪可明显减少CA1区神经元的脱失。结论:脑缺血5min造成海马CA1区神经元选择性迟发性死亡,同时产生空间分辨学习能力的可逆性损害。5mg/kg的氟桂利嗪治疗性用药在一定程度上可以阻止迟发性神经元死亡,同时对学习、记忆等高级神经功能具有保护性作用。

AIM： Flunarizine is a hlocker for calcium overload of cells, which could pass blood brain harrier.In the study.we studied the influence in spatial discrimination learning and the protective effect on neurons in CA1 region of hippocampus in gerhils with delayed neuronal death.METHODS： The experiment was finished in the Laboratory of Physiology,Basic Medical College of Delian Medical University from March to June 2004. A total of 56 healthy gerhils were divided randomly into 3 groups： sham operation group （n=8）, ischemia-reperfusion group （n=24）and flunarizine group （n=24）; gerbils in the later two groups were divided into 2, 7-day reperfusion groups after 5-minute ischemia and Y-maze group（n=8）. Models of ischemia-reperfusion were established in ischemia-reperfusion group and flunarizine group by hlocking hilateral common carotid artery, hut sham operation group only exposed bilateral common carotid artery. Gerbils in flunarizine group received 5 mg/kg flunarizine after operation, once per day lasted to the day hefore death, nther group received 0.5 mL saline. The skill of gerbils in spatial discrimination learning, such as the needed days for learning and the times of conditioned reflex were detected by using Y-maze test at fourth day after operation. The changes of the neuronal microstructure and brain tissue in the CAI region were observed with election microscope and immunohistochemical method.RESULTS： Finally. 56 gerbils were involved in the resah analysis. ① The needed days for learning were more in ischemia-reperfusion .group than those in flunarizine and sham operation groups （5.88±0.99, 2.00~0.76, 2.13~0.64,P 〈 0.01）, but there was no significant difference between flunarizine and sham operation groups （P 〉 0.05）, ② There was no significant difference in the times of conditioned reflex among three groups （P 〉 0.05）. ③ The number of surviving neurons in CA1 region of hippocampus at the seventh day after operation was fewer in ischemia-reperfusion group as compared with that in sham operation and flunarizine groups （8.95±2.29, 65.04~8.09,42.13~5.90, P 〈 0.01）, which was fewer in flunarizine group than that in sham operation group （q = 10.94, P 〈 0.01）. ④ Through electron microscope we could see lots of multi-vesicle bodies formed anddistributed diffusely in CA1 neuron dendrites. It was the early characteritic representation of apoptosis.5 mg / kg dose of flunarizine could decrease the missed neurons in the CA1 region.CONCLUSION： Five-minute cerebral ischemia can induce both selective and delayed death of neurons in CA1 region of hippocampus and reversible damage in spatial discrimination learning, 5mg/kg flunarizine can prevent the happening of delayed neuronal death in some degree and protect the neural function such as learning and memory, etc.