左旋甲氟喹对小鼠全脑缺血诱发的海马CA1区神经元凋亡的保护作用

发布时间：2018-06-22 10:53

本文选题：缺血性中风 + 甲氟喹　；参考：《华东师范大学》2017年硕士论文

【摘要】：缺血性中风是一类严重危害人类身体健康的疾病,会引起不同程度上的脑损伤。在临床上,一般有神经保护以及溶栓和抗凝等治疗手段,但目前对于防治脑缺血所诱发脑功能损伤的药物治疗手段并非十分理想。因此,对脑缺血所导致脑损伤的防治手段尚需进一步研究。目前已知:脑缺血中风导致细胞死亡的一个关键机制在于缺血导致的相关脑区的神经过度兴奋。因此,阻止脑缺血后的神经过度兴奋可望为防治缺血性中风诱发脑细胞死亡的一个有效途径。我们注意到:临床用于抗疟疾的药物甲氟喹是电突触阻断剂;已有大量文献报道,阻断电突触连接可以显著降低神经系统的兴奋度,提示该药物可能可以防止缺血中风诱发的脑细胞死亡。基于这一推测,本研究使用甲氟喹的左旋异构体,在受双侧颈总动脉夹闭14分钟的小鼠全脑缺血中风模型上、通过观察缺血敏感脑区海马CA1的神经元存活情况,探讨了左旋甲氟喹对脑缺血中风诱发脑细胞死亡的可能防治作用。我们首先通过尼氏染色的方法,在上述中风动物模型上观察到海马CA1神经元死亡。为更好地量化神经元的存活情况,我们接下来主要采用免疫荧光染色手段、测量存活神经元的数量。实验观察到:与假手术实验组(即只分离劲总动脉但不结扎缺血)相比,小鼠海马CA1神经元的数目在颈动脉闭合缺血后的1天、3天和7天分别减少27%(P0.001)、50%(P0.001)和36%(P0.001)。随后,我们鉴定了颈动脉闭合缺血后、腹腔即刻注射不同剂量左旋甲氟喹的可能脑保护效果,在缺血后1天的动物模型上发现:与未接受药物注射的模型鼠(即空白溶剂对照组)相比,接受3.5mg/kg、5.5mg/kg和7.5mg/kg左旋甲氟喹注射的小鼠海马CA1神经元数目分别提高了 16%(P=0.0149)、17%(P=0.0161)和23%(P0.001);并且,与非中风模型老鼠(即假手术组)相比,3.5mg/kg给药组神经元数目降低15.2%(P = 0.020),7.5mg/kg给药组神经元数目与假手术组无显著差异(P= 0.413),几乎恢复到正常值。接着,我们观察了给予左旋甲氟喹(7.5mg/kg)后的不同时间、CA1神经元的存活情况,实验发现:缺血给予药物后的1天和3天、小鼠海马CA1神经元存活情况基本一致;其中,在缺血后1天,给药组神经元数目比溶剂组增多了 36%(P0.001),且与假手术组无显著差异(P = 0.298);在缺血后3天,给药组神经元数目比溶剂组增多了 25%(P= 0.001),与假手术组无显著差异(P= 0.368)。最后,为了鉴定在缺血后的一个较长时间给予药物是否依然有效,我们在缺血手术后的1小时腹腔注射7.5mg/kg左旋甲氟喹,也发现CA1神经元数目能基本恢复到正常水平(P= 0.169)。本论文的发现显示左旋甲氟喹可以有效阻止脑缺血诱发的海马CA1神经元死亡,提示该药物对缺血中风具有潜在的脑保护作用,为临床防治缺血性中风诱发的脑损伤提供了一个新的可能途径。
[Abstract]:Ischemic stroke is a kind of disease that seriously endangers the health of human body and can cause brain damage to varying degrees. In clinic, there are neuroprotective, thrombolytic and anticoagulant treatment methods, but at present, the drug therapy is not very ideal for the prevention and treatment of cerebral function injury induced by cerebral ischemia. Therefore, the prevention and treatment of brain injury caused by cerebral ischemia need further study. It is now known that one of the key mechanisms of cell death due to ischemic stroke is the neuronal hyperstimulation in the ischemic-associated brain region. Therefore, the prevention of neuronal hyperstimulation after cerebral ischemia may be an effective way to prevent and treat cerebral cell death induced by ischemic stroke. We note that mefloquine, a clinical drug used against malaria, is an electrical synaptic blocker, and that blocking electrical synaptic connections has been reported to significantly reduce the excitability of the nervous system. It suggests that the drug may prevent brain cell death induced by ischemic stroke. Based on this hypothesis, the survival of hippocampal CA1 neurons in the ischemic sensitive brain region was observed in a mouse model of global cerebral ischemic stroke after bilateral common carotid artery occlusion for 14 minutes, using the levo-isomer of mefloquine. To explore the possible preventive and therapeutic effects of levo-mefloquine on cerebral cell death induced by cerebral ischemic stroke. We first observed hippocampal CA1 neuron death in the model of apoplexy by Nissl staining. In order to better quantify the survival of neurons, we mainly use immunofluorescence staining to measure the number of surviving neurons. It was observed that the number of CA1 neurons in hippocampus decreased by 27% (P0.001) 50% (P0.001) and 36% (P0.001) respectively on the 3rd and 7th day after closed carotid artery ischemia compared with the sham-operated experimental group. Subsequently, we identified the possible protective effects of different doses of levofluraquine immediately after closed carotid ischemia. On the first day after ischemia, the number of CA1 neurons in hippocampus increased by 16% (P0. 0149) 17% (P0. 0161) and 23% (P0. 001) in mice treated with 3.5 mg / kg and 5. 5 mg / kg of 7.5mg/kg, respectively, compared with the control group without drug injection (P0. 0149), and the number of hippocampal CA1 neurons increased by 16% (P0. 0149) and 23% (P0. 001), respectively. The number of neurons in 3.5 mg / kg group decreased by 15.2% (P = 0.020) compared with that in non-apoplexy model mice (P = 0.020). There was no significant difference between the two groups (P = 0.413) and almost returned to normal value. Then, we observed the survival of CA1 neurons at different times after administration of 7.5mg/kg. The results showed that the survival of CA1 neurons in hippocampus of mice was basically the same on the 1st and 3rd day after administration of the drug after ischemia, and the survival of CA1 neurons in hippocampus of mice was basically the same. On the first day after ischemia, the number of neurons in the administration group was 36% higher than that in the solvent group (P = 0.298), and the number of neurons in the administration group was 25% more than that in the solvent group (P = 0.001) on the 3rd day after ischemia, but there was no significant difference between the administration group and the sham-operation group (P = 0.368). Finally, in order to determine whether the drug was still effective for a longer period of time after ischemia, we injected 7.5mg/kg levofloxaquine intraperitoneally at 1 hour after ischemia and found that the number of CA1 neurons returned to normal level (P = 0.169). Our findings suggest that levofluraquine can effectively prevent hippocampal CA1 neuron death induced by cerebral ischemia, suggesting that the drug has a potential cerebral protective effect on ischemic stroke. It provides a new approach for clinical prevention and treatment of cerebral injury induced by ischemic stroke.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

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