【作者】 李鑫；

【导师】 秦新月；

【作者基本信息】 重庆医科大学 ， 神经病学， 2010， 硕士

【摘要】 全脑缺血是在临床中经常发生的事件之一,心跳骤停、溺水、休克、心肺手术引起的低血压等都可引起脑的灌注不足和缺血缺氧性脑损害,进而引起神经元的缺血坏死。而海马等与学习记忆相关的结构对缺血缺氧更敏感,故临床上经常出现继发性的记忆及认知损伤。虽然已有很多研究致力于保护脑缺血后的认知损害,但一直未达到满意的效果。缺血后处理是近年提出的一种新的保护方式,在缺血再灌注前接受短时,轻微的缺血后,组织的损伤程度较直接接受缺血再灌注时明显减轻,说明动物产生了耐受现象。在心血管领域,缺血后处理已被尝试性的开始应用于临床,并在心脏溶栓等临床操作中取得了一定的效果。缺血后处理在脑血管疾病方向的研究刚刚起步,虽然已经证明其在局灶和全脑缺血模型中都减少了缺血再灌注造成的组织结构损伤,且可能是通过减少氧化应激及抑制凋亡等产生了保护作用,但大部分研究仅关注了缺血后处理对组织结构的保护作用,尚未有研究对缺血后处理与认知行为及神经功能的关系进行探讨。而现在越来越多的研究者提出,组织结构的保护不等于功能的保护,只有产生功能保护,才能真正发挥作用。我们选择全脑缺血模型,首先在行为学方面对缺血后处理的功能保护作用进行研究:采用经典的Morris水迷宫研究学习记忆,运用旷场实验(Open-field, OF)以及神经缺损评分两种评价方式,以更完善的方法对缺血后处理的神经功能保护作用进行评价。由于突触的形态可塑性和功能可塑性被认为是学习记忆的基础,又在神经系统的正常功能实现中起着重要作用,故我们推测,缺血后处理对突触形态和长时程增强(Long-term potentiation,LTP)的影响可能其是产生认知和功能保护最重要及最终的影响通路。N-甲基-D-天冬氨酸(N-metheyl-Daspartic-acid ,NMDA)受体开放是LTP的形成过程中的关键环节,故缺血后处理对LTP产生影响很可能是通过对NMDA受体的调控来完成。本实验试图进一步探讨缺血后处理对突触可塑性及NMDA受体的影响,以验证以上两个假设。第一部分缺血后处理对大鼠学习记忆及神经行为的影响目的:通过对大鼠行为学的观察,探讨缺血后处理对学习记忆及神经行为的影响。方法:SD成年雄性大鼠18只被分为假手术组、全脑缺血15 min组、全脑缺血+缺血后处理组,每组6只,全脑缺血15 min后予缺血后处理,处理方式为缺血/再灌注各15 s,循环3次。每组6只以Morris水迷宫观察学习记忆,旷场箱观察自由运动,由专人按神经功能缺损评分(Neurologic Deficit Score, NDS)标准对各组大鼠进行评分。结果:(1)隐藏平台实验:缺血再灌注后第3到5天,后处理组大鼠逃避潜伏期较全脑缺血组大鼠有较明显的缩短(p<0.05),显示缺血后处理在学习记忆的改善上有明显作用。(2)空间探索实验:缺血组大鼠穿台次数较假手术组明显减低(p < 0.001),后处理组大鼠穿台次数较缺血组大鼠升高(p < 0.05)。(3) Open-field评分:缺血组大鼠评分较假手术组升高(p<0.05),缺血再灌注第7天,缺血后处理组大鼠评分较假手术组大鼠评分降低(p < 0.05)。(4)神经功能缺损评分:缺血组大鼠NDS评分较假手术组大鼠升高(p < 0.001),缺血后处理组大鼠的NDS评分较缺血大鼠降低(p<0.05)。结论:缺血后处理后改善了全脑缺血造成的大鼠学习记忆损伤;降低了全脑缺血损伤造成的神经过度兴奋;减少了全脑缺血导致的神经功能缺损。第二部分缺血后处理保护作用与突触可塑性及NMDA受体的关系目的:观察缺血后处理对全脑缺血大鼠神经元形态、LTP以及大脑中NMDA两个主要亚基NR1和NR2B的影响,探讨缺血后处理保护记忆的可能机制。方法:健康清洁级雌性Sprague-Dawley大鼠72只,体重220±20 g,由重庆医科大学实验动物中心提供。随机分为对照组,全脑缺血15min组,全脑缺血后处理组(简称后处理组),每组24只。每组6只以高尔基(golgi)银染观察神经元形态与树突棘密度变化,6只在第7天处死,制作离体脑片,记录强直电刺激后突触后电位(Excitatory Postsynaptic Potential EPSP)斜率和波幅的变化,12只采用RT-PCR技术检测大鼠海马及皮质的NR1mRNA和NR2B mRNA表达水平,6只采用免疫荧光技术检测大鼠大脑中NR1蛋白和NR2B蛋白的表达水平。结果:(1)与假手术组相比,全脑缺血组大鼠缺血海马CA1区神经元形态受损,树突棘密度降低(p<0.01)。后处理组大鼠海马CA1区神经元形态明显改善,树突棘密度较全脑缺血组明显增多(p< 0.01)。(2)全脑缺血大鼠海马CA1区LTP波幅及斜率较假手术组大鼠降低(p<0.01),缺血后处理组大鼠海马CA1区LTP波幅和斜率较全脑缺血组升高(p<0.01 )。(3)缺血再灌注后72h和7d,全脑缺血组大鼠海马及皮质的NR1mRNA表达降低(p<0.01),缺血后处理组大鼠海马及皮质的NR1mRNA表达水平较全脑缺血组大鼠升高(p<0.05)。(4)缺血再灌注后72h和7d,全脑缺血组大鼠海马及皮质的NR2BmRNA表达降低(p<0.01),缺血后处理组大鼠海马及皮质的NR2BmRNA表达水平较全脑缺血组大鼠升高(p<0.05)。(5)缺血再灌注后第7天,全脑缺血组大鼠皮质NR1和NR2B蛋白较对照组降低(p<0.01),缺血后处理组大鼠皮质NR1和NR2B蛋白表达水平较缺血组大鼠升高(p<0.01)。结论:全脑缺血大鼠海马CA1区树突棘密度降低,LTP波幅和斜率降低,海马及皮质的NR1和NR2B降低,与全脑缺血组大鼠相比,缺血后处理增加了大鼠树突棘密度,提高了LTP的波幅和斜率,并增加了NR1和NR2B的表达,这可能是缺血后处理认知功能保护的重要机制之一。更多还原

【Abstract】 Many accidents lead to global ischemia in clinical emergencies, such as cardiac arrest, drowning, shock, and marked hypotension during cardiopulmonary surgery. Global cerebral ischemia may result in a deficit in spatial learning and memory, cause that the CA1 layer of the hippocampus is very vulnerable to ischemia. In the past decades, a lot of researches attempted to provide protection to ischemia/reperfusion injury, but few treatments can be successfully applied in practice so far.Ischemic postconditioning is a new neuroprotective method, which is a series of rapid intermittent interruptions of blood flow in the early phase of reperfusion, mechanically altering the hydrodynamics of reperfusion. In the field of cardiovascular diseases, ischemic postconditioning has been trying to move from labs to clinical applications, and has achieved fine results in cardiovascular such as in thrombolysis. This may be related to its ability of reducing oxidative stress and inhibiting apoptosis. Recently, it has also been proved that ischemic postconditioning can reduce infarct size after focal brain ischemia and reduce neuronal injury after global cerebral ischemia. However, the underlying protective mechanisms of ischemic postconditioning on ischemia are still not clear, and no papers provide its effects and mechanisms on cognitive and behavioral improvement. On the other side, whether reduce infarct size equal to the protection of neurological function has been questioned by many researches.We adopted global cerebral ischemia model, and chose Morris water maze, Open-field, Neurologic deficit score to evaluate the cognitive and behavioral improvement.Then, causing synaptic plasticity was considered as the base of study and memory, and was very important to normal neurologic function, we suppose that the ischemic postconditioning protection may be based on synaptic plasticity, including morphological plasticity and Long-term potentiation. The N-methyl-D-aspartate (NMDA) receptor is required for LTP and memory, we also examined whether ischemic postconditoning influences NMDA receptor.PART 1 The effect of ischemic postconditioning on behavior and memory in rats after global cerebral ischemiaPurpose : We attempt to investigate the effects of ischemic postconditioning on behavior and memory in rats after global cerebral ischemia for confirm whether ischemic postconditioning result protection of neurological functionMethods:18 SD male rats were randomly divided into sham operated group, global cerebral ischemia group and ischemic postconditioning group. The pullsinelli 4 vessel occlusion was applied to produce the models of global cerebral ischemia reperfusion injury, common carotid arteries (CCA) occlusion with 15min and postconditioning with three cycles, of 15sec release and 15 sec occlusion (15s/15s). 6 rats in each group were evaluated by Morris Maze test for the ability of space learning and memory, gauged by Open-field for functional integrity, Neurologic deficit was evaluated by Neurologic deficit score.Results:(1) The mean escape latency of rats in the ischemic postcondtioning group showed significant short compared with the rats in the cerebral ischemia group in the last 3days (p<0.05) .(2) In the probe test, the number of crossing correct platform of rats in the ischemia group was significantly decreased compared with the sham (p<0.001). The number of crossing correct platform of rats in the postconditioning group was significantly increased compared with the cerebral ischemia group (p <0.05).(3) Open-field score of rats in global cerebral group was significantly increased compared with sham group (p<0.05), and score of rats in ischemic postcondtioning group were significantly decreased compared with cerebral ischemia group at 7d (p<0.05).(4) NDS of rats in global cerebral group was significantly increased compared with sham group (p<0.001), and NDS of rats in ischemic postcondtioning group were significantly decreased compared with cerebral ischemia group (p<0.05).Conclusions : Ischemic postcondtioning improved learning and memory deficit in rats caused by ischemia injury; reduced excessive nervous excitement and neurological deficit caused by cerebral ischemia injury.PART 2 The preservation of ischemic postconditioning related to synaptic plasticity and NMDA receptorPurpose:Causing learning and memory were based on synaptic plasticity, ischemic postconditioning may effect cognitive function by dendritic spine and LTP.NMDA receptors, in particular the NR1 receptor and NR2B receptor, are key protein to LTP, So we investigate the effects of ischemic postconditioning on dendritic spine, LTP, NR1 and NR2B, to explore the mechanism of ischemic postconditioning on the preservation of memory.Methods:72 SD male rats were randomly divided into control group, global cerebral ischemia group and ischemic postconditioning group. The treatment like as before.6 rats in each group were evaluated by golgi stain for morphologic change of neuron. LTP was recorded for the evaluation of function; NR1 mRNA and NR2B mRNA expression of cortex and hippocampus were evaluated by RT-PCR;NR1 protein and NR2B protein expression of cortex were evaluated by immunofluorescence.Results:(1) The density of dendritic spine of rats in ischemia group decreased significantly compared with sham group (p<0.01), and the density of dendritic spine of rats in ischemic postcondtioning group was increased significantly compared with ischemia group (p<0.01).(2) The LTP amplitude and slop in CA1 region of hippocamps of rats in ischemia group was decreased significantly compared with sham group (p<0.01). The LTP amplitude and slop in CA1 region of hippocamps of rats in ischemic postconditioning group was increased significantly compared with ischemia group (p<0.01).(3) NR1 mRNA in the hippocampus and cortex of rats were decreased significantly in ischemia group compared with control group at 72h and 7d after cerebral ischemia-reperfusion (p<0.05). NR1 mRNA in the hippocampus and cortex of rats were increased significantly in ischemic postconditioning group compared with ischemia group at 72h and 7d after cerebral ischemia-reperfusion(p<0.05)(4) NR2B mRNA in the hippocampus and cortex of rats were decreased significantly in ischemia group compared with control group at 72h and 7d after cerebral ischemia-reperfusion (p<0.05). NR2B mRNA in the hippocampus and cortex of rats were significantly increased in ischemic postconditioning group compared with ischemia group at 72h and 7d after cerebral ischemia-reperfusion(p<0.05)(5) NR1 protein and NR2B protein in the cortex of rats in ischemic group were significantly decreased compared with the control group (p<0.01). NR1 protein and NR2B protein in the cortex of rats in ischemic postconditioning group were significantly increased compared with the cerebral ischemia group (p<0.01).Conclusions : Compare with rats of ischemia group, Ischemic postconditioning improved the density of dendritic spines, improved the amplitude and slope of LTP, improved the expression of NR1 and NR2B, that may be one of the important mechanism of cognitive functional protection of ischemic postconditioning.更多还原