【作者】 胡平；

【导师】 黄巧冰； 高天明；

【作者基本信息】 第一军医大学 ， 病理生理学， 2002， 硕士

【摘要】 缺血预处理可以使脑组织对随后严重脑缺血具有耐受性(缺血耐受)。3分钟前脑缺血对大鼠海马CA1锥体细胞不产生明显损害，但是可以对随后更长时间的能引起CA1锥体细胞死亡的前脑缺血所致的脑损伤起保护作用。已有实验表明热休克蛋白(heat shock protein，HSP)、B细胞淋巴瘤／白血病-2蛋白(B cell lymphoma/leukemia-2,Bcl-2、活性氧自由基(reactive oxygen species，ROS)、核转录因子-кB(nuclear factor-kappaB，NFкB)的上调参与了预缺血处理调节的神经元保护，但是，确切的机制有待进一步阐明。 大鼠全脑缺血后海马CA1区锥体神经元发生延迟性死亡。兴奋性氨基酸学说即细胞外兴奋性氨基酸过度积累导致钙超载而引起细胞毒性一直被认为是缺血性脑损伤的重要机制。然而，近来运用在体和离体细胞细胞内记录发现，严重脑缺血后CA1锥体细胞的放电频率和兴奋性持续下降。钾通道在控制神经元膜的兴奋性方面发挥重要作用，脑缺血后神经元兴奋性的降低可能是由于钾通道活动的增强所致，最近用膜片钳单通道和全细胞记录确实发现短暂性脑缺血后大鼠CA1区锥体神经元的钾通道电流显著增强。而且在培养的皮层神经元上的研究发现，外向钾电流的增加可以触发细胞的程序性死亡；在体与离体的实验均证明钾通道的阻断剂可以抑制细胞的凋亡。上述结果表明钾通道活动增强可能是全脑缺血后大鼠海马 CA锥体细胞的迟发性死亡的机制之一。 我室最近的研究表明大电导钙依赖性钾通道＊K通道）活动的增加参与了前脑缺血后大鼠海马 CA锥体神经元的凋亡。因此我们推测 BK通道活动的抑制可能参与了脑缺血耐受。在本次研究中，大鼠四血管夹闭3分钟脑缺血为预缺血组，只给予手术而无缺血处理为假手术组，预缺血组3天后再给予四血管夹闭8分钟脑缺血处理分别为缺血耐受组，只给予四血管夹闭8分钟脑缺血处理为严重缺血组。再灌流7天后，预缺血组与假手术组组织学相比无改变。严重缺血组可导致海马 CA 区90％以上的锥体细胞死亡；但是，缺血耐受组海马CAI区89％以上的锥体细胞得以存活。我们运用内面向外式膜片钳技术研究预缺血组缺血后6小时，24小时，48小时以及严重缺血组和缺血耐受组缺血后24小时急性分离的大鼠海马CAI区锥体神经元BK通道活动的变化。实验结果发现预缺血组缺血后第一个24小时内BK通道的开放概率明显下降，而严重脑缺血组缺血后24小时BK通道的开放概率明显增加，经通道动力学分析，预缺血组BK通道开放概率的降低是由于关闭时间的延长，开放频率的降低所致，而与开放时间无关；严重缺血组BK通道开放概率增加是由于开放时间的延长、关闭时间的缩短、开放频率的增加所致；而对 CA区锥体神经元具有明显保护作用的缺血耐受组 BK通道的开放概率与正常对照组无明显变化。缺血前后通道的电导、反转电位无明显改变；因此，上述结果提示BK通道活动降低与脑缺血耐受有关。更多还原

【Abstract】 Preconditioning of the rat brain with a sublethal cerebral ischemia induces resistance to a subsequent lethal ischemia (ischemic tolerance). A 3-min period of forebrain ischemia in rats produces no appreciable neuronal damage in the CA1 subfield of the hippocampus but can protect against neuronal damage following subsequent longer periods of ischemia, which normally kills CA1 neurons in the hippocampus. Preconditioning-mediated neuroprotection has been shown to initiate several defense mechanisms such as up-regulation of heat shock proteins, bcl-2 protein, reactive oxygen species, as well as an activation of NF-KB while the precise mechanism remains to be elucidated.Pyramidal neurons in the CA1 field of the hippocampus occur delayed neuronal death after transient forebrain ischemia. Glutamate excitotoxicity haas been suggested as a potential mediator of postischemic cell injury. However, neuronal hyperactivity as predicted by excitotoxic hypothesis is not evident in CA1 region after ischemia. Both in vitro and in vivo intracellular recording studies have demonstrated that the spontaneous firing rate and neuronal excitability of CA1 neurons progressively decrease following reperfusion. It has been speculated that increased potassium currents may be responsible for the reduced neuronal excitability after ischemia. Indeed, recent studies reported an enhancement in activities of potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia. Moveover, it has been showed recently that the enhancement of outward potassium current mediates apoptotic cell death in cultured cortical neurons,and that the apoptotic cell death induced by serum deprivation could be inhibited by potassium channel blocker in both in vitro and in vivo .It has been suggested that the enhancement in large conductance Ca2+-activated potassium (BK) channel activity is involved in the pathogenesis of ischemic neuronal injury. Therefore,we assumed that the suppression of BK channel activity might be involved in ischemic tolerance induced by a ischemic preconditioning. Rats were subjected to forebrain ischemia induced by 4-vessel occlusion for 3-min and only operation without ischemia as preconditioning group and sham-operation group,respectively.,Rats were subjected to forebrain ischemia induced by 4-vessel occlusion for 8-min with preconditioning and without preconditioning as ischemic tolerance group and lethal ischemic group,respectively. Compared to sham-operation group,no histological neuronal changes in rat hippocampus CA1 subfield 7 days after reperfusion were observed in preconditioning group. More than 90% of the CA1 pyramidal cells were destroyed in lethal ischemic group,but more than 89% of the neurons survived in ischemic tolerance group. To address the hypothesis,we used inside-out configuration of patch clamp techniques to investigate the temporal changes in BK channel activity in hippocampus CA1 pyramidal neurons acutely dissociated from preconditioning group at 6h,24h,and 48h following sublethal ischemia and from lethal ischemia group and ischemic tolerance group at 24h following lethal ischemia. The present study found a decrease in BK channel open probability during the first 24h in preconditioning group,rather than an increase observed in lethal ischemia group. Kinetic analyses showed that the suppression of the BK channel activity in preconditioning group was due to a prolongation of the closed time while there was no significant change in open time and the increase in BK channel activity in lethal ischemia group was due to a prolongation of the open time and shortening of the closed time. There were no differences in channel unitary conductance and reversal potential before and after ischemia in groups. In contrast,no apparent changes in BK channel open probability were observed in ischemic tolerance group. It is suggested that the preconditioning-induced suppression of BK channel activity may be associated with ischemic tolerance.更多还原