【作者】 唐卫华；

【导师】 冯华；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 背景:SAH后脑损伤机制的研究已持续了数十年,至今尚未完全阐明,总体认为损伤机制是多因素和多环节的。既往大部分研究着眼于SAH后颅底大血管的痉挛上,但近来其与SAH后缺血性神经症状的关系受到质疑。提示我们,动脉瘤破裂后血性脑脊液对功能脑区的直接影响很可能是导致临床神经功能障碍的重要原因。已有的研究显示在SAH条件下,存在有钙背景的增强。到目前为止唯一具有循证医学证据的SAH治疗药物是L型钙离子拮抗剂尼莫地平,其具有直接的神经保护作用,显示针对钙信号的干预可以一定程度上改善SAH预后,但其确切疗效仍有争议。T型钙通道的可以介导信号的放大,从而在静息状态发生动作电位及产生动作电流,起到一个类似“扳机样”的作用,广泛参与各种生理及病理生理机制的起动。其中Cav3.3的神经电发放模式为相对较长持续的簇状发放,较多是起到维持T型Ca2+通道的Ca2+“背景电流”的作用,在可兴奋细胞动作电位的触发及去极化的发生上扮演重要的角色。迄今对于T型钙通道在SAH中的作用尚缺乏研究,但SAH环境具有的炎症激活、自由基活化、缺氧等许多因素在其他疾病中已证实能导致T型钙通道功能或表达的改变。围绕SAH的研究也显示在SAH患者及实验性SAH大鼠均存在有扩散性的皮质去极化抑制(Cortical spreading depression-like depolarizations),而海马和下丘脑则是公认的缺血敏感区;SAH对于这三个功能脑区的影响较为显著,而三者的功能状态及其相互作用往往又能决定SAH患者及实验动物的整体神经功能状况;因此T型钙通道在SAH三个功能脑区功能或表达的改变可能参与SAH后脑损伤机制。目的:通过建立的有效的蛛网膜下腔出血的动物模型,在不同层次上观察T型钙通道CaV3.3在正常成年大鼠及SAH大鼠皮质、海马和下丘脑的分布表达。并通过干预CaV3.3的表达及功能,初步探讨T型钙通道CaV3.3在SAH中的意义,从而加深对SAH损伤机制和病理基础的认识。方法:1.建立视交叉前注血及枕大池注血的两种大鼠蛛网膜下腔出血的动物模型,观察其死亡率、生理参数,并结合神经行为学判断(Garcia神经功能评分、Beam Walking任务、Morris水迷宫任务)及尼氏染色评价SAH大鼠的损伤程度及模型的有效性。2.借助常规免疫组化及RT-PCR在不同水平观察CaV3.3在正常成年大鼠的三个脑区的分布,应用免疫荧光多标及激光共聚焦显微镜观察CaV3.3在神经元的表达。3.建立视交叉前注血的大鼠蛛网膜下腔出血的动物模型,借助免疫荧光多标及共聚焦显微镜观察CaV3.3在SAH大鼠的三个脑区的表达;应用T型钙通道相对特异的拮抗剂Mibefradil、L型钙通道拮抗剂Verapamil及CaV3.3反义寡核苷酸进行干预,观察其死亡率、生理参数,并结合神经功能判断评价干预治疗的效果。结果:1.视交叉前注血模型的死亡率为20%;术后Garcia神经功能评分均低于假手术组及对照组,但仅视交叉前注血组与假手术组及对照组比较有显著差异;术后beam walk评分提示SAH大鼠出现精细运动障碍(P<0.01),视交叉前注血模型更为明显; MWM任务显示术后SAH建模大鼠空间学习能力下降(P<0.01),视交叉前注血模型更为明显。枕大池注血组血液在枕骨大孔区及小脑范围内分布较多,视交叉前注血组可见主要分布于前、后颅窝及蝶鞍处。注血组尼氏染色阳性细胞明显减少,其中以皮质及海马脑区最为明显;两种注血方式对比,视交叉前注血组尼氏阳性细胞减少明显。2.常规免疫组化观察到CaV3.3在端脑、间脑、脑干和小脑中都有广泛分布,其中大脑皮质中CaV3.3阳性细胞最为密集,在海马的各区及齿状回也可见不同程度的CaV3.3阳性细胞,而在下丘脑则在乳头体内侧核区多见。在大脑皮质中CaV3.3阳性神经元较为密集。海马区阳性神经元散在分布。下丘脑区则少见CaV3.3阳性神经元,但可见数量较多的CaV3.3阳性的神经纤维样结构。RT-PCR显示大鼠脑皮质、海马、下丘脑均有CaV3.3 mRNA的,其中皮质表达略高海马及下丘脑(P<0.01)。3. SAH建模后3d、7dCaV3.3阳性神经元细胞比率有增多趋势。术后各组Beam walk任务评分均低于假手术组(P<0.01); CaV3.3反义寡核苷酸组、米贝地尔、维拉帕米对SAH后大鼠有治疗作用,可以改善Beam walk评分;MWM任务评分结果类似。其中术后第7天CaV3.3反义寡核苷酸组、米贝地尔治疗效果好于维拉帕米(P<0.01)。CaV3.3反义寡核苷酸可能是在4~7天段时间起效。结论:1.两种大鼠蛛网膜下腔出血的注血模型均能较好模拟SAH,比较之下,大鼠视交叉前注血建模其血块部位更接近wills环,神经功能缺损表现更明显,总体上更为接近临床的真实情况。2. CaV3.3在正常成年大鼠的皮质、海马和下丘脑脑区有均有分布,其中大脑皮质中最为密集;在大脑皮质中CaV3.3阳性神经元较为密集,海马区阳性神经元散在分布。下丘脑区则少见CaV3.3阳性神经元,但可见数量较多的CaV3.3阳性的神经纤维样结构。3. SAH建模后CaV3.3在脑内的表达有增多趋势,应用CaV3.3反义寡核苷酸组、米贝地尔、维拉帕米干预均表现有治疗效果。提示提示T型钙通道及CaV3.3参与了SAH后大鼠CNS的损伤机制。更多还原

【Abstract】 Background: The research of the mechanism of subarachnoid hemorrhage(SAH) induced brain injury has been lasted for several decades but it remaines unclear till now. Previous studies focused on the cerebral vasospasm and little attention have been paid to the changes of function brain region. It is indicated in experimental and clinical studies that nimodipine ,the only effective drugs which is evidence based , could do effect on protection of neurons and meliorate SAH induced demage in some extent or in the early phase.Although the effect and mechanism of nimodipine is still in debate, it means the intervention of calcium sighal would improve the outcome of SAH. The ability of T-type channels to open at similar potentials at which they inactivate suggests that they might generate current under steady-state conditions.This is commonly referred to as a window current,which likely involved in sustained burst firing, is operationally defined as the overlap region between the activation and steady-state inactivation curves,that they play an important role in signal amplification and induce depolarization and AP (action potential) .It is still short of the research in the mechanism of T-type channels in subarachnoid hemorrhage.But previous studies have showed there is inflammation , free radical activation , hypoxia in SAH,which would induce expression alteration and function swtch of T-type channels. Cortical spreading depression-like depolarizations was found in patients or animals post SAH. And Hippocampus and hypothalamus is ischemic sensitive .So, the alteration of T-type channels in these brain regions may make sense after SAH.Object : Reasonable animal SAH model were founded to observe the expression alteration and function swtch of T-type channels Cav3.3 after SAH. And to investigate the contributions of T-Type Channels Cav3.3 in the experimental subarachnoid hemorrhage by means of interven the expression and function of Cav3.3 after SAH .Thus ,clarify the possible mechanism of subarachnoid hemorrhage induced brain injury.Methods:1. SAH was induced in rats via blood injection into the cisterna magna (cisterna magna injection SAH model), or blood injection into the prechiasmatic cistern (prechiasmatic cistern injection SAH model). The mortality rate and physiological parameters was measured. Furthermore, by the means of neuroethology evaluation(Garcia score,Beam Walking Test,Morris water Maze Test)and Nissl’s staining ,the degree of brain after subarachnoid hemorrhage was evaluated .2. Immunohistochemistry and RT-PCR were used to evaluate the expression of T-type channels Cav3.3 in adult rats brain( Cortical,Hippocampus and hypothalamus). By means of immunofluorescence and confocal microscopy to show the expression of Cav3.3 in neurons .3. SAH was induced in rats via blood injection into the cisterna magna into the prechiasmatic cistern. The expression of T-type channels Cav3.3 in SAH rats brain was evaluated by immunofluorescence . Furthermore, Mibefradil, Verapamil and Cav3.3 AS ODN were used to treat SAH. Mortality rate,physiological parameters and neuroethology evaluation were used to evaluate the effect.Results:1. The mortality rate of prechiasmatic cistern injection SAH model was 20%. Garcia score shows that the prechiasmatic cistern injection SAH model was lower than control group and sham group(P<0.01). Beam Walking Test shows that all the SAH groups have poor Skilled Movement score(P<0.01). Morris water Maze Test shows that all the SAH groups have poor space leanning performance., The prechiasmatic cistern injection SAH model is worse in all neuroethology evaluation.;2. Cav3.3 is almost exclusively expressed in adult rats brain and is high in olfactory bulb, striatum, cerebralcerebralcortex, hippocampus, reticular nucleus, lateral habenula,and cerebellum. Few Cav3.3 is found in hypothalamus neurons, But it is abundant in the nerve plexus .And the RT-PCR showed the similar results.3. Beam Walking Test shows that all the SAH groups have poor Skilled Movement score(P<0.01). Mibefradil, Verapamil and Cav3.3 AS ODN would improve the performance in Beam Walking Test . Morris water Maze Test shows the the similar results. Mibefradil and Cav3.3 AS ODN is some better than Verapamil .And Cav3.3 AS ODN may take effect from day3.Conclusion:1. Two SAH model are also reasonable models to mimic the clinical situation .But the prechiasmatic cistern injection SAH model was better .It is more close to the Willis circulation and more poor performance in neuroethology evaluation.It is seemed more close to reality.2. Cav3.3 is expressed in adult rats brain and is high in cerebralcortex, hippocampus. Few Cav3.3 is found in hypothalamus neurons, But it is abundant in the nerve plexus .3. There is overexpression of T-type channels Cav3.3 in SAH rats brain .And by means of interven the expression and function of Cav3.3 would reduce the brain injury after SAH更多还原