【作者】 查晓冰；

【导师】 汪昕； 毛悦时； 徐培康；

【作者基本信息】 复旦大学 ， 神经病学， 2009， 硕士

【摘要】 急性缺血性脑血管病是导致人类死亡的三大主要疾病之一,仅次于心脏病及癌症,但却是病残的主要原因临床上大多数脑梗死是由于脑动脉血栓形成所致,一旦脑动脉阻塞,缺血、缺氧区域的脑组织细胞即刻发生一系烈的“缺血瀑布样反应”,最后导致细胞死亡。缺血性卒中的二级预防目前还没有很好的策略。因此人们对脑卒中的关注从“治”逐渐转移到“防”。脑卒中的一级预防日益引起重视。在尚未出现卒中或有危险因素时即给予干预,降低卒中风险,提高机体自身抵抗能力,以达到避免或减少卒中严重程度的目的。目前已经发现多种预处理方法可以诱导组织细胞对缺血再灌注产生耐受,包括缺血、低氧、HBO、高温、低温、睡眠剥夺、皮层传播抑制以及一些化学物质(如腺苷、内毒素、神经毒素、3—硝基丙酸)等。HBO作为一种预防手段在临床上更有可行性。但有关高压氧诱导脑缺血耐受的机理研究比较缺乏,使其应用缺少必要的理论依据。本课题用线拴法建立大鼠局灶性脑缺血—再灌注模型并经过改良,该模型为脑卒中的常用模型,大鼠与人脑血管结构相似,能很好的模拟人脑缺血再灌的过程。首先探讨HBO预处理诱导脑缺血耐受效应,通过症状学、病理、形态等不同指标分别评价HBO对皮层缺血半暗带和中心坏死区以及海马区的缺血耐受效应。其次探讨高压氧预处理对缺血再灌注脑损伤大鼠神经细胞凋亡以及炎性细胞因子水平的影响。保护作用的机制：抑制由缺血再灌注脑损伤诱导的TNF-α、IL-1β、IL—6过度表达可能是高压氧预处理具有脑保护作用的机制之一。旨在为HBO预处理用于临床提供实验依据。第一部分高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护效应。目的：研究短期的高压氧预处理方法对局灶性脑缺血再灌注损伤是否具有保护效应；同时观察皮层缺血中心区和半暗带以及海马对高压氧预处理不同程度的反应。方法：采用2.5ATA,100%02,每天2次,连续2天的短期高压氧预处理方法,最后一次高氧后次日根据改良的Zea-Longa线栓法制作MCAO再灌注损伤模型。动物分为假手术组、MCAO组,HBO+MCAO组。造模后24小时观察各组动物的一般精神行为状态,体重变化,生存情况,根据Zea-Longa描述的神经学评分方法对神经功能缺损症状评估,测定脑组织含水量,TTC染色,并对脑组织进行灌流切片,行HE、NISSL染色,并在镜下对神经细胞进行计数。结果：高压氧预处理组的动物在次日精神状态较好,毛色光滑,较为活泼,反应迅速,对水和食物的摄取也有较强的主动性,体重下降不如MCAO组明显(23.32±9.38g vs.34.74±8.56g:P<0.01)；生存率在高氧预处理后再受到缺血再灌注损伤打击时得到了显著提高(90%vs.75%；P<0.05)；减轻了神经功能缺失症状(2.29±0.86vs.1.42±0.63；P<0.05)；降低了缺血再灌注损伤后的脑组织含水量(0.76±0.02vs.0.88±0.02；P<0.01)；缩小了梗塞面积(0.15±0.03 Vs.0.22±0.03：P＜0.05)。HE以及NISSL染色均表明HBO对皮层缺血半暗带(102.77±18.63vs.67.36±16.58,p<0.05NISSL)和海马(99.68±19.56vs.34.87±14.75,P<0.01NISSL)有较好的保护作用,但对缺血中心区保护作用不明显(31.66±9.43 vs.28.46±9.64,P>0.05 NISSL).结论：1、MCAO局灶性缺血再灌注模型制作方法相对简单,重复性好,能比较好的应用于预处理效果的比较；且该实验动物模型能提供清楚的缺血中心区和半暗带；2、短期的高压氧预处理方法对大脑中动脉缺血再灌注损伤有保护作用：3、高压氧预处理对缺血皮层半暗带和海马的保护作用优于缺血中心区。第二部分探讨高压氧预处理对缺氧缺血性脑损伤大鼠神经细胞凋亡以及炎性细胞因子水平的影响。方法：按照第一部分的方法进行高压氧预处理和造模。按已确立的24小时后MCAO模型大脑皮层缺血中心区和半暗带的分离方法对皮层和海马分别取材,制作组织匀浆。使用TUNEL法检测细胞凋亡；放免法检测细胞因子TNF-α、IL—1β、IL—6水平。结果：HBO预处理能明显减少缺血半暗带(77.46±22.54vs.117.15±20.52,,P＜0.01)和海马CA1区(68.97±21.36 vs.126.17±22.34,p<0.01)的凋亡阳性细胞数,虽然在缺血中心区比MCAO组的缺血中心区凋亡细胞更多,但两者比较无统计学意义(84.51±18.12vs.64.79±18.34,P>0.05)。HBO预处理明显降低了缺血再灌注大鼠脑组织和血清中TNF-α.IL一1β.IL-6的水平,HBO+MCAO组和MCAO组比较差异明显(均P<0.05)。结论：1、高压氧预处理可明显减少缺氧缺血脑损伤大鼠神经元的凋亡。2高压氧预处理可以有效抑制TNF-α.IL-1β和IL-6炎性细胞因子的释放,可能是高压氧预处理保护作用机制之一。更多还原

【Abstract】 Acute ischemic cerebrovascular disease is one of the three principal diseases causing people to death in which seconded only to heart disease and cancer.but it is the main reason of disabled people.In clinic, most cerebral infarction is caused by cerebral arterial thrombosis.once the cerebral arteries are obstructed.the brain cells in ischemia area will have a series of changes called as"ischemic cascade"at first.then to death.Now days there aren’t good strateges about the secondary prevention of ischemic stroke.The emphases of ischemic cerebrovascular disease are transferred form therapy to prevention gradually and the primary prevention has attracted extensive attention. Interventions means are taken before the episode of stoke or theexistence of risk factors to lower the stroke risk and reinforce the resistance to harmfulstress.The aim is to avoid the happening of stoke or reduce the severity.It has been demonstrated that many pretreatment means can induce tolerance of tissues and cells to ischemia-reperfusion injury,including ischemia, hypoxia, hyperbaric oxygen,chemical agents,conical spreading depression, sleep deprivation, dietary restriction, and both hyperthermia and hypothermia. HBO is more feasible in clinic.Yet the mechanisms on ischemic tolerance induced HBO pretreatment remained poorly defined which has restricted its practical application.We used an established middle cerebral artery occlusion (MCAO) model described initially by Zea-Longa with some improvement.This model is typical for stoke research because vascular construction of Sprague-Dawley rats is similar with that of human being, it can mimic the process of cerebral ischemia-reperfusion in human.First,we investigated the effect of HBO preconditioning on ischemia-reperfusion injury reflected by the means of symptomatology, pathology and morphology to estimate the effect of HBO preconditioning on cortical ischemic penumbra, core or hippocampus.Second,we investigated the effect of HBO preconditioning on inflammatory cytokine and neuronal apoptosis in rat with cerebral ischemia-reperfusion.We hope to provide experimental evidence for the clinical application of HBO preconditioning. Part I The protective effect of HBO preconditioning on transient focal ischemia-reperfusion injury in ratsObjective:To investigate whether the short-term HBO protocol is of protective effect ischemia-reperfusion injury and the degree of response on HBO pretreatment in cortical ischemic penumbra, core or hippocampus respectively.Methods:HBO preconditioning was administered by using 100% oxygen at 2.5 ATA for 1 hour at 12 hours interval for four times in 2 days. The last HBO preconditioning was performed at 24 hours before MCAO produced by the filament model initially reported by Zea-Longa et al with some modifications.Rats were randomly assigned to one of the following three groups:sham group, MCAO group, and HBO plus MCAO group. At 24 hours after the models were made,we observed the general state of spirit and behavior, the change of weight,survival rate,assessed the neurological functional decect by the scales descripted by Johansson and Garcia, determined the brain water content and TTC staining. After perfused and sliced, the sections were performed by HE, NISSL staining and counted under the microscope.Results:During 24h after surgery we observed the rats in MCAO group were depressed and less active, whose coat was in mess.They seemed to lose their interest in food and water. In contrast, the rats in HBO preconditioning group were more vigorous.HBO preconditioning improved survival rate from 75% in MCAO group to 90% (P<0.05) and reduced body weight reduction from 34.74±8.56g in the MCAO group to 23.32±9.38 g (P<0.01).Animals subjected to sham surgery showed no neurobehavioral functional deficit.HBO-PC animals showed better neurological functional recovery at 24 hours when compared with MCAO alone animals(2.29±0.86vs.1.42±0.63;P<0.05).The water content of the MCAO group was significantly higher than the control(P<0.01)and HBO-PC groups(0.76±0.02vs.0.88±0.01;P<0.01).The infarctratio were 0.22±0.03 in MCAO group and 0.15±0.02 in HBO-PC group(P<0.05).There are more intact neurons in the penumbra than in the core at 24 hrs in MCAO and HBO-PC+MCAO groups.The intact neuron numbers per mm~2 in the core of HBO-PC+MCAO group were slightly higher than MCAO group, but not significant(31.66±9.43 vs.28.46±9.64, P>0.05).The intact neuron numbers of the penumbra and the hippocampus were higher in HBO-PC+MCAO group than those of MCAO group (102.77±18.63 vs.67.36±16.58, p<0.05 for penumbra;99.68±19.56vs.34.87±14.75, P<0.01 for hippocampus, respectively).HE also showed the similar results.Conclusion:1 the making method of MCAO models is simple and repeatable. They are suitable for comparing the effect of HBO preconditioning and provide clear ischemic penumbra and core.2 short-term HBO preconditioning protocol is pretective for cerebral ischemia-reperfusion injury.3 the protective effect of HBO preconditioning is better in ischemic penumbra than core. PartⅡ.To investigate the effect of HBO preconditioning on inflammatory cytokine and neuronal apoptosis in rat with cerebral ischemia-reperfusion.Methods:The animal models were made and the HBO was perfomed according to the protocol introduced in Part I.The ischemic core and penumbra were dissected according to well-established protocols in rodent models of unilateral proximal MCAO and the samples were homogenized.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)assay was performed on paraffin-embedded sections according to the manufacture’s instructions.The expression levels of TNF-α、IL-1β、IL-6 were measured with Radioimmunoassay(RIA)Kit.Results:There were more apoptotic neurons in the core of HBO-preconditioning rats than MCAO rats(84.51±18.12vs.64.79±18.34, P>0.05).HBO-preconditioning reduced the number of TUNEL positive cells in the penumbra(77.46±22.54vs.117.15±20.52, P<0.01)and CAl sector (68.97±21.36 vs.126.17±22.34, P<0.01)when compared with MCAO group.At 24 hour after MCAO,HBO-preconditioning significantly reduced the cerebral and serum levels of TNF-α、IL-1β、IL-6 in the rats with Cerebral Ischemia-reperfusion(P<0.05).Conclusion:1 HBO preconditioning could alleviate neuronal apoptosis.2 HBO preconditioning could decrease the level of TNF, IL-1βand IL-6 induced by cerebral ischemia, that may be one of the mechanisms of ischemic tolerance.更多还原