大鼠全脑缺血及再灌流模型的研究

手术阻断大鼠基底动脉及双侧颈总动脉(三动脉阻断)以形成急性或慢性全脑性缺血模型,并在一定时间后解除双侧颈总动脉的阻断,脑血流再通,形成再灌流。用此动物模型观察了脑缺血及再灌流后的脑电图、体感诱发电位的改变,神经病学障碍,脑组织的生化及形态变化。本文讨论了此动物模型的优缺点。     

大鼠脑再灌流综合征模型初探

阻断大鼠双侧椎动脉和颈总动脉,导致急性前脑缺血。在缺血30或60分钟后,再灌流4小时,记录存活率及实验前、后颅顶硬膜外脑电图;测定再灌流后脑组织的水、钠、钾含量,同时取材作光镜和电镜形态学检查。     

茶氨酸对脑缺血再灌注损伤保护作用的实验研究

目的观察茶氨酸对脑缺血再灌注损伤的保护作用,为临床脑缺血再灌注损伤的预防和治疗提供实验依据。方法将24只健康家兔随机分为四组：假手术组、脑缺血组、茶氨酸予处理组和茶氨酸治疗组。麻醉后分离血管,采用基底动脉、双侧颈总动脉结扎法制备急性全脑缺血再灌注动物模型。假手术组仅分离动脉不结扎,予处理组在缺血前应用茶氨酸,治疗组在缺血后应用茶氨酸,缺血组不作特殊药物处理。各组分别在规定时间点即缺血前、再灌注后30min、1h、2h采血测定神经特异性烯醇化酶(NSE)含量,取脑组织活检观察超微结构,处死动物测定脑含水量。结果茶氨酸予处理组和治疗组以上各项指标均较脑缺血组有显著的改善,提示茶氨酸具有神经保护作用。实验还显示茶氨酸予处理组在再灌注30min时NSE含量与假手术组比较无差异,提示用茶氨酸予处理后可能使缺血后脑损害的发生延迟,为进一步的治疗争取了宝贵时间。结论茶氨酸对脑缺血再灌注损伤有保护作用,值得进一步研究。     

急性缺血脑组织的重灌流损伤

中枢神经系统发生缺血性损伤后导致很高的死亡率及致残率。为了有效地防治脑缺血损伤,必须尽快恢复缺血脑组织的血液供应。目前临床常采取溶栓、解痉、血管吻合及心肺脑复苏等疗法使阻断的脑动脉血流再通,改善组织的血液供应,但其疗效仍有争议。因而探讨缺血脑组织重灌流的病理生理改变及重灌流时采取的措施倍受重视。多年的实验研究表     

大鼠脑缺血-再灌流后脑内MPO活性变化及组织损伤病理进观察

目的 :通过测定大鼠局灶性脑缺血 -再灌流后不同时点脑组织中 MPO活性变化 ,探讨炎症反应与脑缺血 -再灌流损伤的关系。方法 :用线栓法制备大鼠大脑中动脉缺血 -再灌流模型 ,检测缺血 3小时后再灌流不同时点脑组织中 MPO活性、脑梗死体积及光镜病理学变化。结果 :缺血组脑组织有 MPO活性升高、中性粒细胞浸润 ,以再灌流后 4 8、 72小时最为明显 ,脑梗死体积、神经元变性程度随再灌流时间延长而加重。结论 :局灶性缺血脑组织中MPO活性与缺血 -再灌流损伤间具有一定的关系 ,炎症反应是加重脑组织损伤的重要因素。     

脑复康对大鼠局灶性脑缺血引起的脑水肿及神经功能变化的影响

目的探讨脑复康(吡拉西坦)对局灶性脑缺血引起的脑水肿及神经学行为的影响.方法阻断大鼠一侧大脑中动脉造成局灶性脑缺血模型.分别通过舌下静脉输注不同剂量的脑复康注射液及甘露醇注射液,观察各组动物在阻断大脑中动脉24h后,动物神经功能、脑梗塞体积及脑组织含水量的变化,并与假手术组及对照组进行比较分析.结果阻断大鼠一侧大脑中动脉引起了动物神经功能的明显变化及阻断侧大脑半球的严重水肿.脑复康注射液剂量依赖性地降低动物的脑梗塞体积及阻断侧大脑半球的含水量,改善其神经功能,1000 mg/kg脑复康注射液的降低脑水含量及脑梗塞体积、改善神经功能的作用与同剂量的甘露醇效果相当.结论较大(250 mg/kg以上)剂量的脑复康注射液对局灶性缺血的脑组织有明显的保护作用.     

不同预缺血时间及间隔时间窗对CA_1区锥体细胞的影响

目的 :探讨预缺血时间及间隔时间窗对CA1区锥体细胞的影响。方法 :钳夹沙土鼠的双侧颈总动脉制造脑缺血模型 ,应用尼氏染色观察迟发性神经元坏死。结果 :3 5min前脑缺血再灌流 7d前 ,1min的预缺血时间没有保护作用 ;2min预缺血时间 ,间隔 6h对脑缺血没有保护作用 ,间隔 1、3、5、7d对脑缺血有保护作用 ,间隔 15d对脑缺血保护作用消失。结论 :2min预缺血时间导致脑内复杂的分子生物学变化 ,间隔时窗 1～ 7d对CA1区锥体细胞有保护作用     

超低温断血流对猴脑氨基酸类神经递质的影响

目的 研究猴脑选择性超深低温断血流复苏过程中脑组织细胞外液的谷氨酸(glutamate, Glu)和γ-氨基丁酸(gamma-aminobutyric acid, GABA)的动态变化.方法 恒河猴7只,随机分为两组:四血管(双侧颈总动脉及椎动脉)阻断冷灌注组(简称四血管组,n=4),两血管(双侧颈总动脉)阻断冷灌注组(简称两血管组,n=3).将微透析管置入右顶叶脑组织内,自缺血前30min开始收集细胞外液,用高效液相色谱-紫外法测定脑选择性超深低温断血流复苏前后脑组织细胞外液Glu及GABA的浓度.结果 四血管组Glu在常温断血流缺血后即迅速升高(P<0.05),低温冷灌注后较常温缺血时明显下降至低于缺血前水平(P<0.05);恢复血流后行再灌注复温,早期即开始升高,迅速达到并超过灌注前水平.GABA则变化不大.两血管组Glu在常温断血流预缺血后迅速升高(P<0.05),低温冷灌注后较常温缺血时明显下降(P<0.05),下降幅度较四血管组明显;恢复血流后行再灌注复温,和行低温冷灌注时相比,Glu变化不明显.GABA在常温断血流预缺血、低温冷灌注及恢复血流复温时均变化不大.结论 脑选择性超深低温断血流复苏通过抑制谷氨酸等兴奋性神经递质的释放而抑制其细胞毒性作用.     

丁苯酞对大鼠脑缺血再灌注损伤后HSP10表达的影响

目的研究丁苯酞干预对大鼠脑缺血再灌注损伤后不同时点脑组织中热休克蛋白10(HSP10)表达的影响,并探讨丁苯酞对缺血性脑血管病保护作用的机制。方法采用夹闭双侧颈总动脉的方法制作大鼠前脑缺血再灌注模型;H&E染色和NSE免疫组化染色神经元,观察脑组织的形态变化和记数各组神经元数目;免疫组织化学检测对照组、缺血再灌注组及丁苯酞干预组大鼠脑组织HSP10的表达水平变化。结果脑缺血再灌注后丁苯酞干预组及缺血再灌注组HSP10表达水平于再灌注后6h开始上调,3d达到高峰,丁苯酞干预组各时间点HSP10阳性表达指数均高于缺血再灌注组,丁苯酞干预组脑组织的损伤程度明显轻于脑缺血再灌注组(P<0.05)。结论丁苯酞可能通过上调大鼠缺血再灌注损伤后脑组织HSP10的表达,从而抑制前脑缺血再灌注损伤后细胞凋亡,减少神经元死亡,减轻缺血再灌注后脑组织的损伤。     

不同药物麻醉后大鼠全脑缺血脑温的变化

目的 了解不同常用麻醉药物后麻醉大鼠全脑缺血脑温的变化。方法 选用乌拉坦（1g/kg)、戊巴比妥钠（40mg/kg)、水合氯醛（400mg/kg)、巴比妥钠（300mg/kg)和苯巴比妥钠（200mg/kg)腹腔麻醉大鼠后全脑缺血测量脑组织温度的动态变化。结果 这些药物麻醉大鼠后行四动脉阻断全脑缺血，脑温进一步下降；但不同药物的作用各异。结论 本实验的结果说明了在脑缺血同一个实验过程中尽量避免使用不同各类的麻醉药物引起的脑温波动，进而影响实验结果的准确性。     

脑缺血再灌注后线粒体氧化应激损伤的动态变化

目的探讨脑缺血再灌注后线粒体氧化应激损伤的动态变化特点。方法利用线栓法制备大鼠大脑中动脉缺血模型,通过线粒体分离技术测定线粒体丙二醛、三磷酸腺苷、线粒体膜电位水平,应用Realtime PCR仪测定线粒体基因(线粒体DNA)拷贝数,分析脑缺血再灌注后不同时间点缺血周围皮质线粒体的氧化应激水平及线粒体失功能的动态变化特点。结果①缺血再灌注后线粒体丙二醛水平呈渐进性增高,6 h为(4.61±0.83)nmol·mg-1 prot,72 h达(6.94±0.96)nmol·mg-1 prot,均较对照组显著升高(P0.05);②缺血再灌注6 h后mtDNA拷贝数开始下降,24 h轻度回升,72 h显著下降水平为对照组的62%(P0.01);③脑缺血再灌注后6 h线粒体三磷酸腺苷水平即显著下降,24 h出现短暂升高,再灌后72 h下降水平为对照组的42%(P0.01);④荧光探针检测显示脑缺血再灌注后线粒体膜电位持续下降,再灌注后72h线粒体膜电位水平显著下降至对照组的43%(P0.01)。结论线粒体损伤随缺血再灌注时间延长而呈渐进性加重,氧化应激损伤是构成其损伤的主要因素。缺血后脑组织线粒体存在短暂的内源性代偿修复机制,但不足以逆转氧化应激对线粒体的持续损伤。     

病变侧亚低温对大鼠局脑缺血再灌注后Akt、Survivin表达的影响

目的 通过检测Akt及Survivin蛋白的表达,探讨亚低温对局脑缺血再灌注后神经元存活的影响.方法 用线拴法制作大鼠大脑中动脉闭塞（MCAO）局脑缺血再灌注模型,将44只SD大鼠随机分成假手术组、常温缺血组和亚低温缺血组,缺血组分别缺血2,6 h再灌注4,24,72 h,1周,2周后处死,亚低温组于缺血后13～14 min实施病灶侧亚低温持续4 h.免疫组织化学法检测Akt、Survivin 蛋白的表达.结果 相同缺血再灌注时间点,亚低温组比常温组缺血侧Akt、Survivin、表达水平显著增高（P＜0.05）.结论 病灶侧亚低温通过促进缺血半暗带脑组织Akt、Survivin表达的核内移,从而抑制神经元凋亡,促进脑缺血后神经功能恢复.     

Changes in striatal dopamine metabolism measured by in vivo voltammetry during transient brain ischemia in rats

In vivo voltammetry was used in rats with brain ischemia induced by four-vessel occlusion to measure changes in dopamine metabolism via measurement of peak 2 (dopamine compounds) in the striatum. Changes in regional cerebral blood flow in the striatum were also assessed by means of a temperature-controlled thermoelectrical device. Peak 2 increased by 600-900% during 30 minutes of four-vessel occlusion, which may have reflected an ischemia-provoked increase in the release of dopamine and a disturbance in the outward transport of its metabolites. Following reperfusion by discontinuation of carotid occlusion, peak 2 rapidly decreased to below control values and then gradually increased, exceeding control values at 180-210 minutes after reperfusion. REgional cerebral blood flow in the striatum decreased to almost 0 ml/100 g/min during the ischemic period, transiently increased to greater than control values after reperfusion, then gradually decreased during the next 240 minutes. Since dopamine is known to have various effects on cerebral metabolism and blood flow, alterations in its behavior may contribute to changes in cerebral blood flow and to postischemic brain damage. In vivo voltammetry may be useful in the investigation of the pathophysiology of brain ischemia.     

Hydroxyl radical production in the cortex and striatum in a rat model of focal cerebral ischemia

BACKGROUND: Increases in hydroxyl radical production have been used as evidence of oxidative stress in cerebral ischemia/ reperfusion. Ischemia can also induce increased dopamine release from the striatum that may contribute to hydroxyl radical formation. We have compared hydroxyl radical production in the cortex and striatum as an index of oxidative stress in a rat model of focal cerebral ischemia with cortical infarction. METHODS: Using a three vessel occlusion model of focal cerebral ischemia combined with bilateral microdialysis, hydroxylation of 4-hydroxybenzoate (4HB) was continuously monitored in both hemispheres in either the lateral striatum or frontoparietal cortex. The ischemia protocol consisted of one hour equilibration, 30 min of three vessel occlusion, then release of the contralateral common carotid artery (CCA) for 2.5 h. RESULTS: Induction of ischemia resulted in a 30-fold increase in dopamine release in the lateral striatum. Compared to the nonischemic striatum, the ratio of the hydroxylation product 3,4-dihydroxybenzoate (34DHB) to 4HB (trapping agent) in the ipsilateral striatum increased significantly 30 min after ischemia induction. In contrast, during the 30 min of three vessel occlusion there was no increase in the ratio in the cortex. Following the release of the contralateral CCA, the ratio from the ischemic cortex increased significantly compared to sham-operated animals. However, under all circumstances, the 34DHB/4HB ratio was greater in the striatum than in the cortex. CONCLUSION: The increase in the 34DHB/4HB ratio in the lateral striatum coincides with the increased dopamine release suggesting a role for dopamine oxidation in the increased production of hydroxyl radicals. The significant increase in the ratio from the ischemic cortex compared to that from the sham-operated animals is consistent with increased oxidative stress induced by ischemia. However, the lower 34DHB/4HB ratio in the cortex which does not receive dopaminergic innervation compared to the striatum suggests a different mechanism for hydroxyl radical production. Such an alternate mechanism may represent a more toxic oxidative insult that contributes to infarction.     

Resveratrol protects against global cerebral ischemic injury in gerbils

Increased oxidative stress has been implicated in the mechanisms of delayed neuronal cell death (DND) following cerebral ischemic insult. In this study, we investigated whether resveratrol, a polyphenolic antioxidant enriched in grape, may ameliorate ischemia-induced neuron cell death. Mongolian gerbils were divided into three groups, namely, sham control, ischemia and ischemia treated with resveratrol. Transient global cerebral ischemia was induced by occlusion of both common carotid arteries (CCA) for 5 min. Resveratrol was injected i.p. (30 mg/kg body weight), either during or shortly after CCA occlusion, and again at 24 h after ischemia. Cerebral blood flow was monitored before and during CCA occlusion using a laser Doppler flowmeter. Brain sections were immuno-stained for neurons, astrocytes and microglial cells. A time course study was also carried out to assess the bioavailability of resveratrol in serum, liver and brain using high performance liquid chromatography (HPLC). Morphometric measurements indicated extensive DND in the hippocampal CA1 region 4 days after ischemia and that neuron cell death was marked by the increase in reactive astrocytes and microglial cells. Administration of resveratrol, either during or after CCA occlusion, significantly (P<0.05) decreased DND as well as glial cell activation. Analysis of resveratrol after i.p. injection indicated its presence in serum, liver and brain with peak activity at 1, 4 and 4 h, respectively. This study demonstrated for the first time that resveratrol, a polyphenolic antioxidant, can cross the blood-brain barrier and exert protective effects against cerebral ischemic injury.     

一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时作用研究

目的：观察一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时的作用方法;民凝大鼠大脑中动脉制成脑缺血模型,选择脑缺血３０ｍｉｎ,６０ｍｉｎ,１２０ｍｉｎ,１８０ｍｉｎ为研究时点,观察各时点用选择性,非选择性一氧化氮合酶抑制剂亚硝酸盐含量测定,缺血脑组织坏死体积测定及损伤海马ＣＡ１电镜观察。     

人工合成E-选择素治疗大鼠局灶脑缺血再灌注损伤的探讨

目的：探讨新的药物治疗脑缺血再灌注损伤。方法：用人工合成 Ｅ－选择素 ２ｍｇ·ｋｇ－１或 １０ ｍｇ·ｋｇ－１溶解于生理盐水中,静脉注入自发性高血压大鼠永久左侧大脑中动脉／颈总动脉（ＭＣＡ／ＣＣＡ）闭塞或ＭＣＡ／ＣＣＡ闭塞２ｈ后ＣＣＡ再灌注模型中。２４ｈ后,脑梗死体积用计算机扫描计算。结果：在永久性ＭＣＡ／ＣＣＡ闭死组中脑梗死体积没有差别,在ＭＣＡ／ＣＣＡ闭死后ＣＣＡ再灌注组中脑梗死体积有意义地缩小（Ｐ＜０．０１）。结论：Ｅ－选择素能够有效地减少大鼠脑缺血再灌注损伤。     

电针预处理对大鼠脑缺血再灌注损伤后EAAT2的表达研究

目的研究电针预处理对脑缺血再灌注损伤后大鼠缺血半暗带兴奋性谷氨酸转运体2(EAAT2)表达的影响,探讨EAAT2在电针预处理诱导脑缺血耐受中的作用。方法 18只SD雄性大鼠随机分为3组(n=6):分别为假手术(Sham)组、右侧大脑中动脉阻闭(MCAO)组、电针预处理(EA)组。假手术组仅分离血管,不进行阻闭,术后24 h检测;MCAO组用MCAO法致缺血120 min后于再灌注24 h检测;EA组大鼠予电针刺激30 min,刺激结束2 h后处理同MCAO组。3组大鼠在观察神经行为学变化后取材,通过2,3,5-氯化三苯四唑(TTC)染色评估梗死面积,并检测EAAT2 mRNA及蛋白表达水平。结果电针预处理能明显降低脑梗死容积百分比(P0.01),提高MCAO大鼠神经行为学评分,诱导脑缺血耐受并抑制脑缺血再灌注损伤后24 h EAAT2表达的下降(P0.01)。结论脑缺血再灌注损伤后,EAAT2表达下降,而电针预处理能显著抑制缺血半暗带EAAT2的表达下调,诱导脑缺血耐受,从而减轻脑缺血再灌注损伤。     

阿司匹林促进大鼠缺血脑组织脑源性神经营养因子的表达

目的:探讨不同剂量阿司匹林(Asp)对脑缺血/再灌注(CI/RP)损伤大鼠的神经保护作用及其对脑源性神经营养因子(BDNF)表达的影响。方法:采用线栓法建立大鼠大脑中动脉CI/RP模型,对CI/RP后大鼠进行肢体神经功能缺损评分:1~3分的48只大鼠入选。入选大鼠分为对照组、Asp小剂量组(20 mg.kg-1)、Asp中剂量组(80 mg.kg-1)和Asp大剂量组(320 mg.kg-1),于CI/RP术后每日腹腔注射Asp或溶媒,并进行神经功能缺损评分,72 h后处死,测定脑梗死体积和BDNF免疫组化检测。结果:CI/RP后24、48和72 h各Asp组大鼠与对照组相比,神经缺损评分明显降低(P<0.05或P<0.01),梗死灶体积显著减小(P<0.01),缺血区域BDNF表达显著增加(P<0.05或P<0.01)。对BDNF表达的作用Asp大剂量组的作用并不优于Asp小剂量组和中剂量组。结论:Asp可以减少CI/RP大鼠的脑梗死体积;促进内源性BDNF的表达可能是其神经保护的机制之一,Asp对BDNF表达的作用并非随Asp剂量的加大而增强。     

大鼠脑缺血再灌注损伤后Cx43蛋白的表达模式

目的：建立大鼠大脑中动脉闭塞（MCAO）缺血再灌注模型,探索再灌注后Cx43蛋白的表达模式。方法75只大鼠随机分为MCAO组（60只）和假手术组（15只）。MCAO组均行MCAO手术,术后根据缺血再灌注时间分为0.5、4、12、24 h 4个亚组,每组15只;假手术组不插入线栓。各组进行改良神经损伤严重程度评分（mNSS）、脑组织TTC染色及神经元尼式染色,Western blot及免疫组织化学方法检测各组脑组织Cx43蛋白的表达并进行统计学分析。结果 MCAO组造模成功率75%。MCAO组大鼠mNSS评分明显高于假手术组（P＜0.05）,脑梗死面积增加,额顶叶皮质区神经元大量损伤。Western blot结果显示：假手术组及MCAO 0.5、4、12、24 h组Cx43表达量分别为0.23±0.12、0.58±0.18、0.78±0.07、0.61±0.05和0.27±0.07, MCAO 0.5、4、12 h组与假手术组差异均有统计学意义（P＜0.05）,而MCAO 24 h组与假手术组差异无统计学意义（P＞0.05）。免疫组化结果显示：MCAO组侧脑室下区Cx43的表达在0.5 h开始升高,4 h达高峰,12 h逐渐下降,直至24 h恢复基线水平;与Western blot结果一致。结论 Cx43蛋白在脑缺血性再灌注损伤发生、发展中起重要作用。