缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系

目的 探讨缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系。方法 实验动物被随机分为手术组、缺血组、预缺血组、预缺血后再缺血组。阴断沙土鼠双侧颈总动脉造成前脑缺血模型。采用细胞特异性抗原胶质纤维酸性蛋白（GFAP）免疫组化法标记星形胶质细胞。结果 预缺血后1－7天,海马CA1区GFAP阳性的星形胶质细胞数轻度增加,至28天时增生非常显著（P＜0．01）。预缺血后1－7天再缺血,海马CA1区存活正常神经元数逐渐下降,预缺血后28天再缺血又显著增加（P＜0．01）。结论 缺血预处理后,神经元可出现迟发性缺血耐受,反应性星形胶质细胞增生可能起了重要作用。     

缺血缺氧对体外培养星形胶质细胞细胞周期和增殖的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期和增殖的影响。方法 用流式细胞仪检测缺血缺氧后不同时问点星形胶质细胞细胞周期变化，并用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)和增殖细胞核抗原(PCNA)的表达水平。结果 体外缺血缺氧损伤后星形胶质细胞S期较正常组明显增高，6h达高峰，而随后则呈下降趋势。PCNA阳性反应损伤后表达均增加，6h表达最高；在缺血缺氧早期，GFAP阳性染色增强，6h最高；缺血缺氧12h后GFAP阳性染色变弱。结论 缺血缺氧损伤后星形胶质细胞活化进入增殖期；PCNA参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的活化密切相关。     

缺血缺氧对体外培养星形胶质细胞细胞周期和周期相关蛋白的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期及细胞周期相关蛋白的影响。方法 用流式细胞仪及Brdu掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力；用荧光免疫细胞化学技术测定增殖细胞核抗原(PCNA)及细胞周期蛋白cyclin D1的表达水平。结果 体外缺血缺氧损伤后S期星形胶质细胞较正常组明显增加，6h达高峰，Brdu掺入法显示损伤后6h星形胶质细胞的增殖活力最高，而随后S期细胞数目及细胞增殖活力都呈下降趋势。PCNA阳性反应损伤后表达增加，6h表达最高，而cyclin D1的表达在损伤后逐渐增加，在24h时达高峰。结论 缺血缺氧损伤激活星形胶质细胞，使其进入新的细胞周期，出现细胞的增殖反应；PCNA及cyclin D1参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的增殖活化密切相关。     

海马CA1区神经元选择易损性组织结构机制的研究

目的 研究海马CA1区神经元选择易损性与此区组织结构特点的关系。方法 用透射电子显微镜观察缺血再灌注12h、24h、36h、48h、72h组CA1区神经元损伤及星形胶质细胞动态变化。结果 在再灌注各组中，星形胶质细胞的超微结构变化早于与之相连的神经细胞，而和不与之相连的神经细胞超微结构变化同步。神经元轴突内超微结构变化早于胞内结构。结论 星形胶质细胞对缺血再灌注损伤具有保护作用。海马CA1区神经元选择易损与此区星形胶质细胞较少从而保护作用小及其轴突长有关。     

海马CA_l区神经元选择易损性组织结构机制的研究

目的　研究海马 CAl 区神经元选择易损性与此区组织结构特点的关系。方法　用透射电子显微镜观察缺血再灌注 12 h、2 4 h、36 h、4 8h、72 h组 CAl 区神经元损伤及星形胶质细胞动态变化。结果　在再灌注各组中 ,星形胶质细胞的超微结构变化早于与之相连的神经细胞 ,而和不与之相连的神经细胞超微结构变化同步。神经元轴突内超微结构变化早于胞内结构。结论　星形胶质细胞对缺血再灌注损伤具有保护作用。海马 CAl 区神经元选择易损与此区星形胶质细胞较少从而保护作用小及其轴突长有关     

人脑梗死后星形胶质细胞活性与凋亡相关蛋白Bcl-2的相关性研究

目的 探讨人脑梗死后星形胶质细胞活性与凋亡的关系。方法 应用10例脑梗死后的尸检全脑标本，用HE和免疫组化方法检测胶质纤维酸性蛋白(GFAP)、凋亡相关蛋白(Bcl-2)，进行定性和定量分析。结果 人脑梗死后以梗死灶为中心，按照组织损伤程度由内到外分为4个区(0-3区)，其中2区为半暗带区，40％以上的神经元和星形胶质细胞形态基本正常，随缺血时间延长，星形胶质细胞增生明显，形态正常神经元的数量减少；3区神经元形态及数量正常，星形胶质细胞反应性增生。GFAP在0区和1区表达很少，2区随缺血时间延长阳性细胞数增多，3区早期即有星形胶质细胞的簇样增生。Bcl-2在0区无表达，1区表达较少，2区于缺血后8h开始出现，23h达高峰，之后开始下降。结论人脑梗死后在半暗带区23h前GFAP和Bcl-2表达呈正相关；23h后呈负相关。2区是临床需要积极抢救的区域，并可应用干预因素上调Bcl-2表达，缩小梗死面积。     

PINK1介导缺氧损伤时星形胶质细胞对神经元的保护作用

目的探究星形胶质细胞内PTEN诱导假定激酶1(PINK1)缺失对缺血时神经保护作用的影响及其作用机制。方法离体培养原代星形胶质细胞,使用小干扰RNA(si RNA)沉默PINK1表达,氧糖剥夺(OGD)建立细胞缺氧模型,分为4组:PINK1沉默组(si RNA+转染剂)、空质粒组(空质粒+转染剂)、转染剂组(只加转染剂)和对照组(星形胶质细胞),各组均与神经元共培养;另设立神经元单独培养组。免疫荧光染色观察神经元凋亡情况。定量PCR及ELISA检测星形胶质细胞促红细胞生成素(EPO)及血管内皮生长因子(VEGF)表达量;Western blot检测星形胶质细胞内缺血诱导因子(HIF)及核因子κB(NF-κB)通路相关蛋白水平。结果 OGD损伤后神经元凋亡率较高,与星形胶质细胞共培养后神经元凋亡率显著降低(P0.05)。PINK1基因沉默后共培养神经元凋亡增加,星形胶质细胞EPO及VEGF分泌量减少、胞内EPO及VEGF转录水平降低(P0.05);HIF-1、HIF-2与NF-κB通路活化水平均显著降低(P0.05)。结论星形胶质细胞对OGD损伤神经元有保护作用,其作用通过EPO及VEGF实现;PINK1基因沉默后星形胶质细胞对缺血神经元保护作用减弱,可能与NF-κB通路活化水平降低、HIF激活受损进而下调EPO和VEGF表达量有关。     

局灶性脑缺血耐受和星形胶质细胞反应

目的　研究短暂性局灶性脑缺血预处理对永久性局灶性脑缺血的保护作用 ,及最佳预处理时间剂量 ,并探讨星形胶质细胞在脑缺血耐受中的反应。方法　采用开颅方法阻断大鼠大脑中动脉 ,通过观察大鼠脑梗死后神经功能损伤状况、脑梗死体积分析及病理形态学变化 ,评价不同的缺血预处理时间剂量 (10分钟、2 0分钟、30分钟 )对永久性局灶性脑缺血的保护作用。采用胶质纤维酸性蛋白 (GFAP)免疫组化法观察星形胶质细胞在脑缺血耐受中的反应。结果　与对照组相比 ,缺血预处理 2 0分钟未引起明显的神经元损伤 ,但使永久性局灶性脑缺血后神经功能损伤减轻 ,梗死体积明显减小 (P <0 .0 1)。免疫组化显示 ,2 0分钟缺血预处理组及重复缺血组星形胶质细胞在损伤预处理侧广泛激活。结论　 2 0分钟局灶性脑缺血预处理能够有效诱导脑缺血耐受。星形胶质细胞的激活可能与脑缺血耐受中神经元的存活相关。     

大鼠脑缺血再灌注后神经细胞的细胞周期特征的比较研究

目的比较观察大鼠局灶性脑缺血后星形胶质细胞和神经元细胞周期的变化特征。方法采用线栓法大鼠大脑中动脉栓塞模型,利用流式细胞技术检测假手术组和缺血再灌注后不同时间点各组大脑皮层和海马中星形胶质细胞和神经元细胞周期的异常激活和动态变化。结果缺血后大脑皮层中神经元24h时即发生明显细胞周期变化,而3d时进入细胞周期的星形胶质细胞才明显增加;海马中星形胶质细胞却先于神经元进入细胞周期,于24h时细胞周期即发生明显变化,与假手术组比较有显著差异(P<0.01)。结论在不同脑区星形胶质细胞和神经元两者对缺血性脑损伤的敏感性互不相同,并且不同脑区的星形胶质细胞对缺血性脑损伤的敏感性也有不同,脑缺血后2种细胞均出现细胞周期的异常激活。     

蒙古沙鼠一过性脑缺血模型迟发性脑梗死的病理学特点

目的 观察一过性缺血后脑组织中嗜酸性神经元、反应性星形胶质细胞和梗死区域的分布,探明脑缺血病理形态学改变的时序性变化.方法 通过2次10 min、间隔5h的单侧颈总动脉夹闭制造蒙古沙鼠脑缺血模型,激光多普勒血流仪检测前部脑皮质血流;于24 h,4d,2、4、16周观察脑组织病理形态学改变.结果 颈总动脉夹闭后激光多普勒血流仪显示前部至后部脑血流量明显降低:分别为22.1％±9.5％,26.3％±4.9％,37.5％±3.5％,F =67.219,P＜0.01;位于前脑部的脑血流量的降低明显高于后脑部.缺血24h后,嗜酸性神经元出现于脑前部皮质中层和深层、4d后遍及整个皮质各层,4d至4周大范围的高密度嗜酸性神经元区域(≥80个/mm2)进展为梗死.脑后部皮质中层和深层进展为低嗜酸性神经元区(＜ 80个/mm2),未进一步进展为梗死.反应性星形胶质细胞分布区域与嗜酸性神经元一致;反应性星形胶质细胞伴随高密度嗜酸性神经元区域在缺血后4d至4周大部分转化为梗死.迟发性星形胶质细胞死亡发生于反应性星形胶质细胞伴随高密度嗜酸性神经元区域.结论 嗜酸性神经元密度是缺血脑组织梗死及迟发性星形胶质细胞死亡的重要标志.     

Inflammatory response and neuronal necrosis in rats with cerebral ischemia

In the middle cerebral artery occlusion model of ischemic injury, inflammation primarily occurs in the infarct and peripheral zones. In the ischemic zone, neurons undergo necrosis and apoptosis, and a large number of reactive microglia are present. In the present study, we investigated the pathological changes in a rat model of middle cerebral artery occlusion. Neuronal necrosis appeared 12 hours after middle cerebral artery occlusion, and the peak of neuronal apoptosis ap- peared 4 to 6 days after middle cerebral artery occlusion. Inflammatory cytokines and microglia play a role in damage and repair after middle cerebral artery occlusion. Serum intercellular cell adhesion molecule-1 levels were positively correlated with the permeability of the blood-brain barrier. These findings indicate that intercellular cell adhesion molecule-1 may be involved in blood-brain barrier injury, microglial activation, and neuronal apoptosis. Inhibiting blood-brain barrier leakage may alleviate neuronal injury following ischemia,     

Paeoniflorin attenuates chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rats

Chronic cerebral hypoperfusion, a mild ischemic condition, is associated with the cognitive deficits of AD. Paeoniflorin (PF), a major constituent of peony root, was proved to be neuroprotective in middle cerebral artery occlusion model. In this study, we investigated whether PF could attenuate chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rat. Seven weeks after permanent bilateral occlusion of the common carotid arteries, the rats were tested in the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglias were labeled with immunocytochemistry in hippocampus. PF at the dose of 2.5 mg/kg ameliorated cerebral hypoperfusion-related learning dysfunction and prevented CA1 neuron damage. Chronic cerebral hypoperfusion increased the immunoreactivity of astrocytes and microglias in hippocampus. The increase was prevented by PF at the dose of 2.5 mg/kg. Cerebral hypoperfusion also increased expression of nuclear factor-kappaB (NF-kappaB), mostly in astrocytes, but not in neurons. With the treatment of PF (2.5 mg/kg), NF-kappaB immunostaining was diminished in hippocampus. Our results demonstrated that PF could attenuate cognitive deficit and brain damage induced by chronic cerebral hypoperfusion and that suppression of neuroinflammatory reaction in brain might be involved in PF-induced neuroprotection.     

Selective cortical neuronal damage after middle cerebral artery occlusion in rats

We studied histopathologic changes in cerebral cortex of 20 rats after middle cerebral artery occlusion by using the Fink-Heimer suppressive silver impregnation method and conventional stains. At 6 hours after occlusion, Fink-Heimer-stained sections revealed abundant coarsely granular, intensely argyrophilic neurons in the ischemic cortex. These distinctive argyrophilic neurons could be clearly differentiated from neurons that suffered postmortem changes; argyrophilic neurons were present in all layers of the lateral parietal cortex but in only the superficial cortical layers II and III in the parasagittal area of the frontoparietal cortex and the temporo-occipital area. At 24 hours after occlusion as the ischemic region progressed to pannecrosis, argyrophilic neurons were still evident in peri-infarct regions, with more prominent neuritic silver deposits but no changes in number or spatial distribution. Over 2-7 days, the argyrophilic neurons gradually disappeared while many fine silver-impregnated degenerating terminals appeared in the peri-infarct regions. At 3-6 weeks after occlusion, no more argyrophilic neurons were seen in the cortex although degenerating axons were still present in the deep white matter. Our results indicate selective neuronal damage in the superficial cortical layers and massive axonal degeneration in the cerebrum surrounding infarcts. The neuronal damage does not appear to progress beyond 6 hours after middle cerebral artery occlusion. The Fink-Heimer method has many advantages over existing conventional stains for documenting selective neuronal damage in focal cerebral ischemia.     

The heterogeneous temporal evolution of focal ischemic neuronal damage in the rat

Summary Male Fisher rats (n=61) underwent permanent focal cerebral ischemia induced by left middle cerebral artery (MCA) occlusion, in conjunction with ipsilateral common carotid artery ligation. The experiments were terminated at time points ranging from immediately following occlusion to 30 days post MCA occlusion. A coronal histological section, in close proximity to the site of the arterial occlusion, was taken from each brain and divided into six areas encompassing the affected cortex and caudate putamen. Each area was analyzed for ischemic damage according to a grading scale that reflects changes in neuronal morphology. Differential neuronal counts were also made on a 0.5-mm² field in each of the six areas. The areas closest to the occluded vessel showed accelerated ischemic damage between 8 and 12 h after occlusion, leaving open the possibility that before 8 h, therapeutic intervention may be effective. After 12 h, changes in these areas progressed to complete necrosis and eventual cavitation with a complete loss of neurons after 10 days. The areas more peripheral to the occluded vessel exhibited mild ischemic damage, with an apparent reversal of damage grading at later time points and no loss of neurons. This reversal of ischemic damage in the peripheral areas is suggestive of a histological equivalent of the penumbra.Supported by NIH Grants NS23393 and NS29463     

eNOS解偶联对大脑中动脉栓塞后大鼠神经血管单元的作用及机制

目的探讨大鼠大脑中动脉栓塞模型中,内皮型一氧化氮合酶(e NOS)解偶联对神经血管单元的作用及机制。方法将80只雄性SD大鼠随机分为假手术组(n=40)和大脑中动脉栓塞(MCAO)组(n=40)。参照Longa法制作MCAO模型。栓塞后6 h,通过蛋白印记法检测缺血区e NOS不同偶联状态的表达情况及3-硝基酪氨酸(3-NT)的水平;通过尼氏染色比较缺血区神经元的形态及存活情况;通过免疫组织化学染色比较缺血区星形胶质细胞、微血管内皮细胞以及血脑屏障紧密连接对应的标记物GFAP、VWF、Occludin的表达水平;通过MRI的T2加权序列和动脉自旋标记(ASL)反应栓塞后脑组织病灶范围及血流情况。结果 MCAO后栓塞部位在T2序列上呈高信号,在ASL序列上血流信号减低。e NOS二聚体/单体的比例明显低于假手术组(P0.05);3-NT的含量明显高于假手术组(P0.05)。尼氏染色显示MCAO后神经元大量空泡形成,核固缩变性,数量显著减少;免疫组化结果显示,VWF、Occludin的表达较假手术组明显减少,而GFAP则明显增多(P0.05)。结论 MCAO会导致e NOS解偶联。e NOS解偶联后NO合成受限,而具有毒性作用的3-NT产生增多,炎症反应加重,从而对神经血管单元结构及功能产生破坏作用。     

Enhanced expression of p53 in reactive astrocytes following transient focal ischemia

The present study used immunohistochemistry to investigate p53 expression in rat brain following transient occlusion of the middle cerebral artery. In the control group, no p53-immunoreactive cells were found in any region of the central nervous system. P53 expression in reactive astrocytes was not obvious in the forebrain one day or three days following ischemic insults. Seven days following ischemic injury, increased expression of p53 was clearly detectable in reactive astrocytes in affected cortical regions, such as forelimb area, hindlimb area, and parietal cortex. At seven days of recirculation, there was also a significant increase in the number of p53-immunoreactive neurons in the cerebral cortex, striatum, and hippocampal CA1-3 regions. Although the present study has not addressed multiple mechanisms contributing to cell death following ischemic injury, the first demonstration of a significant increase in p53 expression in glial cells may prove useful for future investigations of the pathophysiology of ischemia.     

Cortical neurogenesis in adult rats after ischemic brain injury: most new neurons fail to mature

The present study examines the hypothesis that endogenous neural progenitor cells isolated from the neocortex of ischemic brain can differentiate into neurons or glial cells and contribute to neural regeneration. We performed middle cerebral artery occlusion to establish a model of cerebral ischemia/reperfusion injury in adult rats. Immunohistochemical staining of the cortex 1, 3, 7, 14 or 28 days after injury revealed that neural progenitor cells double-positive for nestin and sox-2 appeared in the injured cortex 1 and 3 days post-injury, and were also positive for glial fibrillary acidic protein. New neurons were labeled using bromodeoxyuridine and different stages of maturity were identified using doublecortin, microtubule-associated protein 2 and neuronal nuclei antigen immunohistochemistry. Immature new neurons coexpressing doublecortin and bromodeoxyuridine were observed in the cortex at 3 and 7 days post-injury, and semi-mature and mature new neurons double-positive for microtubule-associated protein 2 and bromodeoxyuridine were found at 14 days post-injury. A few mature new neurons coexpressing neuronal nuclei antigen and bromodeoxyuridine were observed in the injured cortex 28 days post-injury. Glial fibrillary acidic protein/bromodeoxyuridine double-positive astrocytes were also found in the injured cortex. Our findings suggest that neural progenitor cells are present in the damaged cortex of adult rats with cerebral ischemic brain injury, and that they differentiate into astrocytes and immature neurons, but most neurons fail to reach the mature stage.     

银杏叶提取物对脑缺血大鼠脑源性神经营养因子的影响

目的　观察银杏叶提取物 (GBE)对局灶脑缺血大鼠脑源性神经营养因子 (BDNF)表达的影响 ,探讨GBE与缺血损伤神经元可塑性的关系。方法　制作大鼠大脑中动脉闭塞 (MCAO)模型 ,应用免疫组化方法观察不同缺血时间 GBE治疗组及脑缺血组 BDNF阳性细胞数 ,并进行图像分析。结果　坏死灶中心区 GBE组及缺血组BDNF阳性神经元均消失 ,但在坏死灶周围区 ,两组 BDNF阳性细胞均显著增加。两组细胞形态无明显不同 ,但GBE治疗组阳性细胞数又显著高于相应缺血对照组。结论　银杏叶提取物可提高大鼠局灶脑缺血半暗带区 BDNF的表达水平 ,促进神经元的修复及重塑。     

脑缺血对局部超氧化物岐化酶、丙二醛水平的影响

建立大鼠右大脑中动脉栓塞（ＲｉｇｈｔＭｉｄｄｌｅＣｅｒｅｂｒａｌＡｒｔｅｒｙＯｃｌｕｓｉｏｎＲ－ＭＣＡＯ）模型，检测局部缺血后不同时间左右脑组织超氧化物岐化酶（ＳｕｐｅｒｏｘｉｄｅＤｉｓｍｕｔａｓｅＳＯＤ）、丙二醛（ＭＤＡ）的含量，发现Ｒ－ＭＣＡＯ１０分钟始，缺血脑组织ＳＯＤ即降低，至３０分钟后恢复，而ＭＤＡ则在Ｒ－ＭＣＡＯ６０分钟后持续升高。说明缺血后ＳＯＤ活力下降、致ＭＤＡ蓄积，是造成脑组织损伤的机制之一。