大鼠局灶性脑缺血神经细胞凋亡与坏死的研究

用凝胶电泳及原位末端标记研究大鼠脑缺血再灌流后神经元的凋亡与坏死。采用可逆性大脑中动脉阻塞２小时,再灌流０．５～４８小时,造成脑缺血再灌流模型（３０只）,用ＨＥ、原位末端标记（ＴＵＮＥＬ）和琼脂糖凝胶电泳观察神经元的改变。结果：缺血损伤区位于视前区、纹状体和皮质,再灌流０．５小时,视前区的缺血损伤区有较多的凋亡细胞,而皮质、纹状体仅有散在的凋亡细胞。再灌流３～６小时,凋亡细胞数量增多,并可见坏死细     

可逆性大脑中动脉阻塞时神经元凋亡与坏死的形态学特点

目的;观察可逆性大脑中动脉阻塞时神经元凋亡与坏死的形态学特点及其发生过程,探讨缺血性神经元损伤的病理机制。方法 阻塞大鼠大脑中动脉２小时,再灌流０．５￣４８小时制成局灶性脑缺血模型,ＨＥ和ＴＵＮＥＬ染色观察凋亡细胞和坏死细胞形态,图象分析并计数。结果：ＨＥ染色可见凋亡细胞有四种形态;核固缩深染细胞大而肿胀或胞质无明显异常的凋亡细胞,细胞核裂解但胞膜完整的凋亡细胞,凋亡小体,出现于再灌注３小时;再灌     

脑缺血后细胞凋亡与PARP的关系

目的 观察多聚ADP—核糖聚合酶(PARP)在脑缺血再灌流损伤中的表达，探讨PARP在细胞凋亡中的作用。方法 应用线栓法建立大鼠大脑中动脉闭塞再灌流模型(MCAO)。用原位TUNEL和原位杂交技术探求脑缺血后PARP与细胞凋亡的变化关系。结果 缺血再灌流组凋亡细胞主要位于皮质和纹状体的缺血周围区，至24h达高峰。PARP的表达在皮质于脑缺血再灌流6h达高峰，在纹状体于再灌流后2h已达高峰。结论 PARP的mRNA表达发生在细胞凋亡之前，表明PARP促进缺血性脑损伤后神经细胞凋亡。     

大鼠局灶性脑缺血后再灌流时神经元的改变与巨噬细胞应答

取ＳＤ大鼠２９只，制成大鼠局灶性脑缺血模型，观察再灌流０．５～４８小时时神经元的改变与巨噬细胞应答，并探讨两者间的相互关系。结果：再灌流３小时内，神经元主要表现为收缩或肿胀；再灌流６小时组，有散在的红神经元及鬼影细胞；再灌流４８小时组，缺血半影区有大量的凋亡细胞及凋亡小体。凝集素ＧＳＩ－Ｂ４标记巨噬细胞表明，１２小时时出现小胶质细胞活化及来自脑膜的巨噬细胞，２４小时时出现脑实质内血单核细胞浸润；４８小时时脑内巨噬细胞在缺血半影区大量聚集。上述结果表明：①脑缺血后阻止细胞凋亡和减少脑梗塞体积对脑梗塞患者有极重要的意义；②不可逆的神经元损伤早于小胶质细胞活化，来自脑膜的巨噬细胞早于脑实质血单核细胞浸润。该研究结果也为脑梗塞最佳药物治疗时间的选择提供了参考资料。     

全身亚低温对脑缺血再灌流损伤神经细胞凋亡及Caspase-3的影响

目的 探讨亚低温对大鼠局灶性脑缺血再灌流后神经细胞凋亡及Caspase-3基因表达的影响。方法 应用原位末端标记(TUNEL)和原位杂交技术分别观察亚低温组、常温组脑缺血再灌流不同时间点神经细胞凋亡的变化及Caspase-3 mRNA的表达。结果 (1)常温组脑缺血再灌流后凋亡神经细胞主要分布于缺血周围区，随着再灌流时间的延长凋亡细胞数逐渐增加，至24小时达高峰，2天后开始下降，14天时仍高于假手术组；(2)亚低温组脑缺血再灌流后，凋亡神经细胞也主要位于缺血周围区，数量相对较少，其变化规律与常温组相似，同一时间点相比较，亚低温组均显著低于常温组；(3)常温组脑缺血再灌流2小时后，神经细胞Caspase-3 mRNA开始表达，并随着再灌流时间的延长而增强，24小时达高峰，2天后逐渐下降，至14天略高于假手术组；(4)亚低温组脑缺血再灌流后，神经细胞Caspase-3 mRNA的表达也主要位于缺血周围区，其变化规律与常温组相似，同一时间点相比较，亚低温组均显著低于常温组。结论 脑缺血再灌流后。缺血周围区神经细胞的凋亡是一个动态的渐进过程，Caspase-3基因在介导脑缺血损伤神经元凋亡过程中起关键作用。亚低温对短暂性脑缺血后的神经元凋亡有明显的抑制作用，亚低温可能通过Caspase-3 mRNA途径抵抗脑缺血损伤。     

脑缺血后海马CA1区细胞凋亡现象的观察

观察大鼠完全性前脑缺血再灌注损伤过程中海马ＣＡ１区神经元死亡的另一种形式－细胞凋亡。采用琼酯糖凝胶电泳及原位缺口翻译法，观察海马ＣＡ１区神经元是否有凋亡特征性改变。结果；脑缺血损伤后５ｄ，从海马ＣＡ１区神经元提取ＤＮＡ，其琼酯糖凝胶电泳呈现典型梯状结构。而采用原位缺口翻译法，发现缺血损伤后３ｄ，海马ＣＡ１区开始出现胞核染色体阳性的凋亡细胞，缺血损伤后５ｄ，凋亡细胞达到高峰。结论：海马ＣＡ１区尽管     

大鼠局灶性脑缺血后再灌流时神经元的改变与巨噬细胞 …

取ＳＤ大鼠２９只，制成大鼠局灶作脑缺血模型，观察再灌流０．５～４８小时时神经元的改变与巨噬细胞应答，并探讨两者间的相互关系，结果，再灌注３小时，神经元主要表现为收缩或肿胀，再灌流６小时组，有散在的神经元及鬼影细胞，再灌注４８小时组在钙离子致缺血半影区有大量的凋亡细胞及凋亡小体，凝集素ＧＳＩ－Ｂ４标记巨噬细胞表明１２小时时出现小胶质细胞活化及来自脑膜的巨噬细胞，２４小时的出现脑实质内血单核细胞浸润４     

大鼠小脑缺血再灌注损伤的细胞凋亡航尼莫通的保护作用

目的：探讨小脑缺血再灌注损伤神经元的死亡方式及尼莫通的保护作用。方法：将实验用ＳＤ大鼠１５只分为三组，即缺血再灌组、药护组、正常对照组。采用四血管闭塞法制作全脑缺血再灌注动物模型，再灌后４８小时取小脑，石蜡包埋切片。应用末端转移酶介导的缺口末端标记法原位检测凋亡细胞。药护组缺血前３０分钟腹腔注射尼莫通。结果：在小脑皮质及小脑核均见反应阳性的凋亡细胞，药护组的凋亡细胞数显著少于缺血组。结论：细胞凋亡     

碱性成纤维细胞生长因子抗局灶性脑缺血/再灌注引发神经细胞凋亡作用的实验研究

目的 观察碱性成纤维细胞生长因子（ｂＦＧＦ）抗局灶性脑缺血／再灌注引发神经元凋亡作用。方法 采用线栓法制备大脑中动脉缺血／再灌注模型。术后首次即刻分别腹腔注射ｂＦＧＦ和生理盐水，以后每天一次，连用７天。ＴＵＮＥＬ法原位检测缺血后第１、２、３、５、７天脑石蜡切片含ＤＮＡ碎片的凋亡细胞。结果 正常组、假手术组、缺血组对侧半球每张切片有０～８个凋亡细胞，缺血／再灌注后缺血区凋亡细胞明显增多，缺血４８ｈ达     

大鼠局灶性脑缺血再灌注后脑缺血半影区的界定

①目的 界定大鼠局灶性脑缺血再灌注后脑缺血半影区的范围。②方法 应用线栓法建立大鼠大脑中动脉闭塞再灌注模型，氯化三苯基四氮唑(TTC)染色、甲苯胺蓝染色、焦油紫染色和苏木精-伊红染色界定脑缺血半影区和中心区。③结果 脑缺血再灌注2h，缺血病灶位于纹状体外侧区和额顶叶皮质下部，随着缺血再灌注时间的延长病灶范围逐渐扩大；再灌注1～2d最大，波及纹状体内侧区和额顶叶皮质上部以及梨状区皮质，之后逐渐缩小；再灌注7～14d恢复到再灌流2h的水平。缺血病灶与周围正常脑组织之间有一形态和结构过渡区，该区的着色和细胞形态结构界于病灶和正常脑组织之间，即缺血半影区。缺血中心区神经细胞明显减少，呈现坏死特征；缺血半影区细胞主要呈现核固缩、染色质凝聚等凋亡特征。④结论 脑缺血再灌注缺血中心区位于纹状体外侧区和额顶叶皮质下部，缺血半影区位于纹状体内侧区和额顶叶皮质上部以及梨状区皮质。     

Apoptosis and necrosis of neuron after focal ischemia in brain of rats]

This article reports the apoptosis and necrosis of neurons following ischemia-reperfusion in the brain of rats. The focal ischemia model was established by occluding the middle cerebral artery for 2 h and reperfusing for 0.5-48 h. The neuronal changes were investigated by HE staining, TUNEL and agarose gel-electrophoresis. The foci of ischemia were in the preoptic area, striatum and cortex. At 0.5 h of reperfusion, there were quite a few apoptotic neurons in the preoptic ischemic focus, but only a few scattered apoptotic cells were seen in the striatum and cortex. During 3-6 h of reperfusion, the number of apoptotic cells increased and the necrotic cells appeared. The number of apoptotic cells reached a peak at 12-24 h and the in morphology varied, and they were mainly located in the inner boundary of infarct. At 48 h, the apoptotic cells decreased; and the necrotic cells increased. Agarose gel-electrophoresis showed the dense smear and ladder pattern of DNA fragments, the apoptosis and necrosis of neurons coexisted, and apoptosis contributed to the enlargement of the ischemic infarct.     

Protective effect of radix Salviae miltiorrhizae on apoptosis of neurons during focal cerebral ischemia and reperfusion injury.

We have found that Radix Salviae Miltiorrhizae (RSM) plays a protective role in ischemic brain injury, which attracted us to investigate the effect of RSM on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic brains at different reperfusion intervals were tested with the method of TdT-mediated dUTP-DIG nick end labeling (TUNEL), and the effect of RSM on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion in rat models (n = 18). The results showed that few scattered apoptotic cells were observed in right cerebral hemisphere after LMCA occlusion and reperfusion, and that a lot of apoptotic cells were found in left ischemic cerebral cortex and caudoputamen at 12 h reperfusion, and they reached peak at 24-48 h reperfusion. However, in rats pretreated with RSM, the number of apoptotic cells in left cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24 h reperfusion as compared with those of saline-treated rats. From this study, we conclude that administration of RSM can reduce the apoptotic of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.     

Effect of batroxobin on neuronal apoptosis during focal cerebral ischemia and reperfusion in rats.

We have found that Batroxobin plays a protective role in ischemic brain injury, which attracted us to investigate the effect of Batroxobin on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic rat brains at different reperfusion intervals were tested with method of TdT-mediated dUTP-DIG nick end labeling (TUNEL) and the effect of Batroxobin on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion and reperfusion in rat models (n = 18). The results showed that few scattered apoptosis cells were observed in right cerebral hemispheres after LMCA occlusion and reperfusion, and that a lot of apoptosis cells were found in left ischemic cortex and caudoputamen at 12 h reperfusion, and they reached peak at 24 h-48 h reperfusion. However, in the rats pretreated with Batroxobin, the number of apoptosis cells in left cerebral cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24 h reperfusion than that of saline-treated rats. The results indicate that administration of Batroxobin may reduce the apoptosis of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.     

全脑缺血再灌注后神经元凋亡的时相和分布特征

目的探讨大鼠全脑缺血再灌注后神经元凋亡的时相和分布特征,为神经元损伤的早期干预和继发性脑损害的防治提供实验依据.方法建立大鼠全脑缺血模型,采用TTC染色、原位细胞凋亡检测(原位末端标记法,TUNEL)及AO/EB荧光比色法观察脑缺血再灌后海马、皮层凋亡发生的数量和分布,用荧光指示剂Fura-2 AM标记,检测脑海马细胞内游离钙的浓度.结果 TUNEL显示再灌注3 h海马部位即出现散在凋亡细胞,并向皮层区域扩展,24～48 h达高峰;坏死细胞迟于凋亡出现,弥散分布于凋亡细胞周围,再灌注48 h后坏死细胞增多,72 h最多.AO/EB法显示海马、额叶和顶叶凋亡细胞总数分布在再灌注后24 h和72 h达高峰,并显著高于对照组(P＜0.05).TTC染色证实全脑缺血再灌注后不出现梗死灶,而是在海马及大脑皮层有弥散性坏死.胞内[Ca2 ]i各时间点与对照相比匀显著升高(P＜0.01).结论全脑缺血再灌后受累神经元经历了由凋亡到死亡的过程,对缺血敏感的海马神经元首先受损,并向顶叶和额叶皮层扩展,细胞质内[Ca2 ]i增加是主要原因.利用凋亡时间窗进行早期干预可能有益于保护和挽救凋亡前期神经元,减小继发性损害的严重程度.     

脑梗塞后炎性因子与细胞凋亡动态变化的研究

目的：观察局灶性脑缺血大鼠炎性因子与细胞凋亡相关指标的改变,探讨炎性反应与细胞凋亡在脑梗塞后的变化及其内在机制。方法：采用插线法致大脑中动脉栓塞（M CAO）2 h制备局灶性脑缺血大鼠模型,分别于再灌注6、12、24、48、72和168 h取血并处死动物取相应标本。检测腹动脉血液流变学动态变化;采用酶联免疫吸附法（EL ISA）和放射免疫竞争法测定脑组织白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）含量;用苏木素-伊红（HE）染色法观察脑细胞的病理形态学变化并进行细胞计数;用原位末端缺刻标记法检测细胞凋亡情况。结果：与假手术组比较,模型组缺血侧脑组织炎症细胞因子IL-1β水平于再灌注6～24 h明显增高,TNF-α含量于再灌注24～48 h显著增多（P〈0.05）;再灌注6～72 h脑缺血梗死区细胞数量明显减少,12～72 h凋亡细胞大量出现（P〈0.01）。结论：炎症因子IL-1β与TNF-α的生成与释放在6～48 h时增多;细胞凋亡作为缺血损伤后神经元损失的主要形式之一,在24～48 h时最为严重;缺血脑区的形态学变化在缺血后24 h最为严重。     

实验性脑缺血原癌基因表达与神经元凋亡/坏死关系的研究

目的 研究局灶脑缺血／再灌注Wistar大鼠原癌基因c-fos、c-jun表达在缺血性脑损害中可能的作用机制。方法 采用线栓法制备大鼠局灶脑缺血／再灌注模型，于缺血1.5h再灌注4h、24h、72h观察c-fos、c-jun表达及神经元坏死、调亡的变化规律。结果 再灌注4h c-fos、 c-jun及神经元调亡明显升高（P＜0.01），c-fos和c-jun阳性蛋白表达在4h达高峰（P＜0.01），随后在再灌注各时相点无显著性差异（P＞0.05）。结论 c-fos和c-jun异常表达与神经元坏死、凋亡密切相关。     

Functional protection of pentoxifylline against spinal cord ischemia/reperfusion injury in rabbits: necrosis and apoptosis effects

Background Little is known about neuronal death mechanisms following spinal cord ischemia. The present study aimed to investigate the protective effect of pentoxifylline （PTX） against spinal cord ischemia/reperfusion （I/R） injury.
Methods Rabbits sustained spinal cord ischemia following 45 minutes cross-clamping of the infrarenal aorta. Experimental groups were as follows： the first group of animals （sham, n=-8） underwent laparotomy alone and served as the sham group; the second group （I/R, n=20） received carrier （3 ml saline solution） and served as the control group; the third group （PTX-A, n=20） received PTX intravenously 10 minutes prior to ischemia; and the fourth group （PTX-B, n=20） received PTX intravenously at the onset of reperfusion. Rabbits were evaluated for hind-limb motor function with the Tarlov scoring system at 48 hours. Serum was assayed with enzyme-linked immunosorbent assay for tumor necrosis factor α （TNF-α） and spinal cords were harvested for myeloperoxidase （MPO） activity, histopathological analysis, terminal deoxynucleotidyl transferase （TdT）-rnediated dUTP nick end labeling staining, platelet/endothelial ceU adhesion molecule-1 （PECAM-1） and caspase-3 immunohistochemistry, and the number of necrotic and apoptotic neuron were counted and data analyzed at 12, 24, 48 and 72 hours of reperfusion. Spinal cords were studied by electron microscopy.
Results Improved Tarlov scores were seen in PTX-treated rabbits as compared with ischemic control rabbits at 48 hours. A significant reduction was found in TNF-α in serum, activity of MPO and immunoreactivity of the PECAM-1 and caspase-3 in PTX-treated rabbits. There were fewer apoptotic neurons than necrotic neurons （P 〈0.05）. A significant decrease in both necrotic and apoptotic neurons was observed in the PTX-treated groups （PTX-A and PTX-B） compared with the I/R group （P 〈0.05）. Both necrotic and apoptotic neurons were found with the electron microscope.
Conclusions PTX may indu     

大鼠脑缺血再灌流时脑组织病理改变的特点

目的 :观察大鼠脑缺血再灌流时 ,脑组织病理改变的特点和时程。方法 :采用改良的大鼠脑缺血再灌流模型 ,阻塞大脑中动脉 2小时 ,再灌流 0 5～ 48小时 ,TTC和HE染色观察缺血部位、面积 ,神经元渐进性改变的特点。结果 :再灌流 0 5小时有灶性缺血区 ,2 4小时缺血性损伤面积最大 ;6小时内神经元主要为急性期改变即神经元肿胀和浓缩 ;后出现神经元不可逆变性 ,2 4小时形成梗塞区。结论 :本研究结果表明缺血性神经元损伤不仅与缺血的时间、程度且与再灌流时间有关 ,缺血再灌流时神经元经历了可逆变性、不可逆变性和梗塞区形成的变化过程。因此本结果为脑梗塞治疗时间窗的选择提供了参考资料。     

Effects of reperfusion on neuronal changes and macrophagic response after transient focal ischemia--reperfusion of brain in rats]

Male SD rats(n = 29) were subjected to 2 hours' middle cerebral artery occlusion and were killed at various times of reperfusion (0.5-48 hours). The histological features of neuronal changes, the macrophagic response and the relationship between them were investigated. The neurons underwent acute changes(shrinking or swollen), and the pyknotic apoptotic neurons were occasionally seen within 3 hours. The number of apoptotic neurons increased with recirculation time. The scattered red neurons and ghost cells were first found at 6 hours of reperfusion. Many apoptotic neurons, apoptotic bodies were detected in the penumbra of ischemic core at 48 hours. Lectin histochemistry with GSI-B4 was performed to observe the macrophage. The results showed the active microglia and macrophage from meninges presented at 12 hours. The macrophages from parenchyma vessels appeared at 24 hours. Reactive microglia and extrinsic macrophage aggregated into the band of macrophage which phagocytized the apoptotic neurons. The results indicate that, firstly, it is important to prevent the neuronal apoptosis and reduce the volume of infarct in stroke patients; secondly, irreversible neuronal lesions simultaneously stimulate microglial activation and the presence of extrinsic macrophages from meninges was earlier than the appearance of the macrophages from parenchyma blood-monocytes. The authors also suggest that the data on the neuronal changes over time after ischemia and reperfusion be useful reference materials for selecting the most timely medication to treat cerebral infarction.