促红细胞生成素对β淀粉样蛋白诱导大鼠海马CA1区细胞凋亡的保护作用及机制

目的 探讨促红细胞生成素(Erythropoietin,EPO)对阿尔茨海默病(Alzheimer disease,AD)大鼠脑损伤的保护机制.方法 采用大鼠海马内注射B淀粉样蛋白制作AD模型.将SD大鼠随机分为假手术对照组、生理盐水对照组及EPO处理组.大鼠海马内注射Aβ1-40加造模,然后在生理盐水对照组大鼠行脑室立体定向注射生理盐水,EPO处理组则行脑室立体定向注射重组人促红细胞生成素(rHu-EPO).观察手术后24h大鼠海马CA1区抗凋亡蛋白Bcl-xl表达变化,以及术后7d海马CA1区细胞凋亡变化.结果 EPO处理组和生理盐水组海马CA1区大鼠Bcl-xl蛋白表达较假手术对照组减少,但是EPO处理组Bcl-xl蛋白表达高于生理盐水(P<0.05).生理盐水组海马CA1区凋亡细胞明显多于EPO组(P<0.05).结论 EPO可以抑制β淀粉样蛋白诱导海马CA1区细胞凋亡,与其抑制Bcl-xl蛋白表达下降有关.     

促红细胞生成素对β淀粉样蛋白诱导大鼠海马CA_1区锥体细胞线粒体细胞色素C释放的影响

目的探讨促红细胞生成素(EPO)对β淀粉样蛋白诱导大鼠海马CA1区神经元凋亡的影响及可能机制。方法采用大鼠海马内注射β淀粉样蛋白(Aβ)制作阿尔茨海默病(AD)模型。将SD大鼠随机分为假手术组、生理盐水组及EPO处理组。Aβ大鼠海马内注射造模,然后在生理盐水组大鼠行脑室立体定向注射生理盐水,EPO处理组则行脑室立体定向注射重组人促红细胞生成素(rHu-EPO)。观察术后24小时海马CA1区神经元细胞色素C(Cyt C)的变化,以及术后7天海马CA1区细胞凋亡变化。结果 EPO处理组大鼠海马CA1区神经元Cyt C的表达明显高于生理盐水组(P<0.01),EPO处理组大鼠海马CA1区凋亡细胞较生理盐水组减少(P<0.01)。结论 EPO可以通过抑制Aβ1-40诱导海马CA1区锥体细胞线粒体Cyt C释放来抑制神经元凋亡。     

亚低温对缺血性神经元凋亡、细胞色素C释放的影响

通过大鼠短暂全脑缺血模型来探讨亚低温对大鼠脑缺血后细胞色素C(CytochromeC ,CytC)释放及缺血性神经元凋亡的影响 ,揭示亚低温的部分神经保护机制。用原位细胞凋亡检测法 (TUNEL染色 )检测及电镜观察脑缺血后大鼠脑海马CA1区神经元凋亡发生情况 ;免疫组织化学法测定脑缺血后大鼠脑海马区神经元中细胞色素C释放情况。结果显示 :①低温缺血组海马CA1区凋亡神经元数明显少于常温缺血组 (P <0 .0 1) ;②低温缺血3h组海马CA1区神经元CytC阳性表达低于常温缺血 3h组 (P <0 .0 1)。据此认为 ,全脑缺血后的迟发性神经元死亡很可能经由凋亡途径 ,而CytC激活、释放是缺血性神经元凋亡的一个关键事件。亚低温可抑制CytC的释放 ,推测经此途径减少缺血性神经元凋亡而发挥一定的神经保护作用。     

亚低温对缺血性神经元凋亡、细胞色素C释放的影响

通过大鼠短暂全脑缺血模型来探讨亚低温对大鼠脑缺血后细胞色素c(Cytoehrome c,CytC)释放及缺血性神经元凋亡的影响,揭示亚低温的部分神经保护机制.用原位细胞凋亡检测法(TUNEL染色)检测及电镜观察脑缺血后大鼠脑海马CAi区神经元凋亡发生情况;免疫组织化学法测定脑缺血后大鼠脑海马区神经元中细胞色素C释放情况.结果显示:①低温缺血组海马CAi区凋亡神经元数明显少于常温缺血组(P<0.01);②低温缺血3 h组海马CA1区神经元CytC阳性表达低于常温缺血3 h组(P<0.01).据此认为,全脑缺血后的迟发性神经元死亡很可能经由凋亡途径,而CytC激活、释放是缺血性神经元凋亡的一个关键事件.亚低温可抑制CytC的释放.推测经此途径减少缺血性神经元凋亡而发挥一定的神经保护作用.     

促红细胞生成素对大鼠脑缺血再灌注损伤后缺血侧皮层神经元凋亡和Bcl-2表达的影响

目的　探讨促红细胞生成素 (Erythropoietin ,EPO)对大鼠局灶性脑缺血再灌注损伤后的保护作用。方法　采用线栓法阻断大鼠一侧大脑中动脉 (MCA)血流 2h ,再灌注 2 4h制成局灶性脑缺血再灌注损伤模型。将 3 2只雄性SD大鼠随机分成EPO组、缺血再灌注组、假手术组和正常组。于缺血开始时EPO组给EPO 3 0 0 0U/kg腹腔注射 ;缺血再灌注组和假手术组给予等剂量生理盐水。再灌注 2 4h后断头取脑、切片 ,进行HE染色、Bcl -2免疫组化染色和细胞凋亡检测。结果　缺血 2h再灌注 2 4h后 ,EPO组和缺血再灌注组大鼠缺血侧皮层可检测到凋亡细胞 ,且EPO组凋亡细胞数明显少于缺血再灌注组 ,假手术组和正常组未见凋亡细胞 ;EPO组和缺血再灌注组缺血侧皮层Bcl -2阳性细胞数均高于假手术组和正常组 ,与缺血再灌注组相比 ,EPO组Bcl-2蛋白表达显著增高。结论　EPO可抑制缺血再灌注损伤后缺血侧皮层的细胞凋亡 ,其机制可能是通过上调bcl-2基因表达而实现。     

促红细胞生成素对脑缺血再灌注大鼠海马CA1区Bcl-xl表达和认知功能的影响

目的 探讨促红细胞生成素(EPO)对脑缺血再灌注(IR)大鼠海马CA1区Bcl-xl表达和认知功能的影响.方法 将雄性SD大鼠随机分为假手术组、IR组、EPO组,并制作IR模型;制模前3 h,EPO组及IR组大鼠脑室立体定向分别注射重组人EPO(rHu-EPO)及生理盐水.制模24 h后,应用免疫组化法检测各组大鼠海马CA1区Bcl-xl蛋白表达;4周时电迷宫测验各组大鼠学会逃避及记忆再现所需的电击次数.结果 与假手术组比较,制模24 h时,EPO组和IR组海马CA1区Bcl-xl蛋白表达明显减少,但EPO组明显多于IR组 (均P<0.01);制模4周后,EPO组及IR组大鼠学会逃避所需的电击次数明显增多,但EPO组明显少于IR组 (均P<0.01);EPO组及IR组大鼠记忆再现次数明显减少,但EPO组多于IR组(均P<0.01).结论 EPO预处理可以促进IR大鼠海马CA1区Bcl-xl蛋白表达,改善IR大鼠的认知功能损害.     

促红细胞生成素对大鼠脑缺血再灌注损伤后缺血侧皮层神经元凋亡和Bcl-2表达的影响

目的探讨促红细胞生成素(Erythropoietin，EPO)对大鼠局灶性脑缺血再灌注损伤后的保护作用。方法采用线栓法阻断大鼠一侧大脑中动脉(MCA)血流2h，再灌注24h制成局灶性脑缺血再灌注损伤模型。将32只雄性SD大鼠随机分成EPO组、缺血再灌注组、假手术组和正常组。于缺血开始时EPO组给EPO 3000U／kg腹腔注射；缺血再灌注组和假手术组给予等剂量生理盐水。再灌注24h后断头取脑、切片，进行HE染色、Bcl-2免疫组化染色和细胞凋亡检测。结果缺血2h再灌注24h后，EPO组和缺血再灌注组大鼠缺血侧皮层可检测到凋亡细胞，且EPO组凋亡细胞数明显少于缺血再灌注组，假手术组和正常组未见凋亡细胞；EPO组和缺血再灌注组缺血侧皮层Bcl-2阳性细胞数均高于假手术组和正常组，与缺血再灌注组相比，EPO组Bcl-2蛋白表达显著增高。结论EPO可抑制缺血再灌注损伤后缺血侧皮层的细胞凋亡，其机制可能是通过上调bcl-2基因表达而实现。     

局灶性脑缺血大鼠海马CA3区EPO和STAT-5的表达

目的观察局灶性脑缺血后大鼠海马CA3区促红细胞生成素（EPO）和信号传导转录活化因子5（STAT-5）的表达规律，探讨EPO和STAT-5在脑缺血损伤过程中的作用。方法采用线栓法制备大鼠大脑中动脉局灶性脑缺血模型，SD大鼠随机分为假手术组和脑缺血组，后者又分为2、12、24h三个亚组，免疫组化法检测各组大鼠海马CA3区EPO和STAT-5的表达水平。结果（1）脑缺血组各时间点大鼠海马CA3区EPO和STAT-5的表达水平均较假手术组明显增高（P％0．01）；（2）脑缺血组大鼠海马CA3区EPO和STAT-5阳性细胞数在2h明显增加；12h达到高峰，24h下降；（3）脑缺血组各时间点EPO和STAT-5表达水平同步增减。结论EPO有可能是通过EPOR-JAK2-STAT-5信号转导途径对缺血性脑损伤发挥保护性作用。     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

局灶性脑缺血再灌注损伤模型大鼠与促红细胞生成素的神经保护作用

背景：近年来动物实验和体外细胞培养研究证实促红细胞生成素对脑缺血具有神经保护作用，有关促红细胞生成素脑保护的作用机制目前尚未阐明。 目的：通过观察缺血损伤区域脑组织细胞学形态，检测脑组织超氧化物歧化酶、丙二醛浓度，探讨促红细胞生成素对脑缺血再灌注损伤的保护作用。 方法：采用线栓法建立Wistar大鼠局灶性缺血再灌注损伤模型，分别于缺血后2 h腹腔注射生理盐水3 000 U/kg、促红细胞生成素3 000，1 000 U/kg，并设假手术组。缺血再灌注损伤24 h后，应用苏木精-伊红染色法检测大鼠脑组织病理学变化，应用黄嘌呤氧化酶法和硫代巴比妥酸法分别测定超氧化物歧化酶活性和丙二醛浓度。 结果与结论：形态学结果显示促红细胞生成素高剂量组较生理盐水组皮质神经细胞存活数量增多，损伤程度减轻；促红细胞生成素高、低剂量组超氧化物歧化酶活性均明显高于假手术组和生理盐水组(P < 0.05)，丙二醛浓度明显低于假手术组和生理盐水组(P < 0.05)；促红细胞生成素高剂量组超氧化物歧化酶活性明显高于促红细胞生成素低剂量组，丙二醛含量明显低于促红细胞生成素低剂量组(P < 0.05)。提示经腹腔注射促红细胞生成素，可使大鼠脑缺血再灌注损伤区神经细胞存活数量明显增加，可显著改善组织的病理学改变，其保护作用可能是通过促红细胞生成素清除自由基，拮抗过氧化损伤实现的。     

一氧化氮合酶抑制剂对缺血性海马迟发性神经元死亡的保护作用

目的：研究一氧化氮在缺血性海马迟发性神经元死亡中的作用,观察非选择性一氧化氮合酶抑制剂Ｎ＾Ｇ－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ对缺血性海马ＤＮＤ的影响。方法：实验分为假手术组,生理盐水治疗组,Ｌ－ＮＮＡ治疗组。采用大鼠４血管关闭方法制作了全脑缺血再灌流模型,以假手术组为对照,检测了脑缺血１０ｍｉｎ再灌流７２ｈ海马区ＮＯＳ活性的变化并观察计量了海马ＣＡ１区组织病理改变;     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats.

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.     

Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms

Erythropoietin (EPO) is a hormone that is neuroprotective in models of neurodegenerative diseases. This study examined whether EPO can protect against neuronal death in the CA1 region of the rat hippocampus following global cerebral ischemia. Recombinant human EPO was infused into the intracerebral ventricle either before or after the induction of ischemia produced by using the four-vessel-occlusion model in rat. Hippocampal CA1 neuron damage was ameliorated by infusion of 50 U EPO. Administration of EPO was neuroprotective if given 20 hr before or 20 min after ischemia, but not 1 hr following ischemia. Coinjection of the phosphoinositide 3 kinase inhibitor LY294002 with EPO inhibited the protective effects of EPO. Treatment with EPO induced phosphorylation of both AKT and its substrate, glycogen synthase kinase-3beta, in the CA1 region. EPO also enhanced the CA1 level of brain-derived neurotrophic factor. Finally, we determined that ERK activation played minor roles in EPO-mediated neuroprotection. These studies demonstrate that a single injection of EPO ICV up to 20 min after global ischemia is an effective neuroprotective agent and suggest that EPO is a viable candidate for treating global ischemic brain injury.     

缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系初步探讨

目的动态观察缺血预处理后大鼠大脑皮层和海马CA1区神经元凋亡与Fas蛋白表达变化情况,初步探讨缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系。方法四血管阻断法复制全脑缺血模型,动物随机分为非缺血对照组、预处理对照组、缺血预处理组和缺血组。采用尼氏和TUNEL染色法观察皮层及海马CA1区神经元存活数和凋亡细胞数,免疫组化方法检测Fas蛋白在缺血预处理后表达变化情况。结果缺血组缺血6h在皮质及海马CA1区Fas阳性表达细胞计数升高,12h达高峰;缺血预处理组缺血12h阳性细胞计数升高,24h达高峰。缺血组缺血6h出现凋亡细胞,48h凋亡细胞数达到高峰;缺血预处理组凋亡细胞数较缺血组明显减少。缺血组缺血7d神经元数明显减少,12周时神经元大量减少;缺血预处理组缺血7d时神经元数无明显变化,但12周时神经元同样大量减少。结论全脑缺血可能通过诱导Fas蛋白的表达增多,启动细胞凋亡,导致缺血后神经元凋亡的发生;缺血预处理虽可延缓缺血后神经元的凋亡,但无法提供真正的长时期的神经元保护作用,其有限的保护作用可能是通过延缓Fas蛋白的表达而减缓了神经元凋亡的进程。     

Brain lactate uptake increases at the site of impact after traumatic brain injury

Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.