缺血后处理对PC12细胞缺血再灌注损伤致细胞凋亡的作用及其机制研究

目的 探讨缺血后处理对PC12细胞缺血再灌注损伤引起的细胞凋亡的作用及其作用机制。方法 将PC12细胞分为3组:正常组、缺血再灌注组、缺血后处理组。缺血再灌注组予以糖氧剥夺12 h后正常培养,缺血后处理组经糖氧剥夺12 h后予以3个循环的正常培养(10 min)→糖氧剥夺(10 min),再正常培养12 h后通过Hoechst染色检测各组细胞的凋亡情况,应用Westernblot 检测各组细胞Caspase-3活化蛋白及磷酸化NF-κB/p65蛋白表达水平,采用RT-PCR检测各组细胞NF-κB及Caspase-3 mRNA表达水平。结果 Hoechst染色显示缺血后处理可降低缺血再灌注引起的细胞凋亡; 与对照组相比, 缺血再灌注组磷酸化NF-κB/p65和Cleaved caspase-3的蛋白表达水平高; 缺血后处理组磷酸化NF-κB/p65和Cleaved caspase-3的蛋白表达水平明显低于缺血再灌注组; NF-κB和Caspase-3的mRNA表达趋势与蛋白表达基本一致。结论 缺血后处理可以减轻缺血再灌注损伤引起的PC12细胞凋亡,这可能与NF-κB/p65信号通路有关。     

Neuroprotection induced in vitro by ischemic preconditioning and postconditioning: modulation of apoptosis and PI3K-Akt pathways

Preconditioning and postconditioning are mild ischemic exposures before or after severe injurious ischemia, respectively, that elicit endogenous neuroprotective responses. Molecular mechanisms of neuroprotection through preconditioning and postconditioning are not completely understood. Here we optimized the in vitro oxygen and glucose deprivation (OGD) models of preconditioning and postconditioning in primary cortical neuron cultures that allow the studies of the corresponding molecular mechanisms of neuroprotection. We found that the cortical cells preconditioned with a single 45-min OGD treatment administered 24 h prior to injurious 2 h OGD were robustly protected after both 3 h and 16 h of reperfusion. For the postconditioning treatment, we found that three cycles of 15 min OGD followed by 15 min reperfusion, applied immediately after injurious 2 h OGD and prior to complete reperfusion, resulted in effective neuroprotection at both 3 h and 16 h of reperfusion. Using real-time RT–PCR arrays focused on genes of the apoptosis and PI3K–Akt pathways, we found that injurious OGD mainly induced apoptosis-related and repressed PI3K–Akt pathway-related genes after either 3 h or 16 h of reperfusion. Preconditioning treatment resulted in the activation of both pro-survival and anti-apoptotic pathways after 3 h of reperfusion and mainly anti-apoptotic pathway after 16 h of reperfusion. In contrast, the activation of PI3K–Akt pathway mainly contributed to the neuroprotective effect by the postconditioning treatment after 3 h of reperfusion, but differential gene expression likely contributed minimally, if at all, to the neuroprotection observed after 16 h of reperfusion. Among the novel markers of neuroprotection, Nol3 gene upregulation was observed after 3 h of reperfusion following either preconditioning or postconditioning treatments and after 16 h of reperfusion following preconditioning treatment.     

钾通道Kv4.2、Kv1.4在硫化氢后处理对大鼠短暂全脑缺血神经保护中的作用研究

目的研究硫氢化钠(sodium hydrosulfide,Na HS)后处理对短暂全脑缺血大鼠海马中钾通道Kv4.2和Kv1.4 mRNA表达变化的影响及其脑保护作用,从而探讨Na HS对大鼠短暂全脑缺血神经保护作用的机制。方法用4VO方法建立大鼠短暂性全脑缺血(transient global cerebral ischemia,t GCI)模型,大鼠被随机分配到3组,分别为:假手术组(sham)、t GCI组、Na HS后处理组。Na HS后处理组为t GCI之后1 d,给予大鼠腹腔注射Na HS 24μmmol/kg或者180μmmol/kg。通过尼氏染色与Neu N免疫染色确定海马神经元的死亡,通过RT-PCR方法检测海马组织Kv4.2和Kv1.4mRNA水平的表达变化。结果 (1)与t GCI组比较,在t GCI之后1 d给予24μmol/kg Na HS后处理使海马CA1区存活细胞数目显著增加,而高剂量的Na HS(180μmol/kg)后处理对t GCI大鼠海马CA1区则无明显的保护作用。(2)在Re 26 h和Re 48 h,海马组织中Kv4.2、Kv1.4的mRNA表达水平均明显低于假手术组(P<0.05)。在Re 26 h+Na HS组,kv4.2(1.24±0.08)和kv1.4(1.11±0.07)的mRNA表达水平均分别高于Re 26 h组的kv4.2(0.75±0.04)和kv1.4(0.79±0.06),差异均有显著性(P<0.05)。结论外源性Na HS可能通过上调大鼠t GCI后海马区Kv4.2和Kv1.4 mRNA的表达,从而导致膜电位超极化,降低神经元兴奋性和氧耗,继而保护神经元免受脑缺血损伤。     

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid‐mitochondrial apoptotic signaling pathway

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid‐mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA‐074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP₂ and GFAP levels. In OGD‐induced astrocyte injury, CA‐074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD‐induced astrocytes injury, Clik148 or CA‐074Me reversed pMCAO or OGD‐induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome‐c (Cyt‐c) and increase in cytoplastic Cyt‐c and active caspase‐3 at 12–24 h of the late stage of pMCAO or OGD. CA‐074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt‐c and decreased cytoplastic Cyt‐c and active caspase‐3 at 6 h of the early stage of Bid activation. CA‐074Me or Clik148 blocked the pMCAO‐induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase‐3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD‐induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid‐mitochondrial apoptotic signaling pathway. GLIA 2014;62:855–880     

缺血后处理对SH-SY5Y细胞缺血再灌注模型的保护作用

目的研究缺血后处理对SH-SY5Y细胞缺血再灌注损伤模型的保护作用,并初步探讨其作用机制。方法将SH-SY5Y细胞分为三组：正常组、缺血再灌注组、缺血后处理组,缺血再灌注组予以糖氧剥夺12 h后正常培养,缺血后处理组经糖氧剥夺12 h后予以3个循环的正常培养10 min/糖氧剥夺10 min,正常培养12 h后通过光镜、荧光显微镜以及细胞计数法、流式细胞术、SOD活力检测SH-SY5Y细胞的改变。结果缺血后处理组的SH-SY5Y细胞存活率较缺血再灌注组明显增加而凋亡率明显下降（P〈0.01）,经缺血后处理的SH-SY5Y细胞内SOD的活力较缺血再灌注组增加32.53%（P〈0.05）。结论缺血后处理在细胞离体状态下可以发挥保护作用,能够降低SH-SY5Y细胞缺血再灌注损伤引起的细胞凋亡,提高细胞内SOD的活力。     

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38α and p38β activation and confers neuroprotection in the pheochromocytoma PC12 model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38α and β, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

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。     

无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的远隔保护

背景：缺血预处理及缺血后处理是近年来提出减轻缺血再灌注损伤有效方法。 目的：探讨无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的影响及机制。 方法：18只糖尿病SD大鼠数字表法随机分为3组,对照组仅行开腹术;缺血再灌注组仅行胰腺移植;缺血后处理组,移植前行非创伤性双后肢缺血后处理。 结果与结论：缺血再灌注组血糖和胰腺组织中丙二醛水平均高于缺血后处理组(P < 0.01)、而超氧化物歧化酶活性低于缺血后处理组(P < 0.01);与缺血后处理组比较,缺血再灌注组胰腺组织凋亡指数明显增高(P < 0.01)。结果提示,无创伤双后肢缺血后处理对大鼠移植胰的缺血再灌注损伤具有保护作用,机制可能与可通过减少超氧化物歧化酶失活,从而清除氧自由基以及减少胰腺细胞凋亡等有关。     

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.     

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca²⁺]_i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia‐like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia‐induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O₂ (reperfusion‐like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high‐affinity Zn²⁺ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high‐affinity Zn²⁺ probe, we showed that ischemia induced an intracellular Zn²⁺ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia‐induced oligodendrocyte death. Moreover, UO126 blocked the ischemia‐induced increase in poly‐[ADP]‐ribosylation of proteins, and the poly[ADP]‐ribose polymerase 1 (PARP‐1) inhibitor DPQ significantly inhibited ischemia‐induced oligodendroglial cell death—demonstrating that PARP‐1 was required downstream in the Zn²⁺‐ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn²⁺, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia‐reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.     

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38alpha and beta, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

Sevoflurane Postconditioning Ameliorates Oxygen–Glucose Deprivation—Reperfusion Injury in the Rat Hippocampus

Introduction: Sevoflurane is well known to exert a neuroprotective effect through anesthetic preconditioning. However, its effects on postconditioning, a neuroprotective phenomenon following an insult, have not been well studied. Aims: In this study, we examined the ability of sevoflurane to induce postconditioning in rat hippocampal slices, in vitro. Results : 2%, 4%, and 6% sevoflurane reduced neurophysiologic and morphologic neuronal injury following oxygen–glucose deprivation (OGD) and reperfusion. The quantity of damaged neurons was significantly reduced on immunofluorescence staining; excitatory amino acids (Asp, Glu) increased and inhibitory amino acids (GABA) decreased significantly. The effect was concentration‐dependent. Conclusion : Postconditioning with sevoflurane reduces neuronal damage after OGD—reperfusion injury in the CA1 area of rat hippocampus, in vitro.     

MAPK信号转导通路中ERK、JNK和P38在大鼠肝脏缺血再灌注和缺血后处理中表达的变化

背景：近年来,随着器官移植的不断开展和深入研究,人们逐渐认识到供肝原发性无功能是引起肝移植患者早期死亡的主要原因,而缺血再灌注损伤是导致供肝功能不良的重要因素。如何减轻或消除缺血再灌注损伤一直是临床研究的热点。 目的：观察肝缺血再灌注损伤和缺血后处理后MAPK级联通路中P38,JNK和ERK活化的情况。 设计、时间及地点：随机对照动物实验,于2007-05/10在中南大学湘雅医院动物实验中心完成。 材料：102只Wistar大鼠随机分为假手术组6只,缺血30 min再灌注组48只和缺血后处理组48只,后2组又分为0,0.5,1,2,4,8,12,24 h不同时间处理亚组,每亚组6只。 方法：建立大鼠肝脏体内局部缺血再灌注-缺血后处理模型,于复灌后0,0.5,1,2,4,8,12,24 h取肝脏组织。 主要观察指标：应用免疫组织化学的方法对磷酸化的p-P38、p-JNK和p-ERK进行免疫组化检测并作半定量分析。 结果：①p-ERK,p-JNK在缺血后即有轻度增高,但在再灌注后30 min开始增高明显,持续到再灌注后4 h,高峰出现在再灌注后2 h。缺血后处理组p-ERK,p-JNK的表达在再灌注后1,2,4 h较缺血再灌注组增高(P < 0.05)。②p-P38在缺血后即有轻度地增高,但在再灌注后30 min开始增高明显,高峰出现在再灌注后1 h,2 h后开始下降,表达程度维持在缺血后水平。与缺血再灌注组相比,缺血后处理组p-P38的表达增高,但仅在再灌注后1 h明显增高(P < 0.05)。 结论：缺血后处理可通过增高ERK和P38的磷酸化水平,降低JNK的磷酸化水平减轻缺血再灌注导致的肝脏损伤。     

重组人促红细胞生成素后处理缺血/再灌注前后骨骼肌诱导型一氧化氮合酶及一氧化氮的变化

背景：促红细胞生成素对包括心脑在内的多种组织器官的缺血/再灌注损伤有保护作用。 目的：观察重组人促红细胞生成素后处理对家兔后肢腓肠肌缺血/再灌注前后诱导型一氧化氮合酶、一氧化氮和超微结构的影响。 方法：将家兔随机分为空白对照组、模型组和重组人促红细胞生成素组,后2组建立兔左后肢腓肠肌缺血/再灌注实验模型,其中重组人促红细胞生成素组在兔左后肢缺血2 h后静脉注射重组人促红细胞生成素。 结果与结论：再灌注后4,12 h,重组人促红细胞生成素组诱导型一氧化氮合酶表达水平及一氧化氮浓度增高幅度较模型组低(P < 0.05)。电镜下腓肠肌细胞超微结构显示,模型组内皮细胞膜溶解,肿胀明显,肌纤维内线粒体水肿。重组人促红细胞生成素组肌纤维损伤较轻,肌节内Z线及肌节内各带结构基本正常,大部分线粒体结构正常,显示重组人促红细胞生成素组超微结构损伤程度明显轻于模型组。结果证实,重组人促红细胞生成素后处理可通过抑制诱导型一氧化氮合酶蛋白的表达,减少再灌注后过量一氧化氮生成,改善骨骼肌缺血/再灌注损伤。     

褪黑素在培养的小脑颗粒细胞模拟缺血再灌注中的抗氧化作用研究

目的探讨褪黑素(Melatonm,MT)在模拟缺血再灌注中对神经元的保护机制,从而为褪黑素的临床应用提供可靠的理论依据.方法分离SD乳鼠的小脑颗粒细胞进行原代培养,待细胞发育成熟后建立体外模拟缺血再灌注模型.采用相差显微镜进行细胞的形态学观察;通过TBA荧光法和联苯三酚自氧化法来检测模拟缺血再灌注过程中细胞内丙二醛(MDA)生成量、超氧化物歧化酶(SOD)的活性变化以及褪黑素在模拟缺血再灌注过程中对二者的影响.结果模拟缺血(OGD)后再灌注12 h的细胞,胞内MDA的生成比在OGD后再灌注6 h明显增多(P＜0.05),OGD后再灌注12 h MDA生成最多随后降低;经OGD处理后细胞内SOD的活性较正常组明显降低(P＜0.05),OGD后再灌注24 h降到最低随后升高.MT能显著降低细胞内MDA的生成(P＜0.05),并存在着一定的剂量依赖关系.SOD的活性在加入MT后也明显提高(P＜0 05).结论MT可能通过其抗氧化作用在模拟缺血(OGD)再灌注中起到神经保护作用.     

Minocycline protects PC12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation

Minocycline protects animals against cerebral ischemia by inhibiting inflammation. To determine whether minocycline protects PC12 cells from in vitro ischemic-like injury and affects pro-inflammatory 5-lipoxygenase activation, the cell viability and 5-lipoxygenase translocation to nuclear membrane were observed after oxygen-glucose deprivation (OGD). We found that OGD reduced cell viability, which was attenuated by minocycline and 5-lipoxygenase inhibitor caffeic acid. 5-Lipoxygenase protein was detected in PC12 cells by immunohistochemical and Western blot analyses. OGD induced 5-lipoxygenase translocation to nuclear membranes, which was abolished by minocycline and caffeic acid. Thus, minocycline can protect PC12 cells from in vitro ischemic-like injury, and this effect may be partly related to the inhibition of 5-lipoxygenase activation.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Remifentanil postconditioning improves global cerebral ischemia-induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway

Global cerebral ischemia followed by reperfusion, which leads to extensive neuronal damage, particularly the neurons in the hippocampal CA1 region. Apoptosis is one of the major mechanisms that lead to neuronal death after cerebral ischemia and reperfusion. The neuroprotective effects of remifentanil preconditioning against cerebral ischemia/reperfusion injury have been recently reported. Here we investigated whether remifentanil postconditioning exerts neuroprotective effects against global cerebral ischemia/reperfusion injury in rats and its potential mechanisms. Global cerebral ischemia was performed via 10 min of four-vessel occlusion. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. We found remifentanil postconditioning markedly improved the spatial learning and memory as well as attenuated neuronal apoptosis in hippocampus caused by cerebral ischemia/reperfusion injury. In addition, remifentanil postconditioning enhanced the expression of anti-apoptotic gene Bcl-2 while suppressed the expression of pro-apoptotic gene Bax in hippocampal CA1 region. However, the neuroprotective effects of remifentanil postconditioning were abolished by pretreatment of the PI3K inhibitor LY294002. The results suggest that remifentanil postconditioning exhibits neuroprotective effects against global cerebral ischemia/reperfusion injury in rats, and its mechanisms might involve inhibition of neuronal apoptosis through the PI3K pathway.