缺糖缺氧对体外培养海马神经元的影响

目的：观察缺糖缺氧诱导的培养海马神经元损伤。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液在常氧条件下继续培养24h。用0．4％台盼蓝染色,检测神经元坏死,并用TUNEL法检测神经元凋亡,计算存活、坏死和凋亡神经元所占百分率。同时用图像分析仪测定存活、坏死和凋亡神经元的胞体面积、周长和等园直径。结果：培养的海马神经元急性缺糖缺氧后0．5～4h,随缺糖缺氧时间的延长,坏死神经元逐渐增多,缺糖缺氧后0．5～2h再恢复糖和氧供应后24h,凋亡神经元明显增多。图像分析的结果表明,坏死神经元的胞体面积、周长和等园直径均明显大于凋亡神经元。结论：缺糖缺氧可引起海马神经元严重损伤,在急性缺糖缺氧后0．5～4h引起的神经元死亡以坏死为多见,但在缺糖缺氧后0．5～2h再恢复糖和氧供应后24,神经元死亡则以凋亡为多见。     

大鼠海马神经元体外缺糖缺氧模型的建立

目的：建立体外培养大鼠海马神经元缺糖缺氧模型。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液,在常氧下继续培养24h后测定培养液中乳酸脱氢酶活性。测定神经元形态变化,并计算神经元存活百分率。同时用原位末端标记(TUNEL)法检测神经元凋亡。结果：缺糖缺氧后海马神经元胞体逐渐肿胀,培养液中LDH释放量逐渐增多,细胞存活率逐渐减少。恢复糖和氧供应后24h,凋亡神经元百分率明显增多。结论：用改进的无血清、无糖人工脑脊液成功建立了大鼠海马神经元离体缺糖缺氧模型。     

重组人白细胞介素-6对缺氧-复氧后大鼠海马培养神经元Bcl-2、Bax 表达和神经元凋亡的影响

实验在培养的大鼠海马神经元中观察了重组人白细胞介素-6（recombinant human interleukin-6,rhIL-6）对缺氧-复氧后Bcl-2、Bax表达和神经元凋亡的影响。把培养12d的大鼠海马神经元分为对照组和rhIL-6组,同时于缺氧环境（90% N2 10% CO2）中培养2、4h后,再于常氧培养箱内复氧培养24和72h。于不同时间取出,分别用抗Bcl-2和Bax抗血清进行免疫组织化学染色,观察缺氧-复氧后大鼠海马培养神经元Bcl-2和Bax的表达,并用原位末端标记（TUNEL）法和流式细胞术分别检测缺氧-复氧对体外培养海马神经元凋亡的影响。结果可见,与缺氧前相比,缺氧-复氧后24和72h,海马神经元Bal-2表达明显减弱,Bax表达明显增强,凋亡神经元明显增多。经rhIL-6预处理的海马神经元与对照组相比,缺氧-复氧后24和72h,Bcl-2表达明显增强,Bax神经明显减弱,凋亡神经元明显减少。本实验结果提示,rhIL-6对海马神经元缺氧-复氧损伤具有一定的保护作用。     

缺氧/复氧对培养的大鼠海马神经元Fos、Jun表达和神经元凋亡的影响

目的：观察缺氧／复氧对体外培养的海马神经元Fos和Jun表达和神经元凋亡的影响。方法：取培养12d的海马神经元,置2000cm^3的恒温（36℃）密闭容器内,连续充以无氧气体（90％N2、10％CO2）,在缺氧条件下继续培养2、4h后取出,置含10％CO2和空气的培养箱内复氧培养24h和72h。于不同时间取出,观察神经元存活数,分别用抗Fos和抗Jun抗血清进行免疫组织化学染色,计数Fos和Jun表达阳性神经元百分率,并用原位末端标记（TUNEL）法和流式细胞术分别观察和测定缺氧／复氧对体外培养海马神经元凋亡的影响。结果：缺氧／复氧后Fos和Jun表达阳性神经元百分率和凋亡神经元百分率的显著增加。结论：缺氧／复氧后即早反应基因fos在神经元中的持续表达可引起神经元凋亡,原癌基因jun的表达与神经元凋亡的发生有关。     

细胞色素C和BAX在创伤后应激障碍大鼠海马神经元的表达及意义

观察细胞色素C（Cyt C）和凋亡相关基因BAX在创伤后应激障碍（PTSD）大鼠海马神经元中的表达,探讨其在PTSD大鼠海马神经元凋亡中的作用及相互关系。采用国际认定的SPS方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1、4、7、14、28d组和正常对照组。应用免疫组化、免疫荧光双标、激光共聚焦显微镜技术和免疫印迹法检测CytC和BAX蛋白的表达;     

PTSD样大鼠海马神经元凋亡及其ACP变化的研究

目的研究PTSD（posttraumatic stress disorder创伤后应激障碍）样大鼠海马神经元凋亡及ACP（Acid phosphatase酸性磷酸酶）的变化。方法建立大鼠PTSD模型-SPS（single-prolonged stress）,于模型建立后的6h、12h、1d、7d、14d取材;同时取材正常组作为对照,应用Annexin V-F1TC／PI双标记流式细胞术、透射电镜、酶组化方法分别进行各组海马神经元凋亡及ACP表达变化的观察及定量检测。结果模型建立后的6h、12h海马神经元的凋亡细胞增加、ACP活性增强,1d时凋亡细胞增加更为明显、ACP活性更为显著,7d、14d时凋亡细胞逐渐减少、ACP活性减弱。结论PTSD样大鼠海马神经元出现凋亡,凋亡增加的同时ACP酶活性增强,说明ACP酶参与PTSD大鼠海马神经元的凋亡。     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

海马神经元缺血/再灌注后自噬的表达及其作用

目的：研究自噬在大鼠海马神经元缺血缺氧/再灌注过程中的表达及自噬在神经元缺血缺氧/再灌注损伤中的作用。方法：原代培养的大鼠海马神经元经2 h的氧糖剥夺和不同时段的再灌注处理,MTT法检测细胞活性,透射电镜下检测自噬的特异性结构,免疫荧光化学法检测自噬特异性蛋白微管相关蛋白1轻链3（LC3B）的表达。应用自噬抑制剂3-甲基腺嘌呤（3-MA）检测神经元的活性。结果：经氧糖剥夺/再灌注后,海马神经元的活性比未经氧糖剥夺/再灌注组显著地降低。透射电镜和免疫荧光检测,未经氧糖剥夺/再灌注的神经元自噬的发生率极低,氧糖剥夺后和再灌注的不同时间段,均有自噬的发生。应用自噬抑制剂3-MA阻断自噬后,神经元的存活率显著降低。结论：缺血缺氧/再灌注能激活海马神经元的自噬,并可能在缺血缺氧/再灌注过程中起对抗损伤的作用。     

Che-1通过抑制自噬减轻氧糖剥夺所致神经元损伤的研究

目的:研究Che-1蛋白对氧糖剥夺(Oxygen glucose deprivation, OGD)所致神经元损伤的保护作用及机制。方法:OGD处理神经元后,采用免疫荧光染色和免疫印迹法检测Che-1蛋白的表达;慢病毒转染神经元实现Che-1过表达,检测乳酸脱氢酶(Lactate dehydrogenase, LDH)释放量和流式细胞术检测神经元凋亡反映OGD所致神经元损伤程度,采用免疫荧光染色和免疫印迹法检测神经元自噬;使用自噬激动剂雷帕霉素(Rapamycin)处理神经元,并通过检测LDH释放量和流式细胞术研究自噬在Che-1保护作用中的作用。结果:免疫荧光结果显示,OGD后神经元Che-1蛋白表达明显增高;免疫印迹结果显示,OGD后6至48 h神经元Che-1蛋白表达明显增高;慢病毒转染过表达Che-1蛋白后,OGD所致神经元LDH释放量明显减低,且OGD所致神经元凋亡明显减少;过表达Che-1蛋白可显著减少OGD所致神经元Beclin1和LC3II的表达;自噬激动剂Rapamycin可逆转Che-1对OGD所致神经元损伤的保护作用。结论:过表达Che-1蛋白可通过抑制神经元自噬对OGD所致神经元损伤发挥保护作用。     

右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制

摘要 目的：探讨右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制。方法：10只野生型以及20只Sod1KO雄性BALB/c小鼠,12月龄,根据实验目的分为3组：对照组（野生型小鼠）,模型组（氧化应激小鼠模型）和DEX组（氧化应激小鼠模型+50 μg/kg DEX治疗）,每组10只。通过MWM 测试检测小鼠的空间学习和记忆能力。通过免疫染色检测海马中Neu-N+细胞数和PSD-95表达水平。通过蛋白质印迹检测海马中Neu-N、PSD-95、TH、总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平。通过ROS、MDA和SOD检测试剂盒分别检测ROS、MDA和SOD水平。通过 ELISA试剂盒检测NOX2水平。通过RT-qPCR检测IL-1β、IL-6和TNF-α水平。结果：对照小鼠表现出正常的空间学习功能,与对照组小鼠相比,模型组小鼠逃避潜伏期和游泳距离增加（P<0.05）,而DEX治疗能够降低模型组小鼠逃避潜伏期和游泳距离（P<0.05）。三组小鼠平均游泳速度没有统计性差异（P>0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N+细胞数和PSD-95表达水平降低（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N+细胞数和PSD-95表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N、PSD-95和TH蛋白表达水平降低（P<0.05）,总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平升高（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N、PSD-95和TH蛋白表达水平（P<0.05）,降低总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠ROS和MDA水平增加,SOD水平降低（P<0.05）,而DEX治疗能够降低ROS和MDA水平,增加SOD水平（P<0.05）。与对照组小鼠相比,模型组小鼠NOX2水平增加（P<0.05）,而DEX治疗能够降低NOX2水平（P<0.05）。与对照组小鼠相比,模型组小鼠IL-1β、IL-6和TNF-α水平增加（P<0.05）,而DEX治疗能够降低IL-1β、IL-6和TNF-α水平（P<0.05）。结论：DEX对NOX2的抑制可通过抑制小鼠模型中的氧化应激和神经炎症来阻断学习和记忆障碍以及海马神经变性。     

Hypoxia induces an increase in intracellular magnesium via transient receptor potential melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.     

Daphnetin protects hippocampal neurons from oxygen-glucose deprivation–induced injury

Daphnetin, a coumarin derivative extracted from Daphne odora var., was reported to possess a neuroprotective effect. Recently, it has been demonstrated that daphnetin attenuates ischemia/reperfusion (I/R) injury. However, the role of daphnetin in cerebral I/R injury and the potential mechanism have not been fully understood. The present study aimed to explore the regulatory roles of daphnetin on oxygen-glucose deprivation/reoxygenation (OGD/R)–induced cell injury in a model of hippocampal neurons. Our results demonstrated that daphnetin improved cell viability and reduced the lactate dehydrogenase leakage in OGD/R–stimulated hippocampal neurons. In addition, daphnetin inhibited oxidative stress and cell apoptosis in hippocampal neurons after OGD/R stimulation. Furthermore, daphnetin significantly enhanced the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in hippocampal neurons exposed to OGD/R. Knockdown of Nrf2 blocked the protective effect of daphnetin on OGD/R–induced hippocampal neurons. In conclusion, these findings demonstrated that daphnetin attenuated oxidative stress and neuronal apoptosis after OGD/R injury through the activation of the Nrf2/HO-1 signaling pathway in hippocampal neurons. Thus, daphnetin may be a novel therapeutic agent for cerebral I/R injury.     

Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif

The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.     

Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy

The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm² in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm² in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.     

Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting

Postsynaptic density 95 (PSD-95/SAP-90) is a membrane associated guanylate kinase (GK) PDZ protein that scaffolds glutamate receptors and associated signaling networks at excitatory synapses. Affinity chromatography identifies cypin as a major PSD-95-binding protein in brain extracts. Cypin is homologous to a family of hydrolytic bacterial enzymes and shares some similarity with collapsin response mediator protein (CRMP), a cytoplasmic mediator of semaphorin III signalling. Cypin is discretely expressed in neurons and is polarized to basal membranes in intestinal epithelial cells. Overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD-95 and SAP-102, an effect not produced by overexpression of other PDZ ligands. In fact, PSD-95 can induce postsynaptic clustering of an otherwise diffusely localized K+ channel, Kv1.4. By regulating postsynaptic protein sorting, cypin may influence synaptic development and plasticity.     

Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures

Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.     

Distinct Subunit-specific α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking Mechanisms in Cultured Cortical and Hippocampal Neurons in Response to Oxygen and Glucose Deprivation

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca²⁺-impermeable receptors to GluA2-lacking Ca²⁺-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.     

The Selective Antagonism of P2X7 and P2Y1 Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.     

七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用:线粒体KATP通道的作用

利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及该作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATPchannels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoKATP通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30 min,观察OGD损伤14 min复氧1 h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变.结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0.01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71.4%(P＜0.05 vs OGD),相应恢复程度为(61.0±42.3)%和(78.7±21.1)%(P＜0.01),而且6%七氟醚的保护作用可被5-HD取消.OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀.结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoKATP通道有关.     

Knockdown of TRIM32 Protects Hippocampal Neurons from Oxygen–Glucose Deprivation-Induced Injury

Tripartite motif 32 (TRIM32) is a member of TRIM family that plays a potential role in neural regeneration. However, the biological function of TRIM32 in cerebral ischemia reperfusion injury has not been investigated. In the present study, we evaluated the expression level of TRIM32 in hippocampal neurons following oxygen–glucose deprivation/reperfusion (OGD/R). The results showed that TRIM32 expression was significantly elevated in hippocampal neurons subjected to OGD/R as compared to the neurons cultured in the normoxia condition. To further evaluate the role of TRIM32, hippocampal neurons were transfected with TRIM32 small interfering RNA (si-TRIM32) to knock down TRIM32. We found that knockdown of TRIM32 improved cell viability of OGD/R-stimulated hippocampal neurons. Generation of reactive oxygen species was decreased, while contents of superoxide dismutase and glutathione peroxidase were increased after si-TRIM32 transfection. Knockdown of TRIM32 suppressed cell apoptosis, as proved by the increased bcl-2 expression along with decreased bax expression and caspase-3 activity. We also found that TRIM32 knockdown enhanced OGD/R-induced activation of Nrf2 signaling pathway in hippocampal neurons. Furthermore, siRNA-Nrf2 was transfected to knock down Nrf2. SiRNA-Nrf2 transfection reversed the protective effects of TRIM32 knockdown on neurons. These data suggested that knockdown of TRIM32 protected hippocampal neurons from OGD/R-induced oxidative injury through activating Nrf2 signaling pathway.