人重组白细胞介素—6可减少缺氧—复氧后大鼠海马培养神经？…

用原位末端标记（ＴＵＮＥＬ）法观察人重组白细胞介素－６（ｒｈＩＬ－６）对缺氧－复氧后大鼠海马培养神经元的ＤＮＡ损伤的影响。结果显示,海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显增高,经ｒｈＩＬ－６预处理的海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显低于对照组。本结果表明,缺氧－复氧能使体外培养海马神经元发生ＤＮＡ损伤,ｒｈＩＬ－６可减少缺氧－复氧后海马神经元的ＤＮＡ损伤。提示ｒｈＩＬ－６对     

人重组白细胞介素—6对缺氧对后海马培养神经…

用免疫组化方法，观察缺氧诱导体外培养大鼠海马神经细胞Ｆｏｓ表达及人重组白细胞介素－６（ｒｈＩＬ－６＿的影响。结果显示，经ｒｈＩＬ－６卵育的海马神经细胞缺氧后Ｆｏｓ免疫反应一胸核的百分率和Ｆｏｓ反应阳性胸核的平均光密度均明显低于对照组，表明缺氧能诱导体外培养海马神经细胞Ｆｏｓ的表达，ｒｈＩＬ－６能抑制缺氧神经细胞Ｆｏｓ的表达。提示ｒｈＩＬ－６可能参与脑缺氧损伤的调控。     

低氧预处理对大鼠海马神经元缺氧耐性和热休克蛋白表达的影响

采用免疫组织化学方法,观察低氧预处理对大鼠海马培养神经元缺氧耐受性和热休克蛋白（Ｈsp70)表达的影响。结果显示,低氧预处理能诱导海马神经元对Ｈsp70的表达。经低氧预处理的海马神经元缺氧－复氧后Ｈsp70表达较对照组明显增强,神经元损伤程度减轻,神经元存活数明显高于对照组。本结果表明,低氧预处理可诱导海马培养神经元对Hsp７０的表达,并使海马神经元对缺氧产生耐受,减少缺氧－复氧后神经元的死亡。     

人重组白细胞介素—6对大鼠海马神经元Bcl—2表达？…

目的 观察重组人白细胞介素－６（ｒｈＩＬ－６）对体外培养大鼠海马神经元Ｂｃｌ－２表达的影响。方法 取培养３、７、１４、２１和２８天（ｄ）的两组（对照组和ｒｈＩＬ－６组）培养神经元,分别观察其生长发育和神经元活存数,并用抗Ｂｃｌ－２抗血清进行免疫组化染色,观察Ｂｃｌ－２免疫反应（Ｂｃｌ－２－ＩＲ）阳性和阴性神经元数目,计算Ｂｃｌ－２－ＩＲ阳性神经元所占百分率,并在图像分析仪上对Ｂｃｌ－２－ＩＲ神经元     

低氧预处理对大鼠海马神经元缺氧耐受性和热休克蛋白表达的影响

采用免疫组织化学方法 ,观察低氧预处理对大鼠海马培养神经元缺氧耐受性和热休克蛋白(Hsp70 )表达的影响。结果显示 ,低氧预处理能诱导海马神经元对Hsp70的表达。经低氧预处理的海马神经元缺氧 复氧后Hsp70表达较对照组明显增强 ,神经元损伤程度减轻 ,神经元存活数明显高于对照组。本结果表明 ,低氧预处理可诱导海马培养神经元对Hsp70的表达 ,并使海马神经元对缺氧产生耐受 ,减少缺氧 复氧后神经元的死亡     

缺氧-复氧对大鼠海马培养神经元Bcl-2、Bax表达和神经元凋亡的影响

目的 观察缺氧-复氧对体外培养海马神经元Bcl-2和Bax表达和神经元凋亡的影响。方法 取培养12d的海马神经元，置于恒温(36℃)密闭容器内，连续充以无氧气体[90％(体积分数)N2、10％(体积分数)CO2]，在缺氧条件下继续培养4h后，再于常氧培养箱内复氧培养24h和72h。于不同时间观察神经元存活数，并分别用抗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 缺氧-复氧后24～72h,海马神经元对Bcl-2的表达逐渐减弱，对Bax的表达逐渐增强，对Bax／Bcl-2比值逐渐增大，凋亡神经元百分率逐渐增多。结论 缺氧-复氧后24～72h神经元凋亡的发生与神经元Bcl-2表达逐渐减弱，Bax表达逐渐增强，Bax／Bcl-2比值逐渐增大有关。     

氢气预处理对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响

目的探讨氢气对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响。方法原代培养的SD乳鼠海马神经元细胞,随机分成对照组,缺氧/复氧组和氢气预处理组。对照组常规培养;缺氧/复氧组在100%N2中培养15min后复氧30min;氢气预处理组在2%H2+98%N2中培养15min后复氧30min。采用MTT法检测乳鼠海马神经元细胞活力。结果氢气预处理能够增强细胞活力(P<0.05)。结论氢气预处理对海马神经元细胞缺氧/复氧损伤具有保护作用,可能与提高细胞活力有关。     

细胞因子rhIL—1β,rhIL—2及rhIL—6对大鼠海马神经元营养作用的研究

本工作采用显微测量，图像分析和流式细胞分析方法，研究了细胞因子ｒｈＩＬ－１β，ｒｈＩＬ－２及ｒｈＩＬ－６对新生大鼠海马培养神经元突起生长发育，胞体生长状态，神经元存活数及培养过程中蛋白总量影响，结果显示，这３种细胞因子均对海马培养神经元的突起生长，胞体发育以及发育过程中神经元的蛋白质民具有明显的促进作用，并且在提高海马培养神经元存活，延长存活时间方面有较显著的效应，提示白介素类细胞因子ｒｈＩＬ－１     

降钙素基因相关肽对缺氧时大鼠海马培养神经元c—fos表达的影响

采用免疫组化（ABC）方法，观察缺氧诱导体外培养大鼠海马神经元c-fos的表达及降钙素基因相关肽（CGRP）的影响。结果显示，缺氧后海马神经元中Fos-免疫反应（Fos—IR）阳性胞核百分率随缺氧时间的延长而逐渐增多，图像分析的结果显示，缺氧后Fos—IR阳性胞核的平均失密度亦随缺氧时间的延长而逐渐增强。经CGRP孵育的海马神经元缺氧后Fos-IR阳性胞核的百分率和Fos-IR阳性胞核的平均光密度均明显低于非CGRP孵育组。本结果表明，缺氧能诱导体外培养海马神经元。c-fos的表达，神经肽CGRP能抑制缺氧海马神经元c-fos的表达。提示CGRP对海马神经元缺氧损伤可能具有一定保护作用。     

人重组白细胞介素—6对培养的大鼠海马神经元缺氧—复氧后Jun表达的影响

近来的研究表明 ,Ｃ ｊｕｎ原癌基因为脑内即早反应基因 ,能被外界多种刺激因子诱导。低氧、缺血和某些药物等外源性刺激可诱导Ｃ ｊｕｎ表达[1] 。体外培养的交感神经元在去除神经生长因子的条件下可引起Ｃ ｊｕｎ表达增加 ,并证明Ｃ ｊｕｎ过度表达能导致神经元凋亡[2 ] 。但有关缺氧 复氧对体外培养海马神经元Ｊｕｎ表达影响的研究甚少。鉴于我们以前的工作证实人重组白细胞介素 6 (ｒｈＩＬ 6 )能增强海马神经元的抗缺氧能力[3 ] ,但ｒｈＩＬ 6能否影响缺氧 复氧后神经元的Ｊｕｎ表达尚不清楚 ,为此本实验用免疫组织化学方法 ,观察缺…     

缺氧对海马神经元培养的影响及巴曲酶的保护作用—HSP70的表达及细胞形态学的研究

本文采用新生大鼠海马神经元培养技术，以细胞形态学及ＨＳＰ７０免疫阳性细胞表达为指标，首次观察了巴曲酶对缺氧海马神经元损伤的直接保护作用。结果发现：在缺氧前０．５ｈ给予巴曲酶（０．５ＢＵ／ｍｌ），海马神经细胞存活率以及ＨＳＰ７０免疫阳性细胞率均明显高于缺氧对照组（Ｐ＜０．０１），而在缺氧前２４ｈ给予巴曲酶后，二者与缺氧对照组均无明显差别。表明：巴曲酶对缺氧海马神经细胞损伤有直接的保护作用，而且其作用与给药的时机有关。     

低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响

目的 观察低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响。方法 取培养12d的两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12h,分别观察它们的形态变化和神经元存活数,并用抗rhIL-6单克隆抗体进行免疫组化染色,观察缺氧对大鼠海马培养神经元IL-6免疫反应的影响。结果 低氧预处理可增强海马神经元对rhIL-6的免疫反应,经低氧预处理的海马神经元缺氧后神经元存活数和对rhIL-6的免疫反应均明显高于对照组。结论 低氧预处理氧预处理的海马神经元缺氧后神经元存活数和对thIL-6的免疫反应均明显高于对照组。结论 低氧预处理可使体外培养的海马神经元对缺氧产生耐受,其中rhIL-6的免疫反应增加可能是海马神经元对缺氧的一种适应性变化,提示IL-6可能参与脑缺氧耐受性的形成,并在海马神经元缺氧损伤的调控中起重要作用。     

Antioxidants, mitochondrial hyperoxidation and electrical recovery after anoxia in hippocampal slices

Cerebral injury may occur not only during brain ischemia but also during reperfusion afterward. A characteristic event during reperfusion after cerebral ischemia, or reoxygenation after anoxia in hippocampal slices, is hyperoxidation of the electron carriers of the mitochondrial respiratory chain. Earlier studies suggested that mitochondrial hyperoxidation was produced by an oxyradical mechanism and was linked to neuronal damage. Present studies sought to test this hypothesis by determining whether antioxidants could suppress mitochondrial hyperoxidation and improve electrical recovery after anoxia in hippocampal slices. Both 500 μM ascorbate and 50 μM glutathione decreased post-anoxic hyperoxidation of NADH and improved electrical recovery in hippocampal slices. These data support a role of oxygen free radicals in promoting post-anoxic mitochondrial hyperoxidation and electrical failure, and suggest that these effects of anoxia or ischemia may be linked.     

缺氧对海马培养细胞人重组白细胞介素-6免疫组化反应的影响

本文观察了缺氧对体外培养海马细胞中rhIL-6免疫反应的影响。结果显示，体外培养的海马神经元和神经胶质细胞的rhIL-6呈弱阳性免疫反应，缺氧1～4h后神经元和神经胶质细胞的rhIL-6免疫组化反应明显增强。对免疫染色细胞作图像分析结果显示，缺氧后神经元和神经胶质细胞的平均光密度较缺氧前增加，尤以缺氧2h时最明显，之后随缺氧时间延长，神经元和神经胶质细胞的平均光密度又逐渐减弱。上述结果表明，海马脑区存在rhIL-6免疫反应细胞；缺氧条件下rhIL-6免疫组化反应增强，提示rhIL-6可能参与脑缺氧损伤的调控。     

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.     

人重组白细胞介素-6对缺氧后海马培养神经细胞Fos表达的影响

用免疫组化方法，观察缺氧诱导体外培养大鼠海马神经细胞Fos表达及人重组白细胞介素-6（rhIL－6）的影响。结果显示，经rhIL－6孵育的海马神经细胞缺氧后Fos免疫反应阳性胞核的百分率和Fos免疫反应阳性胞核的平均光密度均明显低于对照组，表明缺氧能诱导体外培养海马神经细胞Fos的表达，rhIL－6能抑制缺氧神经细胞Fos的表达。提示rhIL-6可能参与脑缺氧损伤的调控。     

Preliminary evidence of neuronal regeneration in the anoxia tolerant vertebrate brain

Postnatal neurogenesis in response to stroke or ischemia is currently of great medical interest. In this study, we investigated the potential for neurogenesis in an anoxia tolerant vertebrate in response to global ischemia. The results suggest sustained neurogenesis in the turtle that increases after ischemic damage, thus revealing a potential physiological adaptation to repeated anoxia–reoxygenation events. This finding further emphasizes the common vertebrate phenomenon of postnatal neurogenesis, with the capacity for extensive regeneration of neurons apparent in some reptilian species.     

Free radicals are involved in the damage to protein synthesis after anoxia/aglycemia and NMDA exposure

Neuronal protein synthesis is inhibited in CA1 pyramidal neurons for many hours after ischemia, hypoxia or hypoglycemia. This inhibition precedes cell death, is a hallmark characteristic of necrotic damage and may play a key role in the death of vulnerable neurons after these insults. The sequence of events leading to this inhibition remains to be fully elucidated. The protein synthesis failure after 7.5 min anoxia/aglycemia in the rat hippocampal slice can be prevented by blocking N-methyl-D-aspartate receptors in a reduced calcium environment during the insult. In this study, we demonstrate that N-methyl-D-aspartate exposure directly causes a dose-dependent, receptor-mediated and prolonged protein synthesis inhibition in CA1 pyramidal neurons. The free radical scavenger Vitamin E significantly attenuates this damage due to low concentrations of N-methyl-D-aspartate (10 microM). Free radical generation by xanthine/xanthine oxidase (XOD) can directly damage protein synthesis in neurons of the slice. Vitamin E, ascorbic acid and N-acetylcysteine can each prevent the damage due to anoxia/aglycemia and to higher concentrations of N-methyl-D-aspartate (50 microM), provided calcium levels are reduced concomitantly. These findings indicate that both free radicals and calcium play a role in the sequence of events leading to protein synthesis failure after energetic stress like anoxia/aglycemia. They further suggest that the mechanism by which N-methyl-D-aspartate receptor activation damages protein synthesis involves free radical generation.     

Effects of accumulation of phosphocreatine on utilization and restoration of high-energy phosphates during anoxia and recovery in thin hippocampal slices from the guinea pig

Effects of accumulation of phosphocreatine (P-creatine) on utilization and restoration of high-energy phosphates during anoxia and recovery were studied using thin hippocampal slices from the guinea pig. Incubation of slices with creatine (5 to 30 mM) increased the content of tissue P-creatine, in a linear fashion, and these increases depended on the concentration of creatine and the incubation period to as long as 3 h. Concentration of ATP in the slices was not altered. A high concentration of tissue P-creatine (40 to 100 mmol/kg protein) accumulated by incubation with creatine reduced the rate of ATP exhaustion during anoxia whereas P-creatine itself was rapidly decreased. During reoxygenation after anoxia, concentrations of ATP and P-creatine rapidly recovered in slices preincubated with creatine. Concentrations of P-creatine previously exhausted during anoxia increased to 100 mmol/kg protein after 25 min of reoxygenation whereas the concentration was 35 mmol/kg in the control slice. Elevation of the concentration of P-creatine may therefore prolong the survival time of brain tissue during anoxia and facilitate recovery during reoxygenation.