一氧化氮及谷氨酸在内皮素-1诱导神经元凋亡中的作用

目的　观察一氧化氮 (NO)和谷氨酸在内皮素 (ET) 1诱导培养神经元凋亡中的作用。方法　神经元培养取自新生SD大鼠大脑皮质。培养 5天后分 4组 :对照组、ET 1组 (2 0nM)、ET 1 L NAME(N 硝基左旋精氨酸甲酯 ,NO合酶抑制剂 ,1 0 0mM)组和ET 1 APV组 (N 甲基 D 天冬氨酸型受体拮抗剂 ,1 0 0 μM)。培养 2 4h后 ,收集细胞用流式细胞仪定量检测凋亡率。上清液中NO水平通过硝酸还原酶法检测亚硝酸盐浓度反映 ,谷氨酸浓度测定用高压液相法。结果　 2 0nMET 1处理后 2 4h,培养神经元凋亡率较对照组显著增高 (P <0 0 0 1 )。L NAME和APV分别明显阻断ET 1诱导神经元凋亡的作用 ,与ET 1组比较 ,凋亡率降幅分别为 40 % (P<0 0 5)和 80 % (P <0 0 0 1 )。ET 1作用 2 4h后。神经元培养液中NO和谷氨酸浓度较对照组显著增高 (P <0 0 0 1 ) ,L NAME完全抑制了ET 1引起的培养液中NO的升高。结论　NO和谷氨酸参与了ET 1诱导培养大鼠大脑神经元凋亡过程 ,其中谷氨酸更为重要     

大脑皮质神经元细胞的原代培养模型建立及鉴定

目的探讨C57BL/6胎鼠大脑皮质神经元的原代培养方法,并对其纯度进行鉴定。方法用显微解剖方法获取胎鼠大脑皮质组织,通过Papain消化、Hibernate E洗涤、加入含2%B27的无血清的Neurobasal培养液等方法,在体外获得纯化的大脑皮质神经元,并采用NSE免疫荧光染色法检测所培养的神经元细胞的纯度。结果获得的体外培养的皮质神经元细胞生长良好,纯度可达到90%以上。结论本方法利用无血清培养可以获得神经科学所需的高稳定性高纯化的大脑皮质神经元细胞。     

醒脑静对缺氧神经元凋亡的影响研究

目的观察醒脑静对缺氧神经元凋亡的影响。方法利用原代培养的SD大鼠大脑皮质神经元缺氧模型,通过An-nexin V、Hoechest33258染色半定量测定10μg/ml和1.0μg/ml浓度的醒脑静对缺氧神经元凋亡的影响。结果10μg/ml的醒脑静有显著降低缺氧神经元凋亡的作用,但较小剂量醒脑静则无此作用。结论醒脑静对培养大鼠大脑皮质神经元具有保护作用。     

渗透性应激诱导神经元凋亡

目的 :研究渗透性应激对神经元凋亡的诱导。方法 :体外原代培养鸡胚端脑神经元 ,以 1mol·L-1山梨醇处理神经元 1h ,换正常营养液 ,1h后行倒置显微镜、透射电镜形态学观察 ,MTT(噻唑蓝 )细胞活性分析以及DNA琼脂糖凝胶电泳。结果 :神经元活性明显下降 ,DNA电泳及形态学观察显示神经元以凋亡的形式死亡。结论 :渗透性应激可以诱导体外原代培养鸡胚端脑神经元凋亡     

高钾诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst33258 染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现：①高钾诱导神经元死亡呈剂量（50－100mmol/L）和时间依赖性;②神经元死亡呈现明显的凋亡特征：胞体缩小,染色质浓缩,DNA“梯形”条带形成和蛋白质合成抑制剂（cycloheximide,1.0mg/L)可阻断其毒性等;③MK－801（2μmol/L)、尼莫地平（10μmol/L）、硫酸镁（20μmol/L）可阻断高钾的大部分毒性作用。结果提示：高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗料神经元凋亡。     

β-淀粉样多肽诱导神经元凋亡和氧化应激机制的实验观察

现已经明确 ,β 淀粉样多肽 (β ａｍｙｌｏｉｄｐｅｐｔｉｄｅ ,Ａβ)在老年痴呆 (Ａｌｚｈｅｉｍｅｒｄｉｓｅａｓｅ,ＡＤ)成因上起重要作用。有关Ａβ的神经毒性机制有许多假说 ,目前 ,自由基介导的神经元损伤假说倍受重视。本实验进一步观察氧化损伤与Ａβ神经毒性的关系 ,以及自由基清除剂褪黑素 (ｍｅｌａｔｏｎｉｎ ,Ｍｅｌ)的神经保护作用 ,为ＡＤ的抗氧化治疗选择提供实验依据。方法 :(1)选取 30只Ｗｉｓｔａｒ雄性大鼠 ,实验组将 1μｌＡβ2 5～ 35(浓度为 10 μｇ/ μｌ)注射到双侧海马 ,治疗组同时腹腔注射 0 .2 %的Ｍｅｌ溶液 …     

阿魏酸及其酯化产物与体外培养大鼠大脑皮质神经元的存活

背景：大量体外筛选中得到的中药脑神经保护活性成分,多因脂溶性低难以透过血脑屏障而限制了其临床应用。 目的：比较阿魏酸及其酯化产物阿魏酸甲酯、乙酯对原代培养的大鼠大脑皮质神经元体外存活的保护作用。 方法：出生1 d内的乳鼠采用酶消化法分离培养大脑皮质神经元。培养6 d后,分别进行阿魏酸、阿魏酸甲酯及乙酯干预,继续培养1 d。 结果与结论：形态学观察结果显示细胞生长良好,NSE染色检查表明培养6 d的存活细胞大部分均为神经元。MTT比色法测量结果显示,与空白对照组比较,阿魏酸仅在质量浓度为200 mg/L时有明显促进大脑皮质神经元存活的作用,而阿魏酸甲酯、乙酯在质量浓度为0.16~20 mg/L时均能明显促进神经元体外存活,且作用趋势、作用强度相近。表明阿魏酸的酯化产物阿魏酸甲酯和阿魏酸乙酯均能促进原代培养的大脑皮质神经元体外存活,显示出较好的脑神经元保护作用,且活性比阿魏酸更强。     

高钾可诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst332 58染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现 :①高钾诱导神经元死亡呈剂量 ( 50～ 10 0mmol/L)和时间依赖性 ;②神经元死亡呈现明显的凋亡特征 :胞体缩小 ,染色质浓缩 ,DNA“梯形”条带形成和蛋白质合成抑制剂 (cycloheximide ,1.0mg/L)可阻断其毒性等 ;③MK 80 1( 2 μmol/L)、尼莫地平 ( 10 μmol/L)、硫酸镁 ( 2 0mmol/L)可阻断高钾的大部分毒性作用。结果提示 :高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗粒神经元凋亡     

红藻氨酸致惊大鼠海马神经元Caspase—3表达与神经元凋亡

目的：研究Caspase-3在红藻氨酸（Kainate,KA)致惊大鼠海马中的变化及其在海马神经元凋亡中的作用。方法：在KA所致大鼠惊厥模型中，用免疫组织化学方法检测惊厥后不同时间点大鼠海马中Caspase-3的表达，用电子显微镜和原位末端标记法（TUNEL）检测惊厥后不同时间点大鼠海马神经元凋亡。结果：惊厥后1d,大鼠海马内Caspase-3的表达就明显升高，一直持续到惊厥3d；大鼠海马内凋亡细胞从惊厥后3d即明显增多，一直持续到惊厥后7d。结论：KA所致惊厥后，大鼠海马内Caspase-3表达明显升高，神经元凋亡明显增多，而且Caspase-3的变化发生在神经元亡增多之前，提示Caspase-3可能参与了KA致惊厥大鼠海马神经元凋亡的发生。     

Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons

Increased amounts of bilirubin, the end product of heme degradation, are known to be detrimental to the central nervous system, especially in preterm newborns. In an attempt to delineate the cellular mechanisms by which unconjugated bilirubin exerts its toxic effects on neuronal cells in the developing brain, bilirubin (0.25-5 microM) was added to the extracellular medium of 6-day-old primary cultured neurons from the embryonic rat forebrain, and cell alterations were studied over the ensuing 96 h. Bilirubin decreased cell viability dose dependently with an ED(50) around 1 microM. At the dose of 0.5 microM, it triggered delayed cell death that affected 24% of the neurons. Nuclear incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole) depicted the presence of apoptosis (16%). Apoptosis features were confirmed by DNA fragmentation reflected by a progressive loss of [(3)H]thymidine and sequential changes in macromolecular synthesis, as shown by the time course of [(3)H]leucine incorporation, as well as by the beneficial effects of cycloheximide and caspase inhibitors. In parallel, treatments with glutamate receptor antagonists showed that MK-801, but not NBQX, protected neurons against bilirubin neurotoxicity, suggesting a role for NMDA receptors in bilirubin effects. Coupled with previous work about glutamate toxicity in the same culture model, these data support the hypothesis that low levels of free bilirubin may promote programmed neuronal death corresponding to an apoptotic process which involves caspase activation and requires the participation of NMDA receptors, along with bilirubin-induced inhibition of protein kinase C activity.     

血液溶解产物对原代培养的小鼠脑皮层神经元的影响

目的 探讨血液溶解产物对原代培养的小鼠脑皮层神经元的影响及其机制。方法 分离培养新生1~3 d C57BL/6小鼠脑皮层神经元并进行鉴定;加入不同浓度的血液溶解产物,通过细胞形态学观察、细胞计数法评估细胞生长情况;Hochest33342染色法及流式细胞术检测细胞凋亡;Western blot法检测凋亡通路相关蛋白cleaved caspase-3的表达情况。结果 原代培养的小鼠脑皮层神经元NeuN阳性细胞率为（92.24±1.16）%;形态学显示随着血液溶解产物的浓度增加细胞损伤程度增大,贴壁细胞数减少,且Hochest33342染色阳性率增加,细胞凋亡率增加,凋亡通路相关蛋白cleaved caspase-3的表达也增加,且呈浓度依赖性,浓度越高,凋亡蛋白表达越高。结论 血液溶解产物能抑制脑皮层神经元的生长,其机制可能是通过上调凋亡通路相关蛋白的表达而诱导细胞凋亡来实现的。     

Increased cerebrovascular sensitivity to endothelin-1 in a rat model of obstructive sleep apnea: a role for endothelin receptor B

Obstructive sleep apnea (OSA) is associated with cerebrovascular diseases. However, little is known regarding the effects of OSA on the cerebrovascular wall. We tested the hypothesis that OSA augments endothelin-1 (ET-1) constrictions of cerebral arteries. Repeated apneas (30 or 60 per hour) were produced in rats during the sleep cycle (8 hours) by remotely inflating a balloon implanted in the trachea. Four weeks of apneas produced a 23-fold increase in ET-1 sensitivity in isolated and pressurized posterior cerebral arteries (PCAs) compared with PCAs from sham-operated rats (EC₅₀=10^−9.2 mol/L versus 10^−10.6 mol/L; P<0.001). This increased sensitivity was abolished by the ET-B receptor antagonist, BQ-788. Constrictions to the ET-B receptor agonist, IRL-1620, were greater in PCAs from rats after 2 or 4 weeks of apneas compared with that from sham-operated rats (P=0.013). Increased IRL-1620 constrictions in PCAs from OSA rats were normalized with the transient receptor potential channel (TRPC) blocker, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, or the Rho kinase (ROCK) inhibitor, Y27632. These data show that OSA increases the sensitivity of PCAs to ET-1 through enhanced ET-B activity, and enhanced activity of TRPCs and ROCK. We conclude that enhanced ET-1 signaling is part of a pathologic mechanism associated with adverse cerebrovascular outcomes of OSA.     

Dopamine induces apoptosis in cultured rat striatal neurons; possible mechanism of D2-dopamine receptor neuron loss during aging

We examined D₂-dopamine receptor containing neurons in cultures of neonatal rat striatum for apoptosis following dopamine treatment. Exposure to cultures to micromolar concentrations of dopamine resulted in 60–70% killing of D₂-dopamine receptor neurons within 24 hr. We also utilized a double labeling procedure to determine that treatment with dopamine induced apoptosis in D₂-dopamine receptor containing neurons. These results suggest that loss of D₂-dopamine receptor containing neurons during aging could be due to an apoptotic effect of dopamine. J. Neurosci. Res. 47:393–399, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol-induced apoptosis

Ethanol ingestion during pregnancy elicits damage to the developing brain, some of which appears to result from enhanced apoptotic death of neurons. A consistent characteristic of this phenomenon is a highly differing sensitivity to ethanol within specific neuron populations. One possible explanation for this "selective vulnerability" could be cellular variations in glutathione (GSH) homeostasis. Prior studies have illustrated that ethanol elicits apoptotic death of neurons in the developing brain, that oxidative stress may be an underlying mechanism, and that GSH can be neuroprotective. In the present study, both multiphoton microscopy and flow cytometry demonstrate a striking heterogeneity in GSH content within cortical neuron populations. Ethanol differentially elicits apoptotic death and oxidative stress in these neurons. When neuron GSH content is reduced by treatment with butathione sulfoxamine, the ethanol-mediated enhancement of reactive oxygen species is exacerbated. Sorting of cells into high- and low-GSH populations further exemplifies ethanol-mediated oxidative stress whereby apoptotic indices are preferentially elevated in the low-GSH population. Western blot analysis of the low-GSH subpopulations shows higher ethanol-mediated expression of active caspase 3 and 24-kDa PARP-1 fragments compared with the high-GSH subpopulation. In addition, neuronal content of 4-hydroxynonenal adducts is higher in low-GSH neurons in response to ethanol. These studies suggest that GSH content is an important predictor of neuronal sensitivity to ethanol-mediated oxidative stress and subsequent cell death. The data support the proposition that the differences in proapoptotic responses to ethanol within specific neuron populations reflect a heterogeneity of neuron GSH content.     

刺五加多糖对H2O2诱导的大鼠海马神经元凋亡的保护作用

目的研究刺五加多糖（ASPS）对H2O2诱导的海马神经元凋亡的影响及其机制。方法采用H2O2诱导大鼠海马神经元凋亡。采用末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记法检测细胞凋亡率、免疫组化法检测caspase-3蛋白的表达、逆转录PCR法检测caspase-3 mRNA的表达。结果H2O2作用后,海马神经元凋亡率、caspase-3蛋白和mRNA表达水平均显著增高（P〈0.05）;给予ASPS干预后,均显著下降（P〈0.05）;而且,随ASPS剂量增加,作用效果显著增强（P〈0.05）。结论ASPS具有抑制氧化应激损伤诱导神经细胞凋亡作用,其机制与下调caspase-3 mRNA的表达有关。     

Activation of caspase-3 in β-amyloid-induced apoptosis of cultured rat cortical neurons

Amyloid β protein (Aβ) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Aβ-treatment of cultured neurons. Treatment of rat primary cortical culture with Aβ 25–35, an active fragment of Aβ, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Aβ 25–35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Aβ-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and α-fodrin, were produced by Aβ-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Aβ-induced DNA fragmentation and cleavage of α-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH₂-DCB, additionally prevented Aβ-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Aβ-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Aβ-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Aβ-induced neuronal death still occurred with different morphological features.     

Expression of adenosine A1 receptors in cultured neurons from fetal rat brain

The expression of adenosine A₁ receptors was investigated using [³H]2-chloro-N⁶ -cyclopentyladenosine (CCPA) in 8-day-old cultured neurons from fetal rat forebrain grown in serum-free medium. [³H]CCPA bound specifically and with high affinity (Kd = 2.9 nM) to a homogeneous population of sites. Displacement of CCPA binding by various adenosine derivatives indicated that A₁ receptors were selectively labeled. The presence of Gpp(NH)p, a GTP analogue, reduced significantly the binding affinity (Kd = 12.2 nM), suggesting that A₁ receptors detected in intact cultured cells are linked to associated G proteins. © 1993 Wiley-Liss, Inc.     

Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain

Neurons from various brain regions of postnatal (15 days after birth) and fetal (16 days gestation) rats were cultured in the presence of basic fibroblast growth factor (bFGF). bFGF increased the survival of neurons from postnatal septum, striatum, midbrain, and hippocampus. Fetal neurons derived from cerebral cortex, septum, striatum, midbrain, thalamus, and colliculus were far more dependent on bFGF for survival in comparison with postnatal neurons. In contrast, cerebellum neurons of postnatal and fetal rat brain did not respond to bFGF. The increase of postnatal and fetal neuronal survival with bFGF treatment (0.01–10 ng/ml) was dose-dependent and reached 2–4-fold and 5–10-fold more than the control, respectively. Fetal cortical neurons showed almost complete dependence on bFGF since almost all neurons died in control cultures. Nerve growth factor was slightly effective only on postnatal septal and striatal neurons, being ineffective on the other neurons tested. These results indicate that bFGF can function as a neurotrophic factor not only on fetal but also on postnatal neurons of the central nervous system, and that bFGF has great potential for application in vivo.