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首页> 外文期刊>Neuroscience: An International Journal under the Editorial Direction of IBRO >Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.
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Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.

机译：离子型谷氨酸盐受体和谷氨酸盐转运蛋白参与了由海马切片培养物中氧葡萄糖剥夺引起的坏死性神经元细胞死亡。

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摘要

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzyloxyaspartate in concentrations of 25 microM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen-glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10 microM) dose of DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation (but not during the next 48h after oxygen-glucose deprivation) significantly reduced the oxygen-glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen-glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen-glucose deprivation-induced necrotic cell de
机译：器官型海马切片培养物是研究脑缺血和离子型谷氨酸受体在氧葡萄糖剥夺引起的神经变性中作用的可行模型。本文的第一部分介绍了新结果和对现有数据的回顾。本文最后一部分综述了谷氨酸转运蛋白的作用，特别是关于在正常和能量衰竭（缺血样）条件下抑制谷氨酸转运蛋白的最新结果。实验工作基于源自7日龄大鼠的海马切片培养物，并生长约3周。在这种文化中，我们调查了亚场神经元对氧-葡萄糖剥夺的敏感性，诱导的细胞死亡的类型以及离子型谷氨酸受体的参与。在本文的最后部分，我们对谷氨酸转运蛋白的研究中也使用了海马切片培养物。通过细胞摄取碘化丙啶，微管相关蛋白2的免疫细胞化学染色消失和Fluoro-Jade B染色来监测和/或显示神经退行性变。为了区分坏死性和凋亡性神经元细胞死亡，我们使用了免疫细胞化学染色来检测活性胱天蛋白酶图3（细胞凋亡指示剂）和Hoechst 33342染色核染色质。我们对氧葡萄糖剥夺的实验研究证实，CA1锥体细胞最容易出现这种缺血样情况。根据碘化丙锭的摄取判断，在暴露于氧-葡萄糖剥夺30分钟的培养物中，选择性CA1病变仅对CA3产生较小影响。 Hoechst 33342的核染色质染色和活性caspase-3染色表明，氧葡萄糖剥夺仅导致坏死细胞死亡。加入10 microM的N-甲基-D-天冬氨酸谷氨酸受体拮抗剂MK-801和20 microM的N-甲基-D-天冬氨酸谷氨酸受体拮抗剂2,3-二羟氧基-6-硝基-7-氨磺酰基-苯并（F）喹喔啉在培养基中的分布证实了N-甲基-D-天门冬氨酸和非N-甲基-D-天冬氨酸的离子型谷氨酸受体均参与了氧葡萄糖剥夺诱导的细胞死亡。谷氨酸通常通过钠依赖性谷氨酸转运蛋白从细胞外空间快速去除。本文的最后部分回顾了通过添加DL-苏-β-苄氧基天冬氨酸来阻断转运蛋白的作用。在正常情况下，向未经处理的海马切片培养物中添加浓度为25 microM或更高的DL-苏-β-苄氧基天冬氨酸会诱导神经元细胞死亡，这可以通过添加2,3-二羟基6-硝基-7-氨磺酰基来预防。苯并（F）喹喔啉和MK-801。在能量衰竭的情况下，例如脑缺血和氧葡萄糖剥夺，转运蛋白被认为会逆转谷氨酸并释放到细胞外空间。在氧葡萄糖剥夺期间（但在氧葡萄糖剥夺后的下一个48小时内）亚毒性（10 microM）剂量的DL-苏-β-苄氧基天冬氨酸对转运蛋白的阻滞显着降低了氧葡萄糖剥夺引起的碘化丙啶摄取，提示在这些条件下剥夺氧-葡萄糖期间，DL-苏-β-苄氧基天冬氨酸逆向转运蛋白活性具有神经保护作用。除此之外，我们实验室的其他结果表明，用神经胶质细胞系衍生的神经营养因子对切片培养物进行预处理可以上调谷氨酸转运蛋白。作为合乎逻辑的，但在某些神经胶质细胞系衍生的神经营养因子疗法相关的情况下，明显有害的结果是，在神经胶质细胞系衍生的神经营养因子治疗后，氧葡萄糖剥夺诱导的谷氨酸受体介导的细胞死亡的敏感性增加。总而言之，我们得出结论，离子型谷氨酸受体和谷氨酸转运蛋白均参与氧葡萄糖剥夺引起的坏死性细胞凋亡。

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