Neuroprotection for Cell Death of Retinal Ganglion Cells and Molecular Biological Study for Nervi-Regeneration

视网膜神经节细胞细胞死亡的神经保护和神经再生的分子生物学研究

• 批准号: 11470364
• 负责人: MISHIMA Hiromu
• 金额: $ 8.38万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470364/
• 关键词: retinal ganglion; glutamate neurotoxity; intracellular Ca^<2+> concentration; glutamate receptor; cDNA microarray; novel gene; nitric oxide; nitric oxide synthase; 網膜; differential display

NMDA, AMPA and kainate receptors were confirmed to exist on MACS-separated cultured RGC. Moreover, 20-HE inhibited NMDA receptor-mediated currents most prominently and AMPA- and kainate-mediated currents moderately.cDNA arrays were used to detect highly expressed mRNA in the mouse eye. We focused among them on a novel gene, ODAG (GenBank ; Accession No.AB047921), which was downregulated at P10. Mouse ODAG cDNA encodes a protein of 266 amino acids. Human ODAG cDNA and genomic structure were identified by BLAST analysis of the GenBank database with mouse ODAG. Mouse ODAG-specific mRNA expression was detected in various mouse tissues within the eye at P2 and P7, whereas it was not detected anywhere at P14.Expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) induced by glutamate stimulation were investigated. Immunohistochemical analysis revealed nNOS and eNOS expressed in r … More etinal ganglion cells. Intracellular NO levels in cultured RGCs showed fluctuation during a 20-minute observation. The presence of a specific nNOS inhibitor significantly inhibited the increase of intracellular NO after the introduction of glutamate to fee medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs, and demonstrated that NO is produced by nNOS mainly that is stimulated by glutamate in cultured RGCs.Glutamate caused cell death to retinal ganglion cell for 24 hrs in a concentration-dependent manner. Glutamate increased in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in a concentration-dependent manner. NMDA and KCl caused the same results. Betaxolol inhibited the KCl-induced increase in [Ca^<2+>]_i to 25 %. On the other hand, betaxolol significantly inhibited glutamate-induced RGCs death. An increase in [Ca^<2+>]_i is considered the first signal of glutamate neurotoxicity. Betaxolol produced the neuroprotective effects by inhibiting voltage-dependent calcium channels. This method is probably useful for searching neuroprotective drugs and new glaucoma therapy. Less

分离培养的RGC上存在NMDA、AMPA和红藻氨酸受体。此外，20-HE对NMDA受体介导的电流的抑制作用最显著，对AMPA和红藻氨酸介导的电流的抑制作用中等。我们集中在其中一个新的基因，ODAG（GenBank ; Accession No.AB047921），这是在P10下调。小鼠ODAG cDNA编码266个氨基酸的蛋白质。用小鼠ODAG对GenBank数据库进行BLAST分析，鉴定人ODAG cDNA和基因组结构。在P2和P7时，小鼠眼内不同组织中检测到ODAG特异性mRNA的表达，而在P14时则未检测到ODAG特异性mRNA的表达。研究了谷氨酸刺激对大鼠视网膜神经节细胞（RGCs）中一氧化氮合成酶（NOS）亚型的表达和一氧化氮（NO）的合成的影响。免疫组织化学分析显示，nNOS和eNOS在r-myc和r-myc中表达。 ...更多信息 视网膜神经节细胞在培养的RGCs细胞内的NO水平在20分钟的观察期间表现出波动。一种特异性nNOS抑制剂的存在下，显着抑制细胞内NO的增加后，谷氨酸的饲料培养基。本研究发现视网膜神经节细胞（RGCs）中存在NOS的所有组成型异构体，并证明培养的RGCs中NO主要由nNOS产生，而nNOS主要受谷氨酸的刺激，谷氨酸可引起视网膜神经节细胞24小时的死亡，且呈浓度依赖性。谷氨酸浓度依赖性地增加细胞内Ca^2+浓度（[Ca^2+] i）。NMDA和KCl引起相同的结果。倍他洛尔抑制KCl诱导的[Ca^<2+>]_i升高至25%。另一方面，倍他洛尔显著抑制谷氨酸诱导的RGC死亡。[Ca^<2+>]_i的增加被认为是谷氨酸神经毒性的第一个信号。倍他洛尔通过抑制电压依赖性钙通道发挥神经保护作用。这一方法可能有助于寻找神经保护药物和新的青光眼治疗方法。少

项目成果

Yuichi Tsumamoto: "In Situ localization of nition oxide synthase and direct evidence of NO production in rat retinal ganglion cells"Brain Research. (印刷中).

Yuichi Tsumamoto：“一氧化氮合酶的原位定位和大鼠视网膜神经节细胞中 NO 产生的直接证据”大脑研究（正在出版）。

Satoshi Mukai: "Existince of unotropir Glutamiste Receptor Subtypes in Cultined Rat Retinal Ganglion cell Sortor Method and Inhibitory Effects of 20 Hydroxyeclsonl a Neulrosteroid on the Response"Japanese Journal of Pharmacology. (印刷中).

Satoshi Mukai：“培养大鼠视网膜神经节细胞分选方法中乌诺托吡谷氨酰胺受体亚型的存在以及 20 羟基神经类固醇对反应的抑制作用”《日本药理学杂志》（出版中）。

S. Mukai, H. K. Mshima, K. Shoge, M. Shinya, K. Ishihara and M. Sasa: "Existence of Ionotropic Glutamate Receptor Subtypes in Cultured Rat Retinal Ganglion cells obtained by magnetic cell sorter method and inhibitory effects of 20-hydroxyecdysone, a neuro

S. Mukai、H. K. Mshima、K. Shoge、M. Shinya、K. Ishihara 和 M. Sasa：“通过磁性细胞分选仪方法获得的培养大鼠视网膜神经节细胞中离子型谷氨酸受体亚型的存在以及 20-羟基蜕皮激素的抑制作用，