Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine
Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine
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DOI：
10.1006/exer.2001.1009
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发表时间：
2001-07-01
影响因子：
3.4
通讯作者：
Smith, SB
中科院分区：
文献类型：
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作者：
Moore, P;El-Sherbeny, A;Smith, SB
同型半胱氨酸是一种兴奋性氨基酸，是半胱氨酸的同系物，通过刺激N-甲基-D-天冬氨酸（NMDA）受体诱导脑神经细胞死亡。它还选择性地激活视网膜神经节细胞的NMDA受体，但尚不清楚高水平的同型半胱氨酸是否对这些细胞有毒。本研究的目的是确定是否增加同型半胱氨酸水平导致神经节细胞层的神经元死亡：如果是这样，这种死亡是否通过凋亡机制发生，并确定同型半胱氨酸和谷氨酸（一种已知的视网膜兴奋毒素）同时升高对神经节细胞层神经元活力的影响。C57 BL/6小鼠玻璃体内注射同型半胱氨酸或谷氨酸/同型半胱氨酸组合（终浓度：25、75和200 μ M）：注射谷氨酸（25和200 μ M）作为阳性对照。在注射后5-6天收获眼睛并制备冷冻切片。注射高半胱氨酸的小鼠视网膜的系统形态测量分析表明，暴露于200 μ M高半胱氨酸后，神经节细胞层中的细胞总数减少约23%。为了确定神经节细胞层的神经元是否因细胞凋亡而死亡，使用TUNEL方法，并通过已知在视网膜神经节细胞凋亡期间以高水平表达的胱天蛋白酶-3的免疫组织化学研究来证实。显微镜分析显示，注射同型半胱氨酸的眼睛比对侧PBS注射的眼睛神经节细胞层中的TUNEL阳性细胞显著更多。注射75和200 μ M同型半胱氨酸的视网膜在神经节细胞层中显示出比注射PBS的视网膜（0.25）显著更多的TUNEL阳性神经元（分别为2和2.9）。在同时注射同型半胱氨酸/谷氨酸的眼睛中，神经节细胞层中凋亡细胞的数量几乎是单独注射同型半胱氨酸或谷氨酸的两倍。活化的caspase-3的免疫组织化学分析显示，许多阳性标记的神经元在同型半胱氨酸和同型半胱氨酸/谷氨酸注射的眼睛，但不是在PBS注射的眼睛的神经节细胞层。对这些数据的定量分析显示，注射75和200 μ M同型半胱氨酸的视网膜神经节细胞层中的caspase-3阳性神经元的数量（分别为2.9和4.4）明显多于注射PBS的视网膜（0.5）。这证实了神经节细胞层中的神经元死亡是通过细胞凋亡发生的。本研究提供了第一个证据表明，同型半胱氨酸是有毒的神经节细胞层的神经元。此外，它提供的证据表明，这些视网膜神经元死亡的细胞凋亡，它首次证明，兴奋性毒性损伤的神经节细胞层的神经元是由同时升高的同型半胱氨酸和谷氨酸增强。这些发现与糖尿病视网膜病变的视网膜神经节细胞死亡特征相关，其被认为是由NMDA受体的过度刺激介导的。(C)北京：科学出版社.
Homocysteine, an excitatory amino acid and a homolog of cysteine, induces neuronal cell death in brain via stimulation of N-methyl-D-aspartate (NMDA) receptors. It also selectively activates NMDA receptors of retinal ganglion cells, but it is not known if high levels of homocysteine are toxic to these cells. The purpose of this study was to determine whether increased levels of homocysteine caused death of neurons in the ganglion cell layer: if so whether this death occurred via an apoptotic mechanism and to determine the consequences of simultaneous elevation of homocysteine and glutamate, a known retinal excitotoxin, on the viability of neurons of the ganglion cell layer. C57BL/6 mice were injected intravitreally with either homocysteine or glutamate/homocysteine combined (final concentrations: 25, 75, and 200 muM): injection of glutamate (25 and 200 muM) served as a positive control. Eyes were harvested and cryosections prepared 5-6 days post-injection. Systematic morphometric analysis of retinas of mice injected with homocysteine indicated that the total number of cells in the ganglion cell layer decreased by about 23 % following exposure to 200 muM homocysteine. To determine whether the neurons of the ganglion cell layer were dying by apoptosis, the TUNEL method was used and was confirmed by immunohistochemical studies of caspase-3, known to be expressed at high levels during retinal ganglion cell apoptosis. Microscopic analysis revealed significantly more TUNEL-positive cells in the ganglion cell layer in homocysteine-injected eyes than in contralateral PBS-injected eyes. Retinas injected with 75 and 200 muM homocysteine displayed significantly more TUNEL-positive neurons in the ganglion cell layer (2 and 2.9, respectively) than PBS-injected retinas (0.25). In eyes injected simultaneously with homocysteine/glutamate, the number of apoptotic cells in the ganglion cell layer almost doubled that for homocysteine or glutamate injections alone. Immunohistochemical analysis of activated caspase-3 revealed numerous positively labelled neurons in the ganglion cell layer in homocysteine and homocysteine/glutamate-injected eyes, but not in PBS-injected eyes. Quantification of this data revealed a significantly greater number of caspase-3-positive neurons in the ganglion cell layer of retinas injected with 75 and 200 muM homocysteine (2.9 and 4.4, respectively) than for PBS-injected retinas (0.5). This confirms that death of neurons in the ganglion cell layer is occurring by apoptosis. The present study provides the first evidence that homocysteine is toxic to neurons of the ganglion cell layer. In addition, it provides evidence that these retinal neurons are dying by apoptosis and it demonstrates for the first time that excitotoxic damage to neurons of the ganglion cell layer is potentiated by simultaneous elevation of homocysteine and glutamate. These findings are relevant to retinal ganglion cell death characteristic of diabetic retinopathy, which is thought to be mediated by overstimulation of the NMDA receptor. (C) 2001 Academic Press.