Experimental induction of retinal ganglion cell death in adult mice.
Experimental induction of retinal ganglion cell death in adult mice.
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发表时间：
1999-04
影响因子：
4.4
通讯作者：
Yan Li;C. L. Schlamp;R. Nickells
中科院分区：
文献类型：
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作者：
Yan Li;C. L. Schlamp;R. Nickells
目的视网膜神经节细胞在发育过程中以及轴突损伤和暴露于兴奋性毒素等创伤后因细胞凋亡而死亡。细胞凋亡与调节该过程的基因表达的变化有关。视网膜神经节细胞中调节细胞凋亡的基因尚未得到表征，主要是因为之前的研究仅限于基因功能不易操纵的动物模型。为了克服这一限制，通过视神经挤压和玻璃体内注射谷氨酸类似物 N-甲基-D-天冬氨酸 (NMDA) 来表征小鼠视网膜神经节细胞死亡的速度和机制。方法 为了使视网膜神经节细胞 (RGC) 暴露于兴奋毒素，成年 CB6F1 小鼠的一只眼睛玻璃体内注射 NMDA。在物理损伤视神经轴突的替代方案中，使用自闭式细镊子压碎神经。每只动物的一只眼睛都接受了一种或另一种手术。以相对于未治疗的另一只眼睛损失的细胞百分比来监测 RGC 的损失。使用原位杂交检查 Thy1 表达。使用末端转移酶-dUTP 缺口末端标记 (TUNEL) 监测垂死细胞中的 DNA 片段化。结果 根据核形态和神经节细胞标记 Thy1 mRNA 的存在，RGC 包含对照小鼠神经节细胞层 (GCL) 中 67.5% +/- 6.5%（平均值 +/- SD）的细胞。视神经挤压后一周，这些细胞开始死亡，到 3 周时 GCL 中的细胞损失最大为 57.8% +/- 8.1%。 NMDA 注射后的细胞损失呈剂量依赖性，注射 10 纳摩尔的 NMDA 对注射 160 纳摩尔 NMDA 后 6 天内 GCL 中细胞最大损失 72.5% +/- 12.1% 几乎没有影响。视神经挤压和注射NMDA后，细胞死亡表现出细胞凋亡的特征，包括固缩核的形成和TUNEL染色。结论 使用两种不同的信号通路可以在小鼠中诱导定量的 RGC 死亡，从而可以使用转基因动物测试基因在此过程中的作用。
PURPOSE Retinal ganglion cells die by apoptosis during development and after trauma such as axonal damage and exposure to excitotoxins. Apoptosis is associated with changes in the expression of genes that regulate this process. The genes that regulate apoptosis in retinal ganglion cells have not been characterized primarily because previous studies have been limited to animal models in which gene function is not easily manipulated. To overcome this limitation, the rate and mechanism of retinal ganglion cell death in mice was characterized using optic nerve crush and intravitreal injections of the glutamate analog N-methyl-D-aspartate (NMDA). METHODS To expose retinal ganglion cells (RGCs) to excitotoxins, adult CB6F1 mice were injected intravitreally in one eye with NMDA. In an alternative protocol to physically damage the axons in the optic nerve, the nerve was crushed using self-closing fine forceps. Each animal had one or the other procedure carried out on one eye. Loss of RGCs was monitored as a percentage of cells lost relative to the fellow untreated eye. Thy1 expression was examined using in situ hybridization. DNA fragmentation in dying cells was monitored using terminal transferase-dUTP nick-end labeling (TUNEL). RESULTS RGCs comprise 67.5% +/- 6.5% (mean +/- SD) of cells in the ganglion cell layer (GCL) of control mice based on nuclear morphology and the presence of mRNA for the ganglion cell marker Thy1. One week after optic nerve crush, these cells started to die, progressing to a maximum loss of 57.8% +/- 8.1% of the cells in the GCL by 3 weeks. Cell loss after NMDA injection was dose dependent, with injections of 10 nanomoles having virtually no effect to a maximum loss of 72.5% +/- 12.1% of the cells in the GCL within 6 days after injection of 160 nanomoles NMDA. Cell death exhibited features of apoptosis after both optic nerve crush and NMDA injection, including the formation of pyknotic nuclei and TUNEL staining. CONCLUSIONS Quantitative RGC death can be induced in mice using two distinct signaling pathways, making it possible to test the roles of genes in this process using transgenic animals.