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.