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Updated: Mar 1, 2026
Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression
Published on: January 12, 2024
Experimental induction of retinal ganglion cell death in adult mice
Y Li1, C L Schlamp, R W Nickells
1Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison 53792, USA.
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.
Insights
Retinal ganglion cell (RGC) death was induced in mice via optic nerve crush or NMDA excitotoxicity. This study quantifies RGC loss and characterizes apoptosis, enabling future genetic studies of RGC survival.
Area of Science:
- Neuroscience
- Ophthalmology
- Cell Biology
Background:
- Retinal ganglion cells (RGCs) undergo apoptosis during development and after injury.
- Understanding RGC apoptosis is crucial for treating vision loss.
- Previous studies were limited by animal models with difficult genetic manipulation.
Purpose of the Study:
- To characterize the rate and mechanism of RGC death in mice.
- To establish reliable methods for inducing RGC apoptosis in a genetically tractable model.
- To enable future studies on the genetic regulation of RGC survival.
Main Methods:
- RGC apoptosis was induced using optic nerve crush or intravitreal injection of N-methyl-D-aspartate (NMDA).
- RGC loss was quantified by comparing treated eyes to untreated fellow eyes.
- Apoptosis was confirmed by DNA fragmentation (TUNEL staining) and morphological changes.
Main Results:
- Optic nerve crush led to a maximum RGC loss of 57.8% within 3 weeks.
- NMDA injection caused dose-dependent RGC loss, reaching 72.5% with 160 nanomoles.
- Cell death exhibited apoptotic features, including pyknotic nuclei and TUNEL staining.
Conclusions:
- Quantitative RGC death can be reliably induced in mice.
- Two distinct signaling pathways (axonal injury and excitotoxicity) effectively trigger RGC apoptosis.
- These methods facilitate the use of transgenic mice to investigate genes controlling RGC survival.
