An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Summary

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Abstract

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.

The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.

Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

Protocol

All equipments and reagents used are sterile. All animal experiments were approved by the Animal Care and Use Committee (ACUC) at the NEI/NIH (animal study protocol NEI-570), and were performed according to the NIH guidelines and regulations.

1. Baseline Retrograde Labeling of Retinal Ganglion Cells (RGCs) At Day 1

The purpose of this procedure is to label the retinal ganglion cells retrogradely by injecting a neural tracer dye into the superior colliculus of the mice three days before the optic nerve crush injury. The dye will be retrogradely taken up by the retinal ganglion cells and provides a marker for the li....

Discussion

The optic nerve crush injury murine model is useful to study the process of RGC death and survival. This model is also often used to investigate the effects of different reagents and genes on RGC apoptosis and survival. One advantage of this model is that it has a high degree of reproducibility with minimal variations.

However, special care is required in several steps in this model. First, when making the optic nerve crush injury, it is essential not to use too much force and not to crush for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotaxic apparatus	ASI Instruments	SAS-4100
Dissecting microscope	World Precision Instruments, Inc.	PZMIV-BS
Scalpel handle	Fine Science Tools	10003-12
Scalpel Blades #15	Fine Science Tools	10015-00
Microdrill	Fine Science Tools	18000-17
Suture needle	Fine Science Tools	12050-03
Spring scissors	Fine Science Tools	16144-13
Dumont #5 forceps	Fine Science Tools	11252-50
Hamilton syringe	Hamilton Co	88400
Gauze sponges	Office Depot	674889
Fluorochrome	Sigma-Aldrich	39286
Alcohol	Sigma-Aldrich	459844	70%
H2O2	Sigma-Aldrich	H3410	3%