Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Summary

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Abstract

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS ^1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs (siRNAs), plasmids, or viral vectors to the cut end of the optic nerve ^5-7 or injecting vectors into their target, the superior colliculus ⁸. This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy ^9-11. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for experimental manipulations directed against pathways involved in apoptosis.

Protocol

1. Surgical Technique

1. Experiments should be carried out using aseptic technique and following the animal use protocols of your specific institution. Instruments and materials (solutions, test substances, tracers, needles, etc.) coming into contact with living tissue must be sterile to prevent infection and adverse impacts on animal welfare and potential negative impacts on the study.

2. Anesthesia

1. Rats will be anaesthetized using a veterinary isoflurane vaporizer system. Use medical grade oxygen at a rate of 0.8 L/min to vaporize the isoflurane gas. Place the animal in the attached anesthesia box and....

Discussion

There are many variations of this surgical procedure and several of the steps in this protocol are not necessary. It is only necessary to retract the muscles that overlie the optic nerve in order to gain access to the nerve. However, this results in a very limited working space around the nerve making the critical final stages of transection more difficult. In certain situations it is desirable to transfect the cells from the optic nerve stump and the increased access space afforded by retracting all of the extraocular m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotaxic Frame	Stoelting Co.
Rat Gas Mask	Stoelting Co.
Anesthesia System	VetEquip	901806
Isoflurane (PrAErrane)	Baxter Internationl Inc.	DIN 02225875
Surgical Microscope	WPI, Zeiss, Leica
Fluorogold -(Hydroxystilbamidine bis(methanesulfonate)	Sigma-Aldrich	39286
Gelfoam	Pharmacia Corporation (Pfizer)
Tears Naturale P.M.	Alcon
Proviodine	Medline Industries	MDS093945H
Vannas spring scissors	Fine Science Tools	15000-00
Fine tip Dumont forceps	Fine Science Tools	11252-00
Micro surgical hook	Fine Science Tools	10062-12
Eye dressing serrated forceps	Fine Science Tools	11152-10
Dumont #7b sharp curved serrated forceps	Fine Science Tools	11270-20
Cauterizer	Fine Science Tools	18010-00