Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Summary

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

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. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells ^5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site ^{6, 7, 13-15}. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve ^16-19 or injecting vectors into their target the superior colliculus ¹⁰. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. 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 window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

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 dial in a....

Discussion

Optic nerve transection is a highly reproducible model of adult CNS neuron apoptosis. The experimental manipulations demonstrated in this manuscript permit the study of the mechanisms of RGC apoptosis after injury.

Intraocular injections are useful for global targeting of the retina. This procedure requires some practice, as it is critical not to injure the lens with the tip of the glass pipette. Lens damage has been shown to cause the release of growth factors, altering cell survival and rege.......

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
Alcaine Eye Drops	Alcon
Tears Naturale P.M.	Alcon
Fine tip Dumont forceps	Fine Science Tools	11252-00
10 μl Hamilton Syringe (1701RN; 26s/2”/2)	Hamilton Co	80030
1/16 inch Compression Fittings	Hamilton Co	55751-01
1/16 inch OD, 0.010 inch ID, PEEK Tubing	Supelco, Sigma-Aldrich	Z226661
Dual RN Glass Coupler	Hamilton Co	55752-01
Mineral Oil Priming Kit: includes syringe, needles, rubber septa	Hamilton Co	PRMKIT