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.
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.
1. Surgical Technique
2. Anesthesia
3. Syringe Preparation for Intraocular Injections
4. Intraocular Injection Procedure: Targeting the Retina Globally
5. Selectively Targeting Retinal Ganglion Cells from the Optic Nerve Stump
6. Representative Results:
Intraocular injections target all cells in the retina globally, and work well for the delivery substances to injured RGCs since ganglion cells are located in the innermost neuronal layer of the retina, adjacent to the vitreous chamber. RGCs can also be directly targeted by delivering substances to the transected optic nerve stump. Fluorogold retrogradely labeled RGCs can be targeted using peptides, drugs, vectors, plasmids, or siRNAs from the nerve stump as illustrated in Figure 1. Figure 1A-C demonstrates the localization of a Cy3 labeled peptide in the somata of RGCs that were pre-labeled by injecting Fluorogold into the superior colliculus. Cy3 labeled peptides were injected into the optic nerve immediately after axotomy, and the retina was imaged alive in order to prevent the peptides from leaching out of the tissue during fixation. Figure 1D-F demonstrates the localization of Cy3 labeled siRNAs and the retrograde tracer Fluorogold in axotomized RGCs. Cy3 labeled siRNAs were injected into the optic nerve stump immediately after axotomy, and the retina was imaged alive.
Figure 1. Epifluorescence micrographs of RGCs in flatmounted retinas at 1 day after axotomy and injection of either labeled peptides or siRNAs into the optic nerve stump. (A) Fluorogold retrograde labeling in axotomized RGCs at 1 day postaxotomy (B) Cy3 fluorescence in RGCs following retrograde transport of labeled peptides, 1 day after axotomy and peptide injection into the optic nerve. (C) Overlay of (A) and (B) demonstrating the selective localization of Cy3 labeled peptides in Fluorogold labeled RGCs. (D-F) The cell bodies of Fluorogold labeled RGCs (D) are also filled with Cy3 labeled siRNAs (E) injected into the optic nerve stump 24 hours earlier, as shown in the overlay (F). Scale bar in A-C is 50 μm. Scale bar in D-F is 20 μm.
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 regeneration 20, 21. It is also important to carefully insert and withdraw the glass pipette parallel to the direction of the tip. Any lateral force on the tip of the glass pipette can cause a glass fragment to enter the vitreous chamber damaging the lens or retina. Using a pipette with a tip that is too fine may not permit the delivery of viscous solutions. Furthermore, an extremely fine tip does not give tactile feedback when the sclera is punctured increasing the chance of accidentally puncturing the lens. The lens should remain clear and free from any puncture marks when observed under the microscope. If the lens is damaged, a cataract will often form and the lens will cloud over and these experimental results should be excluded.
The syringe system works best when there are no air bubbles present along the tract. Air can expand and compress decreasing the responsiveness of solution withdrawal or delivery. If air bubbles are visible, flush them out with the priming syringe and mineral oil. The Dual RN glass adapter with compression fittings makes changing the pipette efficient between different animals, treatments, or in the case of a breakage. This system is robust and will last many years before the ferrules need to be replaced, as long as pipettes are carefully inserted.
Directly targeting RGCs by injecting the optic nerve is a fairly quick procedure with a few caveats. If the optic nerve stump is too short, it makes the injections difficult. A short stump also increases the chance that the needle will damage the retinal vessels near the optic nerve head as it is inserted to the level of the bevel. Thus, an optic nerve stump of approximately 2 mm in length is desirable when performing nerve injections with this technique. The injections work best when the bevel of the syringe is completely enclosed within the optic nerve. Care must be taken not to pass the needle tip through the side of the nerve as this creates a region of low resistance, allowing fluid to exit the injection site thereby decreasing the effectiveness of the injection.
When working with potential biohazards such as viral particles or transformed cells, it is important to follow institutional guidelines and safety procedures.
The authors have nothing to disclose.
PDK is supported by a CIHR operating grant (MOP 86523)
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Stereotaxic Frame | Stoelting, Kopf, WPI | |||
Rat Gas Mask | Stoelting, Kopf, WPI | |||
Anesthesia System | VetEquip | 901806 | ||
Isoflurane (PrAErrane) | Baxter Corp | 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 Syringe Co. | 80030 | ||
1/16 inch Compression Fittings | Hamilton Syringe Co. | 55751-01 | ||
1/16 inch OD, 0.010 inch ID, PEEK Tubing | Supelco, Bellefonte, PA | Z226661 | ||
Dual RN Glass Coupler | Hamilton Syringe Co. | 55752-01 | ||
Mineral Oil Priming Kit: includes syringe, needles, rubber septa | Hamilton Syringe Co. | PRMKIT |