Transconjunctival Approach for Injection into the Rat Optic Nerve

This research aims to develop more effective methods for delivering therapy to the optic nerve in patients with neurofibromatosis type 1. Our protocol focuses on exploring better techniques for accessing the rat optic nerve for experimental study. This protocol offers the advantage of being minimally invasive, eliminating the need for cutaneous incision.

[Demonstrator] To begin, grasp the conjunctiva in the inferotemporal quadrant using toothed forceps. Use Westcott scissors to create a radial conjunctival incision, and then open tendons fascia in the inferonasal quadrant. Use blunt dissection to remove overlying orbital tissue. Rotate the eye nasally using toothed forceps. Have the assistant insert a spinal cord hook into the inferotemporal quadrant to isolate the optic nerve. Using blunt dissection with forceps or a cotton tipped applicator, remove the remaining orbital tissue and tendons fascia to expose the optic nerve and sheath. Using a 33 gauge needle connected to either a 10-microliter or a 1-milliliter syringe, pierce the optic nerve sheath. Inject varying concentrations of adeno-associated virus EGFP or India ink dye into the optic nerve under direct visualization to ensure that the material is injected directly into the nerve. Re-approximate the conjunctiva to the limbus to cover the incision site. This figure illustrates the outcomes of successful and unsuccessful adeno-associated virus EGFP delivery into the rat optic nerve using a transconjunctival injection technique. The successful optic nerve injection resulted in a widespread green fluorescent signal within the nerve tissue, indicating effective penetration of the adeno-associated virus EGFP vector. The unsuccessful injection showed a green signal only along the outer edge of the optic nerve, suggesting the material leaked and did not enter the nerve tissue.