A Surgical Approach for Optic Nerve Crush in a Rabbit Model

Hello, and welcome. In this video, we'll demonstrate a protocol for performing optic nerve crush, or ONC, in a rabbit model using standard ophthalmic surgical techniques. This experimental technique generates controlled optic nerve injury models essential for studying optic neuropathies and exploring therapeutic interventions. By applying ONC in rabbits, we aim to improve our understanding of optic nerve injuries in neurodegenerative diseases, and develop novel therapeutic strategies. Let's proceed to the detailed protocol section. First, place a partial thickness corneal suture through the superior peripheral cornea near the limbus. Perform a superior conjunctival from 10 to two o'clock using Westcott scissors and .12 millimeter forceps. Carefully dissect Tenon's capsule to expose the underlying superior rectus and superior optic muscles. Use a muscle hook to isolate the superior rectus muscle. Now, the double-armed Vicryl suture will be passed full thickness through the center of the superior rectus. This is tied with a surgeon's knot. The needle is passed partial thickness through the length of the muscle, from the center to the muscle edge. The needle is loaded backend, and then passed full thickness from the underside of the muscle, forward. The needle holder is passed through the loop, and the needle is grasped. When the needle is pulled, the suture will lock that half of the muscle. The second needle is used to secure the other half of the muscle in a similar fashion. Use Westcott scissors to detach the superior rectus muscle from the globe, making sure not to cut the sutures. Gently infraduct the globe with a corneal suture and retract the muscle suture superiorly. Gently dissect the surrounding orbital tissue to fully expose the optic nerve. Avoid cutting through tissue, and only use blunt dissection. Caution should be taken to avoid damaging the vortex vein. A muscle hook may be used to isolate the optic nerve. The optic nerve of the rabbit is slightly superior and surrounded by retro-orbital fats. The nerve is white and about one millimeter in diameter. Clamp the optic nerve using a needle holder for 60 seconds to induce crush injury. The crush site is about three millimeters posterior to the optic nerve head to avoid damaging the ophthalmic artery. Now, reapproximate the superior rectus muscle back to its original location on the globe. Pass the two needles, which were previously used, to secure the superior rectus muscle partially through the original muscle insertion. Tighten the suture and place a surgeon's knot. Complete the procedure by suturing the conjunctiva to the limbus with three neuroptive sutures. During recovery and the postop period, provide analgesia and supportive care to the animal. To assess visual function, we measure the ratio of pupil diameter to white-to-white diameter under mesopic and flash lighting conditions. Before ONC, the pupil constricted significantly in response to light. As early as day one after ONC, the pupil diameter showed little to no change, indicating significant loss of visual function. We also recorded flash visual evoked potentials, VEPs, from both the optic nerve crush and untreated contralateral eyes. Flash VEP measurements reflect the response from the retina, through the optic nerve, to the brain. While P1 latency remained consistent, P1 amplitude decreased significantly in the ONC eyes, indicating significant loss of visual function. To assess axon loss after the ONC procedure, we used fluorescence imaging of longitudinal optic nerve sections. In the untreated group, the anterograde axon tracer CTB showed continuous labeling throughout the optic nerve. However, in the crushed group, axon labeling stopped at the crush site, indicating nerve injury. The fluorescence intensity profiles quantitatively confirmed this, showing a significant drop in average CTB-labeled axon intensity distal to the crush site. In conclusion, we presented a step-by-step guide for the ONC procedure in a rabbit model, ensuring reliable replication. We observed no complications. The effectiveness of the ONC technique in creating optic nerve injury and visual impairment was validated through pupillary response and VEP evaluations. This rabbit model offers a cost-effective alternative to larger animal models, with closer anatomical and physiological resemblance to humans. Thank you for watching.