A Surgical Approach for Optic Nerve Crush in a Rabbit Model

Summary

This paper presents detailed instructions to perform optic nerve crush in a rabbit model using standard ophthalmic surgical techniques.

Abstract

Optic nerve crush (ONC) is an experimental technique used to model and study the mechanisms of optic neuropathies and potential therapeutic interventions. ONC studies have predominantly utilized rodent models; however, these models exhibit notable anatomical and physiological differences from humans, potentially limiting translatability. Here, we describe the surgical technique to achieve ONC model in rabbits, a species with globe dimensions more similar to human eyes. The developed surgical technique utilizes ophthalmic surgical techniques used in strabismus and oculoplastic surgeries and does not require the creation of bone windows or canthotomies. The model was validated through assessments of pupillary response under different lighting conditions and visual evoked potentials (VEPs) pre- and post-ONC. The results demonstrated a significant decrease in both pupillary response and VEP amplitude in the ONC eyes, compared to untreated contralateral eyes, indicating loss of visual function after injury, as expected. This surgical technique may provide models for exploring optic nerve injuries and testing potential therapeutic strategies, with broader implications for understanding and treating ocular neurodegenerative diseases.

Introduction

The optic nerve crush (ONC) technique is an experimental approach for generating controlled optic nerve injury models required for investigating optic neuropathies^1,2,3. By inducing localized damage to the optic nerve, leading to axonal injury and retinal ganglion cell degeneration, the ONC model simulates some of the pathological characteristics of various optic neuropathies, such as traumatic optic neuropathy and glaucoma^4,5,6,7. This controlled model then enables a detailed exploration of the mechanisms underlying nerve damage, degeneration, and potential regeneration within ophthalmology and neuroscience^{8,9,10,11,12,13,14}.

The application of the ONC technique and associated research has been extensively applied using rodent models, with their advantages in terms of size, ease of handling, and low maintenance costs. Additionally, rodents offer a wide array of genetic manipulation tools, which are essential for gene therapy and molecular interventions^12,14,15. Despite these benefits, the significant anatomical and physiological differences between rodents and humans have potential limitations in translatability. Treatment techniques developed in rodents may not be translated to humans due to the large discrepancy in eye size (rat axial length 6.3 mm¹⁶ compared with 22.5 mm in humans). Rabbit eyes more accurately mimic human eye size (rabbit axial length 15.3 mm^17,18), thereby providing a better model for the development of surgical techniques and pharmacokinetic studies than rodents. Rabbits have been used in ONC models, but current reports do not adequately describe the surgical technique, and/or use canthotomy or bone windows^{19,20,21,22,23,24,25}. There are anatomical differences between rabbits and human eyes, namely the presence of myelinated nerve, relatively avascular retina, and lack of collagenous lamina cribrosa, that limit full translatability; nevertheless, the rabbit may still serve as a stepping-stone for eventual human translation.

While ONC techniques have been reported in larger animal models such as pigs^4,6, goats^5,9, and non-human primates^5,26, the complexity of the procedure and/or the cost of these models limit their applicability. Thus, the rabbit offers a middle ground that is both economical and has a large enough eye.

Addressing this need, our study introduces a surgical ONC technique refined for rabbit models. By applying the ONC technique in rabbits, we aim to bridge the current gap in animal models, enhance our understanding of optic nerve injuries and neurodegenerative diseases, and develop novel therapeutic strategies.

Protocol

In this research, New Zealand white rabbits (4.4 - 4.8 kg) were utilized. The design of animal experiments adhered to the ARVO guidelines for the use of animals in ophthalmic and vision research. The Stanford University Institutional Animal Care and Use Committee reviewed and granted approval for these experiments (protocol number: APLAC-33781).

1. ONC

1. Anesthetize the rabbit (n = 4) following the Veterinary Service Center (VSC) guidelines to ensure the rabbit is adequately sedated and pain-free throughout the procedure. Briefly, administer ketamine (30 mg/kg) and xylazine (3 mg/kg) subcutaneously.
   NOTE: Surgical plane of anesthesia is confirmed by assessing multiple anesthetic depth parameters, including toe pinch and jaw tone.
2. At the start of the procedure, administer buprenorphine-extended release (0.15 mg/kg) subcutaneously for analgesia and maropitant citrate (2 mg/kg) for smoother recovery. Monitor the animal's vital signs and anesthetic depth every 5-15 min to ensure health.
3. Provide a heat source such as an electric heating pad or circulating water blanket, and adjust settings based on body temperature.
4. Provide 1-4% isoflurane or 1-6% sevoflurane by face mask to maintain anesthesia as needed.
5. Administer a combination of proparacaine (0.5%), phenylephrine (2.5%), and tropicamide (1%) eye drops approximately 5-15 min prior to the procedure.
   NOTE: The eye drops provide local anesthesia and pupil dilation²⁷.
6. Position the rabbit on its side with the right eye exposed (Figure 1). Apply 5% betadine solution to the eye, lashes, and periocular skin for sterilization using standard aseptic technique.
7. Administer proparacaine eye drops once more for additional local anesthesia.
8. To maintain sterility, drape the animal with cling wrap²⁸, leaving a small eye-sized opening over the right eye (Figure 1).
   1. Make sure to handle only the top and bottom edges of each cling wrap sheet, as they are adhered to the underside of the table and do not interfere with sterility.
   2. Wear sterile gloves and ensure that all autoclaved surgical instruments remain within the sterile field during the procedure. Use a new set of instruments for each animal.
9. Place a partial-thickness corneal traction suture (8-0) through the superior peripheral cornea near the limbus to gently infraduct the eye, facilitating better access to the optic nerve, which is located superiorly in a rabbit (Figure 2A).
10. Perform a conjunctival peritomy (i.e., an incision of the conjunctiva around the cornea)²⁹ from 10 to 2 o'clock using Westcott scissors and 0.12 mm forceps.
11. Carefully blunt dissect the Tenon's capsule using Wescott scissors to expose the underlying superior rectus and superior oblique muscles.
12. Use a muscle hook to isolate the superior muscles (Figure 2B).
13. Secure the hooked muscles with a double-armed suture (8-0) using standard strabismus techniques³⁰ (Figure 2C). Briefly, pass the suture full-thickness through the center of the superior muscles and tie it using a surgeon's knot. Then, pass the needle partial thickness through the length of the muscle from the center to the muscle edge. Load the needle backhand and pass it full-thickness from the underside of the muscle forward. Pass the needle holder through the loop to grasp the needle.
    NOTE: Pulling the needle tightens and locks that half of the muscle. The second needle is then used to secure the remaining half of the muscle in a mirror image of the first half.
14. Use Wescott scissors to detach the muscles from the globe, making sure not to cut the sutures placed on the muscles.
15. Gently infraduct the globe with the corneal suture and expose the intraconal orbital space by retracting the muscle suture superiorly (Figure 2D).
16. Gently blunt dissect the surrounding orbital tissue to fully expose the optic nerve.
    1. Do not cut any tissue in the orbit and only use blunt dissection. Do not damage the vortex vein (Figure 2D).
    2. Use a muscle hook, if needed, to locate and isolate the optic nerve (Figure 2E).
       NOTE: The optic nerve of the rabbit is slightly superior and surrounded by retro-orbital tissues, including white fat. The nerve is white, about 1 mm in diameter. The long posterior ciliary arteries run along the 3 and 9 o'clock meridians inferior to the optic nerve head and may serve as a guide to localize the nerve³¹.
17. Clamp the optic nerve using a needle holder for 60 s to induce crush injury, after which a visible mark will appear (Figure 2F,G). The crush site is about 3 mm posterior to the optic nerve head to avoid damaging the ophthalmic artery. Use a locking needle holder to ensure consistent force is applied across all ONC treatments, resulting in comparable optic nerve injuries among rabbits.
18. Reapproximate the superior muscles back to their original location on the globe (Figure 2H). Pass the two needles (previously used to secure the superior rectus and superior oblique muscles) partial-thickness through the original muscle insertion. Tighten the suture and place a surgeon's knot³⁰.
19. Complete the procedure by suturing the conjunctiva to the limbus with interrupted sutures (Figure 2I).
20. At the end of the procedure, administer warmed crystalloid fluids (10 mL/kg) and meloxicam (0.5 mg/kg) subcutaneously for analgesia. Recover the animal by turning off the gas anesthesia and administering atipamezole (0.3 mg/kg) intramuscularly to reverse the effects of xylazine. Administer buprenorphine-extended release (0.15 mg/kg) subcutaneously during recovery.
21. Administer meloxicam (0.5 mg/kg) subcutaneously once daily during the 2-3 day postoperative period and more as needed. Provide supportive care, including extra enrichment devices (i.e., toys) and dietary enrichment, during the 2-3 day postoperative period and as needed.
22. Administer additional subcutaneous fluids in the postoperative period as needed. Monitor the animal twice daily for the first three postoperative days, then weekly or as needed thereafter.

2. Pupillary response

NOTE: To assess visual function, test the pupillary response from both eyes pre- (days -3, -2, and -1) and post-ONC (days +1, +2, and +3) using the procedures described below.

1. Using a smartphone camera, photograph both right and left eyes under low-light (mesopic) conditions.
2. Shine a penlight directly into the right eye from a distance of about 6 inches. Photograph the eye and then repeat the same procedure for the left eye.
   NOTE: Pupil tone should be stable within 5-10 s.
3. Using Image J, find the ratio of pupil diameter relative to white-to-white diameter from the captured images.

3. Flash visual evoked potentials (VEP)

NOTE: To evaluate the integrity of the visual pathway, from the retina through the optic nerve to the visual cortex, record VEP waveforms from both eyes pre- (day -2) and post-ONC (days +7 and +14) under dark conditions, using the procedures described below.

1. Anesthetize the rabbit following the VSC guidelines. Briefly, administer ketamine (15 mg/kg), dexmedetomidine (0.05 mg/kg), and midazolam (0.3 mg/kg) intramuscularly.
   NOTE: Gas anesthesia may diminish the VEP signal and should, therefore, be avoided, if possible.
2. Administer a combination of proparacaine (0.5%), phenylephrine (2.5%), and tropicamide (1%) eye drops.
3. Insert three subdermal needle electrodes at specific locations as shown in Figure 3A: between the medial canthi (reference electrode with a black cable), between the scapulae (ground electrode with a green cable), and at the external occipital protuberance (recording electrode with a red cable)⁶.
4. Place an eyelid retractor on the left eye and cover the right eye to block any light exposure.
5. Record the VEP waveforms using a handheld device (Figure 3B,C) as a light stimulus is flashed (8.0 cd*s/m², 0.99 Hz).
   NOTE: Prior to recording, ensure the noise level is < 20 µV.
6. Repeat this recording process at least three times.
   NOTE: Triplicate measurements are averaged and treated as a single data point.
7. Follow the same procedure for the right eye, ensuring that the left eye is now covered to prevent light exposure.
8. From the collected VEP waveforms, determine the P1 latency and amplitude. P1 latency is defined as the time of the first positive peak (P1) following the initial negative peak (N1), typically occurring around 60 ms. P1 amplitude is calculated as the difference between the amplitudes at P1 and N1.

4. Axon labeling and imaging

1. At one week post-ONC, inject the anterograde axon tracer cholera toxin B (CTB; conjugated with Alexa Fluor 488) intravitreally into both eyes (50 µL of 1 µg/µL CTB in PBS per eye; sterile filtered) using a 29 G insulin syringe.
2. At two weeks post-ONC, euthanize the rabbits with intravenous pentobarbital sodium (100 mg/kg) and phenytoin sodium (12.8 mg/kg), and confirm death by cardiac auscultation.
3. Enucleate the eyeballs, remove residual tissues using Westcott scissors, and fix the globes in 4% paraformaldehyde (in PBS) for 24 h at 4 °C.
4. After washing the eyeballs with PBS for 4 h, dissect the fixed optic nerves and cryosection them longitudinally.
5. Wash the sections with PBS 3 times for 5 min each.
6. Incubate the section in PBS containing 4',6-diamidino-2-phenylindole (DAPI; 1:1,000) for 5 min.
7. Wash the sections with PBS, 3 times for 5 min each.
8. Mount the sections in mounting medium and image them using a confocal microscope.

Results

To assess visual function, the pupillary response was quantified by finding the ratio of the pupil diameter (solid yellow line in Figure 4A) relative to the white-to-white diameter (dashed yellow line in Figure 4A) under mesopic and flash lighting conditions, as shown in Figure 4. Prior to the ONC procedure, substantial constriction of the pupil was observed in response to light. However, 1-3 days after ONC, the pup...

Discussion

This study introduces a detailed protocol for performing the ONC procedure in rabbits, emphasizing a less invasive approach for accessing the optic nerve. Previous reports lacked comprehensive descriptions, visual guidance, or relied on more invasive techniques, requiring implants, lateral canthotomy, and/or bone resection^{19,20,21,22,23,}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8-0 Coated Vicryl Violet Suture TG140-8 Double Armed	Ethicon	J547G	Used for optic nerve crush procedure
Antisedan (atipamezole hydrochloride 5.0 mg/mL solution)	Zoetis	NDC:54771-6293	Used for operative application
Bonn Tying Forceps Angled Tip 0.2mm, Titanium	Titan Medical	TMF502	Used for optic nerve crush procedure
Castroviejo Needle Holder Curved, Titanium	Titan Medical	TMH105	Used for optic nerve crush procedure
Castroviejo Suturing Forceps 1x2 0.12mm Straight, Titanium	Titan Medical	TMF411	Used for optic nerve crush procedure
Cerenia (maropitant citrate 10 mg/mL solution)	Zoetis	NDC:54771-8179	Used for operative application
Disposable Stainless Steel Subdermal Needle Electrodes	Natus	019-476600	Used for visual evoked potential testing
Euthasol (390 mg/mL pentobarbital sodium and 50 mg/mL phenytoin sodium)	Virbac	NDC: 51311-050	Used for euthanasia
Graefe Muscle Hook, Titanium	Titan Medical	TME133	Used for optic nerve crush procedure
Insulin Syringe 1/2 mL, 29 G, 1/2"	Monoject	8881600350	Used for intravitreal injection
Jameson Muscle Hook	Titan Medical	TME122	Used for optic nerve crush procedure
McPherson Tying Forceps Straight Tip 0.2mm, Titanium	Titan Medical	TMF501	Used for optic nerve crush procedure
Normosol-R (isotonic solution of balanced electrolytes; crystalloid fluid)	ICU Medical Inc.	NDC:0990-7670	Used for operative application
Ofloxacin Ophthalmic Solution 0.3%	Bausch & Lomb	60505-0560-0	Used for post-operative application
Olympus FLUOVIEW FV3000	Olympus	FLUOVIEW FV3000	Confocal laser scanning microscope
Phenylephrine HCl 2.5% Ophthalmic Solution	Bausch & Lomb	82260-102-10	Used for pre-operative application
ProLong Gold Antifade Mountant	Invitrogen	P36934	Mounting medium
Proparacaine HCl 0.5% Ophthalmic Solution	Bausch & Lomb	24208-730-06	Used for pre-operative application
RETeval Visual Electrodiagnostic Device	LKC Technologies, Inc.	RETeval	Used for visual evoked potential testing
Tropicamide 1.0% Ophthalmic Solution	Bausch & Lomb	24208-585-64	Used for pre-operative application
Westcott Scissors Curved	Titan Medical	TMS401	Used for optic nerve crush procedure