Nictitating Membrane Removal: A Surgical Procedure to Remove Nictitating Membrane from Rabbit Eye

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Removal of the nictitating membrane

NOTE: For simplicity, the technique for the right eye is described below. Complete this procedure on the left eye in an identical manner. Remove the nictitating membrane bilaterally during the acclimation period (usually the first week).

1. Place rabbit in an appropriately sized restraining bag.
2. Administer a subcutaneous injection of acepromazine (1 mg/kg) over the shoulders using a 1 cc syringe and 26 G needle to sedate the rabbit. The endpoint for this mild sedation is when the animal maintains a relaxed head position without normal scanning movements and its ears are no longer fully upright.
3. Using a micropipette, apply 25 µL of preservative-free lidocaine (1%) to the eye. Insert a wire lid speculum between the eyelids.
4. Grasp the nictitating membrane at its apex with 0.3 forceps (or equivalent) and pull it over the corneal surface. Inject 1% lidocaine with 1:100,000 epinephrine into the subconjunctival space of the nictitating membrane using a 26 G needle. Inject approximately 0.3 mL to form a modest-sized bleb over the nictitating membrane. Injection volumes over 1 mL are well within a safe dose range for rabbits (2−4 mg/kg).
5. Remove the wire speculum. Wait approximately 5 min for the lidocaine and epinephrine to take effect. During this time, perform the same procedure in the fellow eye.
6. Replace the wire lid speculum. Grasp and extend the nictitating membrane over the corneal surface using 0.3 forceps. Cut the membrane at its base with tenotomy scissors or equivalent.
   NOTE: Bleeding is usually minimal but keep a high-temperature battery cautery unit nearby and use as needed to minimize bleeding. Direct pressure over the cut base of the nictitating membrane can also be used to stop small bleeding if it occurs.
7. Remove the wire lid speculum. Place topical antibiotic ointment (neomycin, polymyxin, bacitracin, and hydrocortisone) over the corneal surface.
8. Perform an identical procedure to the fellow eye as indicated by the protocol.
9. Place animals back in individual cages and allow to heal for at least one week, or until the conjunctival surface has healed completely from a clinical standpoint, before performing any further assays or interventions.
   NOTE: Complete clinical healing is indicated by a lack of any swelling, injection or discharge from the conjunctival surfaces. Animals should keep their eyes open normally, without the presence of protective ptosis.