A total of 56 New Zealand Albino rabbits weighing 2.5 to 3.5 kg were used. All animal experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were anesthetized with ketamine HCl (Ketalar, 50 mg/kg; Pfizer, Sandwich, UK) injected intramuscularly in combination with the relaxing agent xylazine (0.5 mg/kg; VMD, Arendonk, Belgium). Local anesthetic drops (benoxinate HCl 0.4%; Fisher Pharmaceuticals, Tel-Aviv, Israel) were administered. Ocular mustard injury was induced as previously described.
10 Briefly, NM (mechlorethamine; Sigma-Aldrich, St. Louis, MO), at a concentration of 2% in saline, was applied to the cornea of one eye of each animal (the experimental eye) for 5 minutes within a trephine. Immediately after application, NM was quickly absorbed from within the trephine with small Weck-cell sponges (Invotec International, Inc., Jacksonville, FL), followed by washing of the eye with copious amounts of normal saline. Local treatment was immediately initiated according to the protocol described in the next section. In
control eyes (four additional animals, eight eyes), saline solution (instead of NM) was applied to the cornea for 5 minutes, also within the trephine. In
control experiments, the vehicle (saline) was used as eye drops after exposure to NM. During the experiment and follow-up, intramuscular dipyrone injections (10 mg/kg) were given to animals showing pain or distress. This was necessary mainly during the first 5 to 10 days after exposure.