Rabbit Conjunctival Flap Model

Thirty New Zealand albino rabbits, weighting 2.2-2.5 kg each, of either sex were housed and fed separately and were randomly divided into 5 groups (3^rd, 7^th, 14^th, 21^st, and 28^th day group which correspond to the day the rabbits' eyes would be harvest after the surgery), with 6 animals in each group. For each rabbit, one eye was used as the control eye while the other eye defined as the experimental eye. After anesthetized rabbit with phenobarbital (30 mg/kg, IV), a rabbit conjunctival flap model was created in both eyes through the incision of the conjunctiva between 10 o'clock and 2 o'clock in the upper limbus of the cornea with the fornix as the base, as previously described[28]. The scleral tissue under the conjunctiva flap was exposed thoroughly first and then pressed with a cotton swab to stop bleeding. Four to six stitches with 8-0 nylon threads were applied to suture the wound and restore the consistency of the eye. A 500 µL solution of 1% sterilized olmesartan (olmesartan acid-active metabolite, later referred to as olmesartan) in PBS was injected to subconjunctival (at 12 o' clock to the base of the conjunctiva flap) to the experimental eye, while only PBS was injected to subconjunctival to the other eye (control) in exactly the same location by the end of the surgery. Topical antibiotic eye drops were applied to both eyes and then during the following 3d. The same subconjunctival injection of olmesartan and PBS was repeated on the 5^th postoperative day. On the 7^th day postoperatively, the stitches were removed under topical anesthetic eye drops. On the 3^rd, 7^th, 14^th, 21^st and 28^th days after surgery, all 6 rabbits from each group were firstly observed under slit-lamp for anterior segment of eyes, fundoscopy for retina, and then euthanized, and the eyes were enucleated and washed with icy PBS. All of the animal procedures complied with the ARVO Statement and were approved by our Institutional Review Board.