Five days after subconjunctival injection, corneal neovascularization was induced by alkali burn, according to the method devised by Ormerod and collegues with modification [16 (link)]. Briefly, after general anesthesia with an intraperitoneal injection of a combination of xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (35 mg/kg; both purchased from HangZhou Peak Chemical Corp., Zhejiang, China), and topical anesthesia with a drop of 0.5% proparacaine hydrochloride (Alcaine eye drops; Alcon Inc., Fort Worth, TX), filter paper (2.5 mm diameter) was immersed in 2.5 μl 0.1 M NaOH and then placed centrally on the mouse cornea for 30 s. The alkali-treated cornea was then irrigated with 60 ml of normal saline. Subsequently, corneal and limbal epithelia were scraped off with a surgical blade under a microscope. Erythromycin ophthalmic ointment was administered immediately after epithelial denudation.
The area of corneal neovascularization was quantified by photographic documentation every 3 days for a total duration of 9 days. Three mice of each group were sacrificed on days 3, 6, and 9 after corneal injury for immunohistochemistry. Corneas from ten mice of each group were procured on days 5, 8, and 14 after alkali treatment for western blot analysis. On day 9 after alkali treatment, corneas from the remaining mice were procured for real-time quantitative reverse transcription PCR (RT–PCR) analysis.
The area of corneal neovascularization was quantified by photographic documentation every 3 days for a total duration of 9 days. Three mice of each group were sacrificed on days 3, 6, and 9 after corneal injury for immunohistochemistry. Corneas from ten mice of each group were procured on days 5, 8, and 14 after alkali treatment for western blot analysis. On day 9 after alkali treatment, corneas from the remaining mice were procured for real-time quantitative reverse transcription PCR (RT–PCR) analysis.
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