Doxycycline Inhibits Corneal Angiogenesis after Keratoplasty

BACKROUND: Corneal hemangiogenesis occurs in 40%-60% patients after keratoplasty. Blood vessel is one of the high risk factors for corneal immunological rejection. To inhibit corneal hemangiogenesis would prolong the survival time of the grafts and promote the successful rate of the keratoplasty. OBJECTIVE: To explore the inhibitive effects of doxycycline on corneal angiogenesis after keratoplasty. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the State Key Laboratory of Ophthalmology (No.2006DA105054), Zhongshan Ophthalmic Center, Sun Yat-sen University from March to August 2007. MATERIALS: A total of 48 healthy clean Sprague Dawley rats served as recipients (right eye) and 24 Wistar rats as donors (both eyes). CD31-PEfluorescent antibody was obtained from Sigma, USA. Sandwich enzyme-linked immunosorbent assay (ELISA) kit for vascular endothelial growth factor (VEGF) was brought from RapidBio, USA. METHODS: Corneal allogenic transplantation models were established in rats. Recipients were equally and randomly divided into 2 groups: saline control group and doxycycline group. Twenty minutes prior to surgery, mydriasis was performed using 1% atropine, with a diameter of 2.75 mm of implant and 2.5 mm of implant bed. In the saline control group, conjunctiva of the right eye received saline, three times a day, following surgery. In the doxycycline group, conjunctiva of the right eye received 1% doxycycline, three times a day, till 30 days following surgery. MAIN OUTCOME MEASURES: The following parameters were measured: corneal angiogenesis using immunofluorescence, expression of VEGF protein by using ELISA. RESULTS: Compared with the survival time of saline control group [(9.67±2.73) days], the mean survival time of doxycycline group [(20.67±3.01) days] was significantly prolonged (P＜0.01). The mean percentages of neovascularized corneal area in the saline control group were (4.00±1.00)%, (14.33±4.04)%, (31.33±3.51)% at 3, 7 and 14 days after keratoplasty, respectively. The mean percentages of neovascularized corneal area in the doxycycline group were (1.67±1.15)%, (4.67±1.53)%, (18.33±1.53)% at the same time point respectively. Compared with the saline control group, the mean percentages of neovascularized corneal area of the doxycycline group was significantly reduced at 7 and 14 days after keratoplasty (P＜0.05). The expression of VEGF in the saline control group was (541.00±75.44) pg/mg, (960.00±90.14) pg/mg, (976.00±130.41) pg/mg at 3, 7 and 14 days after keratoplasty, respectively, while expression of VEGF in the doxycycline group was (115.33±9.29) pg/mg, (239.00±41.62) pg/mg, (361.00±65.20) pg/mg, respectively. The difference of VEGF expression at all time points between the two groups was significant (P＜0.05). CONCLUSION: Doxycycline has a significant effect in inhibiting corneal angiogenesis and prolonging survival time of implants after keratoplasty.