Sung Kun Chung

The effect of methotrexate on corneal neovascularization in rabbits.

PURPOSE To determine the efficacy of topical application and subconjunctival injection of methotrexate in the treatment of corneal neovascularization using a rabbit model. METHODS Corneal neovascularization was induced with suturing in 12 rabbits (24 eyes). One week after suturing, 12 rabbits were divided into 3 groups of 4 rabbits each. One group was treated with topical methotrexate 2 mg/mL, a second group with topical methotrexate 4 mg/mL, both 4 times a day, and the third group with a subconjunctival injection of methotrexate 2 mg/mL (0.1 mL). Control rabbits received a balanced salt solution instead of methotrexate. The area of corneal neovascularization was measured using light microscopy, 1 week and 2 weeks after initiation of the treatment. The concentrations of vascular endothelial growth factor and interleukin-6 in corneal tissue were measured 2 weeks after initiation of the treatment. RESULTS The area of neovascularization was significantly reduced in the treatment groups compared with the control group (P < 0.05). No significant difference was found in the area of neovascularization between the groups treated with the 2 doses of topical methotrexate. The concentrations of vascular endothelial growth factor and interleukin-6 were significantly lower in the treatment groups compared with the control group (P < 0.05), but no difference was observed among the treatment groups. The corneal epithelium, stroma, and endothelium showed a normal appearance. CONCLUSIONS Topical and subconjunctival methotrexate application may be useful for treating corneal neovascularization, but further study regarding dosage, route of administration, and indications for methotrexate should be performed in the future.Purpose: To evaluate vitreous and plasma concentrations of vascular endothelial growth factor (VEGF) and apelin and to detect the expressions of VEGF and apelin on epiretinal fibrovascular membranes obtained during vitrectomy in eyes with proliferative diabetic retinopathy after intravitreal bevacizumab injection. Methods: Forty-four eyes of 44 patients affected by active proliferative diabetic retinopathy were investigated. The bevacizumab study group consisted of 20 eyes that received an intravitreal bevacizumab injection (1.25 mg) 7 days before pars plana vitrectomy. The control group included 24 eyes that underwent pars plana vitrectomy without previous intravitreal bevacizumab injection. Using enzyme-linked immunosorbent assay, the concentrations of VEGF and apelin were measured in vitreous and plasma samples. The expressions of VEGF and apelin in the excised epiretinal membranes were examined by fluorescence immunostaining. Results: Vitreous and plasma concentrations of VEGF were significantly lower in the bevacizumab group than in the control group (P < 0.001 and P = 0.003, respectively), while the vitreous and plasma concentrations of apelin did not vary significantly between the 2 groups (P = 0.62 and P = 0.09, respectively). In the epiretinal fibrovascular membranes of both the groups, a colocalization of the endothelial marker CD31 with the markers for apelin was observed. Conclusion: Intravitreal bevacizumab injection may lead to a decrease in the intraocular and systemic concentrations of VEGF, suggesting a local and potentially a systemic effect on VEGF but may have no effect on apelin. Apelin may be associated with the development of epiretinal membranes in proliferative diabetic retinopathy and may not directly correlate with VEGF.

The effect of subconjunctival suramin on corneal neovascularization in rabbits.

Purpose: To evaluate the effect of subconjunctival injection of suramin on corneal neovascularization in rabbits. Methods: Corneal neovascularization was induced by silk suturing of the corneal stroma in 40 eyes of 40 male New Zealand white rabbits. Five days after suture placement, all rabbits were randomly divided into 4 groups of 10 rabbits and were treated subconjunctivally with balanced salt solution 0.1 mL (group 1), suramin 0.1 mL (10 mg/mL and 100 mg/mL, groups 2 and 3, respectively), and bevacizumab 2.5 mg/0.1 mL (group 4). Digital photographs of eyes were obtained and analyzed on days 7, 14, and 28 after subconjunctival injections. In addition, vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay (ELISA) and immunohistochemical analyses were used to estimate the level of VEGF and the expression of VEGF and basic FGF in neovascularized cornea, respectively. Results: The neovascularized area in control was increased significantly for 14 days after subconjunctival injection, but slightly decreased on day 28. On days 7 and 14, group 4 exhibited greater antiangiogenic effect than group 3, but group 3 exhibited greater antiangiogenic effect than group 4 on day 28. VEGF ELISA analysis showed the mean concentration of VEGF in group 4 was significantly lower than with other treatments for the first 14 days, but the mean concentration of VEGF in group 4 was similar to that with group 3 on day 28. Immunohistochemical analysis showed that the expressions of both VEGF and basic fibroblast growth factor (FGF) were reduced in group 3 and that bevacizumab reduced VEGF expression relative to basic FGF on day 28. Conclusion: Subconjunctival suramin 100 mg/mL exhibited less antiangiogenic effect than bevacizumab 2.5 mg during the early period of treatment, but it had a longer effect than that of bevacizumab later. Therefore, the combination of subconjunctival bevacizumab and suramin may provide a more potent effect in early treatment as well as a longer antiangiogenic effect in neovascularized cornea.

Toxicity of intrastromal voriconazole injection on corneal endothelium in rabbits.

Purpose: The aim of this study was to evaluate the toxicity of intrastromally injected voriconazole on corneal endothelial cells in rabbits. Methods: In total, 32 eyes of 16 rabbits (8 eyes for each group) were divided into 4 groups according to the concentration of voriconazole [group A: 50 &mgr;g/0.1 mL (0.05%), group B: 100 &mgr;g/0.1 mL (0.1%), group C: 250 &mgr;g/0.1 mL (0.25%), or group D: 500 &mgr;g/0.1 mL (0.5%)]. Right eyes were injected intrastromally with voriconazole at concentrations of 50 &mgr;g/0.1 mL, 100 &mgr;g/0.1 mL, 250 &mgr;g/0.1 mL, or 500 &mgr;g/0.1 mL. Left eyes were injected intrastromally with isotonic saline as controls. Central corneal thickness and endothelial cell counts were measured before and at 6 hours, 1 day, and 1 week after the injection was given. Corneas were then harvested for transmission electron microscopy. Results: Only the 0.5% group did not significantly recover from corneal edema 1 week after the injection (P = 0.167, P = 0.051, P = 0.086, P = 0.001 in groups A–D, respectively). There were significant differences in endothelial cell counts for the 0.1% and 0.25% groups (P = 0.077, P = 0.019, P = 0.008 in groups A–C, respectively). Transmission electron microscopy evaluation revealed definite necrotic changes in endothelial cells at concentrations of 0.1%, 0.25%, and 0.5%, but only microstructural changes at a concentration of 0.05%. Conclusions: Our results suggest that voriconazole could be injected safely into the intrastromal layer at a concentration of 0.05% with low endothelial cell toxicity. However, injections should be administered with caution because of the risk of microstructural damage.

Effect of topical epigallocatechin gallate on corneal neovascularization in rabbits.

Purpose: The aim of this study was to evaluate the efficacy of topical application of epigallocatechin gallate (EGCG) for the treatment of corneal neovascularization in a rabbit model. Methods: Corneal neovascularization was induced in 12 rabbits by placing a black silk suture in the corneal stroma (24 eyes) for a week. After suturing, 1 randomly chosen eye of the 12 rabbits was treated with topical EGCG at 2 different concentrations: 0.01% (group 1) and 0.1% (group 2), whereas the contralateral eyes were treated with sterilized balanced salt solution as the control. All eye drops were applied for 2 weeks after suturing. The suture materials were removed from all eyes on day 7. The surface area of corneal neovascularization was measured and analyzed in all eyes on days 7 and 14. On day 14, all eyes were extracted to measure the concentrations of vascular endothelial growth factor (VEGF) messenger RNA and cyclooxygenase-2 (COX-2) protein. Results: The surface area of induced corneal neovascularization was significantly smaller only in group 2 compared with that of the control group on days 7 and 14 (P < 0.001). The change in surface area of corneal neovascularization after removal of the suture material was not significantly different between all 3 groups. VEGF messenger RNA levels were significantly lower in group 2 than in the control group (P < 0.001). The concentration of COX-2 was significantly lower in group 2 than in the control group (P = 0.043), but no significant difference was observed between group 1 and the control group. Conclusions: Topical administration of EGCG effectively inhibits corneal neovascularization in rabbits. This inhibitory effect is probably related to the suppression of VEGF and COX-2 meditated angiogenesis.