Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes

Abstract

Purpose: To compare the effect of a rat anti-VEGF antibody, administered either by topical or subconjunctival (SC) routes, on a rat model of corneal transplant rejection.

Methods: Twenty-four rats underwent corneal transplantation and were randomized into four treatment groups (n=6 in each group). G1 and G2 received six SC injections (0.02 ml 10 µg/ml) of denatured (G1) or active (G2) anti-VEGF from Day 0 to Day 21 every third day. G3 and G4 were instilled three times a day with denatured (G3) or active (G4) anti-VEGF drops (10 µg/ml) from Day 0 to Day 21. Corneal mean clinical scores (MCSs) of edema (E), transparency (T), and neovessels (nv) were recorded at Days 3, 9, 15, and 21. Quantification of neovessels was performed after lectin staining of vessels on flat mounted corneas.

Results: Twenty-one days after surgery, MCSs differed significantly between G1 and G2, but not between G3 and G4, and the rejection rate was significantly reduced in rats receiving active antibodies regardless of the route of administration (G2=50%, G4=66.65% versus G1 and G3=100%; p<0.05). The mean surfaces of neovessels were significantly reduced in groups treated with active anti-VEGF (G2, G4). However, anti-VEGF therapy did not completely suppress corneal neovessels.

Conclusions: Specific rat anti-VEGF antibodies significantly reduced neovascularization and subsequent corneal graft rejection. The SC administration of the anti-VEGF antibody was more effective than topical instillation.

Figure 1

Demonstrative cases on slit-lamp examination 21 days after transplantation. Control corneas receiving either topical (A) or SC (C) denatured treatment were fully rejected with a maximum clinical score where clear grafts could be observed after topical (B) or SC (D) administration of active anti-VEGF therapy.

Figure 2

Mean clinical corneal scores observed after treatments versus matched controls. Mean clinical scores (±SEM) were higher in control animals treated with denatured SC injections (A, square line) compared to those treated with SC injections of active rat anti-VEGF (A, triangle line) for the three parameters assessed at every time point. With drop administration (B), the scores were also higher for controls (B, lozenge line), but not significantly after 21 days. The asterisk represents a p<0.05 statistical significance.

Figure 3

Flat mounted corneas stained for new vessels 21 days after allotransplantation. Allografted cornea treated with inactivated (A, B) or active (C) rat anti-VEGF antibody and flat mounted at Day 21; new vessels stained with lectin arise from the limbus toward the grafted area (A, B, C; white arrows). The progression of new vessels crossed the trephination line (A; arrow heads) across the stroma of the button (A, black arrow) between the strand of the running suture (B, black arrow), securing the corneal graft to the recipient bed. It was limited to the recipient area in treated animals (C; white arrows). Li, limbus; IF, interface between donor and recipient (A, C; arrowheads,). Scale Bar=2200 µm. Magnification, A, C 20×, B 100×.