Effects of an anti-VEGF-A monoclonal antibody on laser-induced choroidal neovascularization in mice: Optimizing methods to quantify vascular changes

PURPOSE. The purpose of this study was to evaluate different methods of detecting and quantifying experimentally induced choroidal neovascularization (CNV) and vascular changes induced on CNV by an anti-VEGF-A monoclonal antibody. METHODS. Choroidal neovascularization was induced by 532-nm diode laser in C57BL/6 mice. Ten days after the laser, the following methods were used to detect the new vessels: high-resolution angiography with fluorescein isothiocyanatedextran; immunohistochemistry with biotinylated isolectin, rabbit anti-NG2, rat anti-CD31, rabbit anti-VWF, rat ani-CD105, rabbit anti-collagen IV, rat anti-ICAM-2, rabbit anti desmin, and rat anti-MECA 32; and intravital injection of fluorescein-labeled Lycopersicon esculentum (tomato) lectin. To verify the validity of these staining methods in the quantification of treated CNV, the authors applied the most effective of these techniques to three groups of mice after laser induction of CNV and treatment with an anti-VEGF full antibody (G6-31). RESULTS. Fluorescein isothiocyanate-dextran angiography, rat anti-ICAM-2 immunostaining, and tomato lectin intravital injection resulted in the most effective means of identifying choroidal neovascularization. A certain amount of nonspecific fluorescence was detected in the area of CNV for each method. This fluorescence appeared more intense when fluorescein isothiocyanate-dextran was used. Tomato lectin injection and rat anti-ICAM-2 immunostaining were the methods that better recorded the antiangiogenic drug effect. CONCLUSIONS. Because of easy execution, low background fluorescence, and detailed visualization of new vessels, intravital injection of tomato lectin followed by a quantification based on threshold fluorescence represents the best technique for measuring CNV and the vascular changes induced by anti-VEGF-A monoclonal antibody in mice.