Quantitative Image Analysis of Laser-induced Choroidal Neovascularization in Rat

Abstract

Rodent models of laser-induced choroidal neovascularization (CNV) are now extensively used to identify angiogenic proteins, determine the role of specific genes with knockout mice, and evaluate the efficacy and safety of anti-angiogenic therapies. CNV is typically evaluated by fluorescein angiography or vascular endothelial cell labeling in histologic sections. The current study examined an alternative method using high molecular weight FITC-dextran (MW 2×106) for high resolution angiography in RPE-choroid-sclera flat mounts. At 24hr after lasering, the lesions appeared as a circular weakly fluorescent area of approximately equal diameter to the laser spot. No FITC-dextran labeled blood vessels were visible in the lesion at day 1. Three days after lasering, 47% of the lesions showed FITC-dextran labeling indicative of CNV. The incidence (71%) and extent of CNV increased by day 6, and by day 10 all lesions were vascularized, and the maximal area was attained. No significant change followed day 10, and the neovascular area remained constant through day 31. The highest rate of blood vessel growth (between 3 and 10 days after laser) correlates with the peak expression of VEGF, bFGF, and their receptors shown in previous studies. Morphologic analysis of flat mounts and histologic sections showed that the neovascular plexus in most lesions originates from deeper choroidal vessels in the center of the lesion, grows towards the neural retina, then branches circumferentially to anastamose with uninjured choriocapillaris. The microvessels in these lesions are broad and flat, similar to normal choriocapillaris. In a separate study, rats were treated daily with the angiostatic corticosteroid dexamethasone (20–500μ g kg−1day−1), and CNV was examined at day 10 in FITC-dextran labeled flat mounts and histologic sections. Dexamethasone dose-dependently inhibited CNV, and its highest dose inhibited approximately 95% of CNV labeled by FITC-dextran and resulted in lesions with no detectable Factor VIII immunostaining. High resolution angiography with FITC-dextran is reproducible and quantifiable, and it may accelerate the discovery of therapeutic agents that modulate choroidal neovascularization.