Real-Time Fluorometric Quantification of VEGF-Induced Blood-Retinal Barrier Breakdown in Rats

Abstract

Purpose: : Blood-retinal barrier (BRB) breakdown is a hallmark of background diabetic retinopathy and a leading cause of vision loss. The quantification of this process, however, is challenging in animal models. In our present studies, ocular fluorometry was explored as a real-time measurement of BRB breakdown in rats injected intravitreally with vascular endothelial growth factor (VEGF). Additionally, two topically-administered VEGFR/Src kinase inhibitors (TG100948 and TG100801) were tested for their ability to reduce BRB breakdown in this model.

Methods: : Baseline studies: Animals received an intravitreal VEGF injection (200 ng/3 µl) in the right eye to induce retinal edema and a sham injection in the left eye to serve as a paired control. At 6 and 24 h post-VEGF injection (n=16/timepoint), animals received a bolus intravenous injection of 35 mg/kg disodium fluorescein and ocular fluorometry scans were performed within 15-25 min after fluorescein injection to assess BRB breakdown. Area under the curve (AUC) versus time calculations for retina/vitreous fluorescein concentrations were performed using GraphPad Prism software. Efficacy studies: Animals received topical applications of 0.03% TG100948, 0.03% TG100801, or vehicle (10 µl/eye, n=12/group) at both 6 and 22 h post-VEGF injection, followed by ocular fluorometry at 24 h post-VEGF using the same procedure as in the baseline studies.

Results: : The mean baseline AUCs in VEGF injected eyes were 1.5 and 2.4 fold of that in sham-injected contralateral eyes at 6 and 24 h post-intravitreal VEGF injection, respectively. Topical dosing with TG100948 or TG100801 reduced mean AUC by 65% or 52% at the 24 h timepoint (p<0.05) versus that in matched vehicle-treated groups, respectively.

Conclusions: : Ocular fluorometry allows for non-invasive, real-time quantification of BRB breakdown in rats. The utility of this animal model in preclinical research and the development of pharmacological therapies was demonstrated using TG100948 and TG100801, two small molecule kinase inhibitors that following topical delivery successfully reduced BRB breakdown.

Keywords: diabetic retinopathy • retinal neovascularization • retina