New Approach to Study Choroidal Neovascularization: Correlation of Angiofluoresceinogram and Heparan Sufate Proteoglycans Expression

Abstract

Purpose: : Quantitative assessment of retinal and choroidal morphology is a critical step to study choroidal neovascularization (CNV) in animal model. Therefore, the aim of this study was to describe the use of Heidelberg Retina Angiograph-2 (HRA-2) to evaluate the achievement and behavior of CNV lesion induced by laser in a rat model, and correlate the area of CNV lesion with the expression of molecules involved in angiogenesis as heparan sulfate proteoglycans (HSPG).

Methods: : Four CNV lesions were induced by laser photocoagulation around the optic disc in heterozygous pigmented Zucker rats. The lesions were created using argon laser with a power of 300 mW, spot size of 100 µM and duration of 100 ms. After three weeks, fluorescein angiogram (FA) and autofluorescence exams were performed using the HRA-2 device. The dimensions of laser-induced CNV were measured independently by different retinal specialists. After FA, the eyes were enucleated and the retina mRNA were purified. The mRNA encoding the core protein of heparan sulfate proteoglycans (syndecan 4 and perlecam), VEGF and β-actin were analyzed by quantitative real time PCR. The gene expressions were related to the size of CNV lesion.

Results: : Using HRA-2 we were able to identify and measure laser induced CNV lesions in rats. The measurement of the major and the minor linear dimensions as well as the area, reported by different examiners were statistically similar, considering inter examiners reproducibility. Autofluorescence exams demonstrated hyperautofluorescent points close to CNV complex in 50% of lesions. Real time PCR analysis showed an important increase in expression of perlecan (8 times) and a slight increase in Syn4 (1.4 times) in CNV lesions. The increase in the expression of HSPGs was directly proportional to the area of the CNV lesion. VEGF is over-expressed in the greatest CNV lesions (1.5 times).

Keywords: choroid: neovascularization • proteoglycans/glycosaminoglycans • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)