Gene expression analysis of laser–induced CNV in mice

Abstract

Abstract: : Purpose: To determine the changes in gene expression during development and involution of laser–induced choroidal neovascularisaion (CNV) in the mouse model. Methods: CNV was induced in each eye of 30 mice (C57/BL6 strain) using 4 diode laser burns to the posterior pole. The mice were sacrificed at different time points ranging from 6 hours to 14 days after laser application. The retinal pigment epithelium (RPE) and choroid immediately around the laser spots, and in later stages including the CNV itself, were dissected. Total RNA was extracted, reverse transcribed to cDNA and amplified using MessageAmpTM (Ambion). Each sample was hybridized to a triplicate of gene arrays (Affymetrix, mouse 430A), and "treated" samples were compared to samples from untreated RPE/choroid. The hybridization signals were globally normalized and filtered and data was analysed using the Genetrix software. Real time PCR was used to confirm differential expression of selected genes. Results: Triplicate analyses of each sample were consistent among each other. Overall, more genes were significantly (> 2 fold) down– than upregulated at all time points. During the course of CNV formation and involution in this model, four sequential time–dependent stages of differential gene expression are apparent: 1) chemokine–encoding genes, 2) inflammatory cytokine–encoding genes, 3) angiogenic/anti–angiogenic genes, 4) fibrosis–promoting genes. Conclusions:Gene array analysis is a strong tool for global analysis of genes involved in the course of CNV formation and involution. In this model of laser–induced CNV–formation, prominent alterations in inflammatory gene expression are found.

Keywords: age–related macular degeneration • gene microarray • gene/expression