Long–term modulation of gene expression in the mouse eye following laser photocoagulation

Abstract

Abstract: : Purpose: Laser photocoagulation of the retina has been successfully used for the treatment of diabetic retinopathy. It has been proposed that one of the beneficial effects of laser photocoagulation is the long–term modulation of the gene expression pattern in the retina. The aim of the present study was to investigate whether this pattern is altered by laser photocoagulation and to examine the longevity of those changes. Methods: Ten week old female C57BL/6J wild type mice had 20 Argon laser spots delivered to the superior quadrant of the retina at 120mW, 50µm and 0.05s under anaesthesia without breaching the Bruch’s membrane. Eyes were enucleated 3 days, 28 days and 90 days post–laser photocoagulation, the lens was removed and RNA extracted and processed for microarray analysis using the Affymetrix murine genome U74v2A gene chip. Bioinformatic analysis of the data produced lists of differentially expressed genes which were evaluated using PathwayAssist (IobionLab^TM) and EASE (http://david.niaid.nih.gov/david/) amongst others. Expression levels of selected genes were verified by real–time RT–PCR and Western Blotting. Results: The number of differentially expressed genes decreased dramatically over time (3d: 343 genes; 28d: 173 genes; 90d: 107 genes) with 16 to 21% being unknown genes. The expression of most genes was downregulated. Thrombospondin 1, the TWEAK receptor Fn14, the spindle associated protein Spin, and the telomerase binding protein p23 were consistently differentially expressed across all three timepoints. These proteins have been shown to have anti–angiogenic and anti–proliferative capabilities. In addition, the expression levels of a group of 8 crystallin genes were significantly altered at 90d post treatment and these genes have recently been shown to be expressed in retina. Conclusion: Laser photocoagulation had a profound effect on the expression levels of hundreds of genes in the retina and eye cup. This treatment specifically effects long–term changes in expression levels of genes whose protein products are associated with anti–angiogenic and anti–proliferative processes. As cell proliferation and angiogenesis are cellular events necessary in the development of neovascularization, these data provide the first evidence that laser photocoagulation may indeed act by inhibiting angiogenesis and cell proliferation.

Keywords: gene microarray • diabetic retinopathy • laser