Abstract
Abstract: :
Purpose: Retinitis Pigmentosa (RP) comprises a genetically heterogeneous group of diseases which all demonstrate a progressive loss of photoreceptor cells. The rhodopsin knockout (Rho -/-) mouse provides a model of RP in which to investigate the detailed pattern of retinal degeneration and associated gene expression patterns. Methods: The thickness of specific retinal layers in Rho-/- and wild type 10 to 120 day-old mice was measured by confocal scanning laser microscopy. Horizontal optical slices were acquired at 2µm intervals throughout flat-mounted retinas in peripheral and central locations. Microarray analysis was employed to analyze patterns of gene expression using the NIA Mouse 15K cDNA clone set, developmental gene array supplied by the HGMP, UK. RNA was extracted from retinas of Rho-/- and C57 age matched controls at P15 before the onset of obvious pathology, and P90 when most rod photoreceptors have degenerated. Primers specific for selected genes of interest demonstrating greater than 2 fold change in expression were synthesized and used to confirm the expression changes by quantitative RT-PCR. Results: As suggested by previous studies, investigation of the Rho-/- and C57 retina indicated that degeneration of the photoreceptor layer had already begun by P30 and that by P90 few photoreceptors remained. However, preliminary data suggest that the nuclear layers in the P10 and P20 Rho-/- retinas are thicker than in the wild type C57. Microarray analysis revealed many differentially expressed genes, some more highly expressed in the wild type and some in the rho-/- retina. Of note, two metallothionein isoforms, MT-I and MT-II showed increased expression (~ 4 fold) in the rhodopsin knockout. Conversely, several mitochondrial genes showed a significant decrease (~3 fold) in retinal expression in the knockout. Due to the nature of the array many of the differentially expressed genes are uncharacterised. In all cases tested, RT-PCR confirmed the direction of change in expression. Conclusions: Detailed morphological measurement of the layers of the Rho-/- retina has revealed changes prior to overt loss of photoreceptors. Microarray analysis has enabled identification of many novel genes involved in the process of retinal degeneration.
Keywords: gene microarray • retinal degenerations: cell biology • animal model