Modeling Early Stages of Age-Related Macular Degeneration (AMD) by Inducing Oxidative Stress in the RPE

Abstract

Purpose:: We tested the theory that oxidative damage plays a role in the development of AMD by suppressing the levels of manganese superoxide dismutase (MnSOD), an enzyme that reduces levels of reactive oxygen species (ROS).

Methods:: An active and an inactive version of a ribozyme (Rz432) that targets MnSOD mRNA were cloned in a plasmid containing the CBA promoter and a GFP gene. These constructs were packaged in AAV1 that predominantly transduces the retinal pigment epithelium (RPE) when delivered subretinally. Adult wild type mice were injected in the subretinal space in the right eye with active ribozyme, control ribozyme or GFP only. At 6 weeks after injections, we assayed for levels of MnSOD and at 4 months for markers of oxidative damage such as nitrotyrosine, carboxyethylpyrrole (CEP) and 4-hydroxy-2-nonenal (4-HNE). A separate group of mice were analyzed for levels of lipofuscin components A2E and iso-A2E using HPLC. Retinal response to light was measured by electroretinography. Light and electron microscopy were used to examine changes in retinal histology, and outer nuclear layer thickness was quantitated. TUNEL staining and a nucleosome release assays were performed to measure apoptosis.

Results:: The active Rz caused significant knockdown of MnSOD protein and increase in nitrotyrosine, CEP and 4-HNE modified proteins. Levels of A2E and isoA2E were also elevated in Rz treated eyes. Mice showed a progressive loss of a- and b-wave amplitudes in response to the active Rz that was significant by the 4 month time point. Microscopic analysis of retinas injected with active Rz showed shortening and disorganization of photoreceptor outer and inner segments, condensed chromatin and significant thinning of the outer nuclear layer by 4 months post injection. Increased apoptosis was observed only in the photoreceptor layer of retinas treated with active Rz. The active Rz also caused massive vacuolization of the RPE and deterioration of the RPE basal infoldings. Significant thickening of Bruch’s membrane was also detected by morphometry.

Conclusions:: Our results suggest that the retinal changes we observed were the consequences of RPE damage caused by an increased ROS burden. Thus oxidative damage to the RPE may contribute to early pathogenesis similar to that occurring in AMD. Additional time or other contributing factors may be needed to observe other hallmarks of AMD such as drusen and choroidal neovascularization.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • age-related macular degeneration