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V. Justilien, J. Pang, X. Qi, W.W. Hauswirth, A.S. Lewin; Induction of Oxidative Stress to Recapitulate the Early Stages of Age Related Macular Degeneration. . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5884.
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© ARVO (1962-2015); The Authors (2016-present)
Age–related macular degeneration (AMD) is a degenerative eye disease that accounts for the majority of irreversible severe visual loss in the elderly population of industrialized countries. We propose to test the theory that oxidative damage plays a role in the development of AMD by using Adeno–Associated Virus (AAV) delivered ribozymes (Rz) to the retinas of mice to knock down the levels of manganese superoxide dismutase (MnSOD) that mitigate the levels of reactive oxygen species (ROS).
An active and an inactive version of a ribozyme (Rz432) that targets MnSOD (SOD2) were cloned in a plasmid containing the CMV–beta actin promoter and a GFP marker gene. Constructs of the active and inactive Rz were packaged in AAV1 that has been shown to predominantly transduce the retinal pigment epithelium. RPE–J cells were transfected with active Rz or GFP. At 1, 2 and 4 days post transfection, RT–PCR and western blot analysis were performed to measure SOD2 mRNA and protein levels respectively. Four–week old C57BL/6 mice were injected subretinally in the right eye with active or inactive Rz. At 1, 2 and 4 months post injection, full field scotopic electroretinography was performed. Light and electron microscopy were used to examine changes in retinal histology, and outer nuclear layer thickness was quantitated. TUNEL staining was performed to measure apoptosis.
RPE–J cells treated with Rz432 showed a 32% and 60% reduction in SOD2 mRNA and protein levels, respectively, relative to cells treated with GFP. Virus treated 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. Light and electron microscopy 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. The active Rz also caused massive vacuolization of the RPE as well as thickening and disorganization of the layers of Bruch’s membrane. Increased TUNEL staining was observed only in the photoreceptor layer of retinas treated with active Rz.
Treatment of RPE–J cells with Rz432 caused significant reductions in SOD2 mRNA and protein levels. Expression of active Rz432 in mouse retina results in significant attenuation in the ERG response, substantial histological damage, and cell death. We intend to extend these results by staining for markers of oxidative damage to lipids, DNA and proteins after Rz treatment.
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