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Soojung Seo, Yun-Zheng Le, Mandy N. Conners, Kyle D. Jones, William W. Hauswirth, Alfred S. Lewin; Generation of Early AMD Mouse Model by Induction of Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5229.
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RPE oxidative stress is a major contributor to the development of age-related macular degeneration. Mitochondrial MnSOD is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We propose that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to the early signs of AMD pathogenesis.
To test our hypothesis, two different methods were pursued. First, a ribozyme targeting MnSOD was utilized to induce oxidative damage to RPE of wild type mice. We used the RPE-specific VMD2 promoter for expressing Rz432 and examined long-term phenotypes. In the second approach, we induced disruptive recombination by breeding Cre transgenic mice with floxed-SOD2 mice, and inducing RPE-specific Cre expression with doxycycline. Cre-mediated recombination was confirmed by X-gal staining of ROSA26-lacZ-VMD2-Cre mice. After subretinal injection of ribozyme or induction of Cre, we analyzed functional and morphological changes of the retina by electroretinography, fundus imaging, light and electron microscopy, and SD-OCT measurement of ONL thickness. Oxidative damage to DNA was detected by 8-OHdG staining.
Following injection AAV-SOD2 ribozyme, we measured a progressive decrease in ERG : By 6 months post injection, a-wave amplitude was reduced by 61% and b-wave was reduced by 51%. Fundus imaging revealed focal discoloration of the retina and geographic atrophy coincident with the areas of viral transduction. Fluorescein angiography indicated leakage of deep blood vessels. RPE flat mounts showed that individual RPE cells were enlarged and irregular in shape. Microscopic analysis of cross sections showed that RPE thickness was increased while the thickness of OS, IS, and ONL were decreased. Ultrastructural analysis showed thickening in the layers of Bruch’s membrane and mitochondrial fragmentation and loss of cristae. Similar to the AAV-ribozyme injected mice, induction of SOD2 deletion led to fundus abnormalities and leakage of blood vessels by 2 months following induction of Cre recombinase. ONL thickness was decreased by 7 months. Increased level of 8-OHdG indicated increased oxidative stress in the RPE.
The specific reduction of MnSOD2 in the RPE led to increased oxidative stress in the RPE and histological changes in the retina similar to those seen in AMD. We plan to use these models to test both pharmacological and gene-based treatments for AMD.
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