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A. S. Lewin, S. Gibson, J. Liu, W. W. Hauswirth, Y.-Z. Le; Generation of a Mouse Model of Early AMD by Induction of Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6414.
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RPE oxidative stress is hypothesized to contribute to the development of age-related macular degeneration (AMD). Mitochondrial MnSOD (manganese superoxide dismutase) 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 pathogenesis of the early signs of AMD.
Two different strategies for reducing MnSOD in the RPE were pursued. First, transgenic, floxed-SOD2 mice were subjected to a subretinal injection of AAV1 vectored VMD2-Cre to stimulate recombination resulting in loss of functional MnSOD2 by deletion of exon 3. In the second approach, we bred a Tet-On RPE65Cre mice with floxed-SOD2 mice allowing induction of Cre expression, and thereby recombination, by the introduction of doxycycline to food (200 mg/kg of doxycycline). The recombinant MnSOD2 was evaluated with genomic DNA PCR using specific primer for exon3 of MnSOD2 flanking region. The expression of MnSOD2 was measured by immunohistochemical staining using RPE flat mounts and immunostaining. The level of oxidative stress was examined by using DHE (dihydroethidium), 4-HNE (4-hydroxy nonenal) and DCFDA staining and western for nitrotyrosine. The progression of disease was measured by ERG, fundus, autofluorescence and histological analysis.
In floxed-SOD2 mice, the presence of MnSOD2 was reduced as measured by immunohistochemical staining of retinal flat mounts and frozen sections, and by antibody blot of RPE tissue extracts. Staining by DHE, DCFDA, and 4-HNE revealed increased levels of oxidative stress in the RPE. The intensity of autofluorescence in the RPE and choroid using a lambda scan atexcitation wave 405 and 488 showed was increased. Fundus analysis showed disorganized vessel formation and small spots in SOD2Cre mice by 2 month old. The ERG measurement of SOD2f/f-RPE65-Cre mice showed about 30% reduction by 2 month old compared with control mice. The histological changes of retinas were examined by light and electron microscopic analysis and revealed that by 4 month old, the retinas showed shortened outer photoreceptors, thinning of outer nuclear layer, and RPE damage including subretinal deposits formation in the RPE layer.
The specific reduction of MnSOD2 in the RPE led to increased oxidative stress in the RPE and histological changes of retina similar to those seen in AMD. This approach may prove useful in developing the early mouse model of AMD.
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