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Elena Ivanova, Christopher Yee, Botir T Sagdullaev; A mouse model to monitor oxidative stress in the retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3644.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress is a hallmark of retinal neurological diseases - including diabetic retinopathy, retinitis pigmentosa, and aged-related macular degeneration. To overcome oxidative stress, each cell is equipped with a redox sensitive transcription factor, NF-E2-related factor 2 (Nrf2). During stress, the function of Nrf2 is to initiate the transcription of variety of genes involved in antioxidative activity. The purpose of this study is to develop a mouse model to monitor Nrf2 activation in the retina. This model can be used to determine the role of Nrf2 in variety of retinal diseases and to develop pharmacological and/or genetic treatments.
To create a mouse model for monitoring Nrf2 activity, we expressed a luciferase reporter gene (Smirnova et al., 2011) in variety of retinal cells. The CAG-driven constructs were targeted by the rAAV vector to glial and neuronal retinal cells of wild type mice via intravitreal injections. The expression was confirmed by immunostaining with specific antibodies against luciferase. To probe the model, Nrf2 was activated pharmacologically with dimethyl fumarate (BG-12), an established Nrf2 activator. The activation of the constructs was measured in intact retinal explants using a luminometer and living animals using an IVIS imaging system.
Our data show high expression of the construct in diverse populations of retinal cells. Treatment with BG-12 of intact retinal explants for 3h followed by the addition of luciferin gives 8-9-fold activation over the baseline. Upon lysis of the intact retinal explants, and addition of luciferase assay mix, the luminescence signal is dramatically increased (10-fold) and activation over the baseline is 20-fold, as previously observed with the reporter cell line treated with the same concentration of BG-12 for 3 h (Smirnova et al., 2011). In vivo imaging showed an increase of luminescence after intraocular injections of BG-12.
A newly developed mouse model with Nrf2-luciferase expression in the retina allows high sensitivity measurements of Nrf2 activation in real-time both in vitro and in vivo.
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