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M. d. Cano, M. Fujihara, S. S. Biswal, J. T. Handa; Chronic Cigarette Smoke Causes Oxidative Damage and Apoptosis to Retinal Pigmented Epithelial Cells, in Nrf2-/- Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1933.
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Age-related macular Degeneration (AMD) is the leading cause of blindness in patients 65 years or older. The retinal pigmented epithelium (RPE) is considered to be a target during the development of early disease. Oxidative stress is hypothesized to be a contributing factor to RPE dysfunction in AMD. Cigarette smoke (CS) contains potent oxidants and other damaging electrophiles, and is a major risk factor for AMD. The transcription factor Nrf2 binds to the antioxidant response element (ARE), and activates many cytoprotective enzymes. Previous studies from our laboratory have illustrated ultrastructural changes to the RPE of CS expose mice. The purpose of this study was to investigate the effects of oxidative injury through CS in the RPE of mice that are deficient in Nrf2 (Nrf2-/-) and therefore, are susceptible to oxidative and electrophilic stress.
Nrf2-/- mice were exposed to filtered air or CS 5h/day for 6 months in a smoking chamber. Eyes were fixed in 2% paraformaldehyde and examined for oxidative injury to the RPE by 8-Oxo-7,8-Dihydro-2’-Deoxyguanosine (8-OHdG) immunolabeling and apoptosis by TUNEL labeling. The contralateral eye was fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in 0.08M cacodylate buffer for transmission electron microscopy.
The RPE of Nrf2-/- CS exposed mice show a significant increase in oxidative DNA damage by 8OHdG immunolabeling (78.80%) compared to Nrf2-/- mice raised in air (18.5%; p<0.0000001). There was also increased apoptosis in Nrf2-/- mice raised in CS (9.75%) compared to Nrf2-/- mice raised in air (0%; p<0.001). We also saw increased ultrastructural damage to the RPE of Nrf2-/- mice exposed to CS including loss of basal infoldings and development of cytoplasmic vacuoles, compared to air treated mice.
CS induced marked oxidative damage, ultrastructural derangement, and apoptosis to the RPE of mice deficient in Nrf2 signaling. These findings suggest that oxidative and electrophilic stress appear to be involved at least in part, of CS induced RPE damage, and that loss of Nrf2 signaling impairs the protective response by the RPE.
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