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Riikka Hess, Mulugu V. Brahmajothi, Peter Saloupis, Priyatham S Mettu, Scott W Cousins; Repetitive periocular injection of hydroquinone promotes mitochondrial dysfunction in retinal pigment epithelium of mouse eyes. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1277.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that mouse eyes exposed to repeated periocular injection of the environmental toxicant hydroquinone (HQ) develop cytosolic actin aggregates at the retinal pigment epithelium (RPE) and incipient subRPE deposits. We hypothesize that HQ induces RPE mitochondrial dysfunction, which leads to development of this persistent injury phenotype. In this study, we sought to provide evidence that HQ injury in vivo was related to mitochondria dysfunction by measuring increased flavoprotein autofluorescnce (AF), an indicator of electron transport chain complex II dysfunction.
Two-month old C57BL/6J mice underwent bilateral periocular (subconjunctival) injections every three days with vehicle (PBS), 25 mM HQ, or 75 mM HQ (total five injections per group). At 14 days, animals were euthanized and saline-perfused. Eyes were harvested, and posterior eyecups were dissected with removal of the neurosensory retina. Freshly isolated RPE flatmounts were imaged by confocal microscopy with 488 nm excitation laser and barrier filter settings for oxidized flavoprotein AF. AF intensity was quantified for statistical analysis. In addition, flatmounts were stained with TRITC-phalloidin to evaluate aggregate formation. Preliminary studies correlated aggregate formation and mitochondrial function with pre-treatment with the mitochondrial targeted ubiquinone analog, idebenone.
PBS-exposed mice demonstrated expected low levels of endogenous RPE AF (Fig. 1, left panel). Periocular HQ resulted in increased RPE AF, with dose-dependent increase in RPE AF after repetitive exposure (Fig. 1 middle and right panels). As compared to control, AF intensity was significantly higher in mice injected with 25 mM HQ (p <0.001) and 75 mM HQ (p<0.0001) (Fig. 2). Confocal imaging suggested that AF signal localized to cellular mitochondria, consistent with flavoprotein source. Preliminary data indicates that cyctosolic actin aggregates induced by periocular HQ are diminished by mitochondrial-targeting idebenone.
Repetitive sublethal periocular exposure to HQ increases RPE flavoprotein AF in vivo, indication electron transport chain dysfunction. Ongoing studies will correlate the effect of mitochondrial-targeted treatment with AF intensity. However, detection of flavoprotein AF may serve as biomarker for mitochondrial dysfunction and RPE injury in models of dry AMD.
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