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Larry Koreen, Albert R. Wielgus, Grazia Spiga, Peter Saloupis, Diego G. Espinosa-Heidmann, Melody Chan, Sina Farsiu, Scott W. Cousins; Acute Periocular Exposure of Mouse Eyes to Hydroquinone Induces RPE Cytoskeletal Changes, F-actin Aggregates, and Early Deposit Formation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3190.
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Acute sub-lethal oxidative injury in cultured RPE causes extrusion of intracellular and membranous material (i.e., cell membrane blebbing), which is hypothesized to contribute to drusen formation. Our previous in vitro studies showed that RPE cell blebbing is mediated by cytoskeleton reorganization and F-actin aggregate formation. We sought to test the hypothesis that similar cytoskeletal changes occur in vivo when mouse eyes are exposed to sub-conjunctival sub-lethal doses of the cigarette smoke-associated oxidant, hydroquinone (HQ).
BALB/c mice received sub-conjunctival injection of 75mM HQ or vehicle. At various time points after injection, mouse eyes were harvested, retinas were removed, and 4-quadrant RPE flat mounts were prepared. RPE flat mounts were stained with phalloidin and F-actin aggregates were imaged with confocal microscopy. Computer software was developed to quantify actin aggregate formation. Transmission electron microscopy (TEM) images were obtained from some eyes to evaluate sub-RPE deposit formation.
Periocular HQ exposure caused depolymerization of RPE cell actin filaments and formation of F-actin aggregates in vivo. As we have observed previously in vitro after ARPE-19 cells were exposed to HQ, mouse eyes treated with HQ demonstrated significantly more actin aggregates and cytoskeletal changes compared with vehicle-treated and untreated animals. Aggregate formation began as early as 1 hour after exposure and appeared to maximize after 18 hours. Aggregates accumulated much more in the basal portion of mouse RPE cells than in the apical portion. Preliminary TEM imaging suggested that bleb-like structures could be observed in association with mild transient sub-RPE deposit formation in some eyes.
Local ocular exposure of mouse eyes to HQ causes RPE cell cytoskeleton rearrangement and actin aggregate formation, an early stage in the process of cell membrane blebbing, which may lead to formation of sub-retinal deposits and drusen. This system will be helpful in understanding the in vivo correlation with cell culture data from sub-lethal RPE oxidant injury and blebbing.
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