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Shinwu Jeong, Jasmine Kim, Michelle Ngan, Andrew Webster, Joseph T Barr, Pablo Argueso, M Elizabeth Fini; Mechanism of Ocular Surface Staining by Clinical Dyes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3942.
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While ocular surface (OCS) staining with clinical dyes such as sodium fluorescein (FL) or rose-bengal (RB) is commonly used to assess dry eye, the reasons for staining are not well understood. It was recently shown that application of various stressors to monolayer cultures stimulates FL uptake by individual living cells, resulting in a mosaic of “hyper-staining”, similar to the punctate pattern of OCS staining characteristic of dry eye (PMID: 24332360; PMID: 24489650). Here we investigated mechanisms.
Immortalized cells of a human corneal limbal epithelial (HCLE) line were cultured as monolayers or induced to differentiate into a stratified squamous mucosal epithelium, as described (PMID: 12766048). Cultures were treated for 2 h with 3 mM tert-butyl hydroperoxide (t-BHP) to create oxidative stress. Uptake of FL (30 uM, 10 min) or RB (0.05%) was evaluated quantitatively using a plate reader, or qualitatively by imaging. Statistical analysis was performed using the Student t-test.
All cells in monolayer culture actively take up FL or RB at a low level, however when induced to differentiate, islands of RB exclusion develop. We now report that differentiation also creates a partial barrier to FL uptake. T-BHP treatment of monolayer cultures stimulated an increase in FL dye uptake by 2.2-fold (p<1x10-4), evidenced as a mosaic of individual cell hyper-staining. Cell surface binding of Annexin V (an early sign of apoptosis) was increased in parallel. T-BHP treatment of stratified cultures stimulated dye uptake by only 1.5 fold (p=0.005) and binding of Annexin V was similarly reduced. Cells apparently recover from initial damage, as judged by the lack of trypan blue staining, DNA degradation or TUNEL assay positivity. T-BHP stimulated cells to take up dye into discrete vesicles. The t-BHP stimulated uptake of FL was blocked almost to the untreated level by genistein (p<0.01), an inhibitor of caveolin-mediated endocytosis, and by chlorpromazine (p<1X10-5), an inhibitor of clathrin-mediated endocytosis.
The results demonstrate that oxidative stress-induced, sub-lethal cell damage stimulates clinical dye uptake and that squamous mucosal differentiation provides a partial barrier. They further suggest that dye uptake occurs by caveolin- and clathrin-mediated endocytosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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