June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Mechanism of Ocular Surface Staining by Clinical Dyes
Author Affiliations & Notes
  • Shinwu Jeong
    USC Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
    USC Roski Eye Institute and Department of Ophthalmology, Keck School of Medicine of USC, Los Angeles, California, United States
  • Jasmine Kim
    USC Dornsife College of Letters, Arts & Sciences, University of Southern California, Los Angeles, California, United States
  • Michelle Ngan
    Keck School of Medicine of USC, Los Angeles, California, United States
  • Andrew Webster
    USC Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
  • Joseph T Barr
    The Ohio State University College of Optometry, Columbus, Ohio, United States
  • Pablo Argueso
    The Schepens Eye Research Institute, Massachusetts Eye & Ear and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • M Elizabeth Fini
    USC Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Shinwu Jeong, Proteris Biotech, Inc. (F), University of Southern California (P); Jasmine Kim, None; Michelle Ngan, None; Andrew Webster, None; Joseph Barr, Proteris Biotech, Inc. (C); Pablo Argueso, None; M Elizabeth Fini, Proteris Biotech, Inc (I), University of Southern California (P)
  • Footnotes
    Support  EY026479 (MEF), Research to Prevent Blindness (USC)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3942. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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