Fluorescein staining and physiological state of corneal epithelial cells

Abstract

Purpose: To evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining. Methods: Fluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells - HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidium-iodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software. Results: Healthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0-256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p>0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings. Conclusions: The clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells.