title: Fluorescein staining and physiological state of corneal epithelial cells.
abstract: OBJECTIVE
  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.
authors: Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia. Electronic address: k.bandamwar@brienholdenvision.com.
authors: Kalika L
authors: KL
authors: Bandamwar
authors: Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia; Vision Cooperative Research Centre, Sydney, Australia.
authors: Eric B
authors: EB
authors: Papas
authors: Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia.
authors: Qian
authors: Q
authors: Garrett
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology* / Q000502:physiology*; D019573:Epithelium, Corneal / Q000737:chemistry / Q000166:cytology* / Q000502:physiology*; D019793:Fluorescein / Q000737:chemistry*; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D066298:In Vitro Techniques; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods
chemical_list: D019793:Fluorescein
keywords: Apoptosis; Corneal epithelium; Fluorescein; Live-dead cells; Micropunctate staining