Abstract
Abstract: :
Purpose: Rose Bengal (RB) is commonly used in assessing, diagnosing and monitoring disease and forms a basis for dry eye scoring systems. Remarkably, exactly what this dye stains still remains unclear. Methods: Tear staining was assessed by successive timed Schirmer strip collections after instillation of 1% RB into the lower fornix of normal volunteers. Dye was sequentially extracted in saline, Guanidine Hydrochloride (GuHCl) without and with Dithiotreitol (DTT) and quantified by spectrophotometry. These sequential extractions were also probed for mucins. Cell staining was assessed in stratified corneal and conjunctival epithelia with and without the addition of normal and dry eye surface mucins, after cell damage by NaOH and under alkaline pH. Topical anaesthetic and N-acetylcysteine were added to mimic clinical practice. Confocal microscopy determined the extent of dye penetration. Results: Rose Bengal is found in tears and ocular surface cells as both bound and free forms. Successive tear collections extract less dye. Mucin extracting reagents (GuHCl and DTT) yielded further RB. MUC5AC and MUC2 were identified in these tear collections, their peaks coinciding with the trough of RB. Corneal and conjunctival cells, both living and dead, stain with RB. The dye penetrates multilayered epithelia staining all but cell nuclei. In the absence of epithelial mucin, conjunctiva takes up RB more readily than cornea. Partial barrier to stain is provided by addition of tear mucins, but only in conjunctival cells. Conclusions: Rose Bengal binds both soluble and insoluble mucins, though MUC5AC and MUC2 are not the main binding partners. Addition of tear mucins is a less effective barrier to RB than the presence of cell surface mucins. Subscoring of epithelial staining patterns in the cornea and conjunctiva could refine current grading systems and help monitor and understand dry eye disease.
Keywords: cornea: tears/tear film/dry eye • cornea: surface mucins • cornea: epithelium