Abstract
Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting ‘staining’. Although localized intensely stained regions of the cornea frequently occur after exposure to ‘adverse’ clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells.We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining ‘hyperfluorescent’ cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4°C.We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli.
Highlights
The ocular surface can be affected by a number of external factors which can be examined clinically using a topical application of sodium fluorescein (‘fluorescein’) which may reveal characteristic patterns of fluorescein-associated hyperfluorescence which are generally assumed to indicate deleterious cellular and/or tissue change.Corneal staining can be readily observed at the ocular surface after exposure to toxic agents
Cells were washed with phosphate-buffered saline A (PBS) (Oxoid, Basingstoke, U.K.) before treatment with 0.25% trypsin-EDTA solution (Sigma-Aldrich Company Ltd, Gillingham, U.K.); on resuspension hTCEpi cells were treated with an equal volume of 0.25 mg/ml Soybean Trypsin Inhibitor (Gibco, Invitrogen – Life Technologies, Paisley, U.K.) After centrifugation and resuspension, cell numbers were assessed using a haemocytometer (Hausser Scientific, Horsham, PA, USA), before use in experiments
The differential staining pattern apparent in our cell cultures was found to be comparable in appearance to that of the human cornea after fluorescein staining, including the presence of punctate spots (Fig. 1D)
Summary
Corneal staining can be readily observed at the ocular surface after exposure to toxic agents. Horsley and Kahook reported reduced corneal staining when patients were switched from glaucoma eye drops containing benzalkonium chloride (a common preservative in ocular medications) to a similar drug not containing this agent [1]. Topical anesthetic drops are associated with corneal staining [2]. Corneal staining can be observed following: mechanical insult [5], ocular surface dehydration [6,7], specific physical contact lens interactions [8], ocular surface disease and exposure to contact lens solutions [9,10]
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