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Title: An investigation of solution-induced corneal staining using an in vitro model
Author: Bakkar, May
ISNI:       0000 0004 2729 5646
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Purpose: Solution-induced corneal staining (SICS) has been the subject of much debate in the clinical literature. While it has been suggested that this form of staining indicates toxicity of the cornea, microscopy studies have suggested that cells treated with multi-purpose solutions (MPS) known to produce SICS in vivo are undamaged. There is further debate in the literature as to whether or not sodium fluorescein (‘fluorescein’) actually enters epithelial cells or not. The aim of this work was to investigate the cellular mechanisms involved in SICS by developing an in vitro cell culture model to mimic the clinical presentation of this phenomenon. Methods: An in vitro model of SICS was developed using cultured cells that were exposed overnight to ReNu MultiPlus® (Bausch + Lomb) MPS. After addition of fluorescein, cells were imaged using an automated fluorescence microscope. Hyperfluorescent cells were identified using predetermined threshold of intensity in fluorescence microscope, and confocal microscopy was used to investigate where fluorescein was situated within the cells. The extent of cell toxicity was assessed using propidium iodide and Annexin V. In order to examine the contribution of passive and active transport mechanisms in fluorescein uptake and release, levels of hyperfluorescent staining were measured at 37°C and 4°C. In all described experiments, fluorescein staining was expressed by the proportion of hyperfluorescent cells in the total cells. Results: All cultured cells readily took up fluorescein at room temperature, however a sub-population of cells stained more intensely with fluorescein. These cells were termed ‘hyperfluorescent’ cells. Exposure to ReNu MultiPlus® resulted in a significant increase in the proportion of hyperfluorescent cells compared with control cells. In addition, the staining profiles of individual cells showed no correlation between cell death and hyperfluorescence. The data also showed that hyperfluorescence did not occur extensively in deliberately lysed cells.Addition of fluorescein to the cells at 4°C resulted in very low levels of hyperfluorescence compared to high levels at 37°C. Fluorescein was rapidly released from cells at 37°C but not from those at 4°C. Conclusion: In this work, an effective in vitro model of SICS was developed in order to provide a better understanding of the mechanisms involved in fluorescein staining. This work suggests that corneal fluorescein staining may reflect a simple cellular uptake of fluorescein. Levels of staining in the cells appear to be unrelated to cellular toxicity or cell damage. Staining appears to occur in the cytoplasm and the nucleus of the cells. Finally, fluorescein uptake and release are likely to occur through active transports mechanisms.
Supervisor: Dobson, Curtis; Morgan, Philip; Maldonado-Codina, Carole Sponsor: Jordan University of Science and Technology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cornea ; Solution induced-corneal staining ; fluorescein