Investigation of the biological and chemical basis underpinning contact lens solution induced corneal staining

of Thesis submitted by Tahmina Khan for the Degree of Doctor of Philosophy. Purpose: Sodium fluorescein (‘fluorescein’) is the most commonly used diagnostic ophthalmic dye for clinical assessment of the ocular surface. An increase in fluorescein staining presents after the use of certain multi-purpose solutions (MPS) and contact lenses, which is a clinical phenomenon known as solution induced corneal staining (SICS). The clinical relevance and mechanisms underpinning fluorescein staining however is conflicting in the literature. Currently a number of studies indicate fluorescein localises within cells that are healthy and metabolically active, in contrast to conventional thought that fluorescein staining of MPS treated cells is indicative of cellular toxicity; however the precise mechanism underpinning fluorescein internalisation is unknown. The aim of this doctoral thesis was thus to investigate novel biological mechanisms mediating fluorescein uptake and to determine the chemical inducers of increased fluorescein staining. Methods: Spectrofluorimetry was used to analyse the fluorescence intensity of various fluorescein solutions and of tear samples after fluorescein application to the ocular surface. For assessment of fluorescein uptake in cell cultures exposed to test MPS, fluorescence microscopy was performed; subsequently a high content analysis (HCA) microscopy methodology developed in this thesis, was used to enumerate fluorescein fluorescence intensity. The physiological state of cell cultures treated with a number of MPS and fluorescein were also assessed using propidium iodide (PI), Caspase-3 antibody for apoptosis analysis, metabolic activity assays and confocal microscopy. Endocytosis, macropinocytosis, and receptor-mediated uptake, mechanisms which previous to this work have not been investigated in association with SICS and fluorescein uptake, were studied using chemical inhibitors and serum starvation in cell culture, and then analysed by HCA. To study the hyperfluorescence inducing properties of MPS components, surfactant Tetronic 1107 (an MPS surfactant component typically overlooked and not investigated as an inducer of SICS) and Triton X-100 (a surfactant not found in MPS) were exposed to cell cultures and after fluorescein treatment, were analysed by HCA; metabolic activity assessment of identically treated cultures were also performed. Similarly, HCA and metabolic activity measurements were also performed in cell cultures treated with MPS biocides PHMB and Polyquaternium-1 (PQ-1). Results: Fluorescein fluorescence intensity was not affected by temperature, by alkali solvents, or by different MPS formulations; concentration remains the primary factor by which fluorescein intensity is affected. It was seen that after 1% w/v fluorescein instillation on the ocular surface, fluorescein concentration in tear samples significantly decreased within the first minute and decreased to as little as 0.0001% w/v after 10 minutes. Fluorescence microscopy and HCA techniques revealed that on treatment with Biotrue® (Bausch+Lomb, referred to as P-PQ1-1107) and ReNu Sensitive Multi-purpose Solution® (Bausch+Lomb, referred to as P-1107), fluorescein uptake increased in cell cultures, as typically seen clinically. Also typically seen clinically, cell cultures treated with Opti-free Replenish® (Alcon; PQ1-Aldox-1304) and Complete Revitalens® (AMO; PQ1-Alex-904) did not induce hyperfluorescence. Significantly, only those cells treated with PQ1-Aldox-1304 and PQ1-Alex-904 exhibited an increase in PI and loss of metabolic activity. Additionally, given that no significant Caspase-3 staining in all test MPS cells was seen by fluorescence microscopy and blebbing was only observed by confocal microscopy in P-PQ1-1107 treated cells, increased fluorescein hyperfluorescence is unlikely to be related to apoptosis. Study of various biological internalisation mechanisms revealed that dynamin (a protein associated with endocytosis) inhibition in P-PQ1-1107 or P-1107 treated cells significantly decreases fluorescein uptake with no detrimental effect to cell metabolic activity. Interestingly, on quantifying fluorescein uptake in surfactant treated cells, a significant increase in hyperfluorescence was measured with only Tetronic 1107 at concentration 1% w/v with no significant effect on cellular metabolic activity. In contrast, cell culture treatment with biocides PHMB, and PQ-1 did not induce increased fluorescein uptake comparable to MPS PPQ1-1107 or P-1107; metabolic activity assays revealed that this is likely attributed to the decrease in cell metabolic activity exhibited with these biocides. Conclusions: The differential fluorescein staining associated clinically with four varied MPS was also seen in an unique in vitro model, allowing the subsequent study of the SICS phenomenon and the fundamental basis of fluorescein uptake. Using this in vitro model, it was seen that fluorescein internalisation is associated with the protein dynamin in healthy, metabolically active cells, and is unlikely to be mediated by apoptosis. Moreover, fluorescein uptake is induced by surfactant Tetronic 1107 and not biocides PHMB or PQ-1, which is in contrast to the popular belief that increased hyperfluorescence is induced by the biocide component of MPS.