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Dry eye affects millions of individuals. In experimental models, dry eye disease is associated with T helper cell 17-mediated inflammation of the ocular surface that may cause persistent damage to the corneal epithelium. However, the initiating and perpetuating factors associated with chronic inflammation of the ocular surface remain unclear. The ocular microbiota alters ocular surface inflammation and may influence dry eye disease development and progression. Here, we collected serial samples of tears on awakening from sleep, closed eye tears, during a randomized clinical trial of a non-pharmaceutical dry eye therapy and used 16S rRNA metabarcoding to characterize the microbiome. We show the closed dry eye microbiome is distinct from the healthy closed eye microbiome, and that the microbiome remains distinct despite daily saline eye wash upon awakening. The ocular microbiome was described only recently, and this report implicates a distinct microbiome in ocular disease development. Our findings suggest an interplay between microbial commensals and inflammation on the ocular surface. This information may inform future studies of the pathophysiological mechanisms of dry eye disease.

To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing. Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression. The reconstructed curved corneal epithelium was composed of 3-5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression. The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.

Every night, during sleep, the tears of the closed eye become significantly more inflammatory, with a notable influx of neutrophils. Preliminary studies suggested that these neutrophils were significantly different than blood-isolated neutrophils, with an increased concentration, upregulated surface expression of inflammatory membrane receptors, but inability to respond to inflammatory stimuli. The purpose of this dissertation was to better understand the contribution of this neutrophil population to ocular surface homeostasis and dry eye disease.Chapter One presents an introduction to the tear film and ocular surface, along with an introduction to dry eye disease and neutrophil biology. Chapter One also discusses optimization of methods used in later chapters. In Chapter Two, the phenotype of closed eye neutrophils is observed with varying amounts of sleep, to show that neutrophil phenotype changes between one and seven hours of sleep. Chapter Threeinvestigates the relation of neutrophils in the closed eye, to that of the open eye. Chapter Four presents an investigation of leukocytes in the post-lens tear film of scleral contact lens wearers, to determine if this leukocyte population impacts clinical outcomes for these patients.Chapter Five continues the investigation in Chapter One, by comparing the closed eye neutrophil phenotype and degranulation response between normal and dry eye subjects. Chapter Six runs parallel to Chapter Five, to assess intracellular cytokine production in closed eye neutrophils. Chapter Seven presents a small-scale clinical trial for the assessment of an eyewash, immediately upon awakening, as a potential therapy for dry eye disease.Overall, the results from all chapters are discussed in Chapter 8, and future directions are presented.