Tear Fluid Biomarkers and Ocular Surface Parameters in People with Type 2 Diabetes

It is well-known that diabetes is one of the major causes of dry eye disease (DED). As DED can lead to serious ocular surface complications such as punctate keratitis, corneal erosion, corneal scarring, corneal perforation and visual loss, research is ongoing to investigate ways of improving the diagnosis of DED in patients with diabetes. This thesis investigated the role of inflammatory cytokines and metabolic proteins in tear fluid as a non-invasive, objective measure of inflammation in the diagnosis of type 2 diabetes (T2D)-related DED. In this thesis, a number of studies were performed to investigate potential tear fluid biomarkers and comparing them to the established clinical and laboratory measures used in DED diagnosis. It was important to evaluate the clinical behaviours of eye care professionals, such as ophthalmologists and optometrists with respect to the diagnosis and management of diabetes-related DED (Chapter 3). Thus, the online survey was conducted among 194 eye care professionals who work in the UK and Mongolia, to evaluate and compare their preferences for specific clinical procedures used to diagnose the presence of DED in diabetes. For example, how these may differ, depending on the practitioners' profession (ophthalmologist and optometrist) and the countries they work in. This study is the first time that the awareness of eye care professionals in the diagnosis and the management of diabetes-related DED in different regions of the world has been investigated. The results of the survey showed that eye care professionals in both countries have similar practice behaviours for diagnosing and treating diabetes-related DED. That is, ophthalmologists in the UK and in Mongolia have similar practice behaviours in diagnosing diabetes-related DED, and its severity, as for optometrists in the UK. Before commencing the studies to analyse protein biomarkers in tear fluids, it was important to establish the best possible means of sampling and storing tear samples, along with the optimal method of analysing them (Chapter 4). Therefore, a study was conducted to investigate the methodology of tear sampling, storage, analysis, sample centrifugation duration and speed. For tear biomarker assessment, a pilot study investigating the detectability of metabolic proteins in 1 μl tears was conducted (Chapter 5). This study is the first time that metabolic proteins such as Leptin, Insulin, C-peptide, GIP and active Ghrelin, have been shown to be detected in human tears collected from “healthy” individuals using Luminex Xmap technology. The results of this study showed that Leptin could be used in the future as a potential diagnostic biomarker of ocular surface disorders and, possibly, in determining the effects of metabolic disorders, as it has a low intra-subject confidence interval (CI) and a high intra-class correlation coefficient (ICC). A pilot study investigating the correlation between the presence of tear fluid inflammatory cytokines and tear fluid stability (assessed using tear evaporation rate [TER]; and non-invasive tear break-up time [NITBUT]) in healthy subjects was carried out (Chapter 6). This study also investigated the effect of time of day on TER and NITBUT values, and its possible correlation with tear cytokine concentrations in healthy subjects. The results revealed that the NITBUT and TER values had no significant variability over a day, or on different days, in healthy subjects, when the humidity and temperature of the examination room were constant. However, some tear fluid cytokine concentrations did show inter- and intra-day variability, without any consistent correlation with TER diurnal variation. Before commencing the study in a large cohort, a pilot study investigating the detectability of inflammatory cytokines and metabolic proteins in 1 μl tears collected from patients with type 2 diabetes (T2D; with and without DED) and DED-only (without T2D) was conducted (Chapter 7). The concentrations of 14 inflammatory cytokines, six metabolic proteins, ocular surface parameters, DED-related symptoms and quality of life (QoL) were evaluated and compared in a total of 122 subjects divided into four study groups as: T2D with DED, T2D-only (without DED), DED-only (without T2D) and healthy controls (Chapter 8). Overall, the results of this study showed that the ocular surface parameters and DED-related symptoms were similar for patients in the T2D+DED group and the DED-only group. In addition, patients in the T2D-only group had similar ocular surface parameters to healthy controls. Tear fluid Interleukin (IL)-6 and IL-8 concentrations were significantly higher in the T2D+DED group than the T2D-only group, DED-only group and healthy controls, which suggests that IL-6 and IL-8 could potentially be diagnostic biomarkers of T2D-related DED. These studies show that tear fluid biomarker analysis is feasible and it can be utilised in the objective diagnosis of T2D-related DED. This research helps to improve the understanding of the inflammatory mechanisms and markers of DED in T2D. However this complex process will require further research to identify the most appropriate biomarkers.