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This body of work encompasses two studies: the collection of canine tears via a novel polyester<br />rod and the comparison of VEGF-A concentrations in tears from dogs with normal and<br />vascularized corneas. The first study used polyester rods for tear collection in dogs. Fluid volume and VEGF recovery characteristics, as well as potential binding of VEGF to the rod, were determined. Tears were harvested from normal dogs using rods and glass capillary tubes. Tears were assayed for tear film VEGF using a commercial canine VEGF sandwich ELISA kit. Dilutions of VEGF standard were wicked into the rods or drawn into capillary tubes, eluted, and assayed. Percent volume recovery is adequate for polyester rods as is percent VEGF recovery. VEGF is detectable in normal canine tears.The second study harvested tear samples from eyes of dogs with vascularizing corneal disease, as well as the contralateral unaffected eye of unilaterally diseased dogs, and normal dogs. Vascularization scores were assigned to diseased eyes and tear film VEGF concentration was assayed as above. Mean tear film VEGF concentration of diseased eyes did not differ from control eyes, and was not correlated with disease process, extent of vascularization, or other parameters. Tear film VEGF in unaffected eyes was significantly higher than control and vascularized eyes. Canine tear film VEGF exceeds biologically active concentrations, but does not correlate with state of corneal vascularization. VEGF-related control of corneal vascularization may be mediated by other proangiogenic factors. / Master of Science

The purpose of this study was to determine the effects of three medical treatments, topical oxytetracycline, topical prednisolone acetate, and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. The study design consisted of twelve ophthalmically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (500mg IM bid), topical 1% prednisolone acetate (0.2ml tid), or topical oxytetracycline (0.2ml tid), followed by a 9-day washout period. All topical medications were applied to the left eye and the right eye was treated with a placebo. Tears were collected before the first treatment on day one and the morning following the last treatment on day 5. Tear film proteinase activity was measured using gelatin zymography and measurements of optical density. Statistical analysis of the difference between the treated and untreated eyes and the eyes before and after treatment was performed using mixed effects model for ANOVA. When eyes were compared after treatment, there was no significant difference between treated and placebo eyes for MMP-2 or MMP-9 for any of the treatments. When post-treated eyes were compared to pre-treated eyes, there was a significant decrease in MMP-2 activity in the left eye of horses treated with flunixin meglumine (P=0.0259). There were no differences in MMP-2 and MMP-9 activity for the other treatments. In conclusion, topical 1% prednisolone acetate and topical oxytetracycline did not significantly change MMP-2 or MMP-9 activity in normal equine tear film. Systemic flunixin meglumine had an inhibitory, but questionable, effect on MMP-2 activity in normal equine tear film. This project was funded by Patricia Bonsall Stuart Award for Equine Research. / Master of Science

The cornea is the anterior most surface of the eye and plays a critical role in vision. A thin fluid layer, the tear film, coats the outer surface of the cornea and serves to protect, nourish, and lubricate the cornea. At the same time, the tear film is responsible for creating a smooth continuous surface, where the majority of refraction takes place in the eye. A significant component of vision quality is determined by the shape of the cornea and stability of the tear film. A dual interferometer system for measuring the dynamic corneal topography is designed, built, verified, and qualified by testing on human subjects. The system consists of two coaligned simultaneous phase-shifting polarization-splitting Twyman-Green interferometers. The primary interferometer measures the surface of the tear film while the secondary interferometer tracks the absolute position of the cornea, which provides enough information to reconstruct the absolute shape of the cornea. The results are high-resolution and high-accuracy surface topography measurements of the in vivo tear film and cornea that are captured at standard camera frame rates. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)

Introduction: The measurement of tear film stability/regularity is very critical in the diagnosis of dry eye. The tear breakup time, which is used as a diagnostic tool in diagnosing dry eye, is very subjective in nature and variations among individual clinicians exists. The exact mechanism of the tear breakup is also unclear due to the involvement of so many other factors other than the tear film itself. As the prevalence of dry eye is increasing, the need for an objective technique which can be used universally to differentiate between dry eye and normal values increases. Studies have shown that aberrations can be used as an objective technique in diagnosing dry eye, as there is a direct involvement of the tear film in the optics of the eye. However, very few studies have studied the dynamic nature of the anterior surface using aberrations and suggested using dynamic surface aberrations as an objective measure of surface quality. Hence, a series of studies were conducted to understand the aberrations produced by the anterior surface of the eye (tear film and corneal surface) and to measure objectively the anterior surface quality using surface aberrometry. The objectives of each study chapter are as follows: Chapter 3 i): To obtain the noise associated with the instrument using a non-dynamic measuring surface, and ii) to design appropriate acquisition settings for the measurements with ocular surface. Chapter 4: To determine ??) the spectral characteristics of the Placido disc light sources of two corneal analysers, ????) the thermal characteristic for a variety of inanimate objects, human ocular surface and the adnexa in the presence of Placido disc light source at normal working distance, and ??????) to compare the ocular surface aberrations obtained using both the corneal analysers Chapter 5: To determine i) the optimal method for acquisition with respect to normal physiological processes, by examining the blink regimen and head position that elicits the most consistent response over the largest region on repeated measurement; and i??) the largest region selected for analysis by investigating the effect on the individual and summary aberration metrics of the inclusion of non-measurement areas (i.e. where the Placido disc cannot be projected onto the cornea or contact lens). The proportion of non-measurement area that elicits a significantly different result will be determined. Chapter 6: To evaluate ??) a new method of analyzing dynamic ocular surface aberrations using segmented liner regression, and ????) the inter-ocular characteristics of the dynamic ocular surface aberrations using the segmented linear regression. Methods: Chapter 3: The characteristics of the surface aberrometer and the noise associated with the measurements of surface aberrations were evaluated using a non-dynamic surface (model eye). Measurements were obtained in different frame rates and focus positions to evaluate the optimal acquisition technique. At each focus position, a set of three repeated measurements were obtained to analyse the repeatability of the measurements obtained using a surface aberrometer. Chapter 4: The spectral characteristics of the Placido disc light source were obtained by using a PR650 SpectraScan photometer and the thermal characteristics of the objects were obtained using THI-500 non-contact infrared thermometer. The surface aberration measurements were compared between the corneal analysers. The spectral measures were obtained from the light sources, whereas the thermal measures were obtained from three different surfaces and surface of the eye and adnexa of ten participants. The dynamic anterior surface aberrations were obtained after obtaining the thermal measurements from the surface of the eye. Chapter 5: Twelve participants were enrolled by screening twenty participants. Participants were screened with their habitual lenses for contact lens wettability and non-invasive tear breakup time (NITBUT) without contact lenses. The participants were enrolled according to the inclusion and exclusion criteria and categorized into normal and dry eye group for study visits. The measurements of NITBUT and surface aberrations were obtained with and without contact lenses, and study lens wettability were also obtained in two visits on consecutive days. The surface aberration measurements were obtained in natural and forced blinking condition and in two different head positions. All the measurements were randomized between eye and between instruments. Chapter 6: Seventeen non- symptomatic and non- contact lens participants were recruited in this study. NITBUT and dynamic anterior surface aberration measurements were obtained. The order of the measurements was randomized between the eyes. Two open intervals of at least 10 sec and a maximum of 15 sec were used in the analysis of segmented fit. The dynamic vertical prism coefficients and higher order aberrations were used for the analysis. Results: Chapter 3: i. Data acquisition at an inter-frame interval of 0.25s gave the least number of dropped frames across focus positions, therefore this is the preferred frame rate for data acquisition. ii. Data obtained in the initial ~15s reflects the focusing procedure and needs to be manually removed prior to analysis of tear dynamics. iii. Even in the optimal focus position there were significant (small) differences in the distributions between repeated measures. For this reason repeated samples have to be obtained where possible. iv. The green and red focus positions showed the most consistency within repeated measurements. The variability of the measurements was also more similar between the red and green focus positions than the blue focus positions, both at the extreme positions of defocus and with incremental defocus away from the optimal focus position. When obtaining the dynamic sampling of human ocular surface measurements, the optimal position of focus should be obtained at the blink such that as the tear film dissipates between blinks the measurements are obtained in the (relatively) red focus position. Chapter 4: i. CA200 is the preferred device because of the consistent luminance. ii. Although aberrations were not significantly different between devices, the HOA RMS were higher with the CA200 and, combined with different luminance and possible tear response, indicates the devices are not interchangeable. In both instruments, there was no indication that there was a thermal response induced by the power of the light source. Therefore, this aspect of the source does not likely contribute to any difference in the aberrations measured by the two devices Chapter 5: i. Obtain data in the straight-ahead position, as there is no significant increase in target size with head turn. ii. With the CA100F, the forced blink paradigm is preferred as this enables blink dynamics to be examined. With the CA200F, either forced or natural blink paradigms are interpretable for tear dynamics. iii. Differentiation between dry eye and normal groups was best determined with the slope of the RMS aberrations within a blink. iv. Differentiation between performance with and without a contact lens in the dry eye and normal groups was best determined by analysing the width of the confidence interval of the moving average. Chapter 6: i. The location breakpoints one and two are significantly different between eye, open eye interval and order of the measurements for both vertical prism and HOA RMS values. ii. The highest positive slope for the HOA RMS was, on average, higher in the second eye measured (p= 0.0407) and tended to occur later after the blink (p= 0.0676). iii. The location of breakpoint 2 is not significantly different from the NITBUT values (p>0.05), even though the correlation was found to be low and not significant. iv. The average HOA RMS for segmented fit parameter intervals of vertical prism was found to be higher in the second open eye interval compared to first open eye interval. Conclusion: From the results of each chapter, it was observed that choosing the blink paradigm is very important to obtain and analyse the dynamic anterior surface aberrations. Choosing a forced blink paradigm (chapter 5) was showed to be useful when the information regarding blink location were not available. The repeatability of the measurements using a non-dynamic surface (chapter 3) shows that the measurements of surface aberrations are repeatable and it is important to choose a criterion closer to the natural tear film dynamics to obtain more repeatable measurements of anterior surface aberrations (chapter 4, 5 and 6). It also shows that the three phased segmented linear regression techniques can be used to analyse the anterior surface aberrations. The segmented linear regression technique was able to differentiate different stages of the tear film and the location of the second breakpoint calculated using segmented regression was closer to the clinical values of tear breakup time, indicating a possible use of segmented linear regression as an objective measure of surface quality.

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<p> Hydroxypropyl guar (HPG) is an effective ingredient in lubricant eye drops used by patients with dry eye disease. The overall goal of the work described in this thesis is to understand the physical-chemical properties of HPG in the presence ofmodel surfaces and solutes with view to understanding the behavior of HPG in the tear film. </p> <p> HPG behaviors are complex because borate ions bind to HPG, which converts nonionic HPG into anionic polyelectrolyte, RPG-borate. The borate binding constants are very low, meaning the charges on RPG-borate are labile. Another consequence ofweak binding is that the equilibrium electrolyte concentration with HPG-borate is relatively high. Mathematical models were developed to predict the structure of HPG-borate as functions of pH. </p> <p> This thesis probes the question "When does HPG-borate behave as an anionic polyelectrolyte?" This work shows that HPG-borate exhibits deviant behaviors of an anionic polyelectrolyte: does not interact with cationic surfactants below the CMC; does not interact with lysozyme (cationic protein), and does not adsorb onto cationic liposomes. By contrast, anionic polyelectrolytes such as carboxymethyl guar display generic behaviors. On the other hand, HPG-borate forms polyelectrolyte complexes with cationic polyelectrolytes at low ionic strength and other work from our laboratory has shown that HPG-borate flocculates cationic polystyrene latex. </p> <p> This complex range of RPG-borate behaviors was rationalized by proposing that the labile nature ofthe charge groups means that the charge density on RPG-borate is regulated by the local electrostatic environment. Near a cationic surface HPG-borate charge density increases whereas near an anionic surface the charge density is lower. </p> <p> Anionic liposome interactions with HPG-borate were characterized. HPG concentrations close to clinical levels induced depletion flocculation ofthe anionic liposomes. This is the first example we have found depletion interactions were proposed for the tear film. </p> <p> To summarize the main implications for the ophthalmic application of HPG are: 1) under ophthalmic conditions HPG-borate behaves as a nonionic water soluble polymer; 2) RPG-borate will adsorb onto hydrophobic domains but will not interact with lysozyme; 3) depletion interactions are important and have the potential to stabilize the lipid layer and destabilize emulsion droplets and other dispersed species in the tear film. </p> / Thesis / Doctor of Philosophy (PhD)

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