Optometry and Vision Science

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Antifungal ocular drug delivery via contact lenses using a novel in vitro eye model
(University of Waterloo, 2016-04-21) Phan, Chau-Minh; Jones, Lyndon
Purpose: The purpose of this thesis was to evaluate the potential of contact lenses (CLs) as an antifungal drug delivery device, and to develop an in vitro eye model to test thereof. Methods: The first three chapters focused on developing a CL to function as a drug delivery device for natamycin, the only commercially available antifungal: • In the first experiment (Chapter 3), the in vitro uptake and release characteristics of natamycin from several commercially available CLs were evaluated • In the second experiment (Chapter 4), to improve the release characteristics of natamycin from contact lenses, an attempt was made to incorporate novel drug-encapsulated nanoparticles (Dex-b-PLA) within the CLs • In the third experiment (Chapter 5), an alternative strategy employing the incorporation of cyclodextrin (CDs) within the CL polymer matrix was evaluated as a potential modification to prolong the release of natamycin The second half of the thesis was aimed at developing a sophisticated in vitro ocular model capable of adequately measuring drug release from CLs: • In Chapter 6, the design of a novel in vitro eye model to simulate the physiological ocular environment was outlined • In Chapter 7, this model was used to evaluate the release of the antifungal fluconazole from commercially daily disposable CLs • In chapter 8, as an extension of the developed in vitro eye model, an agar eye model was developed to test the effects of natamycin and fluconazole-releasing CLs on Candida albicans Results Commercial CLs, after drug incubation with natamycin, will release the drug rapidly within the first half hour, followed by a plateau phase. However, when CL materials were loaded instead with natamycin encapsulated within novel Dex-b-PLA nanoparticles, the release duration was extended to 12 hours. Modifying the CL polymer with methacrylated CDs did not significantly improve drug release. On the contrary, high loading of CDs decreased overall drug delivery efficiency, likely resulting from unfavourable arrangements of the CDs within the polymer network. The developed ocular platform, termed Ocuflow, simulates physiological tear flow, tear volume, air exposure and mechanical wear. When this system was used to analyze the release of fluconazole from commercial CLs, the drug release was sustained for up to 24 hours. This observation significantly contrasts drug release observed in a vial, which typically follows a burst-plateau profile. When CLs releasing natamycin and fluconazole were tested on agar eye models that were inoculated with Candida albicans, the growth of the yeast was limited by natamycin-containing CLs. The cell morphology of the yeast also differed noticeably based on drug-lens combinations. Conclusions This thesis details potential strategies to develop novel CLs for antifungal ocular drug delivery. The Ocuflow system developed from this thesis is highly versatile; not only can it be used effectively to measure drug release from CLs, but it can also be applied to other in vitro analyses with CLs.
Aspects of Corneal Fluorescein Staining
(University of Waterloo, 2019-04-25) Woods, Jill; Jones, Lyndon
PURPOSE Evaluating the cornea for epithelial fluorescein staining is a key element of the ocular examination of contact lens wearers and people with dry eye disease. It has long been viewed as a method of visualizing a break in the protective epithelial layer, the integrity of which is regarded as vital for protecting the eye and maintaining good vision. There has been very little reported on the typical staining presentation in dry eye. Understanding more about the distribution of epithelial staining in dry eye disease would be valuable to guide evaluation of a treatment’s physiological efficacy. This thesis aimed to determine whether the corneal staining of subjects with symptomatic dry eye presents in a specific distribution pattern Since 2002, the epithelial staining phenomenon of solution induced corneal staining (SICS) has been investigated. The cause of this staining has been suggested to be due to molecular adhesion rather than physiological damage, but the current evidence is equivocal. More investigation of this phenomenon is warranted to understand the process and the clinical significance of SICS. This thesis aimed to investigate the type, severity and pattern of staining that occurs in SICS, and assess the impact on epithelial cells using in-vivo confocal imaging. METHODS Chapter 2 described the CORE corneal staining scale., which uniquely reports the type and extent of the corneal staining on a scale of 0-100. This was the staining scale used to record the level of solution induced corneal staining in all the clinical trials featured in Chapters 5 and 6. Chapter 3 reported an experiment which was conducted to assess the agreement among fifteen observers who used this scale, in two grading sessions, to grade the corneal staining illustrated in 22 photographic images. Inter- and intra-observer agreement results were calculated. Chapter 4 presented a meta-analysis of the corneal staining observed in 368 subjects, across 13 studies, with symptoms of dry eye. For each subject the corneal zone of worst staining was recorded to analyse which region of the cornea most frequently exhibited the most severe staining. In Chapter 5, 20 subjects were exposed to a lens/solution combination, known to induce SICS, in both eyes for a two hour period. In phase one, one lens was rinsed thoroughly before being worn; in phase two, the eye itself was rinsed thoroughly post lens wear; in phase three, confocal microscopy was conducted on both eyes to look for hyper-reflective epithelial cells. In phases one and two, the epithelium was assessed for staining pre and post lens wear with and without fluorescein. Chapter 6 evaluated aspects of the staining data collected in several SICS-inducing studies. The frequency of the reported ‘donut’ pattern of staining was calculated, relative to a diffuse, pan-corneal staining pattern. Seven subjects were identified that had participated in three or more trials using the same SICS-inducing methodology. The data from these individuals were assessed to determine the repeatability of the level of induced corneal staining in these trials. RESULTS The CORE corneal staining scale agreement experiment, in Chapter 3, supported the benefit of training because the concordance of the naïve observer was markedly worse than the observers who had received prior training. The inter- and intra-observer agreement analyses provided valuable data which can be applied to the development a pictorial reference guide and better instructions. The Chapter 4 meta-analysis of the geographic distribution of corneal staining among subjects with symptomatic dry eye demonstrated that the greatest degree of staining was most frequently in the inferior zone. (52.5%) The zone affected least was determined to be the central zone (12.8%). In the SICS experiment of Chapter 5, rinsing the lens prior to wear and rinsing the eye post lens wear did not result in different staining to the non-rinsed condition. All eyes, irrespective of any rinsing treatment, presented with punctate staining over >84% corneal area. The SICS staining was visible before fluorescein was instilled as ‘white light staining’. Confocal images were obtained from 34 of the 40 eyes, and hyper-reflective cells were visible in 33 of those 34 eyes. The meta-analysis in Chapter 6 concluded that the ‘donut-ring’ staining pattern, which is often described as typical of SICS, was actually far less common than a diffuse pan-corneal staining presentation. When SICS responding eyes were defined as exhibiting staining of 10% extent in at least four of the five corneal zones, 89% were identified as presenting with the pan-corneal pattern i.e. all five zones met the 10% extent criteria. When the SICS definition was tightened to include only those with 50% extent in at least four zones, 76% of subjects still identified as the pan-corneal staining pattern. There was minimal evidence of SICS presenting with a repeatable degree of staining in the same individual across different clinical trials. CONCLUSIONS This thesis investigated several aspects of corneal epithelial fluorescein staining and the chapters have furthered understanding in this field in several ways. The CORE corneal staining scale provides valuable data regarding the percentage of the corneal affected by staining. The results of the Chapter 3 agreement experiment provide useful information for the next steps in the development of this scale which will create a valuable corneal staining assessment tool. The evidence that the most severe corneal staining in patients with symptoms of dry eye most often presents in the inferior zone is invaluable to the design of future clinical trials of dry eye treatments. It highlights the importance of specifically assessing this region and the value in targeting fluorescein staining improvements in this zone as a key outcome measure. SICS has been suggested to be due to adhesion between PHMB (or other care system components) and the epithelial cells. The experiment in Chapter 5 confirmed that rinsing the lens does not remove enough PHMB from the lens to prevent SICS, and rinsing the eye afterwards is not effective at removing the bound molecules from the epithelial cells because SICS is still evident post rinsing. The presence of ‘white light staining’ and hyper-reflective cells on in-vivo confocal microscopy indicate that there are changes to the epithelial cells even before the fluorescein is instilled into the eye. More investigation of changes at the cellular level are required to understand what is happening. The meta-analysis of SICS data was able to provide evidence that SICS most commonly presents as a diffuse punctate staining that affects the entire cornea presenting in a pan-corneal pattern, rather than presenting in the commonly described pattern of a donut-ring, which implies central zone sparing. The examination of SICS in seven subjects across several studies questions the repeatability of the SICS phenomenon. A targeted repeatability trial is required to conclusively answer this question.
Cellular changes at the lid margin
(University of Waterloo, 2017-11-28) Muntz, Alex; Jones, Lyndon; Subbaraman, Lakshman
PURPOSE: The hypothesis underlying this thesis is that CL wear, lid wiper epitheliopathy (LWE), and symptoms of dryness and discomfort may be manifest as cellular changes of the lid marginal epithelium, as a result of mechanical action (e.g. friction). The purpose of this thesis was to elucidate the histology of the lid margin epithelium in relation to CL wear, with a focus on ocular discomfort and dryness. The specific aims of each chapter are outlined below: • Chapter 1: to review the relevant literature and to introduce the reader to the topic area; • Chapter 2: to define the rationale and objectives of this thesis; • Chapter 3: to optimize a method of collecting, staining and imaging cells from the lid margin using impression cytology (IC); • Chapter 4: to assess the utility of the IC method developed in chapter 4, towards characterizing the epithelial cell morphology of the upper lid margin in symptomatic and asymptomatic soft lens (SCL) wearers and non-lens wearers with distinct levels of LWE; • Chapter 5: to assess the lid margins of symptomatic and asymptomatic SCL wearers; • Chapter 6: to assess the lid margins of rigid gas permeable (RGP) and non-CL wearers; • Chapter 7: to cross-compare findings from chapters 5 and 6, and to determine differences between the upper and lower lid margins. • Chapter 8: to conclude the findings and knowledge gained following the above projects, and to point out potential future work directions. METHODS: • Chapter 3: Upon anesthesia (proparacaine hydrochloride, 0.5%), the upper lids of 5 subjects (n=10) were everted and IC was conducted using various membranes (mixed cellulose esters, hydrophilic PTFE, polyethersulfone). Several fixatives (100% methanol, 95% ethanol), cytological stains (Papanicolaou (hematoxylin Gill No.1, OG-6, EA-65), Periodic Acid-Schiff (PAS) and Alcian Blue (AB)) and soak times (1, 3, 5 minutes) were tested. Varying concentrations of fluorescent dyes (Calcein AM, Ethidium homodimer-1, Annexin V) were tested and imaged using confocal laser scanning microscopy (CLSM); • Chapter 4: Fifteen participants were enrolled in three study groups: 5 asymptomatic non-lens wearers with low LWE (average grade of 1.0 or lower in both eyes); 5 adapted, asymptomatic SCL wearers with low LWE; 5 adapted, SCL wearers with high LWE (average grade of 2.0 or higher). Participants completed subjective comfort ratings and LWE was assessed using the Korb Protocol B. IC samples were taken from the upper lid margin using Millicell Cell Culture Inserts and cellular features and sample cellularity evaluated after histochemical and immuno-cytochemical staining as described in the previous chapter; • Chapter 5: Forty adapted SCL wearers were enrolled and equally distributed in two study groups based on self-reported CL-related comfort levels. Comfort was assessed using the Young scheme, the Ocular Surface Disease Index (OSDI), the Contact Lens Dryness Evaluation Questionnaire (CLDEQ-8) and diurnal 0-100 scales for comfort and dryness. LWE was assessed using lissamine green (LG) and IC performed on the upper and lower lid margins as in the previous chapters. The lid wiper (LW) and muco-cutaneous junction (MCJ) cellular areas were defined and dimensioned using a custom programmed software and ImageJ; • Chapter 6: Eighteen RGP wearers and 19 non-lens wearers (nCL) were enrolled in two study groups. Comfort, LWE and IC were assessed as in the previous chapter; • Chapter 7: Study groups analyzed in chapters 5 and 6 were cross-compared (n=77) with regards to clinical signs, comfort scores, LWE and lid margin morphology at both lid margins and width measurements for the LW and MCJ areas. Upper and lower lid margins were also compared. RESULTS: • Chapter 3: IC delivered optimal results using the hydrophilic PTFE membrane. Fixing in 95% ethanol for >20 minutes, then staining in 500µl each of AB, hematoxylin Gill No.1, OG-6 and EA-65 for 3 minutes revealed the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Calcein AM (4µM) and Ethidium (4µM) were combined to successfully show cell esterase activity and compromised cell membranes. Up to 200 microscopy digital images were captured for each sample and stitched into a high-resolution, large scale image of the entire IC span; • Chapter 4: Three distinct cellular morphologies were identified, spanning between the tarsal/marginal conjunctiva, through the LW conjunctiva, to the MCJ at the Marx line. Epithelial cell morphology did not vary with LWE grade or lens wear. Sample cellularity may or may not be altered by lens wear, LWE and/or symptoms. No association was found between LWE and ocular discomfort; • Chapter 5: Average (±SD) upper and lower LWE grades were identical in both groups (0.8 ± 0.7) and did not correlate with any subjective comfort score or other study variable. The average width in the upper LW (415±131 µm) and MCJ (114±43), and lower LW (187±120) and MCJ (90±41) was measured (n=139). Wider LW and MCJ areas correlated with higher LWE grades (p<0.05, r=0.61 to 0.86); • Chapter 6: RGP wearers reported overall similar or better comfort than nCL wearers (p>0.05). Average LWE grades (±SD) were significantly different, for both upper (RGP: 1.66±0.97; nCL: 0.44±0.75; p=0.0002) and lower (RGP: 1.48±0.94; nCL: 0.39±0.49; p=0.0001) lid margins. The average width of the upper (RGP: 666±219 µm; nCL: 265±64; p<0.0001) and lower LW areas (RGP: 518±211; nCL: 224±101; p<0.0001) was significantly higher in RGP wearers, and correlated well with the LWE grade (p<0.01, r=0.78 to 0.89); • Chapter 7: The average (±SD) LWE grade of SCL wearers (0.8 ± 0.8) was greater than in nCL (0.4 ± 0.7, p=0.0125) and lower than in RGP wearers (1.6 ± 0.9, p=0.0015). No significant difference was found between the upper and lower LWE grades in any of the four groups. Longer average CL wear times and older age were correlated with higher LWE grades (Spearman r range: 0.27 to 0.31, p<0.05) and better comfort scores (Spearman r range: 0.25 to 0.44, p<0.05). The width of the upper LW of SCL wearers (415 ± 132 µm) was greater than in nCL (266 ± 64, p=0.0003) and narrower than in RGP wearers (667 ± 219, p=0.0004). The width of the lower LW of SCL wearers (187 ± 120) was up to 2.8 times smaller than in RGP wearers (519 ± 212, p<0.0001), but similar to nCL (225 ± 102, p=0.072). The upper LW was significantly wider than the lower LW in all participants (p<0.05), except for RGP wearers. CONCLUSIONS: A protocol for collecting, staining, imaging and analyzing cells from the lid marginal epithelium was developed and showed appropriate sensitivity for identifying distinct cellular morphology and varying degrees of keratinization. We presented the first account to show a correlation between LWE grade and widths of the LW and MCJ areas after histological inspection. By identifying enlarged areas of keratinization in the LW of LWE versus non-LWE subjects, we provide evidence to support the frictional etiology of LWE and possibly also the Marx line. This is the first study to show that SCL lens wear is associated with enlarged LW areas in the upper and lower lid margins, providing strong evidence that the mechanical interaction with a CL may alter the cyto-morphology of the lid margin epithelium. The effect of RGP lenses is similar and significantly more pronounced. Regardless of CL wear, the LW at the upper lid margin is wider than the lower one, upholding the frictional role of the LW during habitual blinking.
Contact Lenses and Tear Film Lipids.
(University of Waterloo, 2017-12-20) Walther, Hendrik; Jones, Lyndon; Subbaraman, Lakshman
Introduction Lipids are essential tear components that aid the stability of the tear film (TF) to protect it from excess evaporation. The composition, conformation, and function of TF lipids are jeopardized by external factors such as contact lens (CL) wear and environmental elements (i.e. UV radiation, oxidation). Specifically, silicone hydrogel (SiHy) CLs exhibit relatively high deposition of TF lipids that may be associated with visual disturbances and discomfort. Additionally, lipids are degraded by oxidation and may cause alterations of the TF lipid layer, which in turn might be a source for dry eye symptoms. The overall goal of this thesis was to evaluate the quantity and location of lipid deposition on various CL materials over time and also to assess the impact lipid contamination may have on various care products and TF quality measurements. The specific aims of each chapter of this thesis were as follows: • Chapter 3: To determine the efficacy of multi-purpose solutions (MPS) on the removal of cholesterol deposits from SiHy lens materials. • Chapter 4: To analyze the uptake of cholesterol on SiHy and conventional hydrogel (CH) daily disposable (DD) CL materials using an in vitro radiochemical detection method. • Chapter 5: To evaluate the differences in lipid uptake and penetration in DD CL using the conventional “in-vial” method compared to a novel in vitro eye model. • Chapter 6: To develop a novel in vitro model to determine pre-lens non-invasive break- up times (NIBUT) and to subsequently compare the break-up times over five contemporary DD lens materials. • Chapter 7: To optimize and develop a method to determine and quantify lipid peroxidation by-products that indicates oxidative stress in tears. Materials and Methods • Chapter 3: Five contemporary SiHy lens materials were incubated for 7 days using a radiochemical experiment. Additionally, lenses were stored and cleaned in different MPSs using a rub and rinse technique. Lipids were then extracted from lenses with 2:1 chloroform:methanol, analyzed in a beta-particle radiation counter and μg/lens of cholesterol was determined. • Chapter 4: Seven different commercially available DD CLs were incubated for 16 hours to determine the impact of material composition on cholesterol deposition. Subsequent to the incubation, lenses were extracted using 2:1 chloroform:methanol and the extracts were analyzed in a beta-particle radiation counter and (ng/lens) extrapolated from standard curves. • Chapter 5: Seven DD CLs were incubated for 4 and 12 hours in an artificial tear solution (ATS) containing fluorescently-labelled cholesterol (7-nitrobenz-2-oxa-1,3-diazol-4- yl-cholesterol, or NBD-cholesterol). Additionally, CLs were incubated in an “in-vial” condition and compared to a novel in vitro eye platform, designed to simulate physiological tear flow, tear volume, and ‘simulated’ blinking. After the incubation period, the CLs were analyzed using a laser scanning confocal microscope (LSCM), and quantitative analyses for penetration depth and relative fluorescence intensity values were carried out. • Chapter 6: Five DD lens materials were incubated in an artificial tear solution using a model blink cell that mimics intermittent air exposure. CLs were incubated by repeatedly being submerged and exposed to air for up to 16 hours. A corneal topographer (Topcon CA-100) was used to illuminate the lens surfaces with placido rings, which were captured with a digital video camera and from which NIBUTs of the CL materials were determined. • Chapter 7: Tear samples were collected using calibrated disposable capillary tubes and various assays that quantify lipid peroxidation by-products were compared against each other: thiobarbituric acid reactive substances (TBARS) assay, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and oxidized low-density-lipoproteins (OxLDL) enzyme-linked immunosorbent assays (ELISAs). Pooled and individual tear samples were diluted in a wide range to determine the lowest volume of tears that could be used. Subsequently, the fluorescence was measured with a fluorescence spectrophotometer at 530 nm (excitation) and 590 nm (emission), as well as their absorbance at 450 nm. Results • Chapter 3: For all lens materials, only one of the multipurpose solutions removed more cholesterol than any other test solution; however, the amount of cholesterol removed from the individual CLs was statistically significant only for the two lens materials that deposited the most: balafilcon A (0.93±0.02μg/lens) and senofilcon A (0.95±0.01μg/lens). All of the other solutions evaluated showed no significant effect on lipid removal. • Chapter 4: Cholesterol deposited significantly more on SiHy lenses than CHs. The uptake of cholesterol ranged from 22.63 ± 2.98 ng/lens to 97.94 ± 4.18 ng/lens for all lens materials, with narafilcon A accumulating the largest quantity of cholesterol. The accumulation of cholesterol was shown to be continuous throughout the 16 hours of incubation without reaching a plateau. • Chapter 5: The depth of penetration of NBD-cholesterol varied between the vial and the eye-blink platform. In general, SiHy lenses showed higher intensities of NBD- cholesterol than CH materials and the fluorescence intensities also varied between the incubation methods as well as the lens materials. • Chapter 6: Overall, NIBUTs ranged from 26.19 ± 5.79 s to 1.23 ± 0.13 s. After the initial (T0) break-up times were determined, CH CLs revealed significantly longer NIBUTs than SiHy CLs. After 16 hours of incubation, the SiHy lens material delefilcon A had the longest break-up time. Significant changes of NIBUTs within the lens materials varied between the examined time points. After 16 hours, all CLs showed significant reductions in NIBUTs in comparison to T0. • Chapter 7: After tear samples were pooled and concentrated, 0.056±0.004 μM of MDA could be measured using the TBARS assay. After optimizing various ELISAs, OxLDL in individual tear samples (2.5μL) ranged between 45.59 ± 2.95 ng/mL and 28.24 ± 4.66 ng/mL. All measurements using the MDA- and 4-HNE ELISA were below the assays limit of detection. Conclusions • Chapter 3: Lipid-removal efficacy varies depending on the combination of lens material and solution. Only one MPS showed a significant reduction of lipids for any of the tested lens materials. • Chapter 4: For the periods of time that DD lens materials are worn, cholesterol deposits significantly more onto SiHy lenses than CHs. This could have implications for wearers who have higher levels of lipid in their tears that are fitted with SiHy DD materials. • Chapter 5: This study provides a novel in vitro approach to evaluate deposition and penetration of lipids on CLs. We show that the traditional “in-vial” incubation method exposes the CLs to an excessively high amount of ATS, which results in an overestimation for cholesterol deposition. The novel eye-platform, provides a more natural environment for in vitro lens incubation studies which will consequently better elucidate the interactions between CLs and TF components. • Chapter 6: NIBUT values reduced gradually over time and varying levels of deposition on different CLs may impact the measured pre-lens NIBUT of various lens materials. While NIBUT of CH materials are longer than that obtained with SiHy materials immediately out of the blister pack, it appears that after TF exposure, the NIBUTs determined between CH and SiHy DD materials are very similar. • Chapter 7: Assays for oxidative stress were optimized and showed that oxidative stress is detectible in small quantities of tears (2.5 μL). These techniques could be employed to determine oxidative stress in TF lipids, which could potentially help to identify patients with dry eye and CL discomfort. This thesis has provided previously unavailable information on lipid deposition on CLs and its effect on clinically relevant TF quality measurements. The results showed that current CL cleaning solutions fail to efficiently remove lipid contaminations and that DD SiHy lens materials provide options for clinicians to consider when patients experience complications and excess lipid uptake with daily wear lens materials. Furthermore, this thesis has presented novel in vitro methods that will be useful for other researchers and the CL industry to appropriately test and predict CL performance.
Contemporary Diagnosis and Management of Dry Eye
(University of Waterloo, 2016-08-31) Ngo, William; Jones, Lyndon; Srinivasan, Sruthi
Introduction Dry eye (DE) disease is characterized by symptoms including, but not limited to, ocular stinging, burning, and tearing. The symptoms can range from being mildly irritating, to severely debilitating and negatively impacting quality of life. The impairment of the tear film, lacrimal functional unit, and meibomian glands (MGs) causes desiccation of the ocular surface, which in turn promotes and exacerbates inflammation. Dry eye is a multifactorial disease and the many causative factors can be completely exclusive from each other. This necessitates that the management of DE be multi-faceted. Dry eye disease affects millions of people around the world and this number will increase as the elderly population rise over the next few decades. However, emerging technologies are allowing clinicians and scientists to constantly discover new ways to diagnose and manage various aspects of DE. The global aim of this thesis was to evaluate some of the contemporary methods used in the diagnosis and management of DE disease. The specific aims of each chapter were as follows: • Chapter 3: To determine whether an experimental spectral domain ultra-long optical coherence tomographer (UL-OCT) can image MGs, and to compare its performance to the Heidelberg Retina Tomograph 3 (HRT3) with Rostock Cornea Module (RCM) in vivo laser scanning confocal microscope (Heidelberg Engineering GmbH, Heidelberg, Germany) and the Keratograph 5M (K5M) (OCULUS, Wetzlar, Germany). • Chapter 4: To determine the inter- and intra-observer repeatability in using the Keratograph 4 (K4) and K5M to grade MG dropout using meibography grading scales. • Chapter 5: To quantify the association of DE diagnostic tests to DE symptoms in an age-matched female cohort. • Chapter 6: To determine the effectiveness of an eyelid warming device in the management of MG dysfunction (MGD). • Chapter 7: To evaluate the effect of lid debridement-scaling (LDS) on DE signs and symptoms in individuals with Sjӧgren’s syndrome (SS). • Chapter 8: To determine the combined effect of a lubricant eye drop, lid hygiene, and omega 3 fatty acids on DE signs and symptoms. Methods • Chapter 3: The superior eyelids of 12 participants were everted and imaged using the UL-OCT. The inferior and superior eyelids were then everted and imaged using the K5M. Finally, the inferior eyelids were everted and imaged with the HRT3/RCM. • Chapter 4: The inferior and superior eyelids of 40 participants were imaged 3 times each on both the K4 and K5M. The images were split into one training and two study sets; the latter were graded (4-point meibography scale) by two observers on two separate occasions (24hrs apart) to examine repeatability. Semi-objective grading of MG dropout was conducted using ImageJ on K4 and K5M images. A 7-point meibography scale was used to grade a separate set of K5M images. • Chapter 5: Twenty females symptomatic of DE (Ocular Surface Disease Index, OSDI, ≥ 13) were age-matched to 20 asymptomatic females (OSDI < 13). Non-invasive tear breakup time (NIBUT), ocular staining, meibum quality, number of obstructed glands, lid wiper epitheliopathy (LWE), Line of Marx (LOM) placement, eyelid margin score, Schirmer’s test, meibography, and visual acuity were compared between the two groups. • Chapter 6: This was a prospective, randomized, controlled, single-masked, bilateral eye study that enrolled 29 participants. Participants were randomized into either the EyeBag group or the control group. Participants in the EyeBag group were instructed to use the EyeBag 10 minutes 2x/day, and the control group remained on their own DE treatment regimen (if applicable). All participants were seen at baseline, 2 weeks and 4 weeks. At 4 weeks, participants in the EyeBag group were asked to stop using the EyeBag. All participants were seen again at the 8 week mark. Primary outcomes were the OSDI, Current Symptoms Questionnaire (CSQ), MG score (MGS), and non-invasive tear break-up time (NIBUT). • Chapter 7: This prospective randomized controlled study enrolled 14 female participants with SS. Seven participants were randomized into the treatment group, where they were selected to receive LDS, the remainder did not receive LDS and served as controls. Lid debridement-scaling was conducted using a stainless steel golf club spud (Hilco Wilson Ophthalmics, Plainville, MA) on both the upper and lower eyelids of both eyes. Outcome variables were assessed prior to LDS and again 1 month later. The outcome variables were the OSDI, Symptoms iN Assessment of Dry Eye (SANDE) visual analogue scores, Sjӧgren’s International Collaborative Clinical Alliance Ocular Staining Score (SICCA OSS), fluorescein tear breakup time (FLBUT), MGS, MG yielding liquid secretions score (MGYLS), and LOM position. • Chapter 8: This prospective study enrolled 28 DE participants. Participants were instructed to use the TheraTears® Lubricant Eye Drops at least 2-4x a day, TheraTears® SteriLid 1-2x a day, and TheraTears® Nutrition 3 gel caps once a day. Participants were followed up at baseline, 1 month and 3 months. Outcome variables were the OSDI, SANDE, NIBUT, osmolarity, number of MGs blocked (#MG blocked), meibum quality, eyelid margin features, Schirmer’s test, tear film lipid layer thickness (LLT), meniscus height, corneal and conjunctival staining. Results • Chapter 3: All twelve participants (7 female, 5 male) completed the study. The only instrument that was able to successfully image MGs was the K5M. • Chapter 4: When using the 4-point scale, inter-observer mean difference (MD) was 0.08±0.55 on day 1 and 0.13±0.50 on day 2, and the concordance correlation coefficient (CCC) was 0.79 and 0.81 on days 1 and 2 respectively. Intra-observer MD was 0.04±0.54, CCC=0.79 for observer 1, and -0.09±0.60, CCC=0.74 for observer 2. For the 7-point scale, inter-observer MD was 0.05±0.45, CCC=0.89 on day 1 and 0.01±0.41, CCC=0.91 on day 2. Intra-observer MD was -0.10±0.35, CCC=0.93 for observer 1 and -0.06±0.30, CCC=0.95 for observer 2. Percentage dropout measured between the K4 and K5M images showed lack of agreement, with only a 21.8% coefficient of repeatability. • Chapter 5: Twenty participant-pairs completed the study. The age (median/interquartile range(IQR)) of the symptomatic group was (60/15) and the asymptomatic group was (62/15). The diagnostic tests (median/IQR, p-value) that were significantly different between the symptomatic group vs. the asymptomatic group were OSDI (35.4/35.4 vs. 3.1/6.7, p < 0.01), NIBUT (2.1s/0.7s vs. 3.0s/3.0s, p = 0.01), meibum quality (3.0/0.0 grade units vs. 2.0/1.0 grade units, p < 0.01), number of obstructed glands (7.0/2.0 glands vs. 5.0/4.8 glands, p < 0.01), and ocular staining (5.5/3.8 grade units vs. 0.5/1.0 grade units, p < 0.01). The diagnostic tests (area under curve (AUC), odds ratio (OR)) that were most strongly associated with DE symptoms were ocular staining (0.93, 5.0), number of glands obstructed (0.79, 2.6), meibum quality (0.76, 2.4), and NIBUT (0.74, 3.2) (all p < 0.05). There was no significant difference between the two groups for the other DE diagnostic tests (all p > 0.05), and similarly, no significant association to DE symptoms (all p > 0.05). • Chapter 6: Twenty five participants completed the study (mean age 38±15 years, 7 male). There was a significant change in OSDI over time for the EyeBag group (mean values±SD, baseline: 39.1±12.5, week 2: 26.8±11.2, week 4: 26.6±26.6, week 8: 27.7±14.6; p<0.05), but no significant change in the control group. There was a significant improvement in symptoms immediately after conducting the EyeBag based on at-home CSQ scores (Δ=-5.0 points, p<0.05), but no significant change in the control group. There was no significant change in MGS and NIBUT over time for either group. • Chapter 7: There were 13 participants that completed the study. Data from the right eye only were analyzed. For the control group (n=6, mean age=62±12), the pre LDS, post LDS, and significance level (pre-mean±SD, post-mean±SD; p-value) were: OSDI (58.3±22.1, 48.3±29.0; p=0.051), SANDE (77.4±22.1, 89.6±32.6; p=0.20), SICCA OSS (7.0±4.5, 8.2±3.5; p=0.25), MGS (1.3±1.5, 1.0±0.9; p=0.75), MGYLS (0.3±0.5, 0.0±0.0; p=0.50), FLBUT (2.99 ±1.54, 2.85±1.79; p=0.63), LOM (2.0±0.0, 2.0±0.0, p=n/a). For the treatment group (n=7, mean age=58±8), the pre LDS, post LDS, and significance level were: OSDI (63.2±13.3, 46.9±19.4; p=0.04), SANDE (72.6±17.1, 77.0±28.0; p=0.54), SICCA OSS (6.6±2.9, 5.0±3.9; p=0.02), MGS (1.0±1.2, 3.1±1.7; p=0.01), MGYLS (0.0±0.0, 0.6±1.0; p=0.50), FLBUT (3.13±0.81, 3.45±1.03; p=0.53), LOM (0.9±0.9, 1.0±1.0, p=1.00). • Chapter 8: There were 20 participants (mean age = 43, from 23 to 66, 17F, 3M) that completed the study. On average, participants used the Lubricant Eye Drop 2.4x/day, the SteriLid 1.1x/day, and the Nutrition 3 gel caps 1x/day. There was a significant change over time (p<0.05) for OSDI (-21.2 points), SANDE (-32.4 points), NIBUT (+0.43s), eyelid margin features (-1.1 grade), meibum quality (-1.0 grade), and #MG blocked (-4.0 glands). Conclusions • Chapter 3: The UL-OCT was unable to image MGs. The HRT3/RCM imaged structures that resembled dermal rete pegs and papillae. Of the three methods used in this study, the only device that was able to successfully image MGs was the K5M. • Chapter 4: Observers graded from -1 to +1 grade units between and within themselves for a 4-point scale, 95% of the time. Although the inter- and intra-observer repeatability of the K4 and K5M were very similar, a low level of agreement in percentage dropout between K4 and K5M images suggests that the two instruments cannot be interchanged. Using a finer scale may be beneficial for detecting change over time. • Chapter 5: The diagnostic tests that were most strongly associated with DE symptoms in older women were ocular staining, meibum quality, number of glands obstructed, and tear film stability. • Chapter 6: The MGDRx® EyeBag was effective at relieving symptoms in participants with DE, but the effect on MG function and tear stability when used for only 4 weeks was modest. • Chapter 7: This pilot study showed that LDS improved symptoms, ocular staining and MG function for the group that received LDS. This indicates that LDS can aid in the management of SS DE. • Chapter 8: After using a combination of TheraTears® Lubricant Eye Drop, SteriLid, and Nutrition, participants experience significant relief in both DE symptoms and signs. This thesis was able to evaluate and suggest improvements to meibography techniques and grading. In addition, this thesis was also able to evaluate the effectiveness of various contemporary methods for DE treatment. The methods used in this study are clinically accessible and clinicians are free to use these findings and apply them directly to their clinical practice.
Development of an in vitro eye model to better represent in vivo physiological conditions
(University of Waterloo, 2021-06-23) Chan, Vivian; Jones, Lyndon; Ngo, William
Purpose The purpose of this thesis was to optimize a novel in vitro blink model, the OcuBlink, to best mimic the physiological characteristics of the human eye. By improving this in vitro model, the results from in vitro studies using this model can be more representative of in vivo studies. Methods The first experimental chapter of this thesis, Chapter 3, explores the results of active lysozyme deposition on contact lenses using the blink model. An ex vivo active lysozyme deposition study was referenced to determine the ideal flow rate for the blink model. These parameters were used to determine the active lysozyme deposition data for several other lens materials. The blink model was directly compared to a simple vial at 8 hours of incubation to show the difference between the two in vitro models. The experiments in Chapter 3 led to several developmental improvements to the blink model design. Chapter 4 explores the changes to the blink model from its initial design through different iterations to incorporate a heated system to simulate ocular temperatures. Chapter 5 uses the new blink model improvements to study contact lens dehydration. The effect of incubation temperature, incubation solution, and in vitro model design were explored. The comparison between the vial system and the blink model showed a difference in water content patterns over time. Results With ex vivo data as a reference, the blink model was able to replicate the active lysozyme deposition data on etafilcon A lenses. The parameters of the blink model were used to determine active lysozyme deposition on other contact lenses. This study provided the expected range for ex vivo active lysozyme deposition for these lens materials as this data was not available in the literature at the time of the study. The comparison of the in vitro models showed that contact lens material plays a large role in active lysozyme deposition patterns over time. The different iterations of the blink model show how different materials and designs can improve and progress in vitro testing of ocular studies. The most significant addition to the blink model was the incorporation of a heated element to allow studies to be conducted at ocular temperatures. The improved blink model showed a decrease in water content for all lenses for an increase in incubation temperature. The incubation solution did not have an effect on water content for most tested lenses, however, lens material played a major role. All lenses decreased in water content after the first hour of incubation. With the blink model, lenses showed a recovery in water content over 16 hours, however the lenses showed a plateau effect in a simple vial model. The recovery in water content on the blink model has not been seen on other in vitro or in vivo studies to date and will need further testing to better understand the phenomenon. Conclusion Although the data has yet to be validated with in vivo data, this thesis shows that the blink model has promise as a predictive tool for in vivo studies. The advanced in vitro blink model can be adjusted per study as required to fulfil experimental requirements. The ultimate goal of the blink model is to produce results that are more representative of in vivo data compared to more simplistic in vitro models while minimizing the cost and time of animal models and clinical trials where appropriate.
The Effect of Freezing on the Elution of PVA from Contact Lenses
(University of Waterloo, 2024-04-30) Shukla, Manish; Jones, Lyndon; Hui, Alex
Contact lenses are widely used, with over 140 million wearers globally. Wearing contact lenses can cause symptoms of discomfort and dryness, which affect nearly half of all wearers. To address this concern, this thesis explores the release of polyvinyl alcohol (PVA) from contact lenses, aiming to improve comfort through controlled elution. PVA forms a protective film when placed on the ocular surface and serves to reduce ocular discomfort. This research specifically studies the impact of freezing on PVA interaction with various contact lens materials and its subsequent release kinetics. This thesis hypothesizes that freezing enhances the hydrogen bonding of PVA to lens materials, enabling the formation of a surface layer on contact lenses and increasing PVA elution. To investigate this hypothesis, commercial lenses (Acuvue® Oasys – senofilcon A, DAILIES® AquaComfort PLUS® - nelfilcon A, 1-Day Acuvue® Moist® - etafilcon A) were soaked in 2.5% w/v high molecular weight PVA solutions at 37°C for 48 hours, followed by 1 hour at either room temperature or freezing at -80°C. The results demonstrate a significant (p<0.05) increase in the cumulative PVA release from nelfilcon A lenses after 24 hours following freezing at -80°C for one hour, with 55.07 ± 2.46 μg of high molecular weight PVA released in comparison to lenses kept at room temperature which showed 46.16 ± 6.94 μg of PVA release. In contrast to nelfilcon A, etafilcon A and senofilcon A did not show a significant (p>0.05) change in the amount of PVA released after freezing. Etafilcon A lenses released 17.03 ± 3.03 μg and 20.21 ± 2.51 μg (p>0.05), and senofilcon A showed 20.33 ± 6.60 μg and 24.14 ± 2.58 μg (p>0.05) at room temperature and after freezing at -80°C for one hour, respectively, suggesting that freezing enhances these iv effects only for nelfilcon A lenses. To further explore the impact of PVA with lenses, experiments with synthesized lenses (pHEMA and PVA loaded pHEMA) were performed, which demonstrated that the presence of PVA inside the lens significantly (p<0.05) impacts subsequent PVA loading and release and the freezing effect. The cumulative release of PVA over 24 hours from pHEMA lenses were 32.64 ± 5.48 μg and 36.25 ± 6.11 μg (p>0.05), at room temperature and after freezing at -80°C for one hour, respectively. PVA loaded pHEMA lenses, in contrast, showed a significant (p<0.05) increase in the cumulative PVA release over 24 hours after freezing, rising from 42.88 ± 4.96 μg to 47.39 ± 6.26 μg after one hour at -80°C. The study emphasizes the importance of PVA incorporation within contact lenses to observe a substantial impact on release after soaking or freezing. The findings suggest that the freezing technique has potential applications in enhancing the release of comfort agents such as PVA from contact lenses, especially those containing PVA internally. In conclusion, this research provides insights into optimizing contact lens design for improved comfort by utilizing PVA release. The demonstrated impact of freezing on nelfilcon A lenses indicates a promising avenue for enhancing the release of comfort agents.
Examination of Contact Lenses and Dry Eye Using Evaporimetry
(University of Waterloo, 2022-01-14) Wong, Stephanie; Jones, Lyndon; Murphy, Paul
Purpose: Evaporimetry is a non-invasive technique used to assess the stability of the tear film. The test measures the rate of tear evaporation, and has been used to investigate dry eye, contact lenses, and the efficacy of different treatments for dry eye and contact lens (CL) discomfort. There is currently only one modified dermatological instrument available for practitioners, and experts have stated a need to develop evaporimeters suitable for use in clinical practice. The purpose of this thesis was two-fold, namely to (i) evaluate the commercially available evaporimeter, and (ii) describe the design, development, and testing of a novel evaporimeter. The overall aims were (i) to assess the calibration of the only commercially available evaporimeter (Eye-VapoMeter), and to investigate its ability to detect in vitro differences between soft CLs, and (2) to describe the development, in vitro, and in vivo testing of a novel binocular evaporimeter. Methods and Materials: (i) In vitro differences between 7 silicone hydrogel and 9 hydrogel CLs were measured with the Eye-VapoMeter. The change in evaporation rate per minute was calculated from the slope of the evaporation rate over time. Four sequential 10-minute time periods were investigated from 0 to 40 minutes. (ii) Calibration of the Eye-VapoMeter was investigated by simulating evaporation from different ocular surface areas and by modifying the air volume inside the evaporimeter goggle using two types of model eyes. The absolute evaporation rate was determined from the slope of water loss over time. The unadjusted evaporation rate from the instrument was measured with different areas and volumes inside the evaporimeter. A linear regression was used to determine the correction factor for each goggle volume based on the unadjusted evaporation rate and absolute evaporation rate. (iii) A novel binocular evaporimeter was developed to measure the tear evaporation rate (TER) from the ocular surface. In vitro testing of the new evaporimeter was performed using four elliptical model eyes with different surface areas (1 to 2.5 cm² in 0.5 cm² steps) and air volumes within the evaporimeter. Measurements were recorded for each side of the goggle. (iv) In vivo pilot testing was performed by conducting a series of experiments on volunteers to determine the best way of performing evaporimetry with the new instrument. Measurements were taken with the eyes open and closed (n=5), with the effect of a liposomal spray (CALMO® Eye Spray), and with a single application of an artificial lubricant (Refresh Tears®) (n=5). Fixation was tested by comparing evaporation rates with the eyes open, and blinking normally in downgaze, primary gaze, and upgaze (n=1). Optimal blink rate was investigated using blink rates of three or five seconds in volunteers with self-reported dry eye (n=3). (v) The effect of a lipid nano-emulsion was assessed. Thirty-six non-CL wearers were enrolled and screened. Twenty-one participants were suitable and classified as dry eye or non-dry eye using the Ocular Surface Disease Index (OSDI) and non-invasive break-up time (dry eye: OSDI ≥13 and break-up time ≤5 seconds in the worst eye). At the test visit, two baseline TERs were taken, 20 minutes apart. A single dose of Systane® Complete was instilled, and TER assessed at 10, 30, and 60 minutes post-instillation. (vi) The effect of CL wear was assessed. Twenty CL wearers were screened and classified using the Contact Lens Dry Eye Questionnaire (CLDEQ-8) as asymptomatic (CLDEQ-8<12) or symptomatic (CLDEQ-8≥12). Two baseline TERs were recorded after a 15-minute interval. Participants were randomized to wear delefilcon A in one eye and nesofilcon A in the other eye. TER was assessed after 15 minutes and 6 hours of CL wear. Results: (i) In vitro measurements with the Eye-VapoMeter found a significant difference in evaporation rates reported for each 10-minute period for each CL. Evaporation rate varied with CL material, water content, and presence of an internal wetting agent. (ii) Calibration measurements showed that water loss occurred at a linear rate. Correction factors were calculated for the Eye-VapoMeter. All graphs of the correction factor and evaporimeter volume were fit with a second order polynomial non-linear regression. (iii) In vitro measurements with the novel evaporimeter measured a significantly lower evaporation rate with the smallest model eye compared to the larger ones, and a significantly lower evaporation rate for the 10 cm³ volume compared to the 13 and 18.63 cm³ volumes. (iv) Pilot testing demonstrated that the relative humidity (RH) significantly changed in each side of the goggle when the novel evaporimeter was placed over the open and closed eye. No significant differences in RH were detected between the goggles. The TER was significantly lower immediately after application of the liposomal spray compared to the second baseline measurement and 15 minutes after the spray was applied. Use of an artificial lubricant found significantly higher TER in both eyes after instillation compared to the first baseline measurement and 15 minutes post-instillation. Comparison of different positions of gaze revealed less change in RH over time in downgaze. Comparison of blink rate found that 2 out of 3 participants preferred a five second blink rate. (v) Twenty people (10 non-dry eye, 10 dry eye) completed the lipid nano-emulsion study. Changes in TER were observed during the study. Nano-emulsion instillation produced an initial increase in TER after 10 minutes, and a reduction in TER after 30 minutes. (vi) Twenty people (10 asymptomatic, 10 symptomatic) completed the CL study. The TER was significantly higher after 6 hours of CL wear. No significant difference in TER was detected between the two groups, or between CL type (delefilcon A and nesofilcon A) after 6 hours of wear. Conclusions: (1) Using a new in vitro technique, the Eye-VapoMeter was able to detect differences in evaporation rate from a range of CLs differentiated by material, water content, and presence of wetting agent. (2) Calibration of the Eye-VapoMeter found the relationship between correction factor and volume was best fit with a second order non-linear regression. (3) A novel closed-chamber binocular evaporimeter was designed, developed, and tested. (4) In vitro testing of the novel evaporimeter detected lower evaporation rates with a smaller surface area and volume. (5) In vivo testing demonstrated that the novel evaporimeter was able to: (a) measure higher TERs in dry eye participants compared to those without dry eye; (b) measure significant decreases in TER following the instillation of a lipid eye drop; (c) measure significantly higher TERs associated with CL wear.
Extended Release of Ciprofloxacin from Commercial Silicone-Hydrogel and Conventional-Hydrogel Contact Lenses containing Vitamin E Diffusion Barriers
(University of Waterloo, 2024-08-13) Al Atrach, Mehdi; Jones, Lyndon; Phan, Chau-Minh
Abstract Purpose: Contact Lenses (CLs) are ophthalmic devices globally used by more than 140 million people to correct vision problems. To overcome the drawbacks of eye drops, drug-delivering CLs have been given much attention because they increase the bioavailability of ocular drugs, resulting in increasing efficacy and reducing potential side effects. These systems typically have a burst and rapid release. To address these concerns, this study aims to develop a contact lens-based ocular drug delivery system using vitamin E as a diffusion barrier to extend the release duration of ciprofloxacin. Methods: The purpose of the first experiment in the thesis (Chapter 3) was to develop and evaluate drug delivery contact lenses: Four commercial silicone hydrogel lenses (senofilcon A, lotrafilcon B, comfilcon A, and samfilcon A) and one conventional hydrogel lens (etafilcon A) were soaked for 24 hours in various concentrations of vitamin E dissolved in ethanol (0.0125 - 0.2 g/mL). The amount of vitamin E loaded was calculated by measuring the dry lens weight before and after vitamin E loading. The lenses were loaded with ciprofloxacin for 24 hours. The amount of ciprofloxacin loaded was determined using an extraction protocol. The drug release was evaluated in phosphate-buffered saline solution in an amber glass vial, at 37°C with shaking. The amount of ciprofloxacin released was measured using a UV-VIS spectrophotometer at 270 nm. The second experimental chapter (Chapter 4) aimed to evaluate the effect of ciprofloxacin loading duration on the uptake and release profiles of senofilcon A. This was done by applying the same method described in Chapter 3, except ciprofloxacin loading durations for senofilcon A were 24 hours and 7 days. Results: The data showed that there was a decrease in ciprofloxacin loading with increasing amounts of vitamin E loaded into the silicone hydrogel lenses. For each lens type, there was an optimal amount of vitamin E loaded that extended the release duration of the drug from 1 hour (without vitamin E) to as long as 16 hours. Senofilcon A with 0.025 g/mL vitamin E loaded exhibited the longest-sustained release for all lens types, achieving 16 hours of release. In contrast, vitamin E loaded into etafilcon A had no effect on the amount of drug loaded or the release duration. The data obtained in Chapter 4 showed no significant difference in the uptake and release profiles among the different loading durations (24 hours and 7 days), except in lenses with 0.025 and 0.05 g/mL vitamin E concentrations, which absorbed and released more drugs in the 7-day loading duration experiment. In addition, senofilcon A longest release duration (16 hours) achieved in chapter 3 did not further increase in chapter 4 after increasing the drug loading duration to 7 days. Conclusion: Vitamin E can be used as a diffusion barrier with commercial silicone hydrogel lenses to provide sustained release of ciprofloxacin. The results suggest that vitamin E may form blockages in channels within a silicone hydrogel lens material, thereby forcing a longer path for drugs to diffuse into and out of the lens material. There is an optimal amount of vitamin E that needs to be loaded to extend the release duration, and this is lens material dependent. Additionally, increasing the drug loading duration to 7 days was not enough to increase the drug-loaded amount, and subsequently the release duration specifically for senofilcon A loaded with 0.2 g/ml vitamin E concentration.
Fabrication of an enzyme responsive biomaterial for the treatment of recurrent corneal erosion
(University of Waterloo, 2023-04-11) Bose, Susmita; Jones, Lyndon
Purpose: Recurrent corneal erosion (RCE) arising from the loss of superficial corneal epithelial cells causes tremendous ocular pain affecting one’s productivity and quality of life. RCE was traditionally treated with eye lubricants and patching (to stop blinking). Soft bandage contact lenses (BCLs), which enable the use of vision during the healing process, have become more widely used in recent years. Because BCLs do not yet contain the therapeutic components necessary for ocular surface repair, they do not outperform ocular lubricants in terms of effectiveness or recovery time. This is a problem. Multiple studies have shown elevated levels of matrix metalloproteinase (MMP) enzymes, particularly MMP-9 and MMP-2, in RCE. The purpose of this thesis was to develop an enzyme-triggered therapeutic release platform using a unique gelatin methacrylate formulation (GelMA+) and bovine lactoferrin (BLF), a potential corneal wound healing therapeutic. Method: Two different formulations of GelMA+ gels were synthesized: 20% w/v and 30% w/v GelMA+ gels. To determine the effect of polymer concentration on the physical characteristics and cytotoxicity of the gels, physical characterization, including mechanical strength, swelling behaviour, pore size, optical clarity, degradation profile in the enzyme MMP-9 and MMP-8, were evaluated. To evaluate whether GelMA+ is suitable to be a biomaterial, cytotoxicity assays (alamarBlue and live/dead assay) were performed on both the GelMA+ formulations. These physical and biological characterization of GelMA+ gels helped to choose the specific GelMA+ formulation for studying the in vitro release profile of BLF from the GelMA+ matrix. To assess the efficacy and therapeutic window of BLF for corneal epithelial cells wound healing, a scratch induced assay model was used. Results: GelMA showed a tunable profile suitable for the diffusion- and enzyme-mediated controlled release of therapeutics above the molecular weight region of 70 kDa. Owing to the high polymer concentration as opposed to 20% w/v GelMA+, the 30% w/v GelMA+ had a higher crosslinking density, tensile strength, smaller pore size, and lower swelling ratio than the 20% w/v GelMA+ (p<0.05). The degradation rate of the 20% w/v gel was much faster (p<0.001), degrading almost completely after 48 hours at 300 µg/mL of MMP-9 whereas 30% w/v GelMA+ gels took almost 6 days to achieve around 95% degradation in the same MMP-9 concentration. After 5 days, no cytotoxicity was detected in the live/dead staining for either formulation, but the 30% w/v GelMA+ showed significantly higher cell viability (p<0.05). In the BLF release study, no burst release of BLF was observed for the 30% w/v gel, and the therapeutic release was sustained over 5 days. The rate of release from the gel significantly increased with increasing concentrations of MMP-9 (p<0.001) and correlated to the rate of degradation of the gels. BLF at 10 mg/mL induced complete wound closure in four days, and 7.5 mg/mL completely closed the wound in six days, suggesting that BLF might be an excellent therapeutic option for corneal abnormalities brought on by mechanical injury. A concentration of 7.5mg/mL or 10 mg/mL BLF would be an appropriate therapeutic window to be released from 30% w/v GelMA+ for treating RCE within a week. Conclusion: The results showed that the degradation of GelMA+ can be tuned by modifying the crosslinking density or exposure to different concentrations of MMP-9. The release of BLF from 30% w/v GelMA+ is driven by a combination of diffusion and degradation of the material by MMP-9 enzymes. Hence the therapeutic releasing enzyme responsive GelMA+ as a BCL can be potential future treatment for RCE. Future work will focus on optimizing the materials to deliver other therapeutic agents at physiologically relevant concentrations of MMP enzymes found in the tear fluid.
Fabrication of microfluidic chip using 3D printing for ocular cell studies
(University of Waterloo, 2023-09-25) Ramasamy, Megala; Jones, Lyndon; Phan, Chau-Minh
Purpose: This thesis aimed to create a polydimethylsiloxane (PDMS) microfluidic chip utilizing low-cost commercial 3D printers and to integrate human corneal epithelial cells (HCEC) into the fabricated chip. Methods: The purpose of the first experimental chapter (Chapter 3) of this thesis was to develop PDMS devices from 3D printed moulds of two commercially available 3D printers: the Formlabs Form 3B+, which employs Stereolithography (SLA) technology, and the AnyCubic D2 DLP, which utilizes Digital Light Processing (DLP) technology. The fabrication of PDMS from 3D printed moulds can be achieved by introducing three simple post-processing steps: heating, sanding, and nail polish coating. A biomaterial coating was applied to the surfaces of the PDMS devices to render their surfaces hydrophilic. The second experimental chapter (Chapter 4) centred on incorporating HCEC into the PDMS device fabricated using the method described in Chapter 3. Results: Both 3D printers could generate optically clear PDMS devices with smaller channel dimensions of 100 µm, with faster print times and higher accuracy for the DLP 3D printer. Heating the 3D printed moulds produced fully cured PDMS chips with perfect channel edges. The sanding and nail polish coating of the mould produced optically transparent and smooth PDMS devices. Coating the PDMS surface with polydopamine (PDA) improved its surface wettability from 110° to 75°. On the PDA and collagen-coated device, HCEC exhibited significantly better growth than on the untreated PDMS device. HCEC cultured on these PDA/collagen-coated PDMS devices demonstrated 80% cell viability compared to conventional tissue culture plates. Conclusion: This study demonstrated that SLA and DLP printers can be used to produce PDMS microfluidic chips quickly and affordably. Notably, the DLP printer offered better accuracy and faster printing time than the SLA printer. The fabricated PDMS chips were transparent and capable of incorporating HCEC.
Immobilization of Gold Nanoparticles for Colourimetric and Ratiometric Refractive Index Sensing
(University of Waterloo, 2019-12-12) Pollit, Lori; Jones, Lyndon; Gu, Frank
The development of refractive index sensors is an expanding field of research, with applications in fields including medical diagnostics, food safety and public health. Nanotechnology has become widely implemented in various refractive index sensing techniques, resulting in substantial progress in detecting minute changes of refractive index. A literature review of the current refractive index sensing techniques which incorporate nanotechnology demonstrates that two main strategies for refractive index sensors have been developed, those that provide highly sensitive measurements and those that provide visual colourimetric measurements that can be detected by the naked eye. All colourimetric sensors based on gold nanoparticles offer a red to blue shift, however, this thesis outlines the development of a ratiometric colourimetric refractive index sensor that provides a unique blue to red shift. The development began with controlling the deposition of the various gold nanoparticle populations which are immobilized via electrostatic interactions between a weak polyelectrolyte and the gold nanoparticles. It was determined that by altering the pH of the polyelectrolyte as well as the size and concentration of the gold nanoparticles, modulation of the nanoparticle populations could be achieved. The nanoplasmonic surfaces were then shown to be effective sensors for the refractive index range of 1.00 to 1.47, providing both red to blue and blue to red colourimetric shifts, depending on the ratio of the different immobilized nanoparticle ensembles. The sensor surfaces were shown to be reusable, however the electrostatic interactions responsible for immobilizing the nanoparticles were weakened when exposed to various cleaning solutions and common solvents, resulting in nanoparticle loss from the sensor surface. Lastly, the optical response achieved by the refractive index sensors was simulated using COMSOL Multiphysics, which provided insight on the properties that resulted in the unique colourimetric shifts.
In Vitro Release of Inflammatory Cytokines from Contact Lens Materials
(University of Waterloo, 2020-12-22) Nagaarudkumaran, Nijani; Jones, Lyndon
The upregulation of inflammatory mediators in the tear film during contact lens wear indicates that lens wear may induce an inflammatory response on the ocular surface. Previous research has investigated the relationship between lens wear and ocular inflammation through analyses of cytokine levels in tear samples. However, there has been little discussion regarding the interaction between contact lens materials and cytokines, and its role in ocular inflammation. This thesis aimed to study the release of cytokines from contact lens materials. Particularly, to determine whether the release of cytokines differed between silicone hydrogel and conventional hydrogel contact lens materials.
Interaction of Tear Inflammatory Markers with Contact Lens Materials
(University of Waterloo, 2020-01-28) Mirzapour, Parisa; Jones, Lyndon
Biomaterials are natural or synthetic materials that come into contact with biological tissue. Contact lenses are the most commonly used biomaterials, being worn by an estimated 140 million people worldwide. While contact lens wear could be considered successful, up to 50% of patients discontinue contact lens wear, primarily due to the development of contact lens discomfort. Due to the interaction of contact lenses with the ocular surface, the ocular environment is of great interest when considering factors contributing to contact lens discomfort. One of these factors may be cytokines released by human corneal epithelial cells, which have the potential to initiate ocular inflammation. The purpose of the investigations presented in this thesis were to assess cytokine adhesion to various contact lens materials, as an excessive binding of cytokines to contact lenses may contribute to the pathology of contact lens discomfort.
Ocular Effects of Scleral Lens Wear on Dry Eye Patients
(University of Waterloo, 2024-11-14) Otchere, Heinz; Jones, Lyndon; Gorbet, Maud
Purpose: Dry eye disease (DED) is among the most complex ocular surface diseases to treat. Complaints of ocular discomfort and dryness are common in DED patients and in contact lens wearers. The use of SL to restore the integrity of the ocular surface in a more severe DED has become increasingly accepted among eye care practitioners. However, little is known about this treatment regimen and its impact on ocular surface in mild to moderate DED. Also, the potential impact of coated SL designs incorporating Hydra-PEG (polyethylene glycol) technology on ocular surface health has not yet been fully investigated. This thesis aimed to address this gap in knowledge and determine whether SL (uncoated or Hydra-PEG) could be used as a viable option for patients who are experiencing milder forms of DED. Over the course of the study, participants were asked to complete DE questionnaires; lens settling and vision, corneal thickness, osmolarity and matrix metalloproteinase 9 (MMP-9) in the pre-corneal tear film were investigated. The study also examined the relationship between the respective parameters and compared the two lens pairs. Methods and Materials: The study was a prospective, double-masked, randomized, dispensing, crossover clinical trial involving five visits, where 20 subjects with mild to moderate dry eye disease (DED) were enrolled. Eligibility of the participants was confirmed following the Tear Film and Ocular Society (TFOS) Diagnostic Report in the first visit: presence of symptoms using the Ocular Surface Disease Index (OSDI) and Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaires (OSDI ≥ 13, SPEED ≥ 4), plus osmolarity values (> 308 mOsmol/L in either eye or interocular difference of > 8 mOsmol/L) and or a positive MMP-9 result (using InflammaDry® test kit) or both. The second visit involved randomization and dispensing of customized hydra-PEG coated or uncoated SL, where each pair of lenses were worn on a daily wear basis for four weeks. Prior to dispensing the lenses, high contrast (HCVA) and low contrast acuities (LCVA) were recorded. The central corneal clearance (CCC) was measured with VisanteTM OCT to determine lens settling over time. Two standard clinical questionnaires (contact lens impact on quality of life (CLIQ) and contact lens dry eye questionnaire 8 (CLDEQ-8)) and an internally developed wearing habit and subjective rating questionnaire were provided to subjects and completed. The third visit (after 4 weeks) involved follow-up of the first randomized SL, where the lenses were evaluated, and clinical measurements repeated. A wash-out period of at least 48 hours was allowed before dispensing (visit 4) and follow-up (visit 5) of the other pair of SL, using the same procedures, measurement protocols and visit schedule. The SL used in the study were Zen™ RC (Alden Optical, Bausch & Lomb, Lancaster, NY, USA) available in only two diameters (14.80 and 15.40 mm), with a central thickness of 250 µm. Results: Eighteen females and two males, mean age 29.10 ± 7.48 years, participated in the study. The overall OSDI and SPEED scores were 36.45 ± 17.08 and 12.50 ± 4.03 respectively. There was no significant difference in the CLIQ and CLDEQ-8 scores between uncoated and coated SL. In terms of wearing habit and subjective ratings, no statistically significant differences were observed in any of the parameters (wear time, dryness, burning, vision, and comfort). This suggests that the use of hydra-PEG coated SL did not show greater performance compared to the uncoated SL. The central corneal clearance (CCC) at the dispensing visit was 260 ± 40 (range: 200 - 300 µm) for both lens pairs. This significantly reduced to 190 ± 40 µm (lens settling: 70 ± 42 µm) for the uncoated SL and 200 ± 30 µm (lens settling: 70 ± 43 µm) for the coated SL at the follow-up visits, (all, p < 0.001). Comparing the CCC for the two lens pairs at the follow-up visits, no significant difference was observed, thus, the lens pairs settled at similar rates. In terms of HCVA and LCVA between the two lens pairs at both the dispensing and follow-up visits, there were no significant differences between them. Also, no significant correlations were found comparing the CCC with HCVA and LCVA of each lens pair at both visits. The CCT at the baseline and follow-up visits were: baseline: 560.55 ± 32.28 µm, post wear of uncoated SL: 557.65 ± 32.10 µm, and post wear of coated SL: 560.50 ± 34.02 µm. There were no significant differences for either lens type for central corneal thickness (CCT) at the baseline and follow-up visits (all, p > 0.05). Also, no significant correlations between CCC and CCT were observed at baseline or follow-up visits. These results demonstrated there were minimal corneal hypoxic effects when these SL were worn on a daily wear basis. For osmolarity measures, there was a statistically significant difference between the baseline and follow-up visit for the coated lenses (311.00 ± 14.86 vs 302.85 ± 7.96 mOsmol/L; p = 0.04). While this is statistically significant, the clinical relevance of this small difference remains questionable. Comparing the two lens pairs, no significant difference was found at all visits. The inflammaDry® test results indicated that 80 % of the participants tested positive for elevated MMP-9 at the baseline visit. There was a reduction in MMP-9 positive test results following the SL wear, however, there were no significant differences between the baseline and follow-up visits for each lens pair. Comparing the two lens pairs, no significant difference was observed. For either lens, no correlation was observed between osmolarity and MMP-9 test results. Conclusion: There is a potential for using SL to manage symptoms in subjects suffering from mild to moderate DED, as the overall wearing habit and subjective ratings showed > 70 % of lens tolerance among the study cohort. SL wear over the month of wear in this cohort induced little change to the cornea or conjunctival tissue. Furthermore, SL may appear to marginally reduce tear film osmolarity, however, further studies are needed to confirm this result and its impact on subjective dryness. The clinical phenomenon of lens settling needs further investigation, especially on its impact on conjunctival morphology. In this cohort of subjects of mild to moderate DED, the hydra-PEG coating technology did not show superior performance over the uncoated lens for any of the factors assessed. Keywords: dry eye disease. scleral contact lens, hydra-PEG, osmolarity, inflammadry, cornea
The Phenotype of Tear Neutrophils and Their Role in Ocular Homeostasis and Inflammation
(University of Waterloo, 2023-07-31) Jin, Yutong; Gorbet, Maud; Jones, Lyndon
Introduction: Neutrophils (polymorphonuclear neutrophils, PMNs) are part of the innate immune system with potent killing mechanisms against pathogens. Although the anterior segment of the eye has a unique characteristic, immune privilege, a significant influx of PMNs has been detected after a prolonged eye closure at night, such as sleep. However, the functions and roles of tear PMNs in ocular homeostasis and complications remain largely unknown. The overall objectives of this thesis were to examine and compare the phenotype and functions of tear PMNs from healthy participants and participants with ocular allergy. Methods: Participants used a gentle eye wash method to collect tear PMNs from the ocular surface by washing their eyes with sterile phosphate buffer saline (PBS). • Study 1: Tear PMNs were either isolated from the eyewash by using the MACS-Column or EasySep cell separation system or reconcentrated by centrifugation. Stimulated (phorbol-12myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucylphenylalanine (fMLP)) and unstimulated ROS production by both isolated and non-isolated tear PMNs were measured using luminol-enhanced chemiluminescence for 60 min. • Study 2: Tear PMNs were incubated with different medium conditions, Hank’s Balanced Salt Solution (HBSS) only, HBSS with lactoferrin, lysozyme, mucin, albumin, and IgG (ATS), and ATS without lactoferrin and lysozyme (MAI). Unstimulated or PMA-stimulated ROS production by tear PMNs was measured using luminol-enhanced chemiluminescence and flow cytometry with DCFH-DA. • Study 3: Tear leukocytes were collected at four different time points, after 2-hr and 7-hr of sleep at night, after 2-hr sleep during the day, and towards the end of the day (around 5 pm). After stimulation with fMLP, changes in the degranulation (lactoferrin, CD66b, CD63) and cell aging state (CD184) of tear PMNs were measured via flow cytometry. Neutrophil extracellular traps (NETs) were quantified by flow cytometry and visualized by microscopy following staining with myeloperoxidase, citrullinated histones, and CD15. • Study 4: Tear PMNs were collected from participants suffering from ocular allergy on two consecutive days, after a full night of sleep in the morning on Day 1 and at the end of the day, i.e., around 4:30 pm, on Day 2. Tear PMNs were either activated with fMLP or left unstimulated, followed by staining with antibodies against degranulation markers (CD66b and CD63), adhesion markers (CD11b and CD54), eosinophil marker (CD193), aging markers (CD184 and CD62L), as well as with the fluorescent probe DCFH-DA. The intensity of fluorescence was measured via flow cytometry. Results: • Study 1: Tear PMNs have a high level of constitutive/spontaneous ROS production even in the absence of any stimulus. However, tear PMNs failed to appropriately respond to LPS and fMLP, although they were able to produce ROS in response to PMA. Higher ROS generation was observed in isolated tear PMNs which may be caused by priming from the magnetic bead cell separation system. • Study 2: The presence of tear proteins significantly reduced the unstimulated and PMAstimulated ROS production by tear PMNs in HBSS and ATS. However, the findings on ROS production by PMA-stimulated PMNs incubated in MAI were different between the flow cytometry and chemiluminescence, suggesting that lactoferrin and lysozyme may have a greater impact on extracellular ROS production. • Study 3: Significantly more cells were collected from the nighttime compared to the daytime. 2hr EC night tear PMNs were less degranulated than 7hr EC night tear PMNs and possessed a higher activation ratio in response to fMLP. Furthermore, 7hr EC night tear PMNs exhibited hypersegmented nuclei and were prone to aggregation, when compared to 2hr EC night tear PMNs, suggesting an aged and activated phenotype. A significantly increased number of NETs were present in 7hr nocturnal closed-eye tear samples. • Study 4: There were significantly more tear PMNs collected from individuals with ocular allergy compared to healthy participants upon awakening. Tear PMNs from ocular allergy participants exhibited a less activated phenotype but a higher activation potential in response to fMLP compared to healthy participants, which was correlated with their younger maturation state. However, no significant difference in the production of ROS by tear PMNs between these two groups was observed. Conclusion: Tear PMNs become more aged and activated with increasing eye closure time at night, which can potentially aid in ocular surface surveillance. The presence of tear proteins may limit the ROS release by tear PMNs, thereby protecting the ocular environment from potential damage. However, in individuals suffering from ocular allergy, tear PMNs may be constantly recruited to the ocular surface, contributing to symptoms of ocular allergy and development of ocular complications associated with inflammation. This thesis identified some of the functionalities and potential roles of tear PMNs in maintaining ocular homeostasis and regulating inflammation. While further investigation is needed to comprehensively characterize the underlying mechanisms of tear PMNs involved in ocular inflammation, the current results may support the development of new therapeutical strategies to reduce ocular surface inflammation.
Protocol development for reliable isolation of tear-film neutrophils and in vitro functionality testing
(University of Waterloo, 2019-09-19) Jin, Yutong; Gorbet, Maud; Jones, Lyndon
During prolonged eye closure, such as during sleep, leukocytes are recruited to the ocular surface, with polymorphonuclear neutrophils (PMNs) representing the largest population. PMNs are essential inflammatory cells of the innate immune system, and possess several efficient killing mechanisms, such as phagocytosis and release of antimicrobial substances stored in granules, to protect the host tissues from invading pathogens. Tear-film neutrophils collected from the closed-eye environment have been shown to express high levels of degranulation and activation leukocyte markers (such as CD66b and Mac-1). As these cells may play a role in ocular inflammation and disease, it is important to determine their functionality. However, large variations in response and collection numbers have been observed previously. Limited knowledge also exists on the ability of tear film PMNs to respond to cytokines and mount an oxidative response. Thus, the objectives of this thesis were to develop a standard protocol to process tear-film PMNs to reliably conduct functionality tests in vitro and to assess the production of reactive oxygen species (ROS) in tear film PMNs. Specifically, 1) the sensitivity of tear-film PMNs to experimental procedures (fixation, centrifugation and incubation) was investigated in terms of expression of surface receptors via flow cytometry; 2) two different cell collection methods were evaluated, and changes in the expression of surface receptors of tear-film PMNs when exposed to interleukin-8 (IL-8) and phorbol 12-myristate 13-acetate (PMA) were examined through flow cytometry; and 3) the ability of tear-film PMNs to generate ROS was assessed using luminol-enhanced chemiluminescence. The response of tear-film PMNs was also compared to blood-isolated PMNs. Up to 20 participants were recruited in this research to perform cell collection and donate a small blood sample. A gentle eye wash method was used to collect cells from the closed-eye environment, whereby participants washed their eyes with sterile phosphate buffer saline (PBS) upon awaking at home, and collected the runoff into a sterile polypropylene tube, which was then delivered to the lab within two hours. The patch-OSCCA collection was also tested; participants slept at home and covered one of their eyes with a patch. When they woke up in the morning, they came to the lab directly with one eye covered, and cells were collected using the ocular surface cell collection apparatus (OSCCA), which gently irrigates the ocular surface with warm PBS and collects the solution and cells from a funnel into a centrifuge tube. Very few cells were obtained using the patch-OSCCA collection method and thus this method was not pursued further in this research. When assessing the effect of experimental procedures and measuring cell activation upon stimulation with IL-8 and PMA (a PKC activator), changes in the expression of CD11b (activation and adhesion leukocyte membrane receptor), CD16 (degranulation and phagocytosis marker), CD66b (membrane receptor expressed upon cell degranulation), CD45 (leukocyte common antigen) and CD55 (complement activation marker) were characterized by flow cytometry. ROS production in stimulated (PMA, lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear-film PMNs was measured using luminol-enhanced chemiluminescence (CL). In all experiments, blood-isolated PMNs were also used to allow for comparison in phenotype. Fixation with paraformaldehyde (PFA) is an important step in flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (5-fold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, it was found that an additional centrifugation step prior to antibody incubation and long (4hr) incubation at 37oC significantly altered the expression of membrane receptors with significant reduction in expression of CD11b, CD16 and CD55 when compared to control samples. Therefore, to preserve cell phenotype and cell integrity of tear film PMNs, any additional centrifugation and incubation step should be avoided and post fixation staining is recommended. To gain a further understanding on the phenotype of tear-film PMNs, their ability to respond to IL-8, a cytokine present in the tear film of the closed-eye environment, was characterized via flow cytometry. The expression of surface receptors CD11b, CD16, CD55 and CD66b on tear-film PMNs remained relatively unchanged when exposed to IL-8, whereas some changes in the level of expression of surface receptors were observed in response to PMA but in a lower magnitude compared to blood-isolated PMNs. The respiratory burst is one of the essential killing mechanisms for PMNs and is also related to phagocytosis. PMA stimulation was able to induce ROS production (as measured by chemiluminescence) in tear-film PMNs, and two distinct responder groups were observed, where the high responder group produced significantly more ROS than the low responders. LPS and fMLP failed to induce intracellular ROS production in tear-film PMNs although fMLP-stimulated tear-film PMNs generated ROS extracellularly in the first three minutes. These results suggested that the signalling pathways downstream of PKC as well as the NADPH oxidase were functional but that intracellular signalling pathways upstream the PKC were impaired. This thesis contributes new and important knowledge on tear film PMNs. We proved that the gentle eye wash method is currently the most effective at home collection method. In addition, our study demonstrated that experimental procedures can significantly affect the expression of membrane receptor expression on tear-film PMNs, and if these processing steps are not carefully considered, conclusions on the phenotype of tear-film PMNs can be severely impacted. Our findings also suggest that the lack of response to stimuli may be due to an impairment in the receptor-mediated intracellular signalling pathway and/or insufficient substrates within the cell. Finally, our study on respiratory burst identified two type of responders, which could have significant implications for microbial keratitis and contact lens-induced infiltrates. This thesis highlights the potential role of tear film PMNs in ocular health and inflammation but more work is still needed to gain a better understanding of the phenotype of tear film neutrophils in the closed eye environment and their underlying mechanisms of activation.
Studies on the Optimization of Neuropeptides Detection in the Human Tear Film
(University of Waterloo, 2020-05-13) Jabeen, Asiya; Jones, Lyndon
Introduction: Dry Eye Disease (DED) stems from a disruption of the homeostasis of the tear film (TF), a thin layer of fluid covering the ocular surface. The TF consists of numerous constituents that include proteins, lipids, mucins, water, electrolytes, immunoglobulins, vitamins, cytokines, and neuropeptides. An imbalance in any of these constituents could result in an unstable tear film, contributing to the pathophysiology of DED. Among these factors, neuropeptides, small proteinaceous substances produced and released by neurons through regulated secretory routes, may have a role in the pathophysiology of DED. To understand the impact of the disease on the concentrations of calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in the tear film, it is important to first understand the sample collection and quantification methods. The purpose of this thesis was to optimize a method to quantify the concentration of four neuropeptides using enzyme linked immunosorbent assay (ELISA). a common laboratory technique used to quantify the concentration of neuropeptides in the tear film. ELISAs have been used to determine the quantity of different components in blood and other bodily fluids in the human tear film. The aims of each chapter were as follows: Chapter 3: To determine the variability of two tear collection methods, basal tear collection and flush tear collection, for quantifying SP, CGRP, VIP, and NPY, and to quantify the day-to-day variability of these neuropeptides. Chapter 4: To assess the validity of a commercially available ELISA kits for the quantification of neuropeptides. Chapter 5: To examine the measurement variability of two commercially available ELISA kits for the quantification of SP. Methods: Chapter 3: Basal and flush tears (following instillation of 20 μL of saline on the ocular surface) of 8 healthy participants were collected from the right and left eyes respectively, using glass microcapillary tubes on two consecutive days. The concentrations of the four neuropeptides in the tears were determined using ELISA, for both collection methods, and for both days. Chapter 4: Basal tears (5 µL) were collected from the temporal canthus of each eye of 3 healthy participants using glass microcapillary tubes. To assess the validity of the ELISA kit used in Chapter 3, two experiments were performed: a spike and recovery experiment, followed by a serial dilution response. In the spike and recovery experiment, 2 μL of tears from each participant were diluted in 108 μL of three known concentrations of SP, CGRP, and NPY (1 pg/mL, 10 pg/mL, and 100 pg/mL). The concentrations of neuropeptides were quantified using ELISA and the percent recovery was calculated. In the serial dilution response experiment, 4 μL of tears were collected from a single participant and was spiked into a known concentration of NPY (100 pg/mL). Serial dilutions (1:2, 1:4 and 1:8) were conducted and the percent recovery was calculated. Multiple troubleshooting and optimizing experiments to conducted to examine the effect of using a blocking agent, C18 pipette tip column, protease inhibitor, and the effect of freeze storage on neuropeptide quantification. Chapter 5: SP from Phoenix Pharmaceuticals, SP from Cayman Chemicals and SP from Sigma-Aldrich were each formulated at 0.5 mg/mL. Their UV absorbance profile from 200 nm to 300 nm was obtained using SoftMax Pro 5.4.1 software on a SPECTRAmax M5e ROM v2.1.35. The two SP from Phoenix Pharmaceuticals and SP from Sigma-Aldrich were formulated at various concentrations (500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL, 15.6 pg/mL, to 7.8 pg/mL and 3.9 pg/mL) and were quantified using two different ELISA kits (Phoenix Pharmaceuticals, Cayman Chemicals). A Bland Altman plot was used to quantify the agreement of the two SP, quantified by each of the ELISA kits. Results: Chapter 3: There was no significant difference in the concentrations of CGRP, SP, NPY, and VIP between the two collection methods (all P > 0.19). The difference in concentrations of CGRP, SP, NPY, and VIP between the two study days was also not significant (P > 0.06 for all tests). Chapter 4: The percent recovery for spike and recovery experiment ranged between 63,953.15% to 13.74% for CGRP, 676.17% to 5.21% for SP and 412.42% to 7.51% for NPY. The initial concentration of NPY was 208 pg/mL and after the 1:2, 1:4, and 1:8 dilution, the observed recovery was 188.40 pg/mL for the 1:2 dilution, 153.60 pg/mL for the 1:4 dilution, and 204.28 pg/mL for the 1:8 dilution. In the troubleshooting experiments; there were minimal differences in the concentration of SP associated with the use of a blocking agent; there was a reduction in VIP when processed using C18 column pipette tips; using protease inhibitors reduced the amount of VIP recovered; the amount of VIP recovered was reduced in the presence of albumin; a higher amount of SP was recovered in freshly collected tears compared to tears which were stored frozen for four months. Chapter 5: A similar absorbance profile was observed for SP from Phoenix Pharmaceuticals and SP from sigma Aldrich. A trend toward higher variability was observed at lower concentrations of SP. The Bland Altman plot shows a mean difference of -3.36%, and a 95% limits of agreement of [-10.75, 4.01] for the Phoenix Pharmaceuticals kit, and a mean difference was -9.70%, and the 95% limits of agreement were [-14.61, -4.79] for the Cayman Chemicals kit. Conclusion: Chapter 3: It was anticipated that diluting to facilitate collection using the flush tears method would have yielded a lower concentration than the basal tears collection method. However, the ELISA kits found no significant difference between the two collection methods. Chapter 4: High variation was observed in the recovered values of both the spike and recovery and the serial dilution response experiments. The troubleshooting experiments have provided some optimization steps to consider for tear sample collection and processing for the detection for neuropeptides. Chapter 5: The agreements of two SP standards quantified with two different SP ELISA kits were poor. Greater variability in fluorescence and absorbance units was associated with lower concentrations of SP, highlighting the difficulty in quantifying SP near the detection limit of the kits.
Uptake and release of preservatives from soft contact lens materials
(University of Waterloo, 2022-01-13) Yee, Alan; Jones, Lyndon
Purpose: The purpose of this thesis was to examine the uptake and release of myristamidopropyl dimethylamine (MAPD) and polyhexamethylene biguanide (PHMB) from soft contact lens material using various in vitro models to determine the safety of the preservatives. Methods: Chapter 3: The detection of radioactive and non-radioactive MAPD were determined with UV-Vis spectroscopy and a radioactive beta counter. MAPD was prepared in phosphate buffered saline (PBS) solutions and different vial materials (glass and polyethylene). Chapter 4: To determine the uptake and release kinetics of 14C MAPD from reusable soft contact lens materials using radioactive labelling, five contemporary CLs were tested over a 1-day, and 7-day period. MAPD were extracted from contact lenses (CLs) with 2:1 chloroform:methanol at the end of each study. The radioactivity was measured using a beta scintillation counter. Chapter 5: To determine the uptake and release kinetics of 14C PHMB from reusable soft contact lens materials using radioactive labelling, five contemporary CLs were tested over a 1-day, and 7-day period. CLs were soaked in PHMB for 8 hours, followed by a release in PBS for 16 hours. PHMB were extracted from CLs with methanol at the end of each study. The experimental design was similar to Chapter 4. Chapter 6: To evaluate the cytotoxicity of MAPD and PHMB (from Chapter 4 and 5) released from reusable soft CLs on immortalized corneal epithelial cells (ICEC) and human corneal epithelial cells (HCEC). CLs were soaked in PBS containing either PHMB or MAPD for 8 hours. After incubation period, the lenses were placed in fresh PBS for 16 hours. The release media was exposed to ICEC and HCEC for 16 hours. Afterwards, two multipurpose solutions, MAPD (2.5 µg/mL, 5 µg/mL, 10 µg/mL) and PHMB (1 µg/mL, 5 µg/mL, 10 µg/mL) concentrations were tested against ICEC and HCEC. Cell viability was then measured using the alamarBlue™ assay. Results: Chapter 3: A mixture of radioactive and non-radioactive MAPD was able to be detected, resulting in a more cost-effective study. There were no differences in absorbance between PBS solutions. No differences in radioactivity were found between glass and polyethylene vials. However, polyethylene vials showed a more equal distribution of MAPD. The results suggest polyethylene vials are better suited for future radioactive kinetic studies (Chapter 4, Chapter 5). Chapter 4: Silicone hydrogel (SH) materials sorbed significantly more MAPD than the conventional hydrogel (CH) materials. However, the CH materials released a greater amount of MAPD than the SH materials. Over a 7-day period, similar results were found between SH and CH materials. Chapter 5: The CH material (etafilcon A) sorbed significantly more MAPD than the SH material (senofilcon A). Etafilcon A released more PHMB compared to all other lens types within a 24-hr period. Over a 7-day period, all CLs continued to sorb more PHMB, with no signs of saturation. The CH materials released more PHMB than the SH materials. Chapter 6: The amount of PHMB or MAPD released from CLs did not have an impact on corneal epithelial cell viability. PHMB and MAPD at concentrations of 5 µg/mL and higher showed significantly reduced cell viability. Direct exposure to multipurpose solutions also significantly reduced cell viability. Conclusion: This thesis provided a chemical and biological assay for assessing the impact of MAPD and PHMB from CLs. Radioactive labelling provided a sensitive method for assessing the uptake and release of MAPD (Chapter 4) and PHMB (Chapter 5) from reusable soft CLs. The uptake and release of MAPD and PHMB were different based on their chemical structure and properties. In chapter 6, MAPD and PHMB released from CLs were not cytotoxic to corneal epithelial cells. Direct exposure of multipurpose solutions and increasing concentrations of MAPD and PHMB significantly reduced cell viability. These models provide a valuable tool to predict future adverse events for new multipurpose solutions.

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