Optometry and Vision Science

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Browsing Optometry and Vision Science by Author "Gorbet, Maud"

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    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
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    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.
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    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.