Cellular changes at the lid margin

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

Improved Demodex diagnosis in the clinical setting using a novel in situ technique

Purpose: To compare existing and novel diagnostic techniques for confirming ocular Demodex infestation and to recommend the most reliable method for routine use by eye care practitioners, based on yield and clinical applicability. Methods: Fifteen participants with a prior Demodex blepharitis diagnosis or featuring typical cylindrical dandruff (CD) collarettes, and seven healthy controls were enrolled. Demodex presence was assessed using five techniques, applied consecutively, on a minimum of two different eyelashes on each eyelid of every participant, for each test, in situ: 1. using fine-point forceps and 25-40x biomicroscopy magnification, by eyelash rotation as proposed by Mastrota (ROT); 2. by removing cylindrical dandruff and exposing the eyelash insertion point at the lid margin (CDR); and 3. by laterally tensioning the eyelash (LET) following CDR. The typical appearance of cigar-shaped mite tails protruding from each assessed eyelash follicle was observed, and mite tails counted and averaged per participant for each assessment technique. 4. Lash epilation, and mite presence evaluated using bright-field microscopy at 10-40x magnification (EPI). 5. Finally, eyelash follicles were imaged using in vivo confocal microscopy (IVCM) and the images visually inspected for mite presence. Results: In the Demodex group, the highest numbers of mites/eyelash were identified by LET (3.8 ± 1.4), versus CDR (2.4 ± 1.6) and ROT (1.1 ± 1.2), alone (all p < 0.002). An average of 1.0 ± 0.8 mites/lash was identified by EPI. IVCM failed to offer unequivocal evidence of Demodex presence even in confimed cases. Conclusions: A novel technique for the clinical diagnosis and grading of Demodex in situ is described. By removing cylindrical dandruff and applying static, lateral tension to the eyelash without epilation, large numbers of mites are visible at the exposed eyelash follicle. The proposed method is convenient and clinically applicable, requiring only forceps and 25-40x biomicroscope magnification, and allowing rapid, efficient evaluation of large numbers of eyelashes

Impression Cytology of the Lid Wiper Area

Muntz, A., van Doorn, K., Subbaraman, L. N., & Jones, L. W. (2016). Impression Cytology of the Lid Wiper Area. Journal of Visualized Experiments, (114). https://doi.org/10.3791/54261Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy

Systemic risk factors of dry eye disease subtypes:A New Zealand cross-sectional study

PURPOSE: To evaluate systemic risk factors of dry eye disease, aqueous tear deficiency, and meibomian gland dysfunction. METHODS: Three hundred and seventy-two community residents (222 females, 150 males; mean ± SD age, 39 ± 22 years) were recruited in a cross-sectional study. Past medical history, dry eye symptomology, ocular surface characteristics, and tear film quality were evaluated for each participant within a single clinical session. The diagnosis of dry eye disease, aqueous tear deficiency, and meibomian gland dysfunction were based on the global consensus recommendations of the Tear Film and Ocular Surface Society's Dry Eye Workshop II (TFOS DEWS II) and International Workshop on Meibomian Gland Dysfunction. RESULTS: Overall, 109 (29%) participants fulfilled the TFOS DEWS II criteria for dry eye disease, 42 (11%) had aqueous tear deficiency, and 95 (26%) had meibomian gland dysfunction. Multivariate logistic regression analysis demonstrated that systemic rheumatologic disease and antidepressant medication were independently associated with aqueous tear deficiency (both p < 0.05). Significant risk factors for meibomian gland dysfunction included age, East Asian ethnicity, migraine headaches, thyroid disease, and oral contraceptive therapy (all p ≤ 0.01). CONCLUSIONS: Both etiological subtypes of dry eye disease were associated with a number of systemic risk factors. These findings would support routine systemic inquiry of dry eye disease and associated systemic conditions and medications, in order to facilitate opportunistic screening and timely inter-disciplinary referral where necessary

TFOS lifestyle: Impact of the digital environment on the ocular surface

Eye strain when performing tasks reliant on a digital environment can cause discomfort, affecting productivity and quality of life. Digital eye strain (the preferred terminology) was defined as “the development or exacerbation of recurrent ocular symptoms and/or signs related specifically to digital device screen viewing”. Digital eye strain prevalence of up to 97% has been reported, due to no previously agreed definition/diagnostic criteria and limitations of current questionnaires which fail to differentiate such symptoms from those arising from non-digital tasks. Objective signs such as blink rate or critical flicker frequency changes are not ‘diagnostic’ of digital eye strain nor validated as sensitive. The mechanisms attributed to ocular surface disease exacerbation are mainly reduced blink rate and completeness, partial/uncorrected refractive error and/or underlying binocular vision anomalies, together with the cognitive demand of the task and differences in position, size, brightness and glare compared to an equivalent non-digital task. In general, interventions are not well established; patients experiencing digital eye strain should be provided with a full refractive correction for the appropriate working distances. Improving blinking, optimizing the work environment and encouraging regular breaks may help. Based on current, best evidence, blue-light blocking interventions do not appear to be an effective management strategy. More and larger clinical trials are needed to assess artificial tear effectiveness for relieving digital eye strain, particularly comparing different constituents; a systematic review within the report identified use of secretagogues and warm compress/humidity goggles/ambient humidifiers as promising strategies, along with nutritional supplementation (such as omega-3 fatty acid supplementation and berry extracts)

Tear exchange and contact lenses: A review

Tear exchange beneath a contact lens facilitates ongoing fluid replenishment between the ocular surface and the lens. This exchange is considerably lower during the wear of soft lenses compared with rigid lenses. As a result, the accumulation of tear film debris and metabolic by-products between the cornea and a soft contact lens increases, potentially leading to complications. Lens design innovations have been proposed, but no substantial improvement in soft lens tear exchange has been reported. Researchers have determined post-lens tear exchange using several methods, notably fluorophotometry. However, due to technological limitations, little remains known about tear hydrodynamics around the lens and, to-date, true tear exchange with contact lenses has not been shown. Further knowledge regarding tear exchange could be vital in aiding better contact lens design, with the prospect of alleviating certain adverse ocular responses. This article reviews the literature to-date on the significance, implications and measurement of tear exchange with contact lenses

Modifiable lifestyle risk factors for dry eye disease

Purpose: To examine the association between modifiable lifestyle factors and dry eye disease. Methods: Three hundred and twenty-two community residents (186 females, 136 males; mean ± SD age, 41 ± 22 years) with no major systemic or ophthalmic conditions (other than dry eye disease) were recruited in a cross-sectional study. A lifestyle factor questionnaire was administered, and dry eye symptomology, ocular surface characteristics, and tear film quality were evaluated for each participant within a single clinical session, in accordance with the global consensus recommendations of the TFOS DEWS II reports. Results: A total of 111 (34 %) participants fulfilled the TFOS DEWS II diagnostic criteria for dry eye disease. Multivariate regression analysis demonstrated that advancing age, female sex, East Asian ethnicity, and increased digital screen exposure time were positive risk factors for dry eye disease (all p < 0.05), while increased caffeine consumption was a protective factor (p = 0.04). Conclusions: Increased digital screen exposure time and reduced caffeine consumption were modifiable lifestyle factors associated with higher odds of dry eye disease. These findings might contribute to informing the design of future prospective research investigating the efficacy of preventative intervention and risk factor modification strategies

Blinking and upper eyelid morphology

Purpose To explore blinking patterns and sagittal eyelid misalignment in the East Asian eye. Methods Forty-four participants (22 females; age 26 ± 5 years; 52% of East Asian ethnicity) were enrolled in this pilot study and subdivided, based on upper eyelid crease presence and extent, into single (n = 10), partial (n = 11) or double (n = 23) eyelid crease groups. Blinking was filmed surreptitiously with high-speed video simultaneously from an inferior temporal and frontal view. Spontaneous blink rate and type (incomplete, almost complete, or complete) were assessed over a 30 s period. Sagittal misalignment of the lids on closure was graded during complete spontaneous blinks, voluntary lid closure and voluntary maximal lid contraction (squeezing). A 0.15 µL drop of lissamine green was placed on the central lower lid margin and the number and type of blinks required to eliminate the drop informed complete palpebral apposition during blinking. Results Mean ± SD blink rates averaged 16.9 ± 10.5 blinks/minute. The proportion of incomplete blinks was 83 ± 22% in single, 58 ± 35% in partial and 59 ± 30% in double eyelid crease groups. The sagittal misalignment of the lid margins during blinking was limited to approximately one-third of the lid margin width; this was similar for all lid morphologies and blink types. The lissamine green drop was eliminated only by voluntary maximal lid contraction, and was similar in all groups (p = 0.97). Conclusions Incomplete blinking and sagittal lid misalignment of the central eyelid margin predominate in habitual blinking, irrespective of lid morphology