Cellular changes at the lid margin
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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.
Cite this version of the work
Alex Muntz (2017). Cellular changes at the lid margin. UWSpace. http://hdl.handle.net/10012/12672