Culture of primary human meibomian gland cells from surgically excised eyelid tissue.

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 µM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

Diurnal changes and topographical distribution of ocular surface epithelial dendritic cells in humans, and repeatability of density and morphology assessment.

PURPOSE: Dendritic cells (DC) play a crucial role in ocular surface defence. DC can be visualised in vivo by confocal microscopy but have not yet been fully characterised in humans. This study investigated the diurnal variation, topographical distribution and repeatability of DC density and morphology measurements. METHODS: In vivo confocal microscopy (IVCM) was conducted on 20 healthy participants (mean age 32.7 ± 6.4 years, 50% female) at baseline and repeated after 30 minutes, 2, 6 and 24 h. Images were captured at the corneal centre, inferior whorl, corneal periphery, limbus and bulbar conjunctiva. DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed-model analysis, non-parametric analyses, Bland and Altman plots, coefficient of repeatability (CoR) and kappa were used. RESULTS: There were no significant changes in DC density (p ≥ 0.74) or morphology (p > 0.07) at any location over the 24-h period. The highest DC density was observed at the corneal limbus followed by the peripheral cornea (p < 0.001), with the lowest density at the corneal centre, inferior whorl and bulbar conjunctiva. Most DC at the corneal periphery, limbus and bulbar conjunctiva had larger cell bodies compared with the corneal centre (p ≤ 0.01), and the presence of long dendrites was observed mostly at non-central locations. Day-to-day CoR for DC density ranged from ±28.1 cells/mm2 at the corneal centre to ±56.4 cells/mm2 at the limbus. Day-to-day agreement of DC morphology determined by kappa ranged from 0.5 to 0.95 for cell body size, 0.60 to 0.95 for presence of dendrites, and 0.55 to 0.80 for the presence of long dendrites at various locations. CONCLUSIONS: No diurnal changes are apparent in corneal or conjunctival DC. Substantial topographical differences exist in DC density and morphology. IVCM provides good repeatability of DC density and acceptable agreement of DC morphology.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17ß-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

Dry eye symptoms in children: can we reliably measure them?

PURPOSE: Many dry eye questionnaires are available, but these may not be suitable for paediatric eye care. The feasibility of use and repeatability of symptom questionnaires administered to children was examined. METHODS: Participants aged 6-15 years (n = 62; 25M:37F; 40% male) completed six questionnaires twice in random order at a single visit: Symptoms assessment in dry eye (SANDE), ocular surface disease index (OSDI), numerical rating scale (NRS), ocular comfort index (OCI, n = 30), dry eye questionnaire 5 (DEQ-5) and the instant ocular symptoms survey (IOSS). Completion time and need for assistance were recorded and relationships with age examined (Pearson correlation, independent t-test). The number of participants requiring assistance and with which items were compared (linear mixed model, pairwise test). Repeatability (coefficient of repeatability (CoR), limit of agreement, bias) and intraclass correlation coefficient (ICC) were examined. RESULTS: Completion time was ≤2 min for each individual questionnaire. Younger participants took longer to complete (r = -0.43 to -0.60, p ≤ 0.01), and required more assistance (p ≤ 0.001). Forty-eight participants required assistance with at least one questionnaire. Older children (13-15 years) only required assistance with OSDI (p ≤ 0.004) and NRS (p ≤ 0.003). Participants required more assistance with SANDE, OSDI and NRS than with DEQ-5 and IOSS (p ≤ 0.02) and with gritty (OSDI, 77% of participants; OCI, 100%) and foreign body sensation (NRS, 92%) items. CoR was similar for all questionnaires with no evidence of a learning effect (p > 0.05). ICC was moderate to excellent. CONCLUSIONS: Dry eye questionnaires can be used reliably in paediatric eye care; more time and assistance may be required for younger children. The DEQ-5 and IOSS are recommended for use in younger age children.

Alteration of the pattern of regenerative corneal subbasal nerves after laser in-situ keratomileusis surgery.

PURPOSE: Corneal nerves exhibit high plasticity, which allows successful reinnervation after nerve damage caused by laser in-situ keratomileusis (LASIK) surgery. This study aimed to examine corneal subbasal nerve orientation during regeneration after LASIK. METHODS: This study involved 20 healthy, myopic subjects who had undergone bilateral Femto-LASIK 12-16 months prior with a superior hinge position. The corneal subbasal nerve plexus at the central, mid-temporal and mid-superior cornea on the right eye were imaged using in vivo confocal microscopy. Global nerve fibre orientation (indicating the overall pattern) and variation of nerve fibre orientation (indicating the consistency of the orientation) was determined using customised MATLAB™ software (www.mathworks.com/products/matlab.html). Differences in nerve orientation variables between groups were examined using the Mann-Whitney U test. Linear mixed models with Bonferroni adjustment were conducted to examine differences between corneal regions, and over time, after LASIK. RESULTS: There were no differences between post-LASIK and control groups in global nerve orientation at any of the examined corneal regions. The post-LASIK subjects had a greater variation of nerve orientation at the central (p = 0.007) and temporal (p = 0.049) cornea than the controls. There was a difference in global nerve fibre orientation between corneal regions (p < 0.001) in the controls but not in the post-LASIK group. The variation of nerve fibre orientation was higher at the central, compared to the temporal and superior cornea after LASIK (p < 0.001), although there were no differences between corneal regions in controls. CONCLUSIONS: Our results demonstrate that there was an increased variability in the corneal subbasal innervation patterns following LASIK when compared to controls.

Comparative limitations and benefits of liquid chromatography - mass spectrometry techniques for analysis of sex steroids in tears.

Sex steroids impact regulation of the ocular surface tissues and thus influence dry eye. Most tissues in the human body synthesise and metabolise active sex steroids at levels required by the tissue. This is likely to also be the case for humans in ocular surface tissues. This study investigated the presence and quantities of selected sex steroids, in addition to sex steroid precursors and metabolites, in human tears. Detection of sex steroids in tears is challenging due to trace level analyte concentrations and low volumes of available tears. Immunoassays have previously been employed to assess sex steroids in tears, however, this approach only allows a single analyte to be measured and can overestimate concentrations. This study evaluated ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and tandem mass spectrometry (MS/MS) methods for the concurrent detection and estimation of fourteen sex steroid and metabolite compounds in human tears. Basal tears were collected and pooled from 5 healthy pre-menopausal women (total 100 µL). Following protein precipitation and centrifuging, extract volumes equivalent to 14 µL of pooled tears were analysed. A Thermo Scientific Q Exactive™ Plus MS was used to compare novel high-resolution MS atmospheric pressure chemical ionization (APCI) and electrospray ionisation (ESI) methods for detection of fourteen target sex steroids, including dehydroepiandrosterone-sulphate (DHEAS), testosterone, oestradiol and their metabolites, using standards and pooled tears. The MS was programmed to switch between positive and negative polarity ionization modes at designated times through the UPLC run, in order to detect each analyte at optimal sensitivity. Analytes were analysed using APCI in standard mixtures at concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 1000 pg/mL, per component, to determine the limits of detection (LOD) and quantification. Both parallel reaction monitoring (PRM) MS/MS and selected ion monitoring (SIM) MS, were evaluated by plotting narrow range mass-to-charge ratio (m/z) chromatograms for each analyte. An APCI UPLC-MS method to simultaneously measure 14 sex steroids using standards was successfully developed following comparative evaluation of all available LC-MS techniques. Preliminary experiments found APCI to be more sensitive than ESI using standards. Narrow m/z range SIM MS resulted in better sensitivity, in tear samples, than PRM. One sex steroid and two androgen metabolites were detected, with the developed APCI UPLC-MS method, and their concentrations estimated in human tears that had been extracted with a protein crash. Progesterone, androsterone-glucuronide (ADT-G) and 3αDiol-G were successfully detected in the tear extract and their concentrations in the pooled tear sample were estimated (with 95% confidence intervals) to be 0.10 ±â€¯0.03 pg/µL, 30.9 ±â€¯18.3 pg/µL and 9.8 ±â€¯4.3 pg/µL respectively. The concentrations of the remaining 11 sex steroids in the tear sample were below the LODs of the method. This work shows that high mass resolution UPLC-MS can detect certain sex steroids and metabolites in tears, but that sensitivity of the technique and the low available tear volumes limit its application to a broader range of sex steroids. The investigation of sex steroids in tears and ocular surface tissue will aid understanding of the influence of sex steroids on ocular surface tissues facilitating better targeted treatment for dry eye.

Topical Review: Effects of Contact Lens Wear on Corneal, Conjunctival, and Lid Margin Sensitivity.

SIGNIFICANCE: Contemporary soft contact lenses do not affect mechanical sensitivity of the cornea, whereas conjunctival sensitivity is increased compared with nonwearers. Orthokeratology lens wear, however, reduces corneal sensitivity. The effects of contact lenses on lid margin sensitivity are unclear, and the link between ocular surface sensitivity and discomfort requires further exploration.Although up to 50% of contact lens wearers experience discomfort with varying severity, impact, and frequency, the relationship between ocular surface sensitivity and ocular surface discomfort experienced during contact lens wear is unclear. The purpose of this review is to examine and summarize the current evidence for the impact of both historical and contemporary contact lens wear on ocular surface sensitivity, the etiology of changes in ocular surface sensitivity, contact lens wear-related factors associated with changes in ocular surface sensitivity, and the relationship between sensitivity and discomfort. Despite minimal effects on mechanical corneal sensitivity with contemporary soft contact lens wear, orthokeratology reduces corneal sensitivity through pressure-related effects. This review addresses the relevance of conjunctival and potentially lid margin sensitivity in tolerance and discomfort with contemporary lens wear and the impact of instrument and stimulus characteristics. Less invasive techniques particularly for lid margin sensitivity measurements are required. Given the potential interactions between a contact lens and the varied types of ocular surface nociceptors, instruments that allow for exploration of cold and chemical sensitivity particularly may better allow the effects of lens wear to be elucidated compared with those that explore high-threshold mechanical sensitivity alone. A better understanding of the relationships between lens wear and ocular surface sensitivity may result in improved management of contact lens discomfort.

Conjunctival MUC5AC+ goblet cell index: relationship with corneal nerves and dry eye.

PURPOSE: To evaluate the relative proportion of conjunctival MUC5AC+ and MUC5AC- goblet cells in a post-LASIK population and their association with dry eye indicators and corneal nerve morphology using a MUC5AC+ Goblet Cell Index. METHODS: Twenty subjects who had undergone LASIK > 12 months previously and 20 age-matched controls were recruited. Dry eye symptoms, tear breakup time, osmolarity, meniscus area and corneal nerve morphology were examined. Conjunctival impression cytology samples were collected from inferior-temporal bulbar conjunctiva using Millicell® inserts. Total goblet cell density was determined from positive cytokeratin-7 (CK7) immunolabelling; MUC5AC+ goblet cell density was determined from both CK7+- and MUC5AC+-immunolabelled cells. The ratio of MUC5AC+ to total density was defined as the "MUC5AC+ Goblet Cell Index". Differences in variables between groups and the associations between goblet cell variables and clinical assessments were examined. RESULTS: No significant differences in the total and MUC5AC+ goblet cell density and tear film parameters were found between groups, although greater ocular discomfort was reported in the post-LASIK group (P = 0.02). A higher MUC5AC+ Index was associated with worse/greater dry eye symptoms (ρ = 0.55, P = 0.01) and higher nerve tortuosity (ρ = 0.57, P = 0.01) in the post-LASIK group; lower nerve density and thickness was found in controls (ρ > -0.45, P < 0.05), but not associated with tear film parameters. CONCLUSIONS: The MUC5AC+ Goblet Cell Index provides an indicator of mucin secretion for assessing the goblet cell function in dry eye. In the post-LASIK participants, we found an increased MUC5AC+ Index associated with worse dry eye symptoms and adverse changes in corneal nerve morphology.

Corneal Nerve Morphology, Sensitivity, and Tear Neuropeptides in Contact Lens Wear.

PURPOSE: To determine tear neuropeptide levels in contact lens wearers and non-wearers, and to examine relationships with indices of corneal innervation, tear function, and ocular discomfort. METHODS: A cross-sectional, single-visit, investigator-masked pilot study. Assessments included Ocular Comfort Index (OCI), central and mid-peripheral corneal nerve density and morphology (HRT-Rostock), corneal sensitivity (Cochet-Bonnet aesthesiometer), tear Substance P and calcitonin gene-related peptide (CGRP) concentration (ELISA), in situ tear osmolarity (TearLab), tear secretion (Phenol Red Thread), and noninvasive tear break-up time (NITBUT; Keeler Tearscope). Groups were compared using independent t-test or Mann-Whitney U test, and regional differences assessed using paired t-tests. Associations were analyzed using Pearson or Spearman correlation. Significance was determined at P < .05. RESULTS: Twenty contact lens wearers (7M:13F, 32 ± 5 years) and 20 non-wearers (7M:13F, 31 ± 5 years) completed the study. OCI score was numerically higher in lens wearers (32.27 ± 5.33) than non-wearers (27.66 ± 9.94). Tear osmolarity was higher [298.0 (IQR 291.0-309.8) vs. 288.5 (282.3-298.3) mOsmol/L; P = .01] whereas NITBUT was lower (9.8 ± 3.4 vs. 13.8 ± 5.6 s; P = .01) in lens wearers compared with non-wearers. Tear neuropeptide concentrations were not different between groups [Substance P 4.29 ng/ml (IQR 1.57-6.05), CGRP 14.89 ng/ml (5.08-59.26)], and there were no differences in nerve morphology or ocular surface sensitivity. Higher nerve density, interconnections, and tortuosity were observed in the central cornea than mid-peripherally (P < .05). OCI score was moderately associated with nerve tortuosity (r = 0.42, P = .01). CGRP was associated with central nerve density (ρ = 0.38, P = .02), as was tear secretion (r = -0.37, P = .02). Nerve interconnections were strongly associated with corneal sensitivity (ρ = 0.64, P < .001). CONCLUSIONS: Relationships were demonstrated between nerve density, tear CGRP, and corneal sensitivity. Markers of corneal neurobiology and sensory function do not appear to be altered in contact lens wear despite worse tear function (osmolarity and stability) in lens wearers. This suggests that mechanisms other than overt changes in corneal innervation regulate tear function during lens wear. The relationship between nerve tortuosity and ocular discomfort requires elucidation.

Preinflammatory Signs in Established Reusable and Disposable Contact Lens Wearers.

SIGNIFICANCE: Established reusable contact lens (CL) wearers show higher tear inflammatory cytokine concentrations and greater conjunctival metaplasia in the region covered by standard soft CLs. The balance of proinflammatory to anti-inflammatory cytokines, but not individual tear cytokine concentrations, was associated with self-reported CL discomfort. PURPOSE: Daily disposable (DD) lenses are often used to improve CL discomfort, but the effect on ocular inflammatory responses has not been fully investigated. This study aimed to compare the concentrations of tear cytokines and conjunctival cell morphology in healthy habitual DD and reusable soft CL wearers. METHODS: Thirty-six established daily CL wearers, including 14 DD and 24 reusable wearers, were enrolled. Symptoms and ocular surface integrity were evaluated. The concentration of tear cytokines (interleukin 1ß [IL-1ß], IL-6, IL-10, IL-12(p70), IL-17A, and tumor necrosis factor α) were determined using Multiplex assays. The ratios of proinflammatory and anti-inflammatory cytokines were calculated. Impression cytology was performed on the conjunctiva, and goblet cell density and epithelial squamous metaplasia were quantified. Differences in variables by CL replacement schedules and the associations between variables were analyzed. RESULTS: Reusable CL wearers had higher concentrations (in pg/mL) of IL-1ß (26 ± 7 vs. 16 ± 11), IL-6 (42 ± 14 vs. 25 ± 20), IL-10 (83 ± 23 vs. 49 ± 36), IL-12(p70) (145 ± 44 vs. 91 ± 68), IL-17A (93 ± 26 vs. 54 ± 44), and tumor necrosis factor α (312 [171 to 468] vs. 189 [6 to 447]) (all P < .01) and greater conjunctival metaplasia in the region covered by CLs (0.7 [0.2 to 1.6] vs. 0.4 [0.04 to 1.2], P = .01) compared with DD wearers. There was a positive association between CL discomfort and ratios of IL-1ß to IL-10 and IL-12(p70) to IL-10 (ρ = 0.42 and ρ = 0.33, P < .05). CONCLUSIONS: Higher ocular inflammatory responses, as indicated by higher tear cytokine concentrations and higher conjunctival epithelial metaplasia, were found in reusable CL wearers than in DD CL wearers. The balance of proinflammatory and anti-inflammatory cytokines may be helpful to assess the inflammatory status of the eye.

Reduced Corneal Sensitivity and Sub-Basal Nerve Density in Long-Term Orthokeratology Lens Wear.

OBJECTIVES: To investigate changes in corneal sensitivity and nerve morphology in orthokeratology (OK) contact lens wear. METHODS: In a cross-sectional study, 54 subjects (aged 18-45 years) were grouped into three categories: nonlens (NL), soft lens (SCL), and OK lens wearers. Corneal sensitivity was measured at the corneal apex and 2.5 mm temporal to the apex using the Cochet-Bonnet aesthesiometer. Corneal nerve morphology was assessed by sampling a 1 mm area of the corneal sub-basal nerve plexus using the Heidelberg Retinal Tomograph with Rostock Corneal Module at the corneal apex and 2.5 mm temporal to the apex. Nerve fiber density (NFD) was calculated by measuring the total length of nerve fibers per square millimeter using Image-Pro Analyser. Comparisons between groups were made using mixed analysis of variance and post hoc paired t tests with Bonferroni correction or the Kruskal-Wallis test and post hoc Mann-Whitney U tests as appropriate. RESULTS: There was a significant difference in corneal sensitivity between the three groups (P=0.027). Central threshold was significantly higher in the OK than NL group (0.69±0.42 g/mm vs. 0.45±0.12 g/mm; P=0.048). Mid-peripheral threshold was not different between the three groups (P>0.05). There was a significant difference in NFD between the three groups (P<0.001). Central NFD was significantly less in the OK than NL and SCL groups (OK: 17.89±4.42 mm/mm, NL: 25.87±5.00 mm/mm; SCL: 24.52±4.93 mm/mm; P<0.001). Mid-peripheral NFD was not different between the three groups (P>0.05). CONCLUSIONS: Long-term OK lens wear is associated with a decrease in central corneal sensitivity and NFD. The mechanism underlying refractive change during OK treatment seems to impact both corneal sensitivity and nerve morphology.

A Possible Association Between Dry Eye Symptoms and Body Fat: A Prospective, Cross-Sectional Preliminary Study.

OBJECTIVES: To examine the relationship between dry eye symptoms and adiposity in a population study. METHODS: A prospective cross-sectional single-visit preliminary study was designed to sample ocular symptoms and indices of adiposity in the general adult population. Patients recruited from the UNSW optometry clinic, the university campus and surrounding community, and overseas were invited to complete a survey composed of the short form Dry Eye Questionnaire (SFDEQ) (Dry Eye Questionnaire-5 or Contact Lens Dry Eye Questionnaire-8) and the Ocular Comfort Index (OCI) via hardcopy or online. Participants self-measured their weight, height, waist circumference (WC), and body fat % (optional). Associations between measures of adiposity (Body mass index [BMI], WC, and body fat %) and ocular symptoms scores (SFDEQ and OCI) were determined using Spearman correlation coefficients. Group differences were compared using independent t test. RESULTS: Three hundred and five participants including 52 contact lens wearers completed the study and 78 of them measured body fat %. There was a moderate correlation between body fat % and dry eye symptoms (SFDEQ r=0.34, P=0.003; OCI r=0.32, P=0.004). The interaction between body fat % and gender, wear of contact lenses, and older age were shown to be significant predictors of less ocular comfort in multivariate analysis (F(3,74)=12.13, P<0.001). CONCLUSIONS: An association between adiposity measured by body fat % and symptoms of dry eye was demonstrated in the general adult population. Confirmation of these findings in a large study is required.

Changes in Tear Cytokine Concentrations Following Discontinuation of Soft Contact Lenses-A Pilot Study.

PURPOSE: This study investigated changes in selected tear cytokine concentrations (IL-1ß, IL-1Ra, IL-6, IL-10, IL-12 (p70), and TNF-α) after a 1-week washout from soft contact lens wear (CLW), and the repeatability of cytokine measurements using custom multiplex assays. METHODS: A total of 10 subjects completed this 6-visit (immediately following contact lens removal, and after 1, 2, 3, 4, and 7 [±1] days without CLW) pilot study. Approximately 20 to 30 µL of pooled basal tears were collected from both eyes at each visit. Two custom multiplex assays were used by two operators to quantify the concentration of tear cytokines. Tear samples from subjects 1 to 6 were analyzed using the first kit by operator 1. Tear samples from subject 7 to 10 plus additional tear samples from subjects 1 to 5, which were used to determine the between-kit per operator repeatability, were analyzed using the second kit by operator 2. Linear mixed models were used to determine changes in tear cytokine concentrations over time. Between-kit per operator and within-kit per operator repeatabilities were assessed using the Bland and Altman analysis. RESULTS: There were no significant changes in tear cytokine concentrations over a 1-week washout of CLW. More than 99% of the tear samples had detectable levels of cytokines using custom multiplex assays. Within-kit per operator repeatability was good, but between-kit per operator repeatability was poor; likely due to protein degradation, differences in operator experience, and operating procedures. CONCLUSION: A washout period may not be necessary when evaluating changes in tear cytokines with new contact lenses or lens care products. A well-trained operator using standardized operating procedures can produce repeatable measurements using custom multiplex assays.

Structural and functional changes in corneal innervation after laser in situ keratomileusis and their relationship with dry eye.

PURPOSE: The most likely etiology of post-LASIK dry eye is corneal nerve damage; however, no direct relationship between post-LASIK dry eye symptoms and nerve damage has been established, and limited information is available about the relationship between dry eye signs and corneal reinnervation after LASIK. Tear neuropeptides (SP and CGRP) are important in the maintenance of corneal nerve health, but the impact of LASIK has not yet been studied. This study evaluated changes in nerve morphology, tear neuropeptide, and dry eye, so as to establish the relationship between reinnervation and dry eye and to assess the role of tear neuropeptides in reinnervation post-LASIK. METHODS: Twenty non-dry eye volunteers who had undergone bilateral myopic-LASIK completed this study. Corneal nerve morphology (density, width, interconnections, and tortuosity), SP and CGRP concentration, and dry eye were monitored over time prior to, 1 day, 1 week, 1, 3, and 6 months post-LASIK. RESULTS: Dry eye symptoms and tear function, except for osmolarity (P = 0.003), remained unchanged post-LASIK. Corneal nerve morphology decreased immediately, and did not return to preoperative levels by 6 months post-LASIK (P < 0.001). Increased tear SP concentration was observed 3 months post-LASIK (P < 0.001). Associations between reinnervation as measured by increased density and lower tear SP (P = 0.03), and between increased density and decreased dry eye symptoms (P = 0.01) were found post-LASIK. CONCLUSION: An inverse relationship between reinnervation post-LASIK and dry eye symptoms was found, confirming that post-LASIK dry eye is a neuropathic disease. This study is the first to demonstrate an association between tear SP and post-LASIK reinnervation, suggesting that strategies for manipulating neuropeptide concentration to improve reinnervation may improve ocular comfort post-LASIK.

Ocular surface sensitivity repeatability with Cochet-Bonnet esthesiometer.

PURPOSE: To determine the repeatability of ocular surface threshold measurements using the Cochet-Bonnet esthesiometer on the same day and 3 months apart. METHODS: Two separate studies were conducted to determine the repeatability of ocular surface threshold measurements made on the same day (n = 20 subjects) and 3 months apart (n = 29 subjects). The Cochet-Bonnet esthesiometer was used to measure corneal and inferior conjunctival thresholds using the ascending method of limits. The pressure exerted by the Cochet-Bonnet esthesiometer was determined using an analytical balance, for both the 0.08- and 0.12-mm-diameter filaments. This calibration was then used to convert filament length measurements to pressure. Repeatability was determined using a Bland and Altman analysis. RESULTS: The pressure exerted at each filament length differed between the two filament diameters. The measured pressure also differed from values provided by the manufacturer. Repeatability of threshold measurements at the central cornea was shown to be good, with better repeatability for same-day measurements (coefficient of repeatability [CoR] = ±0.23 g/mm²) than for those 3 months apart (CoR = ±0.52 g/mm²). Threshold measurements at the inferior conjunctiva, in contrast, were poorly repeatable (CoR = ±12.78 g/mm²). CONCLUSIONS: Cochet-Bonnet esthesiometry is repeatable when performed on the central cornea on the same day and 3 months apart, but this instrument is not recommended for conjunctival threshold measurements.

Consequences of wear interruption for discomfort with contact lenses.

PURPOSE: To establish whether increased end-of-day discomfort during soft contact lens wear is associated with short-term changes occurring to the lens itself. METHODS: Twenty-seven subjects wore hydrogel lenses (Focus Dailies; Alcon) bilaterally for 10 hours on two separate days. Comfort was reported using 1-100 numerical rating scales (1 = intolerable discomfort, 100 = lens cannot be felt). Day 1 ratings were taken before lens insertion and at 0.05, 5, and 10 hours post-insertion. Day 2 ratings occurred at similar times, but lenses were removed after the 5-hour assessment and either reinserted (n = 14) or newly replaced (n = 12). An additional rating was taken 5 minutes after re-insertion. Wear then continued to the 10-hour point. In a separate study, 24 different subjects repeated these procedures using a silicone hydrogel lens (AirOptix Aqua; Alcon) with wear taking place on 3 days to permit lens replacement to be with existing as well as new lenses in all subjects. RESULTS: For hydrogel lenses, comfort scores (mean ± 95% CI) reported after 10 hours were 79.4 ± 8.3 when lenses were worn un-replaced, compared with 73.2 ± 9.2 for replacement with the existing lens. When replacement was with a brand new lens, the corresponding values were 72.9 ± 10.9 (un-replaced) versus 69.2 ± 12.8 (new lens replacement). For silicone hydrogel lenses, 10-hour comfort was 90.3 ± 3.2 (un-replaced) versus 92.2 ± 2.9 (replacement with existing lens) versus 90.0 ± 3.3 (replacement with new lens). Differences between replacement conditions were not significant in any case (analysis of variance, p > 0.05). CONCLUSIONS: Final comfort was not influenced by replacing lenses midway through the wearing period. Comfort decrements experienced by users of these daily contact lenses towards the later part of the wearing period are not caused by changes occurring to the lenses on this time scale. Possible alternative etiological factors include a fatigue-like response in one or more ocular tissues or stimulation of ocular surface nociceptors induced by the presence of the contact lens.

Corneal sensitivity with contact lenses of different mechanical properties.

PURPOSE: To examine the changes in corneal sensitivity after overnight wear of contact lenses with different mechanical properties. METHODS: Twenty young-adult subjects wore a silicone hydrogel, rigid gas-permeable, or orthokeratology (OK) contact lens in randomized order for a single night of wear in the right eye only. All lenses were matched in Dk/t (∼46 ISO Fatt). Changes in corneal apical radius r(o), asphericity Q, and corneal refractive power (Medmont E300) were measured. Changes in central corneal sensitivity were also measured by a masked investigator using two instruments: Cochet-Bonnet (COBO) aesthesiometer and Non-Contact Corneal Aesthesiometer (NCCA). RESULTS: There were significant differences in corneal topographic change from baseline between the lens types for r(o), Q, and corneal refractive power. There were also significant differences in the change from baseline (mean ± SD) in corneal sensitivity between lens types using the COBO (silicone hydrogel, 0.02 ± 0.17 g/mm(2); rigid gas-permeable, 0.03 ± 0.20 g/mm; OK, 0.22 ± 0.33 g/mm(2)). A significant increase in threshold from baseline was only seen in the OK lenses (p = 0.006). There was no change in sensitivity thresholds from baseline for any lens type using the NCCA (p > 0.05). CONCLUSIONS: Central corneal sensitivity is reduced after a single overnight wear of OK lenses, as measured using the COBO aesthesiometer. This suggests that the mechanical force exerted by contact lenses may influence corneal sensitivity.

Understanding the stimulus of an air-jet aesthesiometer: computerised modelling and subjective interpretation.

PURPOSE: Assessment of corneal sensory function is more frequently being carried out using novel non-contact methods, which promise better stimulus control and a greater intensity range than more traditional techniques. An examination of the characteristics of the air-jet stimulus of these 'non-contact' aesthesiometers is important to enable an understanding of how these instruments stimulate the cornea. The purpose of this study was to model the air-jet stimulus of the CRCERT-Belmonte Aesthesiometer (CBA) and its interaction with the cornea. METHOD: A computerised simulation was used to model the CBA stimulus and this model was then examined in the context of verbal descriptions of the supra-threshold stimulus provided by 27 human subjects with normal corneas. The computational fluid dynamics model and the subjective descriptions considered CBA airflow values between 70 and 400 mL min(-1) . RESULTS: The computational fluid dynamics results showed that the CBA air-jet stimulus behaves as expected in terms of force exerted and stabilises and subsides quickly during the 1 s duration for which it is turned on/off. The computer modelling demonstrated that the stimulus of the CBA consists of a central core of air flow, similar in diameter to the CBA nozzle, which exerts maximum pressure at the corneal apex. This core is surrounded by a less prominent zone of lower pressure, extending out to the corneal periphery at higher flow rates. The computer modelling results were confirmed by the subjective descriptions of intensity and corneal area stimulated, whereas relation to duration of sensation was more equivocal. CONCLUSIONS: The CBA probably stimulates sensory receptors over a greater area of the ocular surface compared to the traditional nylon filament aesthesiometer and may recruit neurons sensitive to additional stimulus modalities. Future computational models should also incorporate temperature as an important aspect of the corneal response. This study has effectively demonstrated that it is possible to create a virtual model of an air-jet aesthesiometer stimulus using computational methods and that this model is corroborated by subjective descriptions provided by subjects.

Smartphone gaming induces dry eye symptoms and reduces blinking in school-aged children.

PURPOSE: Smartphone use by children is rising rapidly, but its ocular surface impact is unknown. This study examined the effect of smartphone use on blinking, symptoms, and tear function in children. METHODS: Prospective intervention study where 36 children aged 6-15years (14 M:22 F) played games on a smartphone continuously for one hour. Symptoms (SANDE, IOSS, NRS) and tear film (lipid layer thickness, tear secretion, stability) were assessed before and after gaming. Blink rate and interblink interval were measured in situ using an eye tracking headset, before (during conversation) and continuously throughout gaming. Symptoms and tear film changes were examined using paired t-tests. Changes in blinking throughout one hour were examined using repeated measures ANOVA, post-hoc comparisons with Bonferroni correction. Associations examined using Pearson bivariate correlation. Significance level was 0.05. RESULTS: Symptoms worsened following one hour smartphone gaming (SANDE + 8.2units, p = 0.01; IOSS + 1.3units, p < 0.001; NRS-average +6.3units, p = 0.03; NRS-comfort +7.6units, p = 0.04; NRS-tiredness +10.1units, p = 0.01), but tear film remained unchanged. Blink rate reduced from 20.8 blinks/min to 8.9 blinks/min (p < 0.001) and interblink interval increased from 2.9 s to 8.7 s (p = 0.002) within the first minute of gaming relative to baseline conversation, and this effect remained unchanged throughout one hour of gaming. CONCLUSIONS: Smartphone use in children results in dry eye symptoms and immediate and sustained slowing of blinking, with no change in tear function evident up to one hour. Given the ubiquitous use of smartphones by children, future work should examine whether effects reported herein persist or get worse over a longer term causing cumulative damage to the ocular surface.