Concentrations of MUC16 and MUC5AC using three tear collection methods.

PURPOSE: To determine the optimal tear collection method for analysis of ocular surface mucins MUC5AC and MUC16. METHODS: Fifteen subjects without ocular surface disease were recruited. Subjects presented for tear collection on three separate days for three different tear collection methods with the order of method randomized. Methods used to collect tears from right and left eyes included Schirmer's strip, basal tear collection, and flush tear collection. All samples from the right eyes were individually analyzed for MUC5AC whereas the left eye samples were individually analyzed for MUC16. For each individual sample, 10 µg of protein were loaded per lane into a 1% (w/v) agarose gel and run in electrophoresis buffer for 2 h. After overnight capillary transfer, membranes were incubated with either MUC5AC antibody CLH2 or MUC16 antibody OC125 for western blot analysis. Blots were developed with enhanced chemiluminescence (ECL) and signals captured with the Odyssey Fc (LI-COR). The relative amounts of MUC5AC and MUC16 were quantified with densitometry using software and compared for statistically significant differences between tear collection methods using the Kruskal-Wallis test in SPSS 22 and GraphPad Prism 7.02. Dunn's multiple comparisons test was used for pairwise post-hoc comparisons. RESULTS: Samples containing less than 10 µg of total protein were not used for analysis which left eight samples (out of 45) unusable. The calculated MUC5AC median signal intensities from Schirmer's strip, basal tears, and flush tears were 2.86 (n = 15, the interquartile range [IQR] = 2.54-3.21), 1.65 (n = 14, IQR = 1.34-3.1), and 1.67 (n = 9, IQR = 1.42-1.72), respectively (H = 9.5, p = 0.009). Post-hoc pairwise comparisons showed a statistically significant difference between Schirmer's strip and flush tears (p = 0.01). The calculated MUC16 median signal intensities from Schirmer's strip, basal tears, and flush tears were 1.88 (n = 14, IQR = 1.43-2.61), 5.24 (n = 15, IQR = 4.16-6.21), and 2.45 (n = 7, IQR = 1.85-2.48), respectively (H = 18.1, p = 0.001). Post-hoc pairwise comparison showed statistically significant differences between basal tears and Schirmer's strip (p = 0.0003) and between basal tears and flush tears (p = 0.006). CONCLUSIONS: MUC5AC and MUC16 are present in human tear fluid and can be captured using various tear collection methods. Although basal tear collection yielded the highest relative concentration of MUC16, Schirmer's strip tear collection yielded the highest MUC5AC concentration. Therefore, the tear collection method chosen depends on the mucin of interest.

Ocular Surface Membrane-Associated Mucins.

Ocular surface epithelial cells produce and secrete mucins that form a hydrophilic barrier for protection and lubrication of the eye. This barrier, the glycocalyx, is formed by high molecular weight heavily glycosylated membrane-associated mucins (MAMs) that include MUC1, MUC4, and MUC16. These mucins extend into the tear film from the anterior surfaces of the conjunctiva and cornea, and, through interactions with galectin-3, prevent penetrance of pathogens into the eye. Due primarily to the glycosylation of the mucins, the glycocalyx also creates less friction during blinking and enables the tear film to maintain wetting of the eye. The secretory mucins include soluble MUC7 and gel-forming MUC5AC. These mucins, particularly MUC5AC, assist with removal of debris from the tear film and contribute to the hydrophilicity of the tear film. While new methodologies and cell culture models have expanded our understanding of mucin structure and function on the ocular surface, there is still a paucity of studies characterizing the glycosylation of MAMs on a normal ocular surface and a diseased ocular surface. Although studies have shown alterations in mucin production and expression in dry eye diseases, the relationship between changes in mucins and functional consequences is unclear. This review focuses on comparing what is known about MAMs in wet-surfaced epithelia of the body to what has been studied on the eye.

Association Between Serum Levels of Testosterone and Estradiol With Meibomian Gland Assessments in Postmenopausal Women.

PURPOSE: The aims of this analysis were to determine if there is an association between serum levels of testosterone and estradiol with meibomian gland (MG) morphology and lipid layer thickness. METHODS: The data used for this analysis were collected from postmenopausal women with and without dry eye disease. Meibography was used to assess MG dropout on the central two-thirds of the eyelid and biomicroscopy was used for assessing MG expressibility and meibum quality. Venous blood samples were drawn for serum hormone level analysis. The Kruskal-Wallis test and Spearman correlations were used for statistical analysis. RESULTS: One hundred ninety-eight postmenopausal women with an average age of 61.2 (± 9.1) years were included in this analysis. Testosterone levels showed significant differences between MG dropout grades 1 and 4 (P = 0.002) and grades 2 and 4 (P = 0.01), whereas estradiol levels were different based on MG dropout (P = 0.53). No significant correlations were found between testosterone (r = 0.10, P = 0.17) or estradiol (r = 0.05, P = 0.45) and lipid layer thickness. CONCLUSIONS: Testosterone levels were increased with MG dropout, which was significant between the mild and severe dropout groups, whereas no significant differences were found with estradiol and any MG assessment. Although the literature suggests an association of serum hormone levels and pathogenesis of dry eye disease in postmenopausal women, analysis of active sex steroid precursors and local tissue hormone levels may prove more useful.

Predicting the necessity of LASIK enhancement after cataract surgery in patients with multifocal IOL implantation.

PURPOSE: To investigate if the parameters measured routinely prior to cataract surgery with multifocal intraocular lens (IOL) implantation can predict the necessity of additional laser in situ keratomileusis (LASIK) to improve visual outcome. METHODS: Records of patients undergoing cataract surgery between January 2008 and December 2009 were reviewed. Individuals satisfied with visual outcome of cataract surgery and not satisfied were grouped (group 1 and 2, respectively). Preoperative data of refractive error, axial length, corneal astigmatism, intraocular pressure, and postoperative uncorrected visual acuity were recorded. Data was available for 62 patients (104 eyes), of which LASIK enhancement was deemed necessary in 21 eyes (20%; group 2). The receiver operator characteristic curves were used to discriminate between the groups and linear regression analysis was performed to predict the postoperative visual outcome. RESULTS: The astigmatism measured preoperatively using manifest refraction had an accuracy of 64% in discriminating between the groups. Age, spherical component of refraction, axial length, corneal astigmatism, and intraocular pressure were very close to chance prediction 59%, 57%, 56%, 51%, and 51%, respectively. The postoperative uncorrected visual acuity had an accuracy of 79% in discriminating the groups. Individuals with uncorrected visual acuity worse than 20/40 after cataract surgery were most likely to undergo LASIK enhancement; however, approximately 20% of group 2 underwent LASIK enhancement despite having visual acuity of 20/30 or better. When combined, preoperative visual acuity accounted for just 7% of variance in postoperative uncorrected visual acuity. CONCLUSION: Requirement of LASIK enhancement after cataract surgery with multifocal IOL implant is complex in nature, and parameters routinely measured before surgery cannot successfully identify the group requiring LASIK enhancement or predict postoperative uncorrected visual acuity.