Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 552 Poster Board Number: B0189Presentation Time: 10:30 AM - 12:15 PMBoric Acid-based Multipurpose Contact Lens Care Solutions(MPSs) Cytotoxicity Effect on Human Corneal Epithelial CellsKissaou T. Tchedre 1 , Masaki Imayasu 2 , Yuichi Hori 3 , H D.Cavanagh 4 . 1 R&D and Innovation Center, Menicon LTD, Le Mans,France; 2 R&D center, Menicon, Co. ltd, Kasugai, Japan;3 Ophthalmology, Toho University Sakura Medical Center, Sakura,Japan; 4 Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas,TX.Purpose: The purpose of this study is to determine whethercommercially available new multipurpose contact lens care solutions(MPSs) have any cytotoxicity effect on human corneal epithelial(HCE-T) cells. MPSs effect on membrane-associated mucins (MUC1and MUC16) expressions in the Rat cornea was also assessed.Membrane-associated mucins are one of the major components of theocular surface that play a vital role in the maintenance of the ocularsurface integrityMethods: HCE-T cells were treated with different concentrations ofMPS-F (1ppm PHMB, no boric acid), MPS-G (1.3ppm PHMB, 1ppmPQ-1, boric acid), MPS-H (1.6 ppm, Alexidine, 3ppm PQ-1, boricacid), MPS-I (1ppm PHMB, boric acid), and MPS-J (5ppm ALDOX,10ppm PQ-1, boric acid): 100% treatment for 30 minutes and 10%treatment for 24 hours. Cell death was measured by using aviability/cytotoxicity assay kit. Winstar Rats were also subjected toMPSs (1 drop in the right eye every 10 minutes for 1 hour). The leftEye was used as control (1 drop of PBS every 10 min for 1 hour).Cornea lysates were subsequently prepared and used for Western blotanalysis for MUC1 and MUC16.Results: The viability/cytotoxicity assay result showed that MPSscontaining boric acid induce cell death in HCE-T cells. The westernblot result showed that boric acid-based MPS down-regulatemembrane-associated mucins in the cornea while MPSs without boricacid had no effect on membrane-associated mucins.Conclusions: The concentration of boric acid used in commerciallyavailable multipurpose contact lens care solutions should be chosencarefully to avoid MPS-related ocular surface damage. Ocular surfacedamage simultaneously promotes microbial pathogens and potentiallyincreases clinical rates of infection.Commercial Relationships: Kissaou T. Tchedre, Menicon, Co. Ltd(E); Masaki Imayasu, Menicon Co., Ltd. (E); Yuichi Hori, None; HD. Cavanagh, Menicon Ltd (C)Program Number: 553 Poster Board Number: B0190Presentation Time: 10:30 AM - 12:15 PMThe Effect of Amniotic Membrane De-epithelialization Methodon its Biological Properties and Ability to Promote LimbalEpithelial Cell CultureGary Hin-Fai Yam 1 , Ting Zhang 1 , Andri K Riau 1 , Roger W.Beuerman 1 , Donald T. Tan 2 , Jodhbir S. Mehta 1, 2 . 1 Singapore EyeResearch Institute, Singapore, Singapore; 2 Singapore National EyeCenter, Singapore, Singapore.Purpose: To characterize the de-epithelialized human amnioticmembrane (HAM) and compare cell attachment and proliferationefficiencies.Methods: HAM was de-epithelialized by 20% ethanol (AHAM), 1.2U/ml Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM) and 5M urea (UHAM), respectively, followed bygentle scrapping with a #15 blade. Surface topology, extracellularmatrix (ECM) and growth factor content were characterized andcompared to intact HAM by electron microscopies (EM), atomicforce microscopy (AFM), immunohistochemistry and westernblotting. Primary human limbal epithelial cells (LEC) attachment andproliferation efficiencies were assayed. Statistical significance wascalculated by SPSS and Fisher’s Least Significant Difference test.Results: EHAM, THAM and UHAM had intact basal lamina andsmooth basement membrane surface shown under transmission andscanning EM and AFM. Cell remnants stayed on AHAM. Disruptedbasement membrane and stroma was found in DHAM.Immunostaining intensity quantification and hierarchical clusteringrevealed that ECM composition of EHAM and UHAM resembled tointact HAM. In contrast, DHAM and THAM had drastic loss of ECMand growth factor content. LEC attachment efficiency at 24 hourspost-seeding was the highest in EHAM (51% as on conventionalculture surface), followed by UHAM and AHAM. However, cellproliferation indices at day 10 of culture were similar among differentHAM substrates, suggesting repair of ECM and basement membraneby growing epithelial cells.Conclusions: Urea denudation preserved the basement membraneintegrity, ECM and growth factor composition, and had higher cellattachment and proliferation efficiencies. With its short processingtime, urea treatment offers a novel alternative for HAM deepithelialization.Commercial Relationships: Gary Hin-Fai Yam, None; TingZhang, None; Andri K Riau, None; Roger W. Beuerman, Allergan(F), SERI (P), Santen (R); Donald T. Tan, Network MedicalProducts (P), Carl Zeiss Meditec (F), Alcon Labs (F), Bausch &Lomb (F), Allergan (F), Santen (F); Jodhbir S. Mehta, NoneSupport: Singapore National Medical Research Council grants IBGand R485/34/2006Program Number: 554 Poster Board Number: B0191Presentation Time: 10:30 AM - 12:15 PMKnockdown of MUC16 Alters Tight Junctions of CornealEpithelial Cells Resulting in Decreased TransepithelialResistanceIlene K. Gipson, Sandra J. Spurr-Michaud, Ann S. Tisdale. HarvardMed Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston,MA.Purpose: MUC16 is a major membrane associated mucin of thehuman corneal epithelium. We have shown that MUC16 is a barrierto rose bengal dye penetrance and pathogen adherence. We alsodemonstrated that MUC16’s cytoplasmic tail associates with actinthrough the ezrin, radixin, moesin family of actin linking proteins.Because MUC16 may be associated with actin filaments that insertinto the apical web of actin filaments that terminate in tight junctions,the purpose of this study was to determine the role of MUC16 in tightjunction formation and function as measured by transepithelialresistance (TER).Methods: An immortalized human corneal limbal epithelial cell line(HCLE) (Gipson et al, 2003) and the HCLE cell line stablytransfected with siRNA to MUC16 in which MUC16 levels werereduced by 70% and a vector control line were used. Cells werecultured for optimal mucin expression and stratification. Assayscomparing the cell lines included measurement of apical cell size,localization and mRNA levels of Z01, a tight junction protein, andTER using an Evum2 Epithelial Voltohmmeter.Results: HCLE cells knocked down (KD) for MUC16 showed asignificant 2-3 fold (p