Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadeviation (SD) at the boxes, and the maximum and minimum values.Commercial Relationships: Nassim S. Calixto, None; Vinicius V.Oliveira, None; Renata Fleming, None; Sebastiao Cronemberger,None; Adalmir Dantas, NoneProgram Number: 1662 Poster Board Number: D0297Presentation Time: 8:30 AM - 10:15 AMWnt5a enhances cell migration through regulation of Cdc42 andRhoA pathway in human corneal endothelial cellsJeongGoo Lee 1 , Daniel Sand 1, 2 , J M. Heur 1, 2 . 1 Ophthalmology,University of Southern California, Los Angeles, CA; 2 Doheny EyeInstitute, Los Angeles, CA.Purpose: Wnt5a activates β-catenin-independent pathways forregulation of cellular functions that play crucial roles in woundhealing process in cornea including cell migration and polarity.Elucidation of Wnt5a signaling may help identify potentialtherapeutic targets for enhanced corneal wound healing; however,Wnt5a signaling in corneal endothelial cells (CEC) has not been wellcharacterized.Methods: Expression and/or activation of Frizzled-5, Cdc42, Rac1were analyzed by immunoblotting. Scratch-induced directionalmigration assay was employed to measure migratory rates. Activationof Cdc42 and Rac1 were determined by GTP pull-down assay. RhoAactivity was measured using RhoA specific G-LISA assay kit.Results: Stimulation of human CECs with Wnt5a alone resulted in50% enhancement in cell migration as measured by scratch inducedmigration assay and this effect could be completely blocked byML141 (Cdc42 inhibitor). Co-treatment of both Wnt5a and Y27632(inhibitor of Rho-associated kinase) resulted in a 62% enhancementof migration. The synergistic effects of Wnt5a and Y27632 on humanCECs migration were partially blocked by ML141 and this, in turn,was completely abolished by treatment with both ML141 and RhoAactivator. Under these conditions, activation of both Cdc42 and Rac1and inactivation of RhoA were observed in the Wnt5a treated cells.We further confirmed that activated Cdc42 negatively regulatesRhoA activity and this regulation plays important role in endothelialmigration.Conclusions: These findings suggest that Wnt5A mediated migrationin human CEC is mediated by activation of Cdc42 and inactivation ofRhoA.Commercial Relationships: JeongGoo Lee, None; Daniel Sand,None; J M. Heur, NoneSupport: Baxter Foundation, Research to Prevent Blindness, andNIH Core Grant EY03040Program Number: 1663 Poster Board Number: D0298Presentation Time: 8:30 AM - 10:15 AMInfluence Of Hydrodynamic Culture Conditions On TheExpression Of Cell Junctions Of Tissue-Engineered HumanCorneal EndotheliumOlivier Roy 1 , Isabelle Brunette 3, 4 , Stephanie Proulx 1, 2 . 1 LOEX/CUO2 Ophtalmologie, Université Laval, Quebec, QC, Canada; 3 Centre derecherche HMR, Montréal, QC, Canada; 4 Ophtalmologie, Universitéde Montréal, Montréal, QC, Canada.Purpose: This study was undertaken in order to evaluate theinfluence of fluid flow and pressure on the expression of celljunctions of tissue-engineered corneal endothelium in vitro.Methods: Cultured human corneal endothelial cells were seeded andcultured on a previously devitalized corneal stroma, then either left instandard culture conditions (static cultures) or placed in an artificialanterior chamber with a perfusion rate of of 5 ul/min and ahydrostatic pressure of 18 mm Hg (hydrodynamic cultures). A higherperfusion rate than normal (2.6 ul/min) was chosen in order tomaintain a constant perfusion pressure and allow for the additionalleak of perfusate through the trabecular meshwork into the dish. After4-6 days, corneas were photographed then fixed in 3.7%formaldehyde for histology, 2.5% glutaraldehyde for transmissionelectron microscopy or 4% paraformaldehyde forimmunofluorescence staining of F-actin and the tight junction proteinZO-1. Controls consisted of devitalized corneas that were not seededwith corneal endothelial cells, and were processed in the samemanner.Results: Macroscopically, corneas maintained in hydrodynamiccultures were more transparent than the static-cultured corneas. Thecorneal stromas in the histology cross-sections were thinner in thehydrodynamic-cultured corneas than in the static-cultured corneas.The mean (±SD) collagen spacing, calculated using transmissionelectron microscopy images, was 27 ±4 nm when corneas were underhydrodynamic culture conditions whereas spacing was 46 ±9 nmwhen corneas were left in static culture conditions. Controldevitalized corneas with no endothelium had a mean collagen spacingof 32 ±6 nm when cultured under the same hydrodynamic conditions.Hydrodynamic-cultured corneal endothelium expressed higheramounts of ZO-1 protein, as assessed by immunostaining.Conclusions: This study shows that corneal endothelial cells respondto an anterior chamber flow and pressure by improving cell junctionexpression in vitro. Ultimately, studying the effect of hydrodynamicculture will enable an improved understanding of morphogenesis andcell junction formation of the corneal endothelium.Commercial Relationships: Olivier Roy, None; Isabelle Brunette,None; Stephanie Proulx, NoneSupport: NSERC, FRQS Vision Research NetworkProgram Number: 1664 Poster Board Number: D0299Presentation Time: 8:30 AM - 10:15 AMThe Quebec Corneal Cell Bank: Update on Culture Success ofPathologic Human Corneal Endothelial Cells (2009-2012)Mathieu Theriault 1 , Olivier Roy 1 , Olivier Rochette-Drouin 1 , Marie-Claude Perron 3 , Isabelle Brunette 3, 4 , Stephanie Proulx 1, 2 .1 LOEX/CUO - Recherche, Centre de recherche du CHU, Quebec,QC, Canada; 2 Ophtalmologie, Université Laval, Quebec, QC,Canada; 3 Centre de recherche HMR, Montréal, QC, Canada;4 Ophtalmologie, Université de Montréal, Montréal, QC, Canada.Purpose: The purpose of this study was to assess the feasibility ofinitiating primary cultures of corneal endothelial cells from patientssuffering from various corneal diseases. We also evaluated whichconditions yielded the best results for culture.Methods: Consenting patients undergoing penetrating keratoplasty orDescemet’s stripping automated endothelial keratoplasty wereenrolled in this study. The cornea (or Descemet’s membrane),removed at the time of surgery, was sent to the laboratory. Uponreceipt, specimens were photographed, endothelial cells were isolatedand cultured, and Descemet’s membranes were fixed in 3.7%formaldehyde for histology. Data collected included diagnosis, age- Recherche, Centre de recherche du CHU, Quebec, QC, Canada;©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand sex of the donor, time spent in Optisol, culture success andnumber of cells obtained after a primary culture. Control healthy EyeBank corneas were processed in a similar manner.Results: A total of 194 specimens were obtained between August2009 and November 2012. Pathologies included Fuchs cornealendothelial dystrophy (FECD; n=93), pseudophakic bullouskeratoplathy (PBK; n=33), FECD+PBK (n=4) and other cornealdisorders (n=64). Overall, 56 of the 194 diseased specimens (FECDn=37; PBK n=3; FECD+PBK n=2; other n=14) and all of the 29healthy corneas successfully initiated an endothelial cell culture.Among the diseased specimens, the mean(±SD) donor age was 66.5±11.9 years for successful cultures and 68.9 ±15.2 years for theunsuccessful cultures. Time spent in Optisol was 2.7 ±1.2 and 3.2±2.6 days for successful and unsuccessful cultures, respectively. Thediseased specimens yielded 120 000 ±86 000 cells and the healthycorneas 122 000 ±55 000 cells.Conclusions: This study shows that successful corneal endothelialcell culture can be generated despite various corneal diseases. FECDallowed the highest success rate. Once culture was initiated, a similarnumber of cells was obtained from FECD, BPK and healthyspecimens. Patient sex and age, and the time spent by the specimen inOptisol did not influence success rate. The Quebec Corneal CellBank will become a useful tool for the study of various cornealendotheliopathies.Commercial Relationships: Mathieu Theriault, None; OlivierRoy, None; Olivier Rochette-Drouin, None; Marie-ClaudePerron, None; Isabelle Brunette, None; Stephanie Proulx, NoneSupport: CIHR, FRQS Vision Health Research NetworkProgram Number: 1665 Poster Board Number: D0300Presentation Time: 8:30 AM - 10:15 AMPerception of Cornea and Glaucoma Subspecialists RegardingPrevalence of Corneal Decompensation with Ex-Press ShuntPlacementShalin Shah 1 , Ngo Yen 1 , Thompson W. Hilary 2 , Jayne S. Weiss 1 .1 Department of Ophthalmology, Louisiana State University EyeCenter, LSU School of Medicine, LSU Health Sciences Center, NewOrleans, LA; 2 Biostatistics Section, School of Public Health,Louisiana State University Health Sciences Center, New Orleans,LA.Purpose: Corneal decompensation is a recognized complicationassociated with anterior chamber insertion of Ahmed, Baerveldt andMolteno (ABM) shunts. By comparison, there are no publicationsaddressing corneal decompensation after Ex-Press shunt placement.The purpose of this study was to assess the prevalence and onset ofcorneal decompensation with ABM shunts and Ex-Press shunts asperceived by cornea and glaucoma specialists.Methods: A survey was distributed to members of the CorneaSociety and Glaucoma Society with questions about frequency andonset of complications after anterior chamber placement of ABM andEx-Press shunts. The individual was requested to rank the followingside effects in order of perceived prevalence: chronic hypotony,corneal decompensation, endophthalmitis, infection, malignantglaucoma, pthisis bulbi, and retinal detachment. Time of onset tocorneal decompensation in Ex-Press shunts was compared to that ofthe ABM group.Results: 17 glaucoma subspecialists and 22 cornea subspecialistsparticipated. Corneal decompensation was listed as the mostprevalent of the seven possible complications by both subgroups (chisquare < .001). 65.0% of cornea subspecialists and 84.6% ofglaucoma subspecialists reported the risk of corneal decompensationto be higher with uncomplicated ABM placement than with Ex-Pressshunts (chi square
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Cornea - ARVO

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