Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To test the hypothesis that miR-205 plays a key role instimulation of growth factors release, TGFA and EGF, in woundedcorneal epithelial cell by targeting 3-UTR of KCNJ10, therebyinhibiting the K channel activity which leads to Ca2+ influx andactivating EGFR/ERK phosphorylation.Methods: The expression level of miR-205 and KCNJ10 wereexamined by stem-loop qRT-PCR and western blot in cultured HCEcells. The wound-healing assay was used to examine the recoveryrate of HCE cells. TGFA and EGF in the media, an index as growthfactor secreted by HCE, were measured by ELISA. We used dualluciferase activity as a report gene to assess the effect of miR-205 onKCNJ10 expression. To study the role of miR-205 and KCNJ10 inregulation of HCE function, the HCE cells were transfected withmiR-205 mimic or KCNJ10 siRNA. The patch-clamp technique wasused to measure the K currents in HCE cells.Results: Mechanic scratching HCE cells stimulated the expression ofmiR-205 by 50% within 1 hour and decreased the expression ofKCNJ10 within 24 hours. Application of miR-205 antagomersignificantly delayed the healing process of HCE, an effect waspartially revered by inhibiting K channels with BaCl2. Moreover,inhibition of kcnj10 channels with shRNA also partially restored thedelayed healing by miR-205 antagomer. The possibility that increasein miR-205 in wounded HCE cells facilitates the cell regeneration byinhibiting inward rectifier K channels was also confirmed by thefinding that miR-205 decreased luciferase report gene activity in cellstransfected with 3’UTR of KCNJ10. Moreover, Western blot andpatch-clamp experiments demonstrated that suppression of theendogenous mir-205 expression enhanced the expression in KCNJ10but not KCNJ16 in plasma membrane and inward K currents in HCEcells by 150%. The possibility that inhibition of K channels by miR-205 may stimulate growth factor release through a depolarizationinducedCa2+ signaling is also suggested by the finding thatinhibition of KCNJ10 by siRNA increased TGFA/EGF release intomedia through upregulation of EGFR/ERK pathway.Conclusions: We conclude that miR-205 is one of the importantfactors stimulating healing of wounded HCE cell by inhibitingKCNJ10 inwardly-rectifying K channels thereby enhancingTGFA/EGF release and facilitating the repair process.Commercial Relationships: Daohong Lin, None; Adna Halilovic,None; Sherin Thomas, None; Kemeng Wang, None; Peng Yue,None; Lars Bellner, NoneSupport: AHA 11SDG7360052Program Number: 3866 Poster Board Number: D0110Presentation Time: 2:45 PM - 4:30 PMEffects of In-vivo Application of Cold Atmospheric Plasma onCorneal Wound Healing in New Zealand White RabbitsRashed Alhabshan 1 , David Belyea 1 , Mary Ann Stepp 1, 2 , JeffreyBarratt 1 , Sanjeev Grewal 1 , Alexey Shashurin 3 , Michael Keidar 3 .1 Department of Ophthalmology, George Washington University,Washington, DC; 2 Department of Anatomy and RegenerativeBiology, George Washington University, Washington, DC;3 Department of Mechanical and Aerospace Engineering, GeorgeWashington University, Washington, DC.Purpose: Cold Atmospheric Plasma (CAP) has been shown to reducecorneal infection but little is known about the impact of CAP onhealthy corneal tissues and their ability to respond to injuries. Toexamine the effect of Cold Atmospheric Plasma (CAP) on woundhealing after corneal epithelial and basement membrane ablation inNew Zealand white rabbits.Methods: Twelve New Zealand white rabbits were assigned into twogroups, Group A (7 rabbits) and Group B (5 rabbits). Five rabbitsfrom each group underwent surgical intervention to the right eye, a 6mm central corneal ablation to the epithelium and stroma using anAlger brush with 0.5 mm burr. After ablation, all rabbits in group Areceived 120s application of CAP. Two rabbits in Group A receivedCAP application without ablation. Eyes monitored for haze,inflammation, and reepithelialization with slit lamp examination. 24hours after ablation, two corneas were harvested from each group.The 20th day, the remaining corneas were harvested, fixed informalin and stained with H&E. In addition, immunofluorescencemicroscopy was performed to assess scar formation using antibodiesagainst fibronectin and αsmooth muscle actin.Results: Corneal reepithelialization on day 1 showed that rabbits ingroup A (treated) had an average epithelial defect of 9.25 mm2 andgroup B (untreated) had a defect of 12.05 mm2, not a statisticallysignificant difference. H & E stained sections showed the expectedresponses of stromal fibroblasts to debridement injury in both groups.Epithelial thickness and stromal cell counts on both groups 20 daysafter injury showed no significant statistical differences. At 24 hoursand 20 days after injury, analyses show that CAP-treatment increasedfibronectin deposition in the anterior stroma and αSMA localizationwithin stromal fibroblasts; quantitative analysis ofimmunofluorescence data will be needed to determine whether thesedifferences are significant.Conclusions: CAP treatment following corneal injury does notinterfere with rate of wound closure or induce increasedinflammation. CAP treatment did affect corneal tissues since αSMAand fibronectin localization within stromal fibroblasts appears toincrease slightly after CAP treatment regardless of whether corneashave been wounded. More studies are needed to evaluate thepotential effects of CAP on the cornea and its possible applications inthe field of Ophthalmology.Commercial Relationships: Rashed Alhabshan, None; DavidBelyea, None; Mary Ann Stepp, None; Jeffrey Barratt, None;Sanjeev Grewal, None; Alexey Shashurin, None; Michael Keidar,NoneSupport: "GWU MFA Award"; "NIH RO1 EY08512 to MAS"; "TheMansour F Armaly Glaucoma Research Fund (fund # ET11161)"Program Number: 3867 Poster Board Number: D0111Presentation Time: 2:45 PM - 4:30 PMCorneal optical density during Hyperbaric Oxygen TherapyKnut Evanger 1 , Guro Vaagbø 2 , Einar Thorsen 2, 3 , Olav H. Haugen 1, 4 .1 Institute of Clinical Medicine, University of Bergen, Bergen,Norway; 2 Hyperbaric Medicine Unit, Dept. of OccupationalMedicine, Haukeland University Hospital, Bergen, Norway; 3 Instituteof Medicine, University of Bergen, Bergen, Norway; 4 Department ofOphthalmology, Haukeland University Hospital, Bergen, Norway.Purpose: To evaluate the influence of hyperbaric oxygen therapy onthe anterior corneal optical density and the refractive index. Little isknown about the effect on the anterior corneal surface when the eyeis exposed to enhanced oxygen concentration and pressure.Methods: 16 patients (32 eyes) were receiving 100% oxygen at apressure of 240 kPa in a monoplace chamber for 90 min daily.Anterior corneal optical density was measured with the densitometryprogram of the Pentacam Scheimpflug imaging system (OculusOptikgeräte GmbH) at baseline and repeated after completed 19 daysof treatment. The 180-degree rotating Scheimpflug camera capturesoptical density data according to the light scattering intensity of theanterior cornea. The density measurement values are standardizedfrom 0 (no clouding) to 100 (tissue completely opaque). TheGladstone-Dale constant was calculated at baseline by using theGladestone-Dale formula, the mean anterior corneal optical densityvalue, and a refractive index of 1.376 of the normal human cornea.Results: Mean anterior corneal optical density and refractive index©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.