Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafor Ophthalmic Sciences, All India Institute of Medical Sciences,New Delhi, India; 5 Department of Pathology, All India Institute ofMedical Sciences, New Delhi, India.Purpose: To evaluate the potential use of decellularized humancorneas as a scaffold for cultivating human corneal endothelial cells.Methods: Human corneal tissues (N=20) not suitable fortransplantation were used. Corneal endothelial cells were isolated byexplant culture method. For preparation of the scaffold, cornealepithelium and endothelium were removed mechanically andremaining corneal stroma was decellularized using enzymaticmethod.The resulting acellular matrices were then subjected toHaematoxylin-Eosin (H&E) staining to visualize cellular remnants;quantitative analysis to determine the DNA content; ScanningElectron Microscopy (SEM) for collagen fibril morphology; Alcianblue staining to analyse extracellular matrix (ECM);Immunohistochemistry for structural proteins collagen type I, II, IV,fibronectin; and cytotoxicity assay of decellularized cornea using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Tensilestrength of corneal tissues &Light transmission ratios was estimatedusing uniaxial load testing equipment &UV-visiblespectrophotometer. Native cornea served as control for all theexperiments. 10,000 human corneal endothelial cells were culturedon decellularized scaffold for 14 days and then analyzed using SEM,histology and Immunocytochemistry for ZO-1, and Na+ /K+-ATPase.Results: H&E staining demonstrated efficient elimination of cellularcomponents. Alcian blue confirmed good preservation of theextracellular matrix and major structural proteins collagen type I, IIIV & fibronectin were retained. The amount of DNA indecellularized cornea was 32+ 7.27ng/mg whereas in native cornea itwas 133+8.3ng/mg(p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: EY08834Program Number: 1659 Poster Board Number: D0294Presentation Time: 8:30 AM - 10:15 AMDisappearance and reappearance of cilia of corneal endotheliumpreserved in corneal preservation mediaHidetoshi Tanioka, Katsuhiko Shinomiya, Satoshi Kawasaki, ShigeruKinoshita. Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,Japan.Purpose: It is known that the primary cilia of cells are present incorneal endothelium. However, the primary cilia of donor cornealendothelium disappear when preserved in corneal preservation media.This study investigated the histological change of the cilia of cornealendothelium preserved in corneal preservation media.Methods: This study involved corneas obtained from Japanese whiterabbits. The corneas were preserved in Optisol-GS (Bausch andLomb, Rochester, NY) corneal preservation media at 4°C for 0, 1 and7 days. The cornea preserved for 7 days was incubated at 37°C inculture media (CnT-20; CELLnTEC Advanced Cell Systems AG,Bern, Switzerland) for 2 days. Corneal endothelia of these corneaswere assessed by immunohistochemistry with Anti-Acetylated alphaTubulin antibody and scanning electron microscopy (SEM).Results: Immediately after isolation, long process was observed bySEM on the corneal endothelium, and it showed positive for primarycilia by Anti-Acetylated alpha Tubulin antibody. No primary ciliawere observed on the corneal endothelium preserved for 1 or 7 days.After 7-days preservation, primary cilia were observed on theendothelium that underwent the 2-day incubation at 37°C.Conclusions: The findings of this study using rabbit corneas suggestthat the primary cilia of the corneal endothelium of human donorcorneas for transplantation also disappear during preserved inpreservation media and reappear after transplantation, and show thatthe existence of primary cilia is correlated with the viability ofcorneal endothelium.Commercial Relationships: Hidetoshi Tanioka, None; KatsuhikoShinomiya, None; Satoshi Kawasaki, None; Shigeru Kinoshita,Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: Grant-in-Aid for Scientific Research (KAKENHI)Program Number: 1660 Poster Board Number: D0295Presentation Time: 8:30 AM - 10:15 AMEndothelial loss and pachymetric change in patients undergoingpenetrating keratoplasty with 3 years of followElisa D. Alegria, Regina Velasco, Oscar Baca, Alejandro Babayan.Cornea, Hosp Nuestra Senora De La Luz IAP, Mexico, Mexico.Purpose: Identify endothelial loss and pachymetric changes inpatients operated penetrating keratoplasty at follow up 3 yearsMethods: Pre surgical endothelial count was obtained by the reportsof the LionsEye Bank Institute in Tampa, Florida.A non contact specular microscopy with a microscope Topcon SP-2000P in the operated was made in the operated eye. Specularmicroscopy was taken only with the image captured by auto focus.EEndothelial cel density was calculated by identify at minimum of 30cels . Specular microscope automatically calculates and displays theresults. We also performed and compare it with ultrasonicpachymetry Accutome Accupach V in the last visit.Results: We analize 20 eyes.with 3 years of follow-up. Endothelialloss is greater than 50% of initial basal count representing a p> 0002.We observe the values of pre-surgical pachymetric compared againstthe post surgical have a grat differenceConclusions: Endothelial loss of patients undergoing penetratingkeratoplasty with follow at three years is more than 50%. Thethickness pachymetric also shows significant variationCommercial Relationships: Elisa D. Alegria, None; ReginaVelasco, None; Oscar Baca, None; Alejandro Babayan, NoneProgram Number: 1661 Poster Board Number: D0296Presentation Time: 8:30 AM - 10:15 AMThe Resting Potential of Rat`s Corneal Endothelial CellsNassim S. Calixto 1 , Vinicius V. Oliveira 2 , Renata Fleming 2 , SebastiaoCronemberger 1 , Adalmir Dantas 2 . 1 Ophthalmology, Federal Univ ofMinas Gerais, Belo Horizonte, Brazil; 2 Ophthalmology, FederalUniversity of Rio de Janeiro, Rio de Janeiro, Brazil.Purpose: The mechanism whereby the cornea maintains itstransparency is not known. This study aims to register the presenceand to quantify the resting potential (RP) of endothelial corneal cells.To the best of our knowledge this seems to be the first work tosystematically measure and quantify the RP of endothelial cells of thecornea.Methods: We performed 30 experiments on corneal preparations ofSprague-Dawley rats (n=30). Immediately after decapitation theeyeballs were removed and sectioned near the limbus. The corneaswere transferred to a chamber and infused with Balanced SaltSolution (BSS) driven by a peristaltic pump in order to maintain theBSS flowing at a rate from 0.8 to 0.85 ml/min. The temperature in thechamber was set at 30 °C by means of a thermostatic bath. BSS iscomposed of (mmol/l): Na + 160.0; Cl - 130.0; - HCO 3 25.0; K + 5.0; -H2PO 4 3.0; Mg ++ 1.0; Ca ++ 1.0; Glucose 5.0, presenting a pH = 7.4and 305 mOsm/Kg. The presence or absence of electrical signal wasdetected by recording its voltage variations through two poreelectrodes delicately introduced into the cells and connected to aGrass polygraph. The statistical analysis was made by Graph PadPrism 6.0.Results: In the 30 experiments (30 eyes), the RP presented a mean of40.60 with a standard deviation of 7.05 and a standard error of 1.29.The graphics below illustrate the findings.Conclusions: Although many studies are still needed to understandthe role of the endothelium in the transparency of the cornea, ourresults demonstrate:1. the presence of the resting potential in the corneal endotheliumcells2. a mean of 40.60±7.05 mV for the resting potential of the rat’scorneal endothelial cells.This graph shows the mean value at the center line, the standard©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.
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