Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaBOOH did not improve with 0.1% AMX administration vs. controlunless the cells were pre-treated with AMX. In more chronic cornealstress, AMX pre-treatment may provide some benefit.uptake/clearance of apoptotic neutrophils.Conclusions: Estrogen orchestrates the inflammatory leukocyteresponse in the cornea, namely by regulating macrophagephagocytosis of apoptotic neutrophils. An essential function forhealthy inflammation is limiting the innate immune response, thedysregulation of which can lead to activation of the adaptive immuneresponse and subsequent chronic disease. This has potentialramifications in the cornea, where chronic inflammation can causeblindness or lead to autoimmune diseases.Commercial Relationships: Samantha B. Wang, None; Kyle M.Hu, None; Yuning Wang, None; David W. Lin, None; JonathanJong, None; Jeremy Lai, None; Karsten Gronert, NoneSupport: EY022208Photographic comparison of mechanical "scratch" healing of AMXtreatedHCE cells vs. controlCommercial Relationships: David V. Dudok, None; KevinCheung, None; Hong Liu, None; Luca Vedovelli, None; EmilianoGhinelli, None; Ken Kenyon, None; Sunil Parapuram, None;Cindy M. Hutnik, NoneSupport: Pilot Fund Grant - Department of Ophthalmology, WesternUniversity CanadaProgram Number: 5237 Poster Board Number: C0156Presentation Time: 2:45 PM - 4:30 PMSex-Specific Differences in the Corneal Inflammatory ReparativeResponse via Estrogen Modulation of PhagocytosisSamantha B. Wang, Kyle M. Hu, Yuning Wang, David W. Lin,Jonathan Jong, Jeremy Lai, Karsten Gronert. Vision ScienceProgram, University of California, Berkeley, Oakland, CA.Purpose: Clearance of apoptotic neutrophils from tissues bymacrophages is a crucial and necessary component of inflammatoryresolution. We previously demonstrated sex-specific differences inself-resolving corneal wound healing responses, which correlatedwith sex-specific differences in macrophage phenotypes. We alsoestablished that estrogen down-regulates intrinsic pro-resolving lipidmediators via ERβ. Pro-resolving lipid mediators such as lipoxin A4regulate macrophage phagocytic capacity. Hence, we investigated ifthere is a sex-specific difference in macrophage phagocytosis and ifthis essential housekeeping function is regulated by estrogen.Methods: Age-matched male and female mice underwent fullcorneal epithelial abrasion. Bone-marrow derived macrophages wereused for in vitro studies. Neutrophils were collected from theperitoneum following zymosan A injection and allowed to apoptosebefore introduction to macrophages. Macrophage phagocytic capacitywas measured using myeloperoxidase assay. Flow cytometry was runto determine macrophage phenotype.Results: Following epithelial abrasion, there was a sex-specificdifference in leukocyte dynamics (i.e. neutrophil recruitment andclearance) in the cornea. Males had higher levels of M2-typemacrophages in the healing corneas compared to their femalecounterparts; however, estrogen did not alter macrophagepolarization into either an M1 or M2 subtype in vitro. In contrastestrogen regulated macrophage function by inhibiting the proresolvingactions of lipoxin A4 (48% inhibition of phagocytosis).Estrogen also regulated both neutrophil apoptosis and macrophage476 Corneal Stroma and KeratocytesWednesday, May 08, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 5238-5258/C0157-C0177Organizing Section: CorneaProgram Number: 5238 Poster Board Number: C0157Presentation Time: 2:45 PM - 4:30 PMKeratocyte-Keratocyte Translamellar Connectivity in the MouseCornea is Revealed using a Novel 3-D Ultrastructural ApproachSamuel D. Hanlon 1 , Nancy C. Shenoi 1 , Paul T. Harris 1 , Paul T.Landry 1 , Ali R. Behzad 2 , Evelyn S. Brown 1 , Margaret M. Gondo 1 ,Alan R. Burns 1, 3 . 1 Research, Univ of Houston College of Optometry,Houston, TX; 2 Imaging and Characterization Core Lab, KingAbdullah University of Science and Technology, Thuwal, SaudiArabia; 3 Leukocyte Biology, Baylor College of Medicine, Houston,TX.Purpose: Like the human cornea, mouse stromal keratocytes liebetween the collagen lamellae and make extensive lateral connectionswith one another forming layers essentially parallel with the cornealsurface. This parallel intralamellar network arrangement is importantto the keratocytes as it allows for cell-cell communication via gapjunctions. The extent to which keratocytes form transverse (i.e.,translamellar) connections is unclear. The purpose of the presentstudy was to use a novel imaging strategy to reconstruct theultrastructural 3-D arrangement of the keratocyte network andspecifically evaluate the distribution of translamellar keratocytekeratocyteconnections.Methods: Corneas from C57BL/6 mice between the ages of 8-12weeks were fixed, heavy metal contrasted and embedded in resinblocks for transverse serial block-face sectioning using a Gatan3view microtome system mounted in an FEI Quanta FEG 200scanning electron microscope. Stacks of 375-700 serial Z images (at100 nm intervals; XYdimensions 28x28 um) were obtained from thestroma in the limbus, paralimbus, and central cornea. Amira 5.2software was used to segment keratocytes for 3-D imagereconstruction.Results: Segmented volumes revealed extensive intralamellar contactbetween keratocytes. Translamellar contact was rare in the centralcornea region with only one translamellar contact observed in a totalof five separate reconstructions. In the paralimbus and limbusregions, translamellar contacts were more common (2.7x10 5 ±1.3x10 5and 6.6x10 5 ±2.2x10 5 per mm 3 , respectively) suggesting there may beas many as 1.2x10 5 keratocyte-keratocyte translamellar contacts in asingle mouse cornea (est. 0.4mm 3 ).Conclusions: Collectively, the 3-D data obtained by serial block-facesectioning show for the first time that in the mouse, interlamellarkeratocyte layers oriented parallel to the corneal surface are indeed©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 - Cornealinked by translamellar keratocyte-keratocyte connections, but thisoccurs predominantly at the peripheral cornea. We propose a modelwhereby keratocytes form a single contiguous 3-D network, ratherthan a series of independent parallel networks, with a higher degreeof translamellar connectivity than previously appreciated.Commercial Relationships: Samuel D. Hanlon, None; Nancy C.Shenoi, None; Paul T. Harris, None; Paul T. Landry, None; Ali R.Behzad, None; Evelyn S. Brown, None; Margaret M. Gondo,None; Alan R. Burns, NoneSupport: NIH Grant EY017120, NIH Grant EY07551Program Number: 5239 Poster Board Number: C0158Presentation Time: 2:45 PM - 4:30 PMEffect of Serum Clot Activator on KeratocytesJi-Eun Lee 1 , Seung Uk Lee 2 , Jong Soo Lee 1 . 1 Ophthalmology, PusanNational University, Pusan, Republic of Korea; 2 Ophthalmology,Kosin University, Pusan, Republic of Korea.Purpose: To evaluate the effect of serum clot activator which couldbe used for making autologous serum eye drop on humankeratocytes.Methods: Cultured human corneal keratocytes were exposed to 10,20, and 30 % SiO2 for 24, 48, and 72 hours, MTT-based calorimetricassay was performed to determine the survival rate of keratocytes andlactate dehydrogenase (LDH) leakage assay to assess thecytotoxicity. Apoptotic response was evaluated with flow cytometricanalysis and fluorescence staining with Annexin V and propiodiumiodide. Cellular morphology was evaluated by inverted phasecontrastlight microscopy and electron microscopy.Results: The survival rate of human keratocytes and cytotoxicityshowed the concentration and time dependent response andsignificant response was found after treating with 30% SiO2 for 72hours. Apoptosis developed in flow cytometry and apoptotic cellswere demonstrated in fluorescent micrograph after treating with 30%SiO2 for 48 hours but cells were non viable after 72 hours. Humankeratocytes were more detached from the bottom of the dish anddamaged cells have degenerative changes like microvillidisappearance, vacuoles formation and chromatin of the nuclearremnant condensed along the nuclear periphery after treating with30% SiO2 for 72 hours.Conclusions: SiO2, the serum clot activator, showed the toxicity onhuman keratocytes at the concentration of 30% after 72 hourstreament. So blood should be extracted with tubes without clotactivator during making the autologous serum eye drop forpreventing the possible cytotoxicity on cornea.Commercial Relationships: Ji-Eun Lee, None; Seung Uk Lee,None; Jong Soo Lee, NoneProgram Number: 5240 Poster Board Number: C0159Presentation Time: 2:45 PM - 4:30 PMCo-cultures of Human Corneal Epithelium and Self-assembledKeratocyte and Fibroblast MatrixAudrey E. Hutcheon 1, 2 , Dimitrios Karamichos 1, 2 , Xiaoqing Q. Guo 1,2 , James D. Zieske 1, 2 . 1 Schepens Eye Research Institute/MEE,Boston, MA; 2 Department of Ophthalmology, Harvard MedicalSchool, Boston, MA.Purpose: During corneal wound repair the epithelium switches frominteracting with a mature matrix to a temporary matrix that ispartially assembled by wound-healing fibroblasts. The goal of thecurrent investigation was to develop and examine models that mimicthe interaction of epithelium interacting with a keratocyte-assembledmatrix versus a fibroblast-assembled matrix.Methods: Stromal cells were isolated from corneal explants byplacing tissue in either 1 or 10% FBS in DMEM. After culture andpassaging, the 1% (keratocyte-like) or 10% (fibroblast-like) cellswere plated in Transwell dishes with stabilized Vitamin C (VitC) ±0.1ng/ml of TGF-β3 and maintained in either 1 or 10% FBS inDMEM for 3 weeks. Immortalized human corneal-limbal epithelialcells (HCLE) were then placed atop the self-assembled matrix andcultured for 4 days; after which, the cultures were airlifted andmaintained for an additional 1 or 2 weeks. The co-cultures wereprocessed and examined by transmission electron microscopy (TEM)and indirect-immunofluorescence (IF) for morphology, extracellularmatrix components and fibrotic markers. Antibodies againstcollagens I, III, and V, thrombospondin-1 (TSP-1), cellularfibronectin (cFN), and smooth muscle actin (SMA) were examined.Results: Cells isolated with 1% serum but cultured without T3 didnot assemble a matrix. As observed by TEM, the morphology of thestromal cells isolated using 1% serum had a dendritic appearance,which is characteristic of keratocytes; whereas, the 10% serum cellswere spindle shaped, a fibroblastic characteristic. This distinctdifference in cell shape was also noted after IF with SMA, which wasfound to be present in both the keratocyte-like and fibroblast-likematrices. Collagens I, III, and V localization were similar in allcultures. Most interestingly, cFN and TSP-1 expression was greatlyreduced in the keratocyte-like co-cultures compared to the fibroblastlikeco-cultures.Conclusions: We have developed models that allow for thecomparison of the interaction of human corneal epithelial cells withkeratocyte-like and fibroblast-like self-assembled matrices. Thefibroblast-like matrix appears to stimulate wound-healing responses(as indicated by TSP-1 and cFN expression) to a greater extent thanthe keratocyte-like matrix.Commercial Relationships: Audrey E. Hutcheon, None; DimitriosKaramichos, None; Xiaoqing Q. Guo, None; James D. Zieske,NoneSupport: NIH Grants EY005665, EY020886, EY03790 (Core) andDOD W81XWH-11-1-0477Program Number: 5241 Poster Board Number: C0160Presentation Time: 2:45 PM - 4:30 PMRNAi Gene Silencing Of TGF-beta Signaling: A PowerfulApproach To Control Corneal FibrosisJason T. Rodier, Ajay Sharma, Ashish Tandon, Audra Stallard, RajivR. Mohan. Mason Eye Institute, University of Missouri-Columbia,Columbia, MO.Purpose: Transforming growth factor β (TGFβ) promotes keratocytetransdifferentiation to myofibroblast and cause corneal fibrosis invivo. Our siRNA studies demonstrated that TGFβ primarily usesSMAD signaling for this transformation in the cornea. The presentstudy tested the hypothesis that SMAD-2, SMAD-3 or SMAD-4 genesilencing is a novel approach to treat corneal scarring using an invitro model. We quantified the potency of SMAD gene silencing toabrogate TGFβ pathology and myofibroblast formation usingSMADs siRNA, and (2) examined whether RNA inference (RNAi) orshort-hairpin RNA (shRNA) is a better modality to achieve sustainedSMAD gene silencing in the cornea for gene therapy using an in vitromodel.Methods: : Human donor corneas were used to obtain human cornalefibroblasts (HCF). TGFβ1 (5ng/ml) was used to induce HCFtransformation myofibroblasts under serum-free conditions.Commercial pre-validated siRNA specific for SMADs were used.The shRNA and RNAi sequences specific for SMAD-2, SMAD-3and SMAD-4 were designed using RNAi software, and cloned intomammalian expression vectors to generated SMAD-shRNA/RNAivector constructs. Lipofectamine-2000 was used for transfection.Quantitative real-time PCR, western blotting and©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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