<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>Methods: Retinas of cbs+/+ (n=21) & cbs-/- (n=22) mice wereharvested at ~3 wks. RNA & protein were isolated & subjected toqPCR & western blotting, respectively to analyze expression of ERstress genes & the proteins they encode including BiP, PERK,pPERK, CHOP, ATF6 & RE1α. Retinal cryosections were subjectedto immunofluorescence to evaluate BiP, CHOP, NMDAr, TNFα andthe oxidative stress marker DACF-2DA. To investigate whether Hcyaltered vascular permeability, human retinal endothelial cells (HREC)were exposed to Hcy-thiolactone (20µM, 50µM, 100µM) in thepresence/absence of the NMDAr inhibitor MK801 (25µM) or the ERstress inhibitor phenylbutyric acid, PBA (10mM) followed byincubation with FITC-dextran as an indicator of leakage.Results: Gene & protein analysis revealed ~40% increase in BiP &PERK expression in cbs-/- retinas compared to cbs+/+; there was nosignificant change in expression of ATF4, ATF6, CHOP, IRE1α .Immunofluorescence studies in retinal cryosections of cbs-/- miceshowed a dramatic increase in BiP & CHOP as well as NMDAr,TNFα & DACF-2A. In vitro studies of Hcy-treated HREC showed a25% increase in FITC-dextran leakage through the HREC monolayer,which was reduced significantly by MK801 and PBA.Conclusions: Retinal neurovascular alterations observed in severelyhyperhomocysteinemic cbs mice are accompanied by increased levelsof ER & oxidative stress as well as activation of NMDAr.Importantly, increased vascular permeability may be associated withHcy-induced leakiness, which can be attenuated by inhibiting ERstress or NMDAr.Commercial Relationships: Amany M. Tawfik, None; ShanuMarkand, None; Sylvia Magyerdi, None; Mohamed A. Al-Shabrawey, None; Sylvia B. Smith, NoneSupport: NIH Grant EYO12830 and RO1 EYO14560Program Number: 2464 Poster Board Number: D0069Presentation Time: 2:45 PM - 4:30 PMA Prospective Study of In-Office Diagnostic Vitreous Sampling inPatients with Vitreoretinal Pathology, 2007-2011Bert M. Glaser, Stephanie M. Ecker, Joshua C. Hines, Ann O. Igbre.Ocular Proteomics, National Retina Institute, Towson, MD.Purpose: This prospective study has been compiled to demonstratethat in-office vitreous sampling can be done safely and efficiently inpatients with a variety of retinal pathologies.Methods: Briefly, a vitreous aspiration was performed before aninjection of Anti-VEGF or of corticosteroid for treatment of retinaldisease. Each sample was taken using a standard technique, whichincludes 1) application of 2% topical liodcaine gel followed byplacement of a soaked cotton-tip applicator with 4% Lidocainesolution (in the inferior temporal quadrant; 2) a Sterile Lid Speculumwas placed followed by a drop of betadine 5%; 3) next, a 25 gauge,5/8 inch Terumo Needle was placed via pars plana approach into themid-vitreous cavity and 0.05 mL to 0.10mL of vitreous fluid wasaspirated into a 1mL syringe. After removal of the needle theintravitreal injection is directed via or close to the same spot. Afterthe injection the patient is discharged with post-injection warningstypical of patients following intra-vitreal injections.Results: As of December 31, 2011, a total of 830 patients wereenrolled in the Western IRB approved Vitreous Proteomics study atour institute. A total of 3,741 attempts were made to acquire avitreous sample, resulting in 3,245 samples, yielding an 86.7% rate ofsuccess over a 4 year period. The majority of the vitreoretinaldiagnoses were AMD, DR and RVO, but there were 93 patients withother retinal diseases that were also sampled in this study. Out of the830 patients in the study, there have been 24 (2.89%) patients thathad a complication. The majority of the complications (79.16%,n=19) were mild vitreous hemorrhages, present at the one monthfollow-up visit. All vitreous hemorrhages resolved without sequelae.The other 5 adverse events consisted of a corneal abrasion (n=1),endophthalmitis (n=1), pseudo-endophthalmitis (n=2) and scleritis(n=1). There were no cases identified of acute posterior vitreousdetachment, retinal tears or retinal detachments.Conclusions: This proves that in-office vitreous sampling of 50-100µl is a safe and reproducible procedure. The low rate of adverseevents and the 4 year duration of this study give solid evidence thatvitreous sampling is in fact a safe in-office procedure.Commercial Relationships: Bert M. Glaser, Ocular Proteomics,LLC (E); Stephanie M. Ecker, Ocular Proteomics LLC (E); JoshuaC. Hines, Ocular Proteomics (E); Ann O. Igbre, NoneProgram Number: 2465 Poster Board Number: D0070Presentation Time: 2:45 PM - 4:30 PMMouse Vitreoretinal ProteomeJessica M. Skeie 1, 2 , Stephen H. Tsang 3 , Vinit B. Mahajan 1, 2 .1 Ophthalmology and Visual Sciences, University of Iowa, Iowa City,IA; 2 Omics Laboratory, University of Iowa, Iowa City, IA; 3 Bernardand Shirlee Brown Glaucoma Laboratory, Department ofOphthalmology, Columbia University, New York, NY.Purpose: To identify the protein profiles of mouse retina andvitreous and determine unique protein pathways and interactionnetworks in these tissues.Methods: Vitreous and retina samples from normal mice werecollected by an evisceration technique developed in our laboratoryand pooled. Samples were analyzed in technical triplicate by LC-MS/MS. The proteins discovered were further analyzed usingbioinformatic software to determine significant pathways present,statistically significant protein expression differences betweenvitreous and retina, gene ontologies, and protein interaction networks.Results: In the retina, we identified 5,729 unique spectra with106,734 peptide hits, corresponding to 1,682 unique proteins and1,085 unique spectra with 45,507 peptide hits, corresponding to 677unique proteins in the vitreous. Unbiased clustering of the proteinsverified that the vitreous and retina were significantly different withdifferent gene ontology distributions. The most highly representedpathways that distinguished the retina from the vitreous (p < 0.05)were neuronal cell synapses (LRRK2), pyruvate metabolism,neurophysiological processes, apoptosis/survival, cytoskeletalremodeling, and G-protein signaling (CFTR). Pathways thatdistinguished the vitreous from the retina (p < 0.05) werephenylalanine metabolism and nitrogen metabolism. Some clusters ofinteracting proteins were identified in both the retina and vitreous.These included crystallin cell regulation and oxidative stress proteins.Conclusions: We identified several unique proteins, pathways, andinteraction networks that distinguish the normal mouse retina andvitreous. This methodology can be applied to mouse models ofhuman vitreoretinal disease.Commercial Relationships: Jessica M. Skeie, None; Stephen H.Tsang, None; Vinit B. Mahajan, NoneSupport: NIH 1F32EY022280-01A1Program Number: 2466 Poster Board Number: D0071Presentation Time: 2:45 PM - 4:30 PMThe novelty of Toll like receptor-4 ligand and RGC degenerationYasunari Munemasa, Kazuhide Takada, Kaori Kojima, Satoki Ueno,Yasushi Kitaoka. Ophthalmology, St Marianna University, Kawasaki,Japan.Purpose: Toll like receptor-4 (TLR-4) has been indicated in responseto various ligands related with cell death signaling, in addition toautoimmune response. The purpose of this study is to investigate thenovelty of TLR-4 in RGC degeneration.©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 <strong>ARVO</strong> Office at arvo@arvo.org.
<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>Methods: Eight week male C3H/HeJ and C3H/HeN mice were usedin this study. C3H/HeJ mice show dysfunction of TLR-4. In contrast,C3H/HeN mice show normal TLR-4 function. To generate opticnerve crush (ONC), the nerve of one eye was exposed and clampedapproximately 0.5 mm from the globe with self-closing forceps for10s, contralateral eye was used as a control. RGC was labeled withfluoro-gold at superior colliculus 5 days before ONC andmorphological analysis was performed with whole mount retina at 2weeks after ONC. Matrix-assisted laser desorption/ionisation-time offlight mass spectrometry (MALDI-TOFMS) was used to detect TLR-4 ligands after immunoprecipitation with TLR-4 antibody andisolation of retinal membrane proteins at 5 days after ONC. Inductionof RGC degeneration with TLR-4 ligand was studied with cellviability assay using RGC-5 cells and intravitreal injection of TLR-4ligand on C3H/HeJ and C3H/HeN mice.Results: A significant decrease in RGC was observed in the retina ofC3H/HeJ mice compared to C3H/HeN mice 2 weeks after ONC(C3H/HeJ 0.24 fold / control vs C3H/HeN 0.40 fold / control, P
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