Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologybundles oriented perpendicular to the vertical axis, and to Tuj1labeled axons (not shown), in a pattern most consistent with theorientation of astrocytic processes in the glial lamina (Figure 1a).Additionally, F-actin labeled the walls of the ONH vascularcomponents. F-actin partially co-labeled with Aqp4, furthersuggesting the astrocytic origin of the dense F-actin bundles (Figure2). Preliminary labeling intensity analysis of 8 glaucoma model eyeswith axonal injury grades from 1 to 4.9 (mean and peak IOP±SDfrom 19.2±1.6 to 29.6±3.9 and 22±3.2 to 50.4±1.3 mmHg,respectively) suggested that astrocytic structural changes, as indicatedby reduced F-actin labeling in eyes with minimal injury (Figure 1b),may precede axonal degeneration. With greater injury (Figure 1c),the ONH actin cytoskeleton became exceedingly disorganized withan apparent increase in F-actin fluorescence intensity, possibly due toastrocytic remodeling in response to injury. In contrast, in transectedONH, F-actin remained relatively preserved, suggesting that thechanges in the F-actin cytoskeleton with IOP elevation were notsimply secondary to non-specific axonal injury.Conclusions: The actin cytoskeleton of the rat ONH is a densestructure that may have a role in early and late ONH remodeling inresponse to glaucomatous injury. F-actin labeling within the ONHappears to highlight the glial lamina more effectively when comparedto Aqp4 labeling. In addition, F-actin labeling highlights the vascularcomponents of the ONH.Rat ONH actin cytoskeletal response to elevated IOPspots on SD-OCT and increased vascular permeability andpathologies of retinal blood vessels on FA. The pathologies of retinalblood vessels include micro- and macroaneurysms, pulling andtraction, dilated capillaries, capillary dropout and tortuosity. TEMrevealed neovascularization in the normally avascular retinal pigmentepithelium (RPE). TEM also showed thickening of the basementmembrane in these blood vessels. Abnormal lipid accumulation wasdetected throughout the retina and in retinal blood vessels byhistochemistry staining with oil red O, then confirmed by TEM.Areas with pathological blood vessels and structural abnormalitieshad increased staining with the markers for activated macrophagesF4/80 and Iba-1. Retinal blood vessels, the photoreceptor innersegments (PIS) and the outer plexiform layer (OPL) also showedstaining for iso[4]levuglandin E2 and carboxyethylpyrrole adducts aswell as 7-ketocholesterol, indicators of elevated oxidative stress.TEM also revealed mitochondrial swelling, degeneration andaggregation. These changes were observed in the RPE, PIS, OPL andinner nuclear layers. Significant alterations were found in the retinalexpression of a number of genes including the cholesterol effluxtransporter Abcg1, the lipoprotein Apob, the cholesterogenic enzymeHmgcr, the regulatory protein Insig1, and the inflammatorycytokines/chemokines Ccl1, Cxcl1, Cxcl2 and IL-6.Conclusions: We propose that combined deficiency of Cyp27a1 andCyp46a1 decreases activation of Lxrβ leading to decreasedexpression of target genes like Abcg1 and almost complete blockageof cholesterol elimination from retinal macrophages and vascularendothelium. The latter causes lipid accumulation, inflammation, andoxidative stress that drive the development of the abnormalities in theCyp27a1 -/- Cyp46a1 -/- retinas.Commercial Relationships: Aicha Saadane, None; Casey Charvet,None; Natalia Mast, None; Wenchao Zheng, None; Timothy S.Kern, Bausch & Lomb (F), PamLab (F); Suber Huang, UniversityHosp Case Medical Center (E), Sequenom (C), Notal Vision (C),Bausch and Lomb (C), Second Sight (C); Irina A. Pikuleva, NoneSupport: NIH RO1 EY018383Co-labeling of rat ONH actin cytoskeleton with aquaporin 4Commercial Relationships: Shandiz Tehrani, None; Elaine C.Johnson, None; William Cepurna, None; Matthew R. Bald, None;John C. Morrison, NoneSupport: NIH Gant EY010145Program Number: 290 Poster Board Number: D0239Presentation Time: 8:30 AM - 10:15 AMMechanistic insights into the link between impaired cholesterolelimination and vascular abnormalities in mouse retinaAicha Saadane 1 , Casey Charvet 1 , Natalia Mast 1 , Wenchao Zheng 1 ,Timothy S. Kern 2 , Suber Huang 3, 1 , Irina A. Pikuleva 1 .1 Ophthalmology and Visual Sciences, CWRU, Cleveland, OH;2 Medicine, CWRU, Cleveland, OH; 3 University Hospital, CWRU,Cleveland, OH.Purpose: To elucidate the mechanisms underlying development ofstructural and vascular abnormalities in the retina of mice lackingCyp27a1 and Cyp46a1, enzymes important for cholesteroleliminationMethods: Evaluations by high-resolution spectral-domain opticalcoherence tomography (SD-OCT), fluorescein angiography (FA), andtransmission electron microscopy (TEM); analysis of gene expressionby PCR arrays and quantitative RT-PCR; immuno- and histochemicalstaining; and sterol profiling by mass spectrometryResults: Cyp27a1 -/- Cyp46a1 -/- retinas have multiple hyperreflectiveProgram Number: 291 Poster Board Number: D0240Presentation Time: 8:30 AM - 10:15 AMTHERAPEUTIC EFFICACY OF MELATONIN IN REDUCINGRETINAL DAMAGE IN AN EXPERIMENTAL MODEL OFEARLY TYPE 2 DIABETES IN RATSRuth E. Rosenstein, Melina P. Bordone, Monica S. Chianelli, MaríaI. Keller Sarmiento, Damian Dorfman, Magdalena Miranda,Ezequiel M. Salido. Dept Human Biochem-Sch Med, University ofBuenos Aires, Buenos Aires, Argentina.Purpose: Diabetic retinopathy is a leading cause of acquiredblindness in adults, mostly affected by type 2 diabetes mellitus(T2DM). We have developed an experimental model of early T2DMin adult rats by combining diet-induced insulin resistance and a slightβ-cell secretory impairment, which mimics some features of humanT2DM at its initial stages, and provokes significant retinal alterations.The aim of the present work was to analyze the effect of melatoninon retinal changes induced by a moderate metabolic derangement.Methods: Adult male Wistar rats received a control diet or 30%sucrose in the drinking water ad libitum. Three weeks after thistreatment, animals were injected with vehicle or streptozotocin (STZ,25 mg/kg). One day after vehicle or STZ injection, animals weresubcutaneously implanted with a pellet of melatonin, which wasreplaced every 15 days. At 12 weeks of treatment, fasting andpostprandial glycemia, and glucose and insulin tolerance tests wereanalyzed. Retinal function (scotopic elctroretinograms), retinal lipidperoxidation (thiobarbituric acid reactive substance levels), NOSactivity (using 3H-arginine), TNFα (enzyme-linked immunosorbent©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.