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• W2955322511 abstract "The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease. The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease. Iron is critical for cellular survival, as it is involved in several essential biochemical and metabolic processes. However, iron loading can cause oxidative injury through production of highly reactive hydroxyl free radicals in the Fenton reaction.1Beard J.L. Iron biology in immune function, muscle metabolism and neuronal functioning.J Nutr. 2001; 131 (discussion 580S): 568S-579SCrossref PubMed Google Scholar, 2Aisen P. Enns C. Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism.Int J Biochem Cell Biol. 2001; 33: 940-959Crossref PubMed Scopus (575) Google Scholar Because of the danger of cellular iron overload, tight iron regulation within all tissues is essential for proper cellular function. The retina, and especially the photoreceptors, is particularly vulnerable to iron-induced oxidative injury because of factors such as the high metabolic rate, high oxygen tension, and the abundance of polyunsaturated fatty acids.3Rogers B.S. Symons R.C.A. Komeima K. Shen J. Xiao W. Swaim M.E. Yuan Y.G. Kachi S. Campochiaro P.A. Differential sensitivity of cones to iron-mediated oxidative damage.Invest Ophthalmol Vis Sci. 2007; 48: 438-445Crossref PubMed Scopus (56) Google Scholar Retinal iron overload has been implicated in the pathophysiology of retinal degenerative diseases, including age-related macular degeneration, the leading cause of irreversible blindness in individuals >50 years old in developed nations.4Hahn P. Milam A.H. Dunaief J.L. Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane.Arch Ophthalmol. 2003; 121: 1099-1105Crossref PubMed Scopus (215) Google Scholar Brain iron overload may also contribute to the development of other neurodegenerative diseases, such as Alzheimer and Parkinson diseases.5Rouault T.A. Iron metabolism in the CNS: implications for neurodegenerative diseases.Nat Rev Neurosci. 2013; 14: 551-564Crossref PubMed Scopus (295) Google Scholar, 6Gröger A. Berg D. Does structural neuroimaging reveal a disturbance of iron metabolism in Parkinson's disease? implications from MRI and TCS studies.J Neural Transm. 2012; 119: 1523-1528Crossref PubMed Scopus (23) Google Scholar, 7Smith M.A. Zhu X. Tabaton M. Liu G. McKeel D.W. Cohen M.L. Wang X. Siedlak S.L. Dwyer B.E. Hayashi T. Nakamura M. Nunomura A. Perry G. Increased iron and free radical generation in preclinical Alzheimer disease and mild cognitive impairment.J Alzheimers Dis. 2010; 19: 353-372Crossref Scopus (291) Google Scholar The observation that iron overload occurs in a variety of neurodegenerative diseases suggests that retinal or brain iron dysregulation may play an important role in the development or exacerbation of neurodegeneration and that the processes that promote iron accumulation within neuronal tissue, which are incompletely understood, need to be more thoroughly explored. Unlike other organs, which are directly exposed to the systemic circulation, the retina should be protected from fluctuations in plasma iron by the presence of the blood-retinal barrier (BRB). Yet, in some human cases and mouse models of hereditary hemochromatosis, a set of genetic diseases associated with high plasma iron levels,8Pietrangelo A. Hereditary hemochromatosis: a new look at an old disease.N Engl J Med. 2004; 350: 2383-2397Crossref PubMed Scopus (816) Google Scholar there are elevated retinal iron levels and retinal degeneration.9Roth A.M. Foos R.Y. Ocular pathologic changes in primary hemochromatosis.Arch Ophthalmol. 1972; 87: 507-514Crossref PubMed Scopus (28) Google Scholar, 10Smith S.B. Liu K. Thangaraju M. Ha Y. Ganapathy V. Martin P.M. Gnana-Prakasam J.P. Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: role of cystine/glutamate exchanger.Biochem J. 2009; 424: 243-252Crossref PubMed Scopus (43) Google Scholar These effects on the retina may result from the high plasma iron, abnormal local iron regulatory mechanisms within the retina, or both. Systemic iron levels are elevated in patients receiving oral or i.v. iron supplementation, common treatments for iron-deficiency anemia. A case study from our laboratory recently demonstrated that the short-term i.v. iron treatment in a 42-year-old patient was associated with the development of early-onset macular degeneration,11Song D. Kanu L.N. Li Y. Kelly K.L. Bhuyan R.K. Aleman T. Morgan J.I.W. Dunaief J.L. AMD-like retinopathy associated with intravenous iron.Exp Eye Res. 2016; 151: 122-133Crossref PubMed Scopus (24) Google Scholar suggesting that high plasma iron levels may be sufficient to cause retinal damage. The goal of this study was to determine whether high plasma iron levels could cause retinal iron overload and degeneration, despite intact local iron regulatory mechanisms within the retina. Regulation of systemic iron levels is accomplished by several iron-handling proteins that regulate the flux of iron into and out of the systemic iron pool. The iron regulatory hormone, hepcidin (Hepc), is instrumental in regulating these processes. Hepc [alias hepcidin antimicrobial peptide (Hamp)], is a 25–amino acid peptide hormone that is produced primarily by hepatocytes and plays an essential role in regulating systemic iron levels by antagonizing the only known mammalian iron exporter, ferroportin (Fpn). Hepc binds to the extracellular domain of ferroportin on the cell surface, leading to its internalization and degradation, effectively preventing cellular iron export and limiting the amount of iron that gets into the serum or extracellular fluid.12Nemeth E. Tuttle M.S. Powelson J. Vaughn M.D. Donovan A. Ward D.M.V. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3487) Google Scholar Hepc serves as the central regulator of systemic iron metabolism by controlling the flux of iron through Fpn from three distinct points, the release of iron from liver stores, the release of iron from macrophages involved in recycling red blood cells, and the transport of iron, absorbed from the diet by enterocytes within the duodenum, into the bloodstream.13Ganz T. Hepcidin in iron metabolism.Curr Opin Hematol. 2004; 11: 251-254Crossref PubMed Scopus (145) Google Scholar When the systemic iron pool is elevated, Hepc production by hepatocytes increases, preventing additional release of iron into the systemic pool. The Hepc/Fpn axis is responsible for mediating systemic iron levels, and dysregulation of this axis, in diseases such as primary hemochromatosis and anemia of chronic inflammation, leads to disruption of iron homeostasis. Although most of Hepc is produced by hepatocytes, small amounts are also produced in organs that do not have any known role in regulating systemic iron levels, including the brain,14McCarthy R.C. Kosman D.J. Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux.J Biol Chem. 2013; 288: 17932-17940Crossref PubMed Scopus (52) Google Scholar heart,15Lakhal-Littleton S. Wolna M. Chung Y.J. Christian H.C. Heather L.C. Brescia M. Ball V. Diaz R. Santos A. Biggs D. Clarke K. Davies B. Robbins P.A. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5: e19804Crossref PubMed Scopus (95) Google Scholar kidney,16Cetin Y. Gehrke S.G. Kulaksiz H. Bachmann S. Stremmel W. Theilig F. Rost D. Janetzko A. The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney.J Endocrinol. 2005; 184: 361-370Crossref PubMed Scopus (174) Google Scholar pancreas,17Kulaksiz H. Fein E. Redecker P. Stremmel W. Adler G. Cetin Y. Pancreatic β-cells express hepcidin, an iron-uptake regulatory peptide.J Endocrinol. 2008; 197: 241-249Crossref PubMed Scopus (78) Google Scholar and retina.18Gnana-Prakasam J.P. Martin P.M. Smith S.B. Ganapathy V. Expression and function of iron-regulatory proteins in retina.IUBMB Life. 2010; 62: 363-370PubMed Google Scholar The role of Hepc produced by some of those organs is largely unknown; however, a recent study demonstrated that cardiomyocyte-produced Hepc is necessary for proper cardiac iron regulation and function.15Lakhal-Littleton S. Wolna M. Chung Y.J. Christian H.C. Heather L.C. Brescia M. Ball V. Diaz R. Santos A. Biggs D. Clarke K. Davies B. Robbins P.A. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5: e19804Crossref PubMed Scopus (95) Google Scholar Our laboratory has previously shown that a systemic Hepc knockout (HepcKO), which leads to high blood iron levels due to unrestricted iron absorption from the diet, results in retinal iron overload and retinal degeneration in mice by the age of 18 months.19Hadziahmetovic M. Song Y. Ponnuru P. Iacovelli J. Hunter A. Haddad N. Beard J. Connor J.R. Vaulont S. Dunaief J.L. Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice.Invest Ophthalmol Vis Sci. 2011; 52: 109-118Crossref PubMed Scopus (71) Google Scholar We also demonstrated that in mice with a mutation in Fpn, which causes resistance to Hepc regulation and subsequent serum iron overload, the mutation leads to a similar retinal iron accumulation as observed in the systemic HepcKO mice.20Theurl M. Song D. Clark E. Sterling J. Grieco S. Altamura S. Galy B. Hentze M. Muckenthaler M.U. Dunaief J.L. Mice with hepcidin-resistant ferroportin accumulate iron in the retina.FASEB J. 2016; 30: 813-823Crossref PubMed Scopus (25) Google Scholar These transgenic models demonstrate that changes in the Hepc/Fpn axis significantly affect retinal iron levels but fail to determine whether the retinal iron overload observed in these models is the result of long-term exposure to high blood iron levels or, rather, changes in the local retinal iron regulation from loss of retina-produced Hepc. To test the role of Hepc in the retina, we analyzed the retinal phenotype in two mouse models: the first with a liver-specific deletion of the Hepc (official name, Hamp) gene and the second with a retina-specific deletion of the Hepc gene. In the first model, a liver-specific Hepc knockout mouse strain (LS-HepcKO) has high systemic iron levels, recapitulating the high serum iron conditions of the systemic HepcKO model, while maintaining the ability of the retina to produce its own Hepc. Using this LS-HepcKO model, it was investigated whether the presence of retina-produced Hepc plays a significant role in regulating retinal iron levels and retinal health by comparing these mice with age-matched controls. In the second model, the retina-specific Hepc knockout (RS-HepcKO), it was tested whether loss of retina-produced Hepc alters retinal iron homeostasis in mice with normal serum iron levels. It is essential to understand how changes in serum iron levels affect retinal health and how the retina can regulate local iron levels when the serum iron levels are normal or elevated. These data will provide a clearer image of how retinal health is affected by fluctuations in serum iron levels and provide insight into the mechanisms that contribute to retinal iron overload in retinal degenerative disease. The liver-specific hepcidin knockout mice were generated using a Hepc-floxed (Hepcflox/flox) strain, which was developed as previously described.15Lakhal-Littleton S. Wolna M. Chung Y.J. Christian H.C. Heather L.C. Brescia M. Ball V. Diaz R. Santos A. Biggs D. Clarke K. Davies B. Robbins P.A. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5: e19804Crossref PubMed Scopus (95) Google Scholar Hepcflox/flox mice were crossed with a strain expressing the Cre-recombinase under the control of the hepatocyte-specific promoter, albumin (Alb-Cre; stock number 003574; Jackson Laboratory, Bar Harbor, ME),21Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic β cell-specific gene knock-outs using Cre recombinase.J Biol Chem. 1999; 274: 305-315Crossref PubMed Scopus (1004) Google Scholar to generate the hepatocyte-specific HepcKO (Hepcflox/flox;Alb-Cre+; referred to herein as LS-HepcKO). The knockout of Hepc in the liver was verified using real-time quantitative PCR (qPCR) analysis of Hepc mRNA levels (Figure 1). Mice were aged to 15 days, 3 months, 6 months, and 12 months and then euthanized. All mice were fed a standard laboratory diet with 300 ppm iron, given free access to water and food, and maintained in a temperature-controlled room at 21°C to 23°C under dim cyclic light (12:12 hours light-dark cycle). Both LS-HepcKO mice and controls were on a C57BL/6J background, and both males and females were used in this study. All mice were negative for the rd1 and rd8 alleles. Experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmology and Vision Research. All protocols were approved by the animal care review board of the University of Pennsylvania (Philadelphia, PA). To generate the retina-specific HepcKO mice (referred to herein as RS-HepcKO), Hepcflox/flox mice were crossed to a strain expressing mRx-Cre. The mRx-Cre mouse strain was supplied by an author of this work (Z.K.). The mRx promoter is expressed in most cells within the neurosensory retina (NSR) and a subset of retinal pigment epithelium (RPE) cells.22Klimova L. Lachova J. Machon O. Sedlacek R. Kozmik Z. Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.PLoS One. 2013; 8: e63029Crossref PubMed Scopus (26) Google Scholar RS-HepcKO experimental mice had the genotype Hepcflox/flox;mRx-Cre+, and the control group had the genotype Hepc+/+;mRx-Cre+. Mice were aged to 6 and 12 months and euthanized. Both RS-HepcKO mice and controls were on a C57BL/6J background, and both males and females were used in this study. All mice were negative for the rd1 and rd8 alleles. Mice were euthanized, and eyes were immediately enucleated. Anterior segments were removed, and retinas were completely dissected away from the underlying RPE. Retinas were then flash frozen and stored at -80°C. RPE cells were isolated from other ocular structures using enzymatic (dispase and hyaluronidase) digestion and mechanical dissection, as previously described.23Wolkow N. Song D. Song Y. Chu S. Hadziahmetovic M. Lee J.C. Iacovelli J. Grieco S. Dunaief J.L. Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium.Am J Pathol. 2012; 180: 1614-1624Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar RNA was isolated according to the manufacturer's protocol (RNeasy Kit; Qiagen, Valencia, CA). cDNA was synthesized with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. Gene expression of Tfrc (transferrin receptor), Dmt1 (divalent metal transporter 1; official name, Slc11a2), and Hepc (hepcidin) was analyzed using qPCR, as previously described.19Hadziahmetovic M. Song Y. Ponnuru P. Iacovelli J. Hunter A. Haddad N. Beard J. Connor J.R. Vaulont S. Dunaief J.L. Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice.Invest Ophthalmol Vis Sci. 2011; 52: 109-118Crossref PubMed Scopus (71) Google Scholar Gene expression assays (TaqMan; Applied Biosystems, Foster City, CA) were used for qPCR analysis. Real-time RT-PCR was performed on a commercial sequence detection system (ABI Prism 7500; Applied Biosystems, Darmstadt, Germany). All reactions were performed in technical triplicates (n = 3 to 10 mice per genotype). Probes used were as follows: Tfrc (Mm00441941_m1), Hepc (Mm04231240_s1), and Dmt1 (Mm01308330_s1). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as an internal control (4352932E). NSR protein lysates were extracted using Laemmli SDS lysis buffer supplemented with protease/phosphatase inhibitor mixture (Cell Signaling Technology, Danvers, MA). Lysates were treated and run, as described previously.23Wolkow N. Song D. Song Y. Chu S. Hadziahmetovic M. Lee J.C. Iacovelli J. Grieco S. Dunaief J.L. Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium.Am J Pathol. 2012; 180: 1614-1624Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Imaging was performed using GE Amersham Imager 600 (GE Healthcare, Chalfont St. Giles, UK). FIJI software (NIH, Bethesda, MD) was used for band densitometry.24Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.-Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (27117) Google Scholar Primary antibodies used were as follows: rat antitransferrin receptor (Serotec, Kidlington, UK), goat antialbumin (Bethyl Laboratories, Montgomery, TX), and rabbit anti-GAPDH (Thermo Fisher Scientific, Philadelphia, PA). Secondary antibodies used were as follows: donkey anti-rabbit (ECL Rabbit IgG, horseradish peroxidase–linked whole antibody) and donkey anti-rat (ECL Rat IgG, horseradish peroxidase–linked whole antibody) (GE Healthcare, Chicago, IL). All primary antibodies were used at a 1:1000 dilution, and all secondary antibodies were used at a 1:5000 dilution. GAPDH served as an internal control. Eyes were enucleated immediately after sacrifice and fixed for 15 minutes in 4% paraformaldehyde, and eyecups were generated by removing the cornea and lens. Eyecups were dehydrated overnight in 30% sucrose and embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA). Immunohistochemistry was performed on cryosections (10 μm thick), as described previously.19Hadziahmetovic M. Song Y. Ponnuru P. Iacovelli J. Hunter A. Haddad N. Beard J. Connor J.R. Vaulont S. Dunaief J.L. Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice.Invest Ophthalmol Vis Sci. 2011; 52: 109-118Crossref PubMed Scopus (71) Google Scholar Antibody used goat antialbumin (dilution 1:200; Bethyl Laboratories) and rabbit antilight ferritin (E17; dilution 1:2500; a gift from Paolo Arosio, Ph.D., University of Brescia, Brescia, Italy). Control sections were treated identically but with omission of primary antibody. Sections were analyzed by fluorescence microscopy using identical exposure parameters across genotype using a Nikon Eclipse 80i microscope (Nikon Instruments, Melville, NY), and images were acquired using NIS-BR Elements software version 4.1 (Nikon Instruments). Pixel density analysis of the l-ferritin (Ft-L) stain was completed using an open-source image-processing package (FIJI software).24Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.-Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (27117) Google Scholar Livers from experimental and age-matched control mice were frozen on dry ice. Dried tissue was digested overnight at 65°C in acid digest solution (0.1% trichloroacetic acid and 0.03 mol/L HCl). After digestion, samples were centrifuged, and supernatant (20 μL) was added to 1 mL of chromogen reagent [2.25 mol/L sodium acetate pretreated with Chelex 100 (Bio-Rad, Hercules, CA), 0.01% bathophenanthroline sulfonate, and 0.1% thioglycolic acid]. The absorbances were read at 535 nm. Iron levels were calculated by comparing absorbances of tissue-chromogen samples with serial dilutions of iron standard (Sigma-Aldrich, St. Louis, MO). NSR and RPE samples were analyzed for iron and zinc using an inductively coupled mass spectrometer (iCP-MS; Nexion 300D; Perkin Elmer, Shelton, CT) at the Pennsylvania Animal Diagnostic Laboratory System (PADLS) New Bolton Center Toxicology Laboratory, University of Pennsylvania, School of Veterinary Medicine (Kennett Square, PA), as described previously.25Sterling J. Guttha S. Song Y. Song D. Hadziahmetovic M. Dunaief J.L. Iron importers Zip8 and Zip14 are expressed in retina and regulated by retinal iron levels.Exp Eye Res. 2017; 155: 15-23Crossref PubMed Scopus (22) Google Scholar NSR and RPE tissues for iCP-MS analysis were collected from mice that had been perfused with saline. Eyes were enucleated, fixed in 2% paraformaldehyde–2% glutaraldehyde overnight, and embedded in JB-4 plastic (Polysciences, Warrington, PA). Sections (3 μm thick) were cut through the sagittal plane and stained with toluidine blue, as previously described.19Hadziahmetovic M. Song Y. Ponnuru P. Iacovelli J. Hunter A. Haddad N. Beard J. Connor J.R. Vaulont S. Dunaief J.L. Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice.Invest Ophthalmol Vis Sci. 2011; 52: 109-118Crossref PubMed Scopus (71) Google Scholar Stained sections were observed and imaged using bright-field illumination (model TE300; Nikon, Inc., Tokyo, Japan). To measure outer nuclear layer (ONL) thickness, a retinal section adjacent to the optic nerve head was analyzed (n = 3 per genotype). ONL thickness was determined by counting nuclei per row at 200-μm intervals to 2200 μm superior and inferior to the optic nerve head. To determine the percentage of the retina that contained hypertrophic RPE cells, one slide per retina (n = 3 per genotype) that contained the optic nerve head was chosen. The total length of the RPE in one retinal section was determined. The total length of the RPE that contained hypertrophic RPE (defined as an RPE cell that is >15 μm in height) was determined, and the percentage of the total retinal length that contained hypertrophic RPE cells was calculated. All morphologic analysis was performed by a masked observer (B.H.B. or W.S.). Electron microscopy on retinal samples was performed as described previously.26Dunaief J.L. Zhong Y. Song Y. Hadziahmetovic M. Song D. Systemic administration of the iron chelator deferiprone protects against light-induced photoreceptor degeneration in the mouse retina.Free Radic Biol Med. 2012; 53: 64-71Crossref PubMed Scopus (62) Google Scholar Briefly, after enucleation, eyes were fixed in 2% paraformaldehyde/2% glutaraldehyde overnight at 4°C. The anterior segment was removed, and the posterior portion of each eye was cut into small wedge-shaped pieces and post-fixed in 1% osmium tetroxide/0.1 mol/L sodium cacodylate buffer, dehydrated, and embedded in EMbed-812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (60 to 80 nm thick) were stained and examined with a JEOL1010 transmission electron microscope (JEOL Ltd, Tokyo, Japan). Images were acquired with Advanced Microscopy Techniques Image Capture software version 602 (Advanced Microscopy Techniques Corp., Woburn, MA) and were rotated and cropped with Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA). Perls stain for iron was performed on plastic sections (3 μm thick) or cryosections (10 μm thick), as previously described.27Hadziahmetovic M. Dentchev T. Song Y. Haddad N. He X. Hahn P. Pratico D. Wen R. Harris Z.L. Lambris J.D. Beard J. Dunaief J.L. Ceruloplasmin/hephaestin knockout mice model morphologic and molecular features of AMD.Invest Ophthalmol Vis Sci. 2008; 49: 2728-2736Crossref PubMed Scopus (111) Google Scholar For cryosections, slides were incubated in 5% potassium ferrocyanide in 5% aqueous hydrochloric acid for 45 minutes at 65°C and then bleached using potassium permanganate and oxalic acid. For plastic sections, slides were bleached using potassium permanganate and oxalic acid and then incubated in 5% potassium ferric ferrocyanide and 5% hydrochloric acid solution for 30 minutes at room temperature. Sensitivity for iron detection was enhanced by subsequent incubation of tissue in purple peroxidase substrate for 25 minutes at room temperature (VIP; Vector Laboratories, Inc., Burlingame, CA). Slides were then washed in 1× phosphate-buffered saline. Slides were examined on a Nikon Eclipse 80i microscope, and images were acquired using NIS-BR Elements software. Blood was collected from anesthetized animals by retro-orbital bleeding, placed into BD microtainer blood collection tubes (BD Biosciences, San Jose, CA), and spun down for 30 minutes at 1000 × g. Serum was collected and stored at −20°C. Serum Fe status was analyzed by quantifying total serum iron and transferrin saturation using an Iron/TIBC testing kit (Pointe Scientific, Inc., Canton, MI). For some of the mice with serum iron overload, the Tf saturation calculation was >100% because of the presence of non-transferrin bound iron. If Tf saturation was >100%, it was recorded as 100% Tf saturation. Mice were anesthetized with an i.p. injection of (in mg/kg body weight): 80 ketamine (Par Pharmaceutical, Spring Valley, NY), 10 xylazine (Lloyd Inc., Shenandoah, IA), and 2 acepromazine (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO). Pupils were then dilated with 1% tropicamide (Akorn, Inc., Lake Forest, IL). Once anesthetized adequately, mice were placed on a metal stage. Color and autofluorescence images were acquired using a fundus camera (Micron III; Phoenix Research Laboratories, Inc., Pleasanton, CA). Electroretinography recordings followed procedures described previously.28Lyubarsky A.L. Falsini B. Pennesi M.E. Valentini P. Pugh E.N. UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.J Neurosci. 1999; 19: 442-455Crossref PubMed Google Scholar, 29Lyubarsky A.L. Lem J. Chen J. Falsini B. Iannaccone A. Pugh E.N. Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy.Vision Res. 2002; 42: 401-415Crossref PubMed Scopus (49) Google Scholar In brief, mice were dark adapted overnight and then anesthetized with an i.p. injection of (in mg/kg body weight): 80 ketamine, 10 xylazine, and 2 acepromazine. Pupils were dilated with 1% tropicamide saline solution (Akorn, Inc., Lake Forest, IL). Two electrodes made of UV transparent plastic with embedded platinum wires were placed in electrical contact with the corneas. A platinum wire loop placed in the mouth served as the reference and ground electrode. The electroretinograms were then recorded (Espion Electrophysiology System; Diagnosys LLC, Lowell, MA). The apparatus was modified by the manufacturer for experiments with mice by substituting light-emitting diodes with emission maximum at 365 nm for standard blue ones. The stage was positioned in such a way that the mouse's head was located inside the stimulator (ColorDome; Diagnosys LLC), thus ensuring uniform full-field illumination. The flash intensities for recordings of rod a- and b-waves were 500 and 0.01 scotopic candelas m−2second, delivered by the white xenon flash and green (510-nm maximum) LE" @default.
• W2955322511 created "2019-07-12" @default.
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• W2955322511 date "2019-09-01" @default.
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• W2955322511 title "Liver-Specific, but Not Retina-Specific, Hepcidin Knockout Causes Retinal Iron Accumulation and Degeneration" @default.
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