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Retinal Histopathologic Manifestations and Iron-Mediated Melanosome Degradation

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Retinal Histopathologic Manifestations and Iron-Mediated Melanosome Degradation LABORATORY SCIENCES Aceruloplasminemia Retinal Histopathologic Manifestations and Iron-Mediated Melanosome Degradation Natalie Wolkow, PhD; Ying Song, MD; Ting-Di Wu, PhD; Jiang Qian, MD, PhD; Jean-Luc Guerquin-Kern, PhD; Joshua L. Dunaief, MD, PhD Objective: To examine the retinal histopathologic mani- nosome poor. Melanosome-rich cells had increased lev- festation of aceruloplasminemia, an autosomal reces- els of iron and melanolipofuscin. The melanolipofuscin sive disease caused by mutation of the ferroxidase ceru- granules were observed in large aggregates, where some loplasmin, resulting in tissue iron overload. of the melanosomes were degrading. Melanosome-poor cells lacked melanosomes, melanolipofuscin, and lipo- Methods: The morphologic features of the human ac- fuscin but contained electron-dense aggregates high in eruloplasminemic retina were studied with light and iron, phosphorus, and sulfur. electron microscopy. Retinal iron accumulation was as- sessed with Perls Prussian blue staining, immunohisto- Conclusions: The findings in the aceruloplasminemic chemistry, and secondary ion mass spectrometry. retina resemble some of those found in age-related macu- lar degeneration. Also, they suggest that melanosomes Results: Light and electron microscopic analysis re- in the RPE can be degraded via iron-mediated reactive vealed several ocular pathologic findings that re- oxygen species production. sembled age-related macular degeneration, including reti- nal pigment epithelium (RPE) depigmentation, atrophy Clinical Relevance: Mechanisms underlying the patho- and hypertrophy, nodular and diffuse drusen, and lipo- fuscin and melanolipofuscin granules. Complement dep- logic mechanisms found in aceruloplasminemia also may osition was detected in drusen. The RPE cells and neu- be important in age-related macular degeneration. ral retina had increased levels of iron. Two major types of RPE cells were observed: melanosome rich and mela- Arch Ophthalmol. 2011;129(11):1466-1474 CERULOPLASMINEMIA IS A roportin, markedly impairs iron export from rare autosomal recessive cells.9 Thus, ferroxidases are necessary com- disease1 caused by muta- ponents for iron export. tion of the ceruloplasmin In retinas with age-related macular de- gene.2 Aceruloplasmin- generation (AMD), iron and transferrin Author Affiliations: F.M. Kirby Center for Molecular Aemia is characterized by a triad of diabe- levels are elevated, suggesting that iron tox- Ophthalmology, Scheie Eye tes mellitus, retinal degeneration, and neu- icity may contribute to the pathogenesis Institute, Perelman School of rodegeneration that usually manifests of AMD.10,11 Furthermore, in mice, com- Medicine, University of between the ages of 30 and 55 years.3-5 The bined knockout of ceruloplasmin and mu- Pennsylvania, Philadelphia disease is caused by iron accumulation in tation of its homologue, hephaestin, causes (Drs Wolkow and Song and the pancreas, liver, brain, and retina due retinal and brain iron accumulation and Dunaief); Institut Curie, to a decrease in iron export.4-6 degeneration.12,13 This retinal degenera- Laboratoire de Microscopie 13 Ionique (Drs Wu and Ceruloplasmin is a ferroxidase protein tion resembles AMD. 3-5 Guerquin-Kern) and Institut that is secreted from cells or is glycosylphos- Pathologic studies of many acerulo- National de la Sante´etdela phatidylinositol linked to the outer sur- plasminemic tissues have been published; Recherche Me´dicale (Drs Wu face of cell membranes.7 Ceruloplasmin in- however, no study has examined retinal his- and Guerquin-Kern), Orsay, teracts with ferroportin, the cellular iron topathologic manifestations, to our knowl- France; and Department of exporter, to export ferrous iron from cells.8 edge. The only published reports focusing Pathology and Laboratory Medicine/Affiliated Pathology As a ferroxidase, ceruloplasmin converts fer- on the retina have been clinical case re- Services, Inc, Albany Medical rous to ferric iron, allowing ferric iron to ports, which have shown pigmentary ab- 8 14,15 College and Center, Albany, bind to transferrin in the plasma. Lack of normalities in the peripheral retina. In New York (Dr Qian). ceruloplasmin, even in the presence of fer- 2005, one of us15 published a clinical re- ARCH OPHTHALMOL / VOL 129 (NO. 11), NOV 2011 WWW.ARCHOPHTHALMOL.COM 1466 ©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 port of an individual with aceruloplasminemia. In the pres- setts) and were rotated and cropped with Adobe Photoshop ent study, we examine the retinal histopathologic features (Adobe Systems Incorporated, San Jose, California). of the same individual at age 60 years. QUANTIFICATION OF MELANOSOMES, METHODS LYSOSOMES, AND COMPLEX GRANULES Images of 16 consecutive melanosome-poor and 16 melanosome- TISSUE PREPARATION rich RPE cells were taken (original magnification ϫ10 000) on a previously unexamined section. The RPE granules were quan- Eyes were obtained from a 60-year-old male donor with aceru- tified as previously described.17 loplasminemia after a 7-hour postmortem interval, in accor- dance with the tenets of the Declaration of Helsinki. At the time of the previous study,15 the donor had iron deficiency anemia, SECONDARY ION MASS SPECTROMETRY long-standing diabetes mellitus, cardiomyopathy, iron accu- mulation in the liver, dementia, and macular degeneration char- Secondary ion mass spectrometry (SIMS) is used to analyze the spatial distribution of elements in a sample at a resolution as acterized by drusen and areas of retinal pigment epithelium 18 (RPE) depigmentation.15 Subsequent to the publication of the high as 50 nm. Samples for SIMS analysis were prepared as report, he had experienced many hypoglycemic episodes, had for transmission electron microscopy but with modifications; developed renal failure, had been hospitalized, had developed samples were not postfixed in osmium tetroxide, and sections aspiration pneumonia and sepsis, and had died. For the pres- were not stained with uranyl acetate or lead citrate. Thin (40- ent study, samples from the anterior segment; retina, choroid, nm) and thick (150-nm) sections were cut and imaged with a and sclera; and optic nerve of his right eye were used. As a nor- JEOL1010 transmission electron microscope and then ana- mal control, an eye from a 60-year-old male donor with a 7-hour lyzed with a NanoSIMS 50 ion microprobe (CAMECA SAS, postmortem interval and no history of retinal disease was ob- Gennevilliers, France) operating in scanning mode. tained from the Lions Eye Bank. A control eye with AMD was obtained from an 86-year-old woman with a history of AMD RESULTS after a postmortem interval of 20 hours from the Complica- tions of Age-Related Macular Degeneration Prevention Trial Eye To determine whether the aceruloplasminemic eye had Donor Program in collaboration with the Foundation Fight- ing Blindness. All eyes were fixed in formalin, embedded in par- any unique pathologic manifestations, it was examined affin, and sectioned at 7-µm thickness. at the gross, light microscopic, and electron micro- scopic levels. At the gross level, the phakic right eye had PERIODIC ACID–SCHIFF STAINING no abnormalities other than macular degeneration, con- sistent with the results of the previous clinical report.15 Paraffin sections from the ocular regions indicated herein were At the light microscopic level, the cornea, iris, ciliary stained using a periodic acid–Schiff stain kit (Polysciences, Inc, body, and optic nerve had normal morphologic findings Warrington, Pennsylvania) as suggested by the manufacturer. (data not shown). The peripheral and macular neural Images were acquired on a Nikon Eclipse TE300 camera (Nikon retina had normal findings (Figure 1A and B), but the Corporation, Tokyo, Japan) using Image-Pro Plus software, ver- RPE cells and Bruch’s membrane contained many patho- sion 6.1 (Media Cybernetics, Inc, Bethesda, Maryland). Histo- 16 logic manifestations on all sections examined, similar to pathologic analysis was performed as previously described. those previously described in AMD.16 Nodular dru- sen16,19-21 were present in the macular, although they were PERLS PRUSSIAN BLUE STAINING FOR IRON much more common in the periphery (Figure 1C). The periphery also had many diffuse drusen16,19,20 (Figure 1D), Paraffin sections were bleached with 0.25% potassium perman- but the macula had none. The macula had subretinal ganate and 0.50% oxalic acid. Then, they were stained with 5% 22,23 potassium ferrocyanide and 5% hydrochloride for 30 minutes. drusenoid deposits (Figure 1E); the periphery did not. The macula and periphery had RPE cells with inclu- 16,21 IMMUNOHISTOCHEMISTRY sions (Figure 1F) and RPE cells that had extruded from their normal layer16,24 (Figure 1G). The macula had sev- 16 Paraffin sections were stained using the Vectastain ABC Alka- eral areas of RPE hypertrophy (Figure 1H) and atro- line Phosphatase for Rabbit and Mouse IgG and Vector BCIP- phy16 (Figure 1I); the periphery did not. The macula and NBT kits (Vector Laboratories, Inc, Burlingame, California). the periphery had areas of depigmented RPE cells that Primary antibodies for L-ferritin (F-17; gift of Paolo Arosio, lacked the brown color of their neighbors and instead PhD, and Paolo Santambrogio, PhD), C5b-9 (M0777; Dako, stained purple (Figure 1J and K); normal control and AMD Carpinteria, California), and vitronectin (MAB1945; Milli- RPE cells lacked this color variation (Figure 1L and M). pore, Billerica, Massachusetts)
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