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Clinicopathologic Reports, Case Reports, and Small Case Series
December 01, 2005

Strong Labeling for Iron and the Iron-Handling Proteins Ferritin and Ferroportin in the Photoreceptor Layer in Age-Related Macular Degeneration

Arch Ophthalmol. 2005;123(12):1745-1746. doi:10.1001/archopht.123.12.1745

Age-related macular degeneration (AMD) is the leading cause of legal blindness among people 65 years and older.1 The cause of AMD is unclear, but oxidative stress may play a role because photoreceptors, which are high in readily oxidized polyunsaturated fatty acids, are exposed to high oxygen tensions and photo-oxidation. Antioxidant vitamins decrease the risk of vision loss in some patients with AMD, supporting the role of oxidative stress in AMD.2 Iron can cause oxidative stress, and we have found that maculae from patients with AMD have higher iron levels in the retinal pigment epithelium (RPE) and Bruch’s membrane than do maculae from age-matched control subjects.3 Although elevated iron levels may be associated with AMD but not necessarily causal, recent evidence supports causality; we have found that retinas from iron-overloaded (ceruloplasmin/hephaestin–deficient) mice have some features of AMD and that a patient with retinal iron overload resulting from the rare hereditary disease aceruloplasminemia had early-onset drusen.4 Furthermore, elevated photoreceptor iron levels are associated with retinal degeneration in the Royal College of Surgeons rat.5 Herein we extend these observations by describing a case of geographic atrophy (GA) in which not only RPE cells but also cells in the photoreceptor layer had elevated iron levels. Cells in the photoreceptor layer also labeled strongly for the iron-handling proteins ferritin and ferroportin, the levels of which are up-regulated by iron.

Report of a Case

The macula from a 72-year-old white male postmortem eye donor with advanced GA (Figure 1A) was labeled for iron using the 3,3′-diaminobenzidine–enhanced Perls stain as previously described.3 Strong labeling was evident in the photoreceptor and internal limiting membrane regions (Figure 1C and D). This is significant because none of the 9 postmortem retinas from elderly donors with normal eyes had detectable iron in the neurosensory retina when stained using the same technique.3

Figure 1.
Iron in a postmortem macula with geographic atrophy. A, Photograph of the transilluminated eye cup, with the area of atrophy demarcated by arrows. The optic nerve is the white circle on the left. B, Fluorescence photomicrograph of the macula labeled by immunohistochemistry as previously described. Photoreceptors are labeled with anti-rhodopsin (red), nuclei are labeled with 4",6-diamidino-2-phenylindole (blue), and retinal pigment epithelial (RPE) cells are autofluorescent (yellow). The photoreceptors label with anti-rhodopsin in the outer nuclear layer (ONL) and the nerve fiber layer of Henle (H). As expected, cells in the inner nuclear layer (INL) are negative for rhodopsin. C, The photoreceptor layer, particularly in the vicinity of the nerve fiber layer of Henle (H), is positive for the 3,3′-diaminobenzidine–enhanced Perls stain for iron. The section was bleached with hydrogen peroxide as previously described and counterstained with Richardson (methylene blue/azure II) stain. Br indicates Bruch’s membrane; GCL, ganglion cell layer. D, 3,3"-Diaminobenzidine-enhanced Perls stain labeling both the photoreceptor and the internal limiting membrane regions.

Iron in a postmortem macula with geographic atrophy. A, Photograph of the transilluminated eye cup, with the area of atrophy demarcated by arrows. The optic nerve is the white circle on the left. B, Fluorescence photomicrograph of the macula labeled by immunohistochemistry as previously described.6 Photoreceptors are labeled with anti-rhodopsin (red), nuclei are labeled with 4",6-diamidino-2-phenylindole (blue), and retinal pigment epithelial (RPE) cells are autofluorescent (yellow). The photoreceptors label with anti-rhodopsin in the outer nuclear layer (ONL) and the nerve fiber layer of Henle (H). As expected, cells in the inner nuclear layer (INL) are negative for rhodopsin. C, The photoreceptor layer, particularly in the vicinity of the nerve fiber layer of Henle (H), is positive for the 3,3′-diaminobenzidine–enhanced Perls stain for iron. The section was bleached with hydrogen peroxide as previously described3 and counterstained with Richardson (methylene blue/azure II) stain. Br indicates Bruch’s membrane; GCL, ganglion cell layer. D, 3,3"-Diaminobenzidine-enhanced Perls stain labeling both the photoreceptor and the internal limiting membrane regions.

Because increased intracellular iron causes an increase in ferritin and ferroportin in nonocular tissues,7 we sought to determine whether the elevated photoreceptor iron level may be associated with an increase in these iron-handling proteins in the macula with GA compared with a normal macula from a 65-year-old white donor. We found that anti-ferroportin strongly labeled cells in the photoreceptor layer and along the internal limiting membrane (probably Müller cell end feet) in the macula with GA (Figure 2A). In comparison, photoreceptor label in the normal macula had a more limited distribution in the outer plexiform layer (Figure 2B). Similarly, label for the ferritin was present in the photoreceptor and internal limiting membrane regions in the macula with GA (Figure 2C), but only weakly labeled the normal macula (Figure 2D).

Figure 2.
A macula with geographic atrophy (GA) (A and C) and a normal macula (B and D) immunolabeled with anti-ferritin and anti-ferroportin. A, The macula with GA labeled with anti-ferroportin (red). The nuclei are labeled with DAPI (blue). The retinal pigment epithelial cells are autofluorescent (yellow). GCL indicates ganglion cell layer; ONL, outer nuclear layer. B, The normal macula labeled with anti-ferroportin. C, The macula with GA labeled with anti-ferritin. D, The normal macula labeled with anti-ferritin.

A macula with geographic atrophy (GA) (A and C) and a normal macula (B and D) immunolabeled with anti-ferritin and anti-ferroportin. A, The macula with GA labeled with anti-ferroportin (red). The nuclei are labeled with DAPI (blue). The retinal pigment epithelial cells are autofluorescent (yellow). GCL indicates ganglion cell layer; ONL, outer nuclear layer. B, The normal macula labeled with anti-ferroportin. C, The macula with GA labeled with anti-ferritin. D, The normal macula labeled with anti-ferritin.

Comment

We report a case of GA exhibiting strong label for iron in the neurosensory retina. This retina also labeled strongly for ferroportin and ferritin, both of which have been found to be up-regulated in response to elevated iron levels in the mouse retina.8,9 The mechanism and role of iron export in AMD needs further investigation, but this report provides evidence that iron overload can occur in AMD not only in the RPE and Bruch’s membrane but also in the neurosensory retina. Because iron can cause oxidative stress, it may be toxic to both the RPE and photoreceptors, cells in which cell death leads to blindness in AMD. Iron chelation therapy may one day prove useful in the prevention of vision loss due to AMD.

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Article Information

Correspondence: Dr Dunaief, Scheie Eye Institute, 51 N 39th St, Philadelphia, PA 19104 (jdunaief@mail.med.upenn.edu).

Financial Disclosure: None.

References
1.
Klein  RWang  QKlein  BEMoss  SEMeuer  SM The relationship of age-related maculopathy, cataract, and glaucoma to visual acuity. Invest Ophthalmol Vis Sci 1995;36182- 191
PubMed
2.
Age-Related Eye Disease Study Research Group, A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report No. 8. Arch Ophthalmol 2001;1191417- 1436
PubMedArticle
3.
Hahn  PMilam  AHDunaief  JL Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch’s membrane. Arch Ophthalmol 2003;1211099- 1105
PubMedArticle
4.
Dunaief  JLRicha  CFranks  EP  et al.  Macular degeneration in a patient with aceruloplasminemia, a disease associated with retinal iron overload. Ophthalmology 2005;1121062- 1065
PubMedArticle
5.
Yefimova  MGJeanny  JCKeller  N  et al.  Impaired retinal iron homeostasis associated with defective phagocytosis in Royal College of Surgeons rats. Invest Ophthalmol Vis Sci 2002;43537- 545
PubMed
6.
Dunaief  JLDentchev  TYing  G-SMilam  AH The role of apoptosis in age-related macular degeneration. Arch Ophthalmol 2002;1201435- 1442
PubMedArticle
7.
Rouault  TA Iron on the brain. Nat Genet 2001;28299- 300
PubMedArticle
8.
Hahn  PQian  YDentchev  T  et al.  Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Proc Natl Acad Sci U S A 2004;10113 850- 13 855
PubMedArticle
9.
Hahn  PDentchev  TQian  YRouault  THarris  ZLDunaief  JL Immunolocalization and regulation of iron handling proteins ferritin and ferroportin in the retina. Mol Vis 2004;10598- 607
PubMed
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