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. 2008 Jun;49(6):2728-36.
doi: 10.1167/iovs.07-1472. Epub 2008 Mar 7.

Ceruloplasmin/hephaestin Knockout Mice Model Morphologic and Molecular Features of AMD

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Free PMC article

Ceruloplasmin/hephaestin Knockout Mice Model Morphologic and Molecular Features of AMD

Majda Hadziahmetovic et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: Iron is an essential element in human metabolism but also is a potent generator of oxidative damage with levels that increase with age. Several studies suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). In prior studies, both iron overload and features of AMD were identified in mice deficient in the ferroxidase ceruloplasmin (Cp) and its homologue hephaestin (Heph) (double knockout, DKO). In this study, the location and timing of iron accumulation, the rate and reproducibility of retinal degeneration, and the roles of oxidative stress and complement activation were determined.

Methods: Morphologic analysis and histochemical iron detection by Perls' staining was performed on retina sections from DKO and control mice. Immunofluorescence and immunohistochemistry were performed with antibodies detecting activated complement factor C3, transferrin receptor, L-ferritin, and macrophages. Tissue iron levels were measured by atomic absorption spectrophotometry. Isoprostane F2alpha-VI, a specific marker of oxidative stress, was quantified in the tissue by gas chromatography/mass spectrometry.

Results: DKOs exhibited highly reproducible age-dependent iron overload, which plateaued at 6 months of age, with subsequent progressive retinal degeneration continuing to at least 12 months. The degeneration shared some features of AMD, including RPE hypertrophy and hyperplasia, photoreceptor degeneration, subretinal neovascularization, RPE lipofuscin accumulation, oxidative stress, and complement activation.

Conclusions: DKOs have age-dependent iron accumulation followed by retinal degeneration modeling some of the morphologic and molecular features of AMD. Therefore, these mice are a good platform on which to test therapeutic agents for AMD, such as antioxidants, iron chelators, and antiangiogenic agents.

Figures

Figure 1
Figure 1
The Cp−/−Heph−/− (DKO) ciliary body had iron accumulation and increased L-ferritin. WT (A) and DKO (B) ciliary body Perls' stained (purple) for iron. The 7-month-old DKO ciliary body had detectable iron but the 16-month-old WT control did not. Arrows: Perls' label in the nonpigmented ciliary epithelium and in the RPE. Fluorescence photomicrographs of WT (C), Cp−/− (D), Heph−/− (E), and DKO (F) ciliary bodies immunolabeled for L-ferritin (red), counter-stained with DAPI (blue) and imaged with identical exposure parameters. Scale bar: 50 μm. CB, ciliary body; I, iris; R, retina.
Figure 2
Figure 2
Levels of TfR decreased in DKO retinas. Fluorescence photomicrographs of WT retina (A) immunolabeled with anti-TfR antibodies (red), showed strong immunoreactivity present in all retinal cell layers. In contrast, except for a thin line of immunoreactivity near the junction of the photoreceptor inner and outer segments, immunoreactivity in the remainder of the retina was weak in the 6-month-old DKO (C) and was minimal in the 9-month-old DKO (E) mice. (B, D, F) Matching controls for WT and 6- and 9-month-old DKO mice without primary antibodies. Identical exposure parameters were used. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 μm.
Figure 3
Figure 3
Graphs of iron and isoprostane quantification in the retinas and RPE/choroid of DKO and wild-type eyes with age. Total iron in nanograms per neurosensory retina (A) measured by atomic-absorption spectrometry (AAS) is shown for age and genotype. Total iron in nanograms per RPE/choroid measured by AAS (B) is shown for age and genotype. Isoprostane F2α-VI levels (C) measured by mass spectrometry are shown for 6-month-old DKO in comparison to 6-month-old wild-type neurosensory retinas. *Significant difference (P < 0.05).
Figure 4
Figure 4
DKO mice had age-dependent retinal degeneration with neovascularization. Bright-field micrographs of plastic sections show that relative to 16-month-old WT mice (A) 7-month-old DKO mice had focal areas of RPE hyperplasia (B, arrow) and focal photoreceptor degeneration consisting of thinning of the outer nuclear layer (ONL), inner segment vacuolization, and loss of outer segments. Macrophage infiltration was also present (C, arrow) in 7-month-old DKO. DKO mice at the age of 9 months (D, E) had focal areas of significantly hypertrophic RPE cells with loss of overlying photoreceptor outer segments and thinning of the ONL. Nine-month-old DKOs had more macrophage infiltration than did the 7-month-old DKOs (E, arrow). Twelve-month-old DKOs (F) had hypertrophic RPE cells, loss of inner and outer segments, and thinning of the ONL. Epifluorescence microscopy of eyecups from DiI-perfused mice show that 9-month-old wild-type mice had no neovascularization (G), whereas age-matched DKO (H) had focal areas of hyperfluorescence (arrows). These areas correspond to focal RPE disruption, with a vessel passing though the photoreceptor layer, as seen in a 7-month-old DKO retina (I, arrow). RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar, 50 μm.
Figure 5
Figure 5
Macrophage infiltration in DKO retinas. Fluorescence photomicrograph of 9-month-old DKO (A) immunolabeled with an anti-F4/80 antibody shows subretinal immunoreactivity (arrow, red fluorescence) specific for this glycoprotein expressed by macrophages. Bright-field photomicrographs of 9- and 13-month-old DKO retinas (B, C) labeled with anti-CD11b antibodies show Mac-1-positive cells (arrows, blue chromogen) specific for macrophages. RPE, retinal pigment epithelium; ONL, outer nuclear layer. Scale bar: (A) 50 μm; (B, C) 25 μm.
Figure 6
Figure 6
Lipofuscin accumulated in the DKO RPE with age. Whereas the aged-matched wild-type retinas had no detectable autofluorescent lipofuscin in the RPE (A, C), DKOs had RPE autofluorescence (B, D, E, arrows). Focal areas of autofluorescent hypertrophic RPE were present in 7-month-old (B, arrow) and 9-month-old (D, arrow) DKO retinas. In 12-month DKOs (E, arrow) most of the RPE cells were autofluorescent. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 μm.
Figure 7
Figure 7
Activated complement components were present in DKO Bruch's membrane. Fluorescence photomicrograph of 9-month-old WT immunolabeled with anti-C3b/iC3b/C3c antibodies showed no immunoreactivity, whereas there was sub-RPE immunoreactivity (arrow) in a 9-month-old DKO RPE, retinal pigment epithelium. ONL, outer nuclear layer. Scale bar, 50 μm.

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