Ceruloplasmin/Hephaestin Knockout Mice Model Morphologic and Mechanistic Features of Amd

Abstract

Purpose: : Several studies suggest that iron accumulation with age may be a factor in age related macular degeneration (AMD). We have previously identified both iron overload and some features of AMD in mice deficient for the ferroxidase ceruloplasmin (Cp) and its homolog hephaestin (Heph) (double knockouts, DKO). In this study, we perform a more detailed analysis of the location and timing of iron accumulation, the rate and reproducibility of retinal degeneration, and investigate the mechanisms of the degeneration.

Methods: : C57BL/6 wild type and mice with a targeted mutation in Cp and naturally occurring sla mutation in the Heph gene were used. Histochemical iron detection was done by Perls’ staining in JB4 sections which also were used for morphologic analysis. Immunohistochemistry was performed on cryosections using anti-C3b/iC3b/C3c, anti- transferrin receptor, and anti-L-ferritin antibodies. Tissue iron levels were measured by graphite furnace atomic absorption spectrophotometry. Isoprostane F2α-VI was quantified in the tissue by gas chromatography/mass spectrometry. QRT-PCR was done to quantify VEGF gene expression in DKO and WT.

Results: : DKOs exhibit age-dependent iron overload with subsequent retinal degeneration with some features of AMD, including RPE hypertrophy and hyperplasia, photoreceptor degeneration, RPE lipofuscin accumulation, oxidative stress and complement activation. Similarly to AMD, DKO mice show subretinal neovascularization and have increased VEGF expression in comparison to the WT.

Conclusions: : DKOs have age-dependent iron accumulation followed by retinal degeneration modeling a number of morphological and mechanistic features of AMD.

Keywords: age-related macular degeneration • transgenics/knock-outs