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Rebecca Kapphahn, Heidi Roehrich, Deborah Ferrington, Frederik van Kuijk; Zinc Staining of sub-RPE Deposits in Murine Models of Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5009.
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One of the hallmarks of age-related macular degeneration (AMD) is the accumulation of sub-retinal pigment epithelial (RPE) deposits, including drusen and basal laminar deposits, in Bruch’s membrane. We have previously shown that these sub-RPE deposits contain high concentrations of zinc in human donors with AMD and that these zinc-enriched deposits were especially prevalent in the macula (Lengyel et al, 2007). Since sub-RPE deposits may contribute to AMD pathology, zinc-containing sub-RPE deposits may be an important characteristic for animal models used to investigate AMD mechanisms or test therapeutic interventions. The purpose of this study was to determine if zinc-enriched sub-RPE deposits are present in animal models of retinal degeneration.
The presence of zinc-enriched sub-RPE deposits was assessed in multiple murine models that exhibit retinal degeneration (sod1-/-, mice carrying the rd8 mutation, old C57BL6). In order to visualize sub-RPE zinc deposits, the anterior segment was removed from enucleated globes. The retina and RPE were then removed using zinc-free PBS and the unfixed eye cup was flat mounted on a glass slide without a cover slip. A Leica DM4000B microscope was used to visualize the autofluorescence associated with the optic nerve. Exposure time was then decreased to determine the exposure parameters where autoflourescence was no longer visible. These parameters were used to capture all subsequent images. Membrane permeable fluorescent zinc stain, Zinpyr-1 (Strem Chemicals, Newburyport, MA) (10uM diluted in PBS) was then applied to sub-RPE flat mounts and incubated for 5 min. Excess Zinpyr-1 was removed by rinsing with zinc-free PBS and the sections were cover slipped. The fluorescence associated with zinc deposits was immediately visualized.
High levels of zinc-containing sub-RPE deposits were observed in sod1-/- mice, mice carrying the rd8 mutation, and old (15 months) but not young (3 months) C57BL6 mice. The presence of high levels of zinc-enriched sub-RPE deposits in the sod1-/- mouse, proposed as a model for AMD, further validates its use to investigate AMD disease pathology and test interventions.
Membrane permeable fluorescent zinc staining provides a quick assessment of sub-RPE deposits that can be easily quantified. Staining of sub-RPE deposits helps to validate this AMD-associated phenotype in animal models used in AMD research.
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