Artikel
Detection of sub-retinal epithelial cell layer deposits in mice by zinc staining using ex-vivo and in-vivo methods
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Autoren
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Scott McPherson
- Minneapolis/USA
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H. Roehrich
- Minneapolis/USA
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F.J. van Kuijk
- Minneapolis/USA
| Veröffentlicht: | 7. September 2023 |
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Gliederung
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Background: The relationship between zinc and age-related macular degeneration (AMD) has only been explored with epidemiological and in-vitro studies. Ex-vivo and in-vitro assays associated with animal models are crucial for understanding zinc’s role in AMD. Here we report detection and quantification of sub-retinal epithelial zinc using ex-vivo analysis and in-vivo injection of zinc probes.
Methods: We employed mice deficient in superoxide dismutase-type 1 (CuZn-SOD) or -type 2 (Mn-SOD). Zinc and sub-retinal epithelial deposits were detected using ZPP1 or a novel probe (ZQuinT) that fluoresce upon binding zinc. Retinal flat mounts were stained ex-vivo with zinc probe or examined directly without additional staining following in-vivo injection of probe. Imaging and analysis were done by fluorescence microscopy.
Results: Sub-retinal epithelial cell layer deposits in CuZn-SOD and Mn-SOD deficient mice were detected in flat mounts following in-vivo injection of ZPP1 and ZQuinT. Analysis of probe concentration and incubation time revealed that micromolar concentrations of ZPP1 and millimolar concentrations of ZQuinT allowed to circulate ten minutes were sufficient to detect zinc and sub-retinal epithelial cell layer deposits. Examination of flat mounts showed detection of sub-retinal epithelial cell layer deposits from mice receiving in-vivo zinc probe was similar to detection by ex-vivo staining of retinal flat mounts. We observed increased levels of sub-retinal zinc in normally housed (normal light) CuZn-SOD deficient mice compared to wild type mice, suggesting zinc accumulation can be linked to risk factors associated with AMD.
Conclusions: Zinc dysregulation is a crucial initiating factor in AMD. Animal models and in-vivo assays allows for analysis of retinal zinc in response to genetic and environmental factors associated with AMD. Detection of sub retinal epithelial cell layer deposits after in-vivo zinc probe injection is an essential step in developing clinical and research assays that can detect the earliest, sub-clinical cellular changes associated with AMD.
