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K. B. Phan, Y. Han, N. Tsivkovskaia, S. Narayan, T. V. Bui, N. L. Mata; Characterization of Fluorophores in the Diseased Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4171.
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Excessive accumulation of lipofuscin is an early event in the pathogenesis of age-related macular degeneration. The intrinsic autofluorescence which is associated with lipofuscin precedes death of RPE cells and is, therefore, a good predictor of impending retinal degeneration. A2E has been implicated as the source of lipofuscin autofluorescence. Recently, we have detected a > 500 nm autofluorescence in post-mortem AMD donor eyes and in an AMD animal model which is not attributable to A2E or related fluorophores. Here, studies were conducted to identify and characterize the underlying fluorescing compound(s).
Post-mortem AMD donor eyes were obtained from a local eye bank and processed for histological examination within 24 hours of procurement. Eye globes from abca4-/- mice and "iron overload" ceruloplasmin/hephaestin (Cp-/-, Heph-/Y) double knockout mice were also prepared for histology. The histological preparations were examined by routine cellular staining, immunohistochemistry, and by confocal fluorescence microscopy. Fluorescence emission spectra were obtained with 488 nm excitation. Fluorophores contained within the mouse eye globe extracts were also analyzed by HPLC and spectrofluorometry.
Fluorescence emission spectra from the RPE of abca4-/- mice were dominated by A2E and related fluorophores (emission maximum was ~ 580 nm). The fluorescence emission from the RPE of Cp-/-,Heph-/Y mice was distinct with a maximum at ~ 550 nm. There were also regional differences in the localization of the fluorophores between these two animal models. Interestingly, post-mortem AMD eyes contained both of these fluorescing species. The AMD tissue also contained massive amounts of iron. Because iron is known to interact with melanin and facilitate its oxidation, we theorized that the ~ 550 nm fluorescence observed in the Cp-/-,Heph-/Y mice and in AMD eyes may be attributable to oxidized melanin. Indeed, iron-mediated oxidation of synthetic melanin was found to produce an emission spectrum which is indistinguishable from the ~ 550 nm fluorophore.
We have identified a distinct fluorophore in the "iron overload" AMD animal model and in post-mortem AMD donor eyes. The co-localization of this fluorophore with iron deposits within the RPE is consistent with the theory that this fluorophore is the result of iron-mediated oxidation of melanin. In vitro studies employing synthetic melanin support this theory. Oxidized melanin rapidly decomposes and loses its ability to protect against reactive oxygen species. Thus, findings of the present study provide further impetus for the development of an anti-oxidant therapeutic.
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