Two-photon Microscopy and Fluorescence Lifetime Analysis of Lipid Peroxidation Product in Photoreceptor Outer Segment and in Retinal Pigment Epithelial Cell

Abstract

Purpose: Lipid peroxidation products as 4-hydroxynonenal (4-HNE) are known to form fluorescent protein adducts, and considered to be involved in the lipofuscinogenesis in retinal pigment epithelial (RPE) cell. We have previously shown that acute oxidative stress induces the appearance of lipofuscin-like bright fluorescent inclusions in RPE cell. The purpose of this study is to elucidate the pathogenesis of these inclusions in relation to the fluorescence properties of associated lipid peroxidation products.
Methods: RPE-choroid tissue was isolated from porcine eye and preserved in the culture medium. Lipid peroxidation was induced by exposing tissue to ferrous sulfate and laser photocoagulation (Nd.YAG laser, λ=532 nm, 0.1 s, 300 µm) on the RPE. RPE-autofluorescence (AF) was investigated with two-photon microscopy combined with fluorescence lifetime measurement (FLIM). AF of photoreceptor outer segments (POS) from porcine retina was also investigated under normal and lipid peroxidation conditions to elucidate its association with the AF in RPE cells. After examination of RPE-AF, the tissue was fixed and immunolocalization of opsin, cellular retinalaldehyde binding protein (CRALBP) and 4-HNE adducts were examined by multi-staining immunofluorescent method.
Results: Laser photocoagulation and exposure to ferrous ion induce the appearance of bright AF granules inside and around RPE cells. The emission maximum of the AF granules is at 450-500 nm with a two-photon excitation maximum at 710-750 nm. Their mean fluorescence lifetime (FL) is 0.655 ns. Addition of ferrous ion to POS increases the intensity of their AF and shifts the emission maximum from 380-450 nm to 450-500 nm and the mean FL from 1.3 ns to 0.65 ns. The extent of FL reduction is correlated with the extent of intensity increase. Immunolocalization of bright AF granules inside and around the RPE cells is coincident with the localization of opsin and CRALBP, respectively, and also coincident with the strong staining of 4-HNE.
Conclusions: These results suggest that heat- and ferrous ion-induced bright AF granules inside and around RPE cells detected with two-photon microscopy are phagocytised POS and retinoid-storing inclusions under lipid peroxidation, respectively. Moreover, it is suggested that 4-HNE adducts may be one of the main fluorophores of these AF granules.
OriginalspracheEnglisch
PublikationsstatusVeröffentlicht - 06.03.2013

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