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M. Hammer, N. Mata, K. Kobuch, C. Biskup, S. Schenke, D. Schweitzer; Characterization of Lipofuscin-Like Fluorophores Accumulated in an Organotypic Perfusion Culture of Porcine Fundi Under Blue Light Irradiation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5065.
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The accumulation of lipofuscin-like fluorescent material in porcine fundi, kept in an organotypic perfusion culture and irradiated with blue light, has been reported recently. This makes the irradiated perfusion culture a suitable model to study lipofuscinogenesis, a process taking years in vivo. Knowledge of the mechanisms of lipofuscin accumulation is important since it is a precursor of RPE cell dysfunction and apoptosis in AMD and hereditary macular degeneration. In this study, we provide chemical and morphological characterization of fluorescent structures arising in the organ culture under blue light irradiation.
Explanted porcine fundi were transferred into a culture chamber and perfused with DMEM, supplemented with 15% porcine serum, 25 mM HEPES, and 1% penicillin-streptomycin, kept at 37°C, and exposed to a circadian blue light irradiation (0,41 mW/mm2, peak wavelength: 467 nm) onto the retina. All samples were paralleled by non-irradiated control cultures. After culttivation for 7 through 21 days, tissue specimen were inspected by histology, conventional as well as laser scanning fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence spectroscopy. Chloroform/Methanol extracts of the tissues were analyzed by HPLC and mass spectrometry (LC/ESI-MS).
Histology revealed morphological preservation of the tissues. Fluorophores accumulated in granular structures in the RPE resembling lysosomes. Furthermore, an increase of fluorescence was found in the neural retina. HPLC and mass spectrometry indicated the abundance of the pyridinium bisretinoid A2E, its precursors A2PE and A2PE-H2, as well as oxidation products of these compounds in the RPE. From the retina, two fluorophores were found with shorter retention times and fluorescence emission maxima at 520 nm and 530 nm respectively upon excitation at 440 nm.
The appearance of A2E, A2PE, A2PE-H2, and their oxidation products in ocular fundus perfusion cultures under blue light irradiation indicates lipofuscinogenesis. This makes that organ culture an interesting model to study light-induced alterations of fundus autofluorescence which may indicate early pathologic alterations in AMD. Interestingly, such alterations take place not only in the RPE but also in the retina. Fluorescences found there may be attributed to the oxidation of poly-unsaturated fatty acids.
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