Abstract

SA.21.09

Changes of ocular fundus auto-fluorescence induced by photo-oxidative stress

Martin Hammer1, Silvio Quick1, Anett Eitner2, Susanne Jentsch1, Dietrich Schweitzer1
1Klinik für Augenheilkunde, Friedrich-Schiller-Universität, Jena, 2Institut für Anatomie, Universitätsklinikum Jena, Jena

Objective
Pathologic alterations at the ocular fundus, particularly in age-related macular degeneration (AMD), may change the auto-fluorescence of retina and retinal pigment epithelium (RPE). Besides fluorescence intensity, the emission spectrum as well as the fluorescence lifetime may be altered. In this study, fluorescence lifetimes and spectra were measured from native porcine ocular fundus as well as from tissue cultures under photo-oxidative stress.
Methods
Upon excitation at 488 nm, fluorescence spectra of the photoreceptors as well as RPE were recorded by confocal laser scanning microscopy. Fluorescence lifetimes were measured in two spectral channels (500 – 560 nm and 560 – 700 nm) by a Heidelberg Retina Angiograph equipped with picosecond pulse laser and time –correlated single photon counting. Investigations were performed in native tissue as well as in tissue cultured for up to three days. Photo-oxidative stress was exerted in the culture by blue light (468 nm, 0,41 mW/mm2) irradiation.
Results
Irradiation of the cultured ocular fundus resulted in an increase of fluorescence lifetimes as well as in a hypsochromic shift of the emission spectra. While mean fluorescence lifetimes of 255±69 ps (500-560 nm) and 237±32 ps (560-700 nm) respectively were measured for the native fundus, a highly significant (p<0.0005) increase to 559±110 ps and 430±80 ps was found after seven hours of culture under irradiation. After three days in culture, RPE auto-fluorescence with an emission maximum at 590 nm was measured while the fluorescence spectrum of the photoreceptors peeked at 530 nm.
Conclusions
Confocal microscopy enables the separation of the fluorescence of the RPE and the photoreceptors. The latter show a fluorescence maximum equivalent to FAD. This may hint at a shift in the redox equilibrium FAD/FADH2 which could indicate impairment of the mitochondria. The extension of fluorescence lifetimes under oxidative stress correlate well with in-vivo findings: Patients suffering from non-exudative AMD showed significantly longer fluorescence lifetimes compared to age matched controls. Oxidative damage, induced in cultured retina and RPE, can be observed by fluorescence microscopy with spectral as well as temporal resolution.

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