Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals

Abstract

Background: A wide variety of pathological pathways may result in age-related macular degeneration. Because of its complexity, there is no comprehensive model of the disease yet. One key feature is the accumulation of the autofluorescent pigment lipofuscin in the retinal pigment epithelium (RPE). Thus, we developed an organotypic perfusion culture model of the porcine ocular fundus, generating lipofuscin under exposure to blue light and hydrogen peroxide.

Methods: Porcine fundi (choroid, Bruch's membrane, RPE, and retina) were explanted in toto, transferred into a perfusion culture chamber, perfused with cell culture medium and kept at 37 degrees C. Free radical stress was induced by supplementation of H(2)O(2), and/or the specimens were exposed to blue light, or kept untreated as controls. After a culture period of 7 days, the specimens were subject to microscopic inspection, histology, fluorescence microscopy, and measurement of fluorescence spectra as well as fluorescence decay times.

Results: Histology showed atrophic ganglion cells and rod outer segments. All other tissue structures were morphologically intact. Compared to the controls, RPE and retina exposed to light showed increased fluorescence, which was shifted towards shorter wavelengths. The fluorescence spectra and decays resembled that of lipofuscin granules isolated from human donor eyes. HPLC analysis revealed the abundance of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2E), its precursor products, as well as two new, green-emitting fluorophores.

Conclusions: Porcine ocular fundi were successfully preserved in an organotypic perfusion culture for 7 days, and exhibited remarkable autofluorescence after light and free radical exposure, making the model suitable for investigations of lipofuscinogenesis.