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

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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°C. Free radical stress was induced by supplementation of H2O2, 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.

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Acknowledgments

This work was supported by the German Ministry of Education and Research under grant No. 01EZ0309. The authors are grateful to Prof. Elizabeth Gaillard from Northern Illinois University for the gift of lipofuscin granules isolated from human donor eyes.

Conflict of interests

Hammer: none, Richter: none, Kobuch: none, Mata: employee of Sirion Therapeutics Inc., Schweitzer: none

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Authors and Affiliations

  1. Department of Ophthalmology, University of Jena, Bachstr. 18, 07740, Jena, Germany

    Martin Hammer, Sandra Richter & Dietrich Schweitzer

  2. Department of Ophthalmology, University of Regensburg, F-J Strauss - Allee 11, 93053, Regensburg, Germany

    Karin Kobuch

  3. SIRiON Therapeutics, 11408 Sorrento Valley Rd, San Diego, CA, 92121, USA

    Nathan Mata

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  1. Martin Hammer
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Correspondence to Martin Hammer.

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Hammer, M., Richter, S., Kobuch, K. et al. Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals. Graefes Arch Clin Exp Ophthalmol 246, 979–988 (2008). https://doi.org/10.1007/s00417-008-0789-4

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  • DOI: https://doi.org/10.1007/s00417-008-0789-4

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