Auf dem Weg zur verbesserten Früherkennung neurodegenerativer Erkrankungen. Nicht-invasive Untersuchungen zum retinalen Stoffwechsel mittels Fluoreszenzlebensdauer und Anisotropieanalyse des zellulären Redoxzustandes
Zusammenfassung der Projektergebnisse
This project aimed to investigate the protein binding of NADH and FAD in retinal cells and tissues using Fluorescence lifetime and anisotropy decay measurements. It’s based on five hypothesis which can be addressed as follows: 1. Fluorescence spectra and lifetimes are suitable to distinguish NADH and FAD from other fluorophores at the ocular fundus. 2. The fluorescence lifetime and anisotropy (with limitations) detection with sub-cellular resolution enables the observation of NADH and FAD specifically in mitochondria. 3. Free and protein bound NADH was distinguished by fluorescence lifetime. 4. The measurement of fluorescence anisotropy decays is possible but limited by depolarisation in the microscope objective. 5. The NADH- and flavin fluorescence shows typical changes under hypoxic and oxidative stress but not under hyperglycaemic conditions. The results of this project support the interpretation of in vivo fluorescence lifetime ophthalmoscopy (FLIO). In general, the goal of investigating the redox state and differentiating free and protein bound NAD(P)H and flavines in conditions, possibly underlying neurodegeneration, has been achieved using fluorescence lifetime and anisotropy measurements. We found a shift form free to protein-bound NADH in mitochondria of retinal Müller cells under oxidative stress as well as a hyperpolarization of their inner membrane. Hypoxia, on the other hand, resulted in an increase of NAD(P)H. This may indicate a hypoxic inhibition of respiratory chain complex I and an activation of complex II. Fluorescence lifetime as well as spectra measurements were performed in an organotypic culture model of the porcine ocular fundus using two- as well as one-photon excitation. These investigations revealed the contribution of NAD(P)H, flavines, melanin, and eventually lipofuscin specifically to the fluorescence of single cellular layers of the retina and the retinal pigment epithelium. Some limitations and problems were encountered during the project: A long term organotypic culture of porcine ocular fundi was possible, however deep tissue two-photon imaging was prevented due to retinal blurring later than 3 hours post mortem. Measurement of the fluorescence anisotropy decay was possible. Due to depolarization at optical interfaces and components, specifically by high NA objectives, in our scanning microscope, the degree of polarization of the excitation light at the sample was lower than reported in the literature for non-scanning systems. Supplemental to the research, funded under this grant, clinical in-vivo investigations of retinal fluorescence lifetimes in retinal degeneration were performed. These revealed, for the first time, the contribution of the macular pigment Xanthophyll to the retinal autofluorescence, a differentiation of the fluorescence of Lipofuscin in the retinal pigment epithelium and that of Lipoproteins in drusen, a hallmark of retinal neurodegeneration, specifically age related macular degeneration (AMD). Furthermore, an increase of lifetimes in AMD points to an alteration of the fluorophore composition of Lipofuscin in this disease. Fluorescence lifetimes in the retina and lens of patients, suffering from diabetic retinopathy, may indicate protein glycation. In conclusion, the results from this project contribute to the understanding of alterations of the cellular energy metabolism, which might be implicated in neurodegfeneration, and, thus, help to interpret clinical fluorescence lifetime ophthalmoscopy data in related diseases.
Projektbezogene Publikationen (Auswahl)
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(2017) Hypoxia-induced redox signalling in Müller cells. Acta ophthalmologica 95 (4) e337-e339
Griebsch, Max; Klemm, Matthias; Haueisen, Jens; Hammer, Martin
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"Agreement Between Eyes in Wide-Field Fluorescence Lifetime Imaging Ophthalmoscopy Measurements at the Human Retina in Healthy Volunteers", in International Conference for Innovation in Biomedical Engineering and Life Sciences. vol. 56, F. Ibrahim, J. Usman, M. S. Mohktar, and M. Y. Ahmad, Eds., ed: Springer Singapore, 2015, pp. 298-301
M. Klemm, E. Nagel, A. Dietzel, K. W. Lai, E. Supriyanto, and D. Schweitzer
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"FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye", PLoS One, vol. 10, p. e0131640, 2015
M. Klemm, D. Schweitzer, S. Peters, L. Sauer, M. Hammer, and J. Haueisen
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"Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy", Journal of Biomedical Optics, vol. 20, p. 061106, 2015
D. Schweitzer, L. Deutsch, M. Klemm, S. Jentsch, M. Hammer, S. Peters, et al.
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"Impact of Macular Pigment on Fundus Autofluorescence Lifetimes", Invest Ophthalmol Vis Sci, vol. 56, pp. 4668- 79, 2015
L. Sauer, D. Schweitzer, L. Ramm, R. Augsten, M. Hammer, and S. Peters
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"Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model", Biomedical Optics Express, vol. 7, pp. 3198-3210, 2016
M. Klemm, J. Blum, D. Link, M. Hammer, J. Haueisen, and D. Schweitzer
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"Effects of short term changes in the blood glucose level on the autofluorescence lifetime of the human retina in healthy volunteers", in Ophthalmic Technologies XXVI, SPIE Photonics West, San Francisco, 2016, pp. 96931R-96931R-5
M. Klemm, E. Nagel, D. Schweitzer, S. Schramm, and J. Haueisen
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"Fundus autofluorescence lifetimes are increased in non-proliferative diabetic retinopathy", Acta Ophthalmologica, 2016
J. Schmidt, S. Peters, L. Sauer, D. Schweitzer, M. Klemm, R. Augsten, et al.
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"Hydrogen peroxide modulates energy metabolism and oxidative stress in cultures of permanent human Müller cells MIO-M1", Journal of Biophotonics, 2016
S. Peters, M. Griebsch, M. Klemm, J. Haueisen, and M. Hammer
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"Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy", Acta Ophthalmologica, 2016
L. Sauer, S. Peters, J. Schmidt, D. Schweitzer, M. Klemm, L. Ramm, et al.
