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Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro

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Abstract

Background

Early and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H.

Methods

Using non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally.

Results

Decrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models.

Conclusions

Fluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.

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References

  1. Nishida T, Chikama T, Morishige N, Yanai R, Yamada N, Saito J (2007) Persistent epithelial defects due to neurotrophic keratopathy treated with a substance p-derived peptide and insulin-like growth factor 1. Jpn J Ophthalmol 51:442–447

    Article  PubMed  CAS  Google Scholar 

  2. Haruta Y, Ohashi Y, Matsuda S (1997) Corneal epithelial deficiency induced by the use of beta-blocker eye drops. Eur J Ophthalmol 7:334–339

    PubMed  CAS  Google Scholar 

  3. Dua HS, Saini JS, Azuara-Blanco A, Gupta P (2000) Limbal stem cell deficiency: concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol 48:83–92

    PubMed  CAS  Google Scholar 

  4. Chen JJ, Tseng SC (1990) Corneal epithelial wound healing in partial limbal deficiency. Invest Ophthalmol Vis Sci 31:1301–1314

    PubMed  CAS  Google Scholar 

  5. Huttmann G, Lankenau E, Schulz-Wackerbarth C, Muller M, Steven P, Birngruber R (2009) Optical coherence tomography: from retina imaging to intraoperative use - a review. Klin Monatsbl Augenheilkd 226:958–964

    Article  PubMed  CAS  Google Scholar 

  6. Erie JC, McLaren JW, Patel SV (2009) Confocal microscopy in ophthalmology. Am J Ophthalmol 148:639–646

    Article  PubMed  Google Scholar 

  7. Radosevich AJ, Bouchard MB, Burgess SA, Chen BR, Hillman EM (2008) Hyperspectral in vivo two-photon microscopy of intrinsic contrast. Opt Lett 33:2164–2166

    Article  PubMed  CAS  Google Scholar 

  8. Steven P, Müller M, Koop N, Rose C, Hüttmann G (2009) Comparison of cornea module and DermaInspect noninvasive imaging of ocular surface pathologies. J Biomed Optics 14:064040

    Article  Google Scholar 

  9. Paulsen FP, Woon CW, Varoga D, Jansen A, Garreis F, Jager K, Amm M, Podolsky DK, Steven P, Barker NP, Sel S (2008) Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds. J Biol Chem 283:13418–13427

    Article  PubMed  CAS  Google Scholar 

  10. Bird DK, Yan L, Vrotsos KM, Eliceiri KW, Vaughan EM, Keely PJ, White JG, Ramanujam N (2005) Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res 65:8766–8773

    Article  PubMed  CAS  Google Scholar 

  11. Chia TH, Williamson A, Spencer DD, Levene MJ (2008) Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatically distinct NADH binding. Opt Express 16:4237–4249

    Article  PubMed  CAS  Google Scholar 

  12. Kable EP, Kiemer AK (2005) Non-invasive live-cell measurement of changes in macrophage NAD(P)H by two-photon microscopy. Immunol Lett 96:33–38

    Article  PubMed  CAS  Google Scholar 

  13. Li D, Zheng W, Qu JY (2009) Two-photon autofluorescence microscopy of multicolor excitation. Opt Lett 34:202–204

    Article  PubMed  CAS  Google Scholar 

  14. Piston DW, Masters BR, Webb WW (1995) Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy. J Microsc 178:20–27

    Article  PubMed  CAS  Google Scholar 

  15. Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, White JG, Ramanujam N (2007) In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci U S A 104:19494–19499

    Article  PubMed  CAS  Google Scholar 

  16. Vishwasrao HD, Heikal AA, Kasischke KA, Webb WW (2005) Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J Biol Chem 280:25119–25126

    Article  PubMed  CAS  Google Scholar 

  17. Szaszak M, Steven P, Shima K, Orzekowsky-Schroder R, Huttmann G, Konig IR, Solbach W, Rupp J (2011) Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell. PLoS Pathog 7:e1002108

    Article  PubMed  CAS  Google Scholar 

  18. Huang S, Heikal AA, Webb WW (2002) Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J 82:2811–2825

    Article  PubMed  CAS  Google Scholar 

  19. Yu Q, Heikal AA (2009) Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level. J Photochem Photobiol B 95:46–57

    Article  PubMed  CAS  Google Scholar 

  20. Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML (1992) Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A 89:1271–1275

    Article  PubMed  CAS  Google Scholar 

  21. Anderson RA (1977) Actin filaments in normal and migrating corneal epithelial cells. Invest Ophthalmol Vis Sci 16:161–166

    PubMed  CAS  Google Scholar 

  22. Crosson CE, Klyce SD, Beuerman RW (1986) Epithelial wound closure in the rabbit cornea. A biphasic process. Invest Ophthalmol Vis Sci 27:464–473

    PubMed  CAS  Google Scholar 

  23. Kubawara T, Perkins DG, Cogan DG (1976) Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol Vis Sci 15:4–14

    Google Scholar 

  24. Rasband WS (1997–2011) ImageJ. U. S. National Institutes of Health, Bethesda, Maryland, USA. <http://rsbweb.nih.gov/ij>

  25. Schneckenburger H, Wagner M, Weber P, Strauss WS, Sailer R (2004) Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode. J Fluoresc 14:649–654

    Article  PubMed  CAS  Google Scholar 

  26. Erecinska M, Nelson D, Deas J, Silver IA (1996) Limitation of glycolysis by hexokinase in rat brain synaptosomes during intense ion pumping. Brain Res 726:153–159

    Article  PubMed  CAS  Google Scholar 

  27. Sen S, Riaz SS, Ray DS (2008) Temperature dependence and temperature compensation of kinetics of chemical oscillations; Belousov-Zhabotinskii reaction, glycolysis and circadian rhythms. J Theor Biol 250:103–112

    Article  PubMed  CAS  Google Scholar 

  28. Somero GN (1975) Temperature as a selective factor in protein evolution: the adaptational strategy of "compromise". J Exp Zool 194:175–188

    Article  PubMed  CAS  Google Scholar 

  29. Konig K, Riemann I (2003) High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8:432–439

    Article  PubMed  Google Scholar 

  30. Steven P, Rupp J, Huttmann G, Koop N, Lensing C, Laqua H, Gebert A (2008) Experimental induction and three-dimensional two-photon imaging of conjunctiva-associated lymphoid tissue. Invest Ophthalmol Vis Sci 49:1512–1517

    Article  PubMed  Google Scholar 

  31. Gehlsen U, Huttmann G, Steven P (2010) Intravital multidimensional real-time imaging of the conjunctival immune system. Dev Ophthalmol 45:40–48

    Article  PubMed  CAS  Google Scholar 

  32. Steven P, Hovakimyan M, Guthoff RF, Huttmann G, Stachs O (2010) Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements. J Cataract Refract Surg 36:2150–2159

    Article  PubMed  Google Scholar 

  33. Steven P, Bock F, Hüttmann G, Cursiefen C (2011) Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels. PLoS One 6(10):e26253. doi:10.1371/journal.pone.0026253

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to acknowledge Inke König, (Institute of Medical Biometry and Statistics, University of Luebeck) for substantial advice regarding statistical analysis and Daniela Rieck and Reinhard Schultz for superb technical support regarding tissue culturing and design of the custom made specimen holder. This study was supported by the University of Luebeck, Medical Faculty Grants (to PS).

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Authors and Affiliations

  1. Department of Ophthalmology, University Hospital of Cologne, Kerpenerstr. 62, 50937, Cologne, Germany

    Uta Gehlsen & Philipp Steven

  2. Institute of Anatomy, University of Luebeck, Luebeck, Germany

    Uta Gehlsen & Andrea Oetke

  3. Institute of Medical Microbiology and Hygiene, University of Luebeck, Luebeck, Germany

    Márta Szaszák

  4. Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany

    Norbert Koop & Gereon Huettmann

  5. Institute of Anatomy and Cell Biology, University of Erlangen, Erlangen, Germany

    Friedrich Paulsen

  6. Institute of Anatomy II, University of Jena, Jena, Germany

    Andreas Gebert

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  1. Uta Gehlsen
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  2. Andrea Oetke
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Correspondence to Philipp Steven.

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The authors have full control of the primary data, and agree to allow Graefe's Archive for Clinical and Experimental Ophthalmology to review the data on request.

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Gehlsen, U., Oetke, A., Szaszák, M. et al. Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro. Graefes Arch Clin Exp Ophthalmol 250, 1293–1302 (2012). https://doi.org/10.1007/s00417-012-2051-3

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  • DOI: https://doi.org/10.1007/s00417-012-2051-3

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