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Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

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Abstract

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time.

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Acknowledgments

We appreciate the support of University of Wisconsin Milwaukee RGI 6 Grant, Clinical and Translational Science Institute KL2 Grant: NIH 8Kl2TR000056, Wisconsin Applied Research grant (Wi-ARG), NIH Grants HL-24349 (SHA), HL 49294 (ERJ), and the Department of Veterans Affairs that provided resources essential to the completion of these investigations. We acknowledge the technical help from Dr. Steven Haworth in the Clement J. Zablocki VA, Milwaukee, WI.

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Authors and Affiliations

  1. Biophotonics Lab, Department of Electrical Engineering, University of Wisconsin Milwaukee, 3200 N Cramer St., Milwaukee, WI, 53211, USA

    Kevin Staniszewski, Reyhaneh Sepehr & Mahsa Ranji

  2. Department of Biomedical Engineering, Marquette University, 1515 West Wisconsin Avenue, Milwaukee, WI, 53233, USA

    Said H. Audi

  3. Research and Development, Clement J. Zablocki VA Medical Center, 5000 W. National Avenue, Milwaukee, WI, 5329, USA

    Elizabeth R. Jacobs

  4. Medical College of Wisconsin, Milwaukee, WI, USA

    Elizabeth R. Jacobs

Authors
  1. Kevin Staniszewski
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  2. Said H. Audi
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  3. Reyhaneh Sepehr
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  4. Elizabeth R. Jacobs
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  5. Mahsa Ranji
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Corresponding author

Correspondence to Mahsa Ranji.

Additional information

Associate Editor Ender A Finol oversaw the review of this article.

Kevin Staniszewski, Said H. Audi, and Reyhaneh Sepehr contributed equally to this work.

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Staniszewski, K., Audi, S.H., Sepehr, R. et al. Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs. Ann Biomed Eng 41, 827–836 (2013). https://doi.org/10.1007/s10439-012-0716-z

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  • DOI: https://doi.org/10.1007/s10439-012-0716-z

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