Volume 13, Issue 1 pp. 41-47

High-Resolution Intravital NADH Fluorescence Microscopy Allows Measurements of Tissue Bioenergetics in Rat Ileal Mucosa

JIM ROSE,

JIM ROSE

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

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CLAUDIO MARTIN,

Corresponding Author

CLAUDIO MARTIN

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Faculty of Medicine, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

Richard Ivey Critical Care Trauma Centre, London Health Sciences Centre, London, Ontario, Canada

Address correspondence to Dr. Claudio Martin, London Health Sciences Centre, 800 Commissioners Road East, London, Ontario N6A 4S2, Canada. E-mail: [email protected]Search for more papers by this author

TAMMY MACDONALD,

TAMMY MACDONALD

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

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CHRIS ELLIS,

CHRIS ELLIS

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

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JIM ROSE,

JIM ROSE

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

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CLAUDIO MARTIN,

Corresponding Author

CLAUDIO MARTIN

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Faculty of Medicine, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

Richard Ivey Critical Care Trauma Centre, London Health Sciences Centre, London, Ontario, Canada

TAMMY MACDONALD,

TAMMY MACDONALD

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

Search for more papers by this author

CHRIS ELLIS,

CHRIS ELLIS

Department of Physiology, University of Western Ontario, London, Ontario, Canada

Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

Centre for Critical Illness Research, London Health Sciences Centre, London, Ontario, Canada

Search for more papers by this author

First published: 26 January 2010

https://doi.org/10.1080/10739680500383472

Citations: 11

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ABSTRACT

Objectives: NADH fluorescence microscopy has been used as an index of the metabolic state of tissue but is associated with various obstacles such as low spatial resolution and quenching effects of blood pigments that prevent reliable monitoring of tissue bioenergetics. The objective of this study was to develop a system to monitor tissue bioenergetics in vivo using NADH fluorescence microscopy in the rat ileal mucosa.

Materials and Methods: Using an inverted microscope with an epifluorescence unit and an intensified charge-coupled device camera, NADH fluorescence images were visualized. Fluorescence intensity was measured of β -NADH solutions at varying concentration (n = 6) and pH (n = 3) and in ex vivo (n = 6) and in vivo (n = 6) preparations of ileal mucosa of Sprague-Dawley rats anesthetized with isoflurane.

Results: Intravital fluorescence microscopy reveals a map of the microcirculation that permits visualization of NADH fluorescence and intercapillary areas. The system was adjusted so a linear relationship between physiological concentrations of β -NADH and fluorescence was achieved (r² = 0.98, p < .0001). Decreasing the pH of the solution had no effect on fluorescence intensity and fluorescence intensity in an anoxic ex vivo ileal segment was similar to that of the in vivo ileum after ischemia. Ischemia also resulted in spatial heterogeneity that was abolished by the addition of a 550-nm LP filter.

Conclusions: With this system, intravital NADH fluorescence microscopy provides the high resolution necessary to reliably monitor tissue bioenergetics in the rat ileal mucosa.

REFERENCES

1 Antonsson, J B, Boyle, C C, Kruithoff, K L, Wang, H L, Sacristan, E, Rothschild, H R, Fink, M P. Validation of tonometric measurement of gut intramural pH during endotoxemia and mesenteric occlusion in pigs. Am J Physiol 1990 259: G519–G523 [PUBMED] [INFOTRIEVE] [CSA].
CAS PubMed Web of Science® Google Scholar
2 Baum, T D, Wang, H, Rothschild, H R, Gang, D L, Fink, M P. Mesenteric oxygen metabolism, ileal mucosal hydrogen ion concentration, and tissue edema after crystalloid or colloid resuscitation in porcine endotoxic shock: comparison of Ringer's lactate and 6% hetastarch. Circ Shock 1990 30: 385–397 [PUBMED] [INFOTRIEVE] [CSA].
CAS PubMed Web of Science® Google Scholar
3 Cicutti, N, Rakusan, K, Downey, H F. Coronary artery occlusion extends perfusion territory boundaries through microvascular collaterals. Basic Res Cardiol 1994 89: 427–437 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1007/BF00788280
CAS PubMed Web of Science® Google Scholar
4 Coremans, J MCC, Ince, C, Bruining, H A, Puppels, G J. (Semi-) Quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart. Biophys J 1997 72: 1849–1860 [PUBMED] [INFOTRIEVE] [CSA].
10.1016/S0006-3495(97)78831-3
CAS PubMed Web of Science® Google Scholar
5 Duboc, D, Abastado, P, Muffat-Joly, M, Perrier, P, Toussaint, M, Marsac, C, Francois, D, Lavergne, T, Pocidalo, J J, Guerin, F, Carpentier, A. Evidence of mitochondrial impairment during cardiac allograft rejection. Transplantation 1990 50: 751–755 [PUBMED] [INFOTRIEVE] [CSA].
CAS PubMed Web of Science® Google Scholar
6 Farquhar, I, Martin, C M, Lam, C, Potter, R, Ellis, C G, Sibbald, W J. Decreased capillary density in vivo in bowel mucosa of rats with normotensive sepsis. J Surg Res 1996 61: 190–196 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1006/jsre.1996.0103
CAS PubMed Web of Science® Google Scholar
7 Fink, M. Cytopathic hypoxia: mitohondrial dysfunction as a potential mechanism contributing to organ failure in sepsis. Acta Anaesthesiol Scand Suppl 1997 110: 87–95 [PUBMED] [INFOTRIEVE] [CSA].
10.1111/j.1399-6576.1997.tb05514.x
CAS PubMed Google Scholar
8 Golub, A S, Popel, A S, Zheng, L, Pittman, R N. Analysis of phosphorescence decay for nonuniform oxygen tension: consequences of finite excitation flash duration. Adv Exp Med Biol 1999 471: 649–659 [PUBMED] [INFOTRIEVE] [CSA].
10.1007/978-1-4615-4717-4_75
CAS PubMed Web of Science® Google Scholar
9 Ince, C, Ashruf, J F, Sanderse, E A, Pierik, E GJM, Coremans, J MCC, Bruining, H A. In vivo NADH and Pd-porphyrin video fluori-/phosphorimetry. Adv Exp Med Biol 1992 317: 267–275 [PUBMED] [INFOTRIEVE] [CSA].
10.1007/978-1-4615-3428-0_29
CAS PubMed Web of Science® Google Scholar
10 Ince, C, Coremans, J MCC, Bruining, H A. In vivo NADH fluorescence. Adv Exp Med Biol 1992 317: 277–296 [PUBMED] [INFOTRIEVE] [CSA].
10.1007/978-1-4615-3428-0_30
CAS PubMed Web of Science® Google Scholar
11 Ince, C, Vink, H, Wieringa, P A, Giezeman, M, Spaan, J AE. Heterogeneous NADH fluorescence during post-anoxic reactive hyperemia in saline perfused rat heart. Adv Exp Med Biol 1990 277: 477–482 [PUBMED] [INFOTRIEVE] [CSA].
10.1007/978-1-4684-8181-5_53
CAS PubMed Web of Science® Google Scholar
12 Jobsis, F, Duffield, J C. Oxidative and glycolytic recovery metabolism in muscle: fluorometric observations on their relative contributions. J Gen Physiol 1967 50: 1009–1047 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1085/jgp.50.4.1009
CAS PubMed Web of Science® Google Scholar
13 Kampp, M, Lundgren, O, Nilsson, N J. Extravascular shunting of oxygen in the small intestine of the cat. Acta Physiol Scand 1968 72: 396–403 [PUBMED] [INFOTRIEVE] [CSA].
10.1111/j.1365-201X.1968.tb10849.x
CAS PubMed Web of Science® Google Scholar
14 Kelly, J J, Rorvick, D A, Richmond, K N, Barlow, C H. Videofluorometer for imaging tissue metabolism. Rev Sci Instrum 1989 60: 3498–3502 [CROSSREF] [CSA].
10.1063/1.1140500
CAS Web of Science® Google Scholar
15 Menger, M D, Vollmar, B. In vivo documentation of an arteriovenous shunt in rat pancreatic acinar tissue. Pancreas 1996 13: 125–129 [PUBMED] [INFOTRIEVE] [CSA].
10.1097/00006676-199608000-00002
CAS PubMed Web of Science® Google Scholar
16 Nuutinen, E M. Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart. Basic Res Cardiol 1984 79: 49–58 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1007/BF01935806
CAS PubMed Web of Science® Google Scholar
17 Rose, J, Pitt-Hyde, M, Martin, C. Cellular dysoxia exists in the villus tips of rats with normotensive sepsis. Crit Care Med 2001 29 12 Suppl A26 (Abstr)[CSA].
Google Scholar
18 Temmesfeld-Wollbruck, B, Mayer, K, Grimminger, F. Assessment of intestinal tissue oxygenation: the canary sings—but what does the twitter tell us?. Intensive Care Med 2000 26: 1025–1027 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1007/s001340051314
CAS PubMed Web of Science® Google Scholar
19 Temmesfled-Wollbruck, B, Szalay, A, Olschewski, H, Grimminger, F, Seeger, W. Advantage of buffered solutions or automated capnometry in air-filled balloons for use in gastric tonometry. Intensive Care Med 1997 23: 423–427 [CROSSREF] [CSA].
10.1007/s001340050351
PubMed Web of Science® Google Scholar
20 Tugtekin, I F, Radermacher, P, Theisen, M, Matejovic, M, Stehr, A, Ploner, F, Matura, K, Ince, C, Georgieff, M, Trager, K. Increased ileal mucosal arterial PCO₂ gap is associated with impaired villus microcirculation in endotoxemic pigs. Intensive Care Med 2001 27: 757–766 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1007/s001340100871
CAS PubMed Web of Science® Google Scholar
21 Vollmar, B, Burkhardt, M, Minor, T, Klauke, H, Menger, M D. Technical report: High-resolution microscopic assessment of hepatic NADH fluorescence for in vivo monitoring of tissue oxygenation during hemorrhagic shock and resuscitation. Microvasc Res 1997 54: 164–173 [PUBMED] [INFOTRIEVE] [CROSSREF] [CSA].
10.1006/mvre.1997.2028
CAS PubMed Web of Science® Google Scholar
22 Wakita, M, Nishimura, G, Tamura, M. Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J Biochem (Tokyo) 1995 18: 1151–1160 [CSA].
10.1093/oxfordjournals.jbchem.a125001
Google Scholar

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Volume13, Issue1

January‐February 2006

Pages 41-47

High-Resolution Intravital NADH Fluorescence Microscopy Allows Measurements of Tissue Bioenergetics in Rat Ileal Mucosa

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High-Resolution Intravital NADH Fluorescence Microscopy Allows Measurements of Tissue Bioenergetics in Rat Ileal Mucosa

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