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Applied Optics

Applied Optics


  • Editor: James C. Wyant
  • Vol. 47, Iss. 22 — Aug. 1, 2008
  • pp: 4164–4176

Simultaneous measurement of changes in light absorption due to the reduction of cytochrome c oxidase and light scattering in rat brains during loss of tissue viability

Satoko Kawauchi, Shunichi Sato, Hidetoshi Ooigawa, Hiroshi Nawashiro, Miya Ishihara, and Makoto Kikuchi  »View Author Affiliations

Applied Optics, Vol. 47, Issue 22, pp. 4164-4176 (2008)

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We performed the simultaneous measurement of intrinsic optical signals (IOSs) related to metabolic activity and cellular and subcellular morphological characteristics, i.e., light scattering for a rat global ischemic brain model made by rapidly removing blood by saline infusion. The signals were measured on the basis of multiwavelength diffuse reflectances in which 605 and 830 nm were used to detect the IOSs that are thought to be dominantly affected by redox changes of heme a a 3 and CuA in cytochrome c oxidase (CcO), respectively. For measuring the scattering signal, the wavelength that was found to be most insensitive to the absorption changes, e.g., 620 nm , was used. The measurements suggested that an increase in the absorption due to reduction of heme a a 3 occurred soon after blood clearance, and this was followed by a large triphasic change in light scattering, during which time a decrease in the absorption due to reduction of CuA occurred. Through the triphasic scattering change, scattering signals increased by 5.2 ± 1.5 % ( n = 5 ), and the increase in light scattering showed significant correlation with both the reflectance intensity changes at 605 and 830 nm . This suggests that morphological changes in cells correlate with reductions of heme a a 3 and CuA. Histological analysis of tissue after the triphasic scattering change showed no alteration in either the nuclei or the cytoskeleton, but electron microscopic observation revealed deformed, enlarged mitochondria and expanded dendrites. These findings suggest that the simultaneous measurement of absorption signals related to the redox changes in the CcO and the scattering signal is useful for monitoring tissue viability in the brain.

© 2008 Optical Society of America

OCIS Codes
(000.1430) General : Biology and medicine
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: November 1, 2007
Revised Manuscript: May 29, 2008
Manuscript Accepted: July 7, 2008
Published: July 30, 2008

Virtual Issues
Vol. 3, Iss. 9 Virtual Journal for Biomedical Optics

Satoko Kawauchi, Shunichi Sato, Hidetoshi Ooigawa, Hiroshi Nawashiro, Miya Ishihara, and Makoto Kikuchi, "Simultaneous measurement of changes in light absorption due to the reduction of cytochrome c oxidase and light scattering in rat brains during loss of tissue viability," Appl. Opt. 47, 4164-4176 (2008)

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  1. D. E. Griffiths and D. C. Wharton, “Studies of the electron transport system. XXXV. Purification and properties of cytochrome oxidase,” J. Biol. Chem. 236, 1850-1856 (1961).
  2. D. C. Wharton and Q. H. Gibson, “Spectrophotometric characterization and function of copper in cytochrome c oxidase,” in Biochemistry of Copper, J. Peisach, ed. (Academic, 1966), pp. 235-244.
  3. B. Chance and G. R. Williams, “A method for the localization of sites for oxidative phosphorylation,” Nature 176, 250-254(1955). [CrossRef]
  4. M. K. Wikstrom, “Energy-linked change in the redox state and absorption spectrum of cytochrome a in situ,” Biochim. Biophys. Acta 283, 385-390 (1972). [CrossRef]
  5. P. Hellwig, S. Grzybek, J. Behr, B. Ludwig, H. Michel, and W. Mantele, “Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes,” Biochemistry 38, 1685-1694 (1999). [CrossRef]
  6. H. Miyake, S. Nioka, A. Zaman, D. S. Smith, and B. Chance, “The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia,” Anal. Biochem. 192, 149-155 (1991). [CrossRef]
  7. C. E. Cooper and R. Springett, “Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy,” Philos. Trans. R. Soc. Lond. B 352, 669-676 (1997). [CrossRef]
  8. A. Matsunaga, Y. Nomura, S. Kuroda, M. Tamura, J. Nishihira, and N. Yoshimura, “Energy-dependent redox state of heme a+a3 and copper of cytochrome oxidase in perfused rat brain in situ,” Am. J. Physiol. 275, C1022-C1030 (1998).
  9. F. Fujii, Y. Nodasaka, G. Nishimura, and M. Tamura, “Anoxia induces matrix shrinkage accompanied by an increase in light scattering in isolated brain mitochondria,” Brain Res. 999, 29-39 (2004). [CrossRef]
  10. G. Nollert, T. Shin'oka, and R. A. Jonas, “Near-infrared spectrophotometry of the brain in cardiovascular surgery,” Thorac. Cardiovasc. Surg. 46, 167-175 (1998).
  11. Y. Kakihana, A. Matsunaga, K. Tobo, S. Isowaki, M. Kawakami, I. Tsuneyoshi, Y. Kanmura, and M. Tamura, “Redox behavior of cytochrome oxidase and neurological prognosis in 66 patients who underwent thoracic aortic surgery,” Eur. J. Cardio-Thorac. Surg. 21, 434-439 (2002). [CrossRef]
  12. M. M. Tisdall, I. Tachtsidis, T. S. Leung, C. E. Elwell, and M. Smith, “Near-infrared spectroscopic quantification of changes in the concentration of oxidized cytochrome c oxidase in the healthy human brain during hypoxemia,” J. Biomed. Opt. 12, 024002 (2007). [CrossRef]
  13. I. Tachtsidis, M. Tisdall, T. S. Leung, C. E. Cooper, D. T. Delpy, M. Smith, and C. E. Elwell, “Investigation of in vivo measurement of cerebral cytochrome-c-oxidase redox changes using near-infrared spectroscopy in patients with orthostatic hypotension,” Physiol. Meas. 28, 199-211 (2007). [CrossRef]
  14. C. E. Cooper, M. Cope, R. Springett, P. N. Amess, J. Penrice, L. Tyszczuk, S. Punwani, R. Ordidge, J. Wyatt, and D. T. Delpy, “Use of mitochondrial inhibitors to demonstrate that cytochrome oxidase near-infrared spectroscopy can measure mitochondrial dysfunction noninvasively in the brain,” J. Cereb. Blood Flow Metab. 19, 27-38 (1999). [CrossRef]
  15. R. Springett, J. Newman, M. Cope, and D. T. Delpy, “Oxygen dependency and precision of cytochrome oxidase signal from full spectral NIRS of the piglet brain,” Am. J. Physiol. Heart Circ. Physiol. 279, H2202-2209 (2000).
  16. M. Tsuji, H. Naruse, J. Volpe, and D. Holtzman, “Reduction of cytochrome aa3 measured by near-infrared spectroscopy predicts cerebral energy loss in hypoxic piglets,” Pediatr. Res. 37, 253-259 (1995).
  17. B. M. Salzberg, A. L. Obaid, and H. Gainer, “Large and rapid changes in light scattering accompany secretion by nerve terminals in the mammalian neurohypophysis,” J. Gen. Physiol. 86, 395-411 (1985). [CrossRef]
  18. R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, “Noninvasive detection of changes in membrane potential in cultured neurons by light scattering,” Proc. Natl. Acad. Sci. USA 88, 9382-9386(1991). [CrossRef]
  19. D. Malonek and A. Grinvald, “Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping,” Science 272, 551-554 (1996). [CrossRef]
  20. E. Gratton, S. Fantini, M. A. Franceschini, G. Gratton, and M. Fabiani, “Measurements of scattering and absorption changes in muscle and brain,” Philos. Trans. R. Soc. Lond. B 352, 727-735 (1997). [CrossRef]
  21. A. Villringer and B. Chance, “Non-invasive optical spectroscopy and imaging of human brain function,” Trends Neurosci. 20, 435-442 (1997). [CrossRef]
  22. B. Chance, Q. Luo, S. Nioka, D. C. Alsop, and J. A. Detre, “Optical investigations of physiology: a study of intrinsic and extrinsic biomedical contrast,” Philos. Trans. R. Soc. Lond. B 352, 707-716 (1997). [CrossRef]
  23. B. Chance, A. Mayevsky, B. Guan, and Y. Zhang, “Hypoxia/ischemia triggers a light scattering event in rat brain,” in Oxygen Transport to Tissue XIX, D. K. Harrison and D. T. Delpy, eds. (Plenum, 1997), pp. 457-467.
  24. T. M. Polischuk, C. R. Jarvis, and R. D. Andrew, “Intrinsic optical signaling denoting neuronal damage in response to acute excitotoxic insult by domoic acid in the hippocampal slice,” Neurobiol. Dis. 4, 423-437 (1998). [CrossRef]
  25. Y. Yamashita, M. Oda, H. Naruse, and M. Tamura, “In vivo measurement of reduced scattering and absorption coefficients of living tissue using time-resolved spectroscopy,” OSA Trends Optics Photonics 2, 387-390 (1996).
  26. M. Kohl, U. Lindauer, U. Dirnagl, and A. Villringer, “Separation of changes in light scattering and chromophore concentrations during cortical spreading depression in rats,” Opt. Lett. 23, 555-557 (1998). [CrossRef]
  27. P. G. Aitken, D. Fayuk, G. G. Somjen, and D. A. Turner, “Use of intrinsic optical signals to monitor physiological changes in brain tissue slices,” Methods Enzymol. 18, 91-103 (1999). [CrossRef]
  28. M. Müller and G. G. Somjen, “Intrinsic optical signals in rat hippocampal slices during hypoxia-induced spreading depression-like depolarization,” J. Neurophysiol. 82, 1818-1831 (1999).
  29. R. D. Andrew, C. R. Jarvis, and A. S. Obeidat, “Potential sources of intrinsic optical signals imaged in live brain slices,” Methods Enzymol. 18, 185-196 (1999). [CrossRef]
  30. C. R. Jarvis, L. Lilge, G. J. Vipond, and R. D. Andrew, “Interpretation of intrinsic optical signals and calcein fluorescence during acute excitotoxic insult in the hippocampal slice,” NeuroImage 10, 357-372 (1999). [CrossRef]
  31. S. Bahar, D. Fayuk, G. G. Somjen, P. G. Aitken, and D. A. Turner, “Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices,” J. Neurophysiol. 84, 311-324 (2000).
  32. M. Haller, S. L. Mironov, and D. W. Richter, “Intrinsic optical signals in respiratory brain stem regions of mice: neurotransmitters, neuromodulators, and metabolic stress,” J. Neurophysiol. 86, 412-421 (2001).
  33. D. Fayuk, P. G. Aitken, G. G. Somjen, and D. A. Turner, “Two different mechanisms underlie reversible, intrinsic optical signals in rat hippocampal slices,” J. Neurophysiol. 87, 1924-1937 (2002).
  34. L. J. Johnson, W. Chung, D. F. Hanley, and N. V. Thakor, “Optical scatter imaging detects mitochondrial swelling in living tissue slices,” NeuroImage 17, 1649-1657 (2002). [CrossRef]
  35. L. Tao, D. Masri, S. Hrabetova, and C. Nicholson, “Light scattering in rat neocortical slices differs during spreading depression and ischemia,” Brain Res. 952, 290-300 (2002). [CrossRef]
  36. A. M. Ba, M. Guiou, N. Pouratian, A. Muthialu, D. E. Rex, A. F. Cannestra, J. W. Chen, and A. W. Toga, “Multiwavelength optical intrinsic signal imaging of cortical spreading depression,” J. Neurophysiol. 88, 2726-2735 (2002). [CrossRef]
  37. T. H. Murphy, P. Li, K. Betts, and R. Liu, “Two-photon imaging of stroke onset in vivo reveals that NMDA-receptor independent ischemic depolarization is the major cause of rapid reversible damage to dendrites and spines,” J. Neurosci. 28, 1756-1772 (2008). [CrossRef]
  38. Y. Hoshi, O. Hazeki, Y. Kakihana, and M. Tamura, “Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy,” J. Appl. Physiol. 83, 1842-1848 (1997).
  39. S. Kawauchi, S. Sato, H. Ooigawa, H. Nawashiro, and M. Kikuchi, “Changes in intrinsic optical signals during loss of tissue viability of brains in rats: effect of brain temperature,” Proc. SPIE 6434, 64341O-1-64341O-4 (2007).
  40. A. J. de Crespigny, J. Rother, C. Beaulieu, M. E. Moseley, and M. Hoehn, “Rapid monitoring of diffusion, DC potential, and blood oxygenation changes during global ischemia. Effects of hypoglycemia, hyperglycemia, and TTX,” Stroke 30, 2212-2222 (1999).
  41. S. Charpak and E. Audinat, “Cardiac arrest in rodents: maximal duration compatible with a recovery of neuronal activity,” Proc. Natl. Acad. Sci. USA 95, 4748-4753 (1998). [CrossRef]
  42. P. van der Zee, “Measurement and modelling of the optical properties of human tissue in the near infrared,” Ph.D. thesis (University College London, 1992), pp. 266-269.
  43. M. Kohl, U. Lindauer, G. Royl, M. Kuhl, L. Gold, A. Villringer, and U. Dirnagl, “Physical model for the spectroscopic analysis of cortical intrinsic optical signals,” Phys. Med. Biol. 45, 3749-3764 (2000). [CrossRef]
  44. P. Lappalainen, R. Aasa, B. G. Malmstrom, and M. Saraste, “Soluble CuA-binding domain from the Paracoccus cytochrome c oxidase,” J. Biol. Chem. 268, 26416-26421 (1993).
  45. J. R. Platt, “Electronic structure and excitation of polyenes and porphyrins,” in Radiation Biology, A. Hollander, ed. (McGraw-Hill, 1956), pp. 71-123.
  46. D. Keilin, “Indophenol oxidase, cytochrome oxidase and cytochrome a3,” in The History of Cell Respiration and Cytochrome, J. Keilin, ed. (Cambridge University, 1966), pp. 224-251.
  47. F. F. Jöbsis, J. H. Keizer, J. C. LaManna, and M. Rosenthal, “Reflectance spectrophotometry of cytochrome aa3in vivo,” J. Appl. Physiol. 43, 858-872 (1977).
  48. N. R. Kreisman, T. J. Sick, J. C. LaManna, and M. Rosenthal, “Local tissue oxygen tension-cytochrome a, a3 redox relationships in rat cerebral cortex in vivo,” Brain Res. 218, 161-174(1981). [CrossRef]
  49. C. A. Piantadosi and F. F. Jobsis-Vandervliet, “Spectrophotometry of cerebral cytochrome a, a3 in bloodless rats,” Brain Res. 305, 89-94 (1984). [CrossRef]
  50. U. Heinrich, J. Hoffmann, and D. W. Lubbers, “Quantitative evaluation of optical reflection spectra of blood-free perfused guinea pig brain using a nonlinear multicomponent analysis,” Pflugers Arch. 409, 152-157 (1987). [CrossRef]
  51. M. Ferrari, D. F. Hanley, D. A. Wilson, and R. J. Traystman, “Redox changes in cat brain cytochrome-c oxidase after blood-fluorocarbon exchange,” Am. J. Physiol. 258, H1706-H1713(1990).
  52. S. Wray, M. Cope, D. T. Delpy, J. S. Wyatt, and E. O. Reynolds, “Characterization of the near infrared absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of cerebral oxygenation,” Biochim. Biophys. Acta 933, 184-192 (1988). [CrossRef]
  53. I. Belevich, D. A. Bloch, N. Belevich, M. Wikstrom, and M. I. Verkhovsky, “Exploring the proton pump mechanism of cytochrome c oxidase in real time,” Proc. Natl. Acad. Sci. USA 104, 2685-2690 (2007). [CrossRef]
  54. K. Kitagawa, M. Matsumoto, M. Niinobe, K. Mikoshiba, R. Hata, H. Ueda, N. Handa, R. Fukunaga, Y. Isaka, K. Kimura, and T. Kamada, “Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage-immunohistochemical investigation of dendritic damage,” Neuroscience 31, 401-411 (1989). [CrossRef]
  55. D. L. Nelson and M. M. Cox, “Oxidative phosphorylation and photophosphorylation,” in Lehninger Principles of Biochemistry, D. L. Nelson and M. M. Cox, eds. (Worth Publishers, 2000), pp. 659-673.
  56. K. A. Hossmann, “Viability thresholds and the penumbra of focal ischemia,” Ann. Neurol. 36, 557-565 (1994). [CrossRef]
  57. A. J. Hansen, “Effect of anoxia on ion distribution in the brain,” Physiol. Rev. 65, 101-148 (1985).
  58. N. J. Allen, R. Karadottir, and D. Attwell, “A preferential role for glycolysis in preventing the anoxic depolarization of rat hippocampal area CA1 pyramidal cells,” J. Neurosci. 25, 848-859 (2005). [CrossRef]
  59. R. D. Andrew and B. A. MacVicar, “Imaging cell volume changes and neuronal excitation in the hippocampal slice,” Neuroscience 62, 371-383 (1994). [CrossRef]
  60. A. J. Hansen and C. E. Olsen, “Brain extracellular space during spreading depression and ischemia,” Acta Physiol. Scand. 108, 355-365 (1980).
  61. T. Takano, G. F. Tian, W. Peng, N. Lou, D. Lovatt, A. J. Hansen, K. A. Kasischke, and M. Nedergaard, “Cortical spreading depression causes and coincides with tissue hypoxia,” Nat. Neurosci. 10, 754-762 (2007). [CrossRef]
  62. N. N. Boustany, R. Drezek, and N. V. Thakor, “Calcium-induced alterations in mitochondrial morphology quantified in situ with optical scatter imaging,” Biophys. J. 83, 1691-1700 (2002).
  63. X. Wang, B. W. Pogue, S. Jiang, X. Song, K. D. Paulsen, C. Kogel, S. P. Poplack, and W. A. Wells, “Approximation of Mie scattering parameters in near-infrared tomography of normal breast tissue in vivo,” J. Biomed. Opt. 10, 051704(2005). [CrossRef]
  64. M. Bartek, X. Wang, W. Wells, K. D. Paulsen, and B. W. Pogue, “Estimation of subcellular particle size histograms with electron microscopy for prediction of optical scattering in breast tissue,” J. Biomed. Opt. 11, 064007 (2006). [CrossRef]
  65. C. E. Cooper, S. J. Matcher, J. S. Wyatt, M. Cope, G. C. Brown, E. M. Nemoto, and D. T. Delpy, “Near-infrared spectroscopy of the brain: relevance to cytochrome oxidase bioenergetics,” Biochem. Soc. Trans. 22, 974-980 (1994).
  66. B. K. Siesjo, “Brain metabolism and anaesthesia,” Acta Anaesthesiologica Scandinavica Supplement 70, 56-59 (1978).
  67. I. Belevich, M. I. Verkhovsky, and M. Wikstrom, “Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase,” Nature 440, 829-832 (2006). [CrossRef]
  68. P. van der Zee, M. Essenpreis, and D. T. Delpy, “Optical properties of brain tissue,” Proc. SPIE 1888, 454-465 (1993). [CrossRef]

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