24 Vikram, Rivera, and Kuppusamy
Room Air-Breathing Carbogen-Breathing
Intensity -> Oxygen (mmHg)
0 20
7.8 mm 10
A B C
Probe Distribution
Fig. 1.10. EPR images of LiPc and O2in the tumor. (a) Distribution of the probe in a RIF-1 tumor. (b) Distribution of O2concentration in the same tumor under room air-breathing conditions. (c) Distribution of O2 concentration in the same tumor under carbogen-breathing conditions. The tumor size at the time of measurement was 8× 10 × 11 mm3 (Reprinted with permission from Ilangovan et al. (7)).
(high specificity), no toxicity, and is not affected by the tis-sue environment. The probes have reasonable half-lives and ade-quate distribution within the tissue. Qualitative as well as quanti-tative pO2 readings can be obtained using this technique. The most important advantage is that repeated and noninvasive measurements of tissue oxygenation over time are possible. How-ever, this is limited by the duration of probe retention in the tissue (65).
Data acquisition time is a major issue in EPR imaging of O2. A reasonable 3D spectral–spatial image takes about 30 min to acquire (10). Soluble probes are used for imaging, which have low sensitivity and specificity when compared to particulate probes.
However, improvements in image reconstruction algorithms have considerably reduced data acquisition time for spatial imaging and these will be extended to spectral–spatial imaging in the near future (66, 67). In biological tissues, the penetration depth of the microwave energy is limited to a few millimeters at 1.2 GHz (L-band), but is higher at lower frequencies.
Imaging of Free Radicals In Vivo 25
Acknowledgments
The EPR imaging results illustrated in the review were com-posed from several publications from our laboratory. We grate-fully acknowledge the publishers of the respective journals for permitting us to reproduce the figures used in this article. The work was supported by the NIH grants CA102264, EB004031, and EB005004.
References
1. Ansari, K.N. (1997) The free radicals – the hidden culprits – an update. Indian J. Med.
Sci. 51, 319–336.
2. Kuppusamy, P. (2004) EPR spectroscopy in biology and medicine. Antioxid. Redox Sig-nal 6, 583–585.
3. Zavoisky, E. (1945) Spin-magnetic resonance in paramagnetics. J. Phys. E. 9, 245–249.
4. Halpern, H.J., Spencer, D.P., Polen, J.V., Bowman, M.K., Nelson, A.C., Dowey, E.M., and Teicher, B.A. (1989) Imaging radiofre-quency electron spin resonance spectrome-ter with high resolution and sensitivity for in vivo measurements. Rev. Sci. Instrum. 60, 1040–1050.
5. He, G., Samouilov, A., Kuppusamy, P., and Zweier, J.L. (2001) In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin. J. Magn. Reson. 148, 155–164.
6. Zweier, J.L. and Kuppusamy, P. (1988) Elec-tron paramagnetic resonance measurements of free radicals in the intact beating heart:
A technique for detection and characteri-zation of free radicals in whole biological tissues. Proc. Natl. Acad. Sci. U.S.A. 85, 5703–5707.
7. Ilangovan, G., Bratasz, A., Li, H., Schmal-brock, P., Zweier, J.L., and Kuppusamy, P.
(2004) In vivo measurement and imaging of tumor oxygenation using coembedded para-magnetic particulates. Magn. Reson. Med. 52, 650–657.
8. Kuppusamy, P., Chzhan, M., Wang, P., and Zweier, J.L. (1996) Three-dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distri-bution during the cardiac contractile cycle.
Magn. Reson. Med. 35, 323–328.
9. Kuppusamy, P., Shankar, R.A., Roubaud, V.M., and Zweier, J.L. (2001) Whole body detection and imaging of nitric oxide gen-eration in mice following cardiopulmonary arrest: Detection of intrinsic nitrosoheme complexes. Magn. Reson. Med. 45, 700–707.
10. Kuppusamy, P., Shankar, R.A., and Zweier, J.L. (1998) In vivo measurement of arte-rial and venous oxygenation in the rat using 3D spectral-spatial electron paramag-netic resonance imaging. Phys. Med. Biol. 43, 1837–1844.
11. Kuppusamy, P., Wang, P., Zweier, J.L., Krishna, M.C., Mitchell, J.B., Ma, L., Trim-ble, C.E., and Hsia, C.J. (1996) Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin. Bio-chemistry 35, 7051–7057.
12. Woods, R.K., Hyslop, W.B., Maar, R.B., and Lauterbur, P.C. (1991) Image recon-struction. In: EPR Imaging and In vivo EPR (G.R. Eaton, S.S. Eaton, and K.
Ohno, Eds.), CRC Press, Boca Raton, FL, pp. 91–117.
13. Kuppusamy, P., Wang, P., and Zweier, J.L.
(1995) Three-dimensional spatial EPR imag-ing of the rat heart. Magn. Reson. Med. 34, 99–105.
14. Deng, Y., He, G., Kuppusamy, P., and Zweier, J.L. (2003) Deconvolution algo-rithm based on automatic cutoff frequency selection for EPR imaging. Magn. Reson.
Med. 50, 444–448.
15. Kuppusamy, P. and Zweier, J.L. (1996) A forward-subtraction procedure for removing hyperfine artifacts in electron paramagnetic resonance imaging. Magn. Reson. Med. 35, 316–322.
16. Kuppusamy, P. and Zweier, J.L. (1996) Hyperfine artifacts in electron paramagnetic resonance imaging. Res. Chem. Intermed. 22, 593–604.
17. Berliner, J.L. and Fujii, H. (1985) Magnetic resonance imaging of biological specimens by electron paramagnetic resonance of nitroxide spin labels. Science 227, 517–519.
18. Berliner, L.J. (1992) Applications of EPR imaging to materials, agriculture and medicine. In: Magnetic Resonance Microscopy:
Methods and Applications in Material Science, Agriculture and Biomedicine (B. Blumich
26 Vikram, Rivera, and Kuppusamy
and W. Kuhn, Eds.), VCH Publishers, Wein-heim, pp. 151–163.
19. Eaton, G.R., Eaton, S.S., and Ohno, K.
(1991) EPR Imaging and In Vivo EPR, CRC Press, Boca Raton, FL.
20. Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D.J., Giannella, E., and Zweier, J.L. (1994) Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: A technique for imaging tis-sue metabolism and oxygenation. Proc. Natl.
Acad. Sci. U.S.A. 91, 3388–3392.
21. Kuppusamy, P., Chzhan, M., Wang, P., and Zweier, J.L. (1996) Three-dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distri-bution during the cardiac contractile cycle.
Magn. Reson. Med. 35, 323–328.
22. Kuppusamy, P., Chzhan, M., and Zweier, J.L. (1995) Development and optimization of three-dimensional spatial EPR imaging for biological organs and tissues. J. Magn. Reson.
B 106, 122–130.
23. Kuppusamy, P., Chzhan, M., Samouilov, A., Wang, P., and Zweier, J.L. (1995) Map-ping the spin-density and lineshape distri-bution of free radicals using 4D spectral-spatial EPR imaging. J. Magn. Reson. B 107, 116–125.
24. Froncisz, W. and Hyde, J.S. (1982) The loop gap resonator, a new microwave lumped cir-cuit ESR sample structure. J. Magn. Reson.
47, 515–521.
25. Sotgiu, A. (1985) Resonator design for in vivo EPR spectroscopy. J. Magn. Reson. 65, 206–214.
26. Chzhan, M., Kuppusamy, P., and Zweier, J.L.
(1995) Development of an electronically tun-able L-band resonator for EPR spectroscopy and imaging of biological samples. J. Magn.
Reson. B 108, 67–72.
27. Chzhan, M., Shteynbuk, M., Kuppusamy, P., and Zweier, J.L. (1993) An optimized L-Band resonator for EPR imaging of biolog-ical samples. J. Magn. Reson. A105, 49–53.
28. Chzhan, M., Kuppusamy, P., Samouilov, A., He, G., and Zweier, J.L. (1999) A tunable reentrant resonator with transverse orientation of electric field for in vivo EPR spectroscopy. J. Magn. Reson. 137, 373–378.
29. Villamena, F.A. and Zweier, J.L. (2004) Detection of reactive oxygen and nitrogen species by EPR spin trapping. Antioxid.
Redox. Signal 6, 619–629.
30. Forman, M.B., Virmani, R., and Puett, D.W.
(1990) Mechanisms and therapy of myocar-dial reperfusion injury. Circulation 81, IV-69–IV-78.
31. Manning, A.S., Coltart, J.D., and Hearse, D.J. (1984) Ischemia and reperfusion-induced arrhythmias in the rat – effect of xanthine oxidase inhibition with allopurinol.
Circ. Res. 55, 545–548.
32. Pogwizd, S.M. and Corr, P.B. (1986) Mech-anisms of arrhythmogenesis during myocar-dial ischemia and reperfusion: A perspective of our current understanding. J. Mol. Cell.
Cardiol. 18(suppl 4), 43–47.
33. Yamada, M., Hearse, D.J., and Curtis, M.J.
(1990) Reperfusion and readmission of oxy-gen. Circ. Res. 67, 1211–1224.
34. Kukreja, R.C., Kearns, A.A., Zweier, J.L., Kuppusamy, P., and Hess, M.L. (1991) Sin-glet oxygen interaction with Ca2+-ATPase of cardiac sarcoplasmic reticulum. Circ. Res. 69, 1003–1014.
35. Halliwell, B., Gutteridge, J.M.C., and Cross, C.E. (1992) Free radicals, antioxidants, and human disease: Where are we now? J. Lab.
Clin. Med. 119, 598–620.
36. Esterbauer, H., Wag, G., and Puhl, H.
(1993) Lipid peroxidation and its role in atherosclerosis. Br. Med. Bull. 49, 566–576.
37. Zweier, J.L., Kuppusamy, P., Williams, R., Rayburn, B.K., Smith, D., Weisfeldt, M.L., and Flaherty, J.T. (1989) Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J.
Biol. Chem. 264, 18890–18895.
38. Hearse, D.J. and Tosaki, A. (1988) Free rad-icals and calcium: Simultaneous interacting triggers as determinants of vulnerability to reperfusion-induced arrhythmias in the rat heart. J. Mol. Cell Cardiol. 20, 213–223.
39. Horton, J.W. and White, D.J. (1995) Role of xanthine oxidase and leukocytes in postburn cardiac dysfunction. J. Am. Coll. Surg. 181, 129–137.
40. Tosaki, A., Blasig, I.E., Pali, T., and Ebert, B. (1990) Heart protection and radical trap-ping by DMPO during reperfusion in iso-lated working rat hearts. Free Radic. Biol.
Med. 8, 363–372.
41. Zweier, J.L., Flaherty, J.T., and Weisfeldt, M.L. (1987) Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc. Natl. Acad. Sci.
U.S.A. 84, 1404–1407.
42. Kuppusamy, P., Wang, P., Samouilov, A., and Zweier, J.L. (1996) Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imag-ing. Magn. Reson. Med. 36, 212–218.
43. Kuppusamy, P., Ohnishi, S.T., Numagami, Y., Ohnishi, T., and Zweier, J.L. (1995) Three-dimensional imaging of nitric oxide production in the rat brain subjected to
Imaging of Free Radicals In Vivo 27
ischemia-hypoxia. J. Cereb. Blood Flow Metab.
15, 899–903.
44. Henry, Y. and Guissani, A. (1994) Nitric oxide: A biological effector. Detection using electron paramagnetic resonance. Transfus.
Clin. Biol. 1, 157–164.
45. Berliner, L.J. and Fujii, H. (2004) In vivo spin trapping of nitric oxide. Antioxid. Redox Signal 6, 649–656.
46. Mordvintcev, P., Mulsch, A., Busse, R., and Vanin, A. (1991) On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spec-troscopy. Anal Biochem. 199, 142–146.
47. Lai, C.S. and Komarov, A.M. (1994) Spin trapping of nitric oxide produced in vivo in septic-shock mice. FEBS Lett. 345, 120–124.
48. Zweier, J.L., Wang, P., Samouilov, A., and Kuppusamy, P. (1995) Enzyme-independent formation of nitric oxide in biological tissues.
Nat. Med. 1, 804–809.
49. Zweier, J.L., Wang, P., and Kuppusamy, P.
(1995) Direct measurement of nitric oxide generation in the ischemic heart using elec-tron paramagnetic resonance spectroscopy. J Biol. Chem. 270, 304–307.
50. Schafer, W.M., Nowak, B., Kaiser, H.J., Block, S., Koch, K.C., vom Dahl, J., and Bull, U. (2001) An approach for com-parative quantification of myocardial blood flow (O-15-H2O-PET), perfusion (Tc-99m-tetrofosmin-SPECT) and metabolism (F-18-FDG-PET). Nuklearmedizin 40, 164–171.
51. Ilangovan, G., Li, H., Zweier, J.L., Krishna, M.C., Mitchell, J.B., and Kuppusamy, P.
(2002) In vivo measurement of regional oxy-genation and imaging of redox status in RIF-1 murine tumor: Effect of carbogen-breathing. Magn. Reson. Med. 48, 723–730.
52. Kuppusamy, P., Li, H., Ilangovan, G., Car-dounel, A.J., Zweier, J.L., Yamada, K., Krishna, M.C., and Mitchell, J.B. (2002) Noninvasive imaging of tumor redox status and its modification by tissue glutathione lev-els. Cancer Res. 62, 307–312.
53. Yamada, K.I., Kuppusamy, P., English, S., Yoo, J., Irie, A., Subramanian, S., Mitchell, J.B., and Krishna, M.C. (2002) Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging. Acta Radiol. 43, 433–540.
54. Krishna, M.C., Subramanian, S., Kuppusamy, P., and Mitchell, J.B. (2001) Magnetic reso-nance imaging for in vivo assessment of tissue oxygen concentration. Semin. Radiat. Oncol.
11, 58–69.
55. Mitchell, J.B., Russo, A., Kuppusamy, P., and Krishna, M.C. (2000) Radiation,
radi-cals, and images. Ann N.Y. Acad. Sci. 899, 28–43.
56. Krishna, M.C., Grahame, D.A., Samuni, A., Mitchell, J.B., and Russo, A. (1992) Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of super-oxide. Proc. Natl. Acad. Sci. U.S.A. 89, 5537–5541.
57. Swartz, H.M. (1990) Principles of the metabolism of nitroxides and their implica-tions for spin trapping. Free Radic. Res. Com-mun. 9, 399–405.
58. Chen, K., Glockner, J.F., Morse, P.D., 2nd, and Swartz, H.M. (1989) Effects of oxygen on the metabolism of nitroxide spin labels in cells. Biochemistry 28, 2496–2501.
59. Kuppusamy, P. and Krishna, M.C. (2002) EPR imaging of issue redox status. Curr. Top-ics Biophys. 26, 29–34.
60. Griffith, O.W. (1982) Mechanism of action, metabolism, and toxicity of buthionine sul-foximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol.
Chem. 257, 13704–13712.
61. Yu, N.Y. and Brown, J.M. (1984) Depletion of glutathione in vivo as a method of improv-ing the therapeutic ratio of misonidazole and SR 2508. Int. J. Radiat. Oncol. Biol. Phys. 10, 1265–1269.
62. He, G., Samouilov, A., Kuppusamy, P., and Zweier, J.L. (2001) In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin. J. Magn. Reson. 148, 155–164.
63. Swartz, H.M. (2004) Using EPR to mea-sure a critical but often unmeamea-sured compo-nent of oxidative damage: Oxygen. Antioxid.
Redox Signal 6, 677–686.
64. Ilangovan, G., Zweier, J.L., and Kuppusamy, P. (2004) Mechanism of oxygen-induced EPR line broadening in lithium phthalocya-nine microcrystals. J. Magn. Reson. 170, 42–
48.
65. Gallez, B., Baudelet, C., and Jordan, B.F.
(2004) Assessment of tumor oxygenation by electron paramagnetic resonance: Prin-ciples and applications. NMR Biomed. 17, 240–262.
66. Ahmad, R., Clymer, B., Deng, Y., He, G., Vikram, D., Kuppusamy, P., and Zweier, J.L. (2006) Optimization of data acquisi-tion for EPR imaging. J. Magn. Reson. 179, 263–272.
67. Deng, Y., He, G., Petryakov, S., Kup-pusamy, P., and Zweier, J.L. (2004) Fast EPR imaging at 300 MHz using spinning mag-netic field gradients. J. Magn. Reson. 168, 220–227.