Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O2•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO 2 maps with minimal errors. A methodology for noninvasive pO 2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T* 2 , which is inversely proportional to pO 2 . Factors affecting T* 2 in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO 2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of ϳ1.0 mm and a temporal resolution of ϳ9 s for 2D pO 2 mapping, with a reliable oxygen resolution of ϳ1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.

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Enhancement of image intensity, using the T 1 -weighted spoiled gradient-echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R 1 , R 2 , and M 0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T 1 . Magn Reson Med 56:240 -246,

Electron paramagnetic resonance (EPR) imaging in the continuous wave (CW) and time-domain modes, as well as Overhauser-enhanced magnetic resonance imaging in vivo is described. The review is based mainly on the CW and time-domain EPR instrumentation at 300 MHz developed in our laboratory, and the relative merits of these methods for functional in vivo imaging of small animals to assess hypoxia and tissue redox status are described. Overhauser imaging of small animals at magnetic fields in the range 10-15 mT that is being carried out in our laboratory for tumor imaging and the evaluation of tumor hypoxia based on quantitative evaluation of Overhauser enhancement is also described. Alternate approaches to spectral-spatial imaging using the transverse decay constants to infer in situ line widths and hence in vivo pO 2 using CW and time-domain EPR imaging are also discussed. The nature of the spin probes used, the quality of the images obtained in all the three methods, the achievable resolution, limitations and possible future directions in small animal functional imaging with these modalities are summarized.

The absolute partial pressure of oxygen (pO 2 ) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1-30 days after implantation with or without control of core body temperature. The pO 2 values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO 2 level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO 2 values showed a strong dependence on the core body temperature of the mice. The pO 2 values were responsive to carbogen (95% O 2 , 5% CO 2 ) breathing even 44 -58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long-term non-invasive assessment of pO 2 levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO 2 level than muscle in female C3H mice.

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and non-invasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP is reduced three to eleven times slower (depending on the tissue) than Tempol in vivo, and that maximum tissue concentration varies substantially between tissues (0.6 mM -7.2 mM.) For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance (EPR) spectroscopy, it was shown that the nitroxide reduction rate depends only weakly on cellular pO 2 in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO 2 is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.

A novel approach to measure the time course of paramagnetic spin probe concentration in the circulating blood of a living mouse using X-band (9.4 GHz) electron paramagnetic resonance spectrometer is described. Using this technique, the pharmacokinetics of several nitroxyl spin probes was examined. The decay profiles were also independently simulated using pharmacokinetic properties as well as redox-mediated factors responsible in converting the nitroxyl radicals to the corresponding hydroxylamines. Finally, suitability of nitroxyl radicals as the probes of in vivo redox status and for radioprotection was described. The studies indicate that the six-member piperidine nitroxyls are suitable for estimating redox status in the circulation, whereas the five-member pyrrolidine nitroxyl radicals are suited for tissue redox status determination. For selective protection against radiation of normal tissues rather than cancer/tumor, efficient reoxidation of the hydroxylamine in normal tissue is preferable. Simulation results showed that for carbamoyl-PROXYL, only administration of the radical form might give radioprotection and not the hydroxylamine. However, the hydroxylamine form of TEMPOL, i.e., TEMPOL-H, may give similar radioprotection as the radical form due to efficient reoxidation in vivo.Nitroxyl radicals have been widely used as spin probes for low-frequency in vivo EPR experiments to estimate the biological redox status in living experimental animals (Berliner and Wan, 1989;Ilangovan et al., 2002;Kuppusamy et al., 2002;Yamada et al., 2002;Kasazaki et al., 2003). When a nitroxyl spin probe is administered to a living animal, in vivo EPR signal intensities of the probe show characteristic signal intensity profiles as a function of time in the animal, depending on the organ investigated. Generally, in vivo EPR signal decay rates are obtained based on a suitable region of its decay curve to estimate redox status in the animal .Several nitroxyl radicals have been used in studies that exhibit EPR signal decay profiles, depending on the spin probe used. The decay constant of a spin probe depends on the route of administration (i.e., intravenous, intraperitoneal) and methodology of the analysis (i.e., one-compartment model, two-compartment model) (Kocherginsky and Swartz, 1995). Moreover, species, strains, and gender of the experimental animal was found to affect the EPR signal decay profiles (Kocherginsky and Swartz, 1995;Matsumoto et al., 2000).The following general observations can be summarized from earlier studies. The in vivo EPR signal decay rates of the spin probes are enhanced by reactive oxygen species (Utsumi et al., 1993;Sano et al., 1998;Phumala et al., 1999;Han et al., 2001), such as hydroxyl radical and superoxide, which reduce nitroxyl radical in presence of H atom donor, such as NAD(P)H or glutathione (Samuni et al., 1988;Krishna et al., 1992;Samuni et al., 2002;Takeshita et al., 2002). In contrast, the decay rates are decreased due to reoxidation of the hydroxylamine to the nitroxyl radical un...