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Purpose: To develop a novel resonator for high-quality fast scan electron paramagnetic resonance (EPR) and EPR/NMR co-imaging of the head and brain of mice at 1.25 GHz., Methods: Resonator dimensions were scaled to fit the mouse head with maximum filling factor. A single-loop 6-gap resonator of 20 mm diameter and 20 mm length was constructed. High resonator stability was achieved utilizing a fixed position double coupling loop. Symmetrical mutually inverted connections rendered it insensitive to field modulation and fast scan. Coupling adjustment was provided by a parallel-connected variable capacitor located at the feeding line at λ/4 distance. To minimize radiation loss, the shield around the resonator was supplemented with a planar conductive disc that focuses return magnetic flux., Results: Coupling of the resonator loaded with the mouse head was efficient and easy. This resonator enabled high-quality in vivo 3D EPR imaging of the mouse head following intravenous infusion of nitroxide probes. With this resonator and rapid scan EPR system, 4 ms scans were acquired in forward and reverse directions so that images with 2-scan 3,136 projections were acquired in 25 s. Head images were achieved with resolutions of 0.4 mm, enabling visualization of probe localization and uptake across the blood-brain barrier., Conclusions: This resonator design provides good sensitivity, high stability, and B 1 field homogeneity for in vivo fast scan EPR of the mouse head and brain, enabling faster measurements and higher resolution imaging of probe uptake, localization, and metabolism than previously possible., (© 2021 International Society for Magnetic Resonance in Medicine.)

Purpose: In continuous wave EPR imaging, the acquisition of high-quality images was previously limited by the requisite long acquisition times of each image projection that was typically greater than 1 second. To accelerate the process of image acquisition facilitating greater numbers of projections and higher image resolution, instrumentation was developed to greatly accelerate the magnetic field scan that is used to obtain each EPR image projection., Methods: A low-inductance solenoidal coil for field scanning was used along with a spherical solenoid air core magnet, and scans were driven by triangular symmetric waves, allowing forward and reverse spectrum acquisition as rapid as 3.8 ms. The uniform distribution of projections was used to optimize the contribution of projections for 3D image reconstruction., Results: Using this fast-scan EPR system, high-quality EPR images of phantoms and perfused rat hearts were performed using trityl or nanoparticulate LiNcBuO (lithium octa-n-butoxy-substituted naphthalocyanine) probes with fast-scan EPR imaging at L-band, achieving spatial resolutions of up to 250 micrometers in 1 minute., Conclusion: Fast-scan EPR imaging can greatly facilitate the efficient and precise mapping of the spatial distribution of free radical and other paramagnetic probes in living systems., (© 2019 International Society for Magnetic Resonance in Medicine.)

Purpose: To improve image quality and reduce data requirements for spatial electron paramagnetic resonance imaging (EPRI) by developing a novel reconstruction approach using compressed sensing (CS)., Methods: EPRI is posed as an optimization problem, which is solved using regularized least-squares with sparsity promoting penalty terms, consisting of the l1 norms of the image itself and the total variation of the image. Pseudo-random sampling was employed to facilitate recovery of the sparse signal. The reconstruction was compared with the traditional filtered back-projection reconstruction for simulations, phantoms, isolated rat hearts, and mouse gastrointestinal (GI) tracts labeled with paramagnetic probes., Results: A combination of pseudo-random sampling and CS was able to generate high-fidelity EPR images at high acceleration rates. For three-dimensional (3D) phantom imaging, CS-based EPRI showed little visual degradation at nine-fold acceleration. In rat heart datasets, CS-based EPRI produced high quality images with eight-fold acceleration. A high resolution mouse GI tract reconstruction demonstrated a visual improvement in spatial resolution and a doubling in signal-to-noise ratio (SNR)., Conclusion: A novel 3D EPRI reconstruction using compressed sensing was developed and offers superior SNR and reduced artifacts from highly undersampled data., (Copyright © 2013 Wiley Periodicals, Inc.)

Background and Purpose: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC)., Experimental Approach: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured., Key Results: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO., Conclusion and Implications: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases., (© 2014 The British Pharmacological Society.)

Purpose: To improve the quality and speed of electron paramagnetic resonance imaging (EPRI) acquisition by combining a uniform sampling distribution with spinning gradient acquisition., Theory and Methods: A uniform sampling distribution was derived for spinning gradient EPRI acquisition (uniform spinning sampling, USS) and compared to the existing (equilinear spinning sampling, ESS) acquisition strategy. Novel corrections were introduced to reduce artifacts in experimental data., Results: Simulations demonstrated that USS puts an equal number of projections near each axis whereas ESS puts excessive projections at one axis, wasting acquisition time. Artifact corrections added to the magnetic gradient waveforms reduced noise and correlation between projections. USS images had higher SNR (85.9 ± 0.8 vs. 56.2 ± 0.8) and lower mean-squared error than ESS images. The quality of the USS images did not vary with the magnetic gradient orientation, in contrast to ESS images. The quality of rat heart images was improved using USS compared to that with ESS or traditional fast-scan acquisitions., Conclusion: A novel EPRI acquisition which combines spinning gradient acquisition with a uniform sampling distribution was developed. This USS spinning gradient acquisition offers superior SNR and reduced artifacts compared to prior methods enabling potential improvements in speed and quality of EPR imaging in biological applications., (Copyright © 2013 Wiley Periodicals, Inc.)

In vivo or ex vivo electron paramagnetic resonance imaging (EPRI) is a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. However, applications of EPRI have been limited by long projection acquisition times and the consequent fact that rapid gated EPRI was not possible. Hence in vivo EPRI typically provided only time-averaged information. In order to achieve direct gated EPRI, a fast EPR acquisition scheme was developed to decrease EPR projection acquisition time down to 10-20 ms, along with corresponding software and instrumentation to achieve fast gated EPRI of the isolated beating heart with submillimeter spatial resolution in as little as 2-3 min. Reconstructed images display temporal and spatial variations of the free-radical distribution, anatomical structure, and contractile function within the rat heart during the cardiac cycle., (Copyright © 2012 Wiley Periodicals, Inc.)

A new low-field electron paramagnetic resonance approach for noninvasive measurements of myocardial oxygen tension and tissue acidity was developed. The approach was applied to monitor myocardial pO(2) and pH in a model of global no-flow ischemia (30 min) and reperfusion in isolated perfused rat hearts. The myocardial oxygen measurements were performed using deuterated Finland trityl radical probe. A rapid decrease in myocardial pO(2) from 160 mmHg to about 2 ± 1 mmHg was observed within the first minute of ischemia followed by incomplete restoration of pO(2) to 50 mmHg during 30 min of reperfusion. The lower oxygen concentration after ischemia was attributed to the 50% reduction in coronary flow after ischemia as a consequence of myocardial ischemia and reperfusion damage. Myocardial pH measurements using a specially designed imidazoline pH-sensitive nitroxide showed severe myocardial acidification to pH 6.25 during 30 min of ischemia. Preconditioning of the hearts with two 5-min periods of ischemia significantly reduced the acidification of myocardial tissue during sustained ischemia. Noninvasive electron paramagnetic resonance monitoring of myocardial oxygenation and pH may provide important insights into the mechanisms of ischemia and reperfusion injury and a background for development of new therapeutic approaches., (Copyright © 2011 Wiley Periodicals, Inc.)

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors., (Copyright © 2011 Wiley Periodicals, Inc.)

Electron paramagnetic resonance imaging (EPRI) is a powerful technique that enables spatial mapping of free radicals or other paramagnetic compounds; however, it does not in itself provide anatomic visualization of the body. Proton magnetic resonance imaging (MRI) is well suited to provide anatomical visualization. A hybrid EPR/NMR coimaging instrument was constructed that utilizes the complementary capabilities of both techniques, superimposing EPR and proton-MR images to provide the distribution of paramagnetic species in the body. A common magnet and field gradient system is utilized along with a dual EPR and proton-NMR resonator assembly, enabling coimaging without the need to move the sample. EPRI is performed at approximately 1.2 GHz/ approximately 40 mT and proton MRI is performed at 16.18 MHz/ approximately 380 mT; hence the method is suitable for whole-body coimaging of living mice. The gradient system used is calibrated and controlled in such a manner that the spatial geometry of the two acquired images is matched, enabling their superposition without additional postprocessing or marker registration. The performance of the system was tested in a series of phantoms and in vivo applications by mapping the location of a paramagnetic probe in the gastrointestinal (GI) tract of mice. This hybrid EPR/NMR coimaging instrument enables imaging of paramagnetic molecules along with their anatomic localization in the body., ((c) 2007 Wiley-Liss, Inc.)

Fast and reliable data acquisition is a major requirement for successful and useful biological electron paramagnetic resonance imaging (EPRI) experiments. Even a technologically advanced and professionally supervised EPRI system can exhibit instabilities initiated by perturbations such as animal motion, microphonics, and temperature changes. As a result, part of an acquired data set may become corrupted with excessive noise and distortions, which in turn may degrade the quality of the reconstructed image. In this work an automated scheme to monitor the system performance and stability over the course of an experiment is demonstrated. This method ensures that the quality of the acquired data is maintained during the experiment. For this purpose, four parameters including noise content and integration of each acquired projection are quantified and measured against those of the zero-gradient (ZG) projection, which is set as a quality benchmark. Projections with parameter values that differ substantially from the expected values are identified as damaged and consequently are reacquired. Therefore, the proposed technique not only effectively monitors the quality of acquisition, it also saves a substantial amount of acquisition time because it eliminates the necessity of repeating the entire experiment in cases in which only a small fraction of the data are corrupted., (Copyright (c) 2006 Wiley-Liss, Inc.)

Proton-electron double resonance imaging (PEDRI) requires a dual EPR/NMR resonator. In order to saturate electron spins, long high-power RF EPR pulses are needed. Dissipated power causes resonator and sample heating, resulting in instability of the resonator frequency and coupling, which in turn causes a reduction in sensitivity. To overcome this problem, we designed, built, and tested a modified Alderman-Grant (MAG) resonator for in vivo and in vitro applications. We improved the temperature stability by using fused quartz in critical structural components of the resonator and employing a design that minimized moving parts. All of the components of the resonator and matching circuit, including coupling capacitors, were rigidly positioned on the surface of the quartz tube. Electrical coupling circuitry between the feeding cable and resonator was employed to provide independent coupling and tuning adjustments, which improved the ease and speed of operation. The equivalent schematic of the matching circuit is presented along with calculations of critical parameters. The resonator exhibits no frequency shift with 20 W of continuous power irradiation, and can handle intermittent power of 60 W for up to 3 min without detuning, enabling stable and reliable data acquisition for PEDRI of biological samples., (Copyright (c) 2006 Wiley-Liss, Inc.)

Proton electron double resonance imaging (PEDRI) is an emerging technique that utilizes the Overhauser effect to enable in vivo and in vitro imaging of free radicals in biological systems. Nitroxide spin probes enable measurement of tissue redox state based on their reduction to diamagnetic hydroxylamines. PEDRI instrumentation at 0.02 T was applied to assess the ability to image the in vivo distribution, clearance, and metabolism of nitroxide radicals in living mice. Using phantoms of 2,2,5,5-tetramethyl-3-carboxylpyrrolidine-N-oxyl (PCA) in normal saline the dependence of the enhancement on RF power and spin probe concentration was determined. Enhancements of up to -23 were obtained in phantoms with 2 mM levels. Maximum enhancement of -7 was observed in vivo. Coronal images of nitroxide-infused mice enabled visualization of the kinetics of spin probe uptake and clearance in different organs including the great vessels, heart, lungs, kidneys, and bladder with an in-plane spatial resolution of 0.6 mm. PEDRI of living mice was also performed using 3-carbamoyl-proxyl and 2,2,6,6-tetramethyl-4-oxopiperidine-N-oxyl to compare the different rate of clearance and metabolism among different nitroxide probes. PCA, due to its intravascular compartmentalization, provided the sharpest contrast for the vascular system and highest enhancement values in the PEDRI images among the three nitroxides., (Magn Reson Med, 2006. (c) 2006 Wiley-Liss, Inc.)

Tumor tissue oxygenation is an important parameter that is positively correlated to the chemo- or radiation treatment outcome of certain tumors. Hence, methods to accurately and noninvasively determine the concentration of oxygen (pO2) in tumors will be valuable. In this study, electron paramagnetic resonance (EPR) spectroscopy, utilizing microcrystalline particulates of lithium phthalocyanine (LiPc), was used to perform repeated measurements of pO2 as a function of tumor growth. We permanently embedded the particulates in the tumor by coimplanting them with RIF-1 tumor cells during inoculation in mice. This procedure enabled repeated measurements of oxygen concentration in the tumor to be obtained for >2 weeks during its growth phase. The particulates were stable and nontoxic to the tumor cells. Both an in vitro clonogenic assay and an in vivo tumor growth rate examination in C3H mice showed no apparent effect on cell proliferation or tumor growth rate. The measurements indicated that the pO2 of the tumor decreased exponentially with tumor growth and reached hypoxic levels ( approximately 4 mmHg) within 4 days after inoculation of the tumor cells. Spatial EPR imaging revealed a nonuniform distribution of the embedded particulates, which were localized mainly in the middle of the tumor volume. Oxygen mapping of the tumor, obtained by spectroscopic EPR imaging, showed significant variation of pO2 within the tumor. In summary, EPR spectroscopy and imaging with an embedded oximetry probe enabled accurate and repeated measurements of pO2 to be obtained in growing tumors under nonperturbing conditions., (Copyright 2004 Wiley-Liss, Inc.)

This review summarizes the development and application of a variety of EPR imaging modalities including spatial, spectral-spatial (spectroscopic), gated-imaging and oxygen mapping to cardiovascular studies. It has been hypothesized that free radical metabolism, oxygenation and nitric oxide generation in biological organs such as the heart may vary over the spatially defined tissue structure. We have developed instrumentation optimized for 3D spatial and 3D or 4D spectral-spatial imaging of free radicals at 1.2 GHz. Using this instrumentation high quality 3D spectral-spatial imaging of nitroxyl (nitroxide) metabolism was performed, as well as spatially localized measurements of oxygen concentrations, based on the oxygen-dependent line-broadening of the EPR spectrum. Both exogenously infused probes and endogenous radicals were used to obtain the images. It is demonstrated that the EPR imaging is a powerful tool which can provide unique information regarding the spatial localization of free radicals, oxygen and nitric oxide in biological organs and tissues., (Copyright 2004 John Wiley & Sons, Ltd.)

Oxygen plays a critical role in the pathophysiology of myocardial injury during both ischemia and subsequent reperfusion (I/R). Thus, oxygen concentration is an important variable to measure during I/R. In the present work, electron paramagnetic resonance (EPR)-based oximetry was used to measure the oxygen concentration during a series of I/R episodes and oxygenation levels were correlated with the contractile and hemodynamic functions of the heart. A custom-developed electronically tunable surface coil resonator working at 1.1 GHz was used to determine tissue pO(2) in the beating heart. Microcrystalline particulate of lithium phthalocyanine was used as an EPR oximetry probe. Isolated and perfused rat hearts were subjected to 1 or 3 hr durations of preischemic perfusion, followed by 15-min I/R cycles. In hearts perfused for 3 hr prior to 15-min I/R cycles, the myocardial pO(2) decreased gradually on subsequent reperfusions of three successive I/R cycles. However, in hearts perfused for 1 hr there was almost 100% recovery of myocardial pO(2) in all three I/R cycles. The extent of oxygenation recovered in each reperfusion cycle correlated with the recovery of hemodynamic and contractile function. The results also showed that the oxygen consumption rate of the heart at the end of each I/R episode decreased in direct proportion to the functional recovery. In summary, it was observed that the amount of myocardial oxygen consumption during I/R could provide a reliable index of functional impairment in the heart., (Copyright 2004 Wiley-Liss, Inc.)

The list of pathophysiological conditions associated with the overproduction of superoxide expands every day. Much of the knowledge compiled on the role of this radical in disease has been gathered using the native superoxide dismutase enzyme and, more recently, by the use of superoxide dismutase knockout models or transgenic models that overexpress the various isoforms of the enzyme. Although the native enzyme has shown promising anti-inflammatory properties in both preclinical and clinical studies, there were drawbacks and issues associated with its use as a therapeutic agent and pharmacological tool. Based on the concept that removal of superoxide modulates the course of inflammation, synthetic, low-molecular-weight mimetics of the superoxide dismutase enzymes that could overcome some of the limitations associated with the use of the native enzyme have been designed. In this review, we will discuss the advances made using various superoxide dismutase mimetics that led to the proposal that superoxide (and/or the product of its interaction with nitric oxide, peroxynitrite) is an important mediator of inflammation, and to the conclusion that superoxide dismutase mimetics can be utilized as therapeutic agents in diseases of various etiologies. The importance of the selectivity of such compounds in pharmacological studies will be discussed.

Proton electron double resonance imaging (PEDRI) is a double resonance technique where proton MRI is performed with irradiation of a paramagnetic solute. A low-field PEDRI system was developed at 20.1 mT suitable for imaging free radicals in biological samples. With a new small dual resonator, PEDRI was applied to image nitroxide free radicals in isolated beating rat hearts. Experiments with phantoms showed maximum image enhancement factors (IEF) of 42 or 28 with TEMPONE radical concentrations of 2-3 mM at EPR irradiation powers of 12W or 6W, respectively. In the latter case, image resolution better than 0.5 mm and radical sensitivity of 5 microM was obtained. For isolated heart studies, EPR irradiation power of 6W provided optimal compromise of modest sample heating with good SNR. Only a small increase in temperature of about 1 degrees C was observed, while cardiac function remained within 10% of control values. With infusion of 3 mM TEMPONE an IEF of 15 was observed enabling 2D or 3D images to be obtained in 27 sec or 4.5 min, respectively. These images visualized the change in radical distribution within the heart during infusion and clearance. Thus, PEDRI enables rapid and high-quality imaging of free radical uptake and clearance in perfused hearts and provides a useful technique for studying cardiac radical metabolism., (Copyright 2003 Wiley-Liss, Inc.)

The large line-width associated with electron paramagnetic resonance imaging (EPRI) requires effective algorithms to deconvolve the true spatial profiles of spins from the measured projection data. The commonly used Fourier transform (FT) deconvolution algorithm is easy to implement but suffers from the division-by-zero problem. As a result, a couple of parameters are used to control the deconvolution performance. However, this is inconvenient and the deconvolution results are subject to the experience of the operators. In the present work we examined FT deconvolution for EPRI, and proposed an automatic algorithm to determine the cutoff frequency by calculating the piecewise variance of the division result of the Fourier amplitude spectra. The deconvolution algorithm and the filtered back-projection image reconstruction algorithm were implemented and validated using 3D phantom and in vivo imaging data. It was clearly observed that the image resolution improved after deconvolution with the proposed algorithm., (Copyright 2003 Wiley-Liss, Inc.)

Reperfusion of ischemic tissue is a common event in the treatment of heart attack and stroke. To study disease pathogenesis, methods are required to measure tissue perfusion and area at risk, as well as localized regions of injury. While histology can provide this information, its destructive nature precludes assessment of time course. Thus, there is a critical need for a noninvasive technique to obtain this information. To map myocardial redox state as a possible index of cellular ischemia and viability, electron paramagnetic resonance (EPR) imaging experiments were performed on isolated rat hearts before and after the onset of regional ischemia using nitroxide spin labels. With coronary artery occlusion, the EPR images clearly showed the risk region as a void of lower intensity that reversed upon reperfusion. The extent of risk region in the heart was similar in EPR imaging and histological measurements. The unique information obtained regarding the time course of changes in redox metabolism of the risk region and normal myocardium can provide important insights regarding the mechanisms of myocardial injury during and following ischemia., (Copyright 2003 Wiley-Liss, Inc.)

Surface coil resonators have been widely used to perform topical EPR spectroscopy. They are usually positioned adjacent to or implanted within the body. For EPR applications these resonators have a number of important advantages over other resonator designs due to their ease of sample accessibility, mechanical fabrication, implementation of electronic tuning and coupling functions, and low susceptibility to sample motions. However, a disadvantage is their B(1) field inhomogeneity, which limits their usefulness for 3D imaging applications. We show that this problem can be addressed by mapping and correcting the B(1) field distribution. We report the use of EPR imaging (EPRI) to map the B(1) distribution of a surface coil resonator. We show that EPRI provides a fast, accurate, and reliable technique to evaluate the B(1) distribution. 3D EPRI was performed on phantoms, prepared using three different saline concentrations, to obtain the B(1) distribution. The information obtained from the phantoms was used to correct the images of living animals. With the use of this B(1) correction technique, surface coil resonators can be applied to perform 3D mapping of the distribution of free radicals in biological samples and living systems., (Copyright 2002 Wiley-Liss, Inc.)

The purpose of this study was to noninvasively monitor tumor oxygenation and redox status during hyperoxygenation treatment, such as carbogen-breathing, in a murine tumor model using in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging techniques. The study was performed using implanted lithium phthalocyanine (LiPc) microcrystals as the oximetry probe and 3-carbamoylproxyl (3-CP) as the redox probe in RIF-1 tumors implanted in the upper hind leg of C3H mice. Repetitive measurements of pO(2) from the same tumors as a function of tumor growth (8-24 mm in size) showed that the tumors were hypoxic and that the tumor pO(2) values were decreasing with tumor growth. Carbogen-breathing mostly showed an increase in the tumor oxygenation, although there were considerable variations in the magnitude of change among the tumors. The pharmacokinetic studies with 3-CP showed a significant decrease in the overall tumor reduction status in the carbogen-breathing mice. Spatially resolved (imaging) pharmacokinetic data over the tumor volume were obtained to visualize the distribution of the redox status within the tumor. The redox images of the tumor in the air-breathing mice showed significant heterogeneity in the magnitude and spatial distribution of reducing equivalents. On carbogen-breathing the tissue reduction status decreased considerably, with a concomitant decrease in the heterogeneity of distribution of the redox status. The results suggest that 1) carbogen-breathing considerably enhances tissue oxygenation and significantly decreases the redox status in RIF-1 tumor, and 2) changes in the magnitude and distribution of the redox status within the tumor volume during carbogen-breathing are correlated with the increased tissue oxygenation., (Copyright 2002 Wiley-Liss, Inc.)

Proton electron double resonance imaging (PEDRI) measures the spatial distribution of paramagnetic species in biological samples using the Overhauser effect. Triaryl methyl (TAM) free radicals have been developed as a spin probe for PEDRI since they have very long T(1e). Therefore, low RF power levels are sufficient to saturate the electron spin system with resultant high NMR enhancement facilitating application of PEDRI in living animals. In this report, PEDRI studies were performed at 0.02 T. The power-dependent image enhancement was studied using phantoms of TAM in saline, then the distribution and pharmacokinetics of TAM in living mice were measured. Following intravenous administration of 0.7 mmol/kg of TAM, enhancements of up to -34 were observed enabling visualization of its distribution within the body. Maximum uptake of TAM in the vascular compartment was seen 1 min postadministration with half-clearance within 5 min. Maximum uptake in the kidneys occurred at 10 min with half-clearance at 26 min and maximum accumulation in the bladder after 50 min. Thus, TAM is initially compartmentalized in the vasculature and this is followed by rapid uptake and excretion by the kidneys. PEDRI enabled rapid imaging of the distribution and clearance of this paramagnetic probe and this information should facilitate the use of TAM as a label for oximetry and other applications. Magn Reson Med 48:530-534, 2002., (Copyright 2002 Wiley-Liss, Inc.)

While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals in the whole body of small animals, it solely visualizes the free-radical distribution and does not typically provide anatomic visualization of the body. However, anatomic registration is often required for meaningful interpretation of the EPRI-derived free-radical images. An approach is reported for whole-body, EPRI-based, free-radical imaging along with proton MRI in mice. EPRI instrumentation with a 750-MHz narrow band microwave bridge and transverse oriented electric field reentrant resonator, with automatic coupling control and automatic tuning control capability, was used to map the spatial distribution of nitroxide free radicals in phantoms and in living mice, while low-field proton MRI at 16 MHz was used to define the anatomic structure to register the EPR images. Small capillary tubes containing an aqueous radical label were used as markers to enable image superimposition. With this coregistration technique, the EPRI free-radical images were precisely registered, enabling assignment of the location of the observed free-radical distribution within the organs of the mice. This technique enabled differentiation of the distribution and metabolism of nitroxide radicals within the major organs and body sites of living mice., (Copyright 2002 Wiley-Liss, Inc.)

Proton electron double resonance imaging (PEDRI) uses the Overhauser effect to image the distribution of free-radicals in biological samples and animals. Standard MRI hardware and software is used, with the addition of hardware to irradiate the free-radical-of-interest's EPR resonance. For in vivo applications it must be implemented at a sufficiently low magnetic field to result in an EPR irradiation frequency that will penetrate the sample but will not cause excessive nonresonant power deposition therein. Many clinical MRI systems use resistive magnets that are capable of operating at 10-20 mT, and which could thus be used as PEDRI imagers with the addition of a small amount of extra hardware. This article describes the conversion of a 0.38 T whole-body MRI system for operation as a 20.1 mT small-animal PEDRI imager. The magnet power supply control electronics required a small modification to operate at the lower field strength, but no permanent hardware changes to the MRI console were necessary, and no software modification was required. Frequency down- and up-conversion was used on the NMR RF system, together with a new NMR/EPR dual-resonance RF coil assembly. The system was tested on phantoms containing free-radical solution, and was also used to image the distribution of a free-radical contrast agent injected intravenously into anesthetized mice., (Copyright 2002 Wiley-Liss, Inc.)

A novel electron paramagnetic resonance (EPR)-based oxygen mapping procedure (EPROM) is applied to cartilage grown in a single-, hollow-fiber bioreactor (HFBR) system. Chondrocytes harvested from the sterna of 17-day-old chick embryos were inoculated into an HFBR and produced hyaline cartilage over a period of 4 weeks. Tissue oxygen maps were generated according to the EPROM technique (Velan et al., Magn Reson Med 2000;43:804-809) by making use of the line-broadening effects of oxygen on the signal generated from nitroxide spin probes. In addition, the effect on oxygen consumption of the addition of cyanide to the tissue was investigated. Cyanide is a potent inhibitor of oxidative phosphorylation, and accordingly, given the constant provision of oxygen to the tissue, it would be expected to increase oxygen levels within the HFBR. The EPROM measurements showed a significant increase in oxygen concentration in the cartilage after the addition of cyanide. In contrast to other methods for studying oxygen in cartilage, EPROM can provide direct, noninvasive visualization of local concentrations in three dimensions., (Copyright 2001 Wiley-Liss, Inc.)

Ischemic tissues generate nitric oxide (NO) by direct reduction of tissue nitrite under the acidic conditions that occur during ischemia. In view of the important implications of this enzyme-independent mechanism of NO generation on the pathogenesis and treatment of tissue injury, the NO formation in mice subjected to cardiopulmonary arrest was measured and imaged. Real-time measurement of NO generation was performed by detection of naturally generated NO-heme complexes in tissues using L-band electron paramagnetic resonance (EPR) spectroscopy. To distinguish NO generated from nitrite, animals were labeled with isotopically enriched (15)N-nitrite. Mice were infused with nitrite (70 mg/kg, intravenous), cardiopulmonary arrest induced by an overdose of phenobarbital, and transferred to the EPR resonator. Measurements of NO generation were performed on the intact animal at the levels of the head, thorax, and abdomen. At the end of 3 hr, major organs were isolated and analyzed for their NO signal. The NO complexes were found to have maximum levels in lung, heart, and liver. Three-dimensional spatial mapping of the NO complex in the intact animal subjected to cardiopulmonary arrest was performed using EPR imaging techniques. The images also confirmed the maximum formation in the lungs, heart, and liver. The present data reveal that mice subjected to cardiopulmonary arrest generate large amounts of NO, which is nitrite mediated. The observed signal was largely due to heme-bound NO, which accounted for the high concentrations found in these organs. This increased NO formation during cardiopulmonary arrest could contribute to the difficulty of resuscitation after long periods of arrest. Magn Reson Med 45:700-707, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Tissue oxygen content is a central parameter in physiology but is difficult to measure. We report a novel procedure for spatial mapping of oxygen by electron paramagnetic resonance (EPR) utilizing a spectral-spatial imaging data set, in which an EPR spectrum is obtained from each image volume element. From this data set, spatial maps corresponding to local spin density and maximum EPR spectral line amplitude are generated. A map of local EPR spectral linewidth is then computed. Because linewidth directly correlates with oxygen concentration, the linewidth image provides a map of oxygenation. This method avoids a difficulty inherent in other oxygen content mapping techniques using EPR, that is, the unwanted influence of local spin probe density on the image. We provide simulation results and data from phantom studies demonstrating the validity of this method. We then apply the method to map oxygen content in rat tail tissue and vasculature. This method provides a new, widely applicable, approach to direct visualization of oxygen concentration in living tissue. Magn Reson Med 43:804-809, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Piperidine nitroxides have considerable clinical potential, both as antioxidant therapeutic compounds and contrast agents in magnetic resonance imaging. However, their development has thus far been limited by their rapid bioreduction in vivo. Recently, it was reported that polynitroxyl albumin (PNA) can reverse the bioreduction of the reduced 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) in the rat heart, enabling the performance of high resolution EPR imaging for prolonged time (Kuppusamy et al., Biochemistry 35, 7051-7057 (1996)). In this report, the efficacy of PNA in maintaining Tempol concentrations in vivo in mice was demonstrated, using L-band (1.25 GHz) EPR spectroscopy and imaging. The EPR signal of intravenous Tempol had a half-life of 1.0+/-0.2 min and became undetectable within 6 min. Subcutaneous Tempol, however, decayed at a slower rate (half-life, 5.0+/-0.5 min) suggesting that Tempol had been bioreduced to the corresponding hydroxylamine form, Tempol-H. Subcutaneously injected PNA restored 20% of the Tempol signal in the vicinity of the PNA deposit. In vivo topical EPR imaging demonstrated that the Tempol signal was restored at the site of PNA injection, but not at locations remote from the PNA injection site. The ability of PNA to maintain Tempol in its paramagnetic state in vivo should enable a wide range of therapeutic and diagnostic applications of piperidinyl nitroxides.

The application of electron paramagnetic resonance imaging (EPRI) to obtain information from biological samples has been limited by the lack of ideal single line radical labels. The commonly used nitroxides exhibit multiple lines causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The use of a novel single-line triarylmethyl paramagnetic label that enables marked enhancement in image quality and resolution is reported. This label exhibits a single line EPR spectrum that is sharp (linewidth approximately 60 mG in the absence of oxygen) and relatively stable in tissues. The potential of this label in enabling high resolution EPR imaging of biological samples was demonstrated in a series of phantoms and isolated biological organs such as the rat kidney. The images demonstrate that resolutions better than 100 microns could be obtained at L-band on samples of up to 20 mm in size.

Recently, it has been shown that rat hearts subjected to global ischemia generate nitric oxide (NO) and that a significant portion of it is generated by the reduction of nitrite under the acidic and reducing conditions that occur during myocardial ischemia [Zweier, Wang, Samouilov, Kuppusamy, Nature Med. 1, 804-809 (1995)]. In the present study it is further attempted to map the spatial distributions of the NO generation in the ischemic myocardium using L-band electron paramagnetic resonance imaging. Rat hearts were loaded with 10 mM nitrite and subjected to global no-flow ischemia, during which time a series of three-dimensional spatial electron paramagnetic resonance (EPR) images of the distribution of NO were obtained using the NO trap bis(N-methyl-D-glucamine dithiocarbamate)iron(II). The images clearly showed that NO is formed throughout the myocardium. Kinetic experiments showed that maximum NO generation and trapping occur at the midmyocardium and spread out to endocardium and epicardium of the left ventricle. The magnitude of generation in the RV myocardium is four- to fivefold lower than in the LV.

In vivo or ex vivo EPR imaging, EPRI, has been established as a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. While instrumentation capable of performing EPR imaging of free radicals in whole tissues and isolated organs has been previously reported, it was not possible to image rapidly moving organs such as the beating heart. Therefore instrumentation was developed to enable the performance of gated-spectroscopy and imaging on isolated beating rat hearts at L-band. A synchronized pulsing and timing system capable of gated acquisitions of up to 256 images per cycle, with rates of up to 16 Hz was developed. The temporal and spatial accuracy of this instrumentation was verified using a specially designed beating heart-shaped isovolumic phantom with electromechanically driven sinusoidal motion at a cycle rate of 5 Hz. Gated EPR imaging was performed on a series of isolated rat hearts perfused with nitroxide spin labels. These hearts were paced at a rate of 6 Hz with either 16 or 32 gated images acquired per cardiac contractile cycle. The image enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.

The potential for using electron paramagnetic resonance (EPR) imaging in biological applications has been limited by the lack of ideal single-line imaging probes. The commonly used nitroxides exhibit multiple lines, causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The application of a numerical method, based on forward-subtraction principles for removing hyperfine artifacts in the measured projections is reported. It is demonstrated by using computer simulations, imaging of phantoms, and imaging of rat hearts, that marked enhancement in image quality and resolution can be obtained by removing the hyperfine-imposed limit on the gradient magnitude and performing postacquisition corrections for removing hyperfine artifacts in the image.

While instrumentation capable of performing three-dimensional EPR imaging of free radicals in whole tissues and isolated organs has been developed at L-band, important questions remain regarding the resolution and image quality that can be obtained in practice using the presently available free radical labels. Therefore, studies were performed applying three-dimensional spatial EPR imaging at L-band to image the distribution of free radical labels in the isolated heart and in phantoms of similar size. With nitroxide labels the obtainable resolution is limited by the presence of hyperfine structure in the EPR absorption function that in turn limits the maximum applicable gradient. The authors observed that with the nitroxide labels, resolutions in the range of 1-2 mm are possible, while with a single line glucose char label, resolutions of 0.2 mm are obtained. With the nitroxides, images were of sufficient resolution to resolve the overall global shape of the heart and the location of the left and right ventricular cavities; however, finer structures could not be resolved. With the glucose char much finer resolution could be obtained enabling visualization of the ventricles, aortic root, and proximal coronary arteries.

[O2] was measured in the embedding material (alginate) in a typical apparatus for conducting studies of viable cells with NMR, using low frequency EPR. In suspension cultures respiration was independent of [O2] in the perfusing media down to about 1 microM while in alginate beads, the comparable value was 70 microM, indicating that the alginate was a very substantial barrier to the free diffusion of oxygen. With knowledge of [O2] in the various compartments, [O2] in the perfusing medium can be increased and the full power of NMR can be used to provide information on metabolism under various conditions. These results also provide evidence supporting the feasibility and usefulness of EPR techniques using nitroxides to measure [O2] in macroscopic samples such as NMR perfusion tubes. This technique is rapid, apparently nonperturbing, and enables one to differentiate between the concentrations of oxygen in different compartments.