Development of a hybrid EPR/NMR coimaging system.

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.     

Three-Dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distribution during the cardiac contractile cycle

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 images enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.     

Spatially resolved time-course studies of free radical reactions with an EPRI/MRI fusion technique.

Electron paramagnetic resonance imaging (EPRI) using nitroxyl radicals is a useful technique for visualizing reactive oxygen species (ROS) and the pharmacokinetics of probes. To unambiguously identify anatomical locations, coregistration of EPRI with images obtained by MRI is necessary. In this study the feasibility of performing reliable EPRI/MRI fusion imaging using nitroxyl radical fiducial markers was tested. The pharmacokinetics of the nitroxyl radicals were observed after oral or intravenous administration in C57BL6 mice. To fuse both images, the nitroxyl radical was used as fiducial markers. The EPR and MR images corresponded well and clearly illustrated minimal changes in pharmacokinetics between carbamoyl-PROXYL and carboxy-PROXYL. These results demonstrate that the EPRI/MRI fused imaging technique is useful for investigating in vivo pharmacokinetics and provides unambiguous anatomic details.     

Fast gated EPR imaging of the beating heart: Spatiotemporally resolved 3D imaging of free‐radical distribution during the cardiac cycle

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. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice.

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.     

Mapping of the B1 field distribution of a surface coil resonator using EPR imaging.

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.     

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging

Purpose:
To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. Material and Methods:
Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. Results:
Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. Conclusion:
Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.     

In vivo imaging of a stable paramagnetic probe by pulsed-radiofrequency electron paramagnetic resonance spectroscopy

Imaging of free radicals by electron paramagnetic resonance (EPR) spectroscopy using time domain acquisition as in nuclear magnetic resonance (NMR) has not been attempted because of the short spin-spin relaxation times, typically under 1 μs, of most biologically relevant paramagnetic species. Recent advances in radiofrequency (RF) electronics have enabled the generation of pulses of the order of 10–50 ns. Such short pulses provide adequate spectral coverage for EPR studies at 300 MHz resonant frequency. Acquisition of free induction decays (FID) of paramagnetic species possessing inhomogenously broadened narrow lines after pulsed excitation is feasible with an appropriate digitizer/averager. This report describes the use of time-domain RF EPR spectrometry and imaging for in vivo applications. FID responses were collected from a water-soluble, narrow line width spin probe within phantom samples in solution and also when infused intravenously in an anesthetized mouse. Using static magnetic field gradients and back-projection methods of image reconstruction, two-dimensional images of the spin-probe distribution were obtained in phantom samples as well as in a mouse. The resolution in the images was better than 0.7 mm and devoid of motional artifacts in the in vivo study. Results from this study suggest a potential use for pulsed RF EPR imaging (EPRI) for three-dimensional spatial and spectral-spatial imaging applications. In particular, pulsed EPRI may find use in in vivo studies to minimize motional artifacts from cardiac and lung motion that cause significant problems in frequency-domain spectral acquisition, such as in continuous wave (cw) EPR techniques.     

Proton electron double resonance imaging (PEDRI) of the isolated beating rat heart.

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.     

Multiparametric imaging of tumor oxygenation,redox status,and anatomical structure using overhauser‐enhanced MRI–prepolarized MRI system

An integrated Overhauser‐enhanced MRI–Prepolarized MRI system was developed to obtain radiobiological information that could be accurately coregistered with diagnostic quality anatomic images. EPR and NMR images were acquired through the double resonance technique and field cycling of the main magnetic field from 5 mT to 0.5 T. Dedicated EPR and NMR coils were devised to minimize radiofrequency power deposition with high signal‐to‐noise ratio. Trityl and nitroxide radicals were used to characterize oxygen and redox sensitivities of multispin echo Overhauser‐enhanced MRI. Oxygen resolution of 3 mmHg was obtained from 2 mM deoxygenated trityl phantoms. Trityl radicals were stable in reducing environments and did not alter the redox‐sensitive decaying rate of the nitroxide signals. Nitroxide radicals had a compounding effect for the trityl oximetry. Tumor oxygenation and redox status were acquired with anatomical images by injecting trityl and nitroxide probes subsequently in murine tumors. The Overhauser‐enhanced MRI–Prepolarized MRI system is ready for quantitative longitudinal imaging studies of tumor hypoxia and redox status as radiotherapy prognostic factors. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

Imaging spin probe distribution in the tumor of a living mouse with 250 MHz EPR: correlation with BOLD MRI.

Electron paramagnetic resonance imaging (EPRI) promises to provide new insights into the physiology of tissues in health and disease. Understanding the in vivo imaging capability of this new modality requires comparison with other physiologically responsive techniques. Here, an initial comparison between 2D EPR spatial imaging of a narrow single line injectable paramagnetic trityl spin probe and 2D slice-selected carbogen subtraction BOLD MRI is presented. The images were obtained from the same FSa fibrosarcoma grown in the leg of a C3H mouse. This tumor was unusual in comparison with others imaged with subtraction BOLD MRI because of its peripheral distribution of intensity. The spatial distribution of the EPR spin probe showed the same peripheral distribution. The pixel resolutions of these images are comparable. These images provide an early in vivo comparison of EPRI with a well-established imaging modality. The comparison validates the in vivo distribution of spin probe as imaged with EPRI, and provides a proof of principle for the comparison of BOLD and EPRI.     

Quantitative tumor oxymetric images from 4D electron paramagnetic resonance imaging (EPRI): methodology and comparison with blood oxygen level-dependent (BOLD) MRI.

This work presents a methodology for obtaining quantitative oxygen concentration images in the tumor-bearing legs of living C3H mice. The method uses high-resolution electron paramagnetic resonance imaging (EPRI). Enabling aspects of the methodology include the use of injectable, narrow, single-line triaryl methyl spin probes and an accurate model of overmodulated spectra. Both of these increase the signal-to-noise ratio (SNR), resulting in high resolution in space (1 mm)(3) and oxygen concentrations (approximately 3 torr). Thresholding at 15% the maximum spectral amplitude gives leg/tumor shapes that reproduce those in photographs. The EPRI appears to give reasonable oxygen partial pressures, showing hypoxia (approximately 0-6 torr, 0-10(3) Pa) in many of the tumor voxels. EPRI was able to detect statistically significant changes in oxygen concentrations in the tumor with administration of carbogen, although the changes were not increased uniformly. As a demonstration of the method, EPRI was compared with nearly concurrent (same anesthesia) T(2)*/blood oxygen level-dependent (BOLD) MRI. There was a good spatial correlation between EPRI and MRI. Homogeneous and heterogeneous T(2)*/BOLD MRI correlated well with the quantitative EPRI. This work demonstrates the potential for EPRI to display, at high spatial resolution, quantitative oxygen tension changes in the physiologic response to environmental changes.     

Slice-selective images of free radicals in mice with modulated field gradient electron paramagnetic resonance (EPR) imaging.

Continuous wave (CW) electron paramagnetic resonance (EPR) imaging can be used to obtain slice-selective images of free radicals without measuring three-dimensional (3D) projection data. A method that incorporated a modulated magnetic field gradient (MFG) was combined with polar field gradients to select a slice in the subject noninvasively. The slice-selective in vivo EPR imaging of triarylmethyl radicals in the heads of live mice is reported. 3D surface-rendered images were successfully obtained from slice-selective images. In the experiment in mice, a slice thickness of 1.8 mm was achieved.     

Three-dimensional spatial EPR imaging of the rat heart

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.     

Positron emission tomography as a diagnostic tool. A reassessment based on literature review.

Currently used clinical diagnostic imaging modalities, such as magnetic resonance imaging (MRI) and x-ray computed tomography (CT) provide predominantly anatomic information. CT images reflect x-ray attenuation distribution in the body, whereas MRI signals depend primarily on proton density and tissue relaxivity. In contrast to these predominantly anatomic modalities, positron emission tomography (PET) reflects tissue physiology and metabolism. Although PET has been used predominantly as a research tool, the clinical use of this technique for the detection, noninvasive characterization, and treatment planning of selected disease processes has been extensively studied in oncology, cardiology, and neurology. The author examined currently available literature to reassess the potential role of PET as a diagnostic tool in the following specific clinical situations: (1) the differentiation of radiation necrosis from tumor recurrence; (2) the characterization of the physiologic significance of coronary stenosis and the evaluation of the myocardial viability; and (3) the localization of the epileptogenic foci.     

Evaluation of the dose distribution gradient in the close vicinity of brachytherapy seeds using electron paramagnetic resonance imaging

Electron paramagnetic resonance (EPR) spectroscopy has been successfully employed to determine radiation dose using alanine. The EPR signal intensity reflects the number of stable free radicals produced, and provides a quantitative measurement of the absorbed dose. The aim of the present study was to explore whether this principle can be extended to provide information on spatial dose distribution using EPR imaging (EPRI). Lithium formate was selected because irradiation induces a single EPR line, a characteristic that is particularly convenient for imaging purposes. ¹²⁵I‐brachytherapy seeds were inserted in tablets made of lithium formate. Images were acquired at 1.1 GHz. Monte Carlo (MC) calculations were used for comparison. The dose gradient can be determined using two‐dimensional (2D) EPR images. Quantitative data correlated with the dose estimated by the MC simulations, although differences were observed. This study provides a first proof‐of‐concept that EPRI can be used to estimate the gradient dose distribution in phantoms after irradiation. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

EPRI to estimate the in vivo intracerebral reducing ability in adolescent rats subjected to neonatal isolation

PURPOSE: To estimate the in vivo intracerebral reducing ability after acute stress in adolescent rats subjected to early neonatal isolation (NI), by performing temporal electron paramagnetic resonance imaging (EPRI) of the brain. MATERIALS AND METHODS: An EPRI system operating at an EPR frequency of 700 MHz was used. The intracerebral reducing ability was estimated based on the halflife of the EPR signal of the blood-brain barrier (BBB)-permeable nitroxide radical. The NI treatment was performed for a period of one hour per day over postnatal days 2-9. Six-week-old rats were exposed to acute stress (immobilization for 90 minutes) prior to the EPRI study. RESULTS: Depletion of the intracerebral reducing ability caused by the acute stress was observed; however, this depletion phenomenon did not occur in animals that were not subjected to NI. CONCLUSION: The results obtained in this study prove that NI induces cerebral vulnerability to acute stress in adolescence.     

In vivo imaging of spin-trapped nitric oxide in rats with septic shock: MRI spin trapping.

This paper reports the first in vivo NMR image of the distribution of NO using the "MRI spin-trapping" technique. NO was complexed with the Fe(II)-chelate spin trap, N-methyl-D-glucamine dithiocarbamate (MGD), verified as (MGD)(2)-Fe(II)-NO by EPR, and the radical distribution was "visualized" by MR images. In rats, the (MGD)(2)-Fe(II)-NO complex was concentrated in the liver displaying significantly enhanced contrast in the vascular structure such as hepatic vein and inferior vena cava. Nitric oxide synthase was verified as the source of NO in rats with septic shock by pre-administration of the competitive inhibitor N-monomethyl-L-arginine, resulting in reduced enhancement. The NO complex was more stable in vivo and a more effective MRI contrast agent than other stable nitrogen containing radicals, such as nitroxides. The MRI spin-trapping method should be a powerful tool for visualizing spatial distributions of free radicals in pathologic organs and tissues when combined with the appropriate radical complexing agent, such as (MGD)(2)-Fe(II) used in these studies. Magn Reson Med 42:235-239, 1999.     

Three-dimensional reconstruction of skeletal muscle from MRI

Three-dimensional (3D) reconstructions of medical images can provide useful information to the radiologist, enabling delineation and spatial correlation of anatomic structures in one image rather than consecutive two-dimensional (2D) images. Magnetic resonance imaging (MRI) is known to be a powerful technique for evaluation of skeletal muscle pathology and physiology. We investigated the role of 3D reconstruction from MRI images o pathological and healthy muscle, using volume rendering. The ability to easily delineate and recognize normal and injured muscle in the 3D images were dependent upon the original contrast between normal and injured muscle, spatial resolution, and anatomic complexity in the original slices, and also on imaging parameters such as volume averaging.     

Whole body MRI: Improved lesion detection and characterization with diffusion weighted techniques

Diffusion‐weighted imaging (DWI) is an established functional imaging technique that interrogates the delicate balance of water movement at the cellular level. Technological advances enable this technique to be applied to whole‐body MRI. Theory, b‐value selection, common artifacts and target to background for optimized viewing will be reviewed for applications in the neck, chest, abdomen, and pelvis. Whole‐body imaging with DWI allows novel applications of MRI to aid in evaluation of conditions such as multiple myeloma, lymphoma, and skeletal metastases, while the quantitative nature of this technique permits evaluation of response to therapy. Persisting signal at high b‐values from restricted hypercellular tissue and viscous fluid also permits applications of DWI beyond oncologic imaging. DWI, when used in conjunction with routine imaging, can assist in detecting hemorrhagic degradation products, infection/abscess, and inflammation in colitis, while aiding with discrimination of free fluid and empyema, while limiting the need for intravenous contrast. DWI in conjunction with routine anatomic images provides a platform to improve lesion detection and characterization with findings rivaling other combined anatomic and functional imaging techniques, with the added benefit of no ionizing radiation. J. Magn. Reson. Imaging 2013;38:253–268. © 2013 Wiley Periodicals, Inc.