Sergey Petryakov

Development of a PEDRI free-radical imager using a 0.38 T clinical MRI system

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‐interests 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. Magn Reson Med 47:181–186, 2002.

In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real‐time assessment of tumor tissue extracellular pH (pHe), 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 pHe 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 pHe 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, pHe 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 pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med, 2011.

In vivo imaging of changes in tumor oxygenation during growth and after treatment.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen‐sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen‐sensing probes, which become permanently embedded in the developed tumor. Radiation‐induced fibrosarcoma (RIF‐1) cells, internalized with nanoprobes of lithium octa‐n‐butoxy‐naphthalocyanine (LiNc‐BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30‐Gy X‐radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen‐sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors. Magn Reson Med 57:950–959, 2007.

EPR oximetry in the beating heart: Myocardial oxygen consumption rate as an index of postischemic recovery

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 pO2 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 pO2 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 pO2 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. Magn Reson Med 51:835–842, 2004.

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 B1 field inhomogeneity, which limits their usefulness for 3D imaging applications. We show that this problem can be addressed by mapping and correcting the B1 field distribution. We report the use of EPR imaging (EPRI) to map the B1 distribution of a surface coil resonator. We show that EPRI provides a fast, accurate, and reliable technique to evaluate the B1 distribution. 3D EPRI was performed on phantoms, prepared using three different saline concentrations, to obtain the B1 distribution. The information obtained from the phantoms was used to correct the images of living animals. With the use of this B1 correction technique, surface coil resonators can be applied to perform 3D mapping of the distribution of free radicals in biological samples and living systems. Magn Reson Med 48:1057–1062, 2002.

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 ∼1.2 GHz/∼40 mT and proton MRI is performed at 16.18 MHz/∼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. Magn Reson Med 58:156–166, 2007.

In Vivo Proton–Electron Double-Resonance Imaging of Extracellular Tumor pH Using an Advanced Nitroxide Probe

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

Variable Field Proton–Electron Double-Resonance Imaging: Application to pH mapping of aqueous samples

A new concept of Variable Field Proton-Electron Double-Resonance Imaging (VF PEDRI) is proposed. This allows for functional mapping using specifically designed paramagnetic probes (e.g. oxygen or pH mapping) with MRI high quality spatial resolution and short acquisition time. Studies performed at 200 G field MRI with phantoms show that a pH map of the sample can be extracted using only two PEDRI images acquired in 140 s at pre-selected EPR excitation fields providing pH resolution of 0.1 pH units and a spatial resolution of 1.25mm. Note that while concept of functional VF PEDRI was demonstrated using the pH probe, it can be applied for studies of other biologically relevant parameters of the medium such as redox state, concentrations of oxygen or glutathione using specifically designed EPR probes.

Variable Radio Frequency Proton-Electron Double-Resonance Imaging: Application to pH Mapping of Aqueous Samples

Proton-electron double-resonance imaging (PEDRI) offers rapid image data collection and high resolution for spatial distribution of paramagnetic probes. Recently we developed the concept of variable field (VF) PEDRI which enables extracting a functional map from a limited number of images acquired at pre-selected EPR excitation fields using specific paramagnetic probes (Khramtsov et al., J. Magn. Reson. 202 (2010) 267-273). In this work, we propose and evaluate a new modality of PEDRI-based functional imaging with enhanced temporal resolution which we term variable radio frequency (VRF) PEDRI. The approach allows for functional mapping (e.g., pH mapping) using specifically designed paramagnetic probes with high quality spatial resolution and short acquisition times. This approach uses a stationary magnetic field but different EPR RFs. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of a pH-sensitive nitroxide is converted to a pH map using a corresponding calibration curve. Elimination of field cycling decreased the acquisition time by exclusion periods of ramping and stabilization of the magnetic field. Improved magnetic field homogeneity and stability allowed for the fast MRI acquisition modalities such as fast spin echo. In total, about 30-fold decrease in EPR irradiation time was achieved for VRF PEDRI (2.4s) compared with VF PEDRI (70s). This is particularly important for in vivo applications enabling one to overcome the limiting stability of paramagnetic probes and sample overheating by reducing RF power deposition.

Organ specific mapping of in vivo redox state in control and cigarette smoke-exposed mice using EPR/NMR co-imaging.

In vivo mapping of alterations in redox status is important for understanding organ specific pathology and disease. While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals, it does not provide anatomic visualization of the body. Proton MRI is well suited to provide anatomical visualization. We applied EPR/NMR co-imaging instrumentation to map and monitor the redox state of living mice under normal or oxidative stress conditions induced by secondhand cigarette smoke (SHS) exposure. A hybrid co-imaging instrument, EPRI (1.2 GHz)/proton MRI (16.18 MHz), suitable for whole-body co-imaging of mice was utilized with common magnet and gradients along with dual EPR/NMR resonators that enable co-imaging without sample movement. The metabolism of the nitroxide probe, 3-carbamoyl-proxyl (3-CP), was used to map the redox state of control and SHS-exposed mice. Co-imaging allowed precise 3D mapping of radical distribution and reduction in major organs such as the heart, lungs, liver, bladder and kidneys. Reductive metabolism was markedly decreased in SHS-exposed mice and EPR/NMR co-imaging allowed quantitative assessment of this throughout the body. Thus, in vivo EPR/NMR co-imaging enables in vivo organ specific mapping of free radical metabolism and redox stress and the alterations that occur in the pathogenesis of disease.