In vivo measurement of regional oxygenation and imaging of redox status in RIF-1 murine tumor: effect of carbogen-breathing.

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.     

EPR/NMR co-imaging for anatomic registration of free-radical images.

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.     

Competition of nitroxyl contrast agents as an in vivo tissue redox probe: comparison of pharmacokinetics by the bile flow monitoring (BFM) and blood circulating monitoring (BCM) methods using X-band EPR and simulation of decay profiles.

Nitroxyl radicals used as tissue redox-sensitive contrast agents in electron paramagnetic resonance (EPR) and/or NMR imaging should satisfy the following two conditions: 1) the molecules disperse into tissues rapidly, and 2) paramagnetic loss occurs by simple reduction of the radical. The pharmacokinetic trends of several nitroxyl contrast agents were compared with the results obtained by bile flow monitoring (BFM) and blood circulation monitoring (BCM) methods using X-band EPR. The nitroxyl radicals (TEMPO, TEMPONE (oxo-TEMPO), and amino-TEMPO) showed additional EPR signals in the bile that were attributed to metabolites formed during transport from blood to bile through the liver. However, the highly hydrophilic CAT-1 (trimethylammonium-TEMPO), which has low membrane permeability, showed minimal concentration in the bile. Probes that have carboxyl moiety, such as carboxy-TEMPO and carboxy-PROXYL, can be transported via anion transporter into hepatic cells. The EPR signal decay profiles of the nitroxyl radicals were simulated based on the experimental data. The simulation, which we previously applied to mouse blood, was modified to simultaneously fit the experimental results of BFM and BCM obtained with rats. The simulation data showed the simplicity/complexity of the pharmacokinetic mechanisms and that carbamoyl-PROXYL and TEMPOL (hydroxy-TEMPO) are suitable contrast agents for assessing tissue redox status.     

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.     

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.     

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.     

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.     

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.     

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.     

Mapping ischemic risk region and necrosis in the isolated heart using EPR imaging.

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.     

Early assessment of neoadjuvant chemotherapy by FEC-courses of locally advanced breast cancer using 99mTc-MIBI

Purpose: 
Response assessment at neoadjuvant (preoperative) chemotherapy of locally advanced breast cancer using clinical examination and mammography is insensitive. Mammoscintigraphy with ^99mTc-MIBI was studied for the prediction of response at such therapy before finishing the chemotherapy cycles. Material and Methods: 
Chemotherapy was given as repeated courses of 5-fluorouracil, epirubicin and cyclophosphamide (FEC). In 1 patient group (n = 23), the tumor uptake relative to surrounding breast tissue and lung tissue at SPECT examination after finishing neoadjuvant chemotherapy was compared with the examination made before chemotherapy. In another group (n = 30), a similar comparison after the first therapy cycle (mean 19 days) with a baseline examination was made. Histologic examination of the resected tumors was made. Results: 
After finishing chemotherapy, there was a strong reduction of the relative tumor activity, while there was no correlation with therapy effect as assessed by histology. After one therapy course, there was no significant reduction of the relative tumor uptake. Conclusion: 
Scintigraphy with ^99mTc-MIBI demonstrated the response after finished neoadjuvant chemotherapy of breast cancer using FEC-courses. It cannot be used to predict a therapy response after one therapy course.     

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.     

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.     

Use of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl as an EPR oximetry probe: potential for in vivo measurement of tissue oxygenation in mouse brain.

Measurement of oxygen concentration and distribution in the brain is essential for understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that potentially can be used to map O(2) concentration in the brain. We examined two nitroxides, 3-methoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [2] and 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [3], as pro-imaging agents to deliver 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] across the blood-brain barrier (BBB). In primary cultured neurons, nitroxide [3] but not [2] was hydrolyzed by intracellular esterases to [1], which, being anionic at physiologic pH, was well retained intracellularly. In contrast, [2] was not well retained by neurons. In vivo pharmacokinetic and pharmacodynamic studies in mice suggested that esterase-labile nitroxide [3] crossed the BBB, and was converted to [1] and retained. Retention occurred in brain tissue and not in the extensive vasculature, as evidenced by the fact that removal of blood by whole-body saline perfusion did not eliminate the nitroxide EPR signal from the brain. The EPR linewidths of [1] and [3] were more O(2)-sensitive than that of the commonly-used oximetry probe 4-oxo-2,2,6,6-tetramethylpiperidine-d(16)-1-(15)N-oxyl [4]. Moreover, we used [3] in vivo to estimate O(2) concentration in mouse brains. These results indicate that nitroxide [3] could be useful for mapping O(2) distribution in the brain following stroke.     

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 (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.     

In vivo oximetry using a nitroxide-liposome system.

Liposomes containing the deuterated, charged, aqueous soluble nitroxide 4-trimethyl-ammonium-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (d-Cat1) were used as probes to measure oxygen concentrations in vivo. Following intramuscular or intraperitoneal injection of the liposome suspension. ICR mice were placed over the surface probe of a low frequency (1.1 GHz) electron paramagnetic resonance (EPR) spectrometer. The linewidth of the deuterated nitroxide is sensitive to changes in the dissolved oxygen concentration: this parameter was calibrated separately so that linewidths measured in the injected mice could be converted into oxygen tensions. This technique detected substantial changes in pO2 as the oxygen content of the breathing gas was changed from 21 to 85 to 0%. Intravenous injection of the liposomes also is possible, and the liposomes accumulate in the liver and spleen, where detectable, oxygen-sensitive EPR signals can be measured.     

Value of micro-CT as an investigative tool for osteochondritis dissecans

Purpose: 
To evaluate micro computed tomography (micro-CT) for the assessment of osteochondritis dissecans in comparison with histology. Material and Methods: 
Osteochondritis dissecans lesions of 3 patients were evaluated using micro-CT (0.125 mA, 40 keV, 60 μm slice thickness, 60 μm isotropic resolution, entire sample) and light microscopy (toluidine blue, 3-5 μm slice thickness). The methods were compared regarding preparation time, detectability of tissue types and morphologic features of bone and cartilage. Results: 
Non-destructive micro-CT imaging of the entire sample was faster than histologic preparation of a single slice for light microscopy. Morphologic features of bone and cartilage could be imaged in a comparable way to histology. It was not possible to image cells or different tissue types of bone and cartilage with micro-CT. Conclusion: 
Micro-CT is a fast, non-destructive tool that may be a supplement or, if detailed histologic information is not necessary, an alternative to light microscopy for the investigation of osteochondritis dissecans.     

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.     

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.     

Application of continuous-wave EPR spectral-spatial image reconstruction techniques for in vivo oxymetry: comparison of projection reconstruction and constant-time modalities.

In this study we report the application of continuous-wave (CW) electron paramagnetic resonance (EPR) constant-time spectral spatial imaging (CTSSI) for in vivo oxymetry. 2D and 3D SSI studies of a phantom and live mice were carried out using projection reconstruction (PR) and constant-time (CT) modalities using a CW-EPR spectrometer/imager operating at 300 MHz frequency. Distortion of line shape, which is inherent in the PR method, was minimized by the CTSSI modality. It was also found that CTSSI offers improved noise reduction, restores a smoother line shape, and gives high convergence of estimated values. Spatial resolution was also improved by CTSSI, although fundamental spectral line-width broadening was observed. Although additional corrections are required for accurate estimations of spectral line width, CTSSI was able to demonstrate distinct differences in oxygen tension between a tumor and the normal legs of a C3H mouse. The PR method, on the other hand, was unable to make such a distinction unequivocally with the triarylmethyl spin probes. CTSSI promises to be a more suitable method for quantitative in vivo oxymetric studies using radiofrequency EPR imaging (EPRI).