Eugene D. Barth

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 (∼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 (∼0–6 torr, 0–103 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. Magn Reson Med 49:682–691, 2003.

Electron paramagnetic resonance oxygen images correlate spatially and quantitatively with Oxylite oxygen measurements.

Tumor oxygenation predicts cancer therapy response and malignant phenotype. This has spawned a number of oxymetries. Comparison of different oxymetries is crucial for the validation and understanding of these techniques. Electron paramagnetic resonance (EPR) imaging is a novel technique for providing quantitative high-resolution images of tumor and tissue oxygenation. This work compares sequences of tumor pO2 values from EPR oxygen images with sequences of oxygen measurements made along a track with an Oxylite oxygen probe. Four-dimensional (three spatial and one spectral) EPR oxygen images used spectroscopic imaging techniques to measure the width of a spectral line in each image voxel from a trityl spin probe (OX063, Amersham Health R&D) in the tissues and tumor of mice after spin probe injection. A simple calibration allows direct, quantitative translation of each line width to an oxygen concentration. These four-dimensional EPR images, obtained in 45 minutes from FSa fibrosarcomas grown in the legs of C3H mice, have a spatial resolution of ∼1 mm and oxygen resolution of ∼3 Torr. The position of the Oxylite track was measured within a 2-mm accuracy using a custom stereotactic positioning device. A total of nine images that involve 17 tracks were obtained. Of these, most showed good correlation between the Oxylite measured pO2 and a track located in the tumor within the uncertainties of the Oxylite localizability. The correlation was good both in terms of spatial distribution pattern and pO2 magnitude. The strong correlation of the two modalities corroborates EPR imaging as a useful tool for the study of tumor oxygenation.

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-D-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO-Fe(MGD)2 and NO-Fe(DTCS)2 in mice in real time.

ELECTRON PARAMAGNETIC RESONANCE OXYGEN IMAGE HYPOXIC FRACTION PLUS RADIATION DOSE STRONGLY CORRELATES WITH TUMOR CURE IN FSA FIBROSARCOMAS

PURPOSE Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

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. Magn Reson Med 47:634–638, 2002.

Measurement of differences in pO2 in response to perfluorocarbon/carbogen in FSa and NFSa murine fibrosarcomas with low-frequency electron paramagnetic resonance oximetry.

We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response.

Comparison of 250 MHz electron spin echo and continuous wave oxygen EPR imaging methods for in vivo applications

PURPOSE The authors compare two electron paramagnetic resonance imaging modalities at 250 MHz to determine advantages and disadvantages of those modalities for in vivo oxygen imaging. METHODS Electron spin echo (ESE) and continuous wave (CW) methodologies were used to obtain three-dimensional images of a narrow linewidth, water soluble, nontoxic oxygen-sensitive trityl molecule OX063 in vitro and in vivo. The authors also examined sequential images obtained from the same animal injected intravenously with trityl spin probe to determine temporal stability of methodologies. RESULTS A study of phantoms with different oxygen concentrations revealed a threefold advantage of the ESE methodology in terms of reduced imaging time and more precise oxygen resolution for samples with less than 70 torr oxygen partial pressure. Above 100 torr, CW performed better. The images produced by both methodologies showed pO2 distributions with similar mean values. However, ESE images demonstrated superior performance in low pO2 regions while missing voxels in high pO2 regions. CONCLUSIONS ESE and CW have different areas of applicability. ESE is superior for hypoxia studies in tumors.

Nitroxide conjugate of a thermally responsive elastin‐like polypeptide for noninvasive thermometry

Hyperthermia, as an adjuvant with radiation and chemotherapy, has shown promise in the treatment of cancer. The relevant biological effects of a hyperthermia treatment are both time and temperature-dependent, creating a need for accurate thermometry. We present a novel noninvasive thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), and nitroxide to produce an ELP-nitroxide conjugate. When examined with electron paramagnetic resonance (EPR) spectroscopy, the ELP-nitroxide conjugate has temperature-dependent spectral line widths whose predictive accuracy is approximately 0.3 degrees C (80 microM). We believe that the temperature-dependent changes observed in the EPR spectrum are due to the combined effect of temperature, viscosity and effective radius on the rotational correlation time of the ELP-nitroxide conjugate.

Electron paramagnetic resonance oxygen imaging of a rabbit tumor using localized spin probe delivery.

PURPOSE Application ofin vivo electron paramagnetic resonance (EPR) oxygen imaging (EPROI) to tumors larger than those of mice requires development of both instrumental and medical aspects of imaging. METHODS 250 MHz EPR oxygen imaging was performed using a loop-gap resonator with a volume exceeding 100cm3. The paramagnetic spin probe was injected directly into the femoral artery feeding the rabbit leg/tumor. RESULTS The authors present continuous wave and electron spin echo EPR oxygen images of a large size (4 cm) VX-2 tumor located on the leg of a New Zealand white rabbit. CONCLUSIONS This study demonstrates the feasibility of continuous wave and electron spin echo oxygen imaging modalities for investigation of volumes of tumor and normal tissue relevant to large animals. The injection of the spin probe directly into the artery feeding a rabbit leg will allow one to reduce, by over one order of magnitude, the amount of spin probe used as compared to whole animal IV injection.

Very-low-frequency electron paramagnetic resonance (EPR) imaging of nitroxide-loaded cells.

Recent advances in electron paramagnetic resonance (EPR) imaging have made it possible to image, in real time in vivo, cells that have been labeled with nitroxide spin probes. We previously reported that cells can be loaded to high (millimolar) intracellular concentrations with (2,2,5,5‐tetramethylpyrrolidin‐1‐oxyl‐3‐ylmethyl)amine‐N,N‐diacetic acid by incubation with the corresponding acetoxymethyl (AM) ester. Furthermore, the intracellular lifetime (t1/e) of this nitroxide is 114 min—sufficiently long to permit in vivo imaging studies. In the present study, at a gradient of ∼50 mT/m, we acquire and compare EPR images of a three‐tube phantom, filled with either a 200‐μM solution of the nitroxide, or a suspension of cells preincubated with the nitroxide AM ester. In both cases, 3‐mm resolution images can be acquired with excellent signal‐to‐noise ratios (SNRs). These findings indicate that cells well‐loaded with nitroxide are readily imageable by EPR imaging, and that in vivo tracking studies utilizing such cells should be feasible. Magn Reson Med 58:850–854, 2007.