Electron Paramagnetic Resonance Imaging Research Articles

In vivo ESR imaging of redox status in mice after X-ray irradiation, measured by acyl-protected hydroxylamine probe, ACP

More detailed investigations on the in vivo redox status are needed to elucidate the mechanisms contributing to damage caused by ionizing radiation. In the present study, the in vivo redox status of mice was examined using in vivo electron spin resonance (ESR) imaging after an intraperitoneal injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) as a probe. ACP is easily hydrolyzed to its hydroxylamine form in the mouse body, and the interconversion between hydroxylamine and the corresponding nitroxyl radical reflects the biological redox status. Liver damage, based on changes in liver weight and plasma aspartate aminotransferase levels, was detected in mice 4 days after X-ray irradiation at 7.5 Gy. ESR imaging showed that the signal intensity of the nitroxyl radical was high at the liver area in both damaged and healthy mice after administration of ACP. Whereas the signal decayed at the liver area for healthy mouse, the decay was negligible in damaged mice. Unlike healthy mouse, signal in the chest for damaged mouse increased with time. The distribution of the sum of hydroxylamine and the nitroxyl radical was similar in damaged and healthy mice. X-ray irradiation slightly lowered the reduction activity of the liver microsomal fraction for the nitroxyl radical. Thiobarbituric acid reactive substances in the liver were higher in damaged mice than in healthy mice; however, no significant differences were noted in reduced glutathione. The present results indicate that the redox status of mice exposed to X-ray irradiation is more oxidative than that in healthy mice.

Design and Fabrication of Compact Arrayed Magnet for Biological EPR Imaging

A portable and compact electron paramagnetic resonance (EPR) imaging magnet was developed for building a 750 MHz EPR imaging system. To realize low-cost fabrication, it was shown that the magnetic homogeneity required for redox EPR imaging using nitroxyl probes can be approximately reproduced by regularly arranging small neodymium magnets that are easy to handle. A five-channel coil driver consisting of operational amplifiers and switching power supplies was developed for field sweep, field modulation, and three field gradients to downsize the imaging system. The EPR imaging of 4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (tempone-d16) using a phantom was successfully performed by employing a 3D radial sequence controlled by a digital EPR console.

Algebraic reconstruction of 3D spatial EPR images from high numbers of noisy projections: An improved image reconstruction technique for high resolution fast scan EPR imaging

A novel method for reconstructing 3D spatial EPR images from large numbers of noisy projections was developed that minimizes mean square error between the experimental projections and those from the reconstructed image. The method utilizes raw projection data and zero gradient spectrum to account for EPR line shape and hyperfine structure of the paramagnetic probe without the need for deconvolution techniques that are poorly suited for processing of high noise projections. A numerical phantom was reconstructed for method validation. Reconstruction time for the matrix of 1283 voxels and 16,384 noiseless projections was 4.6 min for a single iteration. The algorithm converged quickly, reaching R2 ~ 0.99975 after the very first iteration. An experimental phantom sample with nitroxyl radical was measured. With 16,384 projections and a field gradient of 8 G/cm, resolutions of 0.4 mm were achieved for a cubical area of 25 × 25 × 25 mm3. Reconstruction was sufficiently fast and memory efficient making it suitable for applications with large 3D matrices and fully determined system of equations. The developed algorithm can be used with any gradient distribution and does not require adjustable filter parameters that makes for simple application. A thorough analysis of the strengths and limitations of this method for 3D spatial EPR imaging is provided.

Abstract 1648: Multimodal imaging of tumor hypoxia with 18F-misonidazole PET, EPR, and MRI

Abstract Introduction: In tumor mouse models, electron paramagnetic resonance (EPR) imaging has been used to quantify pO2 in-vivo for oxygen-guided radiation therapy. Irradiating the hypoxic tumor sub-regions with a boost of radiation significantly increased survival probability when compared to the boosted radiation of normoxic tumor sub-regions. These results indicate 1) the importance of accurate oxygen imaging in-vivo and 2) an advantage to dose-painting hypoxic tumor sub-regions to improve radiation therapy outcomes of hypoxic tumor treatment. The purpose of this study is to evaluate the accuracy of hypoxia imaging using 18F-misonidazole (FMISO) with PET imaging while using EPR imaging as the reference standard for true hypoxia. We include T2-weighted MRI to define tumor anatomy and dynamic contrast enhanced (DCE)-MRI imaging to model vasculature properties to make FMISO PET more accurate in its measurement of hypoxia. Methods: We used six SCC7 tumor mouse models in C3H mice, grown in the leg in the range of 200-500 mm3. Under minimal anesthesia, each mouse leg was set in a soft vinylpolysiloxane cast with embedded fiducials for registered images. A custom-built PET insert was inserted into a 720 MHz EPR imager for near-simultaneous imaging with PET and EPR, acquired two hours post-injection of FMISO to allow the radiotracer to bind to hypoxic tumor cells. Then, T2-weighted and gadodiamide DCE-MRI images were acquired and Ktrans and ve parameters were fitted to model tumor vasculature properties using the Tofts model. Data from all modalities were registered using fiducials, and resampled to isotropic (0.5 mm)3 voxels. The thresholds for hypoxia were defined as tumor to muscle ratio (TMR) ≥ 2 and pO2 ≤ 10 torr for PET and EPR, respectively. To correct the PET image using DCE-MRI parameters, we first modeled FMISO retention as a logistic function of pO2 to map the EPR image to PET TMR image, so that its sigmoidal point of inflection was at the threshold of retention. Then we estimated optimal weighting coefficients of DCE-MRI parameters to add or subtract voxel by voxel to the PET data so that its definition of hypoxia is more similar to the EPR image's hypoxia definition. The quality of overlap between hypoxic tumor regions as defined by PET and EPR was assessed using the Dice Similarity Coefficient (DSC) and the Hausdorff Distance (HD), before and after applying a correction to the PET data. Results: The DSC between hypoxic regions as defined by PET and EPR, before and after applying a correction to PET data, was 0.53 ± 0.2 and 0.80 ± 0.2, respectively. The HD was respectively 3.8 ± 0.5 and 2.1 ± 0.5 mm. Conclusion: These results indicate that there is poor agreement between FMISO PET hypoxia measurements when compared to hypoxia as defined by EPR pO2 images. Applying our correction method significantly improved the overlap between PET and EPR hypoxic tumor regions. Citation Format: Inna Gertsenshteyn, Boris Epel, Lara Leoni, Xiaobing Fan, Richard Friefelder, Eugene Barth, Heejong Kim, Marta Zamora, Erica Markiewicz, Darwin Bodero, Mohammed Bhuiyan, Anna Kucharski, Hsiu-Ming Tsai, Mellissa Grana, Subramanian V. Sundramoorthy, Gregory S. Karczmar, Chien-Min Kao, Chin-Tu Chen, Howard Halpern. Multimodal imaging of tumor hypoxia with 18F-misonidazole PET, EPR, and MRI [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1648.

Dynamic Electron Paramagnetic Resonance Imaging: Modern Technique for Biodistribution and Pharmacokinetic Imaging

The imaging of the biodistribution and pharmacokinetics is critical in understanding the complexity of drug delivery mechanisms, the status of the disease, and the monitoring of the treatment progr...

Biological validation of electron paramagnetic resonance (EPR) image oxygen thresholds in tissue.

Measuring molecular oxygen levels in vivo has been the cornerstone of understanding the effects of hypoxia in normal tissues and malignant tumors. Here we discuss the advances in a variety of partial pressure of oxygen ( ) measurements and imaging techniques and relevant oxygen thresholds. A focus on electron paramagnetic resonance (EPR) imaging shows the validation of treating hypoxic tumours with a threshold of ≤10Torr, and demonstrates utility for in vivo oxygen imaging, as well as its current and future role in cancer studies.

Detection of Antioxidant Phytochemicals Isolated from Camellia japonica Seeds Using HPLC and EPR Imaging.

In this study, we investigated the formation of stable radicals and compounds related to antioxidants in Camellia japonica seeds using high performance liquid chromatography (HPLC) and X-band electron paramagnetic resonance imaging (EPRI). The C. japonica seed coat extracts exhibited antioxidant activity in both in vitro and cell-based studies. The extracts inhibited reactive oxygen and reactive nitrogen species production in cell-based studies. HPLC chromatograms indicated that hydrophilic antioxidant compounds—namely, gallic acid, gallocatechin, epigallocatechin, caffeine, catechin, epicatechin, and epicatechin gallate—were found in the methanolic extract. Lipophilic antioxidant compounds—including α-tocopherol, γ-tocopherol, β-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotrienol, and δ-tocotrienol—were found in the hexane extract. EPRI primarily detected paramagnetic species in seed coats. These radical species were stable organic radicals based on the peak-to-peak line width and g-values. The signals from these stable radicals were strong and stable with a g-value of 2.002. Noninvasive EPRI of the radicals present in C. japonica seeds indicated that the stable radicals were essentially located in the seed coats. The EPRI of the cotyledon demonstrated that additional radicals were localized at an apex of the cotyledon. The results indicated that the stable radicals detected by EPRI and the hydrophilic and lipophilic antioxidant compounds analyzed by HPLC were related to antioxidant reactants and products.

Molecular Imaging of the Tumor Microenvironment Reveals the Relationship between Tumor Oxygenation, Glucose Uptake, and Glycolysis in Pancreatic Ductal Adenocarcinoma.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. SIGNIFICANCE: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

Multiharmonic electron paramagnetic resonance imaging as an innovative approach for in vivo studies

This work is the first report when multiharmonic analysis (MHA) was applied for electron paramagnetic resonance imaging (EPRI) for in vivo applications. Phantom studies were performed for established methodology, and in vivo imaging was conduct as a proof-of-concept. Phantom studies showed at least six times improvement of the signal – to – noise (S/N) ratio. Application MHA for 3D EPR in vivo imaging provides images of spin probe distribution in mouse head. The EPRI, in combination with nitroxide and trityl spin probe, was performed to obtained 3D EPR in vivo images using MHA. For both used spin probes, MHA provided images with better S/N ratio, especially in the case of nitroxide, where projections obtained using conventional CW did not allow for reconstructing reliable data. Trityl radical exhibited high resolution and quality of obtained images after MHA. The MHA methodology allows the selection of a second modulation amplitude even 40 times higher than the natural EPR linewidth of the spin probe without line shape distortion, which highly improves the sensitivity of the acquired signal and allowing for imaging mice regardless of their size in a routine animal experiment.

Electron Paramagnetic Resonance Imaging of Melanin in Honey Bee

Honey bees play a crucial role in the nature by pollinating wild flowers. Over the past years, there has been an increasing concern regarding the honey bee colony decline. Pesticides or environmental effects targeting the biochemistry of insect chitin and cuticle coating may be in part responsible for honey bee pathologies. We here propose the use of electron paramagnetic resonance imaging (EPRI) as a tool to image the melanin-chitin complexes as part of the exoskeleton of the honey bee. EPRI at 9.65 GHz was applied on intact freeze-dried bees. The imaging data were collected on the melanin peak. High-resolution images revealed that this compound is extensively distributed in the periphery of the animal, data consistent with the localization in the cuticle of the bee. While EPR of melanin has been so far explored in the context of melanoma characterization, it may offer new opportunities in research on honey bees and other insects.

Nondestructive Evaluations of Melanin-related Compounds in the Skin Using Electron Paramagnetic Resonance and Permeability Measurements.

Melanin-related compounds in paraffin-embedded tissue specimens of basal cell carcinoma (BCC), seborrheic keratosis (SK), malignant melanoma (MM), and nevus pigmentosus (NP) were nondestructively investigated using permeability measurements (light penetration into pigmented lesion), X-band (9.4 GHz) electron paramagnetic resonance (EPR), and EPR imaging (EPRI). The paramagnetic species in BCC, SK, MM, and NP specimens were analyzed using intensity, linewidth, spectral pattern, and X-band EPRI. The EPR spectra of BCC, SK, and NP showed a single line pattern. The EPR signal intensities of the BCC, SK, and NP samples corresponded to the permeability values that are directly related to pigment color tone, except for MM. The correlation coefficient between EPR and permeability was supported by the high degree of linear relation in the range. We further analyzed MM and speculated that MM contains an additional signal of the pheomelanin radical. In MM and NP samples, two-dimensional (2D) EPRI revealed paramagnetic species distribution and different magnitudes. The paramagnetic (radical) species are directly related to the pigmented lesion site. To conclude, spectroscopic analyses suggest that pheomelanin-related compounds may exist in malignant melanoma.

A Doubly Constrained TV Algorithm for Image Reconstruction

Purpose. The total variation (TV) minimization algorithm is an effective image reconstruction algorithm capable of accurately reconstructing images from sparse and/or noisy data. The TV model consists of two terms: a data fidelity term and a TV regularization term. Two constrained TV models, data divergence-constrained TV minimization (DDcTV) and TV-constrained data divergence minimization (TVcDM), have been successfully applied to computed tomography (CT) and electron paramagnetic resonance imaging (EPRI). In this work, we propose a new constrained TV model, a doubly constrained TV (dcTV) model, which has the potential to further improve the reconstruction accuracy for the two terms which are both of constraint forms. Methods. We perform an inverse crime study to validate the model and its Chambolle-Pock (CP) solver and characterize the performance of the dcTV-CP algorithm in the context of CT. To demonstrate the superiority of the dcTV model, we compare the convergence rate and the reconstruction accuracy with the DDcTV and TVcDM models via simulated data. Results and Conclusions. The performance-characterizing study shows that the dcTV-CP algorithm is an accurate and convergent algorithm, with the model parameters impacting the reconstruction accuracy and the algorithm parameters impacting the convergence path and rate. The comparison studies show that the dcTV-CP algorithm has a relatively fast convergence rate and can achieve higher reconstruction accuracy from sparse projections or noisy projections relative to the other two single-constrained TV models. The knowledge and insights gained in the work may be utilized in the application of the new model in other imaging modalities including divergence-beam CT, magnetic resonance imaging (MRI), positron emission tomography (PET), and EPRI.

Imaging of Tumor-Specific Hypoxia Dynamics and Its Significance in Radiation Biology

Hypoxia has been known to be a feature associated with tumor radioresistance. So far, clinical strategies to overcome chronic hypoxia due to the limitation of the oxygen diffusion have been designed. However, intermittent or acute/cycling hypoxia, whose frequency can range between a few cycles per minutes to hours, is receiving increased attention, because this type of hypoxia has been reported to have an influence on tumor malignancy as well as treatment resistance via increased expression of pro-survival pathways. Therefore, a priori information on fluctuating hypoxia can be important in clinical treatment planning, but complicated dynamics makes it difficult to elucidate biological significance of intermittent hypoxia.Here, we illustrate the use of pulsed electron spin resonance imaging (ESRI) as a novel imaging method to directly monitor fluctuating oxygenation i.e. cycling hypoxia in transplanted tumors. A common resonator platform for both ESRI and magnetic resonance imaging (MRI) provided pO2 maps with anatomical guidance without positional movement. Oxygen images every 3 min in pO2 could visualize the rapid oxygen fluctuation and distinguish the cycling hypoxia and chronic hypoxia. Furthermore, we have examined the vascular renormalization process by longitudinally pO2 mapping during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation and the decrease of cycling tumor hypoxia were visualized by ESRI 2 to 4 days following antiangiogenic treatments. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth.In conclusion, this ESRI technique combined with MRI, may offer a powerful clinical tool to noninvasively detect variable hypoxic status in tumors and to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs.

Structural Analysis of the Stratum Corneum using EPR and EPR Imaging with Stable Spin Probes.

X-band (9.4 GHz) electron paramagnetic resonance (EPR) and electron paramagnetic resonance imaging (EPRI) were used for elucidating structural aspects of the stratum corneum (SC). We found that psoriasis vulgaris (PV)-SC has a less-ordered structure than that of the control SC, indicating the abnormal architecture of PV-SC. Different spectra were observed for the PV-SC. The three-line spectral pattern suggests that the 5-doxylstearic acid (5-DSA) is mobile or less rigid in the SC. The simulated order parameter (S0) value obtained for 5-DSA in the SC was approximately 0.20. The statistical analysis suggests that the value of the PV-SC is significantly smaller than that of the control (p < 0.01). Thus, we suggest that this EPR assay is of great use for evaluating SC function. In addition, the EPRI of various SC samples provides a useful image concerning SC status. A strong red image was observed for the PV skin. No red lesion region was observed in the control. The EPR images differentiated various sizes and number distribution concerning the disordered states in the SC.

Estimation of pO2 histogram from a composite EPR Spectrum of multiple random implants.

Electron paramagnetic resonance (EPR) spectroscopy using oxygen-sensing implants can provide reliable and repeated measurements of the partial pressure of oxygen (pO2) over a period of months or longer; however, it does not provide accurate information about the distribution of tissue oxygenation. While EPR imaging has the capability to provide spatially resolved oxygen data, it is time-consuming and not optimized for discrete number of implants. Previous reports suggest multi-site algorithms, which would require either the implants to be aligned in a certain way so as to deconvolve them using a linear magnetic field gradient or sparse imaging of the implants from a small number of 3D projections. In this paper, we present a simpler and much faster method to estimate the pO2 histogram from a composite, single-scan EPR spectrum measured without applying field gradients to separate the EPR signals from multiple randomly placed oxygen-sensing implants. The method is optimized for a discrete number of implants, validated using simulations, experimental phantoms and in animal models. The results established the composite spectral fitting algorithm as a reliable and robust tool for multi-site oximetry.

Transplantation of Mesenchymal Stem Cells Improves Amyloid-β Pathology by Modifying Microglial Function and Suppressing Oxidative Stress

Mesenchymal stem cells (MSC) are increasingly being studied as a source of cell therapy for neurodegenerative diseases, and several groups have reported their beneficial effects on Alzheimer’s disease (AD). In this study using AD model mice (APdE9), we found that transplantation of MSC via the tail vein improved spatial memory in the Morris water maze test. Using electron paramagnetic resonance imaging to evaluate the in vivo redox state of the brain, we found that MSC transplantation suppressed oxidative stress in AD model mice. To elucidate how MSC treatment ameliorates oxidative stress, we focused on amyloid-β (Aβ) pathology and microglial function. MSC transplantation reduced Aβ deposition in the cortex and hippocampus. Transplantation of MSC also decreased Iba1-positive area in the cortex and reduced activated ameboid shaped microglia. On the other hand, MSC transplantation accelerated accumulation of microglia around Aβ deposits and prompted microglial Aβ uptake and clearance as shown by higher frequency of Aβ-containing microglia. MSC transplantation also increased CD14-positive microglia in vivo, which play a critical role in Aβ uptake. To confirm the effects of MSC on microglia, we co-cultured the mouse microglial cell line MG6 with MSC. Co-culture with MSC enhanced Aβ uptake by MG6 cells accompanied by upregulation of CD14 expression. Additionally, co-culture of MG6 cells with MSC induced microglial phenotype switching from M1 to M2 and suppressed production of proinflammatory cytokines. These data indicate that MSC treatment has the potential to ameliorate oxidative stress through modification of microglial functions, thereby improving Aβ pathology in AD model mice.

Thiyl Radicals are Co-products of Dinitrosyl Iron Complexes (DNIC) Formation

This work is the first report when multiharmonic analysis (MHA) was applied for electron paramagnetic resonance imaging (EPRI) for in vivo applications. Phantom studies were performed for established methodology, and in vivo imaging was conduct as a proof-of-concept. Phantom studies showed at least six times improvement of the signal – to – noise (S/N) ratio. Application MHA for 3D EPR in vivo imaging provides images of spin probe distribution in mouse head. The EPRI, in combination with nitroxide and trityl spin probe, was performed to obtained 3D EPR in vivo images using MHA. For both used spin probes, MHA provided images with better S/N ratio, especially in the case of nitroxide, where projections obtained using conventional CW did not allow for reconstructing reliable data. Trityl radical exhibited high resolution and quality of obtained images after MHA. The MHA methodology allows the selection of a second modulation amplitude even 40 times higher than the natural EPR linewidth of the spin probe without line shape distortion, which highly improves the sensitivity of the acquired signal and allowing for imaging mice regardless of their size in a routine animal experiment.

Effect of Glutathione and Ascorbic Acid on the Brain Redox Status in Transgenic Mouse Model of Alzheimer’s Disease Evaluated by EPR Imaging

Effect of Glutathione and Ascorbic Acid on the Brain Redox Status in Transgenic Mouse Model of Alzheimer’s Disease Evaluated by EPR Imaging

Investigation of Pigments in Thai Purple Rice Using Electron Paramagnetic Resonance Imaging and HPLC.

Paramagnetic species (radicals) related pigments in Thai purple rice were investigated by electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. The location and distribution of the paramagnetic species in purple and white rice were determined by EPR and EPRI. EPR primarily detected three paramagnetic species in purple rice, which were identified as organic radicals, Mn2+, and Fe3+ based on the g-values and hyperfine components of the EPR signals. Noninvasive two-dimensional (2D) EPRI revealed that these stable radicals are primarily located in the pigmented region of purple rice, while very few radicals were observed in the interior of the rice. HPLC revealed that the major compounds were cyanidin-3-O-glucoside and peonidin-3-O-glucoside. EPR, EPR imaging, and HPLC results indicate that the stable radicals contain the radical state of anthocyanins and are mostly found within the pigmented embryo region of purple rice. They could be either associated with antioxidant activities or could be one of the products of their oxidative decomposition.

Comparison of TVcDM and DDcTV algorithms in image reconstruction

ABSTRACTThe total variation (TV) minimization algorithms is a classical representative of the optimization algorithms and have been widely used for their simpleness, robustness, expandability and their potential of accurately reconstructing images from sparse data or noisy data. The commonly used constrained TV (cTV) program is the data-divergence constrained TV (DDcTV) minimization. The other recently proposed cTV program is the TV constrained data-divergence minimization (TVcDM). In the work, we compare the two programs the convergence rate and the reconstruction accuracy via the sparse-view projection set and the noisy projection set in the context of CT. Further, we compare their reconstruction accuracy via real data in electron paramagnetic resonance imaging (EPRI). The studies show that TVcDM has much faster convergence rate relative to DDcTV and that TVcDM may achieve higher accuracy in most cases no matter in sparse-view projections CT reconstructions or in noisy projections CT reconstructions or in EPRI sparse reconstructions. From a comprehensive perspective, TVcDM has superior convergence and accurate reconstruction performance relative to DDcTV and should be recommended in the application of TV reconstruction algorithms. The knowledge and insights gained in the work may be extended to the performance analysis of other optimization-based reconstruction algorithms.