Electron Paramagnetic Resonance Imaging Research Articles

The optimal 18F-fluoromisonidazole PET threshold to define tumor hypoxia in preclinical squamous cell carcinomas using pO2 electron paramagnetic resonance imaging as reference truth.

To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 [Formula: see text] 10mmHg. Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images fromPET/EPR/MRI were co-registered and resampled to isotropic 0.5mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVmean and SUV [Formula: see text] 0.6 [Formula: see text] SUVmax. Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

Dual-Mode Tumor Imaging Using Probes That Are Responsive to Hypoxia-Induced Pathological Conditions.

Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.

Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x‐ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2‐weighted anatomical images for reference. The differences in the mean pO2 were determined through two‐tailed Student's t‐test and one‐way analysis of variance. The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose‐dependent increase in the hypoxic areas (pO2 < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2 > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation‐based radiation therapy.

Abstract 5974: Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse

Abstract [Purpose] In pancreatic ductal adenocarcinoma (PDAC) which is characterized by an intense desmoplastic feature, the extracellular matrix (ECM) can significantly influence the tumor microenvironment (TME). Hyaluronan (HA), a major component of ECM, is associated with elevated tumor pressure, vascular collapse, and poor perfusion in TME, conferring hypoxia. HA expression is also correlated with poor prognosis in the patients with PDAC. PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan in tumors. The resultant improvement in vascular patency and blood perfusion is expected to increase the delivery of therapeutic molecules. The aim of this study was to investigate the change in physiologic and metabolic profile of the tumor in response to treatment with PEGPH20 using multi-modal imaging techniques. We also investigated the capability of PEGPH20 to enhance treatment effect of radiation. [Methods] Athymic nude mice were inoculated with BxPC3 (human pancreatic adenocarcinoma) tumor cells transduced with hyaluronan synthase 3 (HAS3) to the right tibial periosteum. BxPC3-HAS3 tumor treated with PEGPH20 or control buffer were scanned with Electron paramagnetic Resonance imaging (EPRI), dynamic contrast enhanced (DCE) MRI, ultra-small superparamagnetic iron oxide (USPIO) MRI, Photoacoustic imaging (PAI), and Hyperpolarized 13C-MRI using [1-13C] pyruvate to evaluate intratumor pO2, intratumor perfusion, blood volume, O2 saturation, and glycolysis, respectively. [Results] EPRI showed significantly increased pO2 in PEGPH20 treated group. DCE-MRI and USPIO-MRI showed improved perfusion/permeability and local blood volume, respectively after PEGPH20 treatment, accounting for the increase in tumor oxygenation. PAI provided the evidence of immediate changes in tumor oxygenation after treatment. Hyperpolarized 13C-MRI using [1-13C] pyruvate suggested the decreased glycolytic flux evaluated by lactate/pyruvate ratio after PEGPH20 treatment. Combination of radiotherapy and PEGPH20 synergistically delayed tumor progression and prolonged the survival. [Conclusions] This study examined the effect of PEGPH20 on TME in PDAC xenograft model by using non-invasive multimodal imaging techniques. In summary, the non-invasive imaging modalities were useful in evaluating the changes in hemodynamics and metabolism in TME induced by modulation of ECM such as PEGPH20 treatment. PEGPH20 enhanced treatment effect of radiation therapy. The results validated the utility of the imaging methods to non-invasively monitor the changes in TME and predicted the radiosensitizing effect of hyaluronan depletion. Citation Format: Yu Saida, Tomohiro Seki, Shun Kishimoto, Jeffrey R. Brender, Gadisetti VR. Chandramouli, Yasunori Otowa, Kota Yamashita, Kazutoshi Yamamoto, Nallathamby Devasahayam, Murali C. Krishna. Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5974.

The Release of a Highly Cytotoxic Paullone Bearing a TEMPO Free Radical from the HSA Hydrogel: An EPR Spectroscopic Characterization.

This study shows the potential of a thermally induced human serum albumin (HSA) hydrogel to serve as a drug depot for sustained release of a highly cytotoxic modified paullone ligand bearing a TEMPO free radical (HL). The binding of HL to HSA was studied by electron paramagnetic resonance (EPR) spectroscopy and imaging. The EPR protocol was also implemented for the study of matrix degradation, and ligand diffusion rate, in two additional spin-labeled hydrogels, containing 5-doxylstearate and 3-carbamoyl-proxyl. The results showed that the hydrogel is an efficient HL reservoir as it retained 60% of the ligand during 11 days of dialysis in physiological saline. Furthermore, upon incubation with Colo 205 human colon adenocarcinoma cells for 3 days, the HL/HSA hydrogel did not exhibit cytotoxic activity, demonstrating that it is also an efficient ligand depot in the presence of living cells. It was observed that the percentage of HL release is independent of its initial concentration in the hydrogel, suggesting that HSA possesses a specific binding site for the ligand, most likely Sudlow site 2, as predicted by molecular docking. The intrinsic property of albumin to bind and transport various substances, including hydrophobic drugs, may be fine-tuned by appropriate physical/chemical hydrogel preparation procedures, providing optimal drug delivery.

PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

PurposePEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan, an important component of the extracellular matrix, increasing the delivery of therapeutic molecules. Combinations of chemotherapy and PEGPH20, however, have been unsuccessful in Phase III clinical trials. We hypothesize that by increasing tumor oxygenation by improving vascular patency and perfusion, PEGPH20 will also act as a radiosensitization agent. Experimental DesignThe effect of PEGPH20 on radiation treatment was analyzed with respect to tumor growth, survival time, p02, local blood volume, and the perfusion/permeability of blood vessels in a human pancreatic adenocarcinoma BxPC3 mouse model overexpressing hyaluronan synthase 3 (HAS3). ResultsMice overexpressing HAS3 developed fast growing, radiation resistant tumors that became rapidly more hypoxic as time progressed. Treatment with PEGPH20 increased survival times when used in combination with radiation therapy, significantly more than either radiation therapy or PEGPH20 alone. In mice that overexpressed HAS3, EPR imaging showed an increase in local pO2 that could be linked to increases in perfusion/permeability and local blood volume immediately after PEGPH20 treatment. Hyperpolarized [1-13C] pyruvate suggested PEGPH20 caused a metabolic shift towards decreased glycolytic flux. These effects were confined to the mice overexpressing HAS3 - no effect of PEGPH20 on survival, radiation treatment, or pO2 was seen in wild type BxPC3 tumors. ConclusionsPEGPH20 may be useful for radiosensitization of pancreatic cancer but only in the subset of tumors with substantial hyaluronan accumulation. The response of the treatment may potentially be monitored by non-invasive imaging of the hemodynamic and metabolic changes in the tumor microenvironment.

Harnessing Electron Spin Hyperpolarization in Chromophore-Radical Spin Probes for Subcellular Resolution in Electron Paramagnetic Resonance Imaging: Concept and Feasibility.

Obtaining a subcellular resolution for biological samples doped with stable radicals at room temperature (RT) is a long-sought goal in electron paramagnetic resonance imaging (EPRI). The spatial resolution in current EPRI methods is constrained either because of low electron spin polarization at RT or the experimental limitations associated with the field gradients and the radical linewidth. Inspired by the recent demonstration of a large electron spin hyperpolarization in chromophore-nitroxyl spin probe molecules, the present work proposes a novel optically hyperpolarized EPR imaging (OH-EPRI) method, which combines the optical method of two-photon confocal microscopy for hyperpolarization generation and the rapid scan (RS) EPR method for signal detection. An important aspect of OH-EPRI is that it is not limited by the abovementioned restrictions of conventional EPRI since the large hyperpolarization in the spin probes overcomes the poor thermal spin polarization at RT, and the use of two-photon optical excitation of the chromophore naturally generates the required spatial resolution, without the need for any magnetic field gradient. Simulations based on time-dependent Bloch equations, which took into account both the RS field modulation and the hyperpolarization generation by optical means, were performed to examine the feasibility of OH-EPRI. The simulation results revealed that a spatial resolution of up to 2 fL can be achieved in OH-EPRI at RT under in vitro conditions. Notably, the majority of the requirements for an OH-EPRI experiment can be fulfilled by the currently available technologies, thereby paving the way for its easy implementation. Thus, the proposed method could potentially bridge the sensitivity gap between the optical and magnetic imaging techniques.

Redox-Sensitive Mapping of a Mouse Tumor Model Using Sparse Projection Sampling of Electron Paramagnetic Resonance.

Aims: This work aimed to establish an accelerated imaging system for redox-sensitive mapping in a mouse tumor model using electron paramagnetic resonance (EPR) and nitroxyl radicals.Results: Sparse sampling of EPR spectral projections was demonstrated for a solution phantom. The reconstructed three-dimensional (3D) images with filtered back-projection (FBP) and compressed sensing image reconstruction were quantitatively assessed for the solution phantom. Mouse xenograft models of a human-derived pancreatic ductal adenocarcinoma cell line, MIA PaCa-2, were also measured for redox-sensitive mapping with the sparse sampling technique.Innovation: A short-lifetime redox-sensitive nitroxyl radical (15N-labeled perdeuterated Tempone) could be measured to map the decay rates of the EPR signals for the mouse xenograft models. Acceleration of 3D EPR image acquisition broadened the choices of nitroxyl radical probes with various redox sensitivities to biological environments.Conclusion: Sparse sampling of EPR spectral projections accelerated image acquisition in the 3D redox-sensitive mapping of mouse tumor-bearing legs fourfold compared with conventional image acquisition with FBP. Antioxid. Redox Signal. 36, 57–69.

Mapping the Distribution and the Microstructural Dimensions of Metallic Lithium Deposits in an Anode-Free Battery by In Situ EPR Imaging

Mapping the Distribution and the Microstructural Dimensions of Metallic Lithium Deposits in an Anode-Free Battery by In Situ EPR Imaging

Development and Preclinical Study of Free Radical Imaging Using Field-Cycling Dynamic Nuclear Polarization MRI.

Free radicals, such as metabolic intermediates, reactive oxygen species, and metal enzymes, are key substances in organisms, although they can also cause various oxidative diseases. Thus, in vivo free radical imaging should be considered as the ultimate form of metabolic imaging. Unfortunately, electron spin resonance (ESR) imaging has inherent disadvantages, such as free radicals with large linewidths generating blurred images and the presence of two or more free radicals resulting in a complicated imaging procedure. Dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method to visualize in vivo free radicals, theoretically, with the same resolution as the MRI anatomical resolution, and fixed low-field DNP-MRI provides unique information on oxidative diseases and cancer. However, the large gyromagnetic ratio of the electron spin, which is 660-fold greater than that of a proton, requires field cycling, wherein the external magnetic field should be varied during DNP-MRI observations. This causes difficulties in developing a DNP-MRI system for clinical purposes. We developed a novel field-cycling DNP-MRI system for a preclinical study. In the said system, the magnetic field is switched by rotationally moving two magnets, with a magnetic flux density of 0.3 T for MRI and 5 mT for ESR. The image quality was examined using various pulse sequences and ESR irradiation using nitroxyl radical as the phantom, and the optimum conditions were established. Using the system, we performed a preclinical study involving free radical imaging by placing the free radicals under the palm of a human hand.

Molecular-Level Release of Coumarin-3-Carboxylic Acid and Warfarin-Derivatives from BSA-Based Hydrogels.

This investigation aimed at developing BSA hydrogels as a controlled release system to study the release behavior of spin-labeled coumarin-3-carboxylic acid (SL-CCS) and warfarin (SL-WFR). The release profiles of these spin-labeled (SL-) pharmaceuticals from BSA hydrogels prepared with different procedures are compared in detail. The mechanical properties of the gels during formation and release were studied via rheology, while a nanoscopic view on the release behavior was achieved by analyzing SL-drugs–BSA interaction using continuous wave electron paramagnetic resonance (CW EPR) spectroscopy. The influence of type of drug, drug concentration, duration of gel formation, and gelation methods on release behavior were characterized by CW EPR spectroscopy, EPR imaging (EPRI), and dynamic light scattering (DLS), which provide information on the interaction of BSA with SL-drugs, the percentage of drug inside the hydrogel and the nature and size of the released structures, respectively. We found that the release rate of SL-CCS and SL-WFR from BSA hydrogels is tunable through drug ratios, hydrogel incubation time and gelation procedures. All of the results indicate that BSA hydrogels can be potentially exploited in controlled drug delivery applications.

Three-dimensional electron paramagnetic resonance imaging of mice using ascorbic acid sensitive nitroxide imaging probes

Nitroxide compounds have been used as redox-sensitive imaging probes by electron paramagnetic resonance (EPR) for assessing oxidative stress in vivo. Fast redox reactions of nitroxide radicals are favorable for assessment of higher redox sensitivity; however, a variety of nitroxides have not been trialed for use as imaging probes due to their very rapid in vivo reduction, which cannot be captured at the slow operation speed of existing EPR imagers. To overcome this limitation, we improved our EPR system to provide a stable and highly sensitive imaging operation. We challenged the improved EPR imager to perform three-dimensional (3D) EPR imaging of mouse brain using two useful nitroxide imaging probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) and 2,6-dispiro-4′,4ʺ-dipyrane-piperidine-4-one-N-oxyl (DiPy). The second-order rate constant of DiPy with ascorbic acid is 10 times larger than that of Tempol. The improved EPR imager obtained clear 3D EPR images of mouse brain and demonstrated that Tempol could exist with an unpaired electron. The imager also successfully obtained 3D EPR images of mouse head after administration of DiPy. As 126 projections can be acquired in a period of 6 s, 3D EPR imaging can visualize the sequential process of DiPy entering the brain, being distributed within the brain, and being reduced within the brain. These improvements to the EPR imager will enable useful nitroxide imaging probes that were previously unsuitable as imaging probes due to their rapid reduction to be considered for use for sensitive redox assessment in an in vivo system.

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy.

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

The Development of Time-Domain In Vivo EPR Imaging at NCI

The Development of Time-Domain In Vivo EPR Imaging at NCI

Rapid Scan EPR Oxygen Imaging in Photoactivated Resin Used for Stereolithographic 3D Printing.

Oxygen plays a critical role in the photopolymerization process resulting in the formation of solid structures from liquid resins during three-dimensional (3D) printing: it acts as a polymerization inhibitor. Upon exposure to light, oxygen is depleted. As a result, the polymerization process becomes activated. Electron paramagnetic resonance (EPR) imaging is described as a tool to visualize changes in oxygen distribution caused by light exposure. This nondestructive method uses radio waves and, therefore, is not constrained by optical opacity offering greater penetrating depth. Three proof-of-principle imaging experiments were demonstrated: (1) spatial propagation of the photopolymerization process; (2) oxygen depletion as a result of postcuring; and (3) oxygen visualization in a 3D printed spiral model. Commercial stereolithography (SLA) resin was used in these experiments. Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) probe was mixed with the resin to permit oxygen imaging. Li-naphthalocyanine probes are routinely used in various EPR applications because of their long-term stability and high functional sensitivity to oxygen. In this study, we demonstrate that EPR imaging has the potential to become a powerful visualization tool in the development of 3D printing technology, including bioprinting and tissue engineering.

Nitroxyl Radical as a Theranostic Contrast Agent in Magnetic Resonance Redox Imaging.

Significance: In vivo assessment of paramagnetic and diamagnetic conversions of nitroxyl radicals based on cyclic redox mechanism can be an index of tissue redox status. The redox mechanism of nitroxyl radicals, which enables their use as a normal tissue-selective radioprotector, is seen as being attractive on planning radiation therapy.Recent Advances: In vivo redox imaging using nitroxyl radicals as redox-sensitive contrast agents has been developed to assess tissue redox status. Chemical and biological behaviors depending on chemical structures of nitroxyl radical compounds have been understood in detail. Polymer types of nitroxyl radical contrast agents and/or nitroxyl radical-labeled drugs were designed for approaching theranostics.Critical Issues: Nitroxyl radicals as magnetic resonance imaging (MRI) contrast agents have several advantages compared with those used in electron paramagnetic resonance (EPR) imaging, while support by EPR spectroscopy is important to understand information from MRI. Redox-sensitive paramagnetic contrast agents having a medicinal benefit, that is, nitroxyl-labeled drug, have been developed and proposed.Future Directions: A development of suitable nitroxyl contrast agent for translational theranostic applications with high reaction specificity and low normal tissue toxicity is under progress. Nitroxyl radicals as redox-sensitive magnetic resonance contrast agents can be a useful tool to detect an abnormal tissue redox status such as disordered oxidative stress. Antioxid. Redox Signal. 36, 95–121.

Perchlorinated Triarylmethyl Radical 99% Enriched 13C at the Central Carbon as EPR Spin Probe Highly Sensitive to Molecular Tumbling.

Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging applications. We report the synthesis and characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon, 13C1-PTMTC. The anisotropy of the hyperfine splitting with the 13C1 (Ax = 26, Ay = 25, Az = 199.5 MHz) and the g (gx = 2.0015, gy = 2.0015, gz = 2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determined by spectral simulation or via the line width or the apparent Az after calibration, so the spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity.

Spatial distribution of apoplastic antioxidative constituents in maize root.

Apoplastic antioxidative constituents (enzymes, primary and secondary metabolites, ROS) from different root zones of hydroponically grown maize (Zea mays L.) were investigated using a noninvasive isolation procedure: filter strip method. Filter strips were placed at specific positions on the root surface: apical zone (tip) and basal zone (base) to absorb apoplastic fluid. Three major classes of low-weight metabolites (organic acids, sugars, and phenolics) have been identified by HPLC-ECD. The longitudinal distribution of sugars and organic acids had the same pattern: higher concentration in the tip than the base, while it was vice versa for phenolics. The specific activities of guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase were higher in the apoplastic fluid from the root base than the tip, and their different isoforms were separated by isoelectric focusing. Electron paramagnetic resonance (EPR) spectroscopy coupled with the spin-trapping method using DEPMPO showed a persistent generation of hydroxyl radical in the root tip. In vivo EPR imaging of the whole maize root with membrane-permeable and impermeable aminoxyl spin-probes, enabling real-time detection of ROS formation within and outside the membranes, demonstrated ROS accumulation on the root surface, while endodermis and central cylinder were ROS free. For the first time in plant research, 2D EPR images enabled the direct demonstration of site-specific free radical production along the root. Highly sensitive analytical techniques combined with the filter strips, as a non-invasive tool, have increased our knowledge of metabolic processes occurring in the apoplast and their spatial-temporal changes in small regions of the intact root tissue.

High fidelity triangular sweep of the magnetic field for millisecond scan EPR imaging

Linearity of the magnetic field sweep is important for high resolution continuous wave EPR imaging. Driving the field with triangular wave function is the most efficient way to scan EPR projections. However, the magnetic field sweep profile can be significantly distorted during fast millisecond projection scan. In this work, we introduce a method to generate highly linear and properly symmetrical triangular sweeps of the magnetic field using calibrated harmonics of the triangular wave function. First, the frequency response function of the EPR magnet and its power circuitry was obtained. For this, the field sweeping coil was driven with sinusoidal signals of different frequencies and the actual magnetic field inside the magnet was recorded. To cover wide range of frequencies, the measurements were carried out independently using gaussmeter, Hall-effect linear sensor integrated circuit, and an inductance coil. For each frequency, the system gain and the phase delay were determined. These data were used to adjust the amplitudes and the phases of individual harmonics of the triangular wave function. After the calibration, the maximum deviation of the magnetic field from the linear function was 0.05% of sweep width for 4 ms scan. The maximum discrepancy between the forward and the reverse scan was less than 0.04%. Sweep overhead time for changing the scan direction was 5%. The proposed approach allows generation of high fidelity triangular magnetic field sweeps with accuracy better than 0.1% for the range of the magnetic field sweep widths up to 48 G and scan duration from 10 s down to 1 ms.

Early detection of redox imbalance in the APPswe/PS1dE9 mouse model of Alzheimer’s disease by in vivo electron paramagnetic resonance imaging

Alzheimer’s disease (AD) is a common neurodegenerative disease that causes progressive cognitive decline. Deposition of amyloid-β (Aβ) peptides is the most important pathophysiological hallmark of AD. Oxidative stress induced by the generation of reactive oxygen species (ROS) is a prominent phenomenon in AD and is known to occur early in its course. Several reports have suggested a relationship between changes in redox status and AD pathology, including progressive Aβ deposition, glial cell activation, and inflammation. In the present study, we employed a newly designed three-dimensional continuous-wave digital electron paramagnetic resonance (EPR) imager with a blood-brain barrier (BBB)-permeable redox-sensitive piperidine nitroxide probe, 4-oxo-2,2,6,6-tetramethyl-piperidine-d16-1-oxyl, for early detection of changed brain redox status. Using this system, we noninvasively compared age-matched 7-month-old AD model mice with normal littermates (WT mice). The obtained brain redox images of AD and WT mice clearly showed impaired brain redox status of AD mice compared to WT, suggesting that oxidative damage had already increased in 7-month-old AD mice compared with age-matched WT mice. The pathological changes in 7-month-old mice in this study were detected earlier than in previous studies in which only AD mice older than 9 months of age could be imaged. Since EPR images suggested that oxidative damage was already increased in 7-month-old AD mice compared to age-matched WT mice, we also evaluated antioxidant levels and the activity of superoxide dismutase (SOD) in brain tissue homogenates of 7-month-old AD and WT mice. Compared to WT mice, decreased levels of glutathione and mitochondrial SOD activity were found in AD mice, which supports the EPR imaging results indicating impaired brain redox status. These results indicate that the EPR imaging method developed in this study is useful for early noninvasive detection of altered brain redox status due to oxidative disease.