In Vivo Temporal EPR Measurements in the Lung of Mice by a Selected-Region Intensity Determination Method

Selected-region intensity determination (SRID) is a method for obtaining the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region without complicated procedures used in the previous imaging method. We used an in vivo 700 MHz radiofrequency EPR spectrometer equipped with a bridged loop-gap resonator and the SRID method to perform temporal EPR measurements of the lung area of mice which had received a nitroxide radical (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl) administration via the intravenous route. The half-life and initial level of nitroxide radical in the lung or the mediastinum were calculated from temporal changes in the signal intensity. A mathematical model was devised to determine the nitroxide radical concentration in the lung, which is connected to other organs via the circulatory system. Using this model and the results of the EPR measurements, the degrees of influence of the nitroxide reduction in the lung and other organs were simulated. It was found that the reaction rate (=log2/half-life) obtained from the lung mainly reflected the reduction of nitroxide radical there. Authors' address: Hidekatsu Yokoyama, National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, 463-8560 Nagoya, Japan     

In vivo temporal EPR imaging for estimating the kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats

The kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats were investigated by employing an in vivo EPR imaging system equipped with a surface-coil-type resonator (SCR). The exposed kidney of a living rat was inserted into the single-turn coil of the SCR, with the renal major axis aligned with the direction of alternative magnetic field (B(1)). After the injection of nitroxide radical via the tail vein, EPR measurements were repeated. From the temporal EPR images of the kidney on the 2-D projection to the plane which is perpendicular to the direction of B(1,) the decay rate of nitroxide radical in the renal parenchyma and pelvis was estimated. The parenchymal decay rate was found to be significantly shorter than that for the pelvis.     

Determination of absolute concentration of nitroxide radical by radio-frequency EPR imaging

The absolute concentrations of a nitroxide radical in samples in a loop-gap resonator (LGR) were determined by using a radio-frequency (about 720 MHz) electron paramagnetic resonance (EPR) imaging system. EPR imaging of phantoms containing a nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (carbamoyl-PROXYL), dissolved in various concentrations of an aqueous sodium chloride solution was made to investigate the influence of dielectric losses and sample position within the LGR. As it was found that these influences on the signal intensity were sufficiently small (less than 6%), it is possible to use identical radical solutions in which the radical is dissolved in a known concentration as an internal marker. Two phantoms containing aqueous solutions of 3 mM (as a marker) and 1, 2, 3, 4, or 5 mM (as a sample) carbamoyl-PROXYL were placed together in the LGR. From EPR images of these phantoms, the absolute concentration of the sample could be calculated by using the gray-scale value (i.e., the signal intensity) of the marker and sample within a small margin of error (about 4%).     

In vivo temporal EPR study using a region-selected intensity determination method to estimate cerebral reducing ability in rats treated with olanzapine

Region-selected intensity determination (RSID) is a method for obtaining the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a target region, without the use of complicated procedures employed in the conventional imaging methods. An in vivo 700-MHz radio frequency EPR spectrometer equipped with a bridged loop-gap resonator was used with the RSID method to estimate intracerebral reducing ability in the rat following acute administration of olanzapine (OZP) or haloperidol (HPD). To this end, temporal changes in EPR signal intensity of target regions (the striatum and the prefrontal cortex) of rats which had received a blood-brain-barrier-permeable nitroxide radical (3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl) via an intravenous route were observed. The half-lives of EPR signal intensity in both regions of OZP- or HPD-treated rats were significantly longer than in control animals. This indicated that reducing abilities of the striatum and cerebral cortex decreased in the rats to which either OZP or HPD had been acutely administered.     

EPR imaging to estimate the in vivo intracerebral reducing ability of mature rats after neonatal hypoxic-ischemic brain injury

A rat model of neonatal hypoxic-ischemic encephalopathy (Rice's model) was obtained by unilateral ligation of the common carotid artery of 7-day-old rats with hypoxia (exposure to 8% oxygen). To estimate the in vivo intracerebral reducing ability of the mature rats (8 weeks old) of Rice's model, temporal electron paramagnetic resonance (EPR) imaging of the brain of a rat receiving a blood-brain barrier-permeable nitroxide radical, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, was performed. In this imaging technique, the decay rate of the EPR signal intensity in a selected region of the brain is indicative of region-specific reducing ability. The effect of neonatal treatment of an antioxidant agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), after a hypoxic-ischemic insult was also tested. It was found that the reducing ability had been depleted in the contralateral hemisphere of Rice's model rats; this depletion was suppressed by administering MCI-186.     

Reaction kinetics analysis to estimate in vivo decay rate of EPR signals of a nitroxide radical in the brain and the inferior vena cava of rats

To clarify the degree of the influence of the peripheral organs on the temporal changes in the electron paramagnetic resonance (EPR) signal intensity of a nitroxide radical permeating the blood-brain barrier, 3-hydroxymethyl-2,2,5,5-tetramethylprrolidine-1-oxyl (hydroxymethyl-PROXYL), in the brain, temporal changes in the EPR signal in the brain or inferior vena cava of rats were measured by an in vivo 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator or a surface-coil-type resonator. In all measurements, good linearity was observed on semilogarithmic plots of the signal intensity against time after the hydroxymethyl-PROXYL injection. From these plots, the reaction rate and the initial level of hydroxymethyl-PROXYL in the brain and the vena cava were calculated. A mathematical model expressing the nitroxide radical concentration in the brain, which is connected to other organs via the circulatory system, was made. With this model and the results of the EPR measurements, the degrees of influence of the nitroxide reduction in the brain and the other organs were simulated. It was found that the reaction rate (equal to log2/half-life) of hydroxymethyl-PROXYL observed in the brain reflected the reduction of hydroxymethyl-PROXYL there and was not influenced by the reduction in other organs.     

Influence of the lens effect in a sample with large dielectric constant in a loop-gap resonator on the EPR signal intensity at 700 MHz

The influence of the lens effect on the electron paramagnetic resonance (EPR) signal intensity was investigated in a loop-gap resonator (LGR) with an inner diameter of 41 mm. TheQ- value and EPR signal intensity were measured when the phantoms containing 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidin-l-yloxy dissolved in sodium chloride aqueous solutions were put in the LGR. TheQ- value and signal intensity reduced with increasing concentrations of sodium chloride in the phantom, indicating that the imaginary part of the dielectric constant is larger in the phantom with the higher concentration of sodium chloride. However, relationships betweenQ-values of the resonator and EPR signal intensities were not proportional and signal intensities were relatively higher compared with theQ-values. These findings suggest that the signal reduction due to lowQ is slightly compensated by the lens effect in the sample with the large real part of the dielectric constant. In the distribution of the signal intensities of a pinpoint sample made of diphenylpicrylhydrazyl in the agar medium containing sodium chloride in the LGR, it was found that the signal intensity decreased according to the distance from the center and the difference in the signal intensity within 10 mm from the center was about 20%, indicating the inhomogeneity of the alternating magnetic field at the center and marginal region in the sample with the large dielectric constant caused by the lens effect.     

In vivo temporal EPR imaging of the brain of rats by using two types of blood-brain barrier-permeable nitroxide radicals

In vivo temporal EPR imaging was conducted on the brain of rats that received one of two kinds of blood-brain barrier-permeable nitroxide radicals via the tail vein-one is a water-soluble 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (hydroxymethyl-PROXYL); and the other is a non-water-soluble 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). From temporal EPR imaging data, temporal changes in the distribution of the nitroxide radical in the cerebral cortex, striatum, and hippocampus in the brain were investigated. It was found that the half-lives of the three parts in the brain of hydroxymethyl-PROXYL are longer and their EPR signal intensities are greater than those of PCAM.     

EPR Spectroscopy Using Magnetic Field Gradient Modulated by a Triangular Wave

Magnetic field gradient modulation is one of the techniques to obtain an electron paramagnetic resonance (EPR) spectrum in a selected region of the sample. In this study, the magnetic field gradient modulation using a triangular wave was performed to overcome a problem during the sine wave modulation. Plastic materials were used for the bobbins and cases of the electromagnet to reduce the eddy current loss and drive the gradient coils in three-dimensional directions at a frequency of over 160 Hz. While the EPR signal splitting in a nonselected region, which is a problem in spectral analysis, was observed during the simulation and the actual measurement with the sine wave gradient modulation, the EPR signal broadening without splitting was observed in those with the triangular wave modulation. Thus, it is postulated that the triangular wave is more suitable than the sine one for the field gradient modulation. The spatial resolution was determined to be about 4 or 2 mm at the field gradient of 1 or 2 mT/cm, respectively. The separation of the EPR spectra of two types of radicals was also made by the triangular wave gradient modulation. Authors' address: Hidekatsu Yokoyama, Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1, Kitakanemaru, Ohtawara 324-8501, Japan     

Direct EPR irradiation of a sample using a quartz oscillator operating at 250 MHz for EPR measurements

Direct irradiation of a sample using a quartz oscillator operating at 250 MHz was performed for EPR measurements. Because a quartz oscillator is a frequency fixed oscillator, the operating frequency of an EPR resonator (loop-gap type) was tuned to that of the quartz oscillator by using a single-turn coil with a varactor diode attached (frequency shift coil). Because the frequency shift coil was mobile, the distance between the EPR resonator and the coil could be changed. Coarse control of the resonant frequency was achieved by changing this distance mechanically, while fine frequency control was implemented by changing the capacitance of the varactor electrically. In this condition, EPR measurements of a phantom (comprised of agar with a nitroxide radical and physiological saline solution) were made. To compare the presented method with a conventional method, the EPR measurements were also done by using a synthesizer at the same EPR frequency. In the conventional method, the noise level increased at high irradiation power. Because such an increase in the noise was not observed in the presented method, high sensitivity was obtained at high irradiation power.     

Importance of volume limitation for tissue redox status measurements using nitroxyl contrast agents: a comparison of X-band EPR bile flow monitoring (BFM) method and 300 MHz in vivo EPR measurement

Methods proposed for in vivo redox status estimation, X-band (9.4 GHz) electron paramagnetic resonance (EPR) bile flow monitoring (BFM) and 300 MHz in vivo EPR measurement, were compared. The spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was utilized for both methods, due to its suitable lipophilicity. EPR signal decay of a nitroxyl spin probe in the bile flow and in the liver region (upper abdomen) of several rat groups with different selenium status were measured by both the BFM and the in vivo EPR method, respectively. The nitroxyl radical clearance measured with in vivo EPR method may be affected not only by the redox status in the liver but also by information from other tissues in the measured region of the rat. On the other hand, the time course of nitroxyl radical level in the bile flow of rats was found to be a reliable index of redox status. Measurement site and/or volume limitation, which was achieved by the BFM method in this paper, is quite important in estimating reasonable EPR signal decay information as an index of tissue/organ redox status.     

Coarse and Fine Control of the EPR Frequency of a Loop-Gap Resonator Using a Single-Turn Coil with a Varactor Diode Attached

Coarse control and fine control of the resonant frequency of a loop-gap resonator (LGR) operating at an electron paramagnetic resonance (EPR) frequency of ca. 650 MHz were achieved using a single-turn coil with a varactor diode attached (a frequency shift coil). When the distance between the LGR and the frequency shift coil was changed from 15 to 10 mm under the condition of constant voltage to the varactor diode (0 V), a shift of the resonant frequency of the LGR of ca. 20 MHz was observed (coarse frequency control). When the voltage applied to the varactor diode was changed from 0 to 15 V at the same distance between the LGR and the frequency shift coil (10 mm), a shift of the resonant frequency of the LGR of ca. 200 kHz was observed (fine frequency control). There were no significant changes in sensitivity of EPR measurements of a phantom (comprised of agar with a nitroxide radical and physiological saline solution) without and with the frequency shift coil. The EPR sensitivity did not change discernibly when the resonant frequency was shifted by the frequency shift coil. Furthermore, radio-frequency phase adjustment for homodyne detection could be performed by using the frequency shift coil without applying frequency modulation to the carrier wave.     

Gas diffusion in a pulmonary acinus model: experiments with hyperpolarized helium-3

Diffusion of hyperpolarized helium-3 in epoxy phantoms was experimentally studied by pulsed-gradient nuclear magnetic resonance (NMR). One phantom with a dichotomic branching structure densely filling a cubic volume was built using the Kitaoka algorithm to model a healthy human acinus. Two other phantoms, one with a different size and the other one with a partial destruction of the branched structure, were built to simulate changes occurring at the early stages of emphysema. Gas pressure and composition (mixture with nitrogen) were varied, thus exploring different diffusion regimes. Preliminary measurements in a cylindrical glass cell allowed us to calibrate the gradient intensity with 1% accuracy. Measurements of NMR signal attenuation due to gas diffusion were compared to a classical Gaussian model and to Monte Carlo simulations. In the slow diffusion regime, the Gaussian model was in reasonable agreement with experiments for low gradient intensity, but there was a significant systematic deviation at larger gradient intensity. An apparent diffusion coefficient Dapp was deduced, and in agreement with previous findings, a linear decrease of Dapp/D0 with D0(1/2) was observed, where D0 is the free diffusion coefficient. In the regime of intermediate diffusion, experimental data could be described by the Gaussian model for very small gradient intensities only. The corresponding Dapp/D0 values seemed to reach a constant value. Monte Carlo simulations were generally in fair agreement with the measurements in both regimes. Our results suggest that, for diffusion times typical of medical magnetic resonance imaging, an increase in alveolar size has more impact on signal attenuation than a partial destruction of the branched structure at equivalent surface-to-volume ratio.     

Combining a Magnetic Field Modulation Coil with a Surface-Coil-Type EPR Resonator

It is thought that the design of magnetic field modulation coils is one of the factors limiting enlargement of the sample size in electron paramagnetic resonance (EPR) measurements. In this study, we miniaturized the magnetic field modulation coil and combined it with a surface-coil-type resonator (SCR). The inductor of the SCR was a circular single-turn one-loop coil (diameter, 1 mm), and the magnetic field modulation coil was a twin-loop coil consisting of two solenoid coils each made of 15 turns of copper wire on a cylindrical bobbin with an axial length of 3 mm and an elliptical cross section (major axis, 7 mm; minor axis, 3 mm). The former was located on the latter via a spacer (thickness, 3 mm) in such a way that the directions of their axes coincided. Their combined size was about 10 mm wide, 10 mm deep, and 6 mm high. The transmission lines of the SCR were set on resonance at about 700 MHz. EPR measurements of a phantom (comprising agar that included a nitroxide radical and physiological saline solution), made with a miniaturized modulation coil combined with the SCR, exhibited a sensitivity similar to that for the conventional method. Authors' address: Hidekatsu Yokoyama, Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara 324-8501, Japan     

In VivoLongitudinally Detected ESR Measurements at Microwave Regions of 300, 700, and 900 MHz in Rats Treated with a Nitroxide Radical

A signal detector of longitudinally detected ESR (LODESR) is independent of the resonant frequency. We developed anin vivoLODESR spectrometer operating in the regions of 300, 700, and 900 MHz. Using this apparatus, we estimated signal intensities at different operating frequencies obtained from non- or high-dielectric loss phantoms that contained nitroxide radical solutions and from live rats that had received a nitroxide radical. Our result, higher signal intensities in the high-dielectric loss samples (such as physiological saline solution and animals) at a lower frequency, shows that the influence of a decrease in dielectric loss dominates over the signal reduction caused by smaller Zeeman splitting. We believe that this finding strongly supports anin vivoESR resonant frequency that tends to be low.     

A multilayered element resonator for CW-ESR and longitudinally detected ESR at radio frequency

The design and evaluation of a multilayered element resonator (MLR), which consists of multiple layers of half-loop conductor plates and insulator sheets, are presented. An MLR and a bridge shielded loop-gap resonator (BLGR), which have similar sizes and resonant frequencies, were fabricated to compare their performances. Using the MLR and the BLGR, the modulation field width and signal intensity of a phantom containing a nitroxide radical were measured by employing a continuous-wave electron spin resonance (CW-ESR) technique at a radio frequency of 300 MHz. Using the same resonators, the longitudinally detected ESR (LODESR) signal intensities of the phantom were also compared. The loadedQ values of the resonators were almost the same. The modulation widths in the MLR were significantly wider than those in the BLGR when the modulation coils were driven at the same voltage. The signal intensities of CW-ESR and LODESR from the phantom in the MLR were significantly greater than those from the BLGR. Since eddy currents disturb the penetration of the modulation field in CW-ESR or detection of changes in magnetization in LODESR observations, these results show that, in the MLR, the eddy currents were suppressed to a greater degree than in the BLGR.     

一种新型功能化双自由基的合成及EPR表征

新开发的双自由基(TN3)由全取代三苯甲基自由基(trityl)和氮氧自由基两部分组成.TN3的室温EPR谱图由4条不对称的宽谱线组成,具有典型的双自由基特征.以抗坏血酸作为模型化合物发现该双自由基能用来同时检测体系的氧化还原态和氧气浓度.     

In vivo EPR imaging by using an acyl-protected hydroxylamine to analyze intracerebral oxidative stress in rats after epileptic seizures

EPR imaging by using an acyl-protected hydroxylamine, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP), in the head of a living rat after kainic acid (KA)-induced epileptic seizures was performed. ACP is a stable non-radical compound, but is easily deprotected with intracellular esterase to yield a hydroxylamine, which is oxidized by intracellular oxidative stress to yield an EPR-detectable nitroxide radical. From in vivo image data, the average values of EPR signal intensity from the hippocampus, striatum, and cerebral cortex were computed. There was no significant difference in cortical signal intensity between the control and KA-treated rats. The signal intensities from the hippocampus and striatum for the KA-treated rats were significantly higher than those for the control. The in vitro study showed that almost the same quantity of ACP moved into all regions of the brain of the control and KA-treated rats. These findings indicate that following a KA-induced seizure, the oxidative stress in the hippocampus and striatum is enhanced, but not so in the cerebral cortex.     

Spin-trapped hydroxyl free radicals studied at low field by Field-Cycled Dynamic Nuclear Polarization

The technique of Field-Cycled Dynamic Nuclear Polarization (FC-DNP) involves the EPR irradiation of a free radical solution and the subsequent observation of the NMR signal, the experiment being carried out at a range of magnetic field strengths in order to measure the free radical’s EPR spectrum. In this work FC-DNP has been used to study the EPR spectrum of DMPO spin-trapped hydroxyl free radicals at magnetic field strengths between 0.5 mT and 13.0 mT (5–130 Gauss). The low-field EPR spectrum contains six separate EPR lines, in contrast to the well-known X-band spectrum where only four are seen. Knowledge of the spin-adduct’s EPR spectrum will be of use to workers involved in low-field EPR, especially those conducting biological or in-vivo spin-trapping experiments.     

Spatial-temporal correlation of a diffuse sound field

The spatial correlation function of the sound in a diffuse field is a quantity widely used in many reverbrant room acoustic applications. Although results for the spatial and temporal correlation for pure-tone and band-limited diffuse fields have already been developed, these have not been generalized for other signal types. This work presents a generalized derivation of the diffuse field spatial-temporal correlation which can be used for stationary random signals with given power spectral density. It is shown that the spatial-temporal correlation depends entirely on the temporal correlation of the signal exiting the diffuse field, or alternatively on its power spectral density. A simulation using the plane wave model is presented for tonal and broadband diffuse sound fields.