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.     

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.     

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 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.     

Effect of Dissolved Oxygen on the Reduction of Stable Nitroxide by Ascorbic Acid: In Vitro Model for Acute Hypoxia Ischemia

The reaction between 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM), which is a blood–brain-barrier (BBB) permeable stable cyclic nitroxide, and ascorbic acid was examined in a low dissolved oxygen-phosphate buffer (low DO-PB, 0.1 mM DO) under 8% oxygen in a glove bag or in an ordinal dissolved oxygen-phosphate buffer (ordinal DO-PB, 0.3 mM DO) under 21% oxygen in an open atmosphere with and without diethylenetriaminepentaacetic acid (DTPA). In PB with DTPA, no difference was observed between the results in the low DO-PB and those in the ordinal DO-PB. In the ordinal DO-PB without DTPA, the rate constant of the PCAM decrease was half that in the low DO-PB without DTPA. The present results indicate that high DO and coexisting transition metal ions caused the oxidation of ascorbic acid, which degraded the PCAM reduction. The stable nitroxide-ascorbic acid system without DTPA was found to be applicable to an in vitro model for the acute hypoxia ischemia.     

Noninvasive mapping of the redox status in septic mouse by in vivo electron paramagnetic resonance imaging

Increased reactive oxygen species (ROS) contribute to numerous brain disorders, and ROS generation has been examined in diverse experimental models of lipopolysaccharide (LPS)-induced inflammation. The in vivo electron paramagnetic resonance (EPR)/nitroxide spin probe method has been used to analyze the redox status in animal models modulated by ROS generation. In this study, a blood–brain barrier (BBB)-permeable nitroxide spin probe, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP), was used as a redox-sensitive nitroxide probe. Magnetic resonance images of mouse head after the injection of HMP showed that HMP was distributed throughout all regions of the mouse head including the brain, suggesting that HMP can reveal redox information in all regions of the mouse head. After the injection of HMP through the mouse tail vein 6 h after the injection of LPS, three-dimensional (3D) EPR images were obtained each minute under a field scanning of 0.3 s and with 81 projections. The reduction reaction of HMP in septic mouse heads was remarkably accelerated compared to that in control mice, and this accelerated reaction was inhibited by aminoguanidine and allopurinol, which inhibit enzymatic activities of induced nitric oxide synthase and xanthine oxidase, respectively. Based on the pharmacokinetics of HMP in mouse heads, the half-life mapping of HMP was performed in LPS-treated mouse head. Half-life maps clearly show a difference in the redox status induced by ROS generation in the presence or absence of inhibitors of ROS-generating enzymes. The present results suggest that a 3D in vivo EPR imaging system combined with BBB-permeable HMP is a useful noninvasive tool for assessing changes in the redox status in rodent models of brain disease under oxidative stress.     

An ESR-CT imaging of the head of a living rat receiving an administration of a nitroxide radical.

Three-dimensional ESR imaging of a living rat has been performed by an L-band ESR system, which is composed of an L-band ESR spectrometer, a field gradient coil, and a data processor. The imaging was carried out by Lauterbur's method. A nitroxide, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL), was used as an imaging agent in saline solution at a concentration of 0.2 M and administered intraperitoneally to obtain a constant concentration in the head for about an hour. It took about 40 min to obtain one set of ESR-CT images. The cross-sectional images were made, both as coronal and horizontal images. In the images of the rat head the nitroxide-rich region was clearly distinguished from the deficient region. The nitroxide-deficient areas corresponded well to the brain of the rat.     

A spatiotemporal study on the distribution of intraperitoneally injected nitroxide radical in the rat head using an in vivo ESR imaging system

We have developed a rapid-scan in vivo electron spin resonance (ESR) imaging system operating at 700 MHz based on an air-cored two-coil Helmholtz designed resistive magnet. Using this system, we performed ESR-CT for the intraperitoneally injected nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, in the rat head. The imaging data were collected over the time course range from 5 to 47 min after injection at an interval of 3 min and a series of ESR-CT images were reconstructed at the same slice plane (1 cm anterior to interaural line). The series of ESR-CT images thus obtained by rapid scans provided detailed spatiotemporal information on the distribution of the injected nitroxide radical in the rat head. The brain was imaged as a nitroxide-deficient area while the blood vessels and/or extracranium tissues as a nitroxide-rich area. During periods when high intensities of ESR signals were maintained, spots of nitroxide-accumulation were imaged at the central part of the brain. The spots were assigned to the middle sized blood vessels in the brain.     

Monitoring redox-sensitive paramagnetic contrast agent by EPRI, OMRI and MRI

A comparative study of tissue redox-status imaging using commonly used redox sensitive nitroxides has been carried out using electron paramagnetic resonance imaging (EPRI), Overhauser magnetic resonance imaging (OMRI) and conventional T(1)-weighted magnetic resonance imaging, MRI. Imaging studies using phantoms of different nitroxides at different concentration levels showed that EPRI and OMRI sensitivities were found to be linearly dependent on line width of nitroxides up to 2 mM, and the enhancement in MRI intensity was linear up to 5 mM. The sensitivity and resolution of EPRI and OMRI images depended significantly on the line width of the nitroxides whereas the MRI images were almost independent of EPR line width. Reduction of the paramagnetic 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3CP) by ascorbic acid (AsA) to the diamagnetic by hydroxylamine was monitored from a sequence of temporal images, acquired using the three imaging modalities. The decay rates determined by all the three modalities were found to be similar. However the results suggest that T(1)-weighted MRI can monitor the redox status, in addition to providing detailed anatomical structure in a short time. Therefore, a combination of MRI with nitroxides as metabolically responsive contrast agents can be a useful technique for the in vivo imaging probing tissue redox status.     

Intracerebral Antioxidant Ability of Rats Under Hypoxia Estimated Using the Microdialysis-ESR Method

The intracerebral antioxidant ability of rats in 8 and 21 % oxygen (O₂) atmospheres was estimated from the decay of the electron spin resonance (ESR) signal of 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (PCAM), blood–brain-barrier (BBB)-permeable stable cyclic nitroxide, in the perfusate from the brain of the rats using the microdialysis-ESR method. The decay rate of PCAM in 8 % O₂ was about 1.5 times that in 21 % O₂. The oxidation of the perfusate by potassium hexacyanoferrate(III) [K₃Fe(CN)₆] indicated that PCAM in the rats in 8 % O₂ was more greatly reduced to its hydroxylamine than that in 21 % O₂. These results showed that the intracerebral antioxidant ability of rats in 8 % O₂ was larger than that in 21 % O₂. The metal-catalyzed autooxidation of ascorbic acid should be one of the potent factors affecting the intracerebral antioxidant ability of rats in the atmosphere containing different oxygen concentrations.     

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.     

Modified surface-coil-type resonators for EPR measurements of a thin membranelike sample

Surface-coil-type resonators (SCRs) equipped with a circular single-tum coil (conventional SCR), a circular spiral coil (spiral SCR), and a plate-type single-turn coil (plate-type SCR) were fabricated. By using these SCRs, the electron paramagnetic resonance (EPR) sensitivities of thin membranelike samples were investigated. For a non-dielectric-loss phantom, filter paper containing 1,1-diphenyl-2-picrylhydrazyl was used. For a high-dielectric-loss phantom, gauze containing an aqueous solution of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was used. For a biological sample, a pea leaf impregnated with the carbamoyl-PROXYL solution was used. The sensitivity (signal-to-noise ratio) of the spiral and plate-type SCRs for the non-dielectric-loss phantom was significantly greater than that of the conventional SCR. Under these conditions, the sensitivity of the spiral SCR was relatively higher than that of the plate-type SCR. For the high-dielectric-loss phantom, the sensitivity of the plate-type SCRs was significantly greater than that of the conventional SCR, but there were no differences in sensitivity between the spiral and conventional SCRs. The sensitivity of the plate-type SCR in the EPR measurement of a pea leaf was significantly greater than that of the conventional SCR. These findings show that the spiral and plate-type SCRs are suitable for measuring EPR of thin membranelike samples, especially when the former is used for the non-dielectric-loss sample and the latter for high-dielectric-loss sample, including the leaf.     

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 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.     

Temperature Dependence of Hyperfine Interaction for 15N Nitroxide in a Glassy Matrix at 10–210 K

Principal ¹⁵N hyperfine interaction (hfi) values in ¹⁵N-substituted nitroxide spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl dissolved in nematic liquid crystal 4-pentyl-4′-cyanobiphenyl (5CB) were measured in a wide temperature range of 10–210 K, for 5CB frozen to a glassy state. X-band continuous-wave electron paramagnetic resonance (CW EPR) and pulse X- and Q-band ¹⁵N electron-nuclear double resonance (ENDOR) techniques were employed. To avoid microwave saturation at low temperatures in CW EPR studies, a holmium complex Ho(Dbm)₃Bpy (where Dbm is dibenzoylmethane and Bpy is 2,2′-bipyridine) was added. X- and Q-band ¹⁵N-ENDOR data have shown that the nitroxide hfi tensor is axially symmetric. The combination of data from all techniques allowed us to obtain the temperature dependence of isotropic and anisotropic parts of the nitroxide hfi tensor. Above ~100 K, a linear dependence of the anisotropic hfi value was observed, whereas below 30 K it was found to be nearly temperature independent. Such a behavior can be interpreted using the model of restricted orientational motions (librations) of a spin probe in a glassy matrix, with quantum effects occurring at low temperature (“freezing” of the librations). The energy quantum for the libration motion estimated from the temperature dependence of hfi of the spin probe is 84 cm^?1. Low-frequency Raman spectra of 5CB were also obtained, which provided the mean vibrational frequency of 76 cm^?1 for glassy 5CB.     

Evaluation of nonionic nitroxyl lipids as potential organ-specific contrast agents for magnetic resonance imaging.

Considering their intrinsic properties of accumulation in the hepatic tissue, we have synthesized nitroxyl-containing lipids as potential organ-specific contrast agents for magnetic resonance imaging (MRI). Their resistance to reduction by ascorbate and in liver homogenates, and their relaxivity in different media were investigated and compared to those of free carboxyl-Proxyl (3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl) and Tempamaine (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl). With respect to the reduction rates by ascorbate, the lipid derivatives show the same well-known order of reactivity as carboxy-Proxyl and Tempamine, the five-membered nitroxyls being more stable than the six-membered compounds. However the binding of the piperidinoxyl compounds to the fatty acids confers to those lipid derivatives a markedly increased stability. Similarly, in liver homogenates, the nitroxyl lipids remained unchanged more than 20 min, contrarily to carboxy-Proxyl and Tempamine. The measurements of spin-lattice relaxation time (T1) in biological media have demonstrated a higher relaxivity of nitroxyl lipids, which can be related to their interaction with proteins. Tested in vivo, one of the synthesized compounds (0.75 mmol/kg) produced an enhancement of 44 +/- 12% of the hepatic signal 5 min after intraportal injection in T1-weighted images. The potential applicability of the other nitroxyl lipids as contrast agents for MRI was limited in the in vivo studies by an unexpected toxicity. Work is currently in progress to improve the therapeutic index of the present class of nitroxyl lipids.     

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.     

Overhauser dynamic nuclear polarization and molecular dynamics simulations using pyrroline and piperidine ring nitroxide radicals

The efficiency of Overhauser dynamic nuclear polarization (DNP) depends on the local dynamics modulating the dipolar coupling between the two interacting spins. By attaching nitroxide based spin labels to molecules and by measuring the ¹H DNP response of solvent water, information about the local hydration dynamics near the spin label can be obtained. However, there are two commonly used types of nitroxide ring structures; a pyrroline based and a piperidine based molecule. It is important to know when comparing different experiments, whether changes in DNP enhancements are due to changes in local hydration dynamics or because of the different spin label structures. In this study we investigate the key parameters affecting DNP signal enhancements for 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, a 5-membered ring nitroxide radical, and for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, a 6-membered ring nitroxide radical. Using X-Band DNP, field cycling relaxometry, and molecular dynamics simulations, we conclude that the key parameters affecting the DNP amplitude of the ¹H signal of water to be equal when using either nitroxide. Thus, experiments measuring hydration dynamics using either type of spin labels may be compared.     

A loop resonator for slice-selective in vivo EPR imaging in rats

A loop resonator was developed for 300 MHz continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and imaging in live rats. A single-turn loop (55 mm in diameter) was used to provide sufficient space for the rat body. Efficiency for generating a radiofrequency magnetic field of 38 microT/W(1/2) was achieved at the center of the loop. For the resonator itself, an unloaded quality factor of 430 was obtained. When a 350 g rat was placed in the resonator at the level of the lower abdomen, the quality factor decreased to 18. The sensitive volume in the loop was visualized with a bottle filled with an aqueous solution of the nitroxide spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (3-CP). The resonator was shown to enable EPR imaging in live rats. Imaging was performed for 3-CP that had been infused intravenously into the rat and its distribution was visualized within the lower abdomen.     

几种氮氧自由基与β-环糊精及其衍生物作用的EPR研究

测定了三种氮氧自由基与环糊精相互作用的EPR波谱，计算了波谱参数. 结果表明：与环糊精作用后，自由基的超精细分裂常数(a_n) 值及旋转相关时间(τ_c)都有规律变化，由此探讨了氮氧自由基与环糊精相互作用位点及作用力大小.