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.     

Assessment of ESR-CT imaging by comparison with autoradiography for the distribution of a blood-brain-barrier permeable spin probe, MC-PROXYL, to rodent brain

Blood-brain-barrier (BBB)-permeable, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MC-PROXYL) and BBB-impermeable carbamoyl-PROXYL were used to assess the ESR imaging technique by comparing with autoradiography. For this purpose, spin probes, 14C-labeled at their five-membered ring, [14C]MC-PROXYL and [14C]carbamoyl-PROXYL, were newly synthesized. These probes were i.p. or i.v. injected into rats and autoradiograms were recorded. The autoradiograms of rat head showed that [14C]MC-PROXYL distributed well in the brain compared to [14C]carbamoyl-PROXYL. In vivo ESR spectra and two-dimensional ESR images of isolated rat brain treated with MC- or carbamoyl-PROXYL also indicated the extensive distribution of MC-PROXYL but not carbamoyl-PROXYL in the rat brain. The three-dimensional ESR images of the head of rats and mice were consistent with the fact that MC-PROXYL but not carbamoyl-PROXYL is incorporated into the brain. The ESR-CT images were better for mice than rats. However, the quality of the ESR-CT images was still not satisfactory. Although the resolution and sensitivity of the ESR-CT images were worse than those of the autoradiographic images, the former technique has unique features and advantages; e.g., functional, noninvasive and three-dimensional detection.     

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.     

Kinetics of nitroxide spin label removal in biological systems: An in vitro and in vivo ESR study

A systematic study on the disappearance of the electron spin resonance (ESR) signal of nitroxides based on six- or five-membered ring and bearing various charges was carried out in vitro and in vivo. The second-order kinetic rate constants of the reaction of spin probes with ascorbate were measured in vitro at various temperatures in phosphate buffered saline, and the relative activation energies were calculated. Clearance rates of the nitroxide radicals in rat brain homogenates and in blood indicate that the ascorbate contribution to nitroxide removal is about 50–70% in brain and 50–90% in blood. These rates can be easily calculated on the basis of the ascorbate concentration and of the second-order kinetic rate constants measured in phosphate buffered saline. ESR spectra acquired in vivo in rat head and tail, by an L-band resonator, indicated that the nitroxide decay rate is a first-order kinetic process in both domains and that the positively charged nitroxides are not retained in the brain, whereas the anionic and uncharged nitroxides are. Once nitroxides with piperidine ring enter the brain, their decay appears controlled mainly by ascorbate, while the ascorbate has a negligible influence on disappearance in brain of five-membered ring proxyl nitroxides.     

L波段三维ESR成像系统的研制（Ⅱ）——L波段三维ESR成像系统

叙述了一个L波段（1.05 GHz）用于ESR和ESR成像的装置，用这套自制装置实现了3D ESR成像. 该装置由L波段ESR谱仪、三组梯度场线圈及控制单元和PC机数据采集系统组成. 样品腔是一个3-环2-缝再进入式谐振腔，可放入直径为20 mm、 长30 mm的H₂O样品，空谐振腔的频率是1.05 GHz. 微波振荡频率用自动频率控制（AFC）的方法自动锁在有载腔的频率上. 梯度场线圈沿X-，Y-和Z-轴产生线性梯度场，在中心40 mm球形范围内梯度场强度为2 mT/cm. 依照Lauterbur's方法进行3D ESR 图像重建. 用该系统检测了样品中TEMPO氮氧自由基的3D空间分布. 得到了TEMPO的2D、3D ESR图像、用像素灰度表示的自旋密度分布图及3D ESR-CT图像.     

A CT using longitudinally detected ESR (LODESR-CT) of intraperitoneally injected nitroxide radical in a rat's head

We have developed an in vivo longitudinally detected ESR (LODESR) imaging system operating at 700 MHz based on a loop-gap resonator and a pair of saddle-type pickup coils. A good linear relationship between the LODESR signal intensity and the applied power in a range up to 15.8 W was obtained. The detection of LODESR signals was barely affected by variations in the resonant properties. The characteristic of LODESR is suitable for in vivo examination. Using this system, we succeeded in obtaining LODESR-CT images of the head region of a rat after the intraperitoneal injection of a nitroxide radical.     

Three-dimensional in vivo ESR imaging in rats

The first experiment of tridimensional in vivo ESR imaging at 1.2 GHz is described in this paper. The tails of rats weighing 300-350 grams were visualized using 1 cc of a 50 * 10(-3) M solution of nitroxide free radical injected in the caudal vein. In an even distribution of spin label is assumed this would correspond to a final concentration of about 10(-4) M. A reconstruction from projections was used to obtain the images. The apparatus utilizes stationary field gradients. Projections were obtained by sweeping the main field. For 3D reconstructions, the projections were collected along 32 * 8 field gradient orientations. The whole procedure takes approximately 18 minutes.     

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.     

In vivo sodium-23 magnetic resonance surface coil imaging: Observing experimental cerebral ischemia in the rat

Sodium-23 magnetic resonance imaging can be used to detect and assess experimental cerebral ischemia in the rat. An imaging technique utilizing a surface coil is described to produce sodium magnetic resonance images of good quality and resolution within 10 min. A novel method of hemispheric occlusion showed edema in the right brain of the rat head within 3 hr after injury. The edema was especially pronounced by 12 hr with effects in the right brain, eye and surrounding muscle evident.     

Spatiotemporal measurement using L-band ESR-CT system for water sonolysis in the presence of 1-Hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide

When using ESR to measure the radicals generated by ultrasound, it is necessary to extract a solution and place it in the ESR system. To avoid this process, we incorporated an ultrasonic reaction cell in an L-band ESR-CT system, producing a system that allows the detection of the concentration of radicals during ultrasonic irradiation. This system was used to measure the time and space dependences of OH radicals generated by ultrasonic irradiation. When a 10 ml aqueous solution of 1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide (HTIO) was irradiated with ultrasound, it was found that the generation of radicals was clearly shown in a CT image after a period of 10 min. It was also found that continued irradiation resulted in an increased concentration of radicals. In addition to this system, an X-band ESR system was also used to measure the concentration of OH radicals generated, and the results of both systems were then compared. Both results are very similar, showing that the proposed system, which was realized by incorporating an ultrasonic irradiation cell in the L-band ESR-CT system, operated properly. Because this system allows the measurement of sonochemical reactions in an opaque cell or an opaque solution such as blood and industrial wastewater, it is a very useful measurement system for achieving the applying of sonochemistry to the medical engineering field.     

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.     

Functional magnetic resonance (fMR) imaging of a rat brain tumor model: implications for evaluation of tumor microvasculature and therapeutic response

Functional MR (fMR) imaging techniques based on blood oxygenation level dependent (BOLD) effects were developed and applied to a rat brain tumor model to evaluate the potential utility of the method for characterizing tumor growth and regression following treatment. Rats bearing 9L brain tumors in situ were imaged during inhalation of room air and after administration of 100% oxygen + acetazolamide (ACZ) injected 15 mg/kg intravenously. Pixel-to-pixel fMR maps of normalized signal intensity change from baseline values were calculated from T2 weighted spin echo (SE) images acquired pre- and post- oxygen + ACZ administration. Resultant fMR maps were then compared to gross histological sections obtained from corresponding anatomical regions. Regions containing viable tumor with increased cellular density and localized foci of necrotic tumor cells consistent with hypoxia were visualized in the fMR images as regions with decreased signal intensities, indicating diminished oxyhemoglobin concentration and blood flow as compared to normal brain. Histological regions having peritumor edema, caused by increased permeability of tumor vasculature, were visualized in the fMR images as areas with markedly increased signal intensities. These results suggest that fMR imaging techniques could be further developed for use as a non-invasive tool to assess changes in tumor oxygenation/hemodynamics, and to evaluate the pharmacologic effect of anti-neoplastic drugs.     

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.     

Three-dimensional laser-induced photoacoustic tomography of mouse brain with the skin and skull intact

Three-dimensional laser-induced photoacoustic tomography, also referred to as optoacoustic tomography, is developed to image animal brain structures noninvasively with the skin and skull intact. This imaging modality combines the advantages of optical contrast and ultrasonic resolution. The distribution of optical absorption in a mouse brain is imaged successfully. The intrinsic optical contrast reveals not only blood vessels but also other detailed brain structures, such as the cerebellum, hippocampus, and ventriculi lateralis. The spatial resolution is primarily diffraction limited by the received photoacoustic waves. Imaged structures of the brain at different depths match the corresponding histological pictures well.     

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.     

谱-空间2D ESR成像

在Bruker ER 200D ESR 谱仪上安装一套自制的谱-空间2D ESR成像系统,这套系统由一对梯度场线圈、电源、微机及图像重建程序组成. 用滤波反投影图像重建方法,实现了两种自由基样品的谱-空间2D ESR成像,由2D 图像得到样品中自由基的自旋密度空间分布及相应的波谱参数. 讨论了成像参数与图像精度的关系.     

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.     

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.     

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     

Head and body imaging by hydrogen nuclear magnetic resonance

A hydrogen (¹H) nuclear magnetic resonance (NMR) imaging study of the normal head, thorax, and limbs is reported. The images are 10 to 15 mm thick transverse slices obtained in 2 to 4 min using a two-dimensional Fourier transform technique. Spatial resolution in the imaging plane is about 2 mm, enabling the optic nerve and many small blood vessels to be observed. Thorax scans show details of the cardiac chambers, aorta wall, and lungs without artefacts arising from physiological motion.