Hirotada Fujii

An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals.

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC-FL) is an indirect method that monitors the antioxidants ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC-EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC-FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC-EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC-FL, but absolute values showed significant discrepancies. ORAC-EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.

Consequences of Nitric Oxide Generation in Epileptic- Seizure Rodent Models as Studied by In Vivo EPR

The role of nitric oxide (NO) in epileptogenesis was studied in pentylenetetrazole (PTZ)‐treated animals using in vivo and ex vivo EPR spectroscopy. NO generation was measured directly in the brain of a PTZ‐induced mouse in vivo by an L‐band EPR spectrometer. An elevation in NO production in the brain was observed during convulsions, and more NO was generated in the tonic seizure vs. the clonic seizure. NO content in several brain tissues (including the cerebral cortex (CR), cerebellum (CL), olfactory bulb (OB), hippocampus (HI), and hypothalamus (HT)) of PTZ‐doped rats was analyzed quantitatively ex vivo by X‐band EPR. To test the involvement of NO in seizure development, pharmacological analyses were performed using the NO synthase (NOS) inhibitors NG‐nitro‐L‐arginine (L‐NNA), NG‐monomethyl‐L‐arginine (L‐NMMA), and 3‐bromo‐7‐nitroindazole (3Br‐7NI). All of these inhibitors suppressed the convulsions, holding them at the clonic level, and prevented development of a tonic convulsion in rats doped with up to 80 mg/kg PTZ. 3Br‐7NI completely inhibited NO production, but L‐NNA and L‐NMMA showed only 70% inhibition of NO production in PTZ‐doped rats. In order to examine the contributions of NO in convulsions, rats were treated with anticonvulsants (phenytoin and diazepam) before PTZ treatment. Both drugs completely suppressed tonic convulsion in PTZ‐doped rats at doses up to 80 mg/kg, but NO levels were similar to those detected in a clonic convulsion. These results support the notion that NO does not directly induce a clonic convulsion, but may be generated as a consequence of onset of seizure. Magn Reson Med 48:1051–1056, 2002.

In vivo imaging of spin-trapped nitric oxide in rats with septic shock: MRI spin trapping

This paper reports the first in vivo NMR image of the distribution of NO using the “MRI spin‐trapping” technique. NO was complexed with the Fe(II)‐chelate spin trap, N‐methyl‐d‐glucamine dithiocarbamate (MGD), verified as (MGD)2‐Fe(II)‐NO by EPR, and the radical distribution was “visualized” by MR images. In rats, the (MGD)2‐Fe(II)‐NO complex was concentrated in the liver displaying significantly enhanced contrast in the vascular structure such as hepatic vein and inferior vena cava. Nitric oxide synthase was verified as the source of NO in rats with septic shock by pre‐administration of the competitive inhibitor N‐monomethyl‐l‐arginine, resulting in reduced enhancement. The NO complex was more stable in vivo and a more effective MRI contrast agent than other stable nitrogen containing radicals, such as nitroxides. The MRI spin‐trapping method should be a powerful tool for visualizing spatial distributions of free radicals in pathologic organs and tissues when combined with the appropriate radical complexing agent, such as (MGD)2‐Fe(II) used in these studies. Magn Reson Med 42:235–239, 1999.

THE ENTRY OF MANGANESE IONS INTO THE BRAIN IS ACCELERATED BY THE ACTIVATION OF N-METHYL-D-ASPARTATE RECEPTORS

Manganese-enhanced magnetic resonance imaging (MEMRI) is receiving increased interest as a valuable tool for monitoring the physiological functions in the animal brain based on the ability of manganese ions to mimic calcium ions entering to excitable cells. Here the possibility that in vivo MEMRI can detect the entry of manganese ions (Mn2+) in the brain of rats behaving without intended stimulation is tested. This hypothesis was a result of the unexpected observation that Mn2+-dependent signal enhancement was dramatically suppressed in ketamine-anesthetized rats compared with other anesthetics, such as urethane, pentobarbital and isoflurane. The effects of noncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonists, ketamine and MK-801, on MEMRI for MnCl2 injected rats were examined. Treatment with MK-801 suppressed the signal enhancement more effectively than with ketamine. NMDAR agonists, glutamate (100 mg/kg) and N-methyl-d-aspartate (NMDA) (35 mg/kg), enhanced the signal intensities on MEMRI, and this signal enhancement was completely antagonized by MK-801. The systemic administration of the competitive NMDAR antagonist, D-2-amino-5-phosphono-pentanoate (D-AP5), which does not cross the blood-brain barrier (BBB), showed no effects on the signal enhancement induced by NMDA and glutamate. A selective AMPA receptor (AMPAR) antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), did not block the signal enhancement. These data indicated that the Mn2+-dependent signal enhancement took place as a result of the activation of glutamatergic neurons through NMDAR, but not through AMPAR in the brain.

Magnetic resonance and biochemical studies during pentylenetetrazole-kindling development: The relationship between nitric oxide, neuronal nitric oxide synthase and seizures

The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of pentylenetetrazole (PTZ)-kindling as an animal model of primary generalized epilepsy. The daily administration of PTZ is associated with an increase in the amount of neuronal nitric oxide synthase (nNOS). NO generation was measured directly by in vivo and ex vivo electron paramagnetic resonance on rodents undergoing progressive convulsions. We found that primary generalized epilepsy is caused by NO induction during the persistent up-regulation of nNOS expression, but that NO induction is not associated with severe generalized seizures following long-term kindling phenomena after PTZ withdrawal. Morphological changes in the brain structure of rats were measured by magnetic resonance imaging during epileptic convulsions induced by repetitive administration of PTZ. Cerebellum volume for kindled rats decreased 20% but not in rats treated with the nNOS inhibitor, 3Br-7NI, suggesting that generation of NO in the cerebellum is related to decrease in cerebellum volume following PTZ-kindling.

In Vivo Spin Trapping of Nitric Oxide

The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is complexed with ferrous iron-dithiocarbamate ligand spin trap complexes. Despite the minimally invasive nature of the technique, highly sensitive localized concentrations of NO may be observed (trapped) in vivo by both L-band EPR and magnetic resonance imaging.

Half-Life Mapping of Nitroxyl Radicals with Three-Dimensional Electron Paramagnetic Resonance Imaging at an Interval of 3.6 Seconds

This technical note reports a continuous-wave electron paramagnetic resonance (CW-EPR) imager that can visualize the distribution of free radicals with a half-life of subminutes in three-dimensional (3D) space. A total of 46 EPR spectra under magnetic field gradients, called projections, were obtained for image reconstruction at an interval of 3.6 s. A shortened data-acquisition time was achieved with the use of analog signals that drove field gradient coils in the imager. 3D mapping of the half-lives of nitroxyl radicals (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl) was demonstrated in their reduction reaction with ascorbic acid. Inhomogeneous half-lives were clearly mapped pixel-by-pixel in a sample tube.

Development and testing of a CW-EPR apparatus for imaging of short-lifetime nitroxyl radicals in mouse head.

This article describes a method for reducing the acquisition time in three-dimensional (3D) continuous-wave electron paramagnetic resonance (CW-EPR) imaging. To visualize nitroxyl spin probes, which have a short lifetime in living organisms, the acquisition time for a data set of spectral projections should be shorter than the lifetime of the spin probes. To decrease the total time required for data acquisition, the duration of magnetic field scanning was reduced to 0.5s. Moreover, the number of projections was decreased by using the concept of a uniform distribution. To demonstrate this faster data acquisition, two kinds of nitroxyl radicals with different decay rates were measured in mice. 3D EPR imaging of 4-hydroxy-2,2,6,6-tetramethylpiperidine-d17-1-15N-1-oxyl in mouse head was successfully carried out. 3D EPR imaging of nitroxyl spin probes with a half-life of a few minutes was achieved for the first time in live animals.

Ex vivo EPR detection of nitric oxide in brain tissue

The concentration of nitric oxide (NO) was measured in the brain of septic‐shock animals by electron paramagnetic resonance spectrometry (EPR). NO was spin trapped and quantitated in several regions of the brain (cortex, hippocampus, hypothalamus, cerebellum, and olfactory bulb) as well as other organs (liver, kidney, and heart) of rats induced with lipopolysaccharide (LPS) using Fe(II)/dithiocarbamate complexes containing diethyldithiocarbamate (DETC) or N‐methyl‐D‐glucamine (MGD). The spin trap, (DETC)2‐Fe(II), complexed NO generated in all tissues examined, but (MGD)2‐Fe(II) complex was ineffective in detecting NO in the brain of septic‐shock rats, although identical amounts of NO were detected in the liver with either spin trap. A triplet EPR spectrum of (DETC)2‐Fe(II)‐NO with aN = 12.8 gauss and g = 2.04 was observed in the cortex, hippocampus, hypothalamus, cerebellum, but not the olfactory bulb. The amount of NO in the brain was about 20% of that found in the liver. The (DETC)2‐Fe(II)‐NO signal in all the tissues of septic‐shock rats was markedly suppressed by preadministration of the nitric oxide synthase (NOS) inhibitors, NG‐monomethyl‐L‐arginine (L‐NMMA) or 3‐bromo‐7‐nitroindazole, suggesting that the NO detected from brain tissue was produced enzymatically by NOS. In contrast to previous studies on the liver and other organs, phenyl‐N‐tert‐butyl nitrone (PBN), did not suppress iNOS expression in brain tissue of LPS‐treated rats. This could be due to a totally different regulation system for iNOS in liver versus brain tissue. Magn Reson Med 42:599–602, 1999.

Improvement of temporal resolution for three-dimensional continuous-wave electron paramagnetic resonance imaging

This paper describes improved temporal resolution for three-dimensional (3D) continuous-wave electron paramagnetic resonance (EPR) imaging. To improve temporal resolution, the duration of magnetic filed scanning that is used to obtain an EPR spectrum for each projection was reduced to 40 ms. The Helmholtz coil pair for field scanning was driven by triangular waves. The uniform distribution of projections was also used to reduce the number of projections for 3D image reconstruction. The reduction reaction of 4-hydroxy-2,2,6,6-tetramethyl-piperidinooxy with ascorbic acid was visualized by improved 3D EPR imaging techniques with a temporal resolution of 5.8 s.