Midori Hiramatsu

Mitochondrial Dysfunction in the Senescence Accelerated Mouse (SAM)

Oxidative damage to DNA, proteins, and lipids in mitochondria caused by free radicals may be one factor in aging. Oxidative phosphorylation was estimated in liver mitochondria from senescence accelerated mice (SAMP8) and a senescence resistant substrain (SAMR1). The respiratory control ratio decreased in liver mitochondria of SAMP8 during aging, and it was estimated that at 18 months of age this respiratory control value suggested that it might be insufficient to provide ATP synthesis necessary for normal cell metabolism. In addition, the ADP/O, an index of efficiency of ATP synthesis, was depressed at 18 months of age. Dinitrophenol-dependent uncoupled respiration in liver mitochondria of SAMP8 mice was markedly decreased with aging, suggesting a dysfunctional energy transfer mechanism in mitochondria of aged SAMP8 mice. Active uptake of calcium in liver mitochondria was markedly dysfunctional in SAMP8 mice with aging, and uncoupling of respiration was induced more easily in aged mitochondria. Milder effects on these functional parameters were observed in SAMR1 mice. A similar dysfunction was also observed in heart mitochondria of SAMP8 mice at 12 months of age. The amount of Bcl-x in liver mitochondria was slightly decreased in SAMP8. We suggest that these changes in mitochondrial function may be related to the shorter life span of the senescence accelerated mouse.

Increased Superoxide Dismutase Activity in Aged Human Cerebrospinal Fluid and Rat Brain Determined by Electron Spin Resonance Spectrometry Using the Spin Trap Method

Abstract: Superoxide dismutase (SOD) activity in CSF of patients was determined by electron spin resonance spec‐trometry using the spin trap method. Variation in SOD activity was found among patients. SOD activity in CSF of subjects increased with age and this was identified as Cu,Zn‐SOD activity by electrophoresis. In addition, animal experiments showed that SOD activities were higher in mitochon‐drial and cytosol fractions of aged rats than in those of adult rats. This finding on aged rat brain validates the increase of SOD activity in aged human CSF.

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.

Brain Monoamine Levels and El Mouse Convulsions

Abstract: El mice had higher levels of brain dopamine (DA) and serotonin (5‐HT) and a lower level of brain norepinephrine (NE) compared with the other strains of inbred mice. In the interictal stage of “stimulated” El mouse, the brain DA, NE and 5‐HT levels were significantly lower compared with the non‐stimulated El mouse. The administration of 5‐hydroxytryptophan with MK486 markedly depressed the incidence of seizures in the El mouse. Evidence of abnormal DA, NE and 5‐HT metabolism was found at the development age when the seizures became frequent. These results show that 5‐HT may be closely related to the El mouse seizure susceptibility.

Structure-activity relationships of GABA and its relatives on the excitability of an identified molluscan giant neurone (Achatina fulica férussac)

Abstract 1. A spontaneously firing giant neurone (the TAN, tonically autoactive neurone) sensitive to GABA (inhibition) was identified in the subesophageal ganglia of Achatina fulica Ferussac. The effect of GABA and its 80 relatives were examined (the bath application) on TAN excitability. Among them, GABA showed the strongest inhibitory effect (critical concentration is about 10 −5 g/ml). 4-amino-3-hydroxybutanoic acid, 4-amino-2-hydroxybutanoic acid, 5-amino pentanoic and guanidinoacetic acid at 10 −4 g/ml showed also an inhibitory effect. 2. It was confirmed that the GABA effect on the TAN neuromembrane was due to the direct hyperpolarization, by means of the local GABA administration on the TAN surface (the microdrop application). 3. No antagonistic action of three convulsant alkaloids (bicuculline, strychnine and picrotoxin) to the GABA effect on TAN excitability was detected. 4. To indicate the electrical resistance of the TAN neuromembrane, its current-voltage relationships (I–V curve) were measured, by applying a transmembrane triangular current. The I–V curve measured in the GABA application at 3 × 10 −5 g/ml was concordant in the wide range of potential level with that of the normal state, when two I–V curves were superimposed by using the firing level as the common standard. 5. We identified presence of GABA in the subesophageal ganglia of Achatina fulica . The GABA amount was augmented after hydrolyzation of the ganglionic extract.

Free radical scavenging activity of the Japanese herbal medicine toki-shakuyaku-san (TJ-23) and its effect on superoxide dismutase activity, lipid peroxides, glutamate, and monoamine metabolites in aged rat brain.

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O2−), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.

Catecholamine level in cerebrospinal fluid of epileptics

We estimated catecholamine levels in CSF of 15 epileptics and 75 non-neurological patients utilizing a high performance liquid chromatograph with a highly sensitive fluorometer and found the following results: The dopamine (DA) levels in males were significantly higher than those in females, while norepinephrine (NE) levels in males were the same as in females. The DA levels were significantly lower and NE levels significantly higher in epileptics than in non-neurological patients. DA and NE in petit mal patients were on the average lower than in grand mal patients, but untreated grand mal patients had higher NE levels. These results suggest that epilepsy may be associated with a disturbance of DA and/or NE metabolism or release in the brain.

Biochemical pathogenesis of post-traumatic epilepsy

Head trauma is often followed by epilepsy and may be related to the breakdown of red blood cells and hemoglobin within the CNS. Injection of hemoglobin or iron salts into the rat cortex is known to induce a chronic epileptic focus. We observed the formation of superoxide anion (O2) and hydroxyl radical (·OH) after ferric chloride injection into the rat cerebral cortex and suggest that these radicals, especially ·OH, may be responsible for the initiation of lipid peroxidation in neuronal membranes and for the accelerated production of guanidine compounds in the brain, which may in turn lead to epileptogenicity. Then, we found that treatment with epigallocatechin (EGC) or a phosphate diester of vitamins E and C (EPC), which are potent ·OH scavengers, significantly inhibited the formation of malondialdehyde and epileptic discharges in the iron-induced epileptic focus.

Convulsive Activity of α-Guanidinoglutaric Acid and the Possible Involvement of 5-Hydroxytryptamine in the α-Guanidinoglutaric Acid-Induced Seizure Mechanism

Abstract: α‐Guanidinoglutaric acid (α‐GGA) was first found in cobalt‐induced epileptogenic focus tissue in the cerebral cortex of cats. We examined the effect of α‐GGA on the electroencephalogram and on the brain 5‐hydroxytryptamine (5‐HT) level after intraventricular administration into rats. Sporadic low‐voltage spikes appeared 4 min after the administration of α‐GGA. Spikes increased in voltage 6 min after the administration. Multiple spikes appeared 10 min after the administration, and they reached maximal frequency 30 min after the administration. The epileptic discharges disappeared 100 min after the administration. The 5‐HT level increased in the right and left cortices 3 min after the administration. The 5‐HT level decreased in the mid‐brain 5 min after the administration and subsequently in all regions of the brain 10 min after the administration. No change in the 5‐HT level was found 30 min and 100 min after the administration. These results show that α‐GGA induces epileptic seizures in rats after intraventricular administration. The results also suggest that α‐GGA‐induced seizures are associated with abnormal serotonergic function and that they are initiated by a decrease in the 5‐HT level.

Probucol scavenged 1,1-diphenyl-2-picrylhydrazyl radicals and inhibited formation of thiobarbituric acid reactive substances

Probucol is suggested to have antioxidant properties. The direct scavenging action of probucol on hydroxyl radicals, superoxide and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were examined using electron spin resonance (ESR) spectrometry. Probucol scavenged DPPH radicals dose dependently but showed no effect on hydroxyl radicals and superoxide generated by Fenton reaction and by hypoxanthine-xanthine oxidase system, respectively. It inhibited the formation of thiobarbituric acid reactive substances (TBARS) in rat cortex homogenate induced by ascorbic acid and FeCl2 at low dose, but it increased TBARS formation at high doses. Probucol showed no effect on the carbon centered radicals. Iron injection into the rat cortex, which is an experimental model for traumatic epilepsy, increased TBARS level in the cortex, hippocampus, striatum and cerebellum, but pretreatment with probucol inhibited the increase in these brain parts except for the hippocampus. These results suggest that the antioxidant property of probucol is partly due to its free radical scavenging effect.