Akitane Mori

Post-ischemic administration of the acetylcholinesterase inhibitor ENA-713 prevents delayed neuronal death in the gerbil hippocampus

We examined by morphological methodology the effect of (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713), an acetylcholinesterase (AChE) inhibitor, on ischemia-induced neuronal death in the gerbil hippocampus due to a 5-min ligation of bilateral common carotid arteries after light ether anesthesia. Pyramidal cells had been decreased to 27% of sham-operated controls and the number of hypertrophic astrocytes expressing glial fibrillary acidic protein (GFAP) markedly increased in the hippocampal CA1 subfield 14 days after ischemia. However, post-ischemic administration of ENA-713 (three times 0.2 mg/kg, i.p.) significantly ameliorated this ischemia-induced decrease in the number of pyramidal cells by 47% of sham-operated controls, furthermore, it reduced the ischemia-induced accumulation of GFAP-positive astrocyte in the CA1 region. Together with previous results showing that ENA-713 protected against the ischemia-induced cholinergic abnormalities in the gerbil brain and improved cholinergic dysfunctions in the senescent rat brain, our present findings suggest that ENA-713 prove to be useful for treatment with senile dementia such as cerebrovascular dementia.

ESR demonstration of nitric oxide production from nitroglycerin and sodium nitrite in the blood of rats

Electron spin resonance (ESR) spectra of iron-metal complexes formed by the reaction between nitric oxide (NO) and hemoglobin (Hb), referred to as nitrosylhemoglobin (HB-NO), were observed in rat blood treated in vitro and in vivo with nitroglycerin (GTN) at 77K. The same types of spectra were also detected in rats treated with sodium nitrite (NaNO2). Two types of Hb-NO, which were identified by ESR parameters of g values and superhyperfine coupling constants (shfcc), were the 6- and 5-coordinated complexes. These two types of Hb-NO were generated in a dose-dependent manner in the blood after intraperitoneal administration of 1.5-6 mg of GTN. At the higher dose of GTN (6 mg), the 6-coordinated complex was the major species generated initially, but within 10 min, the 5-coordinated complex increased time-dependently. Quantitative analysis of Hb-NO revealed that when GTN 0.3 mg and 0.6 mg was administered sublingually in rats, the concentration of Hb-NO observed in rat blood was 30% higher than the estimated concentration of GTN. The methemoglobin and peroxide complex of hemoglobin were observed in the blood incubated with GTN at 37 degrees C. These results suggest that the function of GTN was related to oxidative stress with the generation of Hb-NO. Therefore, monitoring of Hb-NO levels may be useful as an indicator of the function of various vasodilators.

Metal ions affect neuronal membrane fluidity of rat cerebral cortex.

The effect of various metal ions on neuronal membrane fluidity was examined using 2-(14-carboxypropyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy, which has been used for the examination of membrane fluidity in hydrophobic areas by electron spin resonance spectrometry. Potassium, cobalt, calcium, magnesium, nickel, copper, ferric, and aluminium ions decreased the membrane fluidity while ferrous ions increased it at each high concentration. Sodium and zinc ions had no effect. Ethylenediaminetetraacetic acid decreased membrane fluidity at high concentrations. Nicardipine lowered membrane fluidity and flunarizine elevated it at each high concentration. There was no change in membrane fluidity by other calcium antagonists, nimodipine and nifedipine.

Cyclosporin A prevents ischemia-induced reduction of muscarinic acetylcholine receptors with suppression of microglial activation in gerbil hippocampus

We previously reported the late onset reduction of muscarinic acetylcholine receptors (LORMAR) which begins 7 days after a 5-min period of experimentally induced forebrain ischemia in the gerbil hippocampus. This study demonstrated that post-ischemic administration of cyclosporin A (CsA) reduced LORMAR 10 days after 5 min of forebrain ischemia in the gerbil hippocampus, suggesting that immunosuppression by CsA may reduce damage to the cholinergic system after ischemia. Microglia positive for HLA-DR class II antigen which presented in the hippocampal CA1 area, the region most vulnerable to ischemia, were also reduced by CsA. CsA may suppress microglial activation especially with regard to the antigen-presenting function, and LORMAR may be attenuated by this modulation of microglial function.

Free radicals, lipid peroxides and antioxidants in blood of patients with myotonic dystrophy.

We studied the levels of free radicals, lipid peroxides and antioxidants, as well as superoxide dismutase (SOD) activity in the blood of six patients with myotonic dystrophy (MyD) (mean age 52.8, SD 5.0 years) and seven controls (mean age 48.8, SD 6.3 years). Electron spin resonance was used to assess the free radicals by the spin-trapping method using 5,5-dimethyl-l-pyrroline-l-oxide. The levels of C centre radical (P < 0.05) and H radical (P < 0.05) in blood from the six MyD patients were significantly higher than those in the seven controls. The SOD activities in red blood cells and serum from the six MyD patients showed no significant difference from those in the seven controls. The serum lipid peroxide concentration was increased in five of the MyD patients and tended to increase further as the disease progressed. The serum vitamin E level was low in two patients and in the low normal range in three. Serum coenzyme Q10 was decreased in four patients. The serum selenium level was decreased in two patients and that of serum albumin was decreased in three. Therefore we conclude that increased levels of free radicals and lipid peroxides and decreased antioxidant levels play an important role in the pathogenesis of MyD.

Posttraumatic Epilepsy, Free Radicals and Antioxidant Therapy

Each year, head injuries from car accidents and other causes result in a large population of patients with epileptic seizure disorders.The development of such posttraumatic epilepsy may be related to the breakdown of red blood cells and hemoglobin within the central nervous system. Subpial injection of hemoglobin or iron salts into rat cortex is known to induce a chronic epileptic focus. We observed the formation of superoxide anion and hydroxyl radical (•OH) after ferric chloride injection into rat cerebral cortex, and suggested that these radicals, especially •OH, may be responsible for the initiation of lipid peroxidation in neuronal membranes and for the accelerated production of endogenous convulsants, i.e., guanidino compounds, in the brain. These disorders may in turn lead to epileptogenicity. We found that treatment with epigallocatechin or a phosphate diester of vitamins E and C, which are potent •OH scavengers, significantly inhibited the formation of thiobarbituric acid-reactive substances and epileptic discharges in the iron-induced epileptic focus.

Free radical scavenging by brain homogenate: Implication to free radical damage and antioxidant defense in brain

To study the mechanisms of free radical-induced brain damage and the antioxidant defense in the brain, we quantified the superoxide and hydroxyl radical scavenging effects of brain homogenate using electron spin resonance spectrometry. Brain homogenate was found to scavenge both superoxide and hydroxyl radicals in concentration-dependent fashion. Heat denaturation significantly decreased these scavenging effects. The ability of brain homogenate to scavenge free radicals implies that brain damage can be induced by free radicals since they are known to react virtually with any type of molecule such as nucleic acids, membrane lipids, and proteins in the brain. On the other hand, some molecules which can be regenerated or repaired after free radical scavenging are considered to be antioxidants which include both enzymatic and non-enzymatic antioxidants. Measurement of the decrease in antioxidant activity following heat denaturation suggests that the contribution of enzymatic antioxidants is about 20-40% in scavenging superoxide radicals and about 10-20% in scavenging hydroxyl radicals.

α‐Guanidinoglutaric Acid, an Endogenous Convulsant, as a Novel Nitric Oxide Synthase Inhibitor

Abstract: The effects of α‐guanidinoglutaric acid (GGA), the levels of which were increased in the cobalt‐induced epileptic focus tissue in the cerebral cortex of cats, on brain nitric oxide synthase (NOS) activity were observed. GGA inhibited NOS activity in a linear mixed manner (Ki = 2.69 µM) and was as effective as NG‐monomethyl‐l‐arginine (MeArg; Ki = 3.51 µM), a well‐known NOS inhibitor. Although MeArg was synthesized by substituting the guanidino nitrogen of l‐arginine (Arg), GGA was a non‐guanidino nitrogen‐substituted guanidino compound. On the other hand, Arg, which is an endogenous NOS substrate, elevates the threshold of seizures induced by GGA. There is evidence that GGA is an endogenous, potent, and non‐guanidino nitrogen‐substituted NOS inhibitor and that suppression of nitric oxide biosynthesis may be involved in GGA‐induced convulsions. Therefore, GGA may be a useful tool in elucidating the chemical nature of NOS and the physiological function of nitric oxide.

Neuropeptide levels in discrete brain regions in the iminodipropionitrile-induced persistent dyskinesia rat model

To clarify the role of neuropeptides in dyskinesia induced by iminodipropionitrile (IDPN), the levels of five representative neuropeptides were examined in discrete regions of the rat brain 4 weeks after intraperitoneal injection of IDPN. The five neuropeptides examined were methionine-enkephalin (Met-Enk), substance P (SP) and somatostatin, which are closely related to extrapyramidal function, and thyrotropin-releasing hormone (TRH) and cholecystokinin octapeptide (CCK-8), which are closely related to the neural mechanism of the dopamine system. IDPN pretreatment significantly increased Met-Enk in the basal ganglia but not SP or somatostatin; however, all three neuropeptide levels were increased in the hindbrain. In IDPN-treated rats, TRH and CCK-8 levels were increased in the nucleus accumbens, and the frontal cortical CCK-8 level was extremely increased. These findings, together with previous reports, suggest that neuropeptides in the basal ganglia, hindbrain and cerebral cortex play important roles in the manifestation of dyskinetic symptoms.

Inhibitory effect of arcaine on nitric oxide synthase in the rat brain.

Nitric oxide is synthesized by nitric oxide synthase (NOS) from L-Arg, which contains a guanidino group. Arcaine is the diguanidino compound and a derivative of Arg. This study was conducted to investigate the effects of arcaine on rat brain NOS activity, using nitrite and nitrate as indicators. Arcaine inhibited NOS activity in a linear mixed manner (K1 = 18.68 microM). Almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, but the guanidino nitrogens of arcaine were not substituted. Arcaine was also reported to be a competitive antagonist of the polyamine site on the N-methyl-D-aspartic acid (NMDA) receptor. Arcaine appears to be an excellent drug to investigate not only the chemical nature of NOS but also the functional and structural relationship between NOS and NMDA receptors.