Tomochika Ohno

Progressive brain dysfunction following intracerebroventricular infusion of beta1–42-amyloid peptide

The behavioral, neurochemical and histological changes of rats subjected to 3 days treatment with intracerebroventricular infusion of beta-amyloid peptides(Abeta)(1-42) were investigated 20 days and 80 days after the surgery. Abeta(1-42) produced a dose-dependent and a time-dependent impairment in the spontaneous alternation performance in the Y-maze (spatial working memory), place navigation task in a water maze (spatial reference memory) and passive avoidance retention (non-spatial long-term memory) at doses of 10 and 20 microg/rat. The learning impairments were more severe at 80 days than 20 days after infusion of Abeta(1-42). At 25 days after the infusion, a significant decrease in hemicholinium-3 (HC-3) binding was observed only in the hippocampus, although choline acetyltransferase (ChAT) activity was unchanged in the brain regions tested as compared with the vehicle (Abeta(40-1)) treatment. In contrast, the reduction in ChAT activity 85 days after Abeta(1-42) infusion was significant in hippocampus and striatum. HC-3 binding was also significantly decreased in the posterior cortex, hippocampus and striatum. In the histological analysis, brain atrophy was observed inasmuch as ventricular enlargement and neuronal damage in the CA1 area of the hippocampus were seen 85 days after Abeta(1-42) infusion. These results suggest that the rats subjected to intracerebroventricular infusion of Abeta(1-42) suffered from progressive brain dysfunction, and could be useful as an animal model for evaluating the developmental processes at the early and/or middle stage of Alzheimers-type dementia.

Pharmacological characterization of nicotinic receptor-mediated acetylcholine release in rat brain : an in vivo microdialysis study

In vivo microdialysis was used to investigate nicotinic receptor-mediated acetylcholine release in the hippocampus, frontal cortex, and striatum of freely moving rats. Intraperitoneal administration of (-)-nicotine increased the release of acetylcholine in the hippocampus and frontal cortex but not in the striatum. (-)-Nicotine exhibited a bell-shaped dose-response relationship, and showed attenuation of response at the highest dose (5.0 mg/kg i.p.) in both the hippocampus and frontal cortex. In the hippocampus, (-)-nicotine (1.0 mg/kg i.p.)-induced increase of acetylcholine release was blocked by pretreatment with the centrally acting nicotinic receptor channel blocker, mecamylamine (1.0 mg/kg i.p.), but not by hexamethonium (5.0 mg/kg i.p.), suggesting that the effects of (-)-nicotine were mediated by the central nicotinic receptor. (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418, 1.0 and 5.0 mg/kg i.p.), reported to be a selective agonist for alpha4beta2 nicotinic receptor subunits, also enhanced the release of acetylcholine in the hippocampus, while 3-(2,4-dimethoxybenzlidene)-anabaseine (GTS-21, 1.0 and 5.0 mg/kg i.p.), which has high affinity for the alpha7 nicotinic receptor subunit, was without effect. The natural alkaloids isolated from plants, (-)-cytisine and (-)-lobeline, had little effect on acetylcholine release from the hippocampus. A competitive antagonist for alpha4beta2 subunits of the nicotinic receptor, dihydro-beta-erythroidine, and a partial agonist for the beta2 subunit-containing nicotinic receptor, (-)-cytisine, inhibited (-)-nicotine-induced increase of acetylcholine release from the hippocampus, whereas a selective antagonist for the alpha7 subunit, methyllycaconitine, and a partial agonist for the alpha3 subunit-containing nicotinic receptor, (-)-lobeline, did not. These results indicate that there are certain differences among brain regions in the response of nicotinic receptor-mediated acetylcholine release and that (-)-nicotine-induced acetylcholine release in the rat hippocampus may be attributed to activation of the alpha4beta2 nicotinic receptor subunits.

Effects of 16,16-dimethyl prostaglandin E2 on ethanol-induced and aspirin-induced gastric damage in the rat. Scanning electron microscopic study.

Ethanol and aspirin were administered orally to fasted rats and the effects of 16,16-dimethyl prostaglandin E2 (given orally as a pretreatment) were studied using scanning electron microscopy. Gastric secretory studies (pylorus ligation for 2 h) showed that 3 and 30 micrograms/kg of 16,16-dimethyl prostaglandin E2 were nonantisecretory doses and that 100 micrograms/kg was an antisecretory dose. 16,16-Dimethyl prostaglandin E2, given orally at 3-100 micrograms/Kg, induced no appreciable changes in the gastric surface epithelial cells. Oral administration of 1 ml of 50% ethanol invariably induced, within 10 min, extensive exfoliation of surface epithelial cells throughout the corpus and antrum and exposed the lamina propria. 16,16-Dimethyl prostaglandin E2, given orally at 3, 30, and 100 micrograms/kg 30 min before ethanol treatment, had no protective effect. Aspirin, given orally at 30 or 100 mg/kg, also damaged the surface epithelium of both the corpus and the antrum within 10 min. This damage ranged from apical cellular erosions to widespread exposure of the lamina propria. 16,16-Dimethyl prostaglandin E2, given orally at 3 or 30 micrograms/kg 30 min before aspirin treatment, significantly inhibited the gastric damage induced by both 30 and 100 mg/kg of aspirin. The inhibition of damage index was about 50%-60% at either 30 or 100 micrograms/kg of 16,16-dimethyl prostaglandin E2. The mechanism of the protection seen with 16,16-dimethyl prostaglandin E2 remains to be determined.

Protective effects of polysaccharide fucoidin on myocardial ischemia-reperfusion injury in rats.

We tested whether polysaccharide fucoidin, which inhibits leukocyte rolling in the mesenteric venule, has protective effects in the rat myocardial 30-min ischemia and 6-h reperfusion injury model. Intravenous infusion of fucoidin (27 microg/kg/min from 10 min before to 6 h after reperfusion) significantly attenuated myocardial infarct size 6 h after reperfusion. In this ischemia and reperfusion heart model, expression of P-selectin (determined immunohistochemically) was observed on the venular endothelial cells in the heart 30 min after reperfusion and also was sustained after 6 h. Neutrophil infiltration as estimated by myeloperoxidase activity significantly increased 2 h after reperfusion and kept increasing with time until 6 h after reperfusion. Four-hour infusion of fucoidin after reperfusion significantly reduced neutrophil infiltration, whereas the 2-h infusion of fucoidin did not. These results indicate that neutrophil infiltration and myocardial injury are attributed to expression of P-selectin after reperfusion, and that one of the inhibitory mechanisms of fucoidin seems to be blockade of P-selectin-mediated neutrophil rolling on the vessel wall.

New 5-HT1A receptor agonists possessing 1,4-Benzoxazepine scaffold exhibit highly potent anti-ischemic effects

A series of new 3-substituted-4-(4-aminobutyl)-1,4-benzoxazepin-5(4H)-one derivatives (1-5) which showed a very high affinity for 5-HT1A receptor with good selectivity over dopamine D2 receptor was synthesized. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1,4-benzoxazepin-5(4H)-one (5: SUN N4057) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.

Antiarrhythmic agents act differently on the activation phase of the ACh‐response in guinea‐pig atrial myocytes

1 Anti‐acetylcholine effects of pilsicainide, flecainide, disopyramide and propafenone on the acetylcholine (ACh)‐induced K+ current (IK.ACh) were examined in dissociated guinea‐pig atrial myocytes under whole‐cell voltage clamp by the use of the ‘concentration‐clamp’ technique. 2 The IK.ACh was activated with a latency of about 100 ms after 1 μm ACh application and desensitized to a steady‐state level. The latent period and the time to peak response were shortened with increasing ACh concentration. 3 The values of half‐maximal inhibition (IC50) on the peak and steady state responses were 25 and 25 μm for pilsicainide, 1.7 and 2.0 μm for disopyramide, 19 and 2.0 μm for flecainide and 0.7 and 0.2 μm for propafenone, respectively. 4 Pilsicainide and disopyramide increased the latent period and the time to peak of IK.ACh in a concentration‐dependent manner. Flecainide and propafenone did not change the latent period, but shortened the time to peak and hastened the decay of IK.ACh in a voltage‐independent manner. 5 The results suggest that the mechanisms underlying the anti‐acetylcholine effect of antiarrhythmic drugs are different among these drugs: i.e., pilsicainide and disopyramide mainly block the muscarinic ACh receptors while flecainide and propafenone inhibit the K+ channel itself as open channel blockers.

Analysis of 6R- and 6S-tetrahydrobiopterin and other pterins by reversed-phase ion-pair liquid chromatography with fluorimetric detection by post-column sodium nitrite oxidation

A rapid and sensitive reversed-phase ion-pair liquid chromatographic system with fluorimetric detection by post-column sodium nitrite oxidation was established for measuring six pterin compounds (6R-5,6,7,8-tetrahydrobiopterin, 6S,5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, biopterin, pterin and D-neopterin). The coefficients of variation for these pterins were 0.705-3.714%, and the minimum detectable amount was ca. 10-20 pg at a signal-to-noise ratio of 3. A linear detector response was also verified. The concentrations of the pterin compounds in rat tissues were measured by the described method. Furthermore, by means of brain microdialysis, the output of pterin compounds from rat striatum was detected. Therefore, these results demonstrate that this system can be applied to analyses not only of various rat tissues but also of dialysates collected in vivo.

Learning deficits after unilateral AF64A lesions in the rat basal forebrain : role of cholinergic and noncholinergic systems

Rats were given unilateral infusions of ethylcholine aziridinium ion (AF64A) into the basal forebrain (BF). BF-lesioned rats had significant acquisition and retention deficits in two different types of learning tasks (water maze and active avoidance). Choline acetyltransferase activity was lower than control in the frontal cortex but not in the hippocampus or striatum. AF64A markedly reduced the levels of norepinephrine, dopamine, and serotonin in all brain regions studied. However, L-glutamic acid decarboxylase activity was not altered by AF64A injection. Cholinergic agents (physostigmine and arecoline) ameliorated the AF64A-induced learning deficits in the water maze task but not in the active avoidance task. Noncholinergic agents (desipramine and L-dopa) ameliorated the AF64A-induced avoidance deficits in the active avoidance task but not in the water maze task. 5-Methoxy-N,N-dimethyltryptamine did not improve either active avoidance or water maze learning. These results suggest that intra-BF injection of AF64A produces extensive brain dysfunction and that different neuronal systems are involved in associative and spatial learning.

Irritative and protective activity of mild irritants in rat stomach.

Exposure of the stomach for 30 min to acidified sodium taurocholate (TC) (1–20 mM) or sodium salicylate (SA) (10–80 mM) caused a reduction of transmucosal PD and an increase of luminal pH in anesthetized rats, in a concentration-related manner. Acidified aspirin (ASA) (10–80 mM) reduced PD in the same manner, without significant effect on pH. Histologically, these agents similarly produced damage to the surface cells. After a 30-min exposure to either 20 mM TC or 40 mM SA, acid secretion ceased and bicarbonate (0.5–1 μmol/10 min) appeared in the lumen, whereas acid secretion persisted in the stomach exposed to 40 mM ASA. However, under cimetidine infusion (8 mg/kg/hr) these agents produced similar degrees of luminal alkalinization (≈1 μmol/10 min). Pretreatment with indomethacin (5 mg/kg, subcutaneously) significantly inhibited the increase of pH seen after exposure to 20 mM TC, but had no effect on the increase of pH caused by 40 mM SA. Concurrent administration of 16,16-dmPGE2 (3 μg/kg, subcutaneously) significantly antagonized the effect of indomethacin in the stomach exposed to 20 mM TC and even increased the pH in the stomach exposed to 40 mM ASA. After a 3-hr exposure to these agents, there was macroscopically apparent damage only in the stomach exposed to ASA, although the PD was similarly reduced in response to either agent. The levels of PGE2 in the corpus mucosa were significantly increased in stomachs exposed to 20 mM TC and 40 mM SA, but decreased in those exposed to 40 mM ASA. Pretreatment with indomethacin significantly blocked the increased formation of PGE2 caused by TC and SA. These results suggest that mucosal damaging agents such as TC and SA reduce the PD (surface cell injury) and act as mild irritants to induce gastric alkaline response and adaptive mucosal protection, unless, as in the case of ASA, they have an inhibitory effect on prostaglandin synthesis.

Selective induction of fibroblast growth factor receptor-1 mRNA after transient focal ischemia in the cerebral cortex of rats

The expression of the mRNA of four members of the fibroblast growth factor (FGF) receptor family, was examined in rats subjected to temporal middle cerebral artery occlusion using an in situ hybridization technique. Fibroblast growth factor receptor-1 (FGFR-1) mRNA was strongly expressed in neurons of the cerebral cortex, whereas mRNAs of the other 3 subtypes of FGFRs (FGFR-2, -3, and -4) were not expressed. After temporal occlusion of the middle cerebral artery, expression of FGFR-1 mRNA in cerebral cortical neurons markedly increased in association with the progressive neuronal death; this increase was evident for at least 5 days after the focal ischemia. In view of the neuroprotective activity of basic FGF reported so far, the present results suggest that FGFR-1 induction may subserve to self-protect neurons in the ischemic penumbral field of the cerebral cortex.