Tokishakuyakusan ameliorates spatial memory deficits induced by ovariectomy combined with ß-amyloid in rats.

Previously, we reported that ovariectomy (OVX) combined with ß-amyloid peptide (Aß) impaired spatial memory by decreasing extracellular acetylcholine (ACh) levels in the dorsal hippocampus. Here, we investigated the effect of tokishakuyakusan (TSS), a Kampo medicine, on the impairment of spatial memory induced by OVX combined with Aß in rats. Repeated administration of TSS (300 mg/kg, p.o.) significantly decreased the number of errors in the eight-arm radial maze test. Though TSS had no effect on extracellular ACh levels at baseline, TSS significantly increased extracellular ACh levels in the dorsal hippocampus. These results suggest that TSS improves the impairment of spatial memory induced by OVX combined with Aß by (at least in part) increasing extracellular ACh levels in the dorsal hippocampus.

Electrophysiological evidence for 4-isobutyl-3-isopropylbicyclophosphorothionate as a selective blocker of insect GABA-gated chloride channels.

Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors.

Competitive antagonism of insect GABA receptors by iminopyridazine derivatives of GABA.

A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100 µM in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100 µM, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10 µM in these receptors, although the inhibition at 100 µM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500 µM using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site.

Ginsenoside Rb1 reduces neurodegeneration in the peri-infarct area of a thromboembolic stroke model in non-human primates.

Ginsenoside Rb1 (GRb1), a major component of the traditional herb ginseng, has been reported to show a neuroprotective effect in a rodent ischemic model. The purpose of this study was to investigate effects of GRb1 on early and delayed brain injuries in a non-human primate thromboembolic stroke model. Thromboembolic stroke was induced by occlusion of the middle cerebral artery by injection of an autologous blood clot into the left internal carotid artery. GRb1 (300 microg/kg per day, i.v.) and vehicle were administered from 7 days before embolization to the day following embolization (total: 8 times). Neurological deficits were observed at 1, 6, and 24 h and at 2, 4, and 7 days after embolization. At 7 days after embolization, neuron damage in the peri-infarct area and core region were assessed by NeuN, TUNEL, and GFAP staining. GRb1 improved the skeletal muscle coordination score of the neurologic deficits (median: GRb1 vs vehicle = 10 vs 12, P<0.05). In the GRb1 group, positive neurons expressed by NeuN staining were noted in the ischemic peri-infarct area, and TUNEL- and GFAP-positive cells significantly decreased, when compared with vehicle. These results demonstrated that GRb1 ameliorated both early and delayed injuries in the thromboembolic stroke model in non-human primates.

Cerebral ischemia combined with beta-amyloid impairs spatial memory in the eight-arm radial maze task in rats.

beta-Amyloid (Abeta), a major component of senile plaques in Alzheimer's disease, has been implicated in neuronal cell death, a characteristic feature of this condition. In our previous experiments using primary cultures of hippocampal neurons, Abeta treatment induced neuronal cell death, displaying morphological characteristics of apoptosis that was significantly enhanced by hypoxia. Based on these results, we developed a simple in vivo rat model of Alzheimer's disease using cerebral ischemia, instead of hypoxia, combined with continuous intracerebroventricular administration of Abeta. The combination of cerebral ischemia and Abeta administration, but not either treatment alone, significantly impaired spatial memory in an eight-arm radial maze. A microdialysis study showed that spontaneous release of acetylcholine (ACh) from the dorsal hippocampus had a tendency to decrease in response to Abeta treatment alone or the combination of ischemia and Abeta. High K(+)-evoked increase in ACh release had a tendency to be inhibited by either ischemia or Abeta treatment alone and was significantly inhibited by the combination of both. Moreover, combination of ischemia and Abeta induced apoptosis of pyramidal neurons in the CA1 region of the hippocampus. Donepezil, a drug currently in clinical use for Alzheimer's disease, improved the impairment of spatial memory induced by cerebral ischemia combined with Abeta. These findings suggest that ischemia is an important factor facilitating the symptoms of Alzheimer's disease, and this model may be useful for developing new drugs for the treatment of Alzheimer's disease.

Effects of intra-arterial urokinase on a non-human primate thromboembolic stroke model.

One of the most important prognostic factors in the thrombolytic treatment of acute ischemic stroke is to re-canalize. The purpose of this study was to evaluate the effectiveness and safety of urokinase in a primate thromboembolic stroke model. Thromboembolic stroke was accomplished via occlusion of the middle cerebral artery (MCA) obtained by injecting an autologous blood clot into the left internal carotid artery in 21 male cynomolgus monkeys. Animals were randomly assigned to the following treatment groups: Group 1: vehicle (saline), Group 2: urokinase (40,000 IU), Group 3: urokinase (120,000 IU,) over 2 or 6 h via intra-internal carotid catheter starting 1 h after embolization, respectively. In the urokinase-treated groups, neurologic deficits were improved in consciousness and skeletal muscle coordination, but not sensory and motor systems. The infarction size in Group 2 (11.9 +/- 3.9% of the hemisphere) and 3 (7.6 +/- 2.5%) were significantly smaller than that (24.7 +/- 3.5%) in Group 1. However, 2 of 5 animals in Group 3 died. In conclusion, urokinase improved neurologic deficits and reduced cerebral infarction on thromboembolic stroke in the cynomolgus monkey.

Protective effect of Toki-shakuyaku-san on amyloid beta25-35-induced neuronal damage in cultured rat cortical neurons.

Amyloid beta protein (Abeta) is the major component of senile plaques, the pathological hallmark of the neurodegeneration associated with Alzheimer's disease (AD). This study investigated the effect of Toki-shakuyaku-san (TSS), a traditional medicine, on Abeta25-35-induced neuronal death and lipid peroxidation assessed by measuring lactate dehydrogenase (LDH) and malondialdehyde (MDA), respectively. Abeta25-35 at 10 microM induced neuronal damage and increased the LDH and MDA. TSS at concentrations of 100 and 300 microg/mL significantly reduced the Abeta25-35-induced neuronal death and the lipid peroxidation. These results suggest that TSS has a protective effect against Abeta25-35-induced neuronal damage. TSS may be beneficial for the treatment of AD.

Post-ischemic treatment with toki-shakuyaku-san (tang-gui-shao-yao-san) prevents the impairment of spatial memory induced by repeated cerebral ischemia in rats.

Previously we have reported that Toki-shakuyaku-san (TSS) ameliorated the impairment of spatial memory induced by single cerebral ischemia (1 x 10 minutes) and scopolamine, a muscarinic receptor antagonist. In this experiment, we studied the effect of TSS on repeated cerebral ischemia (2 x 10 minutes, 1-hour interval) induced impairment of spatial memory and neuronal injury in rats. The 8-day post-ischemic treatment with TSS (30-300 mg/kg) was administered p.o. once per day. TSS dose-dependently prevented the impairment of spatial memory, neuronal death and TUNEL positive cells induced by repeated cerebral ischemia. In order to determine the mechanism of TSS, we also studied the effect of TSS on GluR2 mRNA, one of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor subunits. Repeated cerebral ischemia significantly decreased GluR2 flop mRNA at 1 and 3 days after the occlusion. TSS (300 mg/kg) significantly suppressed the decrease in GluR2 flop at 3 days after repeated cerebral ischemia. These results suggested that the TSS has neuroprotective action which may be indirectly mediated by the AMPA receptor, and TSS may be beneficial for the treatment of cerebrovascular dementia.

Ovariectomy combined with amyloid beta(1-42) impairs memory by decreasing acetylcholine release and alpha 7nAChR expression without induction of apoptosis in the hippocampus CA1 neurons of rats.

In this study, the effect of ovariectomy and amyloid Beta(1-42) (ABeta(1-42))on eight-armed radial maze performance, acetylcholine (ACh) release, Alpha7nACh receptor (Alpha7nAChr), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and apoptosis of CA1 neurons in the dorsal hippocampus were investigated in rat. The results showed that the dorsal hippocampus of sham rats contains 136.7 -/+ 16.7 to 160.4 -/+ 21.1 fmol/microl ACh, and respective 201 -/+ 22.9 and 416.6 -/+ 66.3 expression of mRNA for a7nAChR and GAPDH. Ovariectomy alone, after 4 weeks, did not impair memory, and neither induced apoptosis nor changed the basal ACh release. On the other hand, ABeta(1-42) (600 pmol/10 microl/body/day i.c.v. for 7 days) impaired memory, an effect characterized by increased error choices and reduced (50-59%) ACh release, but only with slight apoptosis. Moreover, ovariectomy combined with ABeta(1-42) induced memory impairment characterized by decreased numbers of correct choices and increased numbers of errors. This effect was accompanied by a decrease of the basal ACh level (67%), a7nAChR mRNA expression (52%) and a7nAChR/GAPDH ratio (44%) without induction of apoptosis in the dorsal hippocampus. The high K+-evoked ACh release was not altered in ovariectomized rats, but was decreased by ABeta(1-42) (43%) and ovariectomy + ABeta(1-42) (80%). These results suggest that ovariectomy-induced hormonal deprivation after 4 weeks, when accompanied by ABeta(1-42) accumulation in the dorsal hippocampus, could impair memory by decreasing ACh release and a7nAChR expression without inducing apoptosis in the CA1 field of the dorsal hippocampus.