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A growing body evidence suggests that selenium (Se) deficiency is associated with an increased risk of developing Alzheimer’s disease (AD). Se-dependent glutathione peroxidase-1 (GPx-1) of a major antioxidant enzyme, and the most abundant isoform of GPx in the brain. In the present study, we investigated whether GPx-1 is protective against memory impairments induced by beta-amyloid (Aβ) (1–42) in mice. As the alteration of protein kinase C (PKC)-mediated ERK activation was recognized in the early stage of AD, we examined whether the GPx-1 gene modulates Aβ (1–42)-induced changes in PKC and ERK levels. We observed that Aβ (1–42) treatment (400 pmol, i.c.v.) significantly decreased PKC βII expression in the hippocampus of mice. Aβ (1–42)-induced neurotoxic changes [i.e., oxidative stress (i.e., reactive oxygen species, 4-hydroxy-2-noneal, and protein carbonyl), reduced PKC βII and phospho-ERK expressions, and memory impairment under Y-maze and passive avoidance test] were more pronounced in GPx-1 knockout than in wild type mice. Importantly, exposure to a GPx-1 gene-encoded adenovirus vector (Adv-GPx-1) significantly increased GPx-1 mRNA and GPx activity in the hippocampus of GPx-1 knockout mice. Adv-GPx-1 exposure also significantly blocked the neurotoxic changes induced by Aβ (1–42) in GPx-1 knockout mice. Treatment with ERK inhibitor U0126 did not significantly change Adv-GPx-1-mediated attenuation in PKC βII expression. In contrast, treatment with PKC inhibitor chelerythrine (CHE) reversed Adv-GPx-1-mediated attenuation in ERK phosphorylation, suggesting that PKC βII-mediated ERK signaling is important for Adv-GPx-1-mediated potentials against Aβ (1–42) insult. Our results suggest that treatment with the antioxidant gene GPx-1 rescues Aβ (1–42)-induced memory impairment via activating PKC βII-mediated ERK signaling.

Dextromethorphan (DM) administered at supra-antitussive doses produce psychotoxic and neurotoxic effects in humans. We administered DM (80 mg/kg) to rats intraperitoneally to determine the ultrastructural change induced by DM, because intraperitoneal route is sensitive for the behavioral responses. Treatment with DM resulted in mitochondrial dysfunction and formation of myelinoid bodies in the hippocampus. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] attenuated DM-induced cytosolic oxidative burdens. However, neither MK-801 nor naloxone affected DM-induced mitochondrial dysfunction and formation of myelinoid bodies, indicating that the neurotoxic mechanism needs to be further elucidated. Therefore, the spectrum of toxicological effects associated with DM need to be reassessed. (C) 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.

We investigated whether protein kinase C (PKC) is involved in trimethyltin (TMT)-induced neurotoxicity. TMT treatment (2.8 mg/kg, i.p.) significantly increased PKCδ expression out of PKC isozymes (i.e., α, βI, βII, δ, and ς) in the hippocampus of wild-type (WT) mice. Consistently, treatment with TMT resulted in significant increases in cleaved PKCδ expression. Genetic or pharmacological inhibition (PKCδ knockout or rottlerin) was less susceptible to TMT-induced seizures than WT mice. TMT treatment increased glutathione oxidation, lipid peroxidation, protein oxidation, and levels of reactive oxygen species. These effects were more pronounced in the WT mice than in PKCδ knockout mice. In addition, the ability of TMT to induce nuclear translocation of Nrf2, Nrf2 DNA-binding activity, and upregulation of γ-glutamylcysteine ligase was significantly increased in the PKCδ knockout mice and rottlerin (10 or 20 mg/kg, p.o. × 6)-treated WT mice. Furthermore, neuronal degeneration (as shown by nuclear chromatin clumping and TUNEL staining) in WT mice was most pronounced 2 days after TMT. At the same time, TMT-induced inhibition of phosphoinositol 3-kinase (PI3K)/Akt signaling was evident, thereby decreasing phospho-Bad, expression of Bcl-xL and Bcl-2, and the interaction between phospho-Bad and 14-3-3 protein, and increasing Bax expression and caspase-3 cleavage were observed. Rottlerin or PKCδ knockout significantly protected these changes in anti- and pro-apoptotic factors. Importantly, treatment of the PI3K inhibitor LY294002 (0.8 or 1.6 µg, i.c.v.) 4 h before TMT counteracted protective effects (i.e., Nrf-2-dependent glutathione induction and pro-survival phenomenon) of rottlerin. Therefore, our results suggest that down-regulation of PKCδ and up-regulations of Nrf2-dependent glutathione defense mechanism and PI3K/Akt signaling are critical for attenuating TMT neurotoxicity.

Platelet-activating factor (PAF), a potent mediator of inflammatory and immune responses, plays various roles in neuronal functions. However, little is known about the role of PAF/platelet-activating factor receptor (PAF-R) in Parkinson's disease. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in significant increases in PAF species in the striatum of wild-type mice. These increases paralleled PAF-R gene expression in wild-type mice. Although nuclear factor kappa B (NF-κB) DNA-binding activity was increased significantly in MPTP-treated wild-type mice, this increase was not significant in PAF-R antagonist ginkgolide B (GB)-treated mice or PAF-R knockout (PAF-R(-/-)) mice. Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, significantly ameliorated the dopaminergic deficits induced by MPTP in wild-type mice. MPTP treatment significantly increased oxidative damage, the immunoreactivity of ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglial cells, and microglial differentiation of the M1 type in the striatum of wild-type mice. Consistently, PDTC significantly attenuated MPTP-induced behavioral impairments in wild-type mice. However, dopaminergic deficits, oxidative damage, reactive microglial cells, and behavioral impairments induced by MPTP were not significantly observed in GB-treated mice or PAF-R(-/-) mice. PDTC did not significantly alter the attenuations evident in MPTP-treated PAF-R(-/-) mice, indicating that NF-κB is a critical target for neurotoxic modulation of PAF-R. We propose for the first time that PAF/PAF-R can mediate dopaminergic degeneration via an NF-κB-dependent signaling process.

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.

We have investigated the radioprotective efficacy of eckol, a component of brown seaweed Ecklonia cava, against the gamma ray-induced damage in vivo. Our results showed that eckol significantly decreased the mortality of lethally irradiated mice. The mechanisms of eckol’s protection were found to include: an improvement in hematopoietic recovery, the repair of damaged DNA in immune cells and an enhancement of their proliferation, which had been severely suppressed by ionizing radiation. Thus, we propose eckol as a candidate for adjuvant therapy to alleviate radiation-induced injuries to cancer patients.

We examined whether (-)-nicotine infusion can affect kainic acid (KA)-induced neurotoxicity in rats. Although treatment with a single nicotine infusion (0.5 or 1.0 microg/side, i.c.v.) failed to attenuate KA-induced neurotoxicity, repeated nicotine infusions (1.0 microg/side/day for 10 days) attenuated the seizures, the severe loss of cells in hippocampal regions CA1 and CA3, the increase in activator protein (AP)-1 DNA binding activity, and mortality after KA administration. alpha-Bungarotoxin and mecamylamine blocked the neuroprotective effects of nicotine. These results suggest that repeated nicotine treatment provides alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against KA toxicity.

To determine the role of ceruloplasmin (Cp) in epileptic seizures, we used a kainate (KA) seizure animal model and examined hippocampal samples from epileptic patients. Treatment with KA resulted in a time-dependent decrease in Cp protein expression in the hippocampus of rats. Cp-positive cells were colocalized with neurons or reactive astrocytes in KA-treated rats and epileptic patient samples. KA-induced seizures, initial oxidative stress (i.e., hydroxyl radical formation, lipid peroxidation, protein oxidation, and synaptosomal reactive oxygen species), altered iron status (increasing Fe2+ accumulation and L-ferritin-positive reactive microglial cells and decreasing H-ferritin-positive neurons), and impaired glutathione homeostasis and neurodegeneration (i.e., Fluoro-Nissl and Fluoro-Jade B staining analyses) were more pronounced in Cp antisense oligonucleotide (ASO)- than in Cp sense oligonucleotide-treated rats. Consistently, Cp ASO facilitated KA-induced lactate dehydrogenase (LDH) release, Fe2+ accumulation, and glutathione loss in neuron-rich and mixed cultures. However, Cp ASO did not alter KA-induced LDH release or Fe2+ accumulation in the astroglial culture, but did facilitate impairment in glutathione homeostasis in the same culture. Importantly, treatment with human Cp protein resulted in a significant attenuation against these neurotoxicities induced by Cp ASO. Our results suggest that Cp-mediated neuroprotection occurs via the inhibition of seizure-associated oxidative damage (including impairment in glutathione homeostasis), Fe2+ accumulation, and alterations in ferritin immunoreactivity. Moreover, interactive modulation between neurons and glia was found to be important for Cp upregulation in the attenuation of epileptic damage in both animals and humans.

We investigated the neuroprotective property of analogs of dextromethorphan (DM) in lipopolysaccharide (LPS) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models to identify neuroprotective drugs for Parkinson's disease (PD). In vivo studies showed that daily injections with DM analogs protected dopamine (DA) neurons in substantia nigra pars compacta and restored DA levels in striatum using two different models for PD. Of the five analogs studied, 3-hydroxymorphinan (3-HM), a metabolite of DM, was the most potent, and restored DA neuronal loss and DA depletion up to 90% of the controls. Behavioral studies showed an excellent correlation between potency for preventing toxin-induced decrease in motor activities and neuroprotective effects among the DM analogs studied, of which 3-HM was the most potent in attenuating behavioral damage. In vitro studies revealed two glia-dependent mechanisms for the neuroprotection by 3-HM. First, astroglia mediated the 3-HM-induced neurotrophic effect by increasing the gene expression of neurotrophic factors, which was associated with the increased acetylation of histone H3. Second, microglia participated in 3-HM-mediated neuroprotection by reducing MPTP-elicited reactive microgliosis as evidenced by the decreased production of reactive oxygen species. In summary, we show the potent neuroprotection by 3-HM in LPS and MPTP PD models investigated. With its high efficacy and low toxicity, 3-HM may be a novel therapy for PD.

The expression of caveolin-1 and -2 in the retina was examined; Western blot analysis showed that both were present. Immunohistochemistry indicated that caveolin-1 was expressed in the majority of retinal layers, including the ganglion cell layer, inner plexiform layer, outer plexiform layer, and in the vascular endothelial cells of the retina. Caveolin-2 was primarily immunostained in the vessels, but in a few other elements as well. This is the first demonstration of caveolin differential expression in the retina of rats, and suggests that caveolin plays an important role in signal transduction in glial cells and neuronal cells.

We examined whether the methanol extract of Opuntia ficus-indica (MEOF) has a neuroprotective action against N -methyl- d -aspartate (NMDA)-, kainate (KA)-, and oxygen–glucose deprivation (OGD)-induced neuronal injury in cultured mouse cortical cells. We also evaluated the protective effect of MEOF in the hippocampal CA1 region against neuronal damage evoked by global ischemia in gerbils. Treatment of neuronal cultures with MEOF (30, 300, and 1000 μg/ml) inhibited NMDA (25 μM)-, KA (30 μM)-, and OGD (50 min)-induced neurotoxicity dose-dependently. The butanol fraction of Opuntia ficus-indica (300 μg/ml) significantly reduced NMDA (20 μM)-induced delayed neurotoxicity by 27%. Gerbils were treated with MEOF every 24 h for 3 days (0.1, 1.0, and 4.0 g/kg, p.o.) or for 4 weeks (0.1 and 1.0 g/kg, p.o.), and ischemic injury was induced after the last dose. Neuronal cell damage in the hippocampal CA1 region was evaluated quantitatively at 5 days after the ischemic injury. When gerbils were given doses of 4.0 g/kg (3 days) and 1.0 g/kg (4 weeks), the neuronal damage in the hippocampal region was reduced by 32 and 36%, respectively. These results suggest that the preventive administration of Opuntia ficus-indica extracts may be helpful in alleviating the excitotoxic neuronal damage induced by global ischemia.

The deposition of beta-amyloid protein (A beta), a 39-43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimer's disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti-inflammatory and antioxidant action. In this study, using whole-cell patch clamp recordings we examined the effects of Fucoidan on A beta-induced whole-cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking A beta neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of A beta(25-35) (1 microM) caused a reduction of the whole-cell currents by 16%. Fucoidan, in a dose-dependent manner, blocks the A beta(25-35) reduction of whole-cell currents. Exposure of A beta(25-35) (20 microM) or A beta(1-42) (20 microM) to rat cholinergic basal forebrain cultures for 48 h resulted in 40-60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1-1.0 microM). Fucoidan also attenuated A beta-induced down-regulation of phosphorylated protein kinase C. A beta(1-42)-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, A beta activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block A beta-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against A beta-induced neurotoxicity in basal forebrain neuronal cultures.

Background: We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment. Methods: First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin. Results: Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice. Conclusions: Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.

1. Disruption of calcium homeostasis during neurodegenerative diseases is known to trigger apoptotic or necrotic death in neuronal cells. Recently, the authors reported that intracellular calcium restriction by NMDA receptor antagonists induces apoptosis in cortical cultures. To evaluate whether further restriction of intracellular free calcium can induce apoptosis or necrosis, we examined the neurotoxic characterization of BAPTA/AM, a permeable free calcium chelator, in mouse cortical cultures. 2. Exposure of mixed (glia and neuron) cortical cultures (DIV 13-16) to 3-10 microM BAPTA/AM (non-toxic concentration for glial cells) for 24-48 hr resulted in delayed and necrotic neuronal death. The necrotic findings included swelling and loss of mitochondria and endoplasmic reticulum (ER) with neuronal membrane rupture 24 hr after treatment with BAPTA/AM. Simultaneously, we observed a few TUNEL-positive cells in the neuronal subpopulation of the same cultures. 3. The neurotoxicity evoked by BAPTA/AM (10 microM) was significantly attenuated by the addition of 0.5 microM cycloheximide (a protein synthesis inhibitor), 10 microM actinomycin D (an RNA transcription inhibitor), a high extracellular potassium concentration (total 15 mM KCl), 100 microM t-ACPD (a metabotrophic agonist), 100 microM alpha-tocopherol (a free radical scavenger), 100 microM deferoxamine (a ferric ion chelator), 100 microM L-NAME (a nitric oxide synthase (NOS) inhibitor), 50 microM DNQX (a non-NMDA receptor blocker), and 3-30 microM esculetin (a lipoxygenase inhibitor). However, 0.3-3 mM ASA (a cyclooxygenase inhibitor), 100 ng/ml nerve growth factor (NGF), 10 microM MK-801 (a NMDA receptor antagonist), 20 microM zVAD-fmk (caspase inhibitor) and 50 U/ml catalase failed to inhibit the injury. 4. However, NGF and catalase blocked the neurotoxicity induced by BAPTA/AM in young neuronal cells (DIV 6). BAPTA/AM (10 microM) did not alter the expression of inducible nitric oxide synthase (iNOS) on glial cells. 5. These results suggest that the feature of neuronal death induced by BAPTA/AM exhibits predominantly delayed necrosis mediated by lipoxygenase-dependent free radicals.

Acculmulating evidence indicates that a marked generation of oxygen free radicals derived from the metabolism of arachidonic acid causes neurodegeneration. Recently, we have demonstrated that the novel antioxidant actions mediated by phenidone, a dual inhibitor of cyclooxygenase/lipoxygenase pathways, may play a crucial role in preventing neuroexcitotoxicity in vitro [Neurosci. Lett. 272 (1999) 91], and that phenidone significantly attenuates kainic acid (KA)-induced seizures via inhibiting the synthesis of Fos-related antigen protein [Brain Res. 782 (1998) 337]. In order to extend our understanding of the pharmacological intervention of phenidone, we evaluated the antioxidant activity of this compound in vivo in the present study. In order to better understand the significance of a blockade of both the cyclooxygenase and lipoxygenase pathways, we studied the effects of aspirin (ASP; a non-selective inhibitor of cyclooxygenase), NS-398 (a selective inhibitor of cyclooxygenase-2), esculetin (an inhibitor of lipoxygenase) and phenidone on lipid peroxidation, protein oxidation, and glutathione (GSH) status in the rat hippocampus after KA administration. ASP (7.5 or 15 mg/kg), NS-398 (10 or 20 mg/kg), esculetin (5 or 10 mg/kg) or phenidone (25, 50 or 100 mg/kg) was administered orally five times every 12 h before the injection of KA (10 mg/kg, i.p.). The KA-induced toxic behavioral signs, oxidative stress (lipid peroxidation and protein oxidation), impairment of GSH status, and the loss of hippocampal neurons were dose-dependently attenuated by the phenidone, NS-398+esculetin, and ASP+esculetin. However, ASP, NS-398 and esculetin alone failed to protect against the neurotoxicities induced by KA. Therefore, the results suggest that protection by blockade of both cyclooxygenase and lipoxygenase pathways against KA-induced neuroexcitotoxicity is via antioxidant actions. However, a novel anticonvulsant/neuroprotective effect mediated by phenidone remains to be further characterized.

The expression of pro-apoptotic molecules p53 and Bax in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) was examined. Apoptosis was confirmed by the terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) method. TUNEL (1) apoptotic cells were mainly either ED1 (1) macrophages or T-cells in the parenchyma of EAE. Western blot analysis showed that both p53 and Bax expression significantly (P , 0:01) increased in the spinal cords of EAE rats at the peak stage, and thereafter declined. An immunohistochemical study showed that inflammatory cells (notably T cells) in the parenchyma express p53 and Bax, while brain cells, including neurons and glia, were devoid of nuclear staining for these molecules. The nuclear expression of p53 largely matches apoptotic cells in the parenchyma of EAE. These findings suggest that the pro-apoptotic molecules p53 and Bax may play an important role in eliminating T cells in the parenchyma in EAE. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

To elucidate whether an inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AMG), affects the expression of iNOS in the spinal cords of rats with experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats, and treated EAE rats with AMG. AMG (200mg/kg administered intraperitoneally from day 0 to day 7 post-immunization) significantly reduced the clinical severity of EAE paralysis. AMG, however did not prevent the occurrence of EAE. Western blot analysis showed that iNOS expression was significantly reduced in the spinal cords of rats with EAE treated with AMG compared with rats treated with the vehicle. This rinding supports the conclusion that the production of nitric oxide by iNOS plays an important role in the induction of EAE. The corollary is that the amelioration of EAE paralysis by the treatment with AMG is associated with the suppression of iNOS expression in the target organ i.e. the spinal cord.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis."

Effects of long-term oral administration of ginseng extract on serum protein profile and immunoglobulin (Ig) isotypes were studied in mice. Ginseng extract was orally administered to healthy female mice for 52 days at doses of 30 and 150 mg/kg per day and serum protein electrophoretograms and Ig isotypes levels were evaluated. Serum level of gamma-globulin was decreased dose dependently to 82% (P0.05) and 56% (P0.01) of control values at the doses of 30 and 150 mg/kg per day, respectively. Levels of total protein, albumin, alpha2- and beta-globulin fractions, as well as the ratio of albumin to globulin (A/G) did not change significantly. However, the alpha1-globulin level increased by 24% (P0.05) at the doses of 30 and 150 mg/kg per day. Among the Ig isotypes, including IgG1, IgG2a, IgG2b, IgG3, IgM and IgA, serum IgG1 was dose dependently decreased to 68% (P0.05) of control values at the dose of 150 mg/kg per day without significant changes in other Ig isotypes. As IgG1 isotype is rarely cytotoxic and can act as a blocking antibody, it is suggested that the selective decrease in serum IgG1 induced by ginseng extract without changes in the cytotoxic antibodies such as IgG2a may be helpful for the prevention and inhibition of cancer.

The distribution and characteristics of cholecystokinin (CCK)-like immunoreactive (LIR) nerve fibers was examined in the cat olfactory bulb using immunohistochemistry. CCK-LIR cell bodies were not found; fine varicose CCK-LIR fibers were observed in the most layers. In the main olfactory bulb, no staining was seen in the olfactory nerve layer and white matter. The directions of CCK-LIR fibers in the glomerular, external plexiform and mitral cell layers were generally perpendicular to the bulbar surface; those in the deep granule cell layer were parallel; and those in the internal plexiform and superficial granule cell layers were mixed. In the accessory olfactory bulb, CCK-LIR fibers were localized only in the granule cell layer. The presence of CCK-LIR fibers of the cat olfactory bulb may be involved in the modulation of olfactory transmission.

We examined the neuroprotective efficacy of eugenol against N-methyl-D-aspartate (NMDA)-, oxygen-glucose deprivation-, and xanthine/xanthine oxidase-induced neurotoxicity in primary murine cortical cultures. Eugenol (100-300 microM) attenuated NMDA (300 microM)-induced acute neurotoxicity by 20-60%. At the same concentration range, eugenol also inhibited NMDA (300 microM)-induced elevation in neuronal 45Ca2+ uptake by 10-30%. In the oxygen-glucose deprivation (50 min) neurotoxicity, eugenol (100-300 microM) prevented acute neuronal swelling and reduced neuronal death by 45-60% in a concentration-dependent fashion. Oxidative neuronal injury induced by xanthine/xanthine oxidase was also significantly reduced (75-90%) by eugenol (100- 300 microM) addition. These results suggest that eugenol may play a protective role against ischemic injury by modulating both NMDA receptor and superoxide radical.

MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclopepten-5,10-imine maleate), a non-competitive NMDA receptor antagonist (0.01-1 micrograms), injected intracerebroventricularly (i.c.v.) dose dependently increased the baseline levels of plasma interleukin-6 in mice. In the 1-h immobilization-stressed animals, MK-801 (1 micrograms) administered i.c.v. produced an additive increase of plasma interleukin-6. NMDA (N-methyl-D-aspartate) (3, 10 ng) administered i.c.v. attenuated dose dependently the 1-h immobilization stress-induced rise in plasma interleukin-6 level. Neither 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (0.01-0.5 micrograms) nor alpha-methyl-4-carboxyphenylglycine (MCPG) (1-20 micrograms), antagonists of non-NMDA and metabotropic glutamate receptors, respectively, i.c.v. administered, affected the basal and stress-induced plasma interleukin-6 levels. These data indicate that NMDA receptors may be involved in the suppressive regulation of the plasma interleukin-6 levels.

We studied the effects of p-synephrine on the immobility behaviors and on the spontaneous motor activity in mice. p-Synephrine at oral doses from 1 to 10 mg/kg significantly decreased the duration of immobility in the tail suspension test and the forced swimming test in mice. At 30 mg/kg, the duration of immobility was returned to control values in both tests. Subcutaneous administration of prazosin hydrochloride (62.5 micrograms/kg), an alpha 1 adrenoceptor antagonist, blocked the p-synephrine (3 mg/kg)-induced decrease in immobility in the tail suspension test. p-Synephrine did not change the spontaneous motor activity at oral doses from 0.3 to 10 mg/kg. These results suggest that p-synephrine elicits an antidepressant-like activity in mouse models of immobility tests, through the stimulation of alpha 1 adrenoceptors.

Staurosporine, a nonselective protein kinase inhibitor, has been shown to induce apoptosis in several different nonneuronal cell types. We tested the hypothesis that staurosporine would also induce apoptosis in central neurons. Exposure of murine cortical cell cultures to 30-100 nM staurosporine induced concentration-dependent selective neuronal degeneration over the following day; at higher concentrations, staurosporine damaged glial cells as well. Staurosporine-induced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and DNA laddering. In contrast, NMDA-induced neuronal death was accompanied by acute cell body swelling without DNA laddering. Staurosporine-induced neuronal death, unlike excitotoxic death, was markedly attenuated by the protein synthesis inhibitor cycloheximide; this protective effect was not reversed by a glutathione synthesis inhibitor, buthionine sulfoximine. Interestingly, the glial cell death induced by 1 microM staurosporine was markedly potentiated by cycloheximide. Staurosporine-induced neuronal death was not accompanied by an increase in intracellular free Ca2+ and was attenuated by 30 mM K+; this protective effect of high K+ was blocked by nimodipine or Co2+. Present data suggest that staurosporine can induce apoptosis in cultured cortical neurons and that this apoptosis can be blocked by raising intracellular Ca2+ or by blocking protein synthesis. Staurosporine exposure may be useful as a model for studying central neuronal apoptosis in vitro.

We examined whether fucoidan affected the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in rats. EAE was induced in Lewis rats that were immunized with guinea-pig myelin basic protein (MBP) and complete Freund's adjuvant. Fucoidan (50 mg/kg, daily) was administered to rats with EAE intraperitoneally, either in the EAE induction phase from either 1 day before immunization to day 7 post-immunization (PI), or the effector phase from day 8 to 14 PI, to test which phase of rat EAE is affected by fucoidan treatment.The onset, severity and duration of EAE paralysis in the fucoidan-treated group in the days 8-14 PI-treated rats, but not in days -1-7 PI-treated rats, were significantly delayed, suppressed and reduced, respectively, compared with the vehicle-treated controls. Treatment with fucoidan reduced the encephalitogenic response and TNF-alpha production during EAE. Moreover, the clinical amelioration coincided with decreased infiltration of inflammatory cells in the EAE-affected spinal cord. The ameliorative effect of fucoidan on clinical paralysis in EAE-affected rats may be mediated, in part, by the suppression of the autoreactive T cell response and inflammatory cytokine production.

Accumulated evidence has indicated that neuroinflammation is one of the important etiologic factors of Parkinson's disease (PD). Earlier studies have employed the inflammogen lipopolysaccharide (LPS) to induce inflammation of dopaminergic neurons. Methamphetamine (MA) dopaminergic toxicity similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity is frequently cited as a model of PD. In the present study, we examined whether striatal LPS exposure potentiates MA-induced dopaminergic toxicity. Combined treatment with LPS and MA significantly potentiates behavioral impairment and dopaminergic deficit. However, this combination did not significantly alter the other monoaminergic systems (e.g., serotonin, norepinephrine, and histamine). Consistently, microglial activation, labeled by F4/80 or Iba-1 in the nigrostriatal region was more pronounced with the combined treatment of LPS and MA compared to either treatment alone, but this combination did not significantly alter the microglial activation in other brain regions (e.g., hippocampus, dorsal raphe nuclei, and locus ceruleus). Furthermore, neuroinflammation, oxidative stress, and pro-apoptotic changes in the striatum were more accentuated with combined treatment of LPS and MA compared to either treatment alone. In addition, it is important that cytoplasmic accumulation of alpha-synuclein was observed in the substantia nigra of mice treated with LPS plus MA, and that L-Dopa treatment significantly attenuated behavioral changes and dopaminergic deficits induced by LPS plus MA. These results suggest that combined treatment of LPS with MA is a potential animal model for PD.

Anti-inflammatory drugs such as ibuprofen appear to prevent the development of Parkinson's disease (PD); however, long-term use has undesirable side-effects. A new strategy for anti-inflammatory drug therapy is using a dual inhibitor of COX and lipooxygenase (LOX). Here, we compared the dopaminergic neuroprotective property of phenidone (a dual COX and LOX inhibitor) with COX or LOX inhibitors including SC-560 (a COX-1 inhibitor), aspirin (a COX-1/2 inhibitor), meloxicam (a preferential COX-2 inhibitor), caffeic acid (a 5-LOX inhibitor), and esculetin (a 5, 12-LOX inhibitor) in our lipopolysaccharide (LPS)-induced PD animal model. Our results show that COX-2 and 5-LOX play a major role in LPS-induced dopaminergic neurotoxicity, as meloxicam and phenidone attenuated LPS-induced oxidative stress and meloxicam, phenidone, and caffeic acid attenuated dopaminergic neurodegeneration, while SC-560, aspirin, and esculetin did not. In addition, phenidone was superior in attenuating LPS-induced dopaminergic neurodegeneration and microglia activation, probably as a result of dual inhibition of COX-2 and LOX. Therefore, dual inhibition of COX and LOX with phenidone represents a promising new candidate for anti-inflammatory drug therapy, and may provide a novel therapeutic approach for inflammation-related neurodegenerative diseases including PD.

Oxidative stress may contribute to epileptogenicity in genetic models of epilepsy. To address this, we examined the enzymatic activity of cytosolic Cu/Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), and glutathione peroxidase (GPx) in the developing hippocampus of genetically epilepsy-prone rats (GEPR-9s). We also measured changes in the GSH/GSSG ratio, lipid peroxidation, and protein oxidation at post-natal days (PD) 7, 30, and 90, respectively. Compared with control Sprague-Dawley (SD) rats, GEPR-9s showed similar SOD-1 and SOD-2 activity but lower GPx activity. Epilepsy-prone rats also showed lower GSH/GSSG ratios than controls, and more lipid peroxidation (as measured by malondialdehyde levels) and protein oxidation (as measured by carbonyl levels). Treatment with kainic acid (KA) resulted in more pronounced seizures, less GPx activity, and lower GSH/GSSG ratios in GEPR-9s than in controls, but KA did not significantly affect SOD-1 or SOD-2 activity, suggesting that GEPR-9s do not compensate for reduced GPx activity by increasing SOD. Moreover, KA treatment resulted in significantly a lower GSH/GSSG ratio and GPx-like immunoreactivity and higher malondialdehyde and carbonyl levels in GEPR-9s than in controls. These findings were more evident in GEPR-9s at PD 90 than at PD 30, indicating that oxidative stress is age-dependent. Double-labeling immunocytochemical analysis demonstrated co-localization of GPx-immunoreactive glia-like cells and reactive astrocytes, as labeled by glial fibrillary acidic protein (GFAP). This suggests that mobilization of astroglial cells for synthesis of GPx protein is a response to KA insult, intended to decrease the neurotoxicity induced by peroxides. These responses were more pronounced in control SD rats than in GEPR-9s. Our results suggest that impairment of the GPx (including glutathione)-mediated antioxidant system contributed to epileptogenesis in GEPR-9s.

This study examined whether phenidone, a dual inhibitor of cyclooxygenase (COX) and lipoxygenase (LOX), affects the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in the rat, and the expression of both COX-1/-2 and 5-LOX in EAE spinal cords. Oral phenidone (200 mg/kg) significantly suppressed the incidence and clinical severity of EAE paralysis. Western blot analysis showed that phenidone significantly inhibited the increases in COX-1/-2 and 5-LOX in the spinal cords of rats with EAE. This finding was paralleled by immunohistochemical observations. Overall, these findings suggest that COX-1/-2 and 5-LOX are important inflammatory mediators in the pathogenesis of EAE, and that the inhibition of both COX and LOX ameliorates the autoimmune disorder of the central nervous system.

The effect of PAP 9704, a traditional prescription in Korea consisting of Polygala tenuifolia, Acorus gramineus, and Poria cocos at a ratio of 1:1:1 (dry weight), on methamphetamine (MA)-induced hyperlocomotion was examined in mice. The increased locomotor activity induced by MA (1 mg/kg/d, i.p. x 7) was significantly attenuated by co-administration with PAP 9704 (100 or 200 mg/kg/d, p.o. x 7) in a dose dependent manner. Consistently, it was found that the hyperlocomotor activity occurred in parallel with the expression of striatal fos-related antigen immunoreactivity. The adenosine A(2A) receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (0.5 or 1.0 mg/kg, i.p.), significantly reversed the pharmacological action of PAP 9704 in a dose related manner, but the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (0.5 or 1.0 mg/kg, i.p.) and the A(2B) receptor antagonist alloxazine (1.5 or 3.0 mg/kg, i.p.) did not significantly affect this pharmacological action. Our results suggest that PAP 9704 prevents MA-induced hyperlocomotion, at least in part, via the stimulation of the adenosine A(2A) receptor.

This study investigated the immunohistochemical localization of nestin and its expression level in the brains of mice infected with scrapie, a disease which is characterized by spongiform neurodegeneration, neuronal vacuolation, and astrogliosis. Western blot analysis showed that the protein level of nestin was significantly increased in scrapie-infected brains compared with those of control brains. Nestin immunoreactivity in vascular endothelial cells was more intense in the brains of scrapie-infected mice compared with those of controls. There was marked nestin immunostaining in reactive astrocytes, but not in the neurons of the scrapie-infected brains. Considering all the findings, this study suggests that the nestin-positive cells, including glial cells and vascular endothelial cells, may have the potential to differentiate into mature glial cells and other neuronal elements, which would replenish lost neurons.

Oxidative stress caused by an elevation in reactive oxygen species (ROS) plays an important role in Alzheimer's disease and other neurodegenerative diseases. In this study, we examined the neuroprotective effect of danthron (1,8-dihydroxyanthraquinone) against neurotoxicities induced by beta-amyloid (25-35), excitotoxins, apoptosis, and oxidative stress in primary cortical cultures. Danthron dose-dependently reduced neuronal injury induced by 30 microM beta-amyloid (25-35). Danthron significantly inhibited oxidative injury induced by 100 microM Fe(3+) and decreased membrane lipid peroxidation induced by 100 microM Fe(3+) as measured by thiobarbituric-acid-reactive substance (TBARS). Danthron (0.5-50 microM) ameliorated the effects of buthionine sulfoximine (BSO, 1 mM), which depletes endogenous glutathione by 10-73%. Danthron also dose-dependently inhibited neuronal injury mediated by nitric oxide (NO) radicals, but failed to inhibit injury due to superoxide radicals (O(2-)). These results suggest danthron treatment may, in part, reduce neurotoxicity related to beta-amyloid protein by both dominant inhibitory effects on membrane lipid peroxidation and glutathione deprivation.

The expression of phospholipase D (PLD) in the hearts of rats with experimental autoimmune myocarditis (EAM) was studied to elucidate the functional role of PLD in the pathogenesis of EAM. Western blot analysis showed that the level of the PLD1 isoform was significantly increased in the hearts of rats with EAM on days 14, 17 and 21 postimmunization (pi) (P < 0.01; control vs EAM at 14 pi, 17 pi and 21 pi). The phenotypes of cells exhibiting increased PLD1 expression were primarily inflammatory cells, including ED1 positive macrophages, in the inflammatory EAM lesions. Some cardiomyocytes also showed increased PLD1 immunoreaction in and around EAM lesions. Some PLD1-positive cells were also positive for proliferating cell nuclear antigen in some cardiomyocytes or terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling in some macrophages, suggesting that PLD1 positive cells have a capacity for proliferation or apoptosis depending on cell types in the target organ. Thus, it is postulated that increased expression of PLD1 in EAM may support an early inflammatory response in proliferating inflammatory cells, and its expression in cardiomyocytes may help them to survive by activation of survival factors in hearts with EAM.

We examined the effects of the antioxidant propolis on seizures induced by kainic acid (KA). Sprague-Dawley rats received propolis (75 and 150 mg/kg, p.o.) five times at 12 h intervals. KA (10 mg/kg, i.p.) was injected 1 h after the last propolis treatment. Pretreatment with propolis significantly attenuated KA-induced seizures and KA-induced increases in hippocampal AP-1 DNA binding activity in a dose-dependent manner. KA induced increases in the levels of malondialdehyde and protein carbonyl, and a decrease in the ratio of GSH/GSSG. These oxidative stresses and neuronal degenerations were significantly attenuated by pretreatment with propolis. The neuroprotective effects of propolis appeared to be counteracted by adenosine receptor antagonists [A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (25 or 50 microg/kg); A2A antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (0.5 or 1 mg/kg); and A2B antagonist, alloxazine (1.5 or 3.0 mg/kg)]. However, this counteraction was most pronounced in the presence of the A1 antagonist. Our results suggest that the protective effect of propolis against KA-induced neurotoxic oxidative damage is, at least in part, via adenosine A1 receptor modulation.

Melatonin (N-acetyl-5-methoxytryptamine), a pineal neurohormone, is a hydroxyl radical scavenger and antioxidant, and plays an important role in the immune system. We studied the effect of exogenous melatonin on the pathogenesis of experimental autoimmune encephalomyelitis (EAE). EAE was induced in Lewis rats by immunization with rat spinal cord homogenates. Subsequent oral administration of melatonin at 5 mg/kg significantly reduced the clinical severity of EAE paralysis compared with administration of the vehicle alone (p

To elucidate the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we analyzed the expression of constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) in the spinal cords of rats with EAE. We further examined the structural interaction between apoptotic cells and spinal cord cells including neurons and astrocytes, which are potent cell types of nitric oxide (NO) production in the brain. Western blot analysis showed that three forms of NOS significantly increased in the spinal cords of rats at the peak stage of EAE, while small amounts of these enzymes were identified in the spinal cords of rats without EAE. Immunohistochemical study showed that the expression of either nNOS or eNOS increased in the brain cells including neurons and astrocytes during the peak and recovery stages of EAE, while the expression of iNOS was found mainly in the inflammatory macrophages in the perivascular EAE lesions. Double labeling showed that apoptotic cells had intimate contacts with either neurons or astrocytes, which are major cell types to express nNOS and eNOS constitutively. Our results suggest that the three NOS may play an important role in the recovery of EAE.

We evaluated whether combined treatment with selegiline, a selective MAO-B inhibitor, and EGb 761, a standard extract of Ginkgo biloba, has synergistic effects against ischemic reperfusion injury (IRI) in gerbils. Interestingly, we observed that pretreatment with EGb 761 significantly attenuated selegiline-induced hyperactivity. This finding paralleled striatal fos-related antigen immunoreactivity (FRA-IR) in mice. Four minutes of bilateral carotid artery occlusion caused substantial cell loss in the CA1 of the hippocampus 5 days post-ischemic insult. Pretreatment with EGb 761, with or without selegiline, significantly attenuated this neuronal loss. Combined treatment with EGb 761 plus selegiline was more efficacious in preventing this loss. Synaptosomal formations of protein carbonyl, lipid peroxidation (malondialdehyde (MDA) + 4-hydroxyalkenal (4-HDA)), and reactive oxygen species (ROS) in the hippocampus remained elevated 5 days post-ischemic insult. The antioxidant effects appeared to be most significant in the group treated with EGb 761 plus selegiline. This combined treatment produced more significant attenuation of IRI-induced alterations in intramitochondrial calcium accumulation, the mitochondrial transmembrane potential, and mitochondrial Mn-superoxide dismutase-like immunoreactivity (Mn-SOD-IR) than either treatment alone. Our results suggest that co-administration of EGb 761 and selegiline produces significant neuroprotective effects via suppression of oxidative stress and mitochondrial dysfunction without affecting neurological function.

The possible role of quercetin, a naturally occurring plant flavonoid, in protecting against oxygen-glucose deprivation (OGD)-, excitotoxins-, and free radical-induced neuronal injury in mouse cortical cell cultures was investigated. Pre- and co-treatment with quercetin (100 microM) inhibited 50 min OGD-, 20 microM N-methyl-D-aspartate (NMDA)-, and 50 microM kainate-induced neurotoxicity by 36, 22, and 61%, respectively. Quercetin significantly ameliorated free radical-induced neuronal injury caused by buthionine sulfoximine, sodium nitroprusside, ZnCl(2), and FeCl(2). These results suggest that quercetin may contribute a neuroprotective action against ischemic neural injury, partially via antioxidant actions.

In order to evaluate the role of transforming growth factor (TGF)-beta3 in the neurodegenerative process, we examined the levels of mRNA and immunocytochemical distribution for TGF-beta3 in the rat hippocampus after systemic kainic acid (KA) administration. Hippocampal TGF-beta3 mRNA level was reduced 3 h after KA injection. However, the levels of TGF-beta3 mRNA were elevated 1 day post-KA and lasted for at least 30 days. A mild TGF-beta3 immunoreactivity (TGF-beta3-IR) in the Ammon's horn and a moderate TGF-beta3-IR in the dentate granule cells were observed in the normal hippocampus. The CA1 and CA3 neurons lost their TGF-beta3-IR, while TGF-beta3-positive glia-like cells proliferated mainly throughout the CA1 sector and had an intense immunoreactivity at 7, 15 and 30 days after KA. This immunocytochemical distribution of TGF-beta3-positive non-neuronal populations was similar to that of glial fibrillary acidic protein (GFAP)-positive cells. Double labeling immunocytochemical analysis demonstrated colocalization of TGF-beta3- and GFAP-immunoreactivity in the same cells. These findings suggest a compensatory mechanism of astrocytes for the synthesis of TGF-beta3 protein in response to KA-induced neurodegeneration. In addition, exogenous TGF-beta3 (5 or 10 ng/i.c.v.) significantly attenuated KA-induced seizures and neuronal damages in a dose-related manner. Therefore, our results suggest that TGF-beta3 plays an important role in protective mechanisms against KA-induced neurodegeneration.

The role of endothelin-I (ET-I) in the development of experimental autoimmune encephalomyelitis (EAE) was studied by the blocking the action of ET-I with a receptor antagonist, BQ-123. Intrathecal administration of BQ-123 significantly ameliorated EAE progression at the peak stage of EAE (p < 0.05). By immunohistochemistry, ED-I-positive macrophages in EAE lesions were identified as major producers of ET-I, whereas the immunoreactivity of ET-I on brain cells, such as astrocytes, was dramatically increased in accordance with the progression of EAE. This study points to a putative pro-inflammatory role for ET-I in the pathogenesis of EAE. One possible application for the ET-I receptor antagonist might be helpful in the therapy of autoimmune neurological disorders.

The abrupt elevation in the levels of cyclooxygenase or lipoxygenase metabolites of arachidonic acid during cerebral ischemia contributes to neuronal injury. Recently, evidence has accumulated that both excitotoxic and apoptotic features can coexist in ischemia models in vitro and in vivo. In this study, we evaluated whether phenidone, an inhibitor of both cyclooxygenase and lipoxygenase, can provide protection against excitotoxin- or ischemia-induced neurotoxicity, including the staurosporine apoptosis model, in mouse cortical cultures. We examined the protective effect of phenidone against free radical injuries induced by arachidonic acid, hydrogen peroxide, xanthine/xanthine oxidase, Fe2+/ascorbic acid. Pre- and post-treatment with phenidone (300 microM for 24 h) moderately attenuated the neuronal injury induced by 50 microM kainate and oxygen/glucose deprivation (45 min) by 33% and 50%, respectively. It had no effect on NMDA induced injury (150 microM for 5 min). The maximum dose of phenidone (300 microM) reduced the oxidative injury induced by arachidonic acid (71% inhibition), hydrogen peroxide (95% inhibition), xanthine/xanthine oxidase (57% inhibition), and Fe2+/ascorbic acid (99% inhibition) neurotoxicity. Phenidone (300 microM) decreased staurosporine (100 nM)-induced apoptosis to 30%. These results suggest that phenidone may contribute to neuronal survival by modulating oxidative stress, which is involved in the excitotoxic and apoptotic processes occurring under ischemic conditions.

Intracerebroventricular injection of lipopolysaccharide (LPS) induces a marked increase in circulating interleukin (IL)-6 levels and in IL-6 mRNA expression in brain and peripheral organs. Recently, it was reported that intraperitoneal administration of α-adrenoceptor antagonists inhibits centrally injected LPS-induced increases in plasma IL-6 levels, suggesting the involvement of the norepinephrine (NE) system in the central LPS-induced IL-6 response. However, the localization (either central or peripheral) of NE involvement in the central LPS-induced IL-6 response has not been characterized. In the present study, mice were pretreated with 6-hydroxydopamine (6-OHDA) administered intracerebroventricularly or intraperitoneally to deplete central or peripheral stores of NE, respectively. Intracerebroventricular LPS (50 ng/mouse) markedly increased plasma IL-6 levels and IL-6 mRNA expression in choroid plexus, hypothalamus, pituitary, adrenals, heart, liver, spleen, and lymph nodes, but with minimal effect in lung, kidney, and testis, as revealed by RT-PCR. Pretreatment, with intracerebroventricular 6-OHDA (50 μg/mouse) decreased the LPS-induced plasma IL-6 levels by 39% and the LPS-induced IL-6 mRNA expression in liver, spleen, and lymph nodes, but not in choroid plexus, hypothalamus, pituitary, adrenals, and heart. Pretreatment with intraperitoneal 6-OHDA (100 mg/kg) decreased the LPS-induced plasma IL-6 levels by 36% and the LPS-induced IL-6 mRNA expression in all the peripheral organs displaying increased IL-6 mRNA. Central LPS-induced increase in plasma corticosterone levels was decreased slightly by central but not by peripheral NE depletion. These results suggest that central NE and peripheral NE are differentially involved in the central LPS-induced IL-6 mRNA expression in peripheral organs.

We studied whether eugenol provides neuroprotection against delayed neuronal death in the hippocampal CA1 region following a 5 min occlusion of the common carotid arteries bilaterally under either free-regulating temperature (TF) or maintained temperature (TM, 37 degrees C) conditions in gerbils. Right after occlusion of the carotid arteries, we injected eugenol intraperitoneally at concentrations of either 50, 100, or 200 mg/kg. There was significant preservation of neuronal cells in the CA1 region in the eugenol-treated groups 7 days after the ischemic insult in the TF condition, with respective survival values of 26, 43, and 68%. In the TM condition, however, significant neuroprotection was only seen with eugenol concentrations of 100 and 200 mg/kg (32% and 52%, respectively). When the rectal temperature was maintained at 38 degrees C for 30 min after occlusion of the carotid arteries, no reduction in CA1 damage was observed with any dose of eugenol. These results suggest that eugenol may provide neuroprotection against ischemic damage by its hypothermic action.

To determine the possible role of cyclooxygenase/lipoxygenase pathway in the regulation of proenkephalin (proENK) and prodynorphin (proDYN) gene expression induced by kainic acid (KA) in rat hippocampus, the effects of esculetin, aspirin, or phenidone on the seizure activity, proENK and proDYN mRNA levels, and the level of fos-related antigene (Fra) protein induced by KA in rat hippocampus were studied. Esculetin (5 mg/kg), aspirin (15 mg/kg), or phenidone (50 mg/kg) was administered orally five times every 12 h before the injection of KA (10 mg/kg, i.p.). Seizure activity induced by KA was significantly attenuated by phenidone. However, neither esculetin nor aspirin affected KA-induced seizure activity. The proENK and proDYN mRNA levels were markedly increased 4 and 24 h after KA administration. The elevations of both proENK and proDYN mRNA levels induced by KA were inhibited by pre-administration with phenidone, but not with esculetin and aspirin. ProENK-like protein level increased by KA administration was also inhibited by pre-administration with phenidone, but not with esculetin and aspirin. The increases of proENK and proDYN mRNA levels induced by KA were well correlated with the increases of Fra protein level. Additionally, the induction of Fra protein was inhibited by pre-administration with phenidone, but not with esculetin and aspirin. The results suggest that blockade of both cyclooxygenase and lipoxygenase pathways appears to be responsible for increases of proENK and proDYN mRNA levels induced by KA via inhibiting the induction of Fra protein in rat hippocampus.

SUMMARY 1. The effect of streptozotocin (STZ), a nitric oxide (NO) donor, on kainic acid (KA)-induced neurotoxicity was examined in Sprague-Dawley rats. 2. The administration of KA (8mg/kg, i.p.) produced significant neurotoxicity accompanied with increased immuno-reactivity for Fos-related antigen in the rat hippocampus. 3. Pretreatment with STZ (15mg/kg, i.m.) significantly blocked the neurotoxicity induced by KA. 4. Thus, the neuroprotective effect of STZ may, at least in part, reflect the role of NO in inhibiting seizures.

1. Dipsacus saponin C (DSC) administered intracerebroventricularly (i.c.v.) showed an antinociceptive effect in a dose-dependent (from 3.75 to 30 micrograms) manner as measured by the tailflick assay. The antinociception induced by DSC at the dose of 30 micrograms was maintained at least 1 h. 2. Sulfated cholecystokinin (CCK, from 0.1 to 0.5 ng); muscimol (a GABAA receptor agonist, from 50 to 200 ng); MK-801 [(+/-)-5-methyl-10, 11-dihydro-5H-dibenzo (a,d) cyclohepten-5, 10-imine maleate, from 0.1 to 1 microgram], a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist; or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, from 0.1 to 0.5 microgram), a non-NMDA receptor antagonist, injected i.c.v. significantly reduced the inhibition of the tail-flick response induced by DSC (30 micrograms) administered i.c.v. However, naloxone (an opioid receptor antagonist, 2 micrograms) or baclofen (a GABAB receptor antagonist, 10 ng) did not affect the inhibition of the tail-flick response induced by DSC. 3. The intrathecal (i.t.) injection of yohimbine (an alpha 2-adrenergic receptor antagonist, from 5 to 20 micrograms) and methysergide (a serotonin receptor antagonist, from 5 to 20 micrograms) but not naloxone (from 2 to 8 micrograms), significantly attenuated inhibition of the tail-flick response induced by DSC (30 micrograms) administered i.c.v. 4. Our results suggest that DSC has an antinociceptive effect when it is administered supraspinally and GABAA, NMDA and non-NMDA receptors, but not opioid and GABAB receptors located at the supraspinal level, may be involved in DSC-induced antinociception. Furthermore, DSC administered supraspinally may produce antinociception by stimulating descending alpha 2-adrenergic and serotonin pathways but not the opioidergic pathway.

1. 1. The antinociceptive effect of morphine (25 μg) administered into the 3rd ventricle was significantly attenuated in streptozotocin-induced diabetic rats as measured by the tail-flick assay. 2. 2. The release of serotonin (5-HT; 5-hydroxytryptamine) from the spinal cord caused by intraventricular injection of morphine (25 μg) was significantly reduced in streptozotocin-induced diabetic rats. 3. 3. No differences of 5-HT contents of the spinal cord (lumbar cord) between streptozotocin-induced diabetic- and vehicle-treated rats were found. 4. 4. It is concluded that the reduction of antinociception produced by intraventricular injection of morphine in streptozotocin-induced diabetic rats might be, at least partly, due to the decrease of 5-HT release from the spinal cord.