Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis.

The specific role of endogenous glutathione in response to neuronal degeneration induced by trimethyltin (TMT) in the hippocampus was examined in rats. A single injection of TMT (8 mg/kg, i.p.) produced a rapid increase in the formation of hydroxyl radical and in the levels of malondialdehyde (MDA) and protein carbonyl. TMT-induced seizure activity significantly increased after this initial oxidative stress, and remained elevated for up to 2 weeks post-TMT. Although a significant loss of hippocampal Cornus Ammonis CA1, CA3 and CA4 neurons was observed at 3 weeks post-TMT, the elevation in the level of hydroxyl radicals, MDA, and protein carbonyl had returned to near-control levels at that time. In contrast, the ratio of reduced to oxidized glutathione remained significantly decreased at 3 weeks post-TMT, and the glutathione-like immunoreactivity of the pyramidal neurons was decreased. However glutathione-positive glia-like cells proliferated mainly in the CA1, CA3, and CA4 sectors and were intensely immunoreactive. Double labeling demonstrated the co-localization of glutathione-immunoreactive glia-like cells and reactive astrocytes, as indicated by immunostaining for glial fibrillary acidic protein. This suggests that astroglial cells were mobilized to synthesize glutathione in response to the TMT insult. The TMT-induced changes in glutathione-like immunoreactivity appear to be concurrent with changes in the expression levels of glutathione peroxidase and glutathione reductase. Ascorbate treatment significantly attenuated TMT-induced seizures, as well as the initial oxidative stress, impaired glutathione homeostasis, and neuronal degeneration in a dose-dependent manner. These results suggest that ascorbate is an effective neuroprotectant against TMT. The initial oxidative burden induced by TMT may be a causal factor in the generation of seizures, prolonged disturbance of endogenous glutathione homeostasis, and consequent neuronal degeneration.

Selegiline potentiates the effects of EGb 761 in response to ischemic brain injury.

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.

Immunocytochemical evidence that amyloid beta (1-42) impairs endogenous antioxidant systems in vivo.

Amyloid beta, the major constituent of the senile plaques in the brains of patients with Alzheimer's disease, is cytotoxic to neurons and has a central role in the pathogenesis of the disease. We have previously demonstrated that potent antioxidants idebenone and alpha-tocopherol prevent learning and memory impairment in rats which received a continuous intracerebroventricular infusion of amyloid beta, suggesting a role for oxidative stress in amyloid beta-induced learning and memory impairment. To test the hypothesis, in the present study, we investigated alterations in the immunoreactivity of endogenous antioxidant systems such as mitochondrial Mn-superoxide dismutase, glutathione, glutathione peroxidase and glutathione-S-transferase following the continuous intracerebroventricular infusion of amyloid beta for 2 weeks. The infusion of amyloid beta (1-42) resulted in a significant reduction of the immunoreactivity of these antioxidant substances in such brain areas as the hippocampus, parietal cortex, piriform cortex, substantia nigra and thalamus although the same treatment with amyloid beta (40-1) had little effect. The alterations induced by amyloid beta (1-42) were not uniform, but rather specific for each immunoreactive substance in a brain region-dependent manner. These results demonstrate a cytological effect of oxidative stress induced by amyloid beta (1-42) infusion. Furthermore, our findings may indicate a heterogeneous susceptibility to the oxidative stress produced by amyloid beta.

Prenatal exposure to magnetic field increases dopamine levels in the striatum of offspring.

1. The putative effects of prenatal exposure to magnetic field (MF) have recently received much interest. In the present study, mice were exposed to a MF of 50 mT during gestation (0-19 days). 2. After the exposure was terminated, mothers and offspring were returned to normal laboratory conditions. We then determined changes in striatal levels of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the offspring. 3. Our results indicate that prenatal exposure to MF increases levels of DA and DOPAC in the striatum at 4, 8 and 12 weeks postnatally.

Carbetapentane attenuates kainate-induced seizures via sigma-1 receptor modulation.

We examined the effects of a non-opioid antitussive, carbetapentane (CB) on kainic acid (KA)-induced neurotoxicity in rats. KA administration (10 mg/kg, i.p.) produced robust behavioral convulsions lasting 4 to 5 h. CB (12.5 and 25 mg/kg. i.p.) pretreatment consistently and in a dose-dependent manner reduced the KA-induced seizures, mortality, and marked loss of cells in regions CA1 and CA3 of the hippocampus. Consistently, CB pretreatment also significantly attenuated the KA-induced increase in Fos-related antigen immunoreactivity in the hippocampus. In contrast, pretreatment with the sigma-1 receptor antagonist BD1047 (1 and 2 mg/kg, i.p.) blocked, in a dose-related manner, the neuroprotection afforded by CB. These results suggest that CB provides neuroprotection against KA insult via sigma-1 receptor modulation.

Dextromethorphan affects cocaine-mediated behavioral pattern in parallel with a long-lasting Fos-related antigen-immunoreactivity.

In order to understand the underlying mechanisms responsible for the behaviors mediated by dextromethorphan (DM), we examined the effects of DM on locomotor activity and locomotor patterns in mice, and Fos-related antigen immunoreactivity (FRA-IR) of mouse brain following repeated administration of cocaine. Combined treatments (30 min prior to each cocaine administration) with DM dose-dependently decreased locomotor activity for high doses of cocaine (20 mg/kg, i.p./day x 7). DM combinations did not significantly affect hyperactivity for 10 mg cocaine/kg, i.p./day x 7. In contrast, combined treatments with DM increased the locomotor activity for 5 mg cocaine/kg, i.p./day x 7. These results were consistent with alterations in marginal activity. Repeated administration with cocaine or DM increased FRA-IR in the nucleus accumbens (NAc) and striatum which lasted for at least 7 days. Our results suggest that DM exhibits biphasic effects on the locomotor stimulation induced by cocaine, and that locomotor activities are in parallel with FRA-IR of the striatal complex. However, the role of FRA-IR regulated by DM or/and cocaine remains to be further determined.

Anticonvulsant effects of new morphinan derivatives.

We synthesized a series of compounds that are modified in positions 3 and 17 of the morphinan ring system, with the intention of developing ideal anticonvulsant agents. We examined the effects of these compounds on kainic acid (KA)-induced seizures, and on locomotor patterns in rats. We found that compounds 5, 6, and 8 exhibit novel anticonvulsant effects, with negligible psychotropic effects.

Effects of dextromethorphan on the seizures induced by kainate and the calcium channel agonist BAY k-8644: comparison with the effects of dextrorphan.

BAY k-8644 (an L-type Ca(2+) channel agonist of the dihydropyridine class) is recognized as a potent convulsant agent. In this study, we used BAY k-8644 to explore the effects of dextromethorphan (DM) and its major metabolite, dextrorphan (DX), on the (pro)convulsant activity regulated by calcium channels. BAY k-8644 (2 mg/kg, s.c) potentiated seizures induced in rats by kainic acid (KA) (10 mg/kg, i.p.). DM appears more efficacious than DX in attenuation of KA-induced seizures. The anticonvulsant effect of a low dose (12.5 mg/kg, s.c.) of DM was reversed by BAY k-8644 (2 mg/kg) challenge. In contrast, BAY k-8644 (1 or 2 mg/kg) did not significantly affect an anticonvulsant effect from a higher dose (25 mg/kg) of either DM or DX. Intracerebroventricular injection of BAY k-8644 (37.5 microg) significantly induced seizures in mice. DM (12.5 or 25 mg/kg) pretreatment more significantly attenuated seizures evoked by BAY k-8644 than did DX (12.5 or 25 mg/kg). Furthermore, seizure activity induced by KA or BAY k-8644 was consistent with respective activator protein-1 DNA binding activity of the hippocampus. Therefore, our results suggest that the anticonvulsant effects of the morphinans involve, at least in part, the L-type calcium channel. They also suggest that DM is a more potent anticonvulsant than DX in the KA and BAY k-8644 seizure models.

Phenidone prevents kainate-induced neurotoxicity via antioxidant mechanisms.

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.

Dual effects of dextromethorphan on cocaine-induced conditioned place preference in mice.

Dextromethorphan (DM) at supra-antitussive doses might produce psychotomimetic effects in humans. In order to understand the underlying mechanisms responsible for the behavior induced by DM, we examined the effects of DM on cocaine-induced conditioned place preference (CPP) and locomotor pattern in mice, and Fos-related antigen immunoreactivity (FRA-IR) in the striatal complex (nucleus accumbens and striatum) of the mouse brain. The effects of DM (20 and 40 mg/kg, i.p.) on the CPP for 2.5, 5, 10, and 20 mg cocaine/kg, i.p. were assessed. Pretreatment with DM dose-dependently decreased the CPP for 20 mg cocaine/kg. Similarly, pretreatment with DM appeared to reduce the CPP for 10 mg cocaine/kg, but increase the CPP for 5 mg cocaine/kg. This finding was more pronounced for 2.5 mg cocaine/kg; DM significantly increased the CPP for 2.5 mg cocaine/kg in a dose-related manner. Furthermore, these results were correlated with alterations in the locomotor pattern (marginal activity) and FRA-IR in the striatal complex. Thus, our results suggest that DM exhibits a biphasic effect on the cocaine-induced CPP and locomotor pattern.

Prolonged exposure to cigarette smoke blocks the neurotoxicity induced by kainic acid in rats.

We examined the effects of cigarette smoke (CS) on three parameters associated with kainic acid (KA)-induced neurotoxicity: seizure activity, cell loss in the hippocampus, and increased Fos-related antigen (FRA) expression. Animals were exposed to the main stream of CS from 15 Kentucky 2R1F research cigarettes containing 28.6 mg tar and 1.74 mg nicotine per cigarette, for 10 min a day, 6 days per week, for 4 weeks, using an automatic smoking machine. KA administration (10 mg/kg, i.p.) produced robust behavioral convulsions lasting 4-5 h. Pre-exposure to CS significantly reduced the seizures, mortality, and severe loss of cells in regions CA1 and CA3 of the hippocampus after KA administration. Consistently, pre-exposure to CS significantly attenuated the KA-induced increased FRA immunoreactivity in the hippocampus. In contrast, pretreatment with central nicotinic antagonist, mecamylamine (2 or 10 mg/kg, i.p.) blocked the neuroprotective effects mediated by CS in a dose-dependent manner. These results indicate that CS exposure provides neuroprotection against the KA insult via nicotinic receptor activation.

An immunocytochemical study of mitochondrial manganese-superoxide dismutase in the rat hippocampus after kainate administration.

We examined the immunocytochemical distribution of mitochondrial Mn-superoxide dismutase (SOD-2) in the rat hippocampus after systemic injection of kainic acid (KA), in order to understand SOD-2-responsible antioxidant defense mechanism during the neurodegenerative process. SOD-2 immunostaining was more intense in CA3 pyramidal neurons than in CA1 neurons in the normal hippocampus. The immunoreactivity in region CA1 was reduced without significant neuronal losses within 12 h of KA injection. The CA1 and CA3 neurons lost their immunoreactivity, whereas SOD-2-positive glia-like cells proliferated, mainly throughout the CA1 sector, and had intense immunoreactivity 3 and 7 days after KA injection. This immunocytochemical distribution of SOD-2-positive non-neuronal elements was similar to that of glial fibrillary acidic protein (GFAP) and S-100 protein-positive cells. Activated microglial cells selectively marked with lectin occurred in the areas affected by the KA-induced lesion. Double-labeling immunocytochemistry showed the co-localization of SOD-2-positive non-neuronal cells and GFAP or S-100 protein-like immunoreactivity in the same cells. This suggests that astroglial cells mobilized to synthesize of SOD-2 protein in a response to KA toxicity designed to reduce the oxidative damage.

Phenidone attenuates oxygen/glucose deprivation-induced neurotoxicity by antioxidant and antiapoptotic action in mouse cortical cultures.

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.

Dextromethorphan modulates the AP-1 DNA-binding activity induced by kainic acid.

We have recently reported that dextromethorphan attenuates the neurotoxicity induced by kainic acid in a dose-related fashion. Pretreatments with dextromethorphan (50 mg/kg, p.o. x2) significantly reduced the activator protein-1 DNA-binding activity and the Fos-related antigen-immunoreactive protein induced by kainic acid (10 mg/kg, i.p.) in the CA1, but not the CA3 or the dentate gyrus sector of the rat hippocampus. Paradoxically, dextromethorphan itself caused an elevated activator protein-1 DNA-binding activity and Fos-related antigen-immunoreactive protein in the CA1 region which lasted for at least 4 days. The results suggest that the CA1 area is the critical site for mediating the putative neuroprotective effect induced by dextromethorphan.

Protection of methamphetamine nigrostriatal toxicity by dietary selenium.

Multiple dose administration of methamphetamine (MA) results in long-lasting toxic effects in the nigrostriatal dopaminergic system. These effects are considered to be primarily due to oxidative damage mediated by increased production of hydrogen peroxide or other reactive oxygen species in the dopaminergic system. The present study was designed to determine the protective effects of dietary antioxidant selenium on MA-induced neurotoxicity in the nigrostriatal dopaminergic system. Male C57BL/6J mice were fed either selenium-deficient (< 0.01 ppm Se) or selenium-replete (0.2 ppm Se) diets for 90 days. MA treatment decreased the dopamine (DA) levels in the striatum and substantia nigra (SN) of both Se-replete and Se-deficient animals. However, in Se-replete animals, this DA depletion was significantly attenuated in both the striatum and SN. A novel observation is that MA administration resulted in increased activity of Cu,Zn-SOD in the brains of both Se-deficient and Se-replete animals. However, MA administration to Se-deficient animals exhibited a higher Cu,Zn-SOD activity in the nigrostriatal system than the control animals. Elevated malondialdehyde (MDA) levels in the striatum and SN were also observed in Se-deficient MA-treated animals. Se repletion significantly increased the glutathione peroxidase (GPx) activity and the ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) in the MA-treated animals. In conclusion, we have shown that dietary Se attenuated methamphetamine neurotoxicity and that this protection involves GPx-mediated antioxidant mechanisms. Even though Cu,Zn-SOD activity was significantly elevated by MA treatment, the role of this enzyme in MA-mediated neurotoxicity is not yet clear.

Phenidone blocks the increases of proenkephalin and prodynorphin gene expression induced by kainic acid in rat hippocampus: involvement of Fos-related antigene protein.

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.

Chongmyungtang attenuates kainic acid-induced seizure and mortal effect in the mouse.

TheChongmyungtang (CMT; the combination ofAcorus gramineus, polygala tenuifolia andPoria cocos) has been recognized to possess the preventive effect against several neurologic disorders in human. In this study, we examined the effect of CMT on the three parameters associated with kainic acid (KA)-induced neurotoxicities; seizure/mortality, increased fos-related antigen (FRA) and glial fibrillary acidic protein (GFAP) expression. KA induced vigorous convulsions lasting 4-6 hr. Pretreatments with CMT before KA injection significantly reduced the seizure intensity as well as the mortality. CMT pretreatments also attenuated the KA-induced increase in FRA/GFAP expression in the hippocampus. These results suggest that CMT has a neuroprotective effect against KA-induced neurotoxicities.

A low dose of streptozotocin prevents kainic acid-induced seizures and lethal effects in the rat.

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 (8 mg/kg, i.p.) produced significant neurotoxicity accompanied with increased immunoreactivity for Fos-related antigen in the rat hippocampus. 3. Pretreatment with STZ (15 mg/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.

A new antithrombotic agent, aspalatone, attenuated cardiotoxicity induced by doxorubicin in the mouse; possible involvement of antioxidant mechanism.

A new antithrombotic agent, aspalatone (APT; acetyl salicylic acid maltol ester), was synthesized by esterification of acetyl salicylic acid (ASP) and maltol (MAL). It was suggested that APT possessed an antioxidant effect in in vitro. To evaluate the putative antioxidant effect of APT in in vivo, we developed doxorubicin (DOX)-related cardiac damage, which might be implicated by oxidative stress. Vitamin E (Vit E) was included in the present study as an example of an antioxidant. Prolonged treatments with APT, MAL and Vit E significantly reduced the mortality in animals receiving multiple dose of DOX (3 mg/kg x 4). The potential role of APT, MAL and Vit E against DOX insult may be explained by the induction of glutathione peroxidase activity accompanied by the inhibition of lipid peroxidation. Prolonged treatments of APT, MAL and Vit E also ablated histopathological evidence of DOX cardiomyopathy. ASP challenge, however, did not affect the mortality, myocardial lesion and antioxidant deficit induced by DOX treatments. In conclusion, the protective effect of APT was equipotent to that of Vit E against DOX cardiotoxicity. The results also suggest that the antiperoxidative effect of APT plays a protective role in DOX-related cardiotoxic side effect.

Aspalatone, a new antiplatelet agent, attenuates the neurotoxicity induced by kainic acid in the rat.

The antioxidant efficacy of aspalatone (APT; acetyl salicylic acid maltol ester), a new antiplatelet agent, has been characterized in vivo as well as in vitro, and several observations indicated that the antioxidant could prevent the neuroexcitation caused by oxidative stress. In this report, the effect of APT was evaluated on kainic acid (KA)-induced neurotoxicity, since the neurotoxicity induced by KA is, at least in part, mediated via the formation of free radicals. The results showed that pretreatments with APT or maltol (MAL) significantly attenuated seizure activity, oxidative stress (lipid peroxidation and protein oxidation) and the loss of hippocampal neurons induced by KA. On the other hand, the pretreatments with aspirin (ASP), ASP together with MAL or vitamin E failed to protect against the toxicity produced by KA suggesting that the mechanism of action for APT on the KA-induced neurotoxicity is different from that of ASP. These finding raise the possibility that salicylmaltol, a metabolite of APT, plays a role in preventing the neurotoxicity evoked by KA. Therefore, our results suggest that an APT-related antioxidant mechanism, which is linked to the MAL moiety, is involved in the neuroprotective effect against KA.