Inhibition of Neuronal Nitric Oxide Synthase by 7-Nitroindazole Protects Against MPTP-Induced Neurotoxicity in Mice

Abstract: Several studies suggest that nitric oxide (NO^•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7-nitroindazole (7-NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7-NI dose-dependently protected against MPTP-induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovanillic acid and 3,4-dihydroxyphenylacetic acid. 7-NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO^• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO⁻), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. Our results suggest that NO^• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.     

Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7-Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase

Abstract: The role of nitric oxide (NO^•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO^• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO^• may be directly involved in dopaminergic terminal damage.     

In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole

The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.     

Spinal cord degeneration in C57BL/6N mice following induction of experimental parkinsonism with MPTP

We examined neurodegeneration in spinal cord (SC) and role of such extra-nigral degeneration in MPTP-induced experimental parkinsonism in C57BL/6N mice. HPLC-photodiode array analysis confirmed presence of the active neurotoxin MPP⁺ in SC after single injection of MPTP (25 mg/kg, i.p.). Mitochondrial enzyme monoamine oxidase-B (MAO-B) responsible for in vivo conversion of MPTP to MPP⁺ was inhibited in SC by pre-treatment with l -deprenyl, a specific inhibitor of MAO-B. Besides in vitro conversion of MPTP to MPP⁺ occurred by SC mitochondrial preparation, which was inhibited by l -deprenyl implicating SC as a specific target of MPTP-neurotoxicity. Double immunofluorescent labeling and spectrofluorimetric assay via kynuramine oxidation showed MAO-B expression and activity in SC neurons. Localization of dopamine transporter immunoreactivity in SC along with specific uptake of ³H-MPP⁺ by SC synaptosomal preparation further confirmed SC as target of MPTP-neurotoxicity. Compared with control, increased neuronal death on the seventh day in SC of mice injected with MPTP (2 × 25 mg/kg, at 6 h interval) strongly suggested SC degeneration in pre-symptomatic phase of MPTP-induced experimental parkinsonism. Such extra-nigral neurodegeneration in Parkinson's disease indicated novel molecular mechanism preceding nigrostriatal degeneration and suggested designing broad therapeutic intervention for this complex movement disorder.     

Studies on the Neurotoxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine: Inhibition of NAD-Linked Substrate Oxidation by Its Metabolite, 1-Methyl-4-Phenylpyridinium

Abstract: The effects of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its 4-electron oxidation product 1-methyl-4-phenylpyridinium (MPP⁺) were studied in isolated mitochondria and in mouse brain striatal slices. ADP-stimulated oxidation of NAD-linked substrates was inhibited in a time-dependent manner by MPP⁺ (0.1–0.5 m M ), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP⁺ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP⁺ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.     

Uptake of 1-Methyl-4-Phenylpyridinium and Dopamine in the Mouse Brain Cell Nuclei

Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP⁺), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [³H]-MPP⁺ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [³H]MPP⁺. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP⁺ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.     

Involvement of a Caspase-3-Like Cysteine Protease in 1-Methyl-4-Phenylpyridinium-Mediated Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Exposure of various neuronal cells or cell lines to high concentrations of 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP⁺ induce apoptosis in susceptible neurons. We have further characterized MPP⁺-mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP⁺ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP⁺ and the tetrapeptide inhibitor of caspase-3-like proteases, acetyl-DEVD-CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase-1-like proteases, acetyl-YVAD-CHO, however, was ineffective against MPP⁺ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP⁺-treated CGNs contain markedly increased protease activity that cleaves the caspase-3 substrate acetyl-DEVD- p -nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time-dependent fashion in MPP⁺-treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP⁺ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase-3-like protease.     

Striatal Malonate Lesions Are Attenuated in Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO^•) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate-induced increases in levels of 2,3- and 2,5-dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate-induced increases in 3-nitrotyrosine, a marker for peroxynitrite-mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO^• produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity.     

Fenfluramine-induced serotonergic neurotoxicity in mice: lack of neuroprotection by inhibition/ablation of nNOS

Previous studies have implicated a role for nitric oxide (NO) and peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity. The present study was undertaken to investigate whether NO is involved in serotonergic neurotoxicity caused by fenfluramine. In the first experiment, the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 25 mg/kg x 4) on fenfluramine (25 mg/kg x 4)-induced serotonergic neurotoxicity in Swiss Webster mice was investigated. In the second experiment, the effect of fenfluramine (25 mg/kg x 4) on nNOS (-/-) and wild-type (WT) mice was investigated. Fenfluramine induced hypothermia in all three mouse strains, and 7-NI had no thermoregulatory effect. Selective depletion of 5-HT and 5-HT transporter binding sites in the striatum, frontal cortex and hippocampus in all three mouse strains was observed, with no evidence of dopaminergic neurotoxicity. In the first experiment, 7-NI did not attenuate serotonergic neurotoxicity in Swiss Webster mice. In the second experiment, nNOS(-/-) and WT mice were equally sensitive to serotonergic neurotoxicity. These findings suggest that NO and peroxynitrite do not mediate fenfluramine-induced serotonergic neurotoxicity, and that NO is a selective mediator of amphetamines-induced dopaminergic neurotoxicity.     

Rat-1 Fibroblasts Engineered with GAD65 and GAD67 cDNAs in Retroviral Vectors Produce and Release GABA

Abstract: The effects of the adenosine A₁ agonist N ₆-cyclohexyladenosine (CHA) on MPTP-induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2–3 mg/kg, s.c.), when given together with MPTP, prevented the toxin-induced DA depletion in a dose-dependent manner. This protective action was apparently mediated by the A¹ receptors, because this effect was selectively antagonized by pretreating the animals with the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg, i.p.) but not with the A₂ antagonist 1,3-dipropyl-7-methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A₁ agonist CHA is potentially useful as a neuroprotective agent against MPTP-induced toxicity.     

Protection and Potentiation of 1-Methyl-4-Phenylpyridinium-Induced Toxicity by Cytochrome P450 Inhibitors and Inducer May Be Due to the Altered Uptake of the Toxin

Abstract: Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP⁺). Incubation of mouse brain slices with various concentrations of MPP⁺ (1–100 µ M ) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP⁺-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP⁺. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP⁺ into the slices. In contrast, MPP⁺ uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP⁺ in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices.     

Fibroblast growth factor 9 prevents MPP+-induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP⁺ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP⁺ intoxication and melatonin protection. We found that MPP⁺ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP⁺-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP⁺-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP⁺, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP⁺ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.     

Neuroprotective Effect of the Iron Chelator Desferrioxamine Against MPP+ Toxicity on Striatal Dopaminergic Terminals

Abstract: Microdialysis was used to evaluate the effect of desferrioxamine (DES) against 1-methyl-4-phenylpyridinium (MPP⁺) toxicity. The presence of DES (40 fmol-40 nmol/15 min for a total of 90 min) in the Ringer solution, coperfused with MPP⁺ (40 nmol/15 min) on day 1, produced on day 2 a higher extracellular dopamine output after perfusion of MPP⁺ than in control MPP⁺ perfusion experiments, in which no DES was administered on day 1. Both Ringer perfusion alone (control Ringer) and coperfusion of 40 nmol DES with 40 nmol MPP⁺ on day 1 produced on day 2 similar increases in extracellular dopamine output after a second MPP⁺ perfusion. In the control Ringer experiment, note that the MPP⁺ on day 2 is the first MPP⁺ perfusion. Perfusion of 800 fmol FeCl₃/15 min along with 40 nmol MPP⁺ and 400 fmol DES on day 1 completely abolished on day 2 the neuroprotective effect found with 40 nmol MPP⁺ and 400 fmol DES; 800 fmol FeCl₃ did not increase the neurotoxic effect of 40 nmol MPP⁺ perfusion. The ability of DES to protect against MPP⁺ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.     

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP⁺ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP⁺, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP⁺, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP⁺ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells.     

Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

Involvement of multiple protein kinases in cPLA2 phosphorylation, arachidonic acid release, and cell death in in vivo and in vitro models of 1-methyl-4-phenylpyridinium-induced parkinsonism – the possible key role of PKG

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP⁺-induced cytosolic phospholipase A₂ (cPLA₂) activation of dopaminergic neurons. MPP⁺ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca²⁺]_i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA₂, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF₃), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA₂ on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA₂ phosphorylation at Ser505. Inhibitors of cPLA₂ and PKG increased viability and reduced MPP⁺-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA₂ phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA₂.     

Dopamine Transporter Is Required for In Vivo MPTP Neurotoxicity: Evidence from Mice Lacking the Transporter

Abstract: The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29-fold increase in the level of GFAP mRNA in the striatum of wild-type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP⁺; 10 m M ), via a microdialysis probe produced a massive efflux of DA in wild-type mice (>320-fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.     

Mechanisms Involved in the Neuroprotective Activity of a Nitric Oxide Synthase Inhibitor During Focal Cerebral Ischemia

Abstract: We have reported previously that posttreatment with N ^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide synthase, reduced the volume of cortical and striatal infarct induced by middle cerebral artery occlusion in rats. In the present study, we investigated the mechanisms by which L-NAME (3 mg/kg i.p.) is neuroprotective in this model of cerebral ischemia. First, we have shown the reversal of the neuroprotective effect of L-NAME by a coinjection of L-arginine. Second, in order to determine by which mechanism nitric oxide exacerbates neuronal damage produced by focal cerebral ischemia, we studied the effect of the inhibition of nitric oxide synthase by L-NAME on the histological consequences of a focal injection of N -methyl-D-aspartate (NMDA) in the striatum, and on the striatal overflow of glutamate and aspartate induced either by K⁺ depolarization or by focal cerebral ischemia. We have found that L-NAME treatment reduced the excitotoxic damage produced by NMDA injection. By using microdialysis, we have shown that the K⁺- and the ischemia-induced glutamate efflux was reduced by 52 and 30%, respectively, after the L-NAME treatment. These results indicate that nitric oxide synthesis induced by the NMDA receptor overstimulation is one of the major events leading to neuronal damage. One possible mechanism by which nitric oxide may contribute to the excitotoxic process is by facilitating the ischemia-induced glutamate overflow.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.