Chuang C. Chiueh

Hemiparkinsonism in monkeys after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Infusion of MPTP (0.2-0.8 mg/kg) into the right internal carotid artery of monkeys produces toxin-induced injury to the right nigrostriatal pathway with sparing of other dopaminergic neurones on the infused side and with negligible or little injury to the opposite, untreated side. There are contralateral limb dystonic postures, rigidity, and bradykinesia, but the animals are able to eat and maintain health without drug treatment. Spontaneous motor activity is attended by circling towards the injured side, whereas treatment with L-DOPA/-carbidopa or apomorphine stimulates circling towards the intact side. Dopamine and dopamine metabolite levels are normal in the left caudate and putamen, but markedly depressed on the right (MPTP-treated) side. This animal hemiparkinsonian model will be useful in studies of volitional movement control, drug treatments of Parkinsons disease, and functional efficacy of brain tissue implants.

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringers solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.

Neurochemical and behavioral effects of systematic and intranigral administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the rat☆

At doses of 5-10 mg kg-1, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP) produces in rats acute immobility, retropulsion, straub tail, piloerection, exophthalmos, salivation and clonic movements of the forepaws. It does not produce analgesia as measured by the tail test, nor does it produce permanent motor impairment after chronic or intranigral administration. The acute retropulsion and immobilizing effects can be blocked by methysergide. Administered acutely, NMPTP doubles levels of serotonin in the raphe nucleus and substantia nigra. At the same time, levels of dopamine increase in the caudate nucleus and decrease in the substantia nigra. The NMPTP-induced decrease in dopamine content of the substantia nigra persists in chronically treated rats, but there is no significant decrease in striatal dopamine. After chronic administration of NMPTP, striatal levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased by about 50%. Intranigral administrations of NMPTP (10 micrograms daily for 5 days) failed to produce a 6-hydroxydopamine-like lesion in the nigrostriatal system. These results indicate that NMPTP in the rat does not cause selective destruction of dopaminergic neurons, but it does produce acute tryptamine-like effects.

IV. Differences in the metabolism of MPTP in the rodent and primate parallel differences in sensitivity to its neurotoxic effects

Primates and rodents show marked differences in sensitivity to the neurotoxic effects of MPTP. We and others have previously shown that the toxic effects of MPTP on nigrostriatal cells are dependent on the oxidative metabolism of MPTP to the quaternary species MPP+. We have therefore compared the distribution and metabolism of MPTP in the monkey and several rodent species. Three major differences have been identified: 1) the primate, but not the rodents, showed a persistently high concentration of MPTP metabolites in the caudate nucleus compared to other brain regions; 2) the rodent brains cleared MPTP and its metabolites much more rapidly than did the monkey, and; 3) the predominant metabolite retained by the monkey brain was MPP+, while MPP+ cannot be detected in rodent brains for more than a few hours after injection. The persistence of MPP+ in the primate brain may explain the heightened toxicity of MPTP in this species.

Neuroprotective Properties of Nitric Oxide

ABSTRACT: The discoveries of physiological roles of nitric oxide (·NO) as the mediator of endothelium‐derived relaxing factor (EDRF) action and the activator of guanylyl cyclase to increase cyclic guanosine monophosphate (cGMP), which lead to vasorelaxation in the cardiovascular system, have been awarded with the 1998 Nobel Prize of Medicine. The present review discusses putative beneficial effects of ·NO in the central nervous system (CNS). In addition to its prominent roles of the regulation of cerebral blood flow and the modulation of cell to cell communication in the brain, recent in vitro and in vivo results indicated that ·NO is a potent antioxidative agent. ·NO terminates oxidant stress in the brain by (i) suppressing iron‐induced generation of hydroxyl radicals (·OH) via the Fenton reaction, (ii) interrupting the chain reaction of lipid peroxidation, (iii) augmenting the antioxidative potency of reduced glutathione (GSH) and (iv) inhibiting cysteine proteases.

Neuroprotection by S-nitrosoglutathione of brain dopamine neurons from oxidative stress

The proposed anti‐ and pro‐oxidant effects of nitric oxide (NO) derivatives, such as S‐nitrosoglutathione (GSNO) and peroxynitrite, were investigated in the rat nigrostriatal dopaminergic system. Intranigral infusion of freshly prepared GSNO (0–16.8 nmol, i.n.) prevented iron‐induced (4.2 nmol, i.n.) oxidative stress and nigral injury, reflected by a decrease in striatal dopamine levels. This neuroprotective effect of GSNO was verified by ex vivo imaging of brain dopamine uptake sites using 125I‐labeled RTI‐55. In addition, in vitro data indicate that GSNO concentration‐dependently inhibited iron‐evoked hydroxyl radical generation and brain lipid peroxidation. In this iron‐induced oxidant stress model, GSNO was approximately 100‐fold more potent than the antioxidant glutathione (GSH). Light‐exposed, NO‐exhausted GSNO produced neither antioxidative nor neuroprotective effects, which indicates that NO may mediate at least part of GSNOs effects. Moreover, GSNO completely (and GSH only partially) inhibited the weak pro‐oxidant effect of peroxynitrite, which produced little injury to nigral neurons in vivo. This study provides relevant in vivo evidence suggesting that nanomol GSNO can protect brain dopamine neurons from iron‐induced oxidative stress and degeneration. In conclusion, S‐nitrosylation of GSH by NO and oxygen may be part of the antioxidative cellular defense system.—Rauhala, P., Lin, A. M.‐Y., Chiueh, C. C. Neuroprotection by S‐nitrosoglutathione of brain dopamine neurons from oxidative stress. FASEB J. 12, 165–173 (1998)

The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells.

Using models of serum deprivation and 1-methyl-4-phenylpyridinium (MPP+), we investigated the mechanism by which thioredoxin (Trx) exerts its antiapoptotic protection in human neuroblastoma cells (SH-SY5Y) and preconditioning-induced neuroprotection. We showed that SH-SY5Y cells are highly sensitive to oxidative stress and responsive to both extracellularly administered and preconditioning-induced Trx. Serum deprivation and MPP+ produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2,3-nonenal (HNE), causing the cells to undergo mitochondria-mediated apoptosis. Trx in the submicromolar range blocked the observed apoptosis via a multiphasic protection mechanism that includes the suppression of cytochromec release (most likely via the induction of Bcl-2), the inhibition of procaspase-9 and procaspase-3 activation, and the elevated level of Mn-SOD. The reduced form of Trx suppresses the serum-free-induced hydroxyl radicals, lipid peroxidation, and apoptosis, indicating that H2O2 is removed by Trx peroxidase. The participation of Trx in preconditioning-induced neuroprotection is supported by the observation that inhibition of Trx synthesis with antisense oligonucleotides or of Trx reductase drastically reduced the hormesis effect. This effect of Trx-mediated hormesis against oxidative stress-induced apoptosis is striking. It induced a 30-fold shift in LD50 in the MPP+-induced neurotoxicity.

Apparent antioxidant effect of l-deprenyl on hydroxyl radical formation and nigral injury elicited by MPP+ in vivo

Using a modified microdialysis procedure, we confirmed that intrastriatal administration of 1-methyl-4-phenylpyridinium ion (MPP+) induced a sustained overflow of dopamine accompanied by increased formation of hydroxyl free radicals (.OH) as reflected by salicylate hydroxylation. Pretreatment with l-deprenyl (selegiline 60 pmol, intrastriatal perfusion) significantly decreased the .OH formation elicited by MPP+ (75 nmol). There was a small decrease of dopamine efflux and an insignificant change of the ratio of 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine following l-deprenyl pretreatment. These in vivo findings support prior in vitro data that an unique antioxidant property of l-deprenyl may be independent of its inhibition of type B monoamine oxidase. In addition, intranigral co-administration of l-deprenyl (4.2 nmol) with MPP+ (4.2 nmol) completely protected nigral neurons from probable oxidative injuries induced by MPP+ (4.2 nmol), as reflected by a near 50% loss of striatal dopamine ipsilateral to the side of infusion of drug into the substantia nigra. This apparent neuroprotective effect of l-deprenyl on midbrain nigral neurons was also confirmed by histological findings. The present in vivo data clearly demonstrate that l-deprenyl can protect nigral neurons against dopamine neurotoxicity produced by MPP+, as suggested by an earlier in vitro study. Thus, l-deprenyl can preserve the function of MPP(+)-damaged nigral neurons perhaps by its apparent antioxidant property in addition to its blockade of the bioactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium metabolites by type B monoamine oxidase.

III. Primate model of parkinsonism: Selective lesion of nigrostriatal neurons by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine produces an extrapyramidal syndrome in rhesus monkeys

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to rhesus monkeys (1.0-2.5 mg/kg i.v.) produces irreversible damage to nigrostriatal neurons. Dopaminergic neurons in the dorsolateral part of striatum were the most vulnerable. The major clinical signs of an extrapyramidal syndrome, but not resting tremor, appeared only in MPTP-treated monkeys suffering from more than 80% reduction in striatal dopamine. No chronic changes in the mesolimbic dopaminergic system were observed. Immunocytochemical staining of the mid-brain with a tyrosine hydroxylase antiserum indicated that MPTP produced a significant decrease of dopaminergic cell bodies in the A9, but not in the A10 ventrotegmental area. Despite greater than 80% decrease in A9 nigral cell bodies, the dopamine content decreased only by 50%. Sprouting of the surviving nigral A9 neurons was observed histologically and neurochemically in the area above substantia nigra. The present behavioral, neurochemical and histological results indicate that MPTP produces an ideal primate model for studying parkinsonism. Selective lesion of more than 80% of the nigrostrial neurons by MPTP is sufficient to produce the major clinical signs of the extrapyramidal syndrome in idiopathic parkinsonism.

In Vivo Generation of Hydroxyl Radicals and MPTP‐Induced Dopaminergic Toxicity in the Basal Ganglia

The in vivo generation of .OH free radicals in specific brain regions can be measured by intracerebral microdialysis perfusion of salicylate, avoiding many of the pitfalls inherent in systemic administration of salicylate. Direct infusion of salicylate into the brain can minimize the hepatic hydroxylation of salicylate and its contribution to brain levels of 2,5-DHBA. Levels of 2,5-DHBA detected in the brain dialysate may reflect the .OH adduct plus some enzymatic hydroxylation of salicylate in the brain. After minimizing the contribution of enzyme and/or blood-borne 2,5-DHBA, the present data demonstrate the validity of the use of 2,3-DHBA and apparently 2,5-DHBA as indices of .OH formation in the brain. Therefore, intracranial microdialysis of salicylic acid and measurement of 2,3-DHBA appears to be a useful .OH trapping procedure for monitoring the time course of .OH generation in the extracellular fluid of the brain. These results indicate that nonenzymatic and/or enzymatic oxidation of the dopamine released by MPTP analogues in the extracellular fluid may play a key role in the generation of .OH free radicals in the iron-rich basal ganglia. Moreover, a site-specific generation of cytotoxic .OH free radicals and quinone/semiquinone radicals in the striatum may cause the observed lipid peroxidation, calcium overload, and retrograde degeneration of nigrostriatal neurons. This free-radical-induced nigral injury can be suppressed by antioxidants (i.e., U-78517F, DMSO, and deprenyl) and possibly hypothermia as well. In the future, this in vivo detection of .OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidants and antioxidants in neurodegenerative disorders during aging. It could also pave the way for the research and development of novel neuroprotective antioxidants and strategies for the early or preventive treatment of neurodegenerative disorders, such as Parkinsons disease (Wu et al., this issue), amyotrophic lateral sclerosis, head trauma, and possibly Alzheimers cognitive dysfunction as well. In conclusion, this in vivo free-radical trapping procedure provides evidence to support a current working hypothesis that a site-specific formation of cytotoxic .OH free radicals in the basal ganglia may be one of the neurotoxic mechanisms underlying nigrostriatal degeneration and Parkinsonism caused by the dopaminergic neurotoxin MPTP. Addendum added in proof: The controversy concerning possible neurotoxic and/or neuroprotective roles of NO. in cell cultures was discussed and debated at the symposium (Wink et al., this issue; Dawson et al., this issue; Lipton et al., this issue).(ABSTRACT TRUNCATED AT 400 WORDS)