Hiroshi Shiono

Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringers solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Pralidoxime Iodide (2-PAM) Penetrates Across the Blood-Brain Barrier

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 ± 0.053 (mean ± SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats

The herbicide paraquat is an environmental factor that could be involved in the etiology of Parkinsons disease. We have previously shown that paraquat penetrates through the blood-brain barrier and is taken up by neural cells. In this study, we examined the in vivo toxic mechanism of paraquat to dopamine neurons. GBR-12909, a selective dopamine transporter inhibitor, reduced paraquat uptake into the striatal tissue including dopaminergic terminals. The subchronic treatment with systemic paraquat significantly decreased brain dopamine content in the striatum and slightly in the midbrain and cortex, and was accompanied by the diminished level of its acidic metabolites in rats. When paraquat was administered through a microdialysis probe, a transitory increase in the extracellular levels of glutamate, followed by long-lasting elevations of the extracellular levels of NO(x)(-) (NO(2)(-) plus NO(3)(-)) and dopamine were detected in the striatum of freely moving rats. This dopamine overflow lasted for more than 24 h after the paraquat treatment. Dopamine overflow was inhibited by N(G)-nitro-L-arginine methyl ester, dizocilpine, 6,7-dinitroquinoxaline-2,3-dione and L-deprenyl. The toxic mechanism of paraquat involves glutamate induced activation of non-NMDA receptors, resulting in activation of NMDA receptor-channels. The influx of Ca(2+) into cells stimulates nitric oxide synthase. Released NO would diffuse to dopaminergic terminals and further induce mitochondrial dysfunction by the formation of peroxynitrite, resulting in continuous and long-lasting dopamine overflow. The constant exposure to low levels of paraquat may lead to the vulnerability of dopaminergic terminals in humans, and might potentiate neurodegeneration caused by the exposure of other substances, such as endogenous dopaminergic toxins.

Nitration of manganese superoxide dismutase in cerebrospinal fluids is a marker for peroxynitrite-mediated oxidative stress in neurodegenerative diseases

Peroxynitrite can nitrate tyrosine residues of proteins. We examined nitrotyrosine‐containing proteins in cerebrospinal fluid of 66 patients with neurogenic disease by immunoblot analysis. Nitrated tyrosine residue–containing protein was observed in the cerebrospinal fluid and was concluded to be manganese superoxide dismutase (Mn‐SOD). The nitrated Mn‐SOD level was strikingly elevated in amyotrophic lateral sclerosis patients and was slightly increased in Alzheimers and Parkinsons disease patients, whereas an elevated Mn‐SOD level was observed only in progressive supranuclear palsy group. Ann Neurol 2000;47:524–527.

Paraquat leads to dopaminergic neural vulnerability in organotypic midbrain culture

Paraquat (1,1-dimethyl-4,4-bipyridinium, PQ) is a herbicide to possibly induce Parkinsons disease (PD), since a strong correlation has been found between the incidence of the disease and the amount of PQ used. In this study, we examined PQ toxicity in rat organotypic midbrain slice cultures. PQ dose dependently reduced the number of dopaminergic neurons in cultured slices. Since this damage was prevented by GBR-12909, the dopamine transporter could be an initial step of the PQ induced dopaminergic neurotoxicity. The sequential treatments with lower PQ and 1-methyl-4-phenyl pyridinium (MPP+) doses, where each dose alone was not lethal, markedly killed dopamine neurons, suggesting that the exposure of a lower dose of PQ could lead to the vulnerability of dopaminergic neurons. This cell death was prevented by the inhibitors of NMDA, nitric oxide synthase (NOS), cycloheximide and caspase cascade. Neurons expressing NOS were identified inside and around the regions where dopamine neurons were packed. The cell death induced by the sequential treatments with PQ and MPP+ was also rescued by L-deprenyl and dopamine D2/3 agonists. These results strongly support that the constant exposure to low levels of PQ would lead to the vulnerability of dopaminergic neurons in the nigrostriatal system by the excitotoxic pathway, and might potentiate neurodegeneration caused by the exposure of other substances and aging.

Reduced dorsal hippocampal glutamate release significantly correlates with the spatial memory deficits produced by benzodiazepines and ethanol.

Memory deficits frequently occur after taking benzodiazepines and ethanol. We studied in vivo hippocampal presynaptic glutamate transmission in conjunction with memory deficits induced by benzodiazepines and ethanol in rats as an animal model of amnesia. These drugs potently impaired spatial memory formation as evaluated by the Morris water maze task, the rank order among tested treatments being the combination of triazolam (20 micrograms/kg) with ethanol (2 g/kg) > or = triazolam (100 micrograms/kg) > ethanol (2 g/kg) > or = triazolam (20 micrograms/kg) > rilmazafone (20 micrograms/kg). On the other hand, these drug treatments also reduced glutamate release in the dorsal hippocampus but not in the cerebellum measured by microdialysis: the combined administration of triazolam with ethanol potently inhibited glutamate release to 60% of basal output in the dorsal hippocampus. These decreases in hippocampal glutamate transmission closely correlated with the extent of impairment of spatial memory performance (r = 0.990). Thus, the present results strongly indicated that presynaptic dysfunction in dorsal hippocampal glutamatergic neurons would be critical for spatial memory deficits induced by benzodiazepines and ethanol.

N-methylation ability for azaheterocyclic amines is higher in Parkinson's disease : nicotinamide loading test

Summary. The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) leads to the hypothesis that Parkinsons disease (PD) is may be initiated or precipitated by endogenous toxins by the mechanism similar to that of MPTP in genetically-predisposed individuals. The higher cerebrospinal fluid levels of N-methylated azaheterocyclic amines, such as β-carboline and tetrahydroisoquinoline, have been found in parkinsonian patients compared with age-matched controls. To estimate the N-methylation ability for azaheterocyclic amines in parkinsonian patient, nicotinamide was dosed with 100 mg to 26 parkinsonians and 20 controls consisted of 16 other neurogenic disease patients and 4 healthy volunteers. The urine was collected for 4 h, and then analyzed urinary its metabolites by an improved HPLC method. Nicotinamide has a pyridine ring in its structure and may be metabolized through the pathways similar to those for the endogenous neurotoxins. The urinary excretions of nicotinamide metabolites were significantly affected by aging. The excretion of N1-methylnicotinamide decreased along with aging both in PD patients and controls. In younger (65 years old or younger) PD patients, the excretion amount of N1-methylnicotinamide was significantly higher than that in younger controls. The decline rate of N1-methylnicotinamide excretion in parkinsonians was significantly greater than that in controls; the rate is more than 2-fold higher in parkinsonian patients. The age-associated de-crease in 1-methyl-2-pyridone-5-carboxyamide excretion was observed only in parkinsonian patients, but not in controls. The total excreted amount of N-methylated metabolites (N1-methylnicotinamide plus 1-methyl-2-pyridone-5-carboxyamide) was also observed the age-related decline in both groups. The urinary excretions of nicotinamide and nicotinamide-N-oxide were not influenced by aging. These results would indicate that the excess N-methylation ability for azaheterocyclic amines before the onset had been implicated in PD. On the other hand, the present results suggested that the contribution of aberrant cytochrome P450 or aldehyde oxidase activity acting on the pyridine ring, that could act as detoxification routes of endogenous neurotoxins, would be small in the etiology of PD.

Tandospirone, a 5-HT1A agonist, ameliorates movement disorder via non-dopaminergic systems in rats with unilateral 6-hydroxydopamine-generated lesions.

Serotonin 1A (5-HT1A) receptors are distributed throughout the brain with their highest concentrations in the frontal cortex, subthalamic nucleus and entopeduncular nucleus as well as the dorsal and median raphe nucleus. There is growing evidence that 5-HT1A receptor agonists have an antidepressant effect in individuals with major depressive disorders. Recent clinical studies suggest that tandospirone, a highly potent and selective 5-HT1A receptor agonist used clinically as an antidepressant in Japan and China, may act as an antiparkinsonian drug. In the present study, we investigated the effect of tandospirone on contralateral rotational behavior in a unilateral hemiparkinsonian rat model produced with 6-hydroxydopamine (6-OHDA). Tandospirone, as well as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), significantly increased contralateral turnings in a dose-dependent manner (0.5-10 mg/kg). Tandospirone also remarkably potentiated the contralateral turning induced by 0.025 mg/kg of apomorphine. Pretreatment with WAY-100635, a 5-HT1A receptor antagonist, almost completely blocked the contralateral turning behavior evoked by tandospirone and 8-OHDPAT, but not that by apomorphine. SCH-23390, a selective dopamine D1 receptor antagonist, did not affect on the tandospirone-induced rotational behavior. These results suggested that tandospirone could act on postsynaptic 5-HT1A receptors and modulate excitatory amino acid pathways in the basal ganglia. Thus, tandospirone could have therapeutic potential for the treatment of Parkinsons disease by modulating neuronal activities of non-dopaminergic pathways.

Forensic diagnosis of death from cold

To clarify the forensic diagnosis of hypothermia, the postmortem changes in the bodies of 211 cases of hypothermia (partly including death from diseases) encountered between January 1990 and January 1999 were studied as follows:i) The rectal temperature had been measured in 83 cases in which the time after death was known relatively clearly, and the rate of decrease in the rectal temperature per hour was evaluated. The rate of decrease in the rectal temperature was 2.3 +/- 1.2 degrees C, 2.7 +/- 1.5 degrees C and 3.6 +/- 1.9 degrees C per hour (mean +/- SD) when the ambient temperature was > or = 0 degrees C, 0 degrees C > to >-10 degrees C and -10 degrees C > or =, respectively. ii) The color of blood collected from the right and left hearts was different in 43 (33.6%) of the 128 cases of death from cold that underwent inquest and in 20 (87.0%) of the 23 cases that underwent forensic autopsy. This color difference was useful for the diagnosis of fatal hypothermia. iii) Gastric or duodenal submucosal bleeding (Wishnewskis spots) was noted in 10 (43.5%) of the 23 cases that underwent forensic autopsy. iv) Paradoxical undressing was observed in 35 (21.3%) of the 164 cases that underwent inquest.

An autopsy case of sudden death due to acute gastric dilatation without rupture

We report the first autopsy case of fatal gastric dilatation without rupture. A 31-year-old woman who lived alone was found dead in her living room. Despite being very thin, she showed marked abdominal distention. Autopsy and histological findings revealed that a severely distended stomach, of which walls notably thin and displayed transmural necrosis, occupied the entire abdominal cavity. Severe congestion was observed in the intestine and cecum. Theses findings suggest that bulimia nervosa together with anorexia nervosa resulted in rapid dilation of the stomach. We conclude that the cause of death was acute circulatory failure from hypovolemic shock that occurred following compression of the inferior vena cava and superior mesenteric vein, and by loss of circulatory volume to the third space.