Takako Gotohda

Immunohistochemical investigation of dopaminergic terminal markers and caspase-3 activation in the striatum of human methamphetamine users

Methamphetamine (METH) has been shown to induce neurotoxicity. In a previous human study using quantitative Western blotting and radioligand binding assay, dopaminergic terminal marker deficits were induced in chronic METH users. In this study, we examined the suitability of the immunohistochemical detection of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicular monoamine transporter-2 (VMAT2) levels, and caspase-3 activation in the striatum to diagnose METH abuse. Decreases in TH immunoreactivity in the nucleus accumbens and DAT in the nucleus accumbens and putamen were induced in METH users, whereas a significant difference of VMAT2 was not evident between METH and control groups. However, in the nucleus accumbens of two METH users, levels of VMAT2, a stable marker of striatal dopaminergic terminal integrity, were reduced remarkably. These findings might indicate that dopaminergic terminal degeneration is induced in the striatum of some METH abusers. On the other hand, we observed little caspase-3 activation, indicative of apoptosis, in the striatal neurons of chronic METH users. Overall, the findings of dopaminergic terminal markers were similar to those in the previous human study. Therefore, it is suggested that immunohistochemical techniques could be used to examine dopaminergic terminal marker levels and could also give useful information on chronic and/or lethal METH use in cases of METH-related death, where METH intoxication may not be toxicologically demonstrated.

Effect of toluene inhalation on astrocytes and neurotrophic factor in rat brain

Toluene, an abused substance in Japan, is a neurotoxic chemical that has been shown to have neurobehavioral and electrophysiological effects. In previous work, both acute and chronic effects of toluene on cells have been studied extensively. However, although glial cells are thought to play an important role in the survival of neurons in the brain, the effect of toluene on glial cell function has not yet been characterized. To elucidate this, the effect of toluene inhalation on astrocytes in rat brain was examined. Toluene exposure (1500 ppm for 4 h on 4-10 days) augmented glial fibrillary acidic protein (GFAP) immunoreactivity, particularly in the hippocampus and cerebellum. Quantitative analysis showed that toluene inhalation markedly enhanced GFAP expression in the hippocampus and cerebellum. In both regions, proliferating cell nuclear antigen (PCNA) showed no obvious changes, but glutamine synthetase (GS)-immunoreactive cells were markedly increased by toluene exposure. Thus, the elevation of GFAP expression was induced by astrocyte activation rather than by cell proliferation. If toluene exposure activates astrocytes, astrocytes may play a role in the neurophysiological changes observed in toluene intoxication. A neurotrophic factor, basic fibroblast growth factor (b-FGF) was observed immunohistochemically in the capillary vessel walls in the hippocampus and the cerebellum of toluene-intoxicated rats. Basic-FGF may have induced GFAP expression both in the hippocampus and the cerebellum. So, other neurotrophic factors may affect the difference of GFAP elevation between the hippocampus and the cerebellum. These differences may relate to neurobehavioral function of each brain part after toluene exposure.

Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells

Progesterone treatment has previously been reported to promote the differentiation of glial cells probably through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated brain‐derived neurotrophic factor (BDNF) gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain and hence has been suggested to improve mood disorders and other symptoms in depressive patients. Based on these previous observations, the effects on glial cells of histone deacetylase (HDAC) inhibitors, which are known as agents promoting cell differentiation, were examined using rat C6 glioma cells as a model for in vitro studies. Consequently, trichostatin A (TSA), sodium butyrate (NaB), and valproic acid (VPA) stimulated glial fibrillary acidic protein (GFAP) gene expression, and their stimulatory effects on GFAP gene expression were inhibited by treatment of these cells with finasteride, an inhibitor of the enzyme producing 5α‐reduced neurosteroids. In addition, HDAC inhibitors enhanced serotonin‐stimulated BDNF gene expression, the enhancement of which could be abolished by the inhibition of 5α‐reduced neurosteroid production in the glioma cells. These results suggest that HDAC inhibitors may be able to promote the differentiation of rat C6 glioma cells through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated BDNF gene expression as a consequence of promoting their differentiation, indicating the possibility that differentiated glial cells may be implicated in preserving the integrity of neural networks as well as improving the function of neuronal cells in the brain.

Toluene inhalation induces glial cell line-derived neurotrophic factor, transforming growth factor and tumor necrosis factor in rat cerebellum

Rats were exposed to toluene (1500 ppm for 4 h per day) for 7 days. After toluene inhalation, only granule cells in the dentate gyrus of the hippocampus were slightly shrunken. In the cerebellum, several Purkinje cells were shrunken and lost, and the white matter was thinner than in controls. Microtubule-associated protein 2 (MAP2)-immunopositive filaments of neuronal processes were slightly disarrayed in the radial layer of the hippocampus, and were fragmented in the molecular layer of the cerebellum. It was considered that toluene induced neuronal changes both in the cerebellum and the hippocampus. To elucidate the effect of neurotrophic factors on those neuronal changes, glial cell line-derived neurotrophic factor (GDNF), transforming growth factor (TGF) and tumor necrosis factor (TNF) in rat brain were examined immunohistochemically. In control rats, TNF-alpha was not stained in either the hippocampus or the cerebellum, while TGF-beta1 was scarcely expressed in the cerebellum. GDNF was minimally expressed in the Purkinje cells in the cerebellum. After toluene-treatment, TGF-beta1 was over-expressed in the endothelium of the capillary vessel walls in both regions. In the cerebellum, TNF-alpha was induced only in the granule cells, while GDNF expression was enhanced in the Purkinje cells. These data suggest that toluene induces astrocyte activation through TGF-beta1 upregulation, which then induces GDNF in the Purkinje cells and TNF-alpha in the granule cells of the cerebellum. The differences in the expression of the neurotrophic factors may account for neurobehavioral changes after toluene exposure.

Immunohistochemical studies on early stage of hepatic damage induced by subacute inhalation of toluene vapor in rats

Toluene is one of the most widely used organic solvents and is commonly recognized as a noxious substance inducing chronically toxic damage to neural, hepatic and renal functions in the workers engaged in printing and painting. Although hepatic cells are generally considered to be vulnerable and susceptible to various organic solvents, particularly chloroform and other halogenated hydrocarbons, the hepatotoxic effects of aromatic hydrocarbons including toluene have not yet been sufficiently characterized. In particular, it still seems unclear whether toluene itself can directly act on hepatic cells, inducing toxic damage to their metabolism and function. To assess the toxic effect of toluene inhalation on rat liver, immunohistochemical analyses of the histological markers for hepatic damage were carried out in animals exposed subacutely to toluene vapor. The immunoreactivities of heat shock proteins (HSP‐70 and HSP‐90) and cytochrome P4502E1 (CYP2E1) in the liver were analyzed to assess the hepatotoxic damage induced by toluene inhalation, and the expression of these histological markers was shown to be substantially enhanced by the subacute exposure to toluene vapor. Toluene inhalation was furthermore shown to enhance the immunoreactivities of α‐smooth muscle actin (α‐SMA), collagen, glucocorticoid receptors (GR) and leptin receptors (Ob‐R) in the liver. Additional studies using human hepatoma HepG2 cells showed that toluene can directly induce toxic damage to cells. These findings suggest that toluene inhalation may primarily induce hepatic damage, which may be secondarily exacerbated by the activation of systemic processes possibly connected with glucocorticoids and leptin. Copyright

An autopsy case of rhabdomyolysis related to vegetamin and genetic analysis of the rhabdomyolysis-associated genes

We report an autopsy case of a man who died 2 days after taking an overdose of vegetamin. The autopsy findings were as follows: the epidermis on the axillary fossa and the inguinal skin had become macerated. Skeletal muscle was discolored. Concentrations of urea nitrogen, creatinine and urine myoglobin were 1.95 g/day, 0.66 g/day and 1100 ng/mL, respectively. Immunohistochemically, myoglobin was strongly stained at the Bowmans capsule, and tubular lumen and epithelium. 8-OH-dG was strongly stained in renal tubular epithelium in which cell nuclei were strongly stained. ORP-150 was observed in intraglomerular cells and renal tubular epithelium. The concentrations of phenobarbital, promethazine and chlorpromazine ranged from therapeutic to toxic levels, from toxic to lethal levels and toxic level, respectively. His cause of death was considered to be vegetamin-induced rhabdomyolysis. In genetic analysis of this subject, there were two heterozygous silent mutations in the three hot-spot regions in the RYR1 gene. In the CPT II gene, the subject was found to be heterozygous for an amino acid substitution in exon 4, (1203)G>A causing a (368)Val>Ile amino acid substitution. There was no mutation in the VLCAD gene or CYP2C19 gene. The subject was heterozygous for CYP2D6*1 and CYP2D6*2.

Neuronal changes in the arcuate and hypoglossal nuclei of brain stem induced by head injury.

Abstract In head injury, assessing the damage not only to the cerebrum and the cerebellum but also to the brain stem is very important. In this paper, we report neuronal changes of the arcuate nucleus (ARC) and the hypoglossal nucleus (HN) in the brain stem. We investigated these changes immunohistochemically with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos), and the 72 kD heat-shock protein (HSP70). We measured the percentage of immunopositive neurons among the total neurons of the ARC and the HN. The investigation of neuronal changes in relation to the type of head injury showed different results. In cases of tonsillar herniation, immunoreactivity to MAP2 and mAChR in the ARC was significantly lower than in the HN (p < 0.01). Moreover, MAP2, HSP70 and c-Fos reactivities in the ARC were significantly lower than in other types of head injuries (p < 0.01). In the HN, diffuse axonal injury produced slightly higher immunoreactivity to mAChR and c-Fos (p < 0.1). Our observations indicate that immunohistochemical examination of brain stem nuclei can provide useful information for estimating damage to the brain stem.

Genetic analysis of ryanodine receptor 1 gene and carnitine palmitoyltransferase II gene: an autopsy case of neuroleptic malignant syndrome related to vegetamin.

We report an autopsy case of a man in his forties who died 2 days after taking an overdose of vegetamin. The autopsy findings were as follows: externally, the upper epidermis of some parts of the body had become loosened. The epidermis was easily detached from the dermis using the fingers. Viscous fluid adhered around the nose and mouth. The brain was edematous and weighed 1520 g. Skeletal muscle was discolored. The urine was a slightly red-tinged yellow. The organs showed congestion. Urine tests: urea nitrogen: 1.95 g/day; creatinine: 0.66 g/day; urine myoglobin: 1100 ng/mL. Blood level of drugs: phenobarbital: 38.2 microg/ml; promethazine: 2.22 microg/ml; chlorpromazine: 0.96 microg/ml. Immunohistochemistry identified myoglobin in the kidney. From these findings, his cause of death was considered to be vegetamin-induced neuroleptic malignant syndrome and rhabdomyolysis. Mutation of the ryanodine receptor 1 gene is associated with malignant hyperthermia. However, there was no mutation which causes amino acid substitution in the three hot-spot regions of the ryanodine receptor 1 gene. Partial deficiency of carnitine palmitoyltransferase II is the commonest cause of recurrent rhabdomyolysis in adults. The subject was found to be heterozygous for an amino acid exchange in exon 4, (1203)G-->A causing a (368)Val-->Ile amino acid substitution. It is necessary to examine other candidate gene mutations.

An autopsy case of severe pleuritis induced by misinsertion of a nasogastric nourishment tube: Diagnostic significance of multinucleated giant cells

An 87-year-old female who had been hospitalized due to pneumonia was administered nourishment through a nasogastric tube. She collapsed as a result of dyspnea after the insertion of a new tube and administration of nourishment. Chest X-rays revealed that the tube was inserted into the left pleural cavity passing the trachea and left bronchi and that the nourishment pooled. In spite of immediate treatment including removal of the tube and insertion of a drain, she died 12 days later. Autopsy findings: Both the left pulmonary and parietal pleurae were thickened and covered with a dirty gray-yellowish moss-like paste. The left lower lobe was softened, and this region was suspected as the ruptured site of the pleura. Histological findings: A part of the thick pleura with inflammatory cells, including multinucleated giant cells, was positive-stained for anti alpha-lactalbumin antibody immunohistochemically. These giant cells are often observed in granulomatous inflammation against a foreign material. It was considered that those in the pleura had been induced by the nourishment, and that those in the pulmonary parenchyma had been affected by the insertion of the tube. The multinucleated giant cells clarified the cause of fatal pleuritis and pneumonia and the misinsertion of the tube.