Katsuji Kobayashi

Apoptosis of astrocytes with enhanced lysosomal activity and oligodendrocytes in white matter lesions in Alzheimer's disease.

Cerebral white matter lesions in Alzheimers disease (AD) consist of subcortical degeneration and ischaemic–hypoxic changes. Glial changes are intimately associated with the white matter lesions, and regressive changes in astrocytes and loss of oligodendroglial cells have been reported. We quantitatively compared glial changes including apoptosis and enhanced lysosomal activity in the frontal and temporal white matter by using terminal dUTP nick end labelling (TUNEL) and immunohistochemistry for glial markers, lysosomes and apoptosis‐regulating proteins in non‐familial AD brains. The degree of myelin pallor and axonal loss varied considerably in both the frontal and temporal white matter but fibrillary gliosis in demyelinated lesions tended to be less prominent in the temporal white matter in AD cases. A morphometric study with planimetric methods for cross‐sectional areas of frontal and temporal white matter revealed that the white matter of AD cases manifested atrophy with significant reduction in frontal (11.9%) and temporal (29.4%) white matter compared to normal controls. Double immunolabelling for glial fibrillary acidic protein (GFAP) and KP1 (CD68) revealed KP1‐positive fragmented structures within the weakly GFAP‐labelled astrocytes. These KP1‐positive structures correspond to process fragmentation and cytoplasmic vacuoles, which in turn indicate enhanced lysosomal activity during regressive changes in astrocytes. The KP1‐modified astrocytes were not found in Picks disease and corticobasal degeneration. The density of apoptotic glial cells, largely oligodendroglial, was significantly higher in the temporal than in the frontal white matter, and most GFAP‐positive astrocytes with regressive changes were apoptotic. GFAP‐positive astrocyte density was statistically the same in the frontal and temporal white matter, but the density of KP1‐modified astrocytes was higher in the temporal than in the frontal white matter. The rate of white matter shrinkage was significantly correlated with the density of apoptotic glial cells and the density of KP1‐modified astrocytes in the temporal lobe in AD cases. An increase in apoptotic glial cell density was found to contribute to GFAP‐positive astrocytes with regressive changes in temporal white matter, while apoptosis of vascular smooth muscle cells did not show topographical accentuation. Astrocytes labelled with beta amyloid protein were not apoptotic, and the density of apoptotic cells labelled with CD95 and caspase‐3 was too low in both types of white matter to be statistically evaluated. Our results imply that regressive changes in astrocytes and glial apoptosis are, to some extent, associated with white matter lesions, particularly of the temporal lobe in AD brains. The presence of apoptotic astrocytes with evidence of regressive change could therefore be a histological hallmark for white matter degeneration in AD.

Neurons, intracellular and extracellular neurofibrillary tangles in subdivisions of the hippocampal cortex in normal ageing and Alzheimer's disease

Unaffected neurons, intracellular neurofibrillary tangles (I-NFTs) and extracellular NFTs (E-NFTs) in six normal subjects and six patients with Alzheimers disease (AD) were morphometrically evaluated in eight subdivisions of the hippocampal cortex, using the Gallyas silver impregnation technique modified by the application of hematoxylin and eosin. The subdivisions examined included CA1-4, prosubiculum (PRO), subiculum and presubiculum (PRE), parasubiculum (PARA) and the entorhinal cortex (ENT). In the AD patients, the number of unaffected neurons in ENT, CA1, PRO and PARA was significantly decreased to one-quarter to two-thirds of that of the normal aged subjects. These four subdivisions in the AD patients had a greater number of both I- and E-NFTs. There were no significant differences in the total number of unaffected neurons, I- and ENTs between the AD patients and normal aged subjects for all the subdivisions. These findings suggest that neuronal loss in the hippocampal cortex in AD is almost entirely due to NFT formation. Furthermore, with regards to neuronal loss and NFT formation, there were two different subdivision groups in the AD patients. One group was composed of severely affected subdivisions (ENT, CA1, PRO and Para) and was distinct from the other group which was composed of mildly affected subdivisions (CA4, CA3, CA2 and PRE). Each subdivision in the normal aged subjects belonged to the mildly affected group as seen in the AD patients. These findings indicate that both neuronal loss and NFT formation associated with normal ageing and with AD are not only quantitatively but also qualitatively different.

Regional cerebral blood flow in schizophrenic disorders

Regional cerebral blood flow was measured using a 133Xe inhalation technique in 16 schizophrenic patients and 20 healthy volunteers. The bilateral frontal blood flows in the patient groups were significantly lower than in the control group. In addition, the patient group having auditory hallucination showed a significantly increased blood flow predominantly in the left temporal region. On the other hand, the patient group without auditory hallucination showed a slightly increased flow in the right temporal region. These findings indicate that there are a hypofrontal activity and also a hypertemporoparietal activity in schizophrenics.

Association of phosphorylation site of tau protein with neuronal apoptosis in Alzheimer's disease

In addition to neuritic changes and amyloid deposits, neuronal and glial cell apoptosis is an important pathological feature of Alzheimers disease (AD). Several factors have been postulated as causes or triggers of cellular apoptotic change. This study focused on a quantifiable relationship between phosphorylation sites of tau protein in the neurofibrillary tangles (NFT) and neuronal apoptosis. Five monoclonal anti-tau antibodies (AT180, AT8, HT7, Tau2 and Tau5) for NFT labeling and TdT-mediated UTP nick-end labeling (TUNEL) for localizing apoptotic change were employed. TUNEL-stained neuronal nuclei showed significantly high density in the entorhinal cortex, cornu ammonis (CA) and the parietal cortex. In all regions, density of TUNEL-stained neuronal nuclei showed significantly direct correlation with that of AT8-, AT180- and Tau2-positive neurons. Correlation of TUNEL-stained neuronal nuclei with tau-positive neurons differed depending on the cerebral regions. Density of TUNEL-stained neuronal nuclei showed inverse correlation with that of both AT8-positive and Gallyas-stained NFT in the CA and showed significantly direct correlation with AT8- and HT7-positive neurons in the frontal cortex. Density of tau-positive and Gallyas-stained NFT was higher than that of TUNEL-stained nuclei. We conclude that phosphorylation sites of tau, 159-163 and 202-205, are probably associated with neuronal apoptosis and apoptotic change follows abnormal phosphorylation of tau.

A quantitative study of neurofibrillary tangles, senile plaques and astrocytes in the hippocampal subdivisions and entorhinal cortex in Alzheimer's disease, normal controls and non‐Alzheimer neuropsychiatric diseases

Abstract  The present quantitative study was performed in order to discriminate pathological substrates for dementia from Alzheimer changes in normal controls (NC) and non‐Alzheimer neuropsychiatric diseases (NAND). Regional densities of senile plaques (SP), neurofibrillary tangles (NFT) and astrocytes in the cornu ammonis (CA), subiculum and entorhinal cortex were measured and differences in these densities among Alzheimers disease (AD), NAND and NC were statistically compared. Densities of NFT in the CA and subiculum were significantly higher in AD than in NAND, and densities of SP in all regions were significantly higher in AD than in NAND. Similarly, NFT density in the subiculum and SP density in all regions were higher in AD than in NC. Regional densities of astrocytes in most regions were closely correlated with those of Alzheimer changes. In conclusion, the attribution of the Alzheimer changes, particularly of NFT, to dementia is neglected when they are confined to the entorhinal cortex. However, the attribution of the Alzheimer changes to dementia should be appreciated when they spread from the entorhinal cortex to the subiculum and/or CA.

Immunohistochemical study of neuronal intranuclear and cytoplasmic inclusions in Machado–Joseph disease

Abstract Machado–Joseph disease (MJD) is a dominantly inherited spinocerebellar disorder, and expansions of trinucleotide (CAG) at chromosome 14 have been shown to be the locus of this disorder. Polyglutamine CAG stretches in the neuronal cytoplasms and nuclei were studied with immunolabeling using 1C2, a monoclonal antibody recognizing polyglutamine stretches, and polyclonal antiubiquitin antibody in six genetically verified cases of MJD. 1C2 clearly labeled two types of neuronal intranuclear inclusions (NII) and neuronal cytoplasmic inclusions (NCI) in the substantia nigra, pontine nucleus, dentate nucleus and spinal anterior horn where NII and NCI were also positive for ubiquitin, as were extracellular dot‐like structures and oligodendroglial inclusions. 1C2‐positive NII and NCI had a lesion‐specific distribution. While the spinal motoneurons contained only 1C2‐positive NCI and lacked 1C2‐positive NII, the ventral pontine nucleus neurons had many 1C2‐positive NII and few 1C2‐positive NCI. Semi‐quantitative examination of NII and NCI positive for 1C2 or ubiquitin demonstrated that there were more 1C2‐positive NII and NCI than ubiquitin‐positive ones. It is noteworthy that the nuclei of the spinal motoneurons lacked 1C2‐positive immunoreactivity, so that ubiquitination of 1C2‐positive structures is presumed to occur late in the course of the disease.

CT Findings of the Interval Form of Carbon Monoxide Poisoning Compared with Neuropathological Findings

Cerebral computed tomography findings were described in 2 clinical cases of the interval form of carbon monoxide poisoning and comparison with postmortem CT finding of an autopsy case was made. There was low density in the bilateral frontal region, centrum semiovale and pallidal parts. In the course of the disease, the degree of low density in the white matter showed a tendency to diminish but it became more apparent in the pallidal parts. Myelinopathic white matter lesion in an autopsy material was recognized as a low density area in CT, which is identical with that seen in clinical cases.

Cytoskeletal protein abnormalities in patients with olivopontocerebellar atrophy — an immunocytochemical and Gallyas silver impregnation study

A highly sensitive silver technique for glial cytoplasmic inclusions (GCI) in olivopontocerebellar atrophy (OPCA) was applied to tissues from 15 patients with neurodegenerative disorders inluding OPCA, Joseph disease, Alzheimers disease (AD), Huntingtons chorea, Pick disease and three control non‐neurological subjects. Brain tissue from both OPCA and AD impregnated positively. Neurons, astroglia and oligodendroglia in the putamen, pontine nucleus and inferior olivary nucleus all impregnated in addition to white matter oligodendroglia. Neuronal inclusions in the pontine nucleus appeared as compact or fibrillary masses, and GCI‐bearing oligodendroglia and astrocytes showed homogeneously impregnated somata. The myelinated pontocerebellar tract and the white matter surrounding the inferior olivary nucleus contained a small number of impregnated nerve fibres with a hollow structure, which resembled the myelin sheath. Immunocytochemical studies to clarify these argyrophilic structures in the OPCA subjects employed paired helical filament (PHF), microtubule associated proteins (MAPS), MAPl, MAPZ, MAPS, tau, ubiquitin, neurofilament (200 or 70 kilodaltons) and myelin basic protein (MBP) antisera. GCI‐bearing white matter oligodendroglia expressed PHF, tau, MAP5 and ubiquitin immunoreactives and non‐argyrophilic astroglia were positive for MAP5 antiserum alone. In the putamen, pontine nuclei and inferior olivary nuclei, impregnated neurons as well as the GCI‐bearing oligodendroglia immunostained with PHF, tau, MAP5 and ubiquitin antisera and impregnated astroglia were also immunoreactive to these antisera except for being tau negative in the putamen. Silver impregnated nerve fibres showed only MBP immunoreactivity. These findings indicate that the argyrophilia in the OPCA subjects closely correlates with PHF and tau immunoreactivities.

Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile

Abstract A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112 mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immunoreactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.

Borrelia burgdorferi-seropositive chronic encephalomyelopathy: Lyme neuroborreliosis? An autopsied report.

A 36-year-old Japanese woman presented with progressive cerebellar signs and mental deterioration of subacute course after her return from the USA. Her serum antibody to spirochete Borrelia burgdorferi was significantly elevated. A necropsy 4 years after her initial neurological signs revealed multifocal inflammatory change in the cerebral cortex, thalamus, superior colliculus, dentate nucleus, inferior olivary nucleus and spinal cord. The lesions showed spongiform change, neuronal cell loss, astrocytosis and proliferation of activated microglial cells. The internal capsule was partially vacuolated and the spinal cord, notably at the thoracic level, was demyelinated and cavitated in the lateral funiculus. Microglial cells aggregated within and around the spongiform lesions and microglial nodules were present in the medulla oblongata. Use of Warthin-Starry stain demonstrated silver-impregnated organisms strongly suggesting B. burgdorferi in the central nervous tissues. The dentate nucleus and inferior olivary nucleus showed the most advanced lesions with profound fibrillary gliosis. Occlusive vascular change was relatively mild, and fibrous thickening of the leptomeninges with lymphocyte infiltrates was localized in the basal midbrain. The ataxic symptoms were due to the dentate and olivary nucleus lesions and mental deterioration was attributable to the cortical and thalamic lesions. Spongiform change, neuronal cell loss, and microglial activation are characteristic pathological features in the present case. The cerebellar ataxia and subsequent mental deterioration are unusual clinical features of Lyme neuroborreliosis. Spirochete B. burgdorferi can cause focal inflammatory parenchymal change in the central nervous tissues and the present case may be an encephalitic form of Lyme neuroborreliosis.