Age-related accumulation of lipofuscin in three different regions of rat brain.

The rate of accumulation of auto-fluorescent granules (lipofuscin) in three different regions of rat brain was investigated at various ages from very young to old animals (1, 2, 3, 6, 12 and 30-34 months of age. The accumulation of lipofuscin increased with age in the three brain regions. The first appearance of lipofuscin granules was at 8 weeks of age in the hippocampus and in the thalamus. In the case of cerebral cortex (laminae III), lipofuscin granules were first found in 3-month-old rats. The rate of lipofuscin accumulation was the highest in the hippocampus (y = 0.286x - 0.099, r = 0.963) among the three regions examined. In the case of cerebral cortex and thalamus, a slower rate of lipofuscin accumulation was observed (y = 0.072x - 0.14, r = 0.797 for cerebral cortex; y = 0.067x - 0.14, r = 0.953 for thalamus). It was noted that the most abundant accumulation and the highest rate of lipofuscin accumulation was in the hippocampus. But the rate and magnitude of lipofuscin accumulation in the hippocampus were low compared with cardiac muscles. From these results, it is suggested that brains have better protective system against oxidative stress than other organs.

Age-related changes in basal dendrite and dendritic spine of hippocampal pyramidal neurons (CA1) among SAMP1TA/Ngs--quantitative analysis by the rapid Golgi method.

It has been confirmed that a substrain of the senescence-accelerated mouse SAMP1TA/Ngs develops learning disturbance-like behavior at 3 months of age, exhibits almost normal behavior at 5 months, and manifests learning disturbance at 7 months. The changes with age in basal dendrites and dendritic spines of CA1 pyramidal neurons were quantitatively evaluated by the Golgi method using male SAMP1TA/Ngs. The correlation between the change in learning ability and the morphometry was examined. The number of dendritic spines in the 3- and 7-month-old groups was significantly lower than that in the 5-month-old group. It is presumed that the disturbance in acquisition of learning ability at 3 months of age is secondary to the immaturity of neurons, while the learning disturbance at 7 months of age is due to neuronal aging. This substrain, which is characterized by the impairment of acquired learning ability due to senescence, is useful as a model for studies on human brain dysfunction associated with senescence.

Quantitative age-related changes in apical dendrites and dendritic spines of CA1 pyramidal neurons among senescence accelerated mice (SAMP1TA/Ngs).

SAMP1TA/Ngs, a substrain of senescence accelerated mouse, is a useful animal model for research on brain dysfunction due to senescence. In a previous study it was reported that the age-related changes in basal dendrites and spines of CA1 pyramidal neurons coincide with the behavioral characteristics found in SAMP1TA/Ngs. The goal of the present study was to investigate morphological changes in apical dendrites and dendritic spines of CA1 pyramidal neurons among 3-, 5-, 7-month-old SAMP1TA/Ngs. Pyramidal neurons of the hippocampus were stained by the rapid Golgi method, and the number of apical dendrites, the number of their spines and the density of the dendritic spines were evaluated. The number and density of the spines of apical dendrites were significantly higher at 5 months than at 3 or 7 months of age. We propose that the low number of dendritic spines in 3-month-old animals was caused by immaturity, while the changes in the density and number of dendritic spines in 7-month-old mice were due to accelerated aging. The data on the morphology of apical dendrites are a useful complement to the results reported previously. The findings of the present study also support the hypothesis that this model mouse demonstrates changes in respective developmental stages, i.e. immaturity, adulthood and senescence. This pattern of postnatal growth has special meaning because it indicates the usefulness of the strain in the study of geriatric disorders in humans.

Late infantile Hirschsprung disease-mental retardation syndrome with a 3-bp deletion in ZFHX1B.

A 48-year-old woman with late infantile onset mental retardation developed megacolon. Although the patient had no typical clinical features of Hirschsprung disease-mental retardation syndrome, a new 3-base pair deletion, eliminating an Asn, was identified in the responsible gene ZFHX1B. This suggests that screening for ZFHX1B mutations is warranted even in the absence of typical clinical features of the syndrome.

Thymolipoma with striated myoid cells. Histological, immunohistochemical, and ultrastructural study.

A large asymptomatic anterior mediastinal thymolipoma, discovered by chest radiograph during a regular check-up for company employees, was excised from a 33-year-old Japanese man. On immunohistochemical and electron-microscopic examination, clusters of myoglobin-positive cells with cytoplasmic Z band structures were found scattered in the medulla. Myoid cells have been previously seen in the normal thymus as well as in thymic hyperplasia, thymoma, and in the thymus of patients with myasthenia gravis. To our knowledge, this is the first reported instance of myoid cells in a thymolipoma.

Herbal medicine and the study of aging in senescence-accelerated mice (SAMP1TA/Ngs).

Two types of herbal medicine, Toki-Shakuyaku-San (TSS) and Boui-Jiou-Tou (BJT), were considered from the view point of their possible preventive effect on senility using the Senescence-Accelerated Mouse (SAMP1TA/Ngs), which is thought to be a useful model of human aging. The investigation of the effects of TSS and BJT on the behavior, learning ability, longevity, and histological changes in animals may be important for basic studies on aging in humans. Pellet feed containing each herbal medicine was given to the treated group ad libitum for 24 weeks starting at the age of six weeks. Learning ability and the longevity of mice were assessed and spontaneous motor activity was also measured. Morphological examinations were performed. On the passive avoidance task, the period until acquisition of standard achievement rate was shorter in the treated groups than in the control groups. The mean number and the density of dendritic spines were higher in the BJT treated group than in the control group. With regard to life span, median survival time tended to be longer in the treated groups than in the control groups. The results of this study suggested that TSS and BJT have some valuable effect for the prevention of senility.

Electrospray ionization-tandem mass spectrometry analysis of peptides derived by enzymatic digestion of oxidized globin subunits: An improved method to determine amino acid substitution in the hemoglobin "core".

The determination of an amino acid substitution in the "core" region of abnormal hemoglobin is technically difficult and is time- and labor-intensive both by conventional and by mass spectrometry techniques. Underivatized subunits cleaved with trypsin and lysyl endopeptidase were analyzed by high-performance liquid chromatography-electrospray ionizationmass spectrometry. The core peptides showed a series of multiply charged ions of homo- and heterodimers. Abnormal peptides in the core region could be detected in dimeric form. The sequence of core peptides was determined by product ion spectra of the peptides from oxidized globin digested with trypsin and lysyl endopeptidase. Oxidation of cysteine residues to cysteic acids in the core region resulted in the strong promotion of y-series ions by product ion analysis with a tryptic peptide from apoprotein B-100 as previously reported by Burlet, Yang, and Gaskell (J. Am. Soc. Mass Spectrom. 1992, 3, 337-344). These techniques were used to prove that substitution of an unstable hemoglobin, known as Hb Santa Ana (ß88 leucine → proline), occurred in a patient with congenital hemolytic anemia. The tandem mass spectrometry analysis with oxidized globin digested with trypsin and lysyl endopeptidase offers a novel method to detect substitutions in the core region of hemoglobin.

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.

Enhanced amyloidogenicity of sulfonated transthyretin in vitro, a hypothetical etiology of senile amyloidosis.

Genetic variants of transthyretin (TTR) cause systemic amyloidosis and wild-type TTR may also in some situations produce amyloid fibrils. We have analyzed wild-type and variant TTRs by mass spectrometry and found that TTR preparations from all individuals demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component which had a molecular mass 80 Da larger than free TTR and was proved to be TTR conjugated with sulfite. Here, the amyloid fibril formation of the TTR isoforms was monitored by the turbidity at 330 nm, and by a Congo red-binding assay as a function of pH, according to the method of Lai et al. The S-sulfonated TTR showed the highest level of amyloid fibril formation. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, showed neither elevation of the turbidity nor the Congo red binding. Commercially purchased TTR without further treatment containing free, S-sulfonated and other species of TTR molecules showed an intermediate elevation. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic. Although further experiments are needed to apply the observation to in vivo phenomenon, exogenous sulfite may be a cause of senile systemic amyloidosis.

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

Primary intestinal T-cell lymphoma resembling lymphomatous polyposis: report of a case.

We report an interesting case of primary intestinal T-cell lymphoma (ITL) resembling lymphomatous polyposis (LP) in a 24-year-old man. The neoplasm macroscopically showed numerous small polyps throughout the colon and microscopically showed diffuse proliferation of small-sized tumor cells with occasionally cleaved or irregularly shaped nuclei. The tumor cells were immunohistochemically positive for CD3, CD8, TIA-1, and CD56, and a polymerase chain reaction study showed a single band, indicating monoclonal rearrangement of the T-cell receptor beta gene. The phenotypic features in the current case are consistent with those of ITL derived from cytotoxic CD56+ CD8+ intraepithelial lymphocytes. This is the second documented case of primary ITL with a morphologic pattern of LP.

Quantification of glycated hemoglobin by electrospray ionization mass spectrometry.

Glycated hemoglobin, considered to be the best index for the treatment of diabetes mellitus, was measured by electrospray ionization mass spectrometry (ESI/MS) according to the method proposed by Morris et al. at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, 1996. They compared the values obtained by MS and affinity chromatography. Here, the values obtained by ESI/MS were compared with those obtained by high-performance liquid chromatography and by latex agglutination immunoassay. Whole blood samples were diluted 500 fold with 0.2% formic acid-50% acetonitrile solution and 5 microliters of the diluted solution was injected with the ESI/MS system (TSQ 7000) via a sample loop. The within-run and between-run relative standard deviations of the ratio of glycated and non-glycated beta-chain were less than 5%. The correlation coefficients between ESI/MS and conventional methods were higher than 0.96. However, considerable discrepancies were observed among methods. ESI/MS will allow reproducible measurements of glycated hemoglobin and will be useful in the quality control of HbA1c measurement by other principles and also in routine clinical laboratory tests.

Simple and defined method to detect the SOD-1 mutants from patients with familial amyotrophic lateral sclerosis by mass spectrometry.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis (FALS). We established a simple and defined method to detect the mutant SODI in erythrocytes by electrospray ionization mass spectrometry (ESIMS) using materials precipitated with specific antiserum. Hemolysate was mixed with anti-SOD1 antiserum and the generated precipitate, which was soluble in the solvent for MS analysis, was injected on to an LC column connected to an ESI-mass spectrometer. MS spectra of the reduced SOD1 prepared from normal individuals showed ion peaks corresponding to free monomer SOD1. The spectra from FALS patients revealed doublet ion peaks corresponding to normal and mutant components. The ratios of mutant to normal SOD1 were about 1/2 in cases of (G37R) and (A4S), and about 0.15 in a case of (H46R). This method provides for the rapid diagnosis using small amount of specimens, and will contribute to elucidate the pathomechanism of FALS through the quantification of SOD1 mutants in erythrocytes and in tissues of nervous systems.

The fates of the callosal neurons in neocortex after bisection of the corpus callosum, using the technique of retrograde neuronal labeling with two fluorescent dyes.

The fate of callosal neurons after callosotomy is yet unclear although this has become a common surgical procedure for intractable generalized epilepsies. Using retrograde neuronal labeling with two fluorescent dyes, we demonstrated that callosal neurons in the parietal cortex of the adult rat survive up to 20 weeks after callosotomy. Our data suggest that these neurons possess numerous ipsilateral axon collaterals with indispensable functions in the ipsilateral hemisphere.

Two types of aggregate in the cerebral cortex of a seizure-sensitive strain of the Mongolian gerbil.

A 70-kDa protein, P70, found mostly in the pyramidal cells of the cerebral cortex of cobalt-induced epileptogenic rats, has been implicated in epileptogenesis. The presence of a P70-like substance was searched for immunohistochemically in the cerebral cortex of MGS/ldr, a seizure-sensitive strain of the Mongolian gerbil (Meriones unguiculatus) that we previously established. Immunoreactive aggregates were observed in the pyramidal neurons of the motor cortex and the primary somatosensory cortex. Analysis using confocal laser scanning microscopy revealed that the aggregates were often colocalized with a second type of aggregate with red autofluorescence at the marginal zone of the cell somata. Both aggregates appeared and increased before the appearance of generalized tonic-clonic convulsion. These may be involved in some change of physiological function of the cerebral cortex but their presence itself is not enough to determine the occurrence of epileptic seizure because the gerbils that showed no such seizure had both aggregates.

Transient and repetitive rises in blood pressure synchronized with plasma catecholamine increases after head injury. Report of two cases.

The authors report two patients with repetitive episodes of acute transient rise in blood pressure synchronized with increases in plasma catecholamine after severe head injury. In both cases, the paroxysmal hypertension occurred suddenly on the 2nd day posttrauma, and its frequency declined gradually, disappearing with time. The pathophysiological basis of this peculiar clinical manifestation is discussed.

Computerized tomography angiography of ruptured cerebral aneurysms: factors affecting time to maximum contrast concentration.

OBJECT: This study was conducted to assess the diagnostic value of three-dimensional computerized tomography (3-D CT) angiography in demonstrating cerebral aneurysms in 42 consecutive patients presenting with acute subarachnoid hemorrhage (SAH). METHODS: To obtain the volume data for selective visualization of the cerebral arteries without enhancement of the venous system, the time delay was established between the injection of contrast medium and the start of scanning by using two different methods. The circulation time was calculated with Schad's formula in the first 13 cases, but the results were not satisfactory. In the 29 subsequent cases the time delay was established using a single-level dynamic CT prescan. The dynamic prescan demonstrated the statistical differences in peak time with regard to patient age, SAH grade, and the postresuscitation state after cardiopulmonary arrest. The 3-D CT angiograms were generated from the volume data by using a voxel transmission method. Computerized tomography angiography obtained after optimally adjusted time delay demonstrated the contour of the cerebral arteries in 97% of cases, and aneurysms were detected in 93%. Enhancement of the cavernous sinus and major cortical veins was avoided. Even in patients who suffered cardiopulmonary arrest, images of the major arteries were clearly demonstrated after resuscitation. CONCLUSIONS: In an emergency situation, CT angiography with a dynamic prescan may be an alternative to magnetic resonance angiography or digital subtraction angiography in the diagnosis of ruptured aneurysms. This modality would also be useful for the precise assessment of small aneurysms, blebs, and aneurysms adjacent to the cavernous sinus.

Meningeal melanocytoma in the left frontal region.

We report a rare case of meningeal melanocytoma in the left frontal region. A 45-year-old man complained of a headache. Magnetic resonance (MR) scanning showed characteristic patterns: a slightly high signal intensity mass in the left frontal region on the T1-weighted image and a low signal intensity on the T2-weighted image. The patient underwent gross total removal of the tumor. The postoperative course was uneventful. After two years, there was a small local recurrence. The histological finding of the tumor showed meningeal melanocytoma. To our knowledge, this is the second published report of a meningeal melanocytoma in the supratentorial region.

Recovery from postoperative hypothermia predicts survival in extensively burned patients.

To clarify the cause of postoperative hypothermia in extensively burned patients, factors affecting postoperative hypothermia were studied in 16 extensively burned adult patients (8 survivors and 8 nonsurvivors) with a burn index greater than 35. Body temperature was monitored continuously in either the urinary bladder or rectum. Hypothermia of less than 35 degrees C occurred in 89% (66 of 74) of the total operations performed in these 16 patients. The rate of temperature rise (RTR) was significantly lower in nonsurvivors (0.4 +/- 0.2 degrees C/h) than in survivors (1.7 +/- 0.9 degrees C/h; p < 0.001). Continuous indirect calorimetry performed in seven patients (four survivors and three nonsurvivors) demonstrated that RTR was determined primarily by heat production. The measured energy expenditure reached only 1.7 +/- 0.2 times the basal energy expenditure during rewarming in nonsurvivors, whereas it was 2.7 +/- 0.9 times the basal energy expenditure in survivors (p < 0.01). Surprisingly, in nonsurvivors, the RTR was significantly decreased even during the first 2 weeks. These findings suggest that those who cannot generate heat well in postoperative hypothermia are unable to produce the additional energy required to overcome sepsis.

Manipulation of the somatosensory cortex modulates stimulus-induced repetitive ear movements in a seizure-sensitive strain of gerbil.

Some Mongolian gerbils (Meriones unguiculatus) respond to stimulation by seizures, the pattern of which changes progressively during development. We previously established a seizure-sensitive strain, MGS/Idr, in which all animals exhibit such stimulus-induced seizures. We have now noted that all adults of this strain also show repetitive backward movements of the ears at the ears at the beginning of stimulus-induced seizures, although the incidence varies with the individual. We examined whether the cerebral cortex was involved in these movements and found that electrical stimulation of an area of the somatosensory cortex elicited strong backward movement of the ear on the contralateral side, and that unilateral application of bicuculline, a GABAA receptor antagonist, induced spontaneous repetitive backward movements of the same ear. In this area, sharp waves appeared in the electrocortigram during the repetitive ear movements induced by seizure-inducing stimuli. Unilateral ablation of this area abolished stimulus-induced repetitive movements of the contralateral ear, but had no effects on those of the ipsilateral ear. These results suggest that, in certain types of seizure-susceptible subjects, it may be possible to modify stimulus-induced repetitive movements by manipulating a certain area of the somatosensory cortex which is related to these movements and that this gerbil strain may be useful in research on this subject.