Evaluation of Deep Thermal Rehabilitation System Using Resonant Cavity Applicator During Knee Experiments.

This paper evaluates experiments on the knee using a new heating rehabilitation system. For effective thermal rehabilitation of osteoarthritis, it is necessary to heat the deep tissue inside the knee joint. Our new rehabilitation system is based on the re-entrant type resonant cavity applicator which was developed for deep hyperthermia treatment in our previous studies. Our experimental results using agar phantoms showed our heating system is able to heat the deep tissue inside the knee without physically contacting the surface skin. In this study, we developed a prototype applicator and experimented on a healthy human subject's knee under clinical conditions. To evaluate heating performance, we conducted heating experiments with our resonant cavity applicator and a conventional microwave diathermy system and compared the results. The experimental results of temperature increase distributions inside the human body were estimated by ultrasound imaging techniques. The estimated results from our knee experiments show that our heating system is able to heat knee tissue more deeply than microwave diathermy systems can and thus would be effective for deep thermal rehabilitation applications in clinics.

Three-dimensional architecture of cerebral microvessels with a scanning electron microscope: a cerebrovascular casting method for fetal and adult rats.

Vascular casting for scanning electron microscopic studies on microvascular architecture is in common use for various visceral organs in the field of anatomy. However, only a few studies have been performed on the brain using the previously reported casting method, and no detailed descriptions deal with suitable methodology for producing brain vascular casts. Our casting method, introduced here, for the CNS from the fetal to the adult stage involves the following modifications: (1) Perfusion fixation of the brain is carried out before injecting the plastic resin for casting into the cerebral blood vessels; (2) digestion of nervous tissue is accomplished with a sodium hydroxide and sodium hypochlorite solution; and (3) vascular casts are dried by a freeze-drying method, while the nondigested brain slices opposite the casts can be investigated with light microscopy and transmission electron microscopy. This modified casting method enables one to represent the microvascular system of the rat brain three-dimensionally from embryonal day 17 onward. It is hoped that this method will prove to be a useful tool in morphological vascular research on the nervous system.

A Creutzfeldt-Jakob disease agent (Echigo-1 strain) recovered from brain tissue showing the 'panencephalopathic type' disease.

We used histologic evidence of degenerative changes in both the gray and white matter of the brain to diagnose a patient as having the panencephalopathic type of Creutzfeldt-Jakob disease (CJD). This type of CJD is relatively common in Japan, but not in North America or Europe. We recovered a transmissible pathogen (Echigo-1 strain) from an autopsy specimen of the patient's brain and passed it serially in Hartley guinea pigs. After a long latent period, it caused degenerative changes, mainly in the thalamic area of the guinea pig brain. On the 4th passage, a substrain emerged with a short latent period. When cross-transmitted to Golden Syrian hamsters, this substrain induced severe degeneration in both the thalamus and cerebral cortex. We compare our results with those for other experimental CJDs produced by other types of this disease.

Hereditary dentatorubral-pallidoluysian atrophy: clinical and pathologic variants in a family.

We describe a family showing dentatorubral-pallidoluysian atrophy. Three patients appeared through three successive generations and displayed a wide variety of clinical pictures. The male proband with onset in childhood showed progressive myoclonus epilepsy syndrome. The father experienced cerebellar ataxia, myoclonus, and mild dementia starting in middle age; the paternal grandmother had progressive symptoms of cerebellar ataxia, choreiform movements, and dementia, but neither myoclonus nor epilepsy in senescence. Neuropathologic examination of two patients, the proband and the paternal grandmother, revealed combined degeneration of the dentatorubral and pallidoluysian systems and obvious degeneration involving the striatum in the proband and the cerebellar cortex in the grandmother. The present study indicates that this disease can include many clinical and pathologic variants even in the same family.

Familial juvenile parkinsonism: clinical and pathologic study in a family.

We describe a family with juvenile-onset parkinsonism, which improved following sleep. Four of the five siblings in this family developed a similar onset of parkinsonism at an early age, and the parents were first cousins. In one of the siblings, a 67-year-old woman, pathologic changes at autopsy were confined to the substantia nigra pars compacta (SNPC) and locus ceruleus. The SNPC revealed obvious neuronal loss and gliosis in the medial and ventrolateral regions. In the remainder of the SNPC and the locus ceruleus, the population of neurons was reduced and there was low melanin content in most of the neurons but no detectable gliosis or extraneuronal free melanin pigment suggestive of a neurodegenerative process. There were no Lewy bodies. The entire pathologic picture was different from that of Lewy body Parkinson's disease.

Creutzfeldt-Jakob disease in a patient with a cadaveric dural graft.

We report a 26-year-old woman with Creutzfeldt-Jakob disease (CJD) who had received cadaveric dural material 33 months before the onset of neurologic symptoms. This is the fourth case in which a dural graft was the putative source of the CJD agent. All four cases had the grafting before changes in the sterilization procedure adopted in 1987 to inactivate the CJD agent.

Uniform tissue distribution of tRNA(Lys) mutation in mitochondrial DNA in MERRF patients.

We documented the presence of a point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA) in various postmortem tissues from two patients with myoclonus epilepsy associated with ragged-red fibers (MERRF). The percentages of the mutant mtDNA were similar (93 to 99%) in both clinically affected and unaffected tissues, suggesting that preferential clinical involvement of certain tissues in MERRF is based not only on the variation of distribution of the mutant mtDNA, but also on other factors such as differences in the threshold in various CNS regions and organs.

Immunohistochemical study on neuroglia identified by the monoclonal antibody against a macrophage differentiation antigen (Mac-1).

We have studied frozen sections of the developing and adult mouse central nervous system (CNS), with or without cold lesions, by immunohistochemical and histochemical methods. Using a monoclonal antibody against a macrophage differentiation antigen (Mac-1), we have shown that some neuroglia in the white matter of adult mice stained positively. In the developing CNS, with or without cold lesioning, Mac-1-positive glia were not detected. In the normal adult CNS, a small number of glia in the white matter stained faintly. After cold injury, the number of Mac-1-positive glia and their staining intensity increased for several months. Mac-1-positive glia were always negative for glial fibrillary acidic protein (GFA). Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells. Considering that the phagocytic activity of glia increases after injury to the CNS (Trachtenberg 1983) and that Mac-1 has been reported to be associated with the complement receptor (Beller et al. 1982), Mac-1-positive glia may play a role in phagocytosis in the damaged CNS.

Construction of spinal cord cDNA library and application for subtractive cloning of spinal cord-specific cDNAs.

Neurodegenerative diseases are characterized by neuronal degeneration of specific neurons, e.g., degeneration of motoneurons in amyotrophic lateral sclerosis. As an approach to understand molecular mechanisms of neuronal degeneration of human spinal cord motoneurons in various motor neuron diseases, we have constructed a human spinal cord cDNA library and developed a strategy for isolating spinal cord-specific genes by subtractive cloning. We constructed human spinal cord and brain cDNA libraries from postmortem human spinal cord and brain. To isolate human spinal cord-specific cDNAs, a spinal cord-enriched [32P]cDNA probe was generated by the phenol emulsion reassociation technique. Forty-eight cDNA clones out of 10,000 colonies gave strong signals with the subtracted probe, and individual spinal cord cDNA clones were isolated. Northern blotting analysis confirmed that two spinal cord cDNA clones are, in fact, more abundant in spinal cord compared to brain.

Stability of messenger RNA in postmortem human brains and construction of human brain cDNA libraries.

We studied stabilities of poly(A)(+)-RNA in postmortem mouse and human brains for up to 12 hours. The yields of total RNA were not changed significantly during postmortem periods either in mouse brains or human brains. Cell-specific cDNA probes were used to evaluate postmortem stability of poly(A)(+)-RNA in each cell type in the central nervous system. We used neuron-specific enolase (NSE), S-100 beta (S-100), and myelin-associated glycoprotein (MAG) for molecular markers of neuron, astrocyte, and oligodendrocyte, respectively. There was no detectable degradation of mRNAs coding for NSE, S-100, and MAG during the postmortem periods on Northern blot hybridization analyses. These results indicate that intact mRNAs expressed in neuron, astrocyte, or oligodendrocyte can be isolated from postmortem brains for up to 12 hours after death. Using poly(A)(+)-RNA thus isolated from two postmortem human brains, we constructed directional cDNA libraries and demonstrated the presence of full-length cDNAs for NSE, S-100, and MAG on Southern blot hybridization analysis. The present data should encourage studies on altered gene expressions in human brain in various neurologic diseases.

Activation of human lysosomal sialidase.

An acid sialidase [EC 3.2.1.18], partially purified from human placenta by Con A-Sepharose adsorption and p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatographies, was activated by incubation at 37 degrees C. This activation showed both time and temperature dependencies, with the most effective activation observed at 37 degrees C in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A, significantly inhibited activation. The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparation. These results suggest the possibility of participation of aminopeptidase function in the activation process of sialidase.

Selective decrease of large neurons in the neostriatum in progressive supranuclear palsy.

In order to evaluate the quantitative changes in the neostriatum in progressive supranuclear palsy (PSP), sections of the caudate head (CN) and putamen (PT) from 4 PSP patients were stained with Klüver-Barrera, and the cell body and nuclear area of the neurons were measured by a digitizer. Obtained results were compared to those of 6 age-matched control and 4 Alzheimer's disease and senile dementia of Alzheimer type (AD/SDAT) subjects which were previously reported. The number of large neurons (nuclear area greater than 101 microns 2) in PSP was about 40% (P less than 0.01) and 30% (P less than 0.01) of that of the controls in CN and PT, respectively. In contrast, the number of small neurons in PSP (nuclear area less than 100 microns 2) was well preserved. The values were quite similar to those in AD/SDAT. The implications and possible significance are discussed.

Selective involvement of large neurons in the neostriatum of Alzheimer's disease and senile dementia: a morphometric investigation.

In order to evaluate the quantitative changes in the neostriatum of Alzheimer type (SDAT), sections of the caudate head (CN) and putamen (PT) from 4 AD/SDAT and 6 age-matched control cases were stained with Klüver-Barrera, and the cell body and nuclear areas of the neurons were measured by a digitizer. This study revealed a significant decrease in the number of large neurons (nuclear area; greater than 101 micron 2) and good preservation of the number of small neurons (nuclear area; less than 100 micron 2) in CN and PT of AD/SDAT.

Correlative decrease of large neurons in the neostriatum and basal nucleus of Meynert in Alzheimer's disease.

A quantitative investigation was performed on the large neurons in the neostriatum and basal nucleus of Meynert (bnM) in patients with Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). The degree of decrease of the large neurons in the neostriatum was quite similar to that in the bnM; these decreases were significantly correlative in AD, but not in PSP. These findings indicate that the large neurons in the neostriatum and bnM, which are considered to be cholinergic and to exclusively possess nerve growth factor receptors in the cerebrum, degenerate simultaneously in an equal ratio in AD.

Large neurons in the neostriatum in Alzheimer's disease and progressive supranuclear palsy: a topographic, histologic and ultrastructural investigation.

Large neurons in the neostriatum of patients with Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) were investigated topographically, histologically and ultrastructurally. The number of large neurons whose nuclear area is greater than 101 microns2 was uniformly decreased in the neostriatum in PSP, but the decrease of these neurons in AD appeared to be more marked in the nucleus accumbens. Most of the remaining large neurons in both diseases contained neurofibrillary tangles (NFTs). In addition, some of the small neurons in PSP were positive for tau-immunostaining. Curly fibers were frequently observed in AD, but were absent in PSP. Ultrastructurally, NFTs in AD were composed mainly of paired helical filaments, whereas those in PSP contained straight tubules.

Specific expression of inositol 1,4,5-trisphosphate 3-kinase in dendritic spines.

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase (IP3K) in the rat cerebral cortex and hippocampus was studied immunohistochemically. In both regions, the major structure expressing a high level of IP3K was the dendritic spines of pyramidal neurons, where immunoreactivity was associated with the spine apparatuses and plasmalemma. The postsynaptic densities showed the most intense labelling. Taking into account the results of our previous observations, which demonstrated the restricted localization of the enzyme in the dendritic spines of Purkinje and basket cells in cerebellum, IP3K may be localized specifically in dendritic spines in various regions of the central nervous system, and involved in synaptic signal transduction at the spines.

Remote astrocytic response of prefrontal cortex is caused by the lesions in the nucleus basalis of Meynert, but not in the ventral tegmental area.

The nucleus basalis of Meynert (nbM) was lesioned by injection of ibotenic acid, in 200 g male Wistar rats. The rats were killed 1, 3, 7 or 21 days after surgery, the brains were removed and the prefrontal cortices were subjected to immunohistochemical and Western blot analysis for the expression of glial fibrillary acidic protein (GFAP). In some rats, vehicle was injected into the nbM and in others 6-hydroxydopamine (6-OHDA) was injected into the ventral tegmental area (VTA). Quantitative Western blot analysis revealed significantly greater immunoreactivity for GFAP in the prefrontal cortex of nbM-lesioned rats. Immunohistochemical examination revealed fibrous and hypertrophic GFAP-positive astrocytes even one day after surgery, and this reaction was stronger at 3 days after surgery. After this peak, GFAP-immunoreactivity of the astrocytes decreased from 7 days to 21 days. In contrast, GFAP-positive astrocytes were not observed in the brains of vehicle-injected or VTA-lesioned rats, even 21 days after surgery. The present results indicate that cortical astrocytes respond to cholinergic deafferentation. In addition, our findings provide new insights into the abnormalities of cortical glial cells after cholinergic deafferentation in Alzheimer's disease.

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex.

Subcellular localization of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex was studied immunohistochemically using a monoclonal antibody. Electron microscopy revealed intense immunoreactivity in the dendritic spines of Purkinje cells forming synapses with the parallel fibers, climbing fibers and recurrent collaterals of Purkinje cell axons. The labelling was associated with the hypolemmal cisternae, surrounding matrix and plasmalemma including the postsynaptic densities. Weaker immunoreactivity was present in the dendritic spines of basket cells and in certain segments of Purkinje cell recurrent collaterals. The postsynaptic regions of the dendritic trunks of Purkinje and basket cells were negative. These results indicate that inositol 1,4,5-trisphosphate 3-kinase is distributed amongst the spines of various synaptic relations with different electrophysiological properties, and that axon terminals of certain cell types are another functional site for the enzyme.

Distinct pattern of neuronal degeneration in the fetal rat brain induced by consecutive transplacental administration of methylmercury.

The transplacental neurotoxicity of methylmercury (MeHg) on the fetal rat brain was studied. Adult female rats were administered 1, 2 or 3 mg/kg/day methylmercury chloride (MMC) orally for either 5 or 12 days, and were then mated. They were subsequently administered MMC in the same manner until the end of gestation. On embryonic day 22, a proportion of the fetal brains were histologically examined. Neuronal degeneration of varying degree was detected consistently in the brain stem, cingulate cortex, thalamus and cerebral basal area, including the hypothalamus. The distribution pattern of neuronal damage was different from those in rats treated with MeHg in the postnatal or adult stages. This finding suggests that pathomechanisms in MeHg intoxication operate distinctively in the fetal brain. The offspring derived from dams treated with 1 mg/kg/day MMC for 5 pregestational days and throughout pregnancy survived with inherent brain lesions. This experimental model could be a useful tool for research on the neurotoxicity of MeHg in the human fetal brain.

Intrauterine methylmercury intoxication. Consequence of the inherent brain lesions and cognitive dysfunction in maturity.

We studied the effects of intrauterine neurotoxicity by methylmercury (MeHg) on the postnatal developing and adult stages of rats. We used offspring delivered from dams that had been given 1 mg/kg/day methylmercury chloride for 5 pregestational days and throughout pregnancy. Histopathological examination of the brains of a proportion of the offspring on postnatal days 1 (P1) and P3 revealed degenerative neurons in the brain stem and the limbic system, including the hippocampus and the amygdala. At P7 and P14, degenerative neurons were indiscernible, but reactive astrocytosis remained in the brain stem. At P70 and P180, the brains seemed to have developed well. However, in behavioral analyses performed at 6 months of age, MeHg-exposed rats showed a significant learning disability in the passive avoidance response compared with controls, but no differences in water maze performance. Furthermore, morphometric analysis of the amygdala and hippocampus revealed significantly fewer neurons in both areas in the MeHg-exposed rats. Thus, chronic intrauterine exposure to low-dose MeHg induces a decrease in neuron population in the limbic system, and the offspring have impaired higher brain function.