Tadahisa Kitamura

Quantitative studies on proliferative changes of reactive astrocytes in mouse cerebral cortex

Cell number and proliferation of reactive astrocytes were studied quantitatively in the stabbed cerebral cortex of adult mice, using immunohistochemistry for glial fibrillary acidic protein (GFAP) and [3H]thymidine autoradiography. GFAP-positive astrocytes increased in cell number gradually from 24 to 96 h after stabbing, and their immunoreactivity became intense. The maximum number of GFAP-positive cells was about 4.5 times normal in the layers II-VI of the cortex, whereas it was only 1.5 times normal in the layer I (molecular layer). In contrast to the gradual increase in cell number, no GFAP-positive astrocytes were labeled with [3H]thymidine prior to 48 h after stabbing, in either the layer I or the layers II-VI. Then 3-5% of them were labeled at 72 and 96 h, but very few again after 6 days. By injecting [3H]thymidine successively for 6 days after stabbing, only 17% of GFAP-positive astrocytes of the layer I or the layers II-VI were labeled. These results reveal that, in the cortical layers II-VI, many GFAP-negative source cells initially express much more GFAP-antigen without proliferation and change into GFAP-positive reactive astrocytes. Proliferation of reactive astrocytes is not the major factor for the marked increase in number of them. The cortical layer I would have few GFAP-negative source cells for reactive astrocytes. These source cells may be protoplasmic astrocytes.

Analysis of DNA ploidy patterns of gastric carcinomas of Japanese

Ploidy patterns of gastric carcinomas of 54 Japanese patients, ranging in age from 28 to 82 years, were determined by cytofluorometry. The gastric cancers were divided into four basic ploidy patterns; the first pattern was a diploid mode, the second was a heteroploid mode, the third was a mosaic pattern of a diploid and a heteroploid mode, and the fourth was a mosaic of two or more different heteroploid modes. The incidence was 68.4%, 14.8%, 12.9%, and 3.7% in the first, second, third, and fourth patterns, respectively. All the cancers contained polyploid populations, whereas the early cancers tended to contain a lesser amount of polyploid cells than the advanced ones. Diffuse‐type cancers and well‐differentiated, intestinal‐type adenocarcinomas were mostly unimodal cancers of the diploidy or heteroploidy, whereas poorly differentiated adenocarcinomas were often mosaic cancers of several different ploidies. There was no correlation between ploidy patterns and ages and sexes of patients. For 11 cases, labeling indices with 3H‐thymidine were determined. Labeling indices of the intestinal‐type and diffuse‐type cancers were 12% to 27% and 7% to 19%, respectively.

Distinctive four promoters collectively direct expression of brain-derived neurotrophic factor gene.

In order to get a deeper insight into comprehensive understanding of gene regulation of brain-derived neurotrophic factor (BDNF), we characterized the transcriptional apparatus of this gene on the basis of the genomic structure. The results in this study revealed that there are at least four distinctive promoters in the BDNF gene; two of them are neuron-specific and the rest are active in some non-neuronal tissues as well as neuronal ones. Although the analyses of the promoter usage pattern clarified many characteristic features in controlling these promoter activities, the most notable finding was that administration of kainic acid resulted in great activation of two out of the four promoters in hippocampal neurons in a regionally different manner and thus indicated the presence of two distinct signal transduction pathways for kainate-induced activation of BDNF gene expression in neurons. The analysis of BDNF gene expression in terms of the promoter usage pattern would provide a new and important insight into understanding a molecular control mechanism of this gene expression.

Reactive proliferation of astrocytes studied by immunohistochemistry for proliferating cell nuclear antigen

Astrocyte proliferation in the stab-wounded cerebral cortex of mice was studied using double immunohistochemistry for proliferating cell nuclear antigen (PCNA) and glial fibrillary acidic protein (GFAP). The number of GFAP-positive astrocytes increased markedly from day 0.5 to day 3 after stab wounding. Some GFAP-positive astrocytes in the immediate vicinity of the wound were found to be positive for PCNA. However, the maximum number of these double positive astrocytes was only 5-6% of the number of GFAP-positive astrocytes. This maximum value was observed on days 2.5 and 3. The present study revealed that astrocytes are able to reactively express PCNA, an intrinsic marker of DNA replication. On the other hand, it is suggested that the proliferation of astrocytes in the wounded cerebral cortex is limited, in contrast with their marked reactive up-regulation of GFAP.

Reactions of S-100-positive glia after injury of mouse cerebral cortex

Reactions of glial cells after stab wounding of mouse cerebral cortex were studied by [3H]thymidine autoradiography combined with immunohistochemistry for S-100 protein. S-100-positive cells in the stabbed cortex had the light and electron microscopic characteristics of astrocytes, and they showed remarkable hypertrophic changes 4 to 5 days after stabbing. There were many cells labeled with [3H]thymidine in the stabbed cortex from 24 h to 8 days after stabbing, and the number of labeled cells was maximum at 48 h. A few of the labeled cells were S-100-positive, and the labeled S-100-positive cells were seen 24 h to 6 days after stabbing, mostly after 72-96 h. By successive injections of [3H]thymidine for 6 days after stabbing, about 90% of labeled cells were S-100-negative, and about 90% of S-100-positive cells were unlabeled with [3H]thymidine. The increase in number of S-100-positive cells by day 6 after stabbing was not statistically significant (P greater than 0.05). These results suggest that reactive proliferation of astrocytes is a minor phenomenon in gliosis of injured cerebral cortex, in contrast with their remarkable reactive hypertrophy.

Up-regulation of cystatin C by microglia in the rat facial nucleus following axotomy

Cystatin C, a cysteine proteinase inhibitor, is expressed in the central nervous system (CNS) as well as many other organs of mammals. However, little is known concerning whether its expression is regulated under pathological conditions of the CNS and what types of cells are responsible for this regulation. We performed differential hybridization screening of cDNA libraries derived from the rat facial nucleus and found a cDNA of rat cystatin C to be up-regulated following facial nerve axotomy. In situ hybridization using an RNA probe for rat cystatin C revealed that cystatin C mRNA in the facial nucleus was markedly increased in amount by day 7 after axotomy and was then decreased to the normal level by day 50. The intense signal for cystatin C mRNA in the damaged facial nucleus was localized in the glial cells which had the morphological characteristics of microglia. Light and electron microscopic immunohistochemistry using a rabbit antibody specific for cystatin C confirmed that microglia in the damaged facial nucleus were strongly positive for cystatin C. The immunoreactivity was also found in the extracellular space, consistent with the fact that cells producing cystatin C generally secrete this protein. These results demonstrate that cystatin C is markedly up-regulated by microglia in response to axotomy and is probably secreted by these cells into the extracellular space, suggesting that this proteinase inhibitor has (a) significant function(s) in the processes of neuronal degeneration, regeneration, and/or repair subsequent to axotomy.

GFA-protein gene expression on the astroglia in cow and rat brains.

The distribution of messenger RNA for glial fibrillary acidic protein (GFA-protein) of cow and rat brains was studied by an in situ hybridization technique using a tritium-labeled cDNA probe and autoradiography. The results were compared to the findings of GFA-protein immunohistochemistry. The signals of GFA-protein mRNA were detected on the perikaryal cytoplasm of astroglia observed in the following areas: the subpial area and the white matter of the cerebrum and cerebellum of the cow and the rat; gray matter of cow spinal cord; the thalamus, pontine reticular formation and white matter of the rat brainstem. All of these areas contain astroglia which are strongly positive for GFA-protein immunohistochemistry. On the other hand, we could detect no GFA-protein mRNA-positive glial cells in the areas where astroglia are negative for this immunostaining. These results indicate that the regional differences in the amount of GFA-protein in astroglia depend primarily on the degree of expression of their GFA-protein gene.

Immunofluorescence studies of the monocytes in the injured rat brain

SummaryRat brain was obtained on the 4th day after its damage by a stab wound. The injured and the normal control brain tissues were stained by the immunofluorescence technique using anti-granulomonocytic rabbit serum. After the fluorescence observation the same tissues were further stained by hematoxylin and eosin (HE) and studied comparatively by light microscopy.The following results were obtained: (1) The normal adult rat brain lacks the cells which react with the antiserum, thus the resting microglia occurring in the normal adult brain are antigenically different from the cells of the monocyte-macrophage system. (2) In the injured brain tissues monocytes extravasate, enter brain parenchyma, and take ameboid forms or become macrophages. (3) Among the reactive cells in the injured brain, all of the brain macrophages and most of the ameboid cells were reactive with the antiserum thereby indicating their monocytic origin.

Immunohistochemical studies of blood monocytes infiltrating into the neonatal rat brain

SummaryBrains of normal rats ranging in age from newborn to adult were observed with immunofluorescence technique using anti-granulomonocyte antiserum.For the first 10 days after birth, many cells with positive fluorescence were found in the white matter, the subependyma, the extra-parenchymal spaces, and the leptomeninx, but very few in the gray matter. They were mononuclear, rich in cytoplasm, and globular or irregular in shape. After about day 10 p.n., the positive cells decreased in number and became slender. However, there was no change in the distribution pattern. After about 3 weeks of age, no positive cells were detected in the brain parenchyma, except for very rare necrobiotic ones.It was suggested that blood monocytes infiltrate into the brain parenchyma of normal neonatal rat, but only for a while in the limited areas (white matter and subependyma). They have the morphology and distribution of the “ameboid microglia” of neonatal brain. These monocytes disappear from the brain finally by the end of month 1 p.n.

Glutamine synthetase immunoreactivity in two types of mouse brain glial cells

The localization and distribution of glutamine synthetase (GS) in the adult mouse brain were studied by immunohistochemistry. GS immunoreactivity was found in two morphologically distinct types of glial cells apart from Bergmann glia, one asteroid and the other ovoid. The light and electron microscopic features of the GS-positive asteroid and ovoid cells were well consistent with those of astrocytes and oligodendrocytes, respectively. The GS-positive asteroid cells were present in the hippocampus, cerebral cortex, neostriatum, and cerebellar granular layer, where many synapse receptors for excitatory amino acids such as glutamate are densely distributed. Weakly GS-positive asteroid cells were also scattered in the white matter. The GS-positive ovoid cells were present throughout the gray matter regions of the brain except for the hippocampus, and they were the predominant type of GS-positive cells in the thalamus and brainstem gray matter where excitatory amino acid receptors are relatively sparse. No GS-positive ovoid cells were found in the white matter. These results suggest that, in the mouse brain, GS is localized in oligodendrocytes of the gray matter and in astrocytes. These two types of GS-positive glial cells may play different roles in the metabolism of glutamate.