50 years of research on the phenomena and epigenetic mechanism of neurogenesis.

In 1960s, histological study on developing CNS led us to a novel finding that the periventricular layer ("Matrix" of W. His) of fetal neocortex is composed solely of the matrix cells. Application of 3H-thymidine autoradiography revealed "elevator movement" of the matrix cells. Following the stage of pure matrix cell proliferation (Stage I), stage of neuron production (Stage II) ensues, and when Stage II is over, stage of gliogenesis (Stage III) follows immediately; first, glioblasts, then astrocytes, oligodendrocytes and microglia differentiate, in sequence.As for the mechanism of the switching, recent progress in molecular research on dynamism of the chromatin and transcription-factors revealed the irreversible epigenetic changes controlling the switch. In Stage I of cytogenesis, axial ectodermal cells escape from irreversible differentiation into epidermis, and change into matrix cells composing the neural plate. In Stage II, some matrix cells, in which proneural genes are activated, exit cell cycle to become neurons. When Stage II ends, the neural-repressor REST/NRSBF is up regulated and occupies RE-1 silencer region to irreversibly inactivate neuron-specific genes including the type II Na+ channel, thereby matrix cells can now only produce non-excitable cells, i.e., glial cells. This is the Stage III of cytogenesis of the CNS.

IFN production ability and healthy ageing: mixed model analysis of a 24 year longitudinal study in Japan.

OBJECTIVE: To track changes in interferon (IFN) production in healthy individuals to shed light on the effect these changes have on the course of healthy ageing. DESIGN: Study is based on data that were collected over 24 years from a cohort of individuals whose IFN-α production was quantified as a part of their annual routine health check-up. SETTING: All individuals in this study underwent regular health check-ups at Louis Pasteur Center for Medical Research. PARTICIPANTS: 295 healthy individuals (159 males and 136 females) without a history of cancer, autoimmune diseases and hepatitis C virus (HCV) whose IFN-α production was quantified more than five times within 24 years were selected. Finally, 29 males and 4 females whose IFN-α production was quantified more than 25 times were selected and their data were analysed using a mixed model. MAIN OUTCOME MEASURES: HVJ stimulated IFN-α  production was quantified. Healthy individual's periodical log transformed IFN-α values (y) were plotted versus age (x) and fitted to linear (y=mx+n) and quadratic formula (y=ax(2)+bx+c) expressions to reveal changes in the IFN-α  production in these healthy individuals. RESULTS: The linear expression showed that log (IFN-α) had a slight tendency to decline (3% over 10 years). However, the quadratic formula analysis showed the quadratic expression to be more positive than negative (a concave U-shaped pattern) which means that individuals' once declining IFN production recovered as they aged. CONCLUSIONS: Although we observed a marginal decline in IFN-α  production, we also observed that IFN production recovered even in individuals in their mid50s to early 60s. These results combined with our previous cross-sectional studies of patients with various diseases suggest that in healthy individuals, the impairment of IFN production is triggered more by the onset of disease (notwithstanding the cause) rather than by ageing.

Use of virtual slide system for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan.

We started to use virtual slide (VS) and virtual microscopy (VM) systems for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan. In the system we used a digital slide scanner, VASSALO by CLARO Inc., and a broadband optic fibre provided by NTT West Japan Inc. with the best effort capacity of 100 Mbps. The client is the pathology laboratory of Yamashiro Public Hospital, one of the local centre hospitals located in the south of Kyoto Prefecture, where a full-time pathologist is not present. The client is connected by VPN to the telepathology centre of our institute located in central Kyoto. As a result of the recent 15 test cases of VS telepathology diagnosis, including cases judging negative or positive surgical margins, we could estimate the usefulness of VS in intra-operative remote diagnosis. The time required for the frozen section VS file making was found to be around 10 min when we use x10 objective and if the maximal dimension of the frozen sample is less than 20 mm. Good correct focus of VS images was attained in all cases and all the fields of each tissue specimen. Up to now the capacity of best effort B-band appears to be sufficient to attain diagnosis on time in intra-operation. Telepathology diagnosis was achieved within 5 minutes in most cases using VS viewer provided by CLARO Inc. The VS telepathology system was found to be superior to the conventional still image telepathology system using a robotic microscope since in the former we can observe much greater image information than in the latter in a certain limited time of intra-operation and in the much more efficient ways. In the near future VS telepathology will replace conventional still image telepathology with a robotic microscope even in quick frozen intra-operative diagnosis.

Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia.

The expression of astrocyte marker proteins (S100beta and GFAP) during infarction and glial scar formation after transient middle cerebral artery (MCA) occlusion was examined using double immunostaining. S100beta immunoreactivity markedly decreased in the core of the injured area when observed immediately after reperfusion and did not increase again. In the periphery, however, S100beta expression increased, showing that S100beta synthesis was up-regulated. S100beta+/iNOS+ astrocytes in the periphery were observed from day 1, when small infarct areas were detectable, up to day 5, when infarct expansion had almost ended. TUNEL+ cells in the periphery were present from days 1 to 5. S100beta+/TUNEL+ cells were observed centrally and around the periphery of the injured area, predicting that cell death contributes to the increase of S100beta concentration in the injured area. Our results suggest that (1) higher concentration of S100beta in the extracellular space due to S100beta leakage from damaged astrocytes leads to up-regulation of S100beta synthesis and induction of inducible nitric oxide synthase (iNOS) synthesis in astrocytes, contributing to infarct expansion that results in DNA damage or cell death via NO and ROS production, and (2) GFAP, but not S100beta, is a main contributor to glial scar formation. On day 1 postreperfusion, the microdiascopic images of the injured areas from the unstained thick sections or the areas detected by S100beta immunoreactivity were larger than those of the infarct areas detected by hematoxylin--eosin (HE)-staining. The difference between these sizes might be useful to predict infarct expansion.

Distribution of MAP1A, MAP1B, and MAP2A&B during layer formation in the optic tectum of developing chick embryos.

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M+ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B+ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A-/MAP2A&B- on day 11 but became MAP1A+/MAP2A&B+ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.

The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system.

In the early 1960s I applied 3H-thymidine autoradiography to the study of the cells constituting the neural tube, and found that its wall was composed solely of one kind of single-layered epithelial cell, which perform an elevator movement between the mitotic and DNA-synthetic zones in the wall in accord with the cell cycle. They were identified as multipotent stem cells of the central nervous sytem (CNS) to which I gave the name of matrix cells. (3)H-thymidine autoradiography also revealed the chronology of development of these matrix cells: At first they proliferate only to expand the population (stage I), then switch to differentiate specific neuroblasts in given sequences (stage II), and finally change themselves into ependymoglioblasts, common progenitors of ependymal cells and neuroglia (stage III). Based on these findings, I proposed a monophyletic view of cytogenesis of the central nervous sytem. This matrix cell theory claiming the existence of multipotent stem cells has long been the target of severe criticism and not been accepted among neuro-embryologists for a long time. Recent findings by experimental and clinical neuroscientists on the importance of stem cells have renewed interest in the nature and biology of the multipotent neural stem cells. The present paper describes how the concept of the matrix cell (multipotent neural stem cells in vivo) emerged and what has come out from this view over the last 45 years, and how the basic concept of the matrix cell theory has recently been reconfirmed after a long period of controversy and neglect.

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer.

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls ( P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.

Growth of Notochord and Formation of Cranial and Mesencephalic Flexures in Chicken Embryo: (flexure formation/proliferative activity/notochord/neural tube/chicken embryo).

The proliferative activity of notochordal cells, neuroepithelial cells and endodermal cells in the chicken embryo is examined during flexure formation along its long axis by using bromodeoxyuridine (BrdU)-immunohistochemistry. White Leghorn chicken embryos, Hamburger and Hamilton stages from 9 to 15 are cumulatively labled with BrdU for 120 min and fixed with Carnoy's fluid. Serial transverse and sagittal sections are immunostained with a monoclonal anti-BrdU antibody. The distribution of labeled and unlabeled cells is determined on various regions of the embryos. Before and during the external flexure development from state 10 to 14, the labeled cells are virtually absent at two regions; The cranial region including the floor of the prosencephalon and pharynx close to the pre-chordal plate and the mesencephalic region comprising the mesencephalic notochord and mid-ventral side of the mesencephalon. At stages 10 and 11, the cranial and mesencephalic flexure become apparent at the respective unlabeled regions. It is concluded that the regional differences in proliferative activity between the notochord, neural tube and endoderm play a causal role in the flexure formation of early morphogenesis in the embryo.

The Microcomputer-based Color Image Analyzer and Its Application to Histochemistry 1.

For quantification of histochemical reactions, color image analysis, as well as cytophotometry, is important. However, the extremely large amount of information, usually more than one million bits per image, involved in one microscopic picture of a colored histochemical specimen, when digitalized, has inhibited attempts to develop a more convenient system of color image analysis. Recent advances in microcomputer technology have now made it possible to build, at a reasonable cost, an intelligent color terminal equipped with array-processing hardware, a sufficiently large video random-access memory to represent 4096 colors in each pixel, and packages of machine language subroutines called "macro-commands" in the form of read-only memory that can process color images within a few seconds. This intelligent terminal can easily be controlled by a small personal computer via an IEEE-488 interface bus. The digitalized color image can be stored in and retrieved from a floppy disk. As the macro-commands in this system are controlled by BASIC program through CALLS with parameters, it is highly flexible and adapts readily to the varied needs of histochemists. Reported on herein will be the instrumentation of the microcomputer-based color image analyzer and a few examples of applications of this system to histochemistry: color intensification of cytochemical reactions, quantification of enzyme (e.g., horseradish peroxidase) reaction products, periodicacid-Schiff staining, collagen fibers visualized by Azan-Mallory stain, etc. Possible future applications of this color image analyzer will also be discussed.

THE TEMPORAL SCHEDULE OF DNA SYNTHESIS AND CELL DIFFERENTIATION.

The temporal schedule of DNA synthesis in cells of developing and adult mice is analysed by means of Feulgen cytofluorometry combined with tritiated thymidine autoradiography. The results obtained with cells taken from liver, esophageal epithelium and mucosae of gastrointestinal tracts seemed to conform to the hypothesis that a cell at a particular state of cytodifferentiation possesses specifically inactivated sets of late replicating genes showing a specific pattern of the temporal schedule of DNA synthesis.

DNA CONTENTS IN PURKINJE CELLS AND INNER GRANULE NEURONS IN THE DEVELOPING RAT CEREBELLUM.

Cytophotometric studies revealed that the content of Feulgen DNA in Purkinje cells in the developing rat cerebellum remains at the diploid level throughout the postnatal life. No evidence was found to suggest resumption of DNA synthesis in the neurons. This finding is in accord with autoradiographic observations that neuroblasts once differentiated from matrix cells do not synthesize DNA nor divide again.