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Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two-step culture in 10 and 70% serum-supplemented media for 5 days. Such multipotent MG, called promicroglioblasts (ProMGBs), formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared to be the source of NG2+ ProMGB aggregates. The aggregates were MG marker+/NG2+/GFAP+/NCAM+/S-100beta- and had alkaline phosphatase activity. The marked accumulation of NG2+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2+ MG in the wounds separated with trypsin-EDTA formed NG2+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing beta-tubulin III+ cells in vitro easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain., (Copyright 2006 Wiley-Liss, Inc.)

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

We report here the identification and characterization of a novel nucleolar RNA binding protein, referred to as Nop25 as based on its predicted molecular size and subcellular location. Nop25 homologues were widely discovered in diverse vertebrate species as hypothetical proteins, but not found in yeasts, plants and prokaryotic organisms. Nop25 was ubiquitously expressed in adult mouse organs and constitutively during mouse embryogenesis. Indirect immunofluorescence analysis with an anti-Nop25 antibody, as well as an experiment using a GFP-fused protein, demonstrated that Nop25 was localized in the nucleolus. Treatment of the cells with a low doses of actinomycin D caused Nop25 to translocate to the periphery of the nucleolus, suggesting that nucleolar localization of Nop25 is associated with rRNA transcription. Treatment of COS7 cells with RNase A resulted in a complete dissociation of Nop25 from the nucleolus, while in vitro binding assay demonstrated that Nop25 could bind directly to single-stranded nucleic acids. Further characterization of associated RNA molecules with Nop25 using immunoprecipitation experiment showed that Nop25 might bind to 28S rRNA. Studies on this novel nucleolar RNA binding protein may provide new information on the intricate nucleolar machinery as related to the transcription and processing of rRNA molecules and/or the subsequent assembly and maturation of ribosomes.

Our purpose of the research is a development of the detecting method of EEG under exercising. Usually, measuring EEG is done in the quiet state. In case of the measuring EEG under exercising, a movement of the body causes vibration of electrodes and artifact for the EEG. Therefore, generally detection of the EEG under exercising is said to be difficult. So, we developed the measuring method of EEG under exercising by using algorithm that we designed. Five normal subjects were tested with our method, and EEG without artifact was able to be measured in all cases.

Although it was reported that protein 4.1 G, a cytoskeletal protein characterized by its general expression in the body, interacts with some signal transduction molecules in the central nervous system (CNS), its distribution and significance in vivo remained to be elucidated. In the present study, we have identified 4.1 G-positive cells in the rodent CNS, and demonstrated its immunolocalization in the developing mouse CNS. In the rodent CNS, 4.1 G was colocalized with markers for microglia, such as CD45, OX-42 and ionized calcium-binding adapter molecule 1 (Iba1), but not with markers for neuronal or other glial cells. Additionally, colocalization of 4.1 G and A1 adenosine receptor was observed in the mouse cerebrum. In a mixed glial culture, most OX-42-positive microglia were positive for 4.1 G, and 4.1 G isoforms of the same molecular weight as in the rat brain were expressed in cultured microglia, where 4.1 G mRNA was detected by RT-PCR. In the developing mouse cerebral cortex, 4.1 G was detected in immature microglia, which were positive for Iba1. These results indicate that 4.1 G in the CNS is mainly distributed in microglia in vivo. Considering the interactions between 4.1 G and the signal transduction molecules, putative roles have been proposed for 4.1 G in microglial functions in the CNS.

There is growing evidence that chemokines play important roles in the immune surveillance of central nervous system (CNS). In the CNS, microglia are primary immune effector cells and secrete various chemokines in response to their microenvironment. Using the RT-PCR procedure and indirect immunofluorescence analysis, we found that CCL6 (known as C10/MRP-1 in mouse) was expressed in rat primary microglia without any stimulation, but not in primary astrocytes, although both cell types expressed CCR1 mRNA, which is a receptor for CCL6. Furthermore, immunohistochemical analysis demonstrated that microglia produced CCL6 protein in a normal brain, suggesting that microglia may be the primary source of CCL6 in a normal brain. Recombinant rat CCL6 mediated the migration of microglia and astrocytes in vitro. The CCL6-mediated cell migration was blocked by treating the cells with LY294002, a PI3-kinase inhibitor and Western blot analysis showed that the phosphorylation of Akt could be induced by treating microglia with a recombinant CCL6, suggesting that CCL6 functions by activating the PI3-kinase/Akt pathway. A proinflammatory cytokine, interferon-gamma enhanced the expression of both CCL6 mRNA and protein in microglia, while other proinflammatory cytokines, interleukin-6 and tumor necrosis factor-alpha and an anti-inflammatory cytokine, transforming growth factor-beta exerted no effect on CCL6 expression in microglia. These findings suggest that CCL6 may be a mediator released by microglia for cell-cell communication under physiological as well as pathological conditions of CNS.

To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.

Our purpose of this research is a development of the method that detects EEG of an athlete under exercising. If EEG under exercising can be measured, we can assess the mental condition of the athlete. Usually, EEG is measured in the shield room, and a subject is required rest in bed while measurement. And it is said that measuring EEG under exercising is difficult. In this paper, we will discus about our new measuring method that can detect EEG under exercising by using independent component analysis. Five normal subjects were tested with our method, and EEG without artifact was able to measured. So, we think our new method will be useful for the research of mental condition of the athlete.

This study examined cerebral deoxygenation during intermittent supramaximal exercise in six healthy male subjects (age: 27.2 +/- 0.6 years (mean +/- S.E.). The subjects performed seven times exercise at an intensity corresponding to 150% of maximal oxygen uptake (VO2max) on cycle ergometer (30 s exercise/15 s rest). Cerebral oxygenation was measured by near-infrared spectroscopy (NIRS). The peak blood lactate concentration after exercise was 15.3 +/- 0.2 mmol/l. Cerebral oxygenation increased in first repetition compared with at rest (+ 5.7 +/- 0.6 microM; P < 0.05), but then decreased with time. Thus, in the last repetition cerebral oxygenation was - 8.5 +/- 0.4 microM (P < 0.05). There was no significant change in arterial oxygen saturation (99.6 +/- at rest, 98.4 +/- 0.2 at the final set of intermittent exercise), and there was no correlated change in end-tidal CO2 concentration with cerebral oxygenation (P > 0.05). These findings suggest that the fatigue resulting from dynamic severe exercise related to a decrease in the cerebral oxygenation level., (Copyright 2003 Elsevier B.V.)

The purpose of this study was to determine whether the onset kinetics of muscle oxygenation in localized working muscle (mOxy) was affected by differences in exercise intensity. Five healthy male subjects exercised for 6 min at 125 W, 150 W, and 175 W, and 1 min at 300 W on a cycle ergometer. mOxy was estimated by near-infrared spectroscopy (NIRS) with a continuous wave photometer. The NIRS probe was positioned on the vastus lateralis muscle of the right leg. The relative change in mOxy was calculated from the relative change of the oxygenated hemoglobin (OxyHb) and deoxygenated hemoglobin (DeoxyHb) concentration from their resting values ([mOxy]=Delta[OxyHb]-Delta[DeoxyHb]). Assuming an exponential time course with time delay, the time constants of the mOxy were 5.7 (SD 2.2) s at 125 W, 5.6 (SD 1.9) s at 150 W, 6.0 (SD 2.2) s at 175 W, and 5.6 (SD 2.1) s at 300 W. The time delays of the mOxy were 6.7 (SD 4.2) s at 125 W, 8.6 (SD 1.6) s at 150 W, 6.4 (SD 3.0) s at 175 W, and 5.4 (SD 2.9) s at 300 W. The mean response times of the mOxy were 12.5 (SD 2.7) s at 125 W, 14.2 (SD 2.4) s at 150 W, 12.4 (SD 4.4) s at 175 W, and 11.0 (SD 3.1) s at 300 W. These results indicate that the kinetics of mOxy were not affected by differences in exercise intensity.

Apolipoprotein E (ApoE), one of the genetic risk factors for Alzheimer's disease, is considered to have a critical role in transporting lipids in the brain. In the present study, we investigated ApoE release in primary rat microglial cultures. Microglial cells released ApoE in response to L-Ser in culture medium, and ApoE-immunoreactivity was detected in granules in the cell periphery and in perinuclear structures. Immunocytochemical studies, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR) results all supported the notion that microglial cells are the potential source of ApoE in the brain. L-Ser enhanced ApoE release in a concentration-dependent manner without upregulating ApoE mRNA expression. Astrocytes presumably enhanced production and release of ApoE by microglial cells through secretion of L-Ser. As revealed by gel chromatography, ApoE was secreted as a component of lipoproteins, and L-Ser enhanced release of cholesterol and triglycerides together with ApoE. Activation of microglial cells by lipopolysaccharides and serum resulted in an overall decrease of the ApoE release. These findings suggest that microglial cells are a significant source of lipoproteins containing ApoE in the brain under physiological conditions, and that L-Ser is an important mediator of the neuron-astrocyte-microglia network in the brain.

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 days, followed by culture in the presence of 70% serum for 2 days. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labeled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes., (Copyright 2003 Wiley-Liss, Inc.)

The purpose of this study was to investigate the relationship between muscle oxygenation level at exhaustion and maximal oxygen uptake (VO2max) in an incremental cycling exercise. Nine male subjects took part in an incremental exhaustive cycling exercise, and then cuff occlusion was performed. Changes in oxy-(deltaHbO2) and deoxy-(deltaHb) hemoglobin concentrations in the vastus lateralis muscle were measured with a near infrared spectroscopy (NIRS). Muscle oxygenation during incremental exercise was expressed as a percentage (%Moxy) of the maximal range observed during an arterial occlusion as the lower reference point. A systematic decrease was observed in %Moxy with increasing intensity. A significant relationship was observed between %Moxy at exhaustion and VO2max (p < 0.01). We concluded that the one of the limiting factor of VO2max is the muscle oxygen diffusion capacity, and %Moxy during exercise could be one of the indexes of muscle oxygen diffusion capacity.

Because of variations in the morphology and function of microglial cells, it has often been claimed that microglial cells should be classified into two or more subtypes. However, such subtypes have not fully been characterized. In the present study, we isolated microglial cells expressing microglia-markers CD11b and CD68 from rat mixed glial cultures on the fifth and on the thirteenth days in vitro (DIV 5 and 13) and demonstrate that these two populations of microglial cells have distinct morphology and function. Microglial cells isolated on DIV 5, which we have termed immature cells, are characterized by the presence of large somata, large peroxidase- and alkaline phosphatase-positive granules, and high proliferative activity and suppressed responsiveness to lipopolysaccharide (LPS). In contrast, the microglial cells isolated on DIV 13, which we have termed mature cells, are devoid of granules, appear to be in a state of cell cycle arrest, and respond to LPS by the induction of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha, and interleukin-6. Isolated immature cells maintained in pure culture failed to express iNOS in response to LPS. However, if these cells were cultured on astrocyte-derived extracellular matrix (AsECM) or pure laminin, the cells exhibited an induction of iNOS in response to LPS. AsECM and laminin were also able to induce a state of cell cycle arrest in cultured isolated immature cells. Thus, classification into two types of microglial cells is possible, but both types are in the same cell lineage, because the immature cells can differentiate into mature microglial cells in the presence of laminin or AsECM. Therefore, "microglioblasts" may be the appropriate term for the immature cells., (Copyright 2003 Wiley-Liss, Inc.)

Aquaporin-4 (AQP4) is located on astrocyte endfeet that face blood vessels in the brain and in the pia. It is thought to play a crucial role in the development of brain edema. To confirm the notion that sex steroids and dexamethasone influence brain edema through AQP4 regulation, we investigated the effects of 17beta-estradiol, testosterone, and dexamethasone on the expression of AQP4 in cultured astrocytes. Testosterone significantly up-regulated AQP4 at the level of both protein and mRNA. At a concentration of 100 nM, testosterone significantly increased AQP4 protein levels and ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Moreover, this effect was attenuated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, which can rapidly decrease AQP4 mRNA expression, indicating that the response was specific. These results indicate that AQP4 can alter the osmotic fragility of astrocytes and that testosterone can influence brain edema through AQP4 regulation, whereas 17beta-estradiol and dexamethasone cannot., (Copyright 2003 Wiley-Liss, Inc.)

Recent research indicates that the novel multimodular adaptor protein EHSH1 plays an important role in a partnership between clathrin-mediated endocytosis and intracellular signaling pathways, including the MAPK pathway, receptor-tyrosine kinase/ras-mediated pathway, and the rho family of the GTPase-dependent pathway. We report the detailed expression pattern of EHSH1 in the rat CNS, using separate cultures of neurons, astrocytes, and microglia, and biochemical and immunohistochemical analyses. Cultured neurons from the cortex express primarily the long isoform EHSH1-l, as well as a small amount of the short isoform EHSH1-s. Cultured astrocytes express EHSH1-s, at a level similar to neurons, and a trace of EHSH1-l. Cultured microglia express only EHSH1-s. Double immunofluorescent staining of cortical sections showed that EHSH1 is expressed predominantly in neurons. These results suggest that EHSH1-l is a primary isoform and that EHSH1-l is highly enriched in neurons in the rat adult CNS. Immunohistochemistry of a series of brain sections revealed widespread distribution of EHSH1 throughout the brain. Particularly intense immunoreactivity was observed in the somatodendritic region of neurons in Layer III of the neocortex, hippocampus, globus pallidus, subthalamic nucleus, and substantia nigra. Interestingly, all pyramidal neurons in Layer III of the neocortex and hippocampus did not necessarily exhibit equal levels of immunostaining. In contrast, little EHSH1 immunoreactivity was detected in septofimbrial nucleus and subfornical organ of the septal region, and solitary tract and external cuneate nuclei of the medulla. Variety in the expression of EHSH1 in neurons of different regions may reflect different conditions in clathrin-mediated endocytosis and the following signal transduction., (Copyright 2002 Wiley-Liss, Inc.)

Microglial cells rapidly become activated in response to even minor damage of neurons, suggestive of the intimate interactions between neurons and microglial cells. Although mediators for microglia-neuron interactions have not been well identified, neurotransmitters are possible candidates transmitting signals from neurons to microglial cells. Among the neurotransmitters, we focused on the effects of norepinephrine and other adrenergic agonists on the functions of rat cultured microglial cells. Reverse transcriptase polymerase chain reaction studies revealed that microglial cells expressed mRNAs encoding alpha1A, alpha2A, beta1 and beta2 receptors. Norepinephrine and a beta2 adrenergic agonist terbutaline elevated intracellular cAMP level of microglial cells. Norepinephrine, an alpha1 agonist phenylephrine, a beta1 agonist dobutamine and terbutaline suppressed the expressions of mRNAs encoding pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor alpha. Release of tumor necrosis factor alpha and nitric oxide was suppressed by norepinephrine, phenylephrine, dobutamine and terbutaline. An alpha2 agonist clonidine and dobutamine upregulated the expression of mRNA encoding catechol-O-methyl transferase, an important enzyme to degrade norepinephrine. Norepinephrine, dobutamine and terbutaline upregulated the expressions of mRNA encoding 3-phospshoglycerate dehydrogenase, an essential enzyme for synthesis of L-serine and glycine, which are amino acids necessary for neuronal survival. Clonidine upregulated the expression of mRNA encoding an anti-apoptotic factor Bcl-xL. These results suggest that norepinephrine participates in the regulation of brain function at least partly by modulating the functions of microglia.

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.

We investigated the effects of inhibitors of cAMP-specific phosphodiesterase type IV (PDE IV) on cultured rat microglial cells. Microglial cells expressed mRNA encoding PDE IV. Rolipram and RO-20-1724, specific inhibitors of PDE IV, elevated the intracellular cAMP level much higher than the other types of PDE inhibitors. cAMP in astrocytes but not in cerebrocortical neurons was similarly increased in response to treatment with PDE IV inhibitors examined. The PDE IV inhibitors, a beta-adrenergic agonist isoproterenol and an adenylyl cyclase stimulant forskolin suppressed the proliferation of microglial cells as revealed by PCNA-immunocytochemical staining. The PDE IV inhibitors suppressed release of TNF alpha and nitric oxide (NO) from lipopolysaccharide-activated microglial cells in pure culture, while they did not affect NO release from microglial cells in neuron-microglia coculture. The PDE IV inhibitors also suppressed superoxide anion production by phorbol ester-treated microglial cells. Isoproterenol and forskolin similarly suppressed the macrophage-like functions of activated microglial cells. However, the PDE IV inhibitors displayed novel effects distinct from those of isoproterenol, forskolin and 8Br-cAMP, regarding expression of mRNAs encoding PDE IV, metallothionein-1 and hemeoxigenase-1. The present data showed that the PDE IV inhibitors can be available to control microglial function and that their effects on glial cells should be taken into account when PDE IV inhibitors are used for treatment of brain diseases, such as multiple sclerosis.