• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.467-477
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• 2001

We examined astrocyte regional heterogeneity in their morphological changes in response to various stimuli. Astrocytes were cultured from six different neonatal rat brain regions including cerebral cortex, hippocampus, cerebellum, mid brain, brain stem and hypothalamus. Astrocyte stellation was induced by serum deprivation and the maximum stellation in different regional astrocytes was achieved after 2 h. After 24 h, in all astrocyte cultures, the level of stellation returned to their original level. Cerebellar or hypothalamic astrocytes were the most or the least sensitive, respectively, to serum deprivation. The order of maximum sensitivity to serum deprivation among different regional astrocytes was: cerebellum＞mid $brain{\ge}hippocampus,\;brain\;stem{\ge}cerebral$ cortex＞hypothalamus. Isoproterenol-induced astrocyte stellation was also examined in different regional astrocytes, and similar order of maximum sensitivity as in serum deprivation was observed. Next a possible developmental effect on astrocyte morphological changes was examined in cerebral cortex and cerebellum astrocytes cultured from postnatal day 1 (P1), P4 and P7 rat brains. A much higher sensitivity of cerebellum astrocytes to serum deprivation as well as isoproterenol treatment was consistently observed in P1, P4 and P7-derived astrocytes compared to cerebral cortex astrocytes. The present study demonstrates different regional astrocytes maintain different levels of morphological plasticity in vitro.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.3
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• pp.285-296
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• 1997

Injury to brain transforms resting astrocytes to their reactive form, the hallmark of which is an increase in glial fibrillary acidic protein (GFAP), the major intermediate filament protein of their cell type. The overall glial response after brain injury is referred to as reactive gliosis. Glial-neuronal interaction is important for neuronal migration, neurite outgrowth and axonal guidance during ontogenic development. Although much attention has been given to glial regulation of neuronal development and regeneration, evidences also suggest a neuronal influence on glial cell differentiation, maturation and function. The aim of the present study was to analyze the effects of glial-hippocampal neuronal co-culture on GFAP expression in the co-cultured astrocytes. The following antibodies were used for double immunostaining chemistry; mouse monoclonal antibodies for confirm neuronal cells, rabbit anti GFAP antibodies for confirm astrocytes. Primary cultured astrocytes showed the typical flat polygonal morphology in culture and expressed strong GFAP and vimentin. Co-cultured hippocampal neurons on astrocytes had phase bright cell body and well branched neurites. About half of co-cultured astrocytes expressed negative or weak GFAP and vimentin. After 2 hour glutamate (0.5 mM) exposure of glial-neuronal co-culture, neuronal cells lost their neurites and most of astrocytes expressed strong CFAE and vimentin. In Western blot analysis, total GFAP and vimentin contents in co-cultured astrocytes were lower than those of primary cultured astrocytes. After glutamate exposure of glial-neuronal co-culture, GFAP and vimentin contents in astrocytes were increased to the level of primary cultured astrocytes. These results suggest that neuronal cell decrease GFAP expression in co-cultured astrocytes and hippocampal neuronal-glial co-culture can be used as a reactive gliosis model in vitro for studying GFAP expression of astrocytes.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.144-153
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• 2021

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Culture-conditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

• Journal of Oriental Neuropsychiatry
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• v.12 no.1
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• pp.137-149
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• 2001

Cytokines are polypeptides which possess various biological properties affecting. host defense function and response to disease. Inflammatory cytokines, tumor necrosis $factor-{\alpha}$(TNF-${\alpha}$), interleukin(IL)-1 and IL-6 induce inflammation, fever, hypotension and pain when injected into animals or human subject. When glial cell cultures were prepared from neonatal mice or rats, astrocytes were reported to produce these inflammatory cytokines to viral infection, lipopolysaccharide(LPS), or cytokines. The purpose of this study was to investigate the regulatory effect of these cytokines secretion from primary cultures of rat astrocytes. Substance P(SP) can stimulate secretion of TNF-${\alpha}$ from astrocytes stimulated with LPS. Sesim-Tang significantly inhibited the TNF-${\alpha}$ secretion by astrocytes stimulated with SP and LPS. IL-1 has been shown to elevate TNF-${\alpha}$ secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore also investigated whether IL-1 mediated inhibition of TNF-${\alpha}$ secretion from primary astrocytes by Sesim-Tang. Treatment of Sesim-Tang to astrocytes stimulated with both LPS and SP decreased IL-1 secretion significantly. The secretion of TNF-${\alpha}$ by LPS and SP in astrocytes was progressively inhibited with increasing amount of IL-1 neutralizing antibody. Furthermore Sesim-Tang inhibited the IL-6 secretion by astrocytes stimulated with SP and LPS. The inhibitory effect of inflammatory cytokines by Sesim-Tang, observed in this study, might reflect an antiinflammatory activity and a reduction of various-type pains, fever etc. in the central nervous system.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.2
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• pp.394-402
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• 2003

This study was designed to investigate the protective effect of Bojungmyunyuk-dan(BJMY-Dan) on the cisplatin-induced cytotoxicity of primary rat astrocytes. BJMY-Dan is an oriental herbal prescription for its ability to recover protective effects against anti-cancer chemotherapies. After astrocytes were treated cisplatin, MTT assay was performed for cell viability test. To explore the mechanism of cytotoxicity, I used the several measures of apoptosis to determine whether this processes was involved in cisplatin-induced cell damage in astrocytes. Also, astrocytes were treated with BJMY-Dan and then, followed by the addition of cisplatin. Cisplatin decreased the viability of astrocytes in a dose and time-dependent manner. BJMY-Dan increased the viability of astrocytes treated cisplatin. Astrocytes treated cisplatin were revealed as apoptosis characterized by nuclear staining and flow cytometry. BJMY-Dan protected astrocytes from cisplatin-induced nuclear fragmentation and chromatin condensation. Also, caspase-3 and caspase-9 proteases were activated in astrocytes by cisplatin. BJMY-Dan inhibited the activation of caspase proteases in cisplatin-treated astrocytes. Cleavage of [poly(ADP-ribose) polymerase](PARP) was occurred at 12hr after treatment of cisplatin in astrocytes. BJMY-Dan recovered the cleavage of PARP in cisplatin-treated astrocytes. Also, BJMY-Dan inhibited the activation of pro-apoptotic factor, Bak by cisplatin. Lastly, astrocytes stained with JC-1 and Rhodamine 123 were photographed by fluorescence microscope to visualize changes of mitochondrial membrane permeability transition(MPT) during treatment with cisplatin for 24hr. BJMY-Dan recovered the change of MPT by cisplatin in astrocytes. According to above results, BJMY-Dan may protect astrocytes from cytotoxicity induced by chemotherapeutic agents, including cisplatin.

• Animal cells and systems
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• v.15 no.4
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• pp.268-273
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• 2011

Transgenic mice expressing green fluorescent protein (GFP) under the control of human glial fibrillary acidic protein promoter (hGFAP) have been utilized for in vivo labeling of astrocytes. Although it has been considered that virtually all astrocytes express GFP in this transgenic mouse, we found that different subsets of GFAP-expressing astrocytes express considerably different levels of GFP in the adult brain. Astrocytes in the spinal cord, the molecular layer of thecerebellum, meninges, white matter, corpus callosum and blood vessels exhibited strong GFP, whereas subsets of astrocytes associated with granule cells in the cerebellum and dentate gyrus did not or only marginally exhibited GFP. We also found that a small subset of GFP-expressing cells in the periglomeruli of the olfactory bulb did not express GFAP immunoreactivity. Collectively, these results suggest that human GFAP promoter-derived GFP expression does not faithfully recapitulate the endogenous GFAP expression in mice, suggesting that upstream regulatory mechanisms controlling GFAP transcription are different in different populations of astrocytes, and may reflect the functional diversity of astrocytes.

• Journal of Oriental Neuropsychiatry
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• v.10 no.1
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• pp.109-119
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• 1999

We investigated whether an aqueous extract of Chong-Myung-Tang inhibits secretion of tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$ from primary cultures of mouse astrocytes. Chong-Myung-Tang dosedependently inhibited the $TNF-{\alpha}$ secretion by astrocytes stimulated with substance P (SP) and lipopolysaccharide (LPS). Interleukin-1 (IL-1) has been shown to elevate $TNF-{\alpha}$ secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore investigated whether IL-1 mediated inhibition of $TNF-{\alpha}$ secretion from astrocytes by Chong-Myung-Tang. Treatment of Chong-Myung-Tang to astrocytes stimulated with both LPS and SP decreased IL-1 secretion. Moreover, incubation of astrocytes with IL-1 antibody abolished the synergistic cooperative effect of LPS and SP. These results suggest that Chong-Myung-Tang may inhibits $TNF-{\alpha}$ secretion by inhibiting IL-1 secretion and that Chong-Myung-Tang has a antiinflammatory activity in the central nervous system.

• Journal of Oriental Neuropsychiatry
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• v.10 no.2
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• pp.59-70
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• 1999

We investigated whether an aqueous extract of Rehmannia glutinosa steamed root (RGAE) inhibits secretion of inflammatory cytolanes from primary cultures of mouse astrocytes. RGAE dose-dependently inhibited the $TNF-{\alpha}$ secretion by astrocytes stimulated with substance P (SP) and lipopolysaccharide (LPS). Interleukin-1 (IL-1) has been shown to elevate $TNF-{\alpha}$ secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore also investigated whether IL-1 mediated inhibition of $TNF-{\alpha}$ secretion from primary astrocytes by RGAE. Treatment of RGAE to astrocytes stimulated with both LPS and SP decreased IL-1 secretion to the level observed with LPS alone. Moreover, incubation of astrocytes with IL-1 antibody abolished the synergistic cooperative effect of LPS and SP. These results suggest that RGAE has an antiinflammatory activity on the central nervous system curing some pathological disease states.

• BMB Reports
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• v.29 no.6
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• pp.530-534
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• 1996

Astrocyte are the major glial cell type in the central nervous system (CNS), and analogous to macrophage, mediates the number of immune responses such as production of cytokines including tumor necrosis factor alpha ($TNF-{\alpha}$) upon activation. $TNF-{\alpha}$ has been implicated in neuroimmunological disorders through killing oligodendrocytes and thus causing demyelination. It has been previously demonstrated that mitogen-activated T cells synthesized a 26 kDa precursor form of $TNF-{\alpha}$ which is bound to the surface of a membrane, and is later secreted as a 17 kDa mature version. In order to examine whether astrocytes would produce the transmembrane form of $TNF-{\alpha}$, astrocytes were stimulated with biological stimuli and the membrane form of $TNF-{\alpha}$ was analyzed by Western blot and FACS analysis. When astrocytes are stimulated with lipopolysaccharide (LPS), $IFN-{\gamma}/LPS$, or $IFN-{\gamma}/IL-1{\beta}$, they were able to express a membrane-anchored $TNF-{\alpha}$ of approximately 26 kDa protein which was immunoreactive to an $anti-TNF-{\alpha}$ antibody, whereas unstimulated astrocytes or astrocytes treated with $IFN-{\gamma}$ or $IL-1{\beta}$ alone was not. Our FACS data were also consistent with the immunoblot analysis. Our result suggests that the membrane form of $TNF-{\alpha}$ expressed by activated astrocytes may cause local damage to oligodendrocytes by direct cell-cell contact and contribute to demyelination observed in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE).

• Journal of Oriental Neuropsychiatry
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• v.10 no.1
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• pp.95-108
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• 1999

We investigated whether an aqueous extract of Polygala tenuifolia root (PTAE) inhibits secretion of inflammatory cytokines from primary cultures of mouse astrocytes. PTAE dose-dependently inhibited the Tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$ secretion by astrocytes stimulated with substance P (SP) and lipopolysaccharide (LPS). Interleukin-1 (IL-1) has been shown to elevate $TNF-{\alpha}$ secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore also investigated whether IL-1 mediated inhibition of $TNF-{\alpha}$ secretion from primary astrocytes by PTAE. Treatment of PTAE to astrocytes stimulated with both LPS and SP decreased IL-1 secretion to the level observed with LPS alone. Moreover, incubation of astrocytes with IL-1 antibody abolished the synergistic cooperative effect of LPS and SP. Reverse transcriptase-polymerase chain reaction analysis demonstrated the significantly reduced level of the $TNF-{\alpha}$ mRNA was expressed in astrocytes treated with PTAE. These results suggest that PTAE has an antiinflammatory activity on the central nervous system curing some pathological disease states.