Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.
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We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.Keywords:
Immunogold labelling
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Neuroglia
Immunofluorescence
Abstract Previous reports of increases in glial cell number and expression of glial fibrillary acidic protein (GFAP) in stimulated brain regions or epileptic tissue have implicated a role for increases in extracellular potassium concentration ([K + ] o ) in glial reactions. We examined the effects of altered [K + ] o on DNA and protein syntheses and GFAP expression of cultured glial cells isolated from the posthatch chick brain stem. [K + ] o was varied by adding both KCl and NaCl to K + , NaCl‐free medium to achieve final [K + ] of 1–50 mM. DNA and protein syntheses were measured by incorporation of 3 H‐thymidine and 3 H‐leucine, respectively, into acid‐insoluble material. GFAP expression was measured by a dot‐immunoblotting assay. DNA synthesis in glial cells cultured in high (5–50 mM) K + o was 45–60% less than that of cells cultured in low (1–3 mM) K + o . Protein synthesis per cell was increased 34–44% in cells cultured in high K + as compared to those cultured in low K + . GFAP expression was inversely related to [K + ] o over the 1–10 mM range. Compared to the baseline of 3 mM K + o , GFAP per cell was increased 65% at 1 mM and decreased 45% at 10 mM. These data suggest that increases in glial cell number and GEAP immunoreactivity found in sites of increased neuronal activity and in pathological tissues may not be caused solely by persistent increases in [K + ] o . Instead, these results suggest that neuronal activity, through the release of K + , may have an inhibitory influence on glial proliferation and GFAP expression. In light of work by others implying a relationship between GFAP immunoreactivity and rigidity of astroglial processes together with the data presented here, we suggest that the elevated [K + ] o accompanying neuronal activity, by inhibiting GFAP expression, may facilitate the morphological plasticity of glial cells. Conversely, conditions of low [K + ] o may contribute to rigidity of astrocytic processes.
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We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.
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Previous studies of human hepatic encephalopathy (HE) have shown decreased levels of glial fibrillary acidic protein (GFAP) in Alzheimer type II astrocytes. In view of the important role of ammonia in the pathogenesis of HE, we carried out immunocytochemical and enzyme-linked immunosorbent assay (ELISA) studies on the effect of ammonium chloride (10 mM) on GFAP content in primary astrocyte cultures. There was a 39% loss of GFAP after a four day treatment. There was no fall in total cell protein. Potential mechanisms for this apparent selective loss of GFAP are discussed.
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Objective To establish a culture method of rat cerebral cortical astrocyte for further research on astrocyte in vitro.Methods Cerebral cortex of postnatal 5 days of SD rats were used basing on McCarthy method.Unicell suspension were prepared and inoculated in culture flask.Changed the culture flask after 1 hour and substratum was replaced after 3 days.Passage was done when cells syncretized to monolayer.Glial fibrillary acidic protein of the specifical sign of astrocyte was monitored by indirect immunofluorescence after three times passage.The masccline cells of glial fibrillary acidic protein were affirmed to be astrocyte.Results All cerebral cortical astrocytes cells which were cultured in vitro underwent adherence,fibroblast removed,depuration and passage three times,the rate of masccline cells of glial fibrillary acidic protein was above 95%.Conclusion Culture of cerebral cortical astrocytes in vitro is successful and there is higher purity.The condition was provided for further research of astrocytes.
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Abstract Immuno‐electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP‐vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP‐vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell. © 1992 Wiley‐Liss, Inc.
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Abstract Neuroepithelial progenitor cells from forebrains of newborn rat pups develop into “mature” astrocytes in an epidermal growth factor‐containing medium free of serum (Von Visger et al: Exp Neurol 128:34, 1994). Eight‐week‐old “mature” astrocyte cultures on poly‐L‐lysine‐coated dishes were exposed to an acidic medium (pH 5.8–6.0) for 2–6 h. Immunoreactivity for glial fibrillary acidic protein (GFAP) dramatically and rapidly increased; this immediate increase was not affected by pretreatment with cycloheximide. In further experiments we found that the increase in GFAP was undiminished for 24–48 h after the acid‐treated astrocytes were returned to normal growth medium. The Ca 2+ channel antagonists nifedipine and diltiazem attenuated the increase in GFAP immunoreactivity. These results suggest that extracellular acidosis may produce a rapid increase in GFAP immunoreactivity in astrocytes independent of de novo protein synthesis, possibly by increasing intracellular levels of free Ca 2+ ions. Copy 1995 Wiley‐Liss, Inc.
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