Effects of chronic ethanol treatment on glial fibrillary acidic protein expression in adult rat optic nerve: an immunocytochemical study

Glial fibrillary acidic protein (GFAP) is used as a marker of astrocyte response to various central nervous system injuries. In the present study, the effects of chronic ethanol administration on GFAP immunoreactivity were evaluated in astrocytes of the adult optic nerve head. The results demonstrated that ethanol exposure significantly and dramatically increases GFAP immunoreactivity and the number of immunoreactive astrocytes (p<0.001). In addition, GFAP immunoreactive cells in the optic nerve showed extensive hypertrophy (p<0.001).     

The expression of glial fibrillary acidic protein in a rat cerebellar cell line

A rat cerebellar cell line, WC5, derived by transformation with Rous sarcoma virus, which is temperature-sensitive for transformation (ts-RSV), can be induced to express glial fibrillary acidic protein (GFAP). Immunofluorescence, radioimmune assay, and electron microscopy studies show that GFAP is expressed in WC5 cells grown at the nonpermissive temperature (NPT), but not at the permissive temperature (PT) for transformation. GFAP is first detectable about 3 days after incubating cells at the NPT, and reaches an apparent plateau by the seventh or eighth day. The expression of GFAP is reversible; shifting cells from the NPT to the PT causes a dramatic decrease in GFAP after 96 hr. In order to determine if the expression of GFAP is linked to the temperature-sensitive transforming activity of the viral src gene product, phenotype revertants of WC5 were established. By the criteria of morphology and growth in agar, the revertant lines, in contrast to the parent cell line WC5, were shown to exhibit a transformed phenotype at both the NPT and PT. Immunofluorescence studies on several of the revertant cell lines show that they do not express GFAP at either the PT or NPT. These findings suggest that the expression of GFAP in WC5 is linked to the expression of the src gene product. The advantage of using ts-RSV to derive neural cell lines which exhibit differentiated properties is discussed.     

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-micron slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semi-thin plastic sections of rat brain.     

Expression of glial fibrillary acidic protein in the developing human brain]

It was shown that the glial fibrillary acidic protein (GFAP) content in developing (fetal) human brain is sharply increased. The expression of GFAP was observed already on the 7th-8th week after gestation, the GFAP concentration being less than 0.05% in comparison with adult brain. GFAP can be immunohistochemically detected in radial glial cells. At early stages of development the presence of antigenic determinants of 68 kDa and 100 kDa polypeptides interacting with monoclonal antibodies alongside with native GFAP (51 kDa) and its low molecular weight forms was demonstrated. These antigenic determinants cannot be detected at later stages of development and are absent in adult brain. The data obtained testify to changes in the gene expression of intermediate filament proteins at early stages of human brain ontogenesis.     

Effect of chronic ethanol consumption on emotional stress in the white rat

Chronic pain emotional stress (PES), paired action of the white noise and electric skin stimulation and chronic (during 7 months) ethanol consumption in white rats were shown to act in the same direction. Hypertension, decrease of respiratory rate and increase of Hildebrandt index were observed as a result of PES, ethanol consumption, and especially under PES during ethanol consumption. Ethanol consumption by the animals led to their growth retardation and increase of the spleen and heart mass. Accidental thymus involution was noted both under ethanol consumption and PES. Activation of lipid peroxidation and decrease of superoxide dismutase activity (of its mitochondrial form especially) as well as of Na+,K+-ATP-ase activity were observed in brain homogenates of the rats after PES, while the general ATP-ase activity remained unchanged. An increase of triiodothyronine level and the tendency to thyroxine level increase as well as a decrease of superoxide dismutase activity were observed in the blood serum of these animals. A tendency towards lipid peroxidation level decrease and to brain superoxide dismutase activity increase, as well as blood antioxidation activity increase (evaluated by transferrin and coeruloplasmin contents and by serum superoxide dismutase activity) and a decrease of thyroxine level were observed as a result of ethanol consumption. The mechanisms are discussed of the "anti-stress" action of short-term ethanol consumption and of the action of its chronic consumption, additive to PES.     

胶质原纤维酸性蛋白的研究进展

星形胶质细胞（astrocyte,AS)约占正常成人中枢神经系统（central nervous system,CNS)细胞总数的40％,其重要功能日益受到重视,AS可特异性表达胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP).GFAP是AS骨架蛋白特有的成分,可作为AS的特异性标记物,本文主要从分子生物学角度,就GFAP在复杂的细胞活动（如细胞骨架重建,髓鞘维持,细胞粘附和信号转导途径等）中的广泛作用,及GFAP转基因动物研究等做一综述。     

Monoclonal antibodies to glial fibrillary acidic protein in the cytologic diagnosis of brain tumors

Monoclonal antibodies were used to immunocytochemically demonstrate the presence or absence of glial fibrillary acidic protein (GFAP) in smear preparations from human intracranial tumors. The results show that this approach may be of great help in the histogenetic classification of such tumors.     

Effect of metallic cations and pH on reassembly of glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP), which was purified from acetone powder of the bovine spinal cord, was reassembled in 0.1 M imidazole HCl buffer containing metallic cations, Ca²⁺, Mg²⁺, Na⁺ or K⁺ at physiological or more acidic pH. An electron microscopy revealed reassembled glial filaments at pH 6.8 without any cations but amorphous aggregates at pH 6.3 which were readily observed as a white precipitate by the naked eye. Under more alkaline pH (pH 7.4) only rod-shaped short filaments were formed. In the presence of mM concentrations of Ca²⁺ or Mg²⁺, thick bundles of glial filaments, detectable by light microscopy, were formed at acidic pH. At pH 7.4 long reassembled filaments could be formed in the buffer containing divalent cations. Na⁺ (0.1 M) made filament-like structures of GFAP but they are rather random compared to the filaments promoted by the divalent cations. K⁺ made only amorphous aggregation of the short filaments. These findings indicate that the reassembly of GFAP at physiological pH requires essentially divalent cations but not ionic strength.     

Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Expression of myelin basic protein and glial fibrillary acidic protein genes in human glial tumors

Analysis of the expression of genes encoding myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in human glial tumors was carried out for determination of the expression specificity of these genes according to tumor types and their malignancy. Low levels of MBP mRNA in astrocytoma specimens of malignancy grades II-IV and significantly higher levels in perifocal zones adjacent to them have been determined by Northern hybridization. Diffuse astrocytomas and anaplastic astrocytomas are characterized mostly by a low level of MBP gene expression and high level of GFAP gene expression, but distinct subtypes of diffuse and anaplastic astrocytomas with a high level of GFAP gene expression can also be detected that may be the reflection of different oncogenic pathways. Very low levels or even absence of MBP mRNA were revealed in oligodendroglioma and all oligoastrocytomas. Thus, Northern hybridization data are correlated with serial analysis of gene expression (SAGE). Obtained results show that MBP is a nonspecific marker for tumors of oligodendroglial origin, but determination of relative levels of MBP and GFAP mRNAs may be useful for glial tumor recognition. In such a way, these two genes together with YKL-40 and TSC-22, which we found previously, can be included into the gene panel for determination of so-called “gene signatures” of brain tumors. However, strict requirements in relation to a clinical value of these “gene signatures” cannot be formulated without verifying them on a large number of clinical samples of tumors and valid control.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Quantification of glial fibrillary acidic protein in developing sheep brain cortex, cerebellum and stem

1. The glial fibrillary acidic protein (GFAP) content of foetal, young (lamb) and adult sheep brain white (stem and cerebellum) and grey (cortex) matter-enriched regions has been determined by means of an improved ELISA using one layer of anti-human GFAP monoclonal antibody. 2. The order of GFAP concentration in brain regions was as follows: brain stem greater than cerebellum greater than cortex. 3. Postnatal brain development accounts for an increase of GFAP in all the regions. The most important increase in GFAP was observed in the adult brain and was proportionally more significant in the grey matter-enriched cortex.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Purification of the glial fibrillary acidic protein by anion-exchange chromatography

A procedure for the isolation of assembly-competent glial fibrillary acidic (GFA) protein from 2 m urea extracts of bovine spinal cord by anion-exchange chromatography is reported. The tissue was previously extracted with low-ionic-strength buffer. The procedure allowed the separation of nondegraded GFA protein from GFA protein comprising degraded species. As previously reported for neurofilament preparations obtained from porcine spinal cord (N. Geisler and K. Weber, J. Mol. Biol., 151, 565–571 (1981)), the procedure also allowed the simultaneous separation of the three neurofilament polypeptides (200,000; 150,000; and 70,000 daltons) contained in the 2 m urea extract. Brain filament proteins sequentially eluted at increasing salt concentration (25–200 mm NaCl) according to their isoelectric point. Proteins with higher pI eluted first. Tubulin eluted between the 200,000- and 150,000-dalton neurofilament polypeptides.     

Specificity of the glial fibrillary acidic protein for astroglia.

Glial fibrillary acidic protein (GFA) is the main constituent of glial filaments and the close similarity of GFA and neurofilament protein has been recently reported. However, the immunofluorescence staining of peripheral nerve which may be observed with GFA antisera is not due to cross-reaction between GFA and neurofilament protein. Staining of peripheral axons was also observed with control sera obtained by injecting the rabbits with nonimmunogenic GFA preparations isolated with the same procedure. Immune GFA antisera and control sera reacted with sodium dodecyl sulfate extracts of sciatic nerve. However, the precipitin line formed with peripheral nerve crossed the line against GFA protein, thus indicating nonidentity between the two antigens. Buffer extract of sciatic nerves that had been incubated with spinal cord reacted by immunodiffusion with GFA antisera, thus indicating that redistribution of GFA occurred under these conditions.     

Sex and estrous cycle-dependent differences in glial fibrillary acidic protein immunoreactivity in the adult rat hippocampus

Sex differences in the morphology and function of the hippocampus have been reported in several species, but it is unknown whether a sexual dimorphism exists in glial fibrillary acidic protein (GFAP) expression in the rat hippocampus. We analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. We found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, but in dentate gyrus, males presented the lowest GFAP content. Interestingly, differences in astrocyte morphology were also found. Rounded cells with numerous and short processes were mainly observed in the hippocampus during proestrus whereas cells with stellate shape with few and long processes were present in the hippocampus of males and diestrus females. The marked sex and estrous cycle-dependent differences in GFAP immunoreactivity density and in astrocyte number and morphology found in the rat hippocampus, suggest the involvement of sex steroid hormones in the sexually dimorphic functions of the hippocampus, and in the change in its activity during the estrous cycle.     

Use of monoclonal antibodies to glial fibrillary acidic protein in the cytologic diagnosis of brain tumors

Monoclonal antibodies were used to immunocytochemically demonstrate glial fibrillary acidic protein (GFAP) in 174 smear preparations of brain tumor tissue in order to investigate the presence and distribution of GFAP in a variety of intracranial tumors and to evaluate the value of this technique in the cytodiagnosis of brain tumors. GFAP-positive cells were found in the astrocytic tumors and in some of the oligodendrogliomas, ependymomas and medulloblastomas. In contrast, schwannomas, meningiomas, a primary lymphoma, a hemangiopericytoma pituitary adenomas, germinomas and metastatic tumors were negative for GFAP. The cytodiagnostic accuracy of the 174 brain tumors was raised from 90.8% to 97.1% when GFAP-immunoperoxidase staining was employed to aid the routine cytologic diagnosis. These findings indicate that immunoperoxidase staining for GFAP can be successfully applied to cytologic specimens and is a useful adjunct to routine cytologic diagnosis.