Gfap, S-100 and vimentin proteins in rat after cerebral post-ischemic reperfusion

In the present study astrocytes reactivity during cerebral post-ischemic reperfusion was evaluated immunocytochemically by using antibodies to vimentin, glial fibrillary acidic protein (GFAP) and S-100 protein. At the 7th day of post-ischemic reperfusion few GFAP-positive cells were observed in the hippocampus and cerebellum, the number of GFAP-positive cells increased slightly after 20 days of reperfusion. This poor GFAP-positivity may be due to the inhibition of GFAP polymerization by S-100; in fact, S-100 immuno-reactivity was already evident from the 7th day. Vimentin immuno-staining was evident both at the 7th and 20th day of reperfusion in microglial cells and in oligodendrocytes, suggesting that these cells are involved in the recovery of neurons following brain injury.     

Granular cell brain tumors of the laboratory rat: an immunohistochemical approach

Summary We have studied paraffin-embedded specimens of 17 rat granular cell brain tumors (GCBT) from four long-term drug safety carcinogenicity studies by peroxidase-antiperoxidase (PAP) immunohistochemistry with either polyvalent or monoclonal antibodies against glial fibrillary acidic protein (GFAP), S-100 protein (S-100), Leu-7 epitopes, vimentin (VIM), keratin, desmin, and myelin basic protein. We have found that 9 of the 17 GCBT contained GFAP-positive, S-100-positive, and VIM-positive astrocytes, while GFAP-positive and VIM-positive granular cells were observed in 5 of these 9 tumors. Our findings indicate that astroglial cells are involved in rat GCBT and suggest that an astrocytic origin should be considered for these neoplasms.     

Glial fibrillary acidic protein (GFAP) in oligodendrogliomas: a reflection of transient GFAP expression by immature oligodendroglia

Fourteen pure oligodendrogliomas were studied by light- and electronmicroscopy and immunohistochemistry to examine glial fibrillary acidic protein (GFAP) positivity in the tumors. To compare the immunohistochemical staining patterns of neoplastic oligodendroglia and immature oligodendroglia, myelination glia in the white matter of eight normal brains from children under 6 months of age were studied. The tumors possessed light microscopic and ultrastructural features characteristic of oligodendrogliomas. Microtubules were found in the cytoplasm of nine tumors on electronmicroscopy. In one, intermediate filaments and microtubules were observed in occasional tumor cells with polygonal crystalline structures in the cytoplasm. Using the peroxidase-antiperoxidase technique, all specimens were stained for GFAP, vimentin, S-100 and neuron-specific enolase (NSE). In nine tumors, variable numbers of cells with an oligodendroglial morphology reacted positively for GFAP. All tumors were positive for S-100 and negative for vimentin and NSE. The myelination glia in the eight normal brains stained positively for GFAP but not for vimentin. Vimentin is expressed by developing, reactive and neoplastic astrocytes. Thus, GFAP positivity combined with vimentin negativity in both neoplastic and immature oligodendroglia suggests that GFAP positivity in oligodendrogliomas may reflect the transient expression of this intermediate filament by immature oligodendroglia.     

Immunohistochemical study of ethylnitrosourea-induced rat gliomas with vimentin and astroprotein (GFAP)

Expression of two different types of intermediate filaments, vimentin filaments and glial filaments, was studied immunohistochemically in experimental rat gliomas. Although vimentin filaments are most commonly seen in mesenchymal cells, recent immunocytochemical study demonstrated that this type of filaments can be recognized also in glial cells during early cell differentiation and in tumor cells of epithelial origin. In the present communication, distribution of vimentin filaments in rat glial tumors was investigated and compared with that of glial filaments by using specific antiserum to each protein subunit, vimentin and astroprotein (GFAP). Ethylnitrosourea (50 mg/kg) was injected subcutaneously into 3 day-old Wistar rats. After four to ten months, brains of animals were removed, fixed in 95% ethanol and embedded in paraffin. Peroxidase-antiperoxidase method was carried out on 6 micron-thick sections. In normal portion of the brain, immunoreaction for vimentin was noted in ependymal cells and in vascular endothelial cells but not in astrocytes. This distribution contrasted with that of astroprotein (GFAP), which distributed in astrocytes but not in normal ependymal cells. These findings confirmed that the two antisera used in the present study do not crossreact to each other. In contrast to the absence of vimentin immunoreaction in normal astrocytes, a number of tumor cells showed positive reaction to the antiserum to vimentin. Mixed glioma with astrocytoma and oligodendroglioma had both astroprotein (GFAP)-positive and negative cells. Well developed cellular processes were noted in astroprotein (GFAP)-positive cells (astrocytoma cells). Weak immunoreaction for vimentin was noted in those cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immuno-electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture.

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.     

Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice

In adult hippocampal neurogenesis, new neurons appear to originate from a cell with astrocytic properties expressing glial fibrillary acidic protein (GFAP). Also, new astrocytes are generated in the adult dentate gyrus. Whereas the putative astrocyte-like progenitor cells are consistently S-100beta-negative, many new astrocytes are S-100beta-positive. Thus, it is unclear whether the GFAP-positive progenitor cells are astrocytes in a general sense or rather neural progenitor cells with certain astrocytic characteristics. We therefore investigated the development of GFAP-expressing cells in the context of adult hippocampal neurogenesis. Proliferating cells could be either GFAP-positive or doublecortin-positive (DCX), but never both, indicating two independent populations of dividing cells in the glial and neuronal lineages. Two distinct populations of cells with astroglial properties were detected-one expressing GFAP, the other co-expressing GFAP and S-100beta. We never found S-100beta-cells to be in S-phase. No overlap between neuronal and glial markers was seen at any time point. Thus, astrogenesis occurred in parallel and to some degree independent of adult neurogenesis. The uninterrupted GFAP expression in this lineage, and neuronal markers in the other lineage, argue against a late common precursor for neurogenesis and gliogenesis in the adult hippocampus. Very few newly generated microglia and no new oligodendrocytes were detected. Environmental enrichment and voluntary wheel running-two experimental paradigms with robust stimulatory effects on adult hippocampal neurogenesis-affected hippocampal astrogenesis differentially: Running, but not enrichment, strongly induced net astrogenesis (GFAP/S-100beta), but also GFAP-positive S-100beta-negative cells, which thus appear to be a transiently amplifiable intermediate population within the glial lineage.     

Purification of astrocytes from adult human optic nerve heads by immunopanning

Most in vitro studies in the CNS require pure cultures of astrocytes. Astrocytes from the human optic nerve head (ONH, type 1B) represent a specialized population of astrocytes. Primary cells grown from human optic nerve head explants were cultured for 3-4 weeks. To select astrocytes by immunopanning, cell suspensions were placed on a P100 panning dish coated with C5 anti-neuroepithelial antibody and allowed to attach for 30 min. Nonadherent cells were plated on a second dish coated with anti-Thy1.1 antibody to deplete microglia and meningeal cells. Finally, remnant nonadherent cells were plated on a noncoated dish. Purified cells were immunostained with astrocyte markers: GFAP, vimentin, Pax2, A2B5, nestin and NCAM. Other cell types were characterized by HLA-DR for microglia and smooth muscle actin for vascular smooth muscle. The proportion of GFAP+ astrocytes in the cultures was determined by flow cytometry. About 95% of the cells that adhered to the C5 dish were GFAP+ astrocytes. GFAP+ astrocytes expressed vimentin, Pax2, nestin and NCAM, but not A2B5. From the Thy1.1 dish, 60-75% cells were GFAP+ astrocytes and the remainder cells were GFAP- cells. Using cloning rings, we eliminated fibroblast-like cells, smooth muscle and meningeal cells from astrocyte cultures. Smooth muscle cells and fibroblasts grew on the noncoated dish. In conclusion, immunopanning is an efficient method to get high yields of viable type 1B astrocytes from adult human ONH. The current described culture system may provide a valuable tool in studying human optic nerve head biology and disease.     

Regulation of GFAP expression in glial-like cells of the rat pituitary intermediate lobe by lactation,salt-loading,and adrenalectomy

Glial-like cells in rat pituitary intermediate lobe were localized and characterized by immunohistochemistry with antisera against glial fibrillary acidic protein (GFAP) and S-100. Individual GFAP immunoreactive (IR) cells possessed several processes that often branched into secondary and tertiary processes, terminating with end-feet. The GFAP-immunopositive cell population was distributed in specific rostrocaudal and dorsoventral patterns. The distribution and numbers of cells differed between male and female rats. Examination of altered physiological states, e.g., adrenalectomy, lactation, and salt-loading, revealed state-specific changes in the appearance and distribution of GFAP-IR cells. Adrenalectomy and lactation increased GFAP-IR glial-like cell numbers, whereas salt-loading decreased their numbers and the typical pattern of distribution. By contrast, S-100–expressing cells were evenly distributed in male and female rats, and its expression was not affected by the experimental conditions. Double-label immunocytochemistry indicated that GFAP-IR cells are a subpopulation of S-100-IR cells. These results suggest that cells normally expressing only S-100 may be induced to express GFAP under altered physiologic conditions. Copy 1995 Wiley-Liss, Inc.     

Expression of vimentin and glial fibrillary acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines

Summary The expression of glial fibrillary acidic protein (GFAP) and vimentin was investigated immuno-histochemically in 104 experimental gliomas induced by transplancental application of ethylnitrosourea (ENU) in CDF rats. Immunoreactivity for vimentin was prominent in many astrocytic tumor cells and especially in small glioma cells forming anaplastic medulloblastoma-like foci in many tumors. The majority of tumor cells in oligodendroglial tumors were vimentin negative, except for some of the large polymorphous oligodendrogliomas which contained intermingled vimentin positive glioma cells. GFAP immunoreactivity was detectable only in a low fraction of tumor astrocytes and in a few exceptional cases some oligodendroglial tumor cells stained positive. Immunohistochemistry with antibodies against neurofilaments and cytokeratins revealed no staining in tumor cells of ENU-induced gliomas, while all oligoden-drogliomatous tumors stained positive for HNK-1. Immunocytological and immunoblot investigations of the two rat glioma cell clones RG2 and F98, which are both derived from ENU-induced gliomas, showed a prominent expression of vimentin in monolayer cultures and in syngeneic intracerebral transplantation tumors. F98 additionally demonstrated a fraction of GFAP positive cells especially in confluent cultures and in intracerebral tumors. RG2, on the other hand, exhibited virtually no GFAP immunoreactivity in culture but showed individual GFAP positive tumor cells in intracerebral tumors. Our results revealed a more precise picture of the cellular differentiation in ENU-induced rat gliomas and in two widely used glioma cell lines. They underline the heterogeneity of experimental rat gliomas which may comprise cells at different stages of differentiation towards the oligodendroglial or astroglial phenotype.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Immunochemistry of ethylnitrosourea-induced rat neurinomas,the RN6 neurinoma cell line and their transplantation tumors

Summary The expression of glial fibrillary acidic protein (GFAP), vimentin, S-100 protein (S-100), HNK-1, myelin basic protein (MBP) and fibronectin was investigated immunohistochemically in 51 ethylnitrosourea (ENU)-induced neurinomas of the rat. Additionally, 90 transplantation tumors derived from ENU-induced neurinomas and the RN6 rat neurinoma cell clone were studied. Vimentin immunoreactivity was shown in 50/51 primary neurinomas and 60/90 transplantation tumors. In contrast, GFAP was expressed in only 23/51 primary tumors and in 5/90 transplantation tumors. In the RN6 neurinoma clone, vimentin and GFAP could be demonstrated both in vivo and in vitro GFAP expression varied depending on the tumor localization, i.e., tumors of distal portions of peripheral nerves were more frequently GFAP positive than tumors of the spinal roots or of cranial nerves. The same tendency was observed for S-100. In the series of transplantation tumors S-100 and GFAP immunoreactivity decreased with increasing numbers of transplantation passages. Only individual cells in 5 primary tumors were HNK-1 positive and no MBP-immunorcactive cells were observed. Our results demonstrate that the expression of differentiation antigens in ENU-induced experimental neurinomas parallels the results reported for human neurinomas.     

Co-expression of glial fibrillary acidic protein- and vimentin-type intermediate filaments in human astrocytomas

Summary The expression of intermediate filament (IF) proteins was studied in 71 cases of malignant human astrocytoma and in 17 cases of reactive gliosis, using immunocytochemical techniques with polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and vimentin. In all cases of astrocytoma, varying in degree of malignancy from grade I to grade IV, co-expression of GFAP and vimentin was found. No change in vimentin- or GFAP-IF expression with increasing anaplasia was seen. In addition astrocytic cells in reactive gliosis showed simultaneous expression of GFAP and vimentin. The intracellular distribution of these IF proteins differed. Vimentin was found to be located in a more juxta-nuclear position, whereas GFAP immunoreactivity showed a more intense staining of the cellular processes. Astrocytes in reactive gliosis behaved more or less like neoplastic cells. However, thin cell processes of reactive astrocytes in the cortex and superficial white matter only contained GFAP immunoreactivity. Simultaneous expression of GFAP and vimentin and their proportion in malignant and reactive glial cells are discussed in the light of earlier reports on the IF content of glial cells during development and maturation, in which vimentin precedes GFAP-expression. The existence of two separate (functional) IF systems in astroglia is suggested.     

The effect of activated microglia on astrogliosis parameters in astrocyte cultures

In the diseased central nervous system, astrogliosis is accompanied by microglial activation. Depending on the context of their activation, reactive astrocytes are involved in neuronal survival and regeneration in an either protective or impedimental way. Major reactive changes of astrocytes in vivo are the upregulation of the intermediate filaments GFAP (glial fibrillary acidic protein) and vimentin with accompanying cellular hypertrophy and/or hyperplasia. To examine the involvement of activated microglia in the onset and maintenance of astrogliosis, we used an in vitro model of purified cultures of astrocytes and assessed as parameters for astrogliosis GFAP, vimentin, astroglial hypertrophy and cell growth after treatment with medium conditioned by LPS (lipopolysaccarides)-stimulated microglia. Furthermore, IL-6 as a typically upregulated cytokine in proinflammatory processes in the brain was determined in treated astrocytes. GFAP, the classical marker for astrogliosis, was downregulated on its protein and in parallel with vimentin on its mRNA level. The expression of actin, another cytoskeleton protein used as control, remained unchanged. Ultrastructural studies of astroglial intermediate filaments supported these findings. No hypertrophy was found. Nevertheless, LPS-activated microglia stimulated astrocytes as demonstrated by an increased cell number and an enhanced mRNA expression of IL-6. Resting microglia did not change any of the determined parameters. Our results suggest that the role of activated microglia in astrogliotic processes following injury of the brain has to be reevaluated, as microglia in their activated state might support the onset of astrogliosis on the one hand, but might delay or reduce subsequent glial scar formation on the other hand.     

The nature of immunohistochemically defined astrocytic cells in rat gliomas

The nature of astrocytic cells in rat gliomas induced by ethylnitrosourea (ENU) was studied by means of immunohistochemically demonstrating glial fibrillary acidic protein (GFAP), vimentin and beta-subunit of S 100 protein (S 100 beta) on paraffin sections. A special attention was paid to elucidating whether astrocytic cells would be neoplastic or not. The astrocytic cells in tumors were compared with astrocytes in normal rat brains and reactive astrocytes around a stab wound for their morphology, distribution and immunohistochemical characteristics. GFAP positive astrocytic cells in tumors were roughly divided into astrocytic cells and hypertrophic cells that showed morphologically similar appearance to fibrous astrocytes and hypertrophic reactive astrocytes around a stab wound respectively. A few multi-nucleated pleomorphic cells regarded as a kind of hypertrophic cells were also noted in gross tumors. GFAP positive cells were diffusely distributed in early neoplastic proliferations and microtumors, while in gross tumors they tended to be localized at the periphery. The GFAP positive cells were scattered among negative cells of tumors, forming no tumor mass. These findings suggest that the GFAP positive cells show no neoplastic growth, even though histopathologically they exhibit atypism or pleomorphism. The quantitative analysis of GFAP positive cells in tumors indicated more than 8% in most tumors in the white matter, whereas less than 8% in tumors in the gray matter and approximately 5% in gross tumors. The predominance of GFAP positive cells in the white matter was also observed in lesions induced by a stab wound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differentiation of the human fetal ependyma: immunocytochemical markers.

The development of the ependyma from 6 weeks (wk) gestation to term was studied in 26 human fetuses and infants for immunocytochemical differentiation using antibodies against vimentin, several cytokeratins, glial fibrillary acidic protein (GFAP) and S-100 protein. Acridine orange-RNA fluorescence was uniform in all differentiated ependymal cells. Marked differences were demonstrated among various anticytokeratin antibodies. Vimentin was demonstrated in undifferentiated cells, particularly during mitosis, and persisted as the ependyma matured. It was strong in floor plate cells and processes forming the ventral median septum. Vimentin and cytokeratin CK-904 coexisted with other immunoreactive proteins but disappeared in a caudorostral gradient with maturation. At 8 wk gestation, GFAP was detected in roof plate cells and their processes forming the dorsal median septum. S-100 protein appeared as early as 6 wk and had a more restricted regional distribution than GFAP at all ages. It was strong in the basal plate ependyma of the spinal cord in young fetuses. The temporal and spatial distributions of the immunoreactive proteins studied correlate with evidence that fetal ependymal cells synthesize compounds that attract or repel axonal growth cones to prevent axons from entering the ventricles or deviating from programmed projection pathways. An additional role may be to induce the transformation of radial glial cells in the subventricular zone.     

Gliosis in the spinal cords of rats with experimental allergic encephalomyelitis: immunostaining of carbonic anhydrase and vimentin in reactive astrocytes

Spinal cord sections from rats sensitized to develop experimental allergic encephalomyelitis (EAE) were immunostained with antibodies against glial fibrillary acidic protein (GFAP), carbonic anhydrase, and vimentin, to see whether the latter two antigens could be detected in GFAP-positive reactive astrocytes. Sixteen days after sensitization (16 dpi) there was intense carbonic anhydrase immunostaining in GFAP-positive cells in the spinal cords of EAE rats, particularly in the white matter. At 13 and 20 dpi carbonic anhydrase immunostaining in astrocytes was less intense, and in the spinal cord white matter of control animals carbonic anhydrase was not detected in the few GFAP-positive cells. In the spinal cords of EAE rats vimentin immunostaining was observed in inflammatory cells and astrocytes. In the latter, GFAP and carbonic anhydrase were colocalized with vimentin. The data suggest that carbonic anhydrase expression in astrocytes is an acute response to injury and that vimentin can be detected in astrocytes, as well as inflammatory cells, as early as 16 dpi.     

Immunohistochemical study of folliculo-stellate cells in humna pituitary adenomas

Summary Folliculo-stellate (FS) cells were studied in 102 human pituitary adenomas by immunohistochemical techniques using antibodies to S-100 protein and intermediate filaments protein. In most pituitary adenomas there were few S-100-positive cells, in contrast, numerous FS cells were found in four of the 54 cases of non-functioning adenomas. Among glial fibrillary acidic protein (GFAP), keratin and vimentin, FS cells showed greatest affinity to vimentin. Stains for desmin or neurofilaments were always negative. Counterstains with GFAP and keratin could demonstrate a small number of double-labelled cells, but mainly disclosed two types of FS cells positive for either GFAP or keratin. Accordingly, FS cells were grossly subdivided into two types: GFAP-positive cells which might be neuroectodermal or glial in origin and keratin-positive cells which might be oral ectodermal or derive from the Rathke's pouch.     

Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.     

A monoclonal antibody against vimentin: characterization

A monoclonal antibody was developed using rat cerebral cortex astrocytes purified in vitro as the antigenic material. Screening was done by labeling antibodies bound to cerebellum slices with fluorescent tagged secondary antibodies. The monoclonal antibody (RBA1) bound to intracellular filaments in all cells examined in culture. The coloration of tissue sections by RBA1 was identical to that described for polyclonal antibodies against the intermediate filament protein vimentin. In the brain this included binding to meninges, blood vessels, ependymal cells, choroid plexus lining cells and a subpopulation of astrocytes. The latter included Bergman glial fibers, white matter astrocytes and tanycytes. Müller cells in the retina and fibroblast-like cells in the skin, tongue and intestine were RBA1-positive. In immunoblots in which purified vimentin and desmin were run on SDS and transferred to nitrocellulose paper, RBA1 bound to vimentin but not desmin. When cultured astrocyte proteins were blotted, the antibody bound to both GFAP and vimentin, but no GFAP staining was observed in any of the tissue section staining. Purified vimentin blocked tissue and cultured cell binding of the antibody. Therefore RBA1 is considered to be an antibody specific for the intermediate filament protein vimentin.