Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury

We have examined the injury response of astrocytes in the immature hamster brain in this study, focusing on alterations in the expression of glial fibrillary acidic protein (GFAP) and vimentin. In the adult CNS these two type III intermediate filament (IF) proteins have been shown to undergo robust increases in expression in response to axonal injury. Since injury to the immature CNS reportedly elicits less glial scar formation than adult brain injury, we examined the possibility that immature astrocytes respond differently than adult astrocytes to CNS injury with respect to IF gene expression. In situ hybridization using a ³⁵S-labeled cDNA GFAP probe was done on brainstem sections obtained 2,7 and 14 days after unilateral transection of the corticospinal tract in P8 hamster pups. The results indicated that substantial increases in GFAP mRNA were associated with the degenerating portion of the corticospinal tract by 2 days after axotomy and that the levels remained elevated for at least 14 days. Double-label immunofluorescence studies of this material suggested that GFAP as well as vimentin protein levels were also increased in many astrocytes in and around the degenerating corticospinal tract 2–14 days after axotomy. Most of the reactive astocytes in the degenerating regions exhibited increases in GFAP and vimentin immunostaining but some vimentin-negative GFAP-positive reactive astrocytes were also observed, particularly in regions surrounding the actual degenerative zones. The results from these experiments revealed that immature astrocytes have the potential for altering their normal developmental program of GFAP and vimentin expression after injury and mount a response that is qualitatively similar to that of astrocytes after CNS injury in the adult animal.     

A comparison of intermediate filament markers for presumptive astroglia in the developing rat neocortex: immunostaining against nestin reveals more detail, than GFAP or vimentin

The present study compares the immunopositive elements in the developing rat cortex between the day of birth (P0) and the 18th postnatal day (P18), after immunostaining against nestin, vimentin and glial fibrillary acidic protein (GFAP). Nestin immunostaining revealed more structural details than either vimentin or GFAP, or they together. While vimentin immunostaining preferred radial glia and GFAP preferred astrocytes, nestin immunostaining detected both. Stellate-shaped astrocyte-like cells were already seen at P0 and cells of typical astrocytic morphology were numerous at P3, and were predominating elements from P7, whereas GFAP-immunopositive astrocytes were very scarce even at P7, and became numerous only by P11, when nestin immunopositivity started to disappear. Nestin immunostaining revealed such structures which were not seen in GFAP- or vimentin immunostained sections: cell body-like structures 'hanging' at the end the radial fibers, seeming to divide with their fibers, or having astrocyte-like processes. Nestin immunostaining is therefore highly recommended for studies of the glial architecture in the early post-natal brain development.     

Acute Exposure to CNTFin VivoInduces Multiple Components of Reactive Gliosis

CNS trauma or disease induces a constellation of changes in the glia comprising the condition known as reactive gliosis. At present, little is known regarding the nature of the injury signals and the specific consequences of their actions. Ciliary neurotrophic factor (CNTF) induces acute phase proteins in liver and increases astrocytic glial fibrillary acidic protein (GFAP) bothin vitroandin vivo.The purpose of the present study was to establish whether CNTF induces other aspects of gliosis. Between 10 and 72 h after 100 ng of recombinant human CNTF was administered into the adult rat neocortex, alterations were observed in a region extending several millimeters in circumference from the injection site. Microglia in this region were more apparent and astrocytes were hypertrophic. Byin situhybridization, mRNAs for GFAP, vimentin, and clusterin were upregulated when compared to the control hemisphere (which received heat-inactivated CNTF). By immunocytochemistry, GFAP, vimentin, glutathione-S-transferase μ, S-100, and OX-42 were elevated by 48 h. By contrast, the oligodendroglial marker GST Yp, the neuronal markers MAP-2 and NSE, the intermediate filament nestin, and the stress protein αB-crystallin were unchanged. In addition, a greater than twofold increase in the number of proliferating cells was observed. Since CNTF induces swelling and multiple “gliotic” genes in astrocytes, increases microglial number, and stimulates cell proliferation, we conclude that CNTF is sufficient to induce multiple aspects of gliosis. These data are consistent with a model whereby CNTF (which is synthesized by astrocytes) would be released when the integrity of the astrocyte membrane is compromised, whereupon it would elicit an inflammatory response.     

Interactions between meningeal cells and astrocytes in vivo and in vitro.

At the interface between the meninges and the central nervous system there is a characteristic structure known as the glia limitans, consisting of many fine interdigitating astrocyte processes which contain both GFAP and vimentin, and a basal lamina. A similar structure is set up after brain injury where meningeal cells invade the lesion. We have experimentally put astrocytes and meningeal cells in contact with one another, both in vivo and in vitro, to see whether this results in the formation of a glia limitans. Cultured meningeal cells were injected into the hippocampus of adult rats, and from 1 to 12 weeks later brains were stained were stained for GFAP and vimentin. One week after injection there was a widespread astrocytic reaction stretching up to 2 mm from the injection, the cells being stained intensely for both GFAP and vimentin. Over the next 4-6 weeks this widespread reaction subsided, the only remaining vimentin stained astrocytes, apart from those at the normal glia limitans, being in contact with the injected meningeal cells, or with meningeal cells which had migrated into the injection needle track. In vitro a structure reminiscent of the glia limitans formed where patches of astrocytes abutted meningeal cells; the astrocytes formed a layer of fine interdigitating processes all running parallel to the interface between the two cell types, and there was heavy staining for laminin and fibronectin. We conclude that a glia limitans forms wherever astrocytes and meningeal cells come into contact.     

Tau accumulation in astrocytes in progressive supranuclear palsy is a degenerative rather than a reactive process

Tau-immunoreactive astrocytes in progressive supranuclear palsy (PSP) have a distinctive morphology and are referred to as tufted astrocytes (TA). We hypothesized that TA may be a degenerative change in reactive astrocytes. To test this hypothesis we examined the relationship of TA to gliosis in PSP. We first examined the distribution of gliosis [glial fibrillary acid protein (GFAP)-positive astrocytes], TA, neurofibrillary tangles (NFT) and pretangles in brain sections of neuropathologically pure PSP cases. Second, we examined PSP cases complicated by infarcts or Alzheimer's disease, since these cases would have reactive astrocytes associated with lesions. We used double immunostaining for GFAP and tau for cases with vascular lesions, and triple immunostaining for GFAP, tau and beta-amyloid protein for sections with senile plaques. There was no correlation between the distribution of gliosis and TA, with gliosis prominent in globus pallidus and subthalamic nucleus, and TA prominent in motor cortex and striatum. On the other hand, gliosis paralleled the distribution of NFT, but not the distribution of pretangles, suggesting that NFT contributes to gliosis in PSP. Although reactive astrocytes were present around infarcts and senile plaques, TA were not associated with these lesions. Tau accumulation in astrocytes in PSP was not preferential to (and was actually independent of) reactive astrocytes. This is consistent with the notion that tau accumulation in astrocytes is a degenerative rather than reactive process. Unlike NFT, astrocytic degeneration does not seem to contribute to gliosis or neuronal loss in PSP, and its clinical significance remains unclear.     

Gliosis in human brain: relationship to size but not other properties of astrocytes

Gliosis is the most frequent and therefore important neurocellular reaction to brain insult occurring in diseases ranging from AIDS to infarction. Neuropathological diagnosis is bases on morphological changes of brain glial cells. Changes commonly agreed to reflect gliosis are qualitative increases in size, number and glial fibrillary acidic protein (GFAP) immunoreactivity of astrocytes. These parameters were morphometrically quantified in brain tissues of 22 individuals who died with 7 diseases and statistically compared to the extent of gliosis independently determined by 3 qualified observers. The data indicate that the extent of gliosis correlated with the increase in size of astrocytes in white matter (π = 0.67) and this relationship was statistically significant (P = 0.0006). In contrast, the extent of gliosis was not correlated with the density of astrocytes nor the intensity of GFAP staining.     

Conditional deletion of β1‐integrin in astroglia causes partial reactive gliosis

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of β1‐integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up‐regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin‐C and the DSD‐1 proteoglycan. In addition, we also observed to a certain degree a non‐cell autonomous activation of microglial cells after conditional β1‐integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of β1‐integrin‐mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron‐specific deletion of β1‐integrin, we conclude that β1‐integrin‐mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in β1‐integrin‐mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury. © 2009 Wiley‐Liss, Inc.     

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.     

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures

In the present study we analyze the events which occur during the early stages of astrogliogenesis by examining the pattern of both GFAP and vimentin gene expression and their corresponding immunoreactive proteins during rat brain development. This study was carried out “in vivo” (whole brain) and “in vitro” (primary culture of radial glia) using immunofluorescence, immunoblotting, and Northern blot analysis. Our results demonstrate that although GFAP immunostaining appeared late in gestation and at day 5 in radial glia cultures, GFAP mRNA expression was first detected, at very low levels, on fetal (F) day 15 and increased to F21. During postnatal development a striking increase in GFAP and its encoding messenger occurs. In contrast, the levels of vimentin and its mRNA expression were very high during the fetal stage (F15 to F21). Thereafter vimentin expression declined during postnatal (P) development until P21 and then remained constant at adult levels. In contrast, an increase in vimentin expression was observed in glial cells throughout the entire culture period. The biological significance of the developmental patterns of GFAP and vimentin expression in astroglial cells during brain development is discussed. © 1995 Wiley-Liss, Inc.     

Glial fibrillary acidic protein expression but no glial demarcation follows the lesion in the molecular layer of cerebellum

The present study investigates the reactive gliosis following a simple stab wound lesion to a brain area in which a characteristic astroglial architecture exists, i.e., the Bergmann-glia in the molecular layer of cerebellum. While in mammalian brain the Bergmann-glia contains glial fibrillary acidic protein (GFAP), in the avian Bergmann-glia, the cytoskeletal protein is vimentin, which is characteristic for immature astroglia in mammals. The operations were performed on chickens and rats under deep anaesthesia, using a sterile disposable needle. After a 1-week survival period, the animals were overdosed with ether and perfused transcardially with 4% buffered paraformaldehyde. Free-floating sections cut with a vibration microtome were processed for immunohistochemistry against GFAP and vimentin. GFAP immunopositivity of Bergmann-glia appeared in chicken and increased in rat in the lesioned area but the lesion was not surrounded by typical astrocytes and no demarcation was formed in the molecular layer, in contrast to the usual appearance of reactive gliosis, which was observed in the granular layer and in the white matter in both species. Vimentin immunopositivity of the Bergmann-glia also increased around the lesion in both species. The results suggest that a highly developed glial architecture fails to re-arrange into a demarcating scar, which offers an interesting model system to study the importance of glial demarcation. The observations also support that the resident glia is the main component of the glial reaction, and prove the capability of avian Bergmann-glia to express GFAP.     

The distribution of glial fibrillary acidic protein and vimentin in postnatal marmoset (Callithrix jacchus) brain

Antisera to glial fibrillary acidic protein (GFAP) and vimentin were used to elucidate the distribution of these intermediate filament proteins in postnatal marmoset brains of various ages. The ependyma of the lateral ventricles was unique in being equally immunoreactive for both GFAP and vimentin at all ages. Vimentin alone was consistently demonstrated in endothelial and leptomeningeal cells at all ages. In neonates, vimentin immunoreactivity greatly exceeded that of GFAP and was located primarily in radial glia in the subependymal plate of the anterior cerebrum. Their vimentin-positive processes formed thick fascicles in the corpus callosum but separated into fine fibres on entering the cortex. GFAP immunoreactivity in these cells and processes was very limited. With age, GFAP-positive cells increased in number and displayed the typical stellate appearance of astroglia. The vimentin-positive radial glial population decreased considerably during this period and by 6 months had virtually disappeared. The GFAP reaction in adult brain was even more widespread, largely due to the increased number of positive astrocytes in the white matter. Vimentin immunoreactivity in the adult was greatly diminished and positive radial glia were not detectable. A major change in intermediate filament protein expression, therefore, occurs in the early postnatal period and probably reflects phases in the differentiation of radial glial precursors into astrocytes.     

MM1‐type sporadic Creutzfeldt‐Jakob disease with 1‐month total disease duration and early pathologic indicators

A 62‐year‐old man presented with abnormal behavior and cognitive impairment. Diffusion‐weighted images (DWI) obtained on MRI showed extensive hyperintense regions in the cerebral cortex and striatum. Myoclonus was recognized, and the patient died 1 month after the onset; his condition did not reach the akinetic mutism state. The brain weighed 1300 g and showed no apparent atrophy. Extensive spongiform changes were observed in the cerebral neocortex, striatum, thalamus and cerebellar cortex, but gliosis was mild or absent. Neuropil rarefaction and neuron loss were not apparent. Mild proliferation of anti‐ GFAP‐positive astrocytes was observed in the cerebral cortex, but unaffected regions were noted. Regions without spongiform changes and GFAP‐positive astrocytes included the hippocampal formation and subiculum. PrP immunostaining showed extensive diffuse synaptic‐type PrP deposition in the gray matter, including the hippocampal region, but it was also mild. PrP gene analysis revealed no mutation with methionine homozygosity at polymorphic codon 129. Western blot analysis of proteinase K‐resistant PrP indicated type 1 PrP^Sc. The clinicopathological findings of the present case confirm several hypotheses: (i) the earliest pathologic evidence observed by HE staining in CJD are spongiform changes; (ii) DWI hyperintense regions indicate these spongiform changes; and (iii) regions without spongiform changes, gliosis and proliferation of GFAP‐positive astrocytes, but with PrP deposition, exist in the early disease stage.     

Reactive gliosis in the brains of Lewis rats with experimental allergic encephalomyelitis

This paper assesses reactive gliosis in the optic tracts and other regions of brain in Lewis rats with experimental autoimmune encephalomyelitis (EAE). Enhanced immunostaining for glial fibrillary acidic protein (GFAP) in brains from rats with EAE occurred primarily in the white-matter tracts and was not restricted to sites of inflammation. Immunocytochemical staining for other putative astrocytic antigens demonstrated glutathione-S-transferase (Yb isoenzyme) to be localized extensively in GFAP-positive cells and vimentin to be present both in inflammatory cells and in some GFAP-positive astroglial cells. Positive staining for carbonic anhydrase and glutamine synthetase was observed in oligodendrocytes. In the optic tracts glutamine synthetase, but not carbonic anhydrase, was also observed in some astrocytes.     

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)     

Palisading pattern of subpial astroglial processes in the adult rodent brain: relationship between the glial palisading pattern and the axonal and astroglial organization

The architectural organization of the subpial astrocyte processes was examined near the brain surface by single immunostaining methods. The astroglial processes were stained on brain sections made parallel to the pial surface. The astroglial glial fibrillary acid protein (GFAP) antigen was used as a specific marker. We show that these subpial astrocyte processes present a well organized palisading pattern in the adult mouse and rat spinal cord, medulla and pons. This adult astrocyte palisading pattern is compared to the palisading radial glia organization we previously demonstrated in the fetal mouse brain. The observed analogies afford a new and strong argument in favor of a derivation of the subpial astrocytes from radial glia. Double immunostaining methods, using GFAP and neurofilament antigens as glial and neuronal markers respectively, show the close relationship existing between the trajectories of axonal and glial processes. Beside the colinearity already observed between the axon trajectories and the glial palisades we demonstrate a new kind of axon/glia relationship. Axons are closely intermingled, within the palisading glial tufts, with the peripheral processes of the subpial astrocytes progressing to the pial surface. The findings suggest that fetal radial glia organization has a direct and indirect influence on the adult astroglial and perhaps the axonal pattern.     

Vimentin isoform expression in the human retina characterized with the monoclonal antibody 3CB2

The antigen recognized by the monoclonal antibody 3CB2 (3CB2-Ag and 3CB2 mAb) is expressed by radial glia and astrocytes in the developing and adult vertebrate central nervous system (CNS) of vertebrates as well as in neural stem cells. Here we identified the 3CB2-Ag as vimentin by proteomic analysis of human glial cell line U-87 extracts (derived from a malignant astrocytoma). Indeed, the 3CB2 mAb recognized three vimentin isoforms in glial cell lines. In the human retina, 3CB2-Ag was expressed in Müller cells, astrocytes, some blood vessels, and cells in the horizontal cell layer, as determined by immunoprecipitation and immunofluorescence. Three populations of astrocytes were distinguishable by double-labeling immunohistochemistry: vimentin+/GFAP+, vimentin-/GFAP+, and vimentin+/GFAP-. Hence, we conclude that 1) the 3CB2-Ag is vimentin; 2) vimentin isoforms are differentially expressed in normal and transformed astrocytes; 3) human retinal astrocytes display molecular heterogeneity; and 4) the 3CB2 mAb is a valuable tool to study vimentin expression and its function in the human retina.     

Glial responses to neonatal hypoxic–ischemic injury in the rat cerebral cortex

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic–ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

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.     

Axonal integrity in the absence of functional peroxisomes from projection neurons and astrocytes

Ablation of functional peroxisomes from all neural cells in Nestin‐Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp‐Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long‐chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron‐specific peroxisome knockout model, NEX‐Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP‐Pex5^−/− mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP‐Pex5^−/− mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning. © 2010 Wiley‐Liss, Inc.