Astrocytic Glial Fibrillary Acidic Protein Research Articles

Monitoring the temporal and spatial activation pattern of astrocytes in focal cerebral ischemia using in situ hybridization to GFAP mRNA: comparison with sgp-2 and hsp70 mRNA and the effect of glutamate receptor antagonists

We investigated the temporo-spatial expression of astrocyte glial fibrillary acidic protein ( gfap) and sulfated glycoprotein 2 ( sgp-2) mRNAs in comparison to 70-kDa heat shock protein ( hsp70) mRNA by in situ hybridisation in rats subjected to permanent occlusion of the middle cerebral artery (MCA). Gfap mRNA started to increase in the cingulate cortex of the lesioned hemisphere 6 h after MCA occlusion and gradually spread over the lateral part of the ipsilateral cortex and the striatum from 12 h to 3 days, peaking at 3 days after MCA occlusion. Gfap mRNA also increased in the contralateral cingulate cortex and corpus callosum at 12 and 24 h. Hsp70 mRNA increased markedly in the ipsilateral cortex adjacent to the ischemic lesion, and slightly within the lesion area from 3 to 24 h and disappeared after 3 days. By 7 days, gfap and sgp-2 mRNAs were increased markedly in the peri-infarct area, and in the ipsilateral thalamus parallel with the delayed neuronal damage, whereas the widespread increase of gfap mRNA in the ipsilateral hemisphere declined. Post-occlusion treatment with the glutamate receptor antagonists MK-801 and NBQX slightly attenuated the induction of gfap but did not qualitatively affect the topical expression pattern. Within the cingulate cortex MK-801 treatment resulted in a significant decrease of the signal intensity at all survival times, reflecting most likely an attenuation of lesion-induced spreading depression like depolarization waves by MK-801. The area of hsp70 expression was reduced by both MK-801 and NBQX, most likely reflecting the decrease of the lesion area by both treatment regimens. Our study thus revealed an early and widespread increase of gfap mRNA in the non-ischemic areas including the contralateral hemisphere starting between 3 and 6 h, and a delayed circumscribed expression in the peri-infarct border zone after 1 week. Comparison with the expression of hsp70 mRNA suggests that the absence of an early gfap mRNA induction in the peri-lesion zone reflects an impairment of astrocytic function which may be of importance for infarct growth during the early evolution of the pathological process.

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.

Scrapie in mice deficient in apolipoprotein E or glial fibrillary acidic protein.

In the prion diseases, extensive reactive gliosis is often found to be out of proportion to the degree of apparent neuronal damage. To evaluate the role of astrocytic gliosis in experimental scrapie of the mouse, we inoculated mice deficient in apolipoprotein E (apoE) or the glial fibrillary acidic protein (GFAP) with mouse prions. The expression of both apoE and GFAP in astrocytes increases as part of the reactive gliosis that accompanies scrapie. Null mice deficient in either apoE or GFAP inoculated with prions exhibited incubation times indistinguishable from untargeted control mice. The level of PrPSc and its regional deposition in the brains of ill mice deficient in either protein were also similar to control mice. Our findings demonstrate that neither apoE nor GFAP participates in the pathogenesis of the disease or in the production of PrPSc.

Chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain.

By 24 h after mechanical trauma to the cerebral cortex, astroglial reaction begins and injury sites are infiltrated by activated mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is little information about cellular interactions between astrocytes and leukocytes during this process. We previously showed that murine astrocytes produce chemokines including monocyte chemoattractant protein-1 (MCP-1) during experimental autoimmune encephalomyelitis. In this study, we asked whether astrocytes produce MCP-1 in the absence of immune mediated inflammation. To address this question, we analyzed the time course and cellular source of MCP-1 in mouse brain after penetrating mechanical injury, with particular focus on early time points before histologic detection of infiltrating mononuclear phagocytes. We observed sharply increased steady state levels of MCP-1 mRNA within 3 h after nitrocellulose membrane stab or implant injury to the adult mouse brain, and MCP-1 protein elevations were documented at 12 h postinjury. In situ hybridization combined with immunohistochemistry for the glial fibrillary acidic protein astrocyte marker showed that astrocytes were the cellular source of MCP-1 mRNA at these early time points after mechanical brain injury. Stab injury to the neonatal brain evoked neither MCP-1 expression nor astrogliosis. These results demonstrate that chemokine gene expression comprises one component of the astrocyte activation program. The data are consistent with a role for MCP-1 in the central nervous system inflammatory response to trauma.

Intermediate filament change in astrocytes following mild cortical contusion

Astrocytes are known to become reactive as a result of various types of lesions. They upregulate astrocytic-specific intermediate filament glial fibrillary acidic protein (GFAP) and show a positive signal for the intermediate filament vimentin, a protein primarily found in developing astrocytes. An animal model for cortical contusions has been developed that manifests many of the neuropathologies seen in human closed head injury. The model involves an electronic controlled pneumatic impact device (ECPI), which can deliver precise and controlled cortical contusions to an animal. This model can be used to study astrocyte response to injury, leading to a better understanding of glial reaction to head trauma in humans. Young adult male Fisher 344 rats were subjected to a mild dorsal lateral cortical impact. The astrocytic intermediate filaments GFAP and vimentin were upregulated in a time-dependent manner 2 days after injury. By 30 days following contusion these intermediate filaments returned to near preinjury levels. Using the same injury model bromodeoxyuridine (BrdU) was injected in additional animals on the day of sacrifice, 1, 2, 4, and 10 days after injury, to label cells synthesizing new DNA. Double labeling utilizing fluorescence immunocytochemistry indicated that on postinjury day 2 very few GFAP-positive cells were double labeled with BrdU. No double-labeled cells were seen at the other time points. These data suggest that astrocyte proliferation may not be a major response to mild cortical contusion and that vimentin expression may not necessarily be associated with astrocytic proliferation in response to injury. © 1996 Wiley-Liss, Inc.

Intermediate filament change in astrocytes following mild cortical contusion.

Astrocytes are known to become reactive as a result of various types of lesions. They upregulate astrocytic-specific intermediate filament glial fibrillary acidic protein (GFAP) and show a positive signal for the intermediate filament vimentin, a protein primarily found in developing astrocytes. An animal model for cortical contusions has been developed that manifests many of the neuropathologies seen in human closed head injury. The model involves an electronic controlled pneumatic impact device (ECPI), which can deliver precise and controlled cortical contusions to an animal. This model can be used to study astrocyte response to injury, leading to a better understanding of glial reaction to head trauma in humans. Young adult male Fisher 344 rats were subjected to a mild dorsal lateral cortical impact. The astrocytic intermediate filaments GFAP and vimentin were upregulated in a time-dependent manner 2 days after injury. By 30 days following contusion these intermediate filaments returned to near preinjury levels. Using the same injury model bromodeoxynridine (BrdU) was injected in additional animals on the day of sacrifice, 1, 2, 4, and 10 days after injury, to label cells synthesizing new DNA. Double labeling utilizing fluorescence immunocytochemistry indicated that on postinjury day 2 very few GFAP-positive cells were double labeled with BrdU. No double-labeled cells were seen at the other time points. These data suggest that astrocyte proliferation may not be a major response to mild cortical contusion and that vimentin expression may not necessarily be associated with astrocytic proliferation in response to injury.

SC1: a marker for astrocytes in the adult rodent brain is upregulated during reactive astrocytosis

Astrocytes are the most abundant cell type in the mammalian central nervous system (CNS), and are involved in many processes critical for normal CNS maintenance and function. We have used double-label immunocytochemistry and in situ analysis to show that the SPARC (secreted protein acidic and rich in cysteine)-related protein SC1, co-localizes with the astrocyte marker glial fibrillary acidic protein (GFAP) in the adult rodent brain. Thus, SC1 is an astrocyte marker that may be used to investigate astrocyte heterogeneity and analyze glial cell lineages during neural development. Consistent with the presence of SC1 and GFAP in astrocytes, both proteins were markedly upregulated following reactive astrocytosis induced by focal mechanical trauma. Therefore, SC1 may play an important role in reactive astrocytosis subsequent to a wide variety of neural trauma, including neurodegenerative diseases and acute neural damage.

Glial fibrillary acidic protein-immunoreactive astrocytosis in elderly patients with schizophrenia and dementia.

Clinical and neuropsychological studies of chronically institutionalized patients with schizophrenia indicate that severe cognitive impairment and functional disability in late life are very prevalent. The biological substrates for this dementia remain unknown. While subtle cytoarchitectural and morphometric abnormalities have been described in patients with schizophrenia and interpreted as reflecting aberrant neurodevelopment, postmaturational injury or neurodegeneration associated with gliosis remain as plausible explanations of at least some of the clinical manifestations of schizophrenia. We monitored astrocytosis and neurofibrillary tangle (NFT) formation in 21 elderly patients with schizophrenia (14 with concurrent dementia, 7 without), and in 12 normal and 5 Alzheimer's disease (AD) control cases. Astrocytes in ventromedial temporal, frontal, and calcarine cortices were immunohistochemically identified with monoclonal antibodies directed at glial fibrillary acidic protein (GFAP) and vimentin, and NFTs were labeled with an anti-tau antibody specific for paired helical filaments. There were no increases in GFAP- or vimentin-immunoreactive astrocyte counts, GFAP optical density, or NFT counts for the schizophrenic group as a whole compared to the non-neuropsychiatric group, while both groups differed from AD. When patients with schizophrenia were divided into demented and non-demented subtypes, those with dementia demonstrated significantly greater numbers of GFAP-positive astrocytes than those without dementia. These data may reflect an up-regulation of GFAP in normal astrocytes or the presence of reactive astrocytosis in a subgroup of schizophrenics. In the absence of any diagnostic neuropathological findings in this subgroup, the implications of these observations for the pathogenesis of schizophrenia remain to be determined.

Analysis of glial fibrillary acidic protein, neurofilament protein, actin and heat shock proteins in human fetal brain during the second trimester

Glial fibrillary acidic protein (GFAP), the 66 kDa neurofilament protein (NF-66), actin, the 27 kDa heat shock protein (HSP27) and the 70 kDa constitutive heat shock protein (HSC70) were analyzed in human fetal brain during the second trimester, from 10 to 24 gestational weeks (GW). By immunohistochemistry, cell-type specific localization of GFAP and NF-66 in astrocytes and neurons, respectively, was confirmed. HSP27 was expressed mostly in the nuclear region of neurons and non-neuronal cells, and HSC70 was widely distributed throughout the tissue. By quantitative immunoblotting, GFAP was not detectable in gray matter of prefrontal cortex prior to 16 GW. Between 16 and 21 GW, the content of GFAP rose slowly. Thereafter, GFAP accumulated rapidly. The content of GFAP in different brain regions (prefrontal, parietal, and occipital cortices) differed significantly at 22 GW. In contrast, NF-66 was already highly expressed at 10 GW, slowly rose to maximal values by 18 GW, and thereafter remained stationary. In contrast to GFAP, the content of NF-66 was similar in different brain regions at 22 GW. Although actin was abundant throughout the second trimester, a sharp drop in its content in the prefrontal cortex was detected at 17 GW. To explain such a decrease, two heat shock proteins were analyzed. HSP27, known to modulate actin polymerization, was found to increase sharply at 16–17 GW. In contrast, HSC70 remained constant during the second trimester and was highly expressed in the fetal brain, at a level comparable to that in the adult brain.

Trimethyltin increases interleukin (IL)-lα, IL-6 and tumor necrosis factor α mRNA levels in rat hippocampus

Within the central nervous system (CNS), cytokines are thought to have active roles in pathophysiological changes seen in various neurological diseases and trauma. The present study was undertaken to examine the early response of pro-inflammatory cytokines following exposure to a specific neurotoxicant (trimethyltin; TMT). mRNA levels for interleukin (IL)-1α, IL-1β, IL-6 and tumor necrosis factor (TNF) α were measured in the hippocampus of adult male Long-Evans hooded rats following an acute injection of trimethyltin hydroxide (8 mg TMT/kg body weight). At various times following exposure (6 h to 8 days), hippocampal tissues were excised and relative changes in cytokine mRNA levels were assessed by reverse transcription and polymerase chain reaction. IL-1α, IL-6 and TNFa mRNA levels in the hippocampus increased within 6 h and remained elevated for 8 days. Quantitative analysis of mRNA transcripts revealed a two-fold increase in both IL-6 and TNFa within 6 h and a continued elevation of TNFa to 9-fold by 12 h. Within 96 h, glial fibrillary acidic protein (GFAP) mRNA levels were elevated in the hippocampus. Histological examination showed sparse individual neuronal necrosis at this time in both the pyramidal and granule cell regions with no increase in astrocyte GFAP immunoreactivity. However, an early, 24 h, response of microglial cells was indicated by increased lectin binding. This morphological profile progressed over time to a profound neuronal loss in the CA3-4 granule cell layer and marked astrocyte hypertrophy. The onset of pro-inflammatory cytokine mRNA expression appears to be temporally associated with histological evidence of elevated microglia in the hippocampus. It is proposed that microglia and pro-inflammatory cytokines play a modulatory role in the early stages of TMT-induced neurotoxicity.

Glial fibrillary acidic protein mRNA increases at proestrus in the arcuate nucleus of mice

Glial fibrillary acidic protein (GFAP) increases during proestrus in astrocytes of the hypothalamic arcuate nucleus (ARC). These changes are associated with altered astrocyte-neuron contacts and synaptic remodelling, during preparation for the preovulatory gonadotrophin surge. This study of young C57BL/6J mice showed transient elevations of GFAP mRNA on proestrus in the ARC by in situ hybridization. Basal GFAP mRNA was regained within 18 h. We hypothesize that changes in astrocytic GFAP on proestrus result from elevations of GFAP mRNA that are, in turn, driven by ovarian secretions of estradiol.

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.

GFAP transfected cells produce laminin, leading to neurite outgrowth promotion.

Accumulating evidence indicates the importance of astrocytes in neuronal development and regeneration. While glial fibrillary acidic protein (GFAP) is believed to mediate the morphology of developing astrocytes, its precise function remains unknown. To analyse the function of GFAP in astrocytes, we established GFAP-expressing cell lines by transfection of mouse GFAP cDNA into mouse fibroblast L cells. Stable transfectants expressed GFAP uniformly in the cytoplasm with no phenotypic changes and exhibited extended processes rich in GFAP. GFAP-expressing cells significantly promoted the neurite outgrowth of rat cerebral cortex neurones in the co-culture system. Analysis of the products of GFAP-expressing cells revealed an increase in production of laminin, but not fibronectin. These results suggest that L cells expressing GFAP increase laminin production, leading to promotion of neurite outgrowth.

The astrocytic response to afferent activity blockade in chick nucleus magnocellularis is independent of synaptic activation, age, and neuronal survival

Astrocytes in nucleus magnocellularis (NM) of the chick respond to afferent activity blockade with increased immunoreactivity for glial fibrillary acidic protein (GFAP). NM neurons respond to the same manipulations with reduced protein synthesis, ribosomal dissociation, and subsequent death of a subset of these neurons. In the present study, we sought to evaluate the relationship between these neuronal and glial responses and to determine if similar activity-dependent mechanisms mediate them. We first examined the anatomical relationship between NM neurons and astrocytic processes by electron microscopy and GFAP immunostaining. Both methods showed that NM neurons deprived of activity for 6 hr were apposed by more glial processes than active NM neurons. However, we found no preferential positioning of GFAP-immunoreactive processes near neurons of the dying or surviving populations, and there were no differences in glial process apposition to dying versus surviving neurons at the EM level. To determine whether the astrocytic response is similar to the neuronal response in age dependence, GFAP immunoreactivity was analyzed in adult chickens following unilateral afferent activity blockade. Unlike the neuronal response to activity blockade, the astrocytic response is equally strong in adult animals. These results imply an independence of the neuronal and astrocytic responses to activity blockade, raising the possibility that these two cell types may be responding to different activity-related signals. This possibility was tested using an in vitro slice preparation. Unilateral stimulation of NM was provided in three ways: orthodromically, antidromically, and orthodromically in a low-calcium medium. The regulation of astrocytic GFAP immunoreactivity by these manipulations of activity was then analyzed. The results of these experiments show that, unlike neuronal protein synthesis, astrocytic GFAP immunoreactivity can be suppressed by either presynaptic or postsynaptic neuronal activity. Therefore, the astrocytes and neurons are regulated by different activity-dependent signals and, by the present measures, their responses to activity blockade appear independent of one another.

Trimethyltin-induced expression of GABA and vimentin immunoreactivities in astrocytes of the rat brain.

Adult Sprague-Dawley rats were given a single dose of trimethyltin chloride (TMT). Three days following treatment, a neuronal alteration was observed in the CA3c pyramidal cell layer of hippocampus which was not accompanied by any apparent astrocyte reaction. At 1 as well as 2 weeks after treatment, a gliosis in hippocampus, piriform, and entorhinal cortices was detected by glial fibrillary acidic protein (GFAP) immunohistochemistry. Concomitant with the enhanced astrocytic GFAP, astrocytes were swollen and expressed immunoreactivity to vimentin and gamma-aminobutyric acid (GABA). The astrocytic GABA immunoreactivity may reflect a trimethyltin-induced alteration in astrocyte phenotype, or alterations in compartmentalization and/or metabolism of GABA.

Expression of glial fibrillary acidic protein and glutamine synthetase genes in the natural scrapie of sheep.

Gene expression of two astroglial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), was investigated in cerebellum and brainstem from scrapie-affected sheep. The GFAP and GFAP-mRNA concentrations were increased in the two cerebral regions studied in the scrapie-affected animals as compared to the controls. The good correlation between the increase in GFAP and GFAP-mRNA concentrations found in scrapie-affected sheep indicates a significant de novo synthesis of GFAP in this pathology. In contrast to these results, in scrapie no significant differences in GS-mRNA content appeared in either brain area from scrapie-affected sheep as compared to the controls. This fact could suggest some specificity of GFAP expression changes in this pathology. The overexpression of GFAP gene could be related to a possible interaction between GFAP and scrapie infectious agent in astrocytes. The relative increase in the GFAP and its encoding message in affected animals was higher in the cerebellum than in the brainstem, which would suggest regional comparative differences in the effect here described.

CHRONIC PRENATAL ETHANOL EXPOSURE DOES NOT AFFECT THE EXPRESSION OF SELECTED GENES IN RAT BRAIN DEVELOPMENT

To investigate possible mechanisms contributing to the teratogenic effects of ethanol on the nervous system, we examined the effects of in utero exposure to ethanol on gene expression in the postnatal brain. Pregnant rats were exposed to ethanol by constant inhalation of ethanol vapor during gestational days 8-21, generating a mean blood alcohol level of approximately 150 mg/100 ml during the last week of gestation. Particular care was given to ensure proper nutrition and weight gain in both control and EtOH-treated groups. At different times after birth, the brains of pups from ethanol-treated dams and from a parallel series of untreated dams were dissected into major regions. Cytoplasmic RNA was assayed for different mRNAs encoding proteins representative of both glial and neuronal cell types, by Northern blot and solution hybridization. In situ hybridization was performed with T alpha-1 tubulin and PLP. There were no significant differences between pups from ethanol-treated and control dams in the amount of mRNAs encoding the myelin protein proteolipid protein (PLP), the astrocyte glial fibrillary acidic protein (GFAP), the neurally enriched T alpha-1 isotype of alpha-tubulin, the nerve terminal components synaptophysin p38 and SNAP 25. The mRNA encoding the alpha 1-subunit of the receptor for gamma-amino butyric acid (GABA) was slightly decreased in expression in the hindbrain but not cortex of pups from EtOH-treated dams. These results suggest that, when weight gain is controlled, chronic exposure of rats to ethanol during the last 2 weeks of gestation has little influence on the postnatal expression of these cell type specific genes.

Gonadal steroids regulate the expression of glial fibrillary acidic protein in the adult male rat hippocampus

This study demonstrates that gonadal steroids (estradiol, testosterone, dihydrotestosterone) can regulate the expression of glial fibrillary acidic protein in the adult male rat brain. Previously, we showed that castration of adult male rats increased glial fibrillary acidic protein messenger RNA in the hippocampus and that this increase was additive with the increase induced by deafferenting entorhinal cortex lesions [ Day el al. (1990) Molec. Endocr. 4, 1995–2002]. We extended these effects of castration and entorhinal cortex lesion to glial fibrillary acidic protein, using immunoassays. Furthermore, we found regional differences in responses to castration and sex steroid replacement. In the hippocampus, glial fibrillary acidic protein expression was enhanced by castration and inhibited by sex steriods. In contrast, hypothalamic glial fibrillary acidic protein expression was inhibited by castration. Similar regional differences were also shown for astrocyte glial fibrillary acidic protein distribution by immunocytochemistry. The regional specificity of glial fibrillary acidic protein expression after castration and sex steroid replacement is pertinent to the role of astrocytes in synaptic plasticity in unlesioned adults as well as in responses to lesions where the steroid milieu has been shown to influence sprouting.

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: 1.(1) astrocytes co-expressing glial fibrillary acidic protein (GFAP) and vimentin2.(2) astrocytes expressing only GFAP3.(3) astrocyte-like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP-positive astrocytes and of those co-expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP-positive astrocytes, 67.2% were also vimentin-positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte-like vimentin-positive cells were located closest to the lesion margins. In comparison, the astrocytes co-expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP. This was considered as an argument against a hypothesis that reactive astrocyte division induces a two-stage increase in the cytoskeletal protein level in which synthesis of vimentin precedes that of GFAP.

Identification of canine glial cells by nonradioactive in situ hybridization.

Studies on the development of the canine central nervous system and on demyelinating diseases demand unequivocal identification of the glial cells. For that reason, nonradioactive in situ hybridization (ISH) was performed in primary dog brain cell cultures (DBCC) and in brain sections of neonatal dogs. Specific RNA probes were used to detect messenger RNA (mRNA) coding for proteolipid protein (PLP), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). PLP and MBP are markers for oligodendrocytes, GFAP for astrocytes. Oligodendrocytes positive for PLP and MBP mRNA were found in both DBCC and brain sections of neonatal dogs. Astrocytes expressing GFAP specific mRNA were detected in DBCC and in brain sections. These cells were evenly distributed in the white matter with additional accumulation in the membrana limitans gliae superficialis, around the ventricles and blood vessels. ISH clearly improves the study of oligodendrocytes in brain sections as, in contrast to the immunohistochemical methods, this technique allows to identify individual cells.