Effect of dexamethasone on glutamine synthetase and glial fibrillary acidic protein in normal and transformed astrocytes

The effect of dexamethasone on astrocyte differentiation was investigated in vitro, using cultures of normal and transformed astrocytes. The astrocyte-enriched proteins glutamine synthetase (GS) and glial fibrillary acidic (GFA) protein were used as markers of astrocyte differentiation. Ethanol, the vehicle for dexamethasone, decreases GS activity and increases GFA protein concentration in cultures of the established cell line U251MG and in the majority of cultures of transformed astrocytes derived from varying grades of astrocytoma. Ethanol has no effect on primary cultures of astrocytes derived from immature rat brain. Dexamethasone increases GS activity and decreases GFA protein concentration in cultures of U251MG and a grade IV astrocytoma-derived culture, in comparison with ethanol control. Our results show differential effects of two factors on cell-specific proteins in normal and transformed astrocytes.     

Cortical expression of glial fibrillary acidic protein and glutamine synthetase is decreased in schizophrenia

Altered expression of structural and functional molecules expressed by astrocytes may play a role in the pathophysiology of schizophrenia. We investigated the hypothesis that the astrocytic enzyme glutamine synthetase, involved in maintaining the glutamate-glutamine cycle, and the cytoskeletal molecule glial fibrillary acidic protein (GFAP) are abnormally expressed in schizophrenia. We used Western blot analysis to measure levels of glutamine synthetase and GFAP in several brain regions of subjects with schizophrenia and a comparison group. We found that glutamine synthetase protein expression was significantly decreased in the superior temporal gyrus, and both glutamine synthetase and GFAP were significantly reduced in the anterior cingulate cortex in schizophrenia. Neither molecule demonstrated altered expression in the dorsolateral prefrontal cortex, primary visual cortex, or hippocampus. Chronic treatment with haloperidol did not alter the expression of these molecules in the rat brain, suggesting that our findings are not due to a medication effect. These data support an astrocytic component to the pathophysiology of schizophrenia and suggest that astrocytic molecules involved in enzymatic activity and cytoskeletal integrity may have a role in disease-related abnormalities in this illness.     

Astroglial cell alteration caused by neurotoxins: immunohistochemical observations with antibodies to glial fibrillary acidic protein, laminin, and tyrosine hydroxylase

Kainic acid or 6-hydroxydopamine (6-OHDA) was injected into rat striatum, and their effects on astrocytes, laminin, and catecholamine fibers were examined temporally by immunohistochemical methods in an attempt to understand the roles of reactive astrocytes and laminin on the restoration of central nervous tissue. Kainic acid injection caused a severe neuronal degeneration in the striatum but catecholamine fibers were spared with only transient loss of tyrosine hydroxylase immunoreactivity. Reactive astrocytes appeared around the lesioned area soon after the kainic acid injection, then migrated into that area, and finally covered the lesioned striatum. Laminin immunoreactivity was found only in the lesioned area before the migration of reactive astrocytes and disappeared when the area was covered by astrocytes. 6-OHDA injection, on the other hand, resulted in a severe degeneration of catecholamine fibers, but striatal neurons were mostly spared. From 7 to 28 days after injection, regenerating fibers were found to enter the affected region. In this period reactive astrocytes were seen in the affected region but were only slightly more numerous than those found in control (saline injected) striatum. Laminin-immunoreactive blood vessels seemed to show a distribution similar to that in control striatum. These observations indicate that reactive astrocytes may play an important role in areas of neuronal cell loss and that laminin may aid their migration into such areas. Laminin and reactive astrocytes may not, however, be essential for the regeneration of dopamine fibers.     

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 significantde 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 over-expression 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.     

An immunohistochemical study of glial fibrillary acidic (GFA) protein and S-100 protein in the colon affected by Hirschsprung's disease

Summary The supportive cells of the enteric nervous system were examined in gut tissues from 15 patients with Hirschsprung's disease by means of immunohistochemistry, utilizing antisera to glial fibrillary acidic (GFA) protein and S-100 protein. In the normoganglionic segment, GFA protein immunoreactivity was predominantly found in association with the myenteric plexus and to a lesser extent in the submucous plexus. On the other hand, the extrinsic, hypertrophic nerve fasciculi were selectively immunostained with GFA protein antiserum throughout the entire length of the aganglionic intestinal walls from all children studied. The large fasciculi were numerous in the distal aganglionic segment and commonly appeared in the intermuscular zone and submucosal connective tissue. Both small-and mediumsized nerve fasciculi with GFA protein immunoreactivity were also encountered within the circular muscle layer of the proximal aganglionic segment. A subpopulation of supportive cells within the hypertrophic nerve fasciculi showed immunoreactivity for GFA protein, while all supportive elements of these fasciculi were stained for S-100 protein. The intrinsic nerve fibers within the circular muscle layer of normoganglionic segments were stained for S-100 protein, but not for GFA protein. The present study supports our previous findings that two types of supportive cells can be differentiated by immunohistochemistry in the enteric nervous system, utilizing antisera to GFA protein and S-100 protein. It is also concluded that the demonstration of GFA protein by immunohistochemical methods favors the diagnosis of aganglionic colons with Hirschsprung's disease, since GFA protein immunoreactivity is confined to the extrinsic, hypertrophic nerve fasciculi characteristic of aganglionic bowels.     

Selective vulnerability in the gerbil hippocampus following transient ischemia

Summary Following brief ischemia, the Mongolian gerbil is reported to develop unusual hippocampal cell injury (Brain Res 239:57–69, 1982). To further clarify this hippocampal vulnerability, gerbils were subjected to ischemia for 3, 5, 10, 20, and 30 min by bilateral occlusion of the common carotid arteries. They were perfusion-fixed after varying intervals of survival time ranging from 3 h up to 7 days. Following brief ischemia (5–10min), about 90% of the animals developed typical hippocampal damage. The lesion was present throughout the extent of the dorsal hippocampus, whereas damage outside the hippocampus was not observed. Each sector of the hippocampus showed different types of cell reaction to ischemia. Ischemic cell change was seen in scattered CA4 neurons, and reactive change was found in CA2, whereas CA1 pyramidal cells developed a strikingly slow cell death process. Ischemia for 3 min did not produce hippocampal lesion in most cases. Following prolonged ischemia (20–30min), brain injury had a wide variety in its extent and distribution. These results revealed that the gerbil brief ischemia model can serve as an excellent, reliable model to study the long-known hippocampal selective vulnerability to ischemia. Delayed neuronal death in CA1 pyramidal cells was confirmed after varying degrees of ischemic insult. These findings demonstrated that the pathology of neuronal injury following brief ischemia was by no means uniform nor simple.     

Transient ischemia stimulates glial fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus.

Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c-fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.     

Co-expression of glial fibrillary acidic protein and vimentin in reactive astrocytes following brain injury in rats.

The immunohistochemical expression of glial fibrillary acidic protein (GFAP) and vimentin (VIM) was studied in reactive astrocytes of the rat cerebral cortex 5 days after a brain injury. Seriated Epon semithin sections were immunostained alternatively for GFAP or VIM. Thereafter, both antigens were detected in consecutive sections of the same cell. Bordering the wound, an inner reactive glial layer 300-350 microns thick, showed positive astrocytes with the two immunohistochemical techniques. In this layer, about 60% of the GFAP positive astrocytes were also positive for VIM. Outside the inner layer, only GFAP positive astrocytes could be found.     

Determination of S-100 and glial fibrillary acidic protein concentrations in cerebrospinal fluid after brain infarction

BACKGROUND AND PURPOSE: We initiated the present study to evaluate the clinical value of consecutive concentration determinations of S-100 and glial fibrillary acidic proteins in cerebrospinal fluid from patients with brain infarction. METHODS: We took sequential samples of cerebrospinal fluid from 28 patients within 48 hours, at 7 days, and at 18-21 days after the ictus. We measured astroglial protein concentrations using an enzyme-linked immunosorbent assay and also determined size of the infarction (computed tomography), clinical state of the patient (simplified activities of daily living test), blood-brain barrier dysfunction (cerebrospinal fluid/serum albumin ratio), and a myelin marker (myelin basic protein). RESULTS: We found a transient increase of both proteins in the cerebrospinal fluid during the first week after the ischemic stroke (p less than 0.05). This increment was significantly correlated with the size of the infarction and the clinical state of the patients. CONCLUSIONS: Transient release of astroglial proteins into the cerebrospinal fluid possibly reflects initial focal ischemic damage and, in the later phase, ongoing destruction of astroglial cells in the penumbra zone. We suggest that determinations of cerebrospinal fluid astroglial protein concentrations can be used to estimate ischemic brain damage, which should be of particular value in clinical trials of pharmacological agents, such as calcium antagonists, on stroke patients.     

Specific alteration in the expression of glial fibrillary acidic protein, glutamate dehydrogenase, and glutamine synthetase in rats with genetic absence epilepsy

Astrocytes play a predominant role in energy metabolism and in the catabolism of gamma-aminobutyric acid (GABA) and glutamate, neurotransmitters critically involved in epileptic processes. We show specific astrocytic alterations in the genetic absence epilepsy rats from Strasbourg (GAERS). Spontaneous absence seizures appear in this strain in the cortex and thalamus after the age of 1 month. In these brain structures, we demonstrate increased GFAP expression in both adult and young GAERS, suggesting that reactive astrocytes are already present before the onset of seizures. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS), which are localized mainly in astrocytes and involved in glutamate catabolism, are shown to be differentially altered. GDH expression was increased in the thalamus of both young and adult GAERS and in the cortex of young GAERS. GS expression was slightly decreased in the thalamus of young GAERS. These astrocytic modifications are not adaptive responses to seizures, as the modifications appear before the development of absence seizures. Thus, astrocytes might be involved in the neuronal processes giving rise to epileptic seizures in this strain.     

Reduced glial fibrillary acidic protein and glutamine synthetase expression in astrocytes and Bergmann glial cells in the rat cerebellum caused by delta(9)-tetrahydrocannabinol administration during development

In this study we analyzed the responses of cerebellar astroglial cells to pre- and perinatal delta(9)-tetrahydrocannabinol (THC) exposure in three postnatal ages and both sexes. To determine whether THC during development directly modifies astroglial growth, this study investigated the effects of THC on astroglial morphological changes and on the expression of specific astroglial markers (glial fibrillary acidic protein: GFAP and glutamine synthetase: GS). Our results demonstrated that the administration of THC during development has deleterious effects on astroglial maturation in the cerebellum. These results also indicate that THC might interfere with astroglial differentiation in a way dependent on sex. The effect of cannabinoids on the development of cerebellar astroglial cells (astrocytes and Bergmann glial cells) is to reduce protein synthesis, since both GFAP and GS decreased in astroglial cells, not only during THC exposure but also in adult ages. Our data suggest that pre- and perinatal THC exposure directly interferes with astroglial maturation by disrupting normal cytoskeletal formation, as indicated by the irregular disposition of GFAP and the lower GFAP expression observed at all the ages studied. THC exposure during development may also modulate glutamatergic nervous activity since GS expression is reduced in THC-exposed brains. GS expression increased progressively after THC withdrawal, but GS expression had still not reached control values two months after THC withdrawal. This indicates that glutamate uptake is lower in glial cells exposed to THC, since GS expression is lower than in older controls. Consequently, glutamatergic neurotransmission may be affected by cannabinoid exposure during gestation. Therefore, cannabinoids exert developmental toxicity, at least on astroglial cells, which could contribute to fetal brain growth retardation.     

Medulloblastoma with glial and rhabdomyoblastic differentiation. A myoglobin and glial fibrillary acidic protein immunohistochemical and ultrastructural study

This is a report of an unusual, densely cellular, midcerebellar neoplasm in a seven-year-old boy. Although clinically consistent with a medulloblastoma, immunohistochemistry and electron microscopy demonstrated glial and rhabdomyoblastic differentiation in the tumor. We discuss the differential diagnosis of this tumor as it relates to glial differentiation in medulloblastomas and the myogenic potential of primitive neuroectoderm.     

Developmental expression of glial fibrillary acidic protein and glutamine synthetase in serum-free aggregating cell cultures of fetal rat telencephalon

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by a combined biochemical and double-labeling immunocytochemical study for the developmental expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). It was found that these two astroglial markers are co-expressed at different developmental stages in vitro. During the phase of cellular maturation (i.e. between days 14 and 34), GFAP levels and GS activity increase rapidly and in parallel. At the same time, the number of immunoreactive cells increase while the long and thick processes staining in early cultures gradually disappear. The present results demonstrate that in this particular cell culture system only one type of astrocytes develops which expresses both GFAP and GS and which attains a relatively high degree of maturation.     

Quercetin inhibits c-fos, heat shock protein, and glial fibrillary acidic protein expression in injured astrocytes

Quercetin, a bioflavonoid, is found widely in many kinds of fruits and vegetables. It is known to engage in many bioactivities, such as interfering with of the progress of stress responses to injury. In the present study, we investigated the effects of quercetin on some injury responses in primary cultures of astrocytes. These injury responses included the elevation of c-fos protein, heat shock protein (HSP70), and glial fibrillary acidic protein (GFAP). After heat shock insult, the levels of c-fos protein and HSP70 in astrocytes increased. With quercetin treatment, these proteins were significantly reduced. The inhibition of these injury responses by quercetin in astrocytes indicated a dose dependency, with the highest effect at 100 microM. We have previously established a scratch injury model in a primary culture of astrocytes. In that model, astrocytes responded to the scratch injury by an elevation in their GFAP level and formation of hypertrophic cell processes, which extend into the scratch areas. Quercetin treatment reduced the number of hypertrophic cell processes being extended into the scratch areas. With 100 microM of quercetin, there was a complete inhibition of the formation of the hypertrophic cell process. Western blot analysis for GFAP indicated that quercetin significantly reduced the induction of GFAP in the scratch model. At 100 microM, the total GFAP content in the injured cultures was reduced to a level lower than that of the control. This implied that quercetin might possess an antigliotic property.     

Effect of methotrexate on glial fibrillary acidic protein content of astrocytes in primary culture.

We have recently reported that methotrexate (MTX) causes degenerative as well as reactive-like astroglial changes and alters the cell cycle kinetics of astrocytes in vitro. To further characterize the nature of the reactive-like changes that were noted by light and electron microscopy following MTX exposure, the glial fibrillary acidic protein (GFAP) content of astrocytes in culture was investigated by enzyme-linked immunosorbent assay, flow cytometry and double-immunofluorescent staining. An increase in GFAP content which did not correlate with drug dosage or DNA synthesis was noted in the MTX-treated cultures. It is postulated that this increase in GFAP content of astrocytes reflects an adaptive response to MTX-induced injury and partly explains the gliosis that is seen in methotrexate encephalopathy.     

Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein

The topographical features of the neuroglial network in the mature rat CNS and the differentiation of fibrous neuroglia in the neocortex and hippocampus have been studied by immunofluorescence using antibodies to the glial fibrillary acidic (GFA) protein. In the mature brain and spinal cord the distribution and appearance of neuroglia as observed with immunofluorescence were similar to those described by Weigert ('95) using his method for astrocytic fibers. In the developing rat immunofluorescence started to appear on the surface of the neocortex in the first week after birth. In 9-day-old rats a continuous external glial membrane had formed by immunofluorescence. Immunofluorescence of the perivascular glial membrane appeared later. Blood vessels completely surrounded by a fluorescent membrane were not observed in the middle cortical layers before the eighteenth day of life. An unexpected finding in the hippocampus was the sudden appearance in the 8-day-old rat of a radial system of immunofluorescent fibers crossing the granular layer and extending in the molecular layer. The radial fibers were still present in the adult rat. However, they became widely spaced and thus less prominent than in the immature animal. In fetal rats immunofluorescent fibers first appeared in the medial wall of the lateral ventricles on the eighteenth post-conceptionaly day. The radial system of glial fibers extending from the ventricles to the surface of the brain and characteristic of this stage of development was not stained by immunofluorescence.