Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in the rat brain

The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of glial filaments, was studied on coronal sections of the globus pallidus, the area CA4 of the hippocampus and the arcuate nucleus of the hypothalamus, 3 estrogen-sensitive areas of the rat brain. The number and the surface density of the GFAP-immunoreactive cells were evaluated in 6 adult ovariectomized rats injected with a single dose (20 mg/kg) of estradiol valerate (OVX + E2 rats) and in 6 ovariectomized littermates injected with vehicle (OVX rats). Two days after the injection, a similar distribution of the GFAP was observed in the arcuate nucleus of OVX + E2 rats when compared to OVX rats, whereas a significantly (P less than 0.001) increased surface density of GFAP immunoreactive material was observed in the globus pallidus and hippocampus of estradiol-treated rats. Since the number of GFAP-positive cells was unchanged by the estradiol injection, the enhanced surface density of GFAP immunoreactive material in the hippocampus and globus pallidus suggest a possible influence of estradiol on GFAP-immunoreactive glial processes.     

Development of glial fibrillary acidic protein immunoreactivity in thyroidectomized rats

The majority of astroglia develop postnatally in rats. GFAP (glial fibrillary acidic protein)-immunoreactivity appears mainly during the 2nd and 3rd postnatal weeks throughout the brain. Hypothyroidism inhibits, among others, the cell proliferation, maturation, and migration of neurons. However, hardly any data on the effect of hypothyroidism on GFAP-immunoreactivity are available in the literature. In our experiments, thyroidectomy was performed between the 3rd and 5th postnatal days. Operated and control animals from the same litter were perfused transcardially and processed for immunohisto-chemistry in parallel after 2, 3, and 4 wk. On the basis of serial sections, the development of GFAP-immunoreactivity was not generally affected by hypothyroidism. We could observe only two phenomena that showed a tendency of retardation in the operated animals: (1) the decrease of the strong GFAP-immunopositivity of white matter tracts (for example, internal capsule and pyramidal tract) and (2) the gradual disappearance of the GFAP-immunoreactive radial fibers (for example, in the neocortex, in the olfactory bulb, and around the 3rd ventricle).     

Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus

The influence of gonadal steroids on the ultrastructure of glial cells and on the immunoreactivity for the specific astrocytic marker glial fibrillary acidic protein (GFAP) has been assessed in the neuroendocrine hypothalamus. The following parameters were analyzed in the arcuate nucleus of adult female rats: the number and the surface density of cells immunoreactive for GFAP, the number of glial profiles showing bundles of glial filaments, the size of the bundles of glial filaments, and the proportion of neuronal perikaryal membrane apposed by glial processes. These parameters were studied during the different phases of the estrous cycle, after ovariectomy, and after the administration of estradiol or progesterone to ovariectomized rats. No significant differences were detected in the number of GFAP-immunoreactive cells among the different experimental groups. The surface density of GFAP-immunoreactive material, the number of glial profiles in the neuropil, and the proportion of neuronal perikaryal membrane covered by glia were increased in the afternoon of proestrus and in the morning of estrus compared with other phases of the estrous cycle or to ovariectomized rats and showed a rapid (5 h) and reversible increase in ovariectomized rats injected with 17β estradiol, with a maximal effect by 24 h after the administration of the hormone. In contrast, the size of the bundles of glial filaments was decreased in the afternoon of proestrus, in the morning of estrus, and by the administration of estradiol to ovariectomized rats. The parameters studied were not affected by the administration of progesterone. However, progesterone (300 μg/rat) blocked the effects of 17β estradiol (1, 10, and 300 μg). The results suggest that glial cells may be actively involved in the modulation of neuroendocrine events by the hypothalamus. © 1994 Wiley-Liss, Inc.     

MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain

Exposure of a limited brain surface to a high potassium (K+) concentration produces an injury limited to the underlying cortex, without apparently affecting other brain areas. Such a treatment produces an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes, as assessed by immunohistochemical techniques, throughout the cortex ipsilateral to K+ exposure. This effect is evident 2 days after treatment and persists up to, at least, day 7. Thirty days after K+ exposure GFAP immunostaining is similar in both hemispheres. Administration of the non-competitive NMDA antagonist MK-801 (4 mg/kg i.p.) prior to the injury prevented the rise in GFAP immunoreactivity (IR) at 2 but not 7 days after the treatment. Administration of MK-801 after the injury appeared to have no effect on GFAP expression. This work confirms that brain injury, associated with spreading depression, can induce a glial response far from the lesion site. Furthermore, the fact that this phenomenon can be modified by an NMDA receptor antagonist suggests that glutamate may play a role, in vivo, in the regulation of astrocytic response to injury and introduces the possibility that brain injury-induced gliosis may be pharmacologically manipulated.     

Extra-neural glial fibrillary acidic protein (GFAP) immunoreactivity in perisinusoidal stellate cells of rat liver

The dendritic processes and perinuclear cytoplasm of stellate-shaped perisinusoidal cells in frozen sections of rat liver were specifically labeled with antisera raised independently to glial fibrillary acidic protein (GFAP), the major component of intermediate filaments in astrocytes. A liver protein co-migrating with authentic GFAP and immunoreactive with GFAP antisera was demonstrated with immunoblots of brain and liver extracts enriched in intermediate filament proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study presents yet another example of immunoreactivity to GFAP, or a highly similar protein localized outside the CNS, in cells of mesenchymal origin exhibiting some morphological features common to astroglia.     

Postnatal development of glial fibrillary acidic protein immunoreactivity in the hamster arcuate nucleus

The postnatal development (from 2 days to 1 year) of glial fibrillary acidic protein (GFAP) immunoreactive cells was studied in the arcuate nucleus of male hamsters. In the first postnatal week, GFAP immunoreactivity was observed in radial glial cells whose cell bodies were located in the ependymal layer. Cell processes of GFAP immunoreactive radial glia crossed the arcuate nucleus and reached the pial surface, where they formed a thin and incomplete external limiting membrane. During the second postnatal week, some immunoreactive cell bodies were also located far from the ependymal layer. Some of these cell bodies presented processes that made contact with the ependymal layer whereas others, probably corresponding to maturing astrocytes, did not show ventricular connections. In the third week, only astrocytes showed GFAP immunoreactive perikarya and their immunoreactive processes reached either the blood vessels to form end-feet, or the basal hypothalamic zone to form the glia limitans. In successive weeks, there was an increase of the amount of GFAP-immunoreactive profiles on the glia limitans and surrounding the arcuate nucleus blood vessels. After the 6th postnatal week we observed some GFAP-immunoreactive cells close to arcuate neurons. The number of these cells increased from the 8th postnatal week. From this age on GFAP immunoreactive astrocytic processes compartimentalized the arcuate nucleus defining several rows of aligned neurons. These results indicate that the cytoarchitectonic organization of GFAP immunoreactive elements and their relationship with neurons, blood vessels and pia is not completed until the first 8 weeks of postnatal life in the arcuate nucleus of the hamster.     

Ovarian cycle-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus

The interpeduncular nucleus (IPN) of female rats was studied across the estrous cycle to observe whether the expression of the astroglial marker, glial fibrillary acidic protein (GFAP) reacts to hormonal changes in an area not belonging to the 'endocrine brain'. A marked reduction of immunoreactive GFAP was observed in estrus as compared to the immunoreactivities in met- and proestrus. This finding is consistent with earlier observations in the endocrine hypothalamus, but also proves that gonadal steroids influence astroglia in brain regions not involved in neuroendocrine regulation. Since cyclic fluctuations of synaptic numbers in the female have been described only for the endocrine hypothalamus, decrease of immunoreactive GFAP in the IPN during estrus may reflect a down-regulation of GFAP synthesis.     

Extracellular ATP induces stellation and increases glial fibrillary acidic protein content and DNA synthesis in primary astrocyte cultures

A number of factors appear to be involved in the proliferative and hypertrophic processes which characterize reactive astrocytosis. We have investigated the possibility that ATP, an agent that is released by injured cells following tissue destruction, may be one such factor. For this purpose, we utilized primary cultures of astrocytes derived from cerebral cortices of neonatal rats to study the effect of extracellular ATP on properties associated with astrògliosis. Light microscopic studies disclosed marked stellation of astrocytes after 30–60 min of exposure to 100 M-1 mM ATP. In addition, the content of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), was increased 35–40% following 60-min exposure to ATP; this effect persisted for 1–3 days of exposure to 100 M ATP. [³H]Thymidine incorportion increased progressively from 1–3 days; a 3.6-fold increase in DNA synthesis was observed following 3 days of exposure to 1 mM ATP, suggesting stimulation of cellular proliferation. These findings show that high micromolar to low millimolar concentrations of extracellular ATP reproduce several features associated with reactive gliosis and suggest that extracellular ATP may be involved in the activation of astrocytes following CNS injury.Supported by the Department of Veterans Affairs (Merit Reviews and GRECC) and the NIH (DK 38153 and NS 30291)     

Immunoreactive glial fibrillary acidic protein in pituicytes of the rat neurohypophysis

The glial fibrillary acidic protein (GFAP) has been associated with glial filaments. Electron microscopic examination of rat pituicytes in our laboratory has revealed few of these 8–9 nm filaments that are present in other astrocytes. Since the literature is inconsistent on the existence of filaments in pituicytes, we investigated the content of GFAP in these cells. Immunocytochemical methods revealed a strong positivity for GFAP in pituicytes. Furthermore, the primary antiserum dilution required for optimal staining suggests that there may be more GFAP in pituicytes than in other glial elements. The significance of immunoreactive GFAP in pituicytes is discussed in terms of possible functions and embryonic origins.     

Colocalization of taurine and glial fibrillary acidic protein immunoreactivity in mouse hippocampus induced by short-term ethanol exposure

Morphological changes of the hippocampus were investigated in mice exhibiting signs of intoxication following short-term exposure to 6% ethanol. These alterations were examined by a double immunofluorescent study using antibodies to taurine and anti-glial fibrillary acidic protein (GFAP) antibody. Antibody-labeled taurine was localized mainly in the astrocytes and endothelial cells of control mice. Ethanol administration resulted in a significant increase in the accumulation of taurine and GFAP immunoreactivity (IR) in the stratum lacunosum-moleculare (sl-m) of the hippocampus. Specifically, the cell bodies of taurine-positive astrocytes were hypertrophied, their processes were elongated in the pericapillary region, and some colocalized with GFAP-IR cells. Furthermore, quantitative analysis revealed that the merged area in ethanol-treated mice was twice that (71.6% vs. 35.8%) of control mice. Since taurine is involved in various neuroprotective functions, the present observations suggest that the expression of taurine IR in reactive astrocytes after ethanol exposure might play an important role in neuroprotective processes.     

Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect.

We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone.     

Colocalization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain

Angiotensinogen is produced in the brain, but its precise localization and the cells in the central nervous system producing it are unknown. We have performed a double staining test for angiotensinogen and glial fibrillary acidic protein in rat brain and report here that these proteins colocalize in astrocytes.     

A method to quantify glial fibrillary acidic protein immunoreactivity on the suprachiasmatic nucleus.

The aim of the present study was to develop a quantitative method to measure the immunoreactivity of the glial fibrillary acidic protein on the suprachiasmatic nucleus of hamster. For this purpose, optical microscopy images from brain sections processed for glial fibrillary acidic protein immunostaining were digitised under different light conditions. Image treatment and immunoreactivity quantification were performed following five different methods using the program Adobe Photoshop. The results were analysed in order to determine the ability of each method to differentiate immunoreactivity levels, and their susceptibility to the light conditions during image acquisition. Four of the five methods were found to be susceptible to the tested light conditions, while the other was not. This last method, which permits detection of differences in immunoreactivity between the different sections, was considered for further quantification of glial fibrillary acidic protein immunoreactivity on the suprachiasmatic nucleus.     

Colocalization of fructose-1,6-bisphosphatase and glial fibrillary acidic protein in rat brain

Immunofluorescence studies of rat brain sections demonstrated an exclusive colocalization of the gluconeogenic key enzyme fructose-1,6-bisphosphatase (FBPase) with the astroglial marker glial fibrillary acid protein, indicating FBPase in brain as an astrocyte-specific enzyme. This conclusion was supported by the presence of FBPase activity in astroglia-rich but not neuron-rich primary cultures derived from rat brain.     

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.