The effect of low doses of ionizing radiation on the intermediate filaments and the Ca2+-activated proteolysis system in the rat brain

The immunochemical methods were used to study the effect of low-level radiation (0.00645 C/kg and 0.0129 C/kg) on the content and polypeptide composition of glial intermediate filament proteins (GIFP) in different rat brain areas. Changes in glial fibrillar acidic protein (GFAP) concentration were more significant with the dose of 0.0129 C/kg than 0.00645 C/kg. It is suggested that soluble GIFP is more susceptible to the effect of Ca(2+)-dependent proteinases, calpains, than the filament one is, and degrades as early as the first few hours following irradiation. However, low radiation doses were ineffective with respect to calpains activity in the animal brain. The increased Ca2+ concentration enhances considerably GFAP degradation under the effect of calpains I and II. It is suggested that with low radiation doses the rearrangements of glial intermediate filaments may occur due to activation of calpains by releasing Ca ions.     

Glial Fibrillary Acidic Protein in the Cytoskeletal and Soluble Protein Fractions of the Developing Rat Brain

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer's disease

Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer's disease and therefore provides an ideal tissue to investigate incipient neurodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two-dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer's disease (AD). Two-dimensional electrophoresis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation.     

The protein of glial intermediate filaments in different areas of the rat brain at experimental neurosis

The content and polypeptide composition of glial fibrillary acidic protein (GFAP) in the rat cerebral cortex, cerebellum, hippocampus, and mesencephalon were studied under conditions of experimental neurosis. Significant changes of the total GFAP content were observed in the hippocampus, mesencephalon, and cerebellum. Both the content and polypeptide composition of soluble GFAP form were markedly modified. These changes of glial filament protein apparently reflect the peculiarities of the reorganization of the astrocyte intermediate filaments at the animal’s long-term neurotization.     

Distribution of Glial Fibrillary Acidic Protein in Gliosed Human White Matter

Glial fibrillary acidic protein (GFAP) in gliosed white matter from multiple sclerosis plaques and cerebral infarcts was examined by polyacrylamide gel electrophoresis and immunoblotting. Using a monoclonal antibody raised against human GFAP, up to 11 GFAP polypeptide bands of molecular weight 37-49 kilodaltons were identified in particulate and supernatant fractions of CNS tissue homogenates. Soluble GFAP constituted about one-quarter of the total GFAP in normal cerebral white matter. In brain lesions in which reactive astrocytes were observed microscopically, the proportion of soluble GFAP was increased, with a greater representation of the lower-molecular-weight forms. In brain chronic sclerotic plaques, almost all of the GFAP was in the particulate form. Purified particulate GFAP was susceptible to proteolysis at acid but not at neutral pH in the presence of CNS homogenates. In tissue autolysis studies, GFAP was stable in situ for periods well in excess of average CNS postmortem times.     

Characteristics of specific intermediate filament proteins in human brain tumors

The content of glial fibrillary acidic protein (GFAP) was measured in human brain tumours with different histological structure, origin and rate of malignancy. The polypeptide composition of CFAP was established in human brain and tumours by SDS polyacrylamide gel electrophoresis followed by immunoblotting. In tumours with an astrocyte type of differentiation, GFAP was revealed as a set of immunologically related and partially degraded polypeptides with a molecular weight of around and below 37 kD. It was assumed that the appearance of intact GFAP polypeptides (49 kD) in some tumours may be considered as a result of penetration of reactive astrocytes into tumour tissue.     

Effect of Vitamin E on the Content and Polypeptide Composition of Glial Fibrillary Acidic Protein from the Rat Brain under Conditions of Aluminum Chloride Intoxication

We studied the effects of isolated and combined chronic (21 days) introductions of aluminum chloride and vitamin E (α-tocopherol) on the polypeptide composition and content of glial fibrillary acidic protein (GFAP) in different brain structures of rats. Injections of AlCl₃ solution (12 mg/kg, i.p., daily) caused the appearance of low-molecular (47 to 38 kdalton) polypeptides and an increase in the content of GFAP in cytoskeletal fractions to 160 to 220%, as compared with the control. Introduction of vitamin E within the same interval provided significant normalization of the GFAP content in the brain of animals injected with AlCl₃ and to a considerable extent prevented the appearance of degraded polypeptides in the GFAP composition. We discuss the prospects of using vitamin E as an antioxidant for the correction of Al³⁺-induced pathological processes in the CNS.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 15–20, January–February, 2005.     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

Alzheimer's disease: the glial reaction is general and severe in all areas of the central nervous system

GFAP (Glial Fibrillary Acidic protein) was quantified in unfractionated homogenates of different brain regions from 10 Alzheimer patients versus 25 controls using immunoblot techniques and anti-human GFAP. There was a strong increase of GFAP in the brain regions that contained the characteristic Alzheimer lesions. This corresponds to the "astrocytic gliosis". Moreover, there was a 11 fold GFAP increase (p less than 0.001) in the other regions of the Alzheimer brains that do not present the Alzheimer pathology, such as caudate nucleus, cerebellum or brain stem. Different from the gliosis, the physiological signification of such an increase in the whole brain is unknown, but it might reflect the prominent part played by astrocytes during Alzheimer's disease (AD).     

Distribution of GFAP immunoreactive structures in the rhombencephalon of the sterlet (Acipenser ruthenus) and its evolutionary implication

Previous studies have revealed that although the brains of cypriniform teleosts (iberian barb, Barbus comiza; carp, Cyprinus carpio; goldfish, Carassius auratus) are rich in glial fibrillary acidic protein (GFAP), they have, however, areas devoid of GFAP immunoreactivity. The largest ones of these are in the rhombencephalon, e.g., the zones of the sensory and motor neurons in the vagal lobe. Our studies in amniotes suggested that the GFAP immunonegative areas could be characteristic of the more advanced brains (avian and mammalian), whereas no similar areas were found in reptiles. A similar tendency was found in the Chondrichthyes, i.e., GFAP immunonegative areas appeared as brain complexity progressed. The question arose whether the GFAP immunonegative brain areas in the Teleostei were also the result of such a tendency. Within the radiation of ray-finned fishes (Actinopterygii), Chondrostei represent a less advanced level as compared to the Teleostei. Therefore, the distribution of GFAP immunoreactivity was investigated in the rhombencephalon of the sterlet (Acipenser ruthenus) as a representative of Chondrostei, and in the carp. Serial vibratome sections were processed according to the avidin-biotinylated horseradish peroxidase method.Several comparable GFAP immunoreactive structures were found in the two species: the dense periventricular ependymoglial plexus, the midsagittal glial septum, the small glial septa separating the nerve fiber bundles, and the wide glial endfeet lining the meningeal surface. In the vagal lobe in the zones adjacent to the meningeal and ventricular surfaces, the glial structures also proved to be similar. In contrast to the carp, however, no areas were found devoid of GFAP immunoreactivity in the sterlet.The results suggest that this trend of glial evolution, i.e., GFAP immunonegative areas appearing as brain complexity progressed, is a common feature shared by Actinopterygii, Amniota, and Chondrichthyes, despite their separate evolutionary histories. J. Exp. Zool. 293:395-406, 2002.     

Alterations in monoamine oxidase activity of the mouse brain and liver after mixed neutron-gamma irradiation

Monoamine oxidase (MAO) plays an important role in the metabolism of neuro-transmitter biogenic amines. Its activity was determined in mouse brain and liver after exposure to different kinds of ionizing radiation and after pretreatment with a radioprotective agent. After a lethal dose of mixed neutron-gamma irradiation the MAO activity decreased in the brain and increased in the liver. In contrast, after a lethal dose of 60Co-gamma irradiation enzyme activity was considerably increased in the brain while in the liver it increased like after mixed neutron-gamma irradiation. AET (S2-aminoethyl-isothiuronium-Br X HBr), when administered in a radio-protective dose, inhibited MAO activity in the brain, while it increased in the liver. Even more marked changes of enzyme activity were observed in both brain and liver after AET pretreatment and mixed neutron-gamma irradiation. On the basis of the results it is suggested that different kinds of ionizing radiation lead to different types of lipid peroxidation in the lipid environment surrounding MAO, an event leading to altered enzyme activity. AET itself inhibited MAO in the brain and increased the activity in the liver but did not prevent the alterations caused by ionizing radiation in enzyme activity.     

Astroglia proteins in the rat brain in experimental chronic hepatitis and 2-oxoglutarate effect

A probable increase of the level of calcium-binding protein S100b and soluble form of glial fibrillary acidic protein (GFAP), as well as reducing the level of filament GFAP in the brain of Wistar rats under hepatic encephalopathy development caused by chronic hepatitis (HP) were shown. Increasing concentrations of S100b may stimulate the disassembly of intermediate filaments of astrocytes. The immunohistochemical analysis helps to reveal that astrocytes in the brain of rats that had HP lose the characteristic stellate shape and swelling. Immunoblotting result have shown the fragmentation of the main filament form of GFAP and appearance of low mass derivates. Application of 2-oxoglutarate (2.28 g/l of drinking water during 10 days after the onset of chronic hepatitis) stabilized the studied proteins and the state of astroglia.     

Enzyme-Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody

Two enzyme-linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP-specific antigenic determinant. One ELISA is a four-layer system working in the concentration range 5-600 ng GFAP/ml. The other ELISA is a five-layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5-60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2-14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.     

Changes in the Solubility of Glial Fibrillary Acidic Protein After Ischemic Brain Damage in the Mouse

Abstract: In the present study, changes in the content of glial fibrillary acidic protein (GFAP) in mouse cortex were investigated at different time intervals after unilateral middle cerebral artery occlusion. The GFAP content was assessed semiquantitatively by ELISA and immunoblotting. GFAP immunoreactivity was determined for each animal separately in protein fractions obtained from the ipsilateral, lesioned cortex and the contralateral, unlesioned cortex. Changes in the GFAP content of the lesioned cortex with respect to that of the unlesioned cortex were calculated for each fraction individually. GFAP was detectable in all protein fractions with a significant amount recovered from the aqueous extracts. A pronounced increase in the GFAP content of the lesioned cortex was observed. As measured by ELISA, this increase was maximal 5 days after injury and significantly more pronounced for the soluble and the Triton X-100-soluble protein fractions (mean increase 7 days after lesion, 281.4 and 240.2%, respectively) than for the crude cytoskeletal fraction (mean increase, 153.3%). A small and transient increase in GFAP immunoreactivity was also found in all protein fractions prepared from the contralateral, unlesioned cortex. These results were confirmed by immunoblotting.     

The effect of long-term lactobacilli (lactic acid bacteria) enteral treatment on the central nervous system of growing rats

The aim of this study was to explore the relationship between consumption of large doses of lactic acid bacteria (LAB) and the behaviour and brain morphobiochemistry of normal growing rats. Four groups of rats were treated with LAB cultures twice daily for 6 months. The control group received 1 ml of saline per treatment, while two experimental groups received 1 ml of living bacteria (Lactobacillus plantarum and Lactobacillus fermentum, respectively) and the remaining group received a heat-treated (inactivated) L. fermentum culture. After 2 and 6 months of treatment, respectively, eight animals from each group were sacrificed, and specimens were taken for further analyses. The behaviour of the rats was evaluated five times in an open-field test at monthly intervals throughout the study. Lactobacilli treatment for 2 months induced changes in the motoric behaviour of the rats. The concentration of the astrocytesoluble and filament glial fibrillary acidic protein (GFAP) decreased in the posterior part of the hemispheres, including the thalamus, hippocampus and cortex of the rats treated with L. fermentum. A greater decrease in filament GFAP (up to 50%) was shown in the group receiving the live form of L. fermentum. In contrast, the GFAP in the live L. plantarum-treated group increased, showing elevated levels of the soluble and filament forms of GFAP in the posterior part of the hemispheres.A 60–66% decrease in the amount of the astrocyte-specific Ca-binding protein S-100b was shown in the posterior parts of the hemispheres and in the hindbrain of rats given LAB for 2 months.Prolonged feeding with LAB for 4 months up to full adulthood led to a further decrease in astrocyte reaction, reflected as an additional decrease in the amount of soluble GFAP and locomotor activity in all experimental groups. The changes in filament GFAP and S-100b appeared to disappear after prolonged feeding (total of 6 months) with LAB.In summary, LAB dietary treatment affected the ontogenetic development of the astrocytes, with the highest intensity observed in the early stages of rat development. It can be postulated that LAB treatment may play a preventive role in neurological diseases by decreasing astrocyte reaction and, consequently, lowering locomotor activity.     

NDRG2与GFAP在不同脑区星形胶质细胞的表达与分布

目的:观察NDRG2(N-myc下游调节基因2)与GFAP(胶质纤维酸性蛋白)在不同脑区星形胶质细胞的表达与分布。方法:利用免疫荧光NDRG2与GFAP双标技术以及Western Blot技术观察皮层、海马及纹状体等不同脑区星形胶质细胞NDRG2和GFAP的表达与分布。结果:免疫荧光结果显示NDRG2阳性细胞广泛而均匀地分布于不同脑区,并与GFAP存在较好的共定位;NDRG2与GFAP标记的星形胶质细胞形态不尽相同。Western Blot结果显示NDRG2在皮层中表达比海马和纹状体多,而GFAP在海马中表达比皮层和纹状体多。结论:NDRG2广泛表达于不同脑区星形胶质细胞,并于GFAP存在较好的共定位。     

Identification of several forms of the glial fibrillary acidic protein, or alpha-albumin, by a specific monoclonal antibody

Abstract: A monoclonal antibody (mAb), termed UIA/ NEU/I/G1 (G1), that reacted with the astroglial marker glial fibrillary acidic protein (GFAP), or α-albumin, is described. It was directed against a structural determinant of GFAP. The G1 mAb could be used for quantitative determination of GFAP in two-site radiometric assays and for histoimmunological demonstration of GFAP. The G1 mAb reacted with the GFAP from rat as well as from man. The presence of several different molecular weight forms of GFAP in aqueous and detergent extracts from human brain was shown with the G1 mAb. The possible meaning of these forms is discussed.     

Expression of glial fibrillary acidic protein in the developing human brain]

It was shown that the glial fibrillary acidic protein (GFAP) content in developing (fetal) human brain is sharply increased. The expression of GFAP was observed already on the 7th-8th week after gestation, the GFAP concentration being less than 0.05% in comparison with adult brain. GFAP can be immunohistochemically detected in radial glial cells. At early stages of development the presence of antigenic determinants of 68 kDa and 100 kDa polypeptides interacting with monoclonal antibodies alongside with native GFAP (51 kDa) and its low molecular weight forms was demonstrated. These antigenic determinants cannot be detected at later stages of development and are absent in adult brain. The data obtained testify to changes in the gene expression of intermediate filament proteins at early stages of human brain ontogenesis.     

Immunoenzyme detection of specific brain antigens as a criterion of the permeability of the hemato-encephalic barrier in rats following acute gamma irradiation

The immunoenzyme detection systems for the measurement of the alpha-2 globulin of the brain (alpha 2M) and glial fibrillary acidic antigens (GFAP) were developed. These systems were used for the study of the penetration through hemato-encephalic barrier in rats subjected to gamma radiation. This method is recommended for the indirect evaluation of the hemato-encephalic barrier functional disorders.     

The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo

Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.