反复前脑缺血大鼠脑白质区胶质纤维酸性蛋白表达变化

目的观察大鼠反复前脑缺血再灌注后不同脑白质区胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）表达的变化，探讨其规律，为对脑缺血后星形胶质细胞的进一步研究提供实验依据。方法反复夹闭大鼠双侧颈总动脉制备前脑缺血再灌注模型，免疫组化法检测脑缺血再灌注后1周、2周、4周胼胝体、内囊和脑室周围GFAP的表达。结果缺血再灌注后，不同部位各时间点GFAP的表达均高于假手术组水平；在胼胝体、内囊GFAP的表达在1周时增加，2周时持续上升，4周时更明显；而脑室周围则在1周时上升，2周时达高峰，4周时回落但仍高于1周时的水平。结论反复前脑缺血后白质区GFAP表达明显升高，但不同脑区变化的规律和幅度略有差异，说明不同脑区对缺血的敏感性不同，星形胶质细胞的反应性略有差异。     

Astroglial alterations in rat hippocampus during chronic lead exposure.

The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low-level lead exposure. The experiments combined immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [1 g% w/v (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother's milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion-fixation at 30, 60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP-positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead-exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.     

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

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

Upregulation of glial clusterin in brains of patients with AIDs

Since clusterin (CLU) production in reactive astrocytes may be neuroprotective, we examined its distribution in AIDS brains where brain injury and reactive astrocytosis are common. The relative area and number of CLU-positive astrocytes, as well as their percent total of all white matter glia, significantly increased in AIDS brains with and without HIV encephalitis (P<0.05). Proliferation markers were absent. In contrast, the relative area and number of GFAP-positive astrocytes and their percent of all white matter glia, increased in some cases but the mean increases were not significant. Clusterin is sensitive marker of glial reactivity in AIDS brains and its enhanced expression was not dependent on increases in GFAP.     

Transfection and overexpression of metallothionein-I in neonatal rat primary astrocyte cultures and in astrocytoma cells increases their resistance to methylmercury-induced cytotoxicity

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.     

Microglia, but not astrocytes, react to sciatic nerve injury in aging rats

Peripheral nerve axotomy activates microglia and astrocytes within regions of brainstem or spinal cord from which the nerve arises. The present study demonstrates that unilateral sciatic axotomy in rats 2 to 18 months of age results in differing responses with age between these two glial populations. By 4 days postaxotomy, both astrocytes and microglia become activated in 2-month-old rats, whereas only the microglial population shows evidence of activation in rats 8 to 18 months of age.     

Monoclonal antibody, KK1, recognizes human retinal astrocytes and distinguishes a subtype of astrocytes in mouse brain

Astrocytes exhibit a diverse morphology and numerous functions in the central nervous system as well as in the retina. In order to obtain markers for the analysis of astrocytes, we prepared monoclonal antibodies that recognized antigens specific to astrocytes. Monoclonal antibody (mAb), designated KK1, reacted with the processes of astrocytes in the nerve fiber layer and the ganglion cell layer in the human retina as detected by indirect immunofluorescence. Normal Müller cells, whose processes are localized vertically in retina, were not labeled by KK1 mAb. In mouse brain, KK1 mAb reacted specifically with astrocytes in the white matter, but not with those in the gray matter. Studies employing a high-resolution confocal laser scanning microscope and double-labeling with KK1 mAb and commercially available anti-glial fibrillary acidic protein (GFAP) mAb (GA5) revealed that KK1 mAb visualized the processes that were not recognized by anti-GFAP rnAb (GA5) in both human retina and mouse brain. In cultured mouse astrocytes. KKI mAb reacted only with anti-GFAP mAb (GA5)-positive cells, but a small percentage of anti-GFAP mAb (GA5)-positive cells were labeled with KK1 mAb. In addition, the subcellular distribution of the KK1 antigen in cultured astrocytes apparently differed from that of GFAP labeled by anti-GFAP mAb (GA5). The antigen that was purified from the normal mouse brain by KK1 mAb-conjugated beads reacted with anti-GFAP mAb(GA5) in immunoblotting. No reactivity of KK1 mAb was observed in immunohistochemical analysis in GFAP − / − mutant mouse brain. These results demonstrate that KK1 mAb specifically recognized an epitope of GFAP that did not react with other anti-GFAP mAb (GA5). Retinal astrocytes and a subtype of astrocytes in the white matter of mouse brain shared the epitope that was recognized by KKI mAb. KKI mAb might be a powerful tool to investigate a subtype of astrocytes.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

星形胶质细胞主要细胞骨架研究的最新进展

星形胶质细胞(Astrocyte,Ast)是中枢神经系统的主要细胞成分之一,约占正常成人中枢神经系统细胞总数的40％,其功能日益受到重视.其细胞骨架主要由微丝(MF)、微管(MT)和中间纤维(IFs)3种蛋白质组成,其细胞骨架的复杂变化在中枢神经系统的生理和病理变化中发挥重要作用,本文就生理病理条件下Ast细胞骨架的复杂变化(细胞骨架的生物学特性、信号转导途径、细胞骨架的构建与其在临床疾病中变化等)作一综述.     

Influence of basic fibroblast growth factor on carbonic anhydrase expression by rat glial cells in primary culture

Modifications of the morphology, the proliferation and the synthesis of carbonic anhydrase of glial cells in primary cultures maintained in defined medium have been investigated under the action of basic fibroblast growth factor. Cultures contained essentially three cell types: astrocytes which expressed glial fibrillary acidic protein, oligodendrocytes which were characterized by the presence of carbonic anhydrase and precursor cells in which these two proteins were detected by immunocytochemistry. In the presence of basic fibroblast growth factor astrocytes and oligodendrocytes underwent morphological changes, characterized by a fibrous aspect; astroglial cells acquired essentially several long processes and oligodendroglial cells formed generally two long processes. The factor increased the proliferation of these two cell types. The quantity of carbonic anhydrase per oligodendrocyte was enhanced in treated cultures. The double-stained precursor cells were present between days 7 and 11 of culture in defined medium, while in the presence of fibroblast growth factor these cells were more numerous and were still present after 14 days. The basic fibroblast growth factor stimulated the proliferation of these young glial cells and modified their morphology. But the differentiation of precursor cells towards one glial cell type appeared to be delayed.     

Regional and temporal progression of reactive astrocytosis in the brain of the myelin mutant taiep rat

Reactive astrocytosis in taiep rats was shown by glial fibrillary acidic protein (GFAP) immunoreactivity measured by means of enzyme-linked immunosorbent assay and indirect immunofluorescence. Increased GFAP immunoreactivity was first observed in the brainstem of 15-day-old taiep rats and was widespread throughout all brain regions at 6 months of age. Characteristically, astrocytes were hypertrophic and displayed strong GFAP fluorescence. The pattern of these reactive cells may correlate with the process of dysmyelination in the taiep rat.     

Analysis of cerebrospinal fluid glial fibrillary acidic protein after seizures in children

PURPOSE: To evaluate pediatric seizure patients for astrocytic injury by measuring cerebrospinal fluid (CSF) glial fibrillary acidic protein (GFAP), determine risk factors for GFAP elevation after seizures, and compare seizure-induced astrocyte injury with neuronal injury by concurrent measurement of CSF neuron-specific enolase (NSE). METHODS: CSF obtained from pediatric patients (n = 52) within 24 h of seizure was assayed for GFAP and NSE. Retrospective chart review was performed for seizure type, duration, and etiology. RESULTS: Overall, children with seizures had elevated CSF GFAP compared with controls (p = 0.0075), but no elevation of NSE (p = 0.1437). No effect of seizure type or etiology was found, but a significant positive effect of seizure duration (p = 0.0010) and status epilepticus (p = 0.0296) was seen on CSF GFAP. Individually, seven children (13%) had elevated GFAP (>440 pg/ml); in five children, the increased GFAP was not accompanied by elevations in NSE (<12 ng/ml). Five children with elevated GFAP had symptomatic etiologies for their seizures, but the etiology of one child with elevated GFAP was cryptogenic, and one had febrile seizures. CONCLUSIONS: Elevation of CSF GFAP after seizures suggests that astrocytic injury may occur in a subgroup of children, primarily in the context of prolonged seizures and symptomatic etiologies. Increased GFAP levels may occur in patients with normal NSE, suggesting that GFAP may be a more sensitive marker of brain injury in some cases.     

The postnatal development of glial fibrillary acidic protein and neurofilament triplet proteins in rat brain stem

Cytoskeletal preparations containing both the glial fibrillary acidic protein and the neurofilament triplet proteins were prepared from brain stems of rats at different ages and the individual peptides separated in polyacrylamide gels. Stained peptide bands were quantitated as the area under peaks generated by densitometric scanning. Peak areas were converted to grams of protein based on total gel dye binding and total protein applied to the gels. Between 5 and 30 days, the concentration of the peptide (g of peptide/mg of tissue protein) of apparent molecular weight 51,000 (corresponding to the glial fibrillary acidic protein), increased 3 fold. The corresponding increase in total concentration of the three peptides corresponding to the neurofilament proteins was 4.5 fold. However, the increase in concentration of the individual neurofilament peptides was each different. Very little of the apparent molecular weight 210,000 neurofilament peptide was present at 5 days and its concentration increased 11 fold by 30 days compared to about 3.5 fold for the other two neurofilament peptides. These results are in general agreement with studies using immunological techniques and the methods have the advantages of using readily available techniques and allowing the simultaneous comparison of both neuronal and glial specific filaments during development.     

Pathological reaction of astrocytes in perinatal brain injury

Summary Astrocytic reaction to various types of pre-and perinatal damage in the brain was studied using the immunohistochemical method for glial fibrillary acidic protein. The reactive gliosis could be detected as early as 20 weeks gestation. Reactive proliferation of the astrocytes could be seen already at 4 days after the insult. In addition to reacting to focal lesions, the astrocytes also proliferated diffusely throughout the white matter. The diffuse proliferation is the most significant finding in the evaluation of the perinatal damage, in both the acute state and in the long-term survivors.Supported in part by NIH Grant NS 06239. Part of the study was carried out while Dr. Roessmann was Visiting Professor at the University of Göttingen, supported by Deutsche Forschungsgemeinschaft, DFG-AZ: GO 76/100-1Presented in part at the IX International Congress of Neuropathology, Vienna, 1982     

Ultrastructural characteristics of glial fibrillary acidic protein expression in epoxy resin-embedded human brain tumors

Summary Thirteen surgically removed, epoxy resin (Durcupan ACM or Epon 812)-embedded human brain tumors were examined for glial fibrillary acidic protein (GFAP) content in semithin and ultrathin sections with the immunogold-silver staining method. Mild aldehyde fixation and the hydrophobic resin embedding did not interfere with the antigenicity, since silver intensification of the immunogold marker provided excellent visualization of the reaction on both light microscopic and ultrastructural levels. The GFAP reaction was usually localized on the glial intermediate filament bundles, usually correlating well with the amount of filaments. The unstained filamental regions of two ependymomas might correspond to the vimentin expression revealed by double labeling in semithin sections. Occasional GFAP immunopositivity without filamental appearance was observed in one of the oligodendrogliomas, as patchy electron-dense cytoplasmic corpuscules, in Rosenthal fibers and in some mainly necrobiotic tumor cells, reflecting a possible connection with glial filaments.     

Reactive astrogliosis after basic fibroblast growth factor (bFGF) injection in injured neonatal rat brain

Reactive gliosis was revealed by immunocytochemistry using antibodies against the glial fibrillary acidic protein (GFAP) after a stab or an electrolytic lesion administered to the cerebral cortex, corpus callosum, striatum, or hippocampus of a 6-day-old rat. The intensity of the gliosis was about the same in the various structures injured and did not change with the delay of 3, 7, or 20 days between the injury and the sacrifice of the animals. When basic fibroblast growth factor (bFGF) was injected in the lesion locus just after the lesion was performed, it resulted (as soon as 3 days after injury) in a strong astrogliosis that was enhanced after a delay of 7 days, the astrocytes in the lesion area exhibiting enlarged cell processes and intense GFAP-positive immunoreactivity. After a delay of 20 days, the astrocytes were not dispersed any more but packed in three or four layers along the borders of the lesion, thus reducing its extension. This suggests a possible role for bFGF in promoting scar formation following brain injury.     

In vivo induction of the growth associated protein GAP43/B-50 in rat astrocytes following transient middle cerebral artery occlusion

Immunohistochemistry was used to investigate the induction of growth-associated protein GAP43/B-50 in the astrocytes of rat cerebrum in vivo following ischemic injury produced by 30 min of transient middle cerebral artery occlusion. Three days after operation, GAP43 immunoreactivity first appeared in some astrocytic populations surrounding the infarcted lesion. Induction of GAP43 in those astrocytes persisted for up to 14 days and disappeared at 30 days postoperation. Double-immunofluorescence staining confirmed that the GAP43-immunoreactive astrocytes examined were all positive for glial fibrillary acidic protein. Our present data suggest that certain astrocytes could be induced to synthesize GAP43 in vivo in response to an ischemic insult in adult rats.     

Developmental patterns of intermediate filament gene expression in the normal hamster brain

We have examined the patterns of expression of the major intermediate filament (IF) protein mRNAs during development of the hamster brain. Quantitative northern blotting was used to examine changes in the levels of mRNAs for the low, middle and high molecular weight neurofilament proteins (NF-L, NF-M, NF-H) as well as peripherin, vimentin and glial fibrillary acidic protein (GFAP). Total RNA was isolated from hamster brains at embryonic (E) days 12 and 14 and postnatal (P) days 1, 3, 5, 7, 9, 11, 13, 15, 20, 28 and 60-90 (adult), and probed with specific IF cDNAs. Northern blotting revealed that NF-L and NF-M mRNAs were present at very low levels in embryonic brain and that significant expression of these genes only occurred postnatally when the levels increased dramatically until P28 and then declined again in the adult. Increases in NF-H mRNA levels were somewhat delayed relative to those of NF-L and NF-M. NF-H mRNA was not seen at embryonic stages and was expressed at very low levels prior to P9; after that time the levels increased rapidly until P28 and then declined in the adult. Two of the type III IF genes, peripherin and vimentin, followed a pattern of expression opposite that of the NF genes. Both peripherin and vimentin mRNAs were present in embryonic brain and were expressed at higher levels during early postnatal stages than at later times. The magnitude and rate of reduction in vimentin gene expression in the postnatal interval was much greater than that of peripherin. GFAP mRNA levels were extremely low prior to P9 after which a robust increase occurred, followed by a decline in the adult. We discuss the implication of the dramatic changes in IF isotype expression in brain to the pathways of both neuronal and glial development in vivo.     

睫状神经营养因子对坐骨神经切断吻合后大鼠脊髓前角胶质纤维酸性蛋白

背景：睫状神经营养因子具有多种生物活性,在神经系统发育、分化和损伤修复中具有重要意义。 目的：观察睫状神经营养因子对坐骨神经切断吻合后大鼠相应脊髓节段前角星形胶质细胞的特异标记物胶质纤维酸性蛋白表达的影响。 方法：将SD大鼠随机分为对照组、模型组、生理盐水组及药物组。除对照组外,对所有大鼠实施双侧坐骨神经切断吻合术,药物组手术区局部注射睫状神经营养因子100 ng/kg,1次/d,生理盐水组局部注射等量生理盐水。术后1,3,7,14,21,28 d取相应脊髓节段,免疫组织化学染色观察胶质纤维酸性蛋白的表达,苏木精-伊红染色、TUNEL染色对脊髓前角神经元进行计数。 结果与结论：大鼠坐骨神经切断吻合后相应脊髓节段星形胶质细胞胞体大,突起分枝多且粗大,神经元数目逐渐减少,凋亡神经元增多,胶质纤维酸性蛋白表达增高。与模型组和生理盐水组比较,药物组神经元存活数目增多,凋亡减少,胶质纤维酸性蛋白表达明显增加(P < 0.05或P < 0.01)。同时,药物组大鼠的运动功能障碍较轻,恢复较快。说明睫状神经营养因子可以通过促进大鼠脊髓前角胶质纤维酸性蛋白的表达起到神经保护作用。 关键词：胶质纤维酸性蛋白;睫状神经营养因子;星形胶质细胞;神经元凋亡;周围神经损伤     

Contrary effect of lactic acid on expression of neuron-specific enolase and glial fibrillary acidic protein in human glioma cells

Summary We examined the effect of lactic acid on cultured human glioma cell lines expressing glial fibrillary acidic protein (GFAP), vimentin and neuronspecific enolase (NSE). The growth of the cells was inhibited by the lactic acid in a dose-dependent manner. At 56 mM of lactic acid, the surviving cells of the KNS-42-c2 cell line developed slender processes and increasingly formed bizzar giant cells. In an immunofluorescence study of the lactic acid-resistant cells, the GFAP-positive cells prominently decreased in number, while the NSE-positive cells clearly increased. The vimentin was not affected throughout the experiment. After removing lactic acid from the medium, the GFAP-positive cells gradually increased in number. The method of dot immunoassay was useful for quantifying GFAP in cellular extracts. It indicated that the amount of GFAP decreased in the cells cultured with lactate-containing media and increased to the primary values after removing the lactic acid. These results may suggest that the morphological and immunochemical diversities of glioma cells are secondarily affected by cellular microenvironments such as lactic acid.Supported in part by Grant-in aid 624380309 from the Ministry of Education, Science and Culture, Japan