Age-related changes in glial fibrillary acidic protein mRNA in the mouse brain

Several RNA sequences were tested for age-related changes in prevalence levels in the mouse cerebral cortex, hippocampus, and cerebellum. In all three regions, there were increased levels of RNA for glial fibrillary acidic protein, an astrocyte-specific protein, by RNA gel-blot analysis and by a solution hybridization assay. There was no change in glutamine synthetase mRNA level, another glial protein. The only other mRNA sequence which changed was Thy-1 antigen, a neuronal protein, which decreased slightly in the hippocampus. We conclude that with age there is an age-related increase in glial fibrillary acidic protein RNA prevalence potentially reflecting an increase in the size, number, and/or fibrous character of astrocytes.     

Distribution of glial fibrillary acidic protein and vimentin-immunopositive elements in the developing chicken brain from hatch to adulthood

 The present study describes the distribution of glial fibrillary acidic protein (GFAP) and vimentin-immunopositive structures in the brain of the domestic chicken (Gallus domesticus) from hatching to maturity. The telencephalon is penetrated by a vimentin-immunopositive radial fibre system, representing a modified form of radial glia, in day-old chicks. Numerous fibres of this system persist until adulthood, mainly in the lobus parolfactorius, lamina medullaris dorsalis and lamina frontalis superior. GFAP immunoreactivity also appears in the course of development in these fibres. The distribution of GFAP-immunopositive astrocytes in the post-hatch telencephalon is like that found in adult chicken, except for the ectostriatum, in which an adult-like GFAP-immunostaining only develops during week three. This delay may be associated with a relatively slow maturation of this visual centre. In the diencephalon and in the mesencephalic tegmentum of day-old chicks GFAP-immunopositive astrocytes are confined to the border zone of several nuclei. In these areas as well as in the pons most GFAP positive astrocytes only appear gradually during the first two post-hatch weeks, although radial fibres occur only sparsely at hatch. Summarizing these results, a gradual replacement of radial fibres by astrocytes, typical of mammals, cannot be found in chicken. In the nucleus laminaris we observed a characteristic palisade of non-ependymal glia, reactive to GFAP but not to vimentin, which almost completely disappears by adulthood. We suggest that this glial system is instrumental in the development of the dendritic organisation of this nucleus. The optic tectum displays a dense array of GFAP-immunopositive radial glia at hatching, similar in this to the situation found in reptiles. However, in the tectum of reptiles this radial glia persists for the lifetime, whereas in the chick it disappears from the superficial tectal layers. This phenomenon may reflect the fact that there is no replacement of tectal cells or regeneration of retinotectal pathways in the chicken. In the early stage, the large cerebral tracts were found to contain dense accumulations of GFAP-positive cells, with peculiarly long outgrowths accompanying nerve fibres. No vimentin-immunopositivity was found in these glial elements; however vimentin was present in the glia situated at the optic chiasm, the anterior commissure and at other decussations. These structures, as well as the raphe, displayed the most intense vimentin-immunopositivity in the post-hatch chicken. This characteristic glial population may represent glial elements that have been reported to regulate fibre-crossing at the midline. Accepted: 18 March 1998     

Cell proliferation and glial fibrillary acidic protein in brain tumors

The results of histoautoradiographic and immunohistochemical studies of biopsy specimens of 15 brain tumours are reported. The specimens were labeled with 3H-thymidine using an in vitro technique. Meningiomas, oligodendrogliomas and well differentiated astrocytomas showed a median S-phase fraction of about 1%. In contrast, the labeling indices of 4 from 7 anaplastic astrocytomas were higher (2.1, 3.0, 3.5, 11.4). With increasing degree of malignancy the proliferative heterogeneity of the tumours increases. In every glioma varying amounts of glial fibrillary acidic protein (GFAP) were detected immunohistochemically (PAP technique). In 3 high-grade gliomas (2 glioblastomas, 1 anaplastic astrocytoma) an inverse relation of the investigated parameters (high S-phase fraction, low GFAP expression) was found. An exact prediction on biological behaviour of an individual tumour by GFAP detection immunohistochemically is not possible, because a high GFAP content can be detected also in some malignant tumours. However, the 3H-thymidine labeling indices of viable parts of the tumours, probably reflecting the growth fraction seem to be clinically important parameters, especially in respect to the prognosis.     

Immunocytochemical demonstration of glial fibrillary acidic protein in mouse tanycytes

Summary Immunohistochemical techniques were used to stain for the astrocytespecific glial fibrillary acidic protein (GFAP) in the cells lining the third ventricle of the developing and mature mouse brain. Before birth immunoreactive tanycytes were only observed in the infundibular recess of the median eminence, where they could first be seen at embryonic day 17. They possessed long processes running towards the ventral surface of the brain. During the early postnatal period GFAP-positive tanycytes gradually appeared throughout the third ventricle, although the ependymal cells themselves remained unstained. The tanycytes retained their immunoreactivity for anti-GFAP serum in the adult, and were also evident in the adult rat third ventricle. It is suggested that the presence of GFAP in these specialised cells of the third ventricle indicates that they, the transient radial glia of the developing cerebral cortex, the persistent Bergmann glia of the cerebellum, similar astrocytes with radial processes in the hippocampal dentate gyrus and conventional astroglia are all closely related cell types.     

Glial fibrillary acidic protein immunoreactivity in normal and diseased human breast

Summary Immunostaining for glial fibrillary acidic protein (GFAP) identifies a minor subpopulation of immunoreactive myoepithelial cells in the normal resting human breast. The GFAP-immunoreactive cells also express a panel of myoepithelial cell markers, including cytokeratin 14 (CK 14), vimentin, smooth-muscle-specific actin isoforms, nerve growth factor receptor (NGFR) and common acute lymphoblastic leukaemia antigen (CALLA). The percentage of GFAP-immunoreactive myoepithelial cells is greatly increased in various neoplastic and non-neoplastic diseases of the breast, being highest in adenomyoepitheliomas. Furthermore, in all the instances of fibroadenoma, phyllodes tumour, epitheliosis and gynaecomastia, a variable number of epithelial cells also acquires immunoreactivity for GFAP, vimentin, CK 14, NGFR and, to a lesser extent, for CALLA. Conversely, GFAP immunoreactivity has never been encountered in the malignant cells of the different types of breast carcinoma. These findings suggest that the expression of GFAP might be a (possibly transient) feature of proliferating epithelial and myoepithelial cells in breast diseases other than carcinomas.     

波形蛋白和胶质细胞纤维酸性蛋白在成年大鼠伸长细胞中的表达

目的：研究波形蛋白(VIM)和胶质细胞纤维酸性蛋白(GFAP)在成年大鼠伸长细胞(TAS)中的表达,观察TAS的特点并探讨胶质细胞的分类。方法：采用免疫组织化学方法,显示第三脑室腹侧壁和正中隆起(ME)TAS的特征。结果：在TAS中VIM的表达强度高,GFAP表达较弱,差异非常显著。对星形胶质细胞(AST)来说,不论使用Triton与否,GFAP均为高强度表达,而VIM为阴性。ME的TAS较第三脑室的突起较粗大分支较多。结论：无Triton存在下,VIM可以明确定位并区分第三脑室区域2种主要的胶质细胞TAS和AST;成年大鼠TAS还保持部分未成熟细胞的特性;ME是大量获得TAS的组织来源。     

Plectin regulates the organization of glial fibrillary acidic protein in Alexander disease

Alexander disease (AxD) is a rare but fatal neurological disorder caused by mutations in the astrocyte-specific intermediate filament protein glial fibrillary acidic protein (GFAP). Histologically, AxD is characterized by cytoplasmic inclusion bodies called Rosenthal fibers (RFs), which contain GFAP, small heat shock proteins, and other undefined components. Here, we describe the expression of the cytoskeletal linker protein plectin in the AxD brain. RFs displayed positive immunostaining for plectin and GFAP, both of which were increased in the AxD brain. Co-localization, co-immunoprecipitation, and in vitro overlay analyses demonstrated direct interaction of plectin and GFAP. GFAP with the most common AxD mutation, R239C (RC GFAP), mainly formed abnormal aggregates in human primary astrocytes and murine plectin-deficient fibroblasts. Transient transfection of full-length plectin cDNA converted these aggregates to thin filaments, which exhibited diffuse cytoplasmic distribution. Compared to wild-type GFAP expression, RC GFAP expression lowered plectin levels in astrocytoma-derived stable transfectants and plectin-positive fibroblasts. A much higher proportion of total GFAP was found in the Triton X-insoluble fraction of plectin-deficient fibroblasts than in wild-type fibroblasts. Taken together, our results suggest that insufficient amounts of plectin, due to RC GFAP expression, promote GFAP aggregation and RF formation in AxD.     

Immunocytochemical demonstration of glial fibrillary acidic protein in the cortical perivascular glial sheat of the cat

The peroxidase-antiperoxidase method was used on paraffin embedded material to demonstrate the distribution of the glial fibrillary acidic protein (GFA protein) in the perivascular glial membrane of the cortical blood vessels of cats. The nature of the dense zones was determined by ultrastructural immunocytochemistry with anti-GFA protein. Immunoreactivity was seen exclusively in the dense zones of the perivascular glia and in the astrocytic processes of the neuropil. The dense zones may exert a stabilizing function on the basal membrane during the changes in the caliber of the vessels.     

大鼠严重烧伤后脑内胶质原纤维酸性蛋白表达变化及意义

目的:探讨严重烫伤早期脑内胶质原纤维酸性蛋白(GFAP)表达变化与血脑屏障通透性变化的关系.方法:SD大鼠随机分为正常对照组、假烫组、烫伤组,其中烫伤组又分为烫伤后3、 6、 12、 24、 48 h等5组,建立30% Ⅲ度烫伤模型.应用免疫组织化学方法检测脑内GFAP表达变化,用化学法测量脑组织内伊文蓝的含量及脑百分含水量,并分析它们之间的关系.结果:烫伤3～6 h GFAP阳性细胞数目明显增多,染色增强,平均积分光密度较假烫组明显升高,烫伤后12～24 h阳性细胞数达高峰,48 h脑组织内阳性细胞数与对照组比较仍明显增多.严重烫伤后脑百分含水量及脑内伊文蓝含量增高,以6～12 h最为显著.结论:严重烫伤后GFAP表达上升可能与血脑屏障通透性增高有关.     

Delta opioid receptor mRNA distribution in the brain: Comparison to delta receptor binding and proenkephalin mRNA

The recent cloning of the mouse delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992) has demonstrated it to be a member of the seven transmembrane G-protein coupled family of neurotransmitter receptors. The present study describes the cellular localization in the central nervous system (CNS) of an mRNA encoding this receptor and compares it with the distribution of delta receptor binding and proenkephalin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. Delta receptor mRNA was visualized with a cRNA probe (472–903 bp) corresponding to transmembrane domains III–VI of the receptor, while proenkephalin mRNA was labeled with a cRNA probe to exon 3 (139–832 bp). A high level of correspondence was observed between the distribution of delta receptor mRNA and delta receptor binding as defined by the selective ligand [³H]d-Pen²-Pen⁵-enkephalin. Delta receptor mRNA and binding were expressed in the neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, diagonal band of Broca, amygdala and the nucleus of the solitary tract. Discrepancies in the distribution of delta receptor mRNA and binding in the olfactory bulb, hippocampus, globus pallidus and substantia nigra pars reticulata, may in part be due to differential receptor synthesis and transport. These results are discussed in relation to the distribution of proenkephalin mRNA and how this may affect our understanding of opioid circuitry in the CNS.     

Expression and localization of cartilage-specific matrix protein chondromodulin-I mRNA in salivary pleomorphic adenomas

Pleomorphic adenoma is the most common epithelial tumor in the salivary glands. This tumor frequently exhibits mesenchyme-like components, including myxoid or chondroid areas. Recently, using immunohistochemical techniques, we reported that cartilage-specific matrix protein, chondromodulin-I (ChM-I), was deposited on the inter-territorial matrix of the chondroid area in salivary pleomorphic adenomas and that ChM-I, which is also a strong angio-inhibitory factor, plays an important role in the avascular nature of the chondroid area and the chondroid formation in this type of tumor. To elucidate which cells express ChM-I mRNA in pleomorphic adenomas, we examined the expression and localization of ChM-I mRNA in this type of tumor using an in situ hybridization technique. Immunoreactivity for ChM-I was observed in the inter-territorial matrix of the chondroid area, especially around the lacunae, and in the cytoplasm of neoplastic myoepithelial cells of the myxoid element of pleomorphic adenomas. On in situ hybridization analysis, strong signals for ChM-I mRNA were detected in the cytoplasm of the lacuna cells of the chondroid element, and moderate to marked signals were observed in the cytoplasm of the neoplastic myoepithelial cells of the myxoid element. Signals for ChM-I mRNA were also seen in the cytoplasm of the spindle-shaped neoplastic myoepithelial cells in the transitional areas between the myxoid and chondroid elements of this tumor. Signals for ChM-I mRNA were not seen in the inner ductal cells or the fibrous element. These findings indicate that lacuna cells and neoplastic myoepithelial cells express ChM-I mRNA and that mature ChM-I, which lacuna cells and neoplastic myoepithelial cells translate, is deposited in the chondroid matrix of pleomorphic adenomas. In conclusion, lacuna cells and neoplastic myoepithelial cells express ChM-I mRNA ectopically in pleomorphic adenoma, and this plays an important role in chondroid formation and hypovascularity in this type of tumor.     

Distribution of phosphorylated glial fibrillary acidic protein in the mouse central nervous system

BACKGROUND: Glial fibrillary acidic protein (GFAP) is the principal component of intermediate filaments (IFs) in mature astrocytes in the central nervous system (CNS). Like other IF proteins, GFAP has multiple phosphorylation sites in the N-terminal head domain. The distribution of phospho-GFAP in vivo has not been elucidated. RESULTS: We generated Gfap(hwt) knock-in mice, in which the coding region for the head domain of GFAP is replaced with the corresponding human sequence. In combination with a series of monoclonal antibodies (mAbs) reactive to human phospho-GFAP, we visualized the distribution of phospho-GFAP in vivo in mice. GFAP phosphorylated at Thr7, Ser8 and/or Ser13 increased postnatally in the CNS of these mice. Limited populations of GFAP-positive astrocytes were labelled with anti-phospho-GFAP mAbs in most brain areas, whereas almost all the astrocytes in the optic nerve and spinal cord were labelled. Astrocytes in the subventricular zone and rostral migratory stream preferentially contained phospho-GFAP. In a cold injury model of the cerebral cortex, we detected phospho-GFAP in reactive astrocytes at 2-3 weeks after the injury. CONCLUSIONS: Phospho-GFAP provides a molecular marker indicating the heterogeneity of astrocytes, and Gfap(hwt) knock-in mice will aid in monitoring intracellular conditions of astrocytes, under various conditions. Our results suggest that the phosphorylation of GFAP plays a role in non-dividing astrocytes in vivo.     

星形细胞瘤的胶质纤维酸性蛋白和波形蛋白免疫组织化学定量研究

应用图像分析仪对２５例Ⅱ～Ⅳ级星形细胞瘤进行ＧＦＡＰ，ＶｌＭ免疫组织化学定量研究。结果表明：２５例星形细胞瘤全部呈ＧＦＡＰ、ＶＩＭ阳性反应，ＧＦＡＰ、ＶＩＭ免疫反应强度与肿瘤组织的分化程度有关，高分化的星形细胞瘤（Ⅱ级），ＧＦＡＰ反应强，ＶＩＭ反应弱，而低分化的星形细胞瘤（Ⅲ、Ⅳ级）中，ＶＩＭ反应强，ＧＦＡＰ反应弱，即ＧＦＡＰ与肿瘤恶性度负相关；ＶＩＭ与肿瘤组织恶性程度正相关。     

Double labeling of mRNA and protein markers in cultured embryoid bodies

Summary In vitro suspension cultures of embryonal carcinoma or embryonic stem cells (EC/ES) generate cell aggregates termed as embryoid bodies (EBs). EBs have been analyzed to study the mechanisms of cellular differentiation in vitro. The multipotency of EC/ES cells to differentiate into various cell types as well as the expression of many marker genes provides a valuable in vitro model system to study the mechanisms of cellular differentiation. Here we present a procedure for a mRNA detection of a specific gene using double labeling-mRNA probe and an antibody against cellular marker proteins. This double labeling analysis in combination with a culture of EBs provides a useful approach to analyze several mechanisms of cellular differentiation from multipotent EC/ES cells.     

脊髓损伤模型大鼠神经胶质细胞增殖与胶质纤维酸性蛋白的表达

背景：星形胶质细胞可以通过细胞裂解释放各种神经营养因子,并可促进损伤脊髓的修复。 目的：观察脊髓损伤模型大鼠神经胶质纤维酸性蛋白的表达及对其后肢功能恢复的影响。 方法：将SD大鼠采用Allen's法撞击T9~10节段致脊髓损伤,造模成功后蛛网膜下腔移植骨形态发生蛋白7,并设置仅蛛网膜下腔移植His蛋白的正常SD大鼠做对照。用BBB评分法评估两组大鼠后肢的运动功能,用免疫组织化学染色法和Western-blot法观察各组神经胶质纤维酸性蛋白的表达。 结果与结论：BBB评分结果显示,模型组大鼠脊髓损伤后下肢功能自行恢复率达68%。模型组脊髓损伤3和7 d,损伤区域神经胶质纤维酸性蛋白表达逐渐增加(P  < 0.05),随后逐渐下降,于脊髓损伤28 d后逐渐恢复到对照组水平(P > 0.05)。脊髓损伤后1~14 d两组胶质纤维酸性蛋白表达逐渐升高(P > 0.05)。结果证实,脊髓损伤后蛛网膜下腔移植骨形态发生蛋白7可诱导星形胶质细胞增殖,神经胶质纤维酸性蛋白的表达增强,进而促进脊髓损伤大鼠后肢功能的恢复。     

Thalamic reticular nucleus parcellation delineated by VIP and TRH gene expression in the rat

The distribution of the mRNAs encoding VIP (vasoactive intestinal peptide) and TRH (thyrotropin releasing hormone) was examined in the thalamic reticular nucleus of the adult rat using hybridization histochemistry with S35-labeled oligoprobes. Low levels of TRH expression were found in a medial tier. High levels of VIP expression were found in neurons located in a lateral shell of the same portion. High levels of TRH expression were found in a tier located dorsally and in a tier located ventrally to the first one. In these regions no VIP expression could be detected. These data suggest a parcellation of this nucleus according to the differential expression patterns of TRH and VIP.     

Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes

—Two antisera to glial fibrillary acidic protein from human brain and an antiserum to a 49 k dalton glial filament protein from human brain detected a cross-reacting antigen in the Schwann cells of the olfactory and vomeronasal nerves. The antigen was demonstrated at light- and electron-microscope levels. It was found throughout the cytoplasm and in association with cytoplasmic filaments of olfactory nerve Schwann cells in intact tissue and in Schwann cells grown in vitro.This observation, together with observations on the ultrastructure of olfactory nerve Schwann cells, relates them to central astroglia and to glial cells of the myenteric plexus, rather than to Schwann cells of other peripheral nerves. The unusual properties of olfactory nerve Schwann cells are of interest in relation to the regenerative abilities of the olfactory nerves.