Immunohistochemical distribution of S-100 protein and glial fibrillary acidic protein in normal and neoplastic salivary glands

Summary Immunohistochemical localization of S-100 protein, its and subunits, and glial fibrillary acidic protein (GFAP) in normal and neoplastic salivary glands was studied by the peroxidase-antiperoxidase method and immunoblot analysis. Positive immunostaining for S-100 protein was observed in pleomorphic adenoma, adenolymphoma, tubular adenoma, adenoid cystic carcinoma, acinic cell tumour, adenocarcinoma and carcinoma in pleomorphic adenoma. S-100 protein was localized in myoepithelial cells, epithelial cells of intercalated ducts and serous acinar cells of normal salivary gland. Both and subunits of S-100 protein showed almost identical distribution in normal and neoplastic salivary glands, but skeletal muscle cells were -positive/-negative whereas Schwann cells and fat cells were -negative/-positive in the stroma and neighbouring tissue. GFAP was only found in pleomorphic adenoma and its malignant counterpart. Immunoblot analysis showed that the GFAP-related antigen consisted of several polypeptide bands with a molecular weight ranging between 35,000 to 50,000 daltons.     

Immunohistochemical demonstration of glial fibrillary acidic protein in scrapie

Although little is known about its metabolism, glial fibrillary acidic (GFA) protein has become widely used as a cell-specific, species-non-specific antigenic marker for normal or pathologically altered astroglia. So far there have been few investigations on GFA protein in relation to scrapie and analogous spongiform encephalopathies although there has been a need for an unequivocal method for the discrimination of different types of glia in these diseases. In the present studies, a commercially available antiserum to GFA protein incorporated in a peroxidase-antiperoxidase procedure was applied to paraffin sections of brain and spinal cord from mice affected with scrapie, avirulent Semliki forest virus and cuprizone encephalopathy, and to tissues from healthy and scrapie-affected sheep. Excellent delineation of GFA protein was obtained in astroglial cell bodies and processes and in fibrils in the glia limitans and in perivascular and subependymal sites. The method was extremely sensitive and selective. A massive increase in GFA protein in scrapie-affected mice paralleled an increase in reactive astrocytes and facilitated the construction of astroglial lesion profiles for scrapie and the other encephalopathies. In sheep, abundant GFA protein occurred in both healthy and in scrapie-affected animals and in the tissues examined the differences were not conclusive.     

Immunohistochemistry of glial fibrillary acidic protein, vimentin and S-100 protein for study of astrocytes in hippocampus of rat

The distribution of astrocytes was studied in the hippocampus of mature Wistar rats. Immunohistochemistry was performed using antibodies against glial fibrillary acidic protein (GFAP), vimentin and S-100 protein and peroxidase anti-peroxidase techniques. Material from fresh-frozen brain, post-fixed in acetone, yielded a complex picture of the glial populations when stained for GFAP. Astrocytes immunoreactive for GFAP were seen in white matter tracts but also in the large dendritic layers of the hippocampus. Frozen material also contained different types of astrocytes following staining with a monoclonal antibody to vimentin. Stellate astrocyte types in the dendritic layers contained vimentin-stained processes. In addition a form of residual radial glia was found in the dentate gyrus. Material from brains perfusion-fixed with formaldehyde remained positive for astrocytic GFAP, but was negative for vimentin. Staining for S-100 protein antibodies revealed numerous astrocytes and diffuse background staining in fixed material. This study allows one to make predictions concerning the use of astrocytic markers in experimental pathological material.     

Altered glial fibrillary acidic protein immunoreactivity in rat brain following chronic hypoxia

Glial fibrillary acidic immunoreactivity in brain was examined in normal animals and in rats subjected to chronic hypoxia. Animals were exposed to a chronic normobaric adaptive hypoxia with decreasing amounts of oxygen (finally 6%) for a period of 59 and 114 days, respectively. In paraffin-embedded sections the glial fibrillary acidic protein immunoreactivity in normal and hypoxic animals was examined at three coronal levels. A mild glial fibrillary acidic protein immunoreactivity in the perivascular glial layer, external glial limitans membrane and periventricular astrocytes, as well as in some areas of hippocampus and cerebellum, was noted in normal animals. Chronic hypoxia for 114 days resulted in a marked increase of glial fibrillary acidic protein immunoreactivity in dentate gyrus of hippocampus, Bergmann glia of the cerebellum, internal capsule and pyramidal tract. On the other hand, the glial fibrillary acidic protein activity following 59 days hypoxic exposure was almost the same as controls. These results show that systemic deep chronic hypoxia (depending on the intensity and the duration) activates the endogenous expression of glial fibrillary acidic protein in astrocytes of specific brain regions. The probable significance of this finding is discussed.     

Localization of S-100 protein and glial fibrillary acidic protein-related antigen in pleomorphic adenoma of the salivary glands

Pleomorphic adenoma of the salivary glands was studied by the peroxidase-antiperoxidase method and Ouchterlony's double diffusion for the presence of so-called nervous system-specific proteins, S-100 protein, glial fibrillary acidic protein (GFAP), and astroprotein. Positive immunostaining for S-100 protein was observed in tumor cells of epithelial, myxomatous, and chondroid areas. An immunodiffusion test confirmed its presence in the tumor. Normal salivary glands were also stained with anti-S-100 serum, but the reaction was less intense than that of tumor. Specific immunostaining for GFAP and astroprotein was found in a small number of cells of myxomatous and chondroid areas and occasionally in cells in epithelial areas. An immunodiffusion test suggests that the GFAP-related antigen of the pleomorphic adenoma showing a minor heterogeneity of antigenic determinants is almost identical with GFAP. Normal salivary glands did not stain with GFAP or astroprotein antisera, nor did they react with anti-GFAP serum by immunodiffusion. Thus, the S-100 protein and GFAP-related antigen may be actively synthesized in adenoma cells during the course of tumor development. In addition, the GFAP-related antigen is considered to be a tumor-associated antigen of pleomorphic adenoma of the salivary glands.     

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.     

宫内感染后低龄大鼠脑组织中胶质纤维酸性蛋白的表达变化

目的:探讨胶质纤维酸性蛋白(GFAP)在宫内感染后低龄大鼠脑组织中的表达变化及其意义。方法: 对孕大鼠子宫内注入大肠杆菌建立宫内感染的大鼠模型,以子宫内注入生理盐水为对照组。两组分别于生后1、3、7、14及21 d取幼鼠脑组织,应用免疫组化方法检测脑组织中不同脑区GFAP的表达。结果: 生后1、3 d龄大鼠仅脑室旁白质区可见少许GFAP阳性细胞,两组细胞数无显著差异(P>0.05),其余脑区未见明显GFAP表达。感染组7日龄大鼠脑室旁白质和海马区GFAP阳性细胞数增多,与对照组比较差异显著(脑室旁白质区:9.73±3.55 vs 5.67±1.90,P<0.05;海马区:7.81±3.61 vs 2.16±1.11,P<0.05)。感染组14 d龄大鼠脑室旁白质、胼胝体及皮层区GFAP阳性细胞数增多,与对照组比较均有显著差异(脑室旁白质区:12.72±1.81 vs 9.00±0.93,P<0.01;胼胝体区:10.98±3.26 vs 4.44±1.15,P<0.01;皮层区:5.43±1.79 vs 2.71±0.67,P<0.01)。两组21 d龄大鼠各脑区GFAP阳性细胞数无显著差异(P>0.05)。结论: 宫内感染后低龄大鼠脑组织中GFAP表达增加。     

Distribution of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the rat brain

Summary The topographical mapping of glial fibrillary acidic protein (GFAP)-immunoreactivity was performed in coronal serial sections of the rat mesencephalon, rhombencephalon and spinal cord. Relative to a background of poor or moderate overall staining of the mesencephalon, the interpeduncular nucleus, substantia nigra and the periaqueductal grey matter were prominent by their intense GFAP-immunoreactivity. The pons and particularly the medulla contained more GFAP-labelled elements compared with the mesencephalon. The spinal trigeminal nucleus and Rolando substance were distinguished by their intense staining. Large fibre tracts were usually poor in immunoreactive GFAP. In a concluding discussion, findings relevant to the GFAP-mapping of the whole rat CNS are evaluated with regard to possible reasons underlying the observed differential distribution of GFAP-immunoreactivity.     

Distribution of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the rat brain

In the first of two papers dealing with the distribution of glial fibrillary acidic protein-(GFAP)-immunoreactive elements in the rat brain, the localization of immunostaining in the forebrain is systematically described. While the limbic cortex was found to contain intensely stained, evenly distributed astrocytes, the neocortex showed clearly stratified GFAP-staining, with substantially less immunoreactivity occurring in the middle layers than in the areas close to the brain surface or the white matter. A remarkably regular staining pattern was observed in the hippocampus and dentate gyrus. The striatum remained unstained in sharp contrast to the pallidum. In the diencephalon, the main thalamic nuclei were poor in GFAP-labelled elements in contrast to the internuclear border zones. In the hypothalamus, nuclei were conspicuous by their GFAP-staining. A consistent differential staining pattern was obtained in the epithalamic structures. The observed distributional pattern of diencephalic GFAP-immunoreactivity is thought to be due to different regional proliferation of the embryonic neuroepithelium of the diencephalon. The uneven distribution of GFAP-immunoreactivity in the forebrain is explained on a mainly developmental basis.     

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-μm slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semithin plastic sections of rat brain.     

Immunohistochemical demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.     

The concentration of glial fibrillary acidic protein increases with age in the mouse and rat brain

The role of aging in the expression of the astrocyte protein, glial fibrillary acidic protein (GFAP), was examined. In both mice and rats the concentration of GFAP increased throughout the brain as a function of aging. The largest increase (2-fold) was observed in striatum for both species. The neuron-specific proteins, synapsin I and neurofilament-200 (Mr 200 kilodaltons), were not altered by aging in any region of the mouse or rat brain. Brains of aged rats, but not mice, showed a decrease in beta-tubulin. The data suggest that astrocytic hypertrophy observed with aging involves an accumulation of glial filaments.     

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.     

Immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) in nasal gliomas

Using the Sternberger method (Immunoluk Histoset KIT) GFAP (glial fibrillary acidic protein) was demonstrated immunohistochemically in 4 nasal gliomas. In these histologically complex tumour-like lesions mesenchymal, epithelial, and neuroglial tissues as well as small groups of scattered glial elements could be differentiated specifically by the highly sensitive GFAP immunoperoxidase technique. GFAP was present in astrocytes and astrocyte-like differentiations. The reactivity of cell processes was essentially lower. The GFAP immunostain does not always correlate with Mallory's phosphotungstic acid hematoxylin (PTAH) stain and Gallyas' silver impregnation method for astrocytes. Additionally the immunohistochemical investigation of semithin sections prepared by the so-called pop off technique after Bretschneider et al. (1981) allows the correct localization of GFAP in astrocytes and their modulations. Furthermore, in this study, the intimate connection of epithelium and glial cells as well as astrocytes containing hemosiderin granules could be demonstrated. The latter findings suggest a possible phagocytotic activity of astrocytes. Our results show that the demonstration of GFAP by the Sternberger method is a valuable aid in establishing astrocytic glial differentiations and modulations in complex tumour-like lesions such as nasal gliomas.     

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.     

An immunohistochemical study on the distribution of glial fibrillary acidic protein, S-100 protein, neuron-specific enolase, and neurofilament in medulloblastomas

In order to clarify the differentiation of medulloblastomas, the authors studied on the morphological features and immunohistochemical expression of glial fibrillary acidic protein (GFAP), S-100 protein, neuron-specific enolase (NSE), and neurofilament (NF) in 31 medulloblastomas. GFAP was detected only in a small number of tumor cells of 5 medulloblastomas; S-100 protein in both small tumor cells and some so-called spongioblastic cells in 16 medulloblastomas; NSE in the more abundant tumor cells and the matrix in 28 medulloblastomas; NF in a few tumor cells of 12 medulloblastomas; GFAP and NF in 2 medulloblastomas, but each of them in different tumor cells. These results suggest that medulloblastomas have a capacity of differentiation along neuronal and/or glial lines. The conventional morphological markers of differentiation in medulloblastomas such as spongioblastic cells and Homer Wright rosettes were not necessarily compatible with expression of immunohistochemical markers such as GFAP or NF. NSE and S-100 protein seem less valuable markers of differentiation because they were detected in both neuronal and glial elements. But NSE, which was observed in most medulloblastomas, might have a value as a marker for medulloblastomas.     

An ELISA for glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of the astrocyte, and body fluid levels of GFAP are an important tool for estimating astrogliosis and astrocytic activation in vivo. This paper presents a new sandwich ELISA allowing quantification of GFAP(SMI26) from the cerebrospinal fluid (CSF). The sensitivity of the GFAP(SMI26) ELISA is 5 pg/ml with a recovery of 94% and a mean within- and between-batch precision of 6% and 10%, respectively. The upper reference value for CSF GFAP(SMI26) levels (9 pg/ml) was defined as the 95% cumulative frequency from 315 CSF samples. Based on this cut-off, a significantly higher proportion of patients with subarachnoid hemorrhage (100%), traumatic brain injury (100%), dementia (76%) and normal pressure hydrocephalus (85%) had pathologically elevated CSF GFAP(SMI26) levels compared to patients with peripheral nervous system disorders (0%). In a critical review of the literature, we compare the analytical and clinical sensitivity of previous GFAP ELISA methods with particular reference to patients with dementia.     

Region specific decrease in glial fibrillary acidic protein immunoreactivity in the brain of a rat model of depression

A growing body of evidence from human postmortem and animal studies suggests that deficits in glial cell (particularly astrocytes) density and function, in limbic regions of the brain contribute to the etiology of depressive disorders. Despite the widespread use of Wistar-Kyoto (WKY) rat strain as a model of depression and stress susceptibility, there is a paucity of data examining whether alterations in brain astrocytic population are present in the model. In the present study, we investigated the expression of the astrocytic markers glial fibrillary acidic protein (GFAP) in various brain regions in WKY rats in comparison to Sprague–Dawley rats. A significant deficit in GFAP-immunoreactive cells was found in the prefrontal cortex region (infralimbic, prelimbic and anterior cingulate cortex), in the basolateral amygdala as well as in the hippocampus (CA3 and dentate gyrus) in WKY rat brain. No statistical difference was found in the other brain regions analyzed (insular cortex, somatosensory cortex, CA1 and callosal white matter). No difference was found in the total density of astrocytes (assessed by s-100β immunoreactivity), neurons (determined by NeuN expression) or in the total number of cells in the regions of interest. A slight increase in the intensity of s-100β immunoreactivity was observed. The lower expression of GFAP in WKY rats was further confirmed by Western-blot analysis. These results suggest that specific astrocytic deficits in GFAP expression in corticolimbic circuits may be a general correlate of depressive-like behavior in animal models in addition to human major depression. Moreover, they suggest that glial physiology may become a therapeutic target in depression and other stress-related conditions.     

Glial fibrillary acidic protein in cryogenic lesions of the rat brain

Histologic sections of rat brains obtained at time intervals ranging from 30 min to 48 h following a cryogenic lesion placed on the surface of the parietal lobe, were immunocytochemically stained for glial fibrillary acidic (GFA) protein (glial filaments), IgG and albumin. Intense staining for the GFA protein and enlargement of astrocytes in the subcortical white matter and corpus callosum, were demonstrated near the lesion at 30 min and spread throughout the white matter up to 48 h. The early reaction of the astrocytes suggests that disassembly of glial filaments or new GFA synthesis may contribute to astrocytic changes.     

Immunocytochemical demonstration of glial fibrillary acidic protein in imprint smears of human brain tumors

Papanicolaou-destained imprint smears from 24 brain tumors were investigated by means of avidin-biotin-peroxidase complex method (ABC) with the use of monoclonal antibodies against glial fibrillary acidic protein (GFAP). Positive staining reaction to GFAP antibody has been demonstrated in cells from the following tumors: astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, mixed glioma, and ependymoma. The reaction for GFAP was negative for the following tumors: medulloblastoma, neurilemmoma, melanoma, hemangioblastoma, and metastatic tumors. In astrocytoma, the cell bodies and processes were positive with delicate fibrillary patterns; in anaplastic astrocytoma, cytoplasm and the processes were intensively stained. In glioblastoma multiforme, the staining patterns were also mixed, and the short, thickened processes were characteristic. Use of both a smear preparation and the immunoperoxidase staining technique is of great value in diagnosis of tumors of the central nervous system.