Significance of immunohistochemistry in neuro-oncology. I. Demonstration of glial fibrillary acid protein (GFAP) in extracranial metastases from primary brain tumors

8 cases were studied to determine whether immunohistochemical investigation with anti-GFAP could contribute to confirming a primary brain tumor origin for an extracranial metastasis. The materials studied consisted of 3 glioblastomas, 3 anaplastic astrocytomas, and 2 medulloblastomas, along with their extracranial metastases. GFAP could be immunohistochemically demonstrated in all 6 primary glial tumors as well as in the metastases of the 3 astrocytomas and of 2 glioblastomas. The medulloblastomas and their metastases were immunohistochemically GFAP-negative. GFAP is thus a marker for extracranial metastases of astrocytomas and glioblastomas. A negative result however does not exclude the possibility that a metastasis is of glial origin as shown by the GFAP-negative metastasis of the one glioblastoma.     

Importance of immunohistochemistry for neuro-oncology. II. Localization of factor VIII-associated proteins and glial fibrillary acidic protein (GFAP) in angiomatous and sarcomatous tumors

Immunhistochemical methods utilizing specific antibodies against Factor VIII-related antigen and glial fibrillary acidic protein were employed in studies of 48 intracranial and intraspinal tumors. Factor VIII-related antigen occurred only in endothelial cells of the vascular wall and is therefore not of importance for the differential diagnosis of CNS tumors. Isolated Factor VIII positive cells in the stroma of hemangioblastomas turned out to be mast cells which may also normally contain this substance. The GFAP positive cells in hemangioblastomas are believed to all be of astrocytic lineage. Many of the multinuclear giant cells present in monstrocellular sarcomas contained GFAP but were Factor VIII negative. Genuine fibroxanthoma of the meninges can apparently exist next to pleomorphic xanthoastrocytomas. As demonstrated by one of our cases, the demonstration of GFAP alone can successfully distinguish between them.     

Importance of immunohistochemistry for neuro-oncology. III. Demonstration of glial fibrillary acidic proteins (GFAP) in anaplastic astrocytomas and glioblastomas

Expression of gliofibrillary acidic protein in 23 anaplastic astrocytomas and 33 glioblastomas has been investigated and correlated with tumor behavior as reflected in both the length of the preoperative history and in the post-operative survival time. Three degrees of positive immunoreactivity to anti-GFAP can be distinguished: positive GFAP reaction in more than 2/3 of cells; in 1/3 to 2/3 of all cells; in less than 1/3 of all cells; negative reaction. All anaplastic astrocytomas and 27 of 33 glioblastomas showed GFAP positive reactions. The proportion of highly reactive tumors is higher by anaplastic astrocytomas than by glioblastomas (7 of 33). For both astrocytomas and glioblastomas there is a tendency for a decrease in the expression of GFAP to be associated with a shorter preoperative history and with a shorter survival time. This is more prominent for astrocytomas than for glioblastomas. This finding supports the opinion expressed in previous publications that the GFAP expression is reversely related to the level of tumor anaplasticity.     

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.     

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.     

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.     

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.     

Altered pattern of immunohistochemical staining for glial fibrillary acidic protein (GFAP) in the forebrain and cerebellum of the mutant spastic rat

The spastic rat is a neurological mutant of the Han-Wistar strain with prominent spasticity, tremor, and ataxia. Neurodegeneration is found in the CA3 sector of the hippocampus and in Purkinje cells of the cerebellum. We examined the forebrain and cerebellum of spastic rats for glial reactions by using immunolabelling for the astrocytic marker, glial fibrillary acidic protein (GFAP). First, a map of the GFAP-distribution was made representing a systematic series of frontal sections in controls. Reactive astrocytes with increased GFAP should occur in the areas with established neuronal degeneration, but they could also demarcate further regions with pathology in this rat strain. Since the baseline levels of GFAP-immunoreactivity differ between brain regions, control rats and clinically normal littermates served as controls to judge relative increases in major structures. In the CA3 sector and hilus of the dorsal hippocampus, a massive gliosis was detected. In the cerebellum, a patchy increase of GFAP labelling in Bergmann glia was found. Further increases of GFAP-labelling in reactive astrocytes occurred in fiber tracts, the ventral thalamic nuclei, medial geniculate nuclei, pontine region and optic layer of the superior colliculus. Inconsistent changes were noted in cortex and pallidum. No defects of glial labelling or malformations in glial architectonics were found. The reactive changes of astroglial cells in hippocampus and cerebellum are in proportion to the neuronal degeneration. The glial reactions in the other brain regions possibly reflect a reaction to fiber degeneration and incipient neuronal degeneration or functional alterations of glial cells in response to neuronal dysfunction.     

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300 mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1–10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.     

Astroglial architecture of the carp (Cyprinus carpio) brain as revealed by immunohistochemical staining against glial fibrillary acidic protein (GFAP)

The present paper is the first comprehensive study on the astroglia of a teleost fish that is based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein, an immunohistochemical marker of astroglia). The ray-finned fishes (Actinopterygii) and their largest group, the Teleostei, represent a separate pathway of vertebrate evolution. Their brain has a very complex macroscopic structure; several parts either have no equivalents in tetrapods or have a very different shape, e.g., the telencephalon. The results show that the teleost brain has a varied and highly specialized astroglial architecture. The primary system is made up of radial glia, which are of ependymal origin and cover the pial surface with endfeet. The tendency is, however, that the more caudal a brain area is, the less regular is the radial arrangement. A typical radial glia dominates some parts of the diencephalon (median eminence, lobus inferior and habenula) and the telencephalon. In the rest of the diencephalon and in the mesencephalon, the course of the glial fibers is modified by brain tracts. The most specialized areas of the teleost brain, the optic tectum and the cerebellum, display elaborate variations of the original radial system, which is adapted to their layered organization. In the cerebellum, an equivalent of the Bergmann-glia can be found, although its fiber arrangement shows meaningful differences from that of mammals or birds. In the lower brain stem radial glia are confined to fibers separating the brain tracts and forming the midline raphe. A dense ependymoglial plexus covers the inner surface of the tectum and the bottom of the rhombencephalic ventricle, intruding into the vagal and facial lobes. The structure and the position of the rhombencephalic plexus suggest that it corresponds to a circumventricular organ that entirely occupies the bottom of the ventricle. Perivascular glia show an unusual form as they consist of long fibers running along the blood vessels. In the large brain tracts long glial fibers run parallel with the course of the neural fibers. At least in the diencephalon, these glial fibers seem to be modified radial fibers. Real astrocytes (i.e., stellate-shaped cells) can be found only in the brain stem and even there only rarely. The glial specialization in the various areas of the teleost brain seems to be more elaborate than that found either in amphibia or in reptiles.     

The glial architecture of the median eminence of the Mongolian gerbil (Meriones unguiculatus); a study of glial fibrillary acidic protein (GFAP) immunoreactivity in semithin sections.

The glial architecture of the median eminence (ME) of the Mongolian gerbil (Meriones unguiculatus) was studied immunohistochemically. For this purpose, semithin sections of the proximal ME were processed according to the PAP technique using antibodies directed against glial fibrillary acidic protein (GFAP). Various glial cells were stained. Their distribution, the arrangement and morphology of their processes, and the spatial relations with adjacent tissue components could be examined in detail. Most of the immunoreactive cells were identified as either tanycytes (present throughout the internal zone, but preferentially located in the ependymal and subependymal layer), or as tanycyte-like cells (present throughout the external zone, but preferentially situated in the reticular layer). The processes of both cell types established numerous contacts with capillaries of the primary portal plexus in the external zone. Moreover, many projections of tanycyte processes to capillaries of the internal zone were revealed, most notably in the subependymal layer. Peculiar uni- and bipolar cells could be detected in the fibre layer of the internal zone, the processes of which were oriented parallel to the course of the axons of the hypothalamo-neurohypophyseal system. It was demonstrated that the methodology used to study the glial cells of the ME was also well applicable to the neural lobe. This technique, therefore, provides a valuable tool for the precise visualization of the majority of glial cells in the whole neurohypophysis of the gerbil. Thus, by sequential immunostaining of serial semithin sections investigations concerning the presence of multiple substances within single neurohypophyseal glial cells become possible.     

Significance of immunohistochemistry in neuro-oncology. V. Keratin as a marker for epithelial differentiation of primary and secondary intracranial and intraspinal tumors

Intermediate filament keratin is regarded as a good marker for epithelial and mesothelial tumors. In the intracranial and intraspinal spaces keratin has been demonstrated only in the endocrine cells of the adenohypophysis, squamous epithelial islands in the pars tuberalis of the hypophysis and in the choroid plexus epithelium. Since gliomas and meningiomas do not express keratin, this marker provides an additional help for differentiating between primary and secondary CNS tumors. Indirect immunofluorescence using an anti-keratin serum was used in a retrospective search for keratin in 80 tumors of the cranium and intraspinal space. Of the primary CNS tumors keratin positivity occurred in craniopharyngiomas, epidermoid tumors, pituitary adenomas, chordomas, a plexus papilloma as well as in the majority of germ cell tumors. Only 3 renal cell carcinoma metastases of 21 metastatic epithelial cell tumors (7 bronchial carcinomas, 6 breast cancers, 6 renal carcinomas, 1 rectum carcinoma, 1 cervix carcinoma) were keratin-negative. Similar findings were made in two melanoma metastases which we examined, whereas in a seminoma metastasis a few keratin expressing cells were found. Primary CNS tumors such as myxopapillary ependymomas, medulloepitheliomas, malignant meningiomas and paragangliomas which are often difficult to distinguish from these metastases proved to be keratin negative.     

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.     

Astroglial architecture of the carp ( Cyprinus carpio ) brain as revealed by immunohistochemical staining against glial fibrillary acidic protein (GFAP)

 The present paper is the first comprehensive study on the astroglia of a teleost fish that is based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein, an immunohistochemical marker of astroglia). The ray-finned fishes (Actinopterygii) and their largest group, the Teleostei, represent a separate pathway of vertebrate evolution. Their brain has a very complex macroscopic structure; several parts either have no equivalents in tetrapods or have a very different shape, e.g., the telencephalon. The results show that the teleost brain has a varied and highly specialized astroglial architecture. The primary system is made up of radial glia, which are of ependymal origin and cover the pial surface with endfeet. The tendency is, however, that the more caudal a brain area is, the less regular is the radial arrangement. A typical radial glia dominates some parts of the diencephalon (median eminence, lobus inferior and habenula) and the telencephalon. In the rest of the diencephalon and in the mesencephalon, the course of the glial fibers is modified by brain tracts. The most specialized areas of the teleost brain, the optic tectum and the cerebellum, display elaborate variations of the original radial system, which is adapted to their layered organization. In the cerebellum, an equivalent of the Bergmann-glia can be found, although its fiber arrangement shows meaningful differences from that of mammals or birds. In the lower brain stem radial glia are confined to fibers separating the brain tracts and forming the midline raphe. A dense ependymoglial plexus covers the inner surface of the tectum and the bottom of the rhombencephalic ventricle, intruding into the vagal and facial lobes. The structure and the position of the rhombencephalic plexus suggest that it corresponds to a circumventricular organ that entirely occupies the bottom of the ventricle. Perivascular glia show an unusual form as they consist of long fibers running along the blood vessels. In the large brain tracts long glial fibers run parallel with the course of the neural fibers. At least in the diencephalon, these glial fibers seem to be modified radial fibers. Real astrocytes (i.e., stellate-shaped cells) can be found only in the brain stem and even there only rarely. The glial specialization in the various areas of the teleost brain seems to be more elaborate than that found either in amphibia or in reptiles. Accepted: 15 May 1998     

星形细胞活性及GFAP在评价脑梗死中的作用及意义

目的:观察星形胶质细胞的超微结构及胶质纤维酸性蛋白(GFAP)脑梗死后的变化,并探讨GFAP在急性脑梗塞后的作用。方法:用双肾双夹易卒中型肾血管性高血压鼠(RHRSP)复制右大脑中动脉闭塞(MCAO)模型,分MCAO后1、3、6、9周末4个时间组,每组均进行脑梗死灶边缘区(A区),近顶叶皮层的远隔区(B区),对侧皮层与A区相对应的镜区(C区)的电镜观察,并测定各区的GFAP阳性细胞数目及光密度值。结果:脑梗死后星形细胞在全脑的增殖均较活跃。在脑梗死后GFAP阳性细胞数在边缘区最高,B区次之,C区最少。在缺血时间上,其改变以MCAO后1周末最高,3-6周末次之,9周末最少。GFAP阳性细胞浆平均光密度值变化情况与GFAP阳性细胞数目变化基本相同。结论:星形胶质细胞增殖程度和脑梗死后脑组织病变程度可用GFAP的表达值进行评价,增殖的星形胶质细胞在脑梗死后脑组织结构的修复中起着重要的作用。     

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.     

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve

Summary Normal and transected rat olfactory nerves were stained immunohistochemically using a monoclonal antibody previously shown to selectively detect GFAP-like immunoreactivity in central astrocytes but not in peripheral Schwann cells. Low levels of central type GFAP were found in the olfactory nerves, presumably in ensheathing cells. The levels of GFAP increased dramatically after nerve transection. A population of strongly GFAP-positive cells was detected at the junction between the olfactory epithelium and initial part of the nerves, of possible relevance to the regenerative abilities of this pathway.