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.     

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.     

Significance of immunohistochemistry for neuro-oncology. VI. Occurrence, localization and distribution of glial fibrillary acid protein (GFAP) in 820 tumors

In a retrospective study 820 tumors were immunohistochemically examined with anti-GFAP. All 224 astrocytomas and 105 of 112 glioblastomas were, at least focally, positive. 72% of ependymomas and 64% of oligodendrogliomas contained tumor cells which expressed GFAP. In such entities the reaction is dependent on the histologic subtype. Only 26 of 114 medulloblastomas (22.8%) demonstrated scattered GFAP positive cells. GFAP was also demonstrated in the CNS in gangliogliomas, monstrocellular sarcomas, 3 of 6 PNET, one non-classifiable tumor in a child, 1 plexus papilloma, in scattered stromal cells in 15 of 26 hemangioblastomas as well as in the mature glial component of intracranial germ cell tumors. Outside of the CNS there was evidence of GFAP in 3 cases with nasal glial heterotopy and in the myxoidal part of a pleomorphic salivary gland adenoma. Neoplasms which proved negative to GFAP in our series included purely neural differentiated tumors meningioma, neurolemmomas, chordomas, paragangliomas, sarcomas, lymphomas, melanomas and carcinoma metastases. Separating GFAP-positive reactive astrocytes from the actual tumor cells has proved to be a problem in the routine use of GFAP in differential diagnosis. Absence of an immunohistochemical response does not exclude a tumor of glial origin. Tissue samples which are too small, particularly in the case of anaplastic astrocytomas and glioblastomas can give false negative results.     

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.     

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。     

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.     

Expression of glial fibrillary acidic protein, actin, fibronectin and factor VIII antigen in human astrocytomas

Frozen sections and cell cultures of 50 human astrocytomas, fetal and adult human brain were examined for immunofluorescence reactivity with antisera to glial fibrillary acidic protein (GFAP), actin, fibronectin and factor VIII antigen. In frozen tissue sections GFAP expression was restricted to normal and neoplastic astrocytes while fibronectin and factor VIII antigen were localized to blood vessels. In primary cell culture, 80-100% neoplastic astrocytes expressed GFAP but not fibronectin or factor VIII antigen while actin was present as diffuse cytoplasmic staining of the cell body and cell processes. By the 5th-6th passage in vitro, GFAP immunoreactivity was lost while fibronectin and actin cables were prominently expressed. Factor VIII antigen remained negative throughout serial subculture. In double fluorochrome experiments, GFAP positive cells did not express fibronectin or actin cables, while GFAP negative cells expressed fibronectin and had prominent actin cables. Our results suggest a change in population of astrocytoma cells with increasing passage in vitro, reflecting either an overgrowth of tumour glioblasts or dedifferentiation of tumour astrocytes.     

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.     

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     

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.     

Glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in dogs infected with canine distemper virus

An experiment based on astrocyte immunoreactivity to glial fibrillary acidic protein (GFAP) was designed to determine whether the astrocyte response in canine distemper encephalitis (CDE) was associated with the age of the animal, type of lesion and the cerebellar region affected. Four histopathological types of CDE lesion were examined, namely acute (11 dogs), acute with necrosis (four dogs), subacute (22 dogs) and chronic (six dogs). The animals were divided into three age groups, namely, 0-2 years (27 dogs), 2.1-4 years (12 dogs), and 4.1-12 years (four dogs). Three different cerebellar regions were evaluated. Cerebellar sections from three healthy dogs were used for control purposes. The highest number of astrocytes occurred in the cerebellar white matter and in dogs with acute distemper encephalopathy. In animals with subacute distemper encephalitis, the numbers of astrocytes appeared to increase with age, but the opposite effect occurred in dogs with acute or chronic encephalitis; age appeared not to influence the astrocyte numbers in dogs suffering from acute encephalitis with necrosis.     

Glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes are increased in the hypothalamus of androgen-insensitive testicular feminized (Tfm) mice.

In the hypothalamus of androgen-insensitive testicular feminized (Tfm) mice the normal pattern of immunohistochemical staining for glial fibrillary acidic protein (GFAP) is markedly different from normal. Along the borders of the third ventricle and in the dorsomedial and arcuate nuclei, the numbers of stained astrocytes are increased. The usual ordered array of tanycytic processes is obscured by a tangle of GFAP-stained stellate glial cells. GFAP immunostaining in other regions of the Tfm forebrain is similar to that in normal mice. These results suggest that the distribution of reactive glia in the hypothalamus may have been changed as a consequence of the genetic defect in Tfm mice.