Specific monoclonal antibodies to glial fibrillary acidic protein (GFAP)

Anti-glial fibrillary acidic protein (GFAP) monoclonal antibody has been obtained by fusing SP2/O myeloma cells with splenic cells from mice immunized with human GFAP. It belongs to the IgG1 class and it recognizes an epitope on GFAP which is shared by each fragment of the protein. Immunohistological studies show that the epitope characterized is absolutely specific for GFAP.     

Phosphorylation of the glial fibrillary acidic protein

The glial fibrillary acidic protein (GFAP) was found to be phosphorylated in vivo after intracerebral injection of [³²P]-orthophosphate, in brain Slices, and in a cell free system. The phosphorylated proteins were separated by two-dimensional gel electrophoresis and then transferred to nitrocellulose sheets. Two isoelectric variants of GFAP were immunochemically identified by monoclonal antibodies. Autoradiography demonstrated that only the more acidic isoelectric variant of GFAP was phosphorylated. Phosphoamino acid analysis revealed that under all conditions GFAP was phosphorylated at serine and threonine residues. Incubation of brain slices with[³²P]-orthophosphate and the protein kinase C activator phorbol 12-myristate 13-acetate or forskolin, an activator of cyclic AMP-dependent protein kinase, stimulated phosphorylation of GFAP. Likewise phosphorylation of GFAP was also accentuated by calcium/phosphatidylserine/diolein and by exogenous cyclic AMP-dependent kinase in a cell free system. These findings announce that protein kinase C and cyclic-AMP dependent kinase may play physiologic roles in the in situ phosphorylation of GFAP. When isolated cytoskeletal preparations were incubated with [γ-³²P] ATP, GFAP was phosphorylated in vitro by two additional protein kinases, a Ca⁺⁺/calmodulin-depen-dent kinase and an effector-independent kinase. The results of these investigations strongly suggest that phosphorylation of GFAP appears to be regulated by multiple second messenger pathways.     

Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein

The topographical features of the neuroglial network in the mature rat CNS and the differentiation of fibrous neuroglia in the neocortex and hippocampus have been studied by immunofluorescence using antibodies to the glial fibrillary acidic (GFA) protein. In the mature brain and spinal cord the distribution and appearance of neuroglia as observed with immunofluorescence were similar to those described by Weigert ('95) using his method for astrocytic fibers. In the developing rat immunofluorescence started to appear on the surface of the neocortex in the first week after birth. In 9-day-old rats a continuous external glial membrane had formed by immunofluorescence. Immunofluorescence of the perivascular glial membrane appeared later. Blood vessels completely surrounded by a fluorescent membrane were not observed in the middle cortical layers before the eighteenth day of life. An unexpected finding in the hippocampus was the sudden appearance in the 8-day-old rat of a radial system of immunofluorescent fibers crossing the granular layer and extending in the molecular layer. The radial fibers were still present in the adult rat. However, they became widely spaced and thus less prominent than in the immature animal. In fetal rats immunofluorescent fibers first appeared in the medial wall of the lateral ventricles on the eighteenth post-conceptionaly day. The radial system of glial fibers extending from the ventricles to the surface of the brain and characteristic of this stage of development was not stained by immunofluorescence.     

An unusual meningioma variant with glial fibrillary acidic protein expression

We studied a recurrent meningioma located in the right frontal lobe. The tumor showed high cellularity and the cells had plump, hyalinous cytoplasm. Immunohistochemically, almost all the tumor cells were positive for epithelial membrane antigen and vimentin, and unexpectedly, glial fibrillary acidic protein (GFAP). Ultrastructural investigation revealed abundant 8- to 10-nm filaments in the cytoplasm. Conspicuous interdigitations with numerous desmosomes were present. Frequently, intracellular and intercellular lumina lined by microvilli were also found. We considered the present case to be an unusual variant of meningioma with GFAP expression. A few cases of meningioma with triple expression of GFAP, vimentin and cytokeratin have been reported previously. However, the present case showed obvious pathological differences from these, and had no immunoreactivity for cytokeratin. Received: 30 December 1996 / Revised, accepted: 20 May 1997     

Measurement of glial fibrillary acidic protein (GFAP) and anti-GFAP antibodies by solid-phase radioimmunoassays

A solid-phase radioimmunoassay was developed for the detection of glial fibrillary acidic protein (GFAP) and GFAP-specific immunoglobulin G (IgG) antibodies. In antibody assays purified GFAP was adsorbed onto polystyrene beads, followed by incubation in dilutions of serum.
(125-I)-labelled human anti-IgG was used to quantify antibodies bound to solid-phase GFAP. GFAP in samples was measured by the inhibition of the binding of anti-GFAP antibody to solid-phase GFAP.
The serum and CSF samples of 19 brain tumor, 40 multiple sclerosis and 66 control patients were assayed for anti-GFAP antibodies. The binding values in the serum and CSF samples of the brain tumor patient group were higher and the binding values in the CSF samples of the multiple sclerosis group lower than those of the control group. The differences were statistically significant.     

Monoclonal antibody to glial fibrillary acidic protein reveals a parcellation of individual barrels in the early postnatal mouse somatosensory cortex

The relative dispositions of cells in immature and mature barrel field cortices that bind antibody to glial fibrillary acidic protein (GFAP) were examined and photographed under the light microscope. Light micrographs demonstrate that radially oriented glial cells are present in the barrel field of postnatal day 6 cortices and that they are located predominantly within the presumptive barrel sides and/or septae, thus sharply delineating individual barrels from each other. The relative dispositions of radial glial fibers observed at this time implicate glia in development of topographic order during early postnatal development of the somatosensory cortex. In contrast, no such delineation could be detected in the cortices of more mature mice, because GFAP-positive astrocytes are present throughout the barrel field and are not confined to barrel sides. This ephemeral nature of the GFAP-delineated barrel field is of interest with respect to the recently reported ephemeral lectin-delineated barrel field.     

Co-expression of glial fibrillary acidic protein and vimentin in reactive astrocytes following brain injury in rats.

The immunohistochemical expression of glial fibrillary acidic protein (GFAP) and vimentin (VIM) was studied in reactive astrocytes of the rat cerebral cortex 5 days after a brain injury. Seriated Epon semithin sections were immunostained alternatively for GFAP or VIM. Thereafter, both antigens were detected in consecutive sections of the same cell. Bordering the wound, an inner reactive glial layer 300-350 microns thick, showed positive astrocytes with the two immunohistochemical techniques. In this layer, about 60% of the GFAP positive astrocytes were also positive for VIM. Outside the inner layer, only GFAP positive astrocytes could be found.     

Development of glial fibrillary acidic protein immunoreactivity in thyroidectomized rats

The majority of astroglia develop postnatally in rats. GFAP (glial fibrillary acidic protein)-immunoreactivity appears mainly during the 2nd and 3rd postnatal weeks throughout the brain. Hypothyroidism inhibits, among others, the cell proliferation, maturation, and migration of neurons. However, hardly any data on the effect of hypothyroidism on GFAP-immunoreactivity are available in the literature. In our experiments, thyroidectomy was performed between the 3rd and 5th postnatal days. Operated and control animals from the same litter were perfused transcardially and processed for immunohisto-chemistry in parallel after 2, 3, and 4 wk. On the basis of serial sections, the development of GFAP-immunoreactivity was not generally affected by hypothyroidism. We could observe only two phenomena that showed a tendency of retardation in the operated animals: (1) the decrease of the strong GFAP-immunopositivity of white matter tracts (for example, internal capsule and pyramidal tract) and (2) the gradual disappearance of the GFAP-immunoreactive radial fibers (for example, in the neocortex, in the olfactory bulb, and around the 3rd ventricle).     

Immunofluorescent staining of frozen sections for glial fibrillary acidic protein

Summary Immunofluorescent staining for glial fibrillary acidic protein (GFAP) has been used as part of the diagnostic evaluation of eleven patients and compared with routine special stains. In one case, a difficult fibrillary neoplasm of the spinal cord, this diagnostic procedure provided rapid, positive identification of the glial nature of the tumor. In all cases, the GFAP reactivity was consistent with staining properties of PTAH and more rapid than PTAH.     

Spreading depression increases immunohistochemical staining of glial fibrillary acidic protein

Reactive astrocytosis is a process by which astrocytes respond to brain injury by showing an increase in glial fibrillary acidic protein (GFAP) staining that is associated with hypertrophy and/or hyperplasia of these cells. Because spreading depression (SD) is a perturbation uncomplicated by neuronal necrosis and is seen in both in vivo and in vitro neural structures, we sought to determine whether SD was a sufficient stimulus to induce enhanced GFAP staining. SD was elicited in anesthetized rats by application of KCI to parietal cortex for 3 hr; equimolar NaCI was applied to contralateral cortex. SD was confirmed by monitoring DC potentials in frontal neocortices. Animals were allowed to recover for 48 hr, and their brains were processed for semiquantitative and computer-based analyses of GFAP staining intensity. Experimental GFAP staining was referenced to contralateral control levels. Neocortical SD (13-37 SDs) was associated with a significant (p less than 10(-4)), 43% increase in GFAP staining intensity, which remained statistically greater than normal for more than 2 weeks. If SD was inhibited by combined hyperoxia and hypercarbia, only a nonsignificant (p greater than 0.20), 7% increase in GFAP staining was seen. Thus, SD may be a useful physiologic process with which to begin to explore the cellular mechanisms that induce the transformation of normal astrocytes into reactive species.     

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.     

Suprasellar meningioma with expression of glial fibrillary acidic protein: a peculiar variant

A 24-year-old female presented with a 3-year history of a suprasellar and intraventricular solid midline process measuring about 3×4 cm. At surgery, this tumour was sharply delineated and of stone-like firmness and was removed completely. Histology suggested meningioma, featuring nests and cords of epithelium-like cells with prominent cytoplasm amidst abundant fibrous stroma with prominent lymphoplasmocellular infiltration. Immunocytochemically, the tumour cells expressed vimentin, S-100 protein, epithelial membrane antigen, cytokeratins, and most surprisingly, glial fibrillary acidic protein (GFAP). Ultrastructural investigation revealed abundant intermediate filaments and occasionally dense secretory granules in tumour cells with short, finger-like cytoplasmic processes joined by very rare small, but well-developed desmosomes. This tumour most likely represents a peculiar variant of meningioma with prominent production of GFAP, as previously described [Budka H (1986) Acta Neuropathol (Berl) 72: 43–54].This observation was presented by H. Budka at the Slide Seminar on Tumours of the XII^th International Congress of Neuropathology, Toronto, September 1994     

Astroglial cell alteration caused by neurotoxins: immunohistochemical observations with antibodies to glial fibrillary acidic protein, laminin, and tyrosine hydroxylase

Kainic acid or 6-hydroxydopamine (6-OHDA) was injected into rat striatum, and their effects on astrocytes, laminin, and catecholamine fibers were examined temporally by immunohistochemical methods in an attempt to understand the roles of reactive astrocytes and laminin on the restoration of central nervous tissue. Kainic acid injection caused a severe neuronal degeneration in the striatum but catecholamine fibers were spared with only transient loss of tyrosine hydroxylase immunoreactivity. Reactive astrocytes appeared around the lesioned area soon after the kainic acid injection, then migrated into that area, and finally covered the lesioned striatum. Laminin immunoreactivity was found only in the lesioned area before the migration of reactive astrocytes and disappeared when the area was covered by astrocytes. 6-OHDA injection, on the other hand, resulted in a severe degeneration of catecholamine fibers, but striatal neurons were mostly spared. From 7 to 28 days after injection, regenerating fibers were found to enter the affected region. In this period reactive astrocytes were seen in the affected region but were only slightly more numerous than those found in control (saline injected) striatum. Laminin-immunoreactive blood vessels seemed to show a distribution similar to that in control striatum. These observations indicate that reactive astrocytes may play an important role in areas of neuronal cell loss and that laminin may aid their migration into such areas. Laminin and reactive astrocytes may not, however, be essential for the regeneration of dopamine fibers.     

Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis

Spinal cord sections from Lewis rats with acute experimental allergic encephalomyelitis (EAE) showed greatly increased staining of astrocytes when stained immunocytochemically for glial fibrillary acidicc protein (GFAP). Fibrous processes in white matter were heavily stained early in the course of the disease when paralysis was first evident (10–12 days after injection of guinea pig spinal cord myelin), then protoplasmic astrocytes were stained in the gray matter and became more heavily stained at 20 dats post-injection. The stained astrocytes were evenly distributed throughout the tissue, and did not correspond to the sites of the lesions. Spinal cord slices of control and EAE rats were incubated with [³H]amino acids, then cytoskeletal proteins were prepared in an enriched fraction, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands counted for radioactivity. In the EAE rat all cytoskeletal proteins, including the neurofilaments, vimentin, microtubules, GFAP and actin, showed increased uptake of radioactive amino acids. Immunoprecipitation of GFAP with specific antiserum showed increased radioactivity in the complex beginning at day 10 when cellular infiltration was beginning in the EAE animals. As the disease became acute, the radioactivity in the immunoprecipitated GFAP increased, in some cases to very high levels, then by day 18 when recovery was underway, the radioactivity had fallen to normal levels. Possible agents causing metabolic activation of protein synthesis in EAE animals include stimulating substances elaborated by infiltrating lymphoid scells, and the generalized edema accompanying the demyelinative condition. The activation of GFAP protein staining and metabolism in EAE might serve as a model for the activated growth of astrocyte processes which cause the severe gliosis seen in multiple sclerosis.     

Hippocampal kindling alters the concentration of glial fibrillary acidic protein and other marker proteins in rat brain

The effect of hippocampal kindling on neuronal and glial marker proteins was studied in the rat by immunochemical methods. In hippocampus, pyriform cortex and amygdala there was an increase in glial fibrillary acidic protein (GFAP), indicating reactive gliosis, and an increase in the glycolytic enzyme NSE, suggesting increased anaerobic metabolism. Neuronal cell adhesion molecule (NCAM) decreased in pyriform cortex and amygdala of kindled rats, indicating neuronal degeneration.     

Quantification of regional glial fibrillary acidic protein levels in Alzheimer's disease

OBJECTIVES: Our objectives were to quantify glial fibrillary acidic protein (GFAP) in brains of Alzheimer's disease (AD) cases, and non-AD controls to determine the regions with the most severe gliosis in AD. MATERIAL AND METHODS: In a case control design, we used an enzyme-linked immunosorbent assay (ELISA) to quantify GFAP in frozen brain from four areas of neocortex in 10 AD cases, 10 age-matched controls, and 10 younger controls from the Honolulu-Asia Aging Study autopsy archive. RESULTS: Median age at death was 83.5 years for cases and age-matched controls, and 77 years for younger controls. For the AD cases compared with the age-matched controls, levels of GFAP in occipital (P=0.01), parietal (P=0.028), and temporal lobes (P=0.004) (but not frontal) were significantly higher in the cases. The median GFAP excess in AD cases compared with age matched controls was highest in the temporal lobe. CONCLUSIONS: Regional quantification of GFAP reveals that the glial response is most prominent in the temporal lobe in AD.