A. Bignami

Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence

Abstract The glial fibrillary acidic (GFA) protein, a brain specific protein extracted from severely gliosed human tissue, is not species specific; cross-reaction occurs between anti-human GFA protein antibodies and brain extracts of rabbit, guinea pig, rat and dog. Using anti-GFA protein antiserum, astrocytes are selectively stained with the indirect immunofluorescence technique in both normal and pathological (gliosed) brain tissue.

THE ASTROGLIAL RESPONSE TO STABBING. IMMUNOFLUORESCENCE STUDIES WITH ANTIBODIES TO ASTROCYTE-SPECIFIC PROTEIN (GFA) IN MAMMALIAN AND SUBMAMMALIAN VERTEBRATES

The astroglial response t o stabbing. Immunofluorescence studies with antibodies to astrocyte‐specific protein (GFA) in mammalian and sub‐mammalian vertebrates

Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Abstract Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro . Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro .

Differentiation of astrocytes in the cerebellar cortex and the pyramidal tracts of the newborn rat. An immunofluorescence study with antibodies to a protein specific to astrocytes

An automatic machine for assembling and banding together the components making up an expansion shell so that the assembly can be conveniently packaged and shipped. In one embodiment, the machine contains a series of work stations arranged to assemble the bolt components within a die. The expansion shell halves are staked to a common strap and the components then passed through a combination forming gage and banding mechanism which aligns the parts in assembly and bands them together using a plastic sleeve. In a second embodiment of the invention, the expansion shells are staked to the strap prior to being loaded into the die.

Specificity of the glial fibrillary acidic protein for astroglia.

Glial fibrillary acidic protein (GFA) is the main constituent of glial filaments and the close similarity of GFA and neurofilament protein has been recently reported. However, the immunofluorescence staining of peripheral nerve which may be observed with GFA antisera is not due to cross-reaction between GFA and neurofilament protein. Staining of peripheral axons was also observed with control sera obtained by injecting the rabbits with nonimmunogenic GFA preparations isolated with the same procedure. Immune GFA antisera and control sera reacted with sodium dodecyl sulfate extracts of sciatic nerve. However, the precipitin line formed with peripheral nerve crossed the line against GFA protein, thus indicating nonidentity between the two antigens. Buffer extract of sciatic nerves that had been incubated with spinal cord reacted by immunodiffusion with GFA antisera, thus indicating that redistribution of GFA occurred under these conditions.

The cellular reaction to wallerian degeneration in the central nervous system of the cat

Abstract The fine structural features of Wallerian degeneration have been examined by electron microscopy of the cat central nervous system from 1 to 104 days following lesions. In the early stages of Wallerian degeneration, increased electron density is evident in the axoplasm of many axons. Myelin sheaths are occasionally seen to surround cells resembling oligodendroglia. Mitotic figures are present, occasionally in cells with glial filaments. Phagocytosis is carried out by cells resembling those found in the region of blood vessels. The phagocytes take up degenerating myelin and later the ingested myelin loses its characteristic structure and density and appers to be broken downto form lipid droplets. After 6–8 weeks, the phagocytes containing myelin remnants and lipid droplets are seen to abut capillaries. Later, the nervous parenchyma becomes filled with fibrillary glial processes, and phagocytes within the glial scar assume the morphological characteristics of microlia seen by light microscopy. Neither astroglial nor oligodendroglial cells appear to participate in the phagocytosis of degenerating myelin.

Glial Fibrillary Acidic Protein (GFA) in Normal Neural Cells and in Pathological Conditions

Publisher Summary This chapter discusses glial fibrillary acidic (GFA) protein in normal neural cells and in pathological conditions. A review of the literature on GFA protein indicates that it has found its major application as an immunohistochemical marker for astroglia, especially under conditions where identification of the cell type is difficult, that is, during development, in culture, in cellular fractions and, more recently, in human brain tumors. Laboratory procedures have been developed to facilitate production of the antisera. The solubility of GFA protein in aqueous solutions and the in vitro formation of filaments from purified GFA protein preparations suggest that glial filaments, like other constituents of the cytoskeleton in nonmuscle cells, namely, microtubules and actin microfilaments, may assemble and disassemble in the cytoplasm. The extensive remodeling of the glial framework during brain development and the rapid change in cell shape occurring in cultured astrocytes following serum withdrawal and the administration of cyclic AMP make this hypothesis particularly attractive. The original isolation of GFA protein from gliosed human brain required relatively simple procedures such as buffer extraction and ammonium sulfate precipitation to obtain purified preparations. The immunogenicity of GFA protein varies considerably depending on the degree of degradation. Nondegraded or partially degraded preparations are nonimmunogenic or weakly immunogenic whereas strong antisera are obtained with degraded antigen. Immunohistological studies with GFA protein antisera provide new data in three critical areas of research on the development of neuroglia: (1) myelination gliosis, (2) time of appearance of Bergmann fibers in the cerebellum, and (3) the nature of radial glia.

Immunochemical and immunofluorescence studies of the glial fibrillary acidic protein in vertebrates

Abstract Phosphate buffer extracts (pH 8.0) of the brain and the spinal cord of various vertebrates, mammalian and submammalian, cross-reacted with antihuman glial fibrillary acidic (GFA) protein serum by Ouchterlony double diffusion. Mammalian brain extracts, as previously reported, gave an immunodiffusion pattern of complete identity with human GFA protein. In submammalian vertebrates (shark, goldfish, turtle and chicken) strong spurs were observed at the junction with human GFA protein, suggesting incomplete identity. GFA protein from mammalian and submammalian vertebrates had the same extraction properties, immunoelectrophoretic mobility and molecular weight as human GFA protein. By immunofluorescence with antihuman GFA protein, serum astrocytes were selectively stained in cryostat sections of the brain and the spinal cord in all vertebrates studied, mammalian and submammalian, with the exception of the lamprey. No immunofluorescence was observed in invertebrate ganglia. Some antomical observations on the astrocytic framework of various vertebrates, as observed with immunofluorescence, are reported.

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera.

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.

Characteristics of the rat C-6 glioma maintained in organ culture systems. Production of glial fibrillary acidic protein in the absence of gliofibrillogenesis

Abstract Cells from a cloned rat C-6 glioma cell line were cultured on collagen-coated coverslips for up to 30 days and on organ culture systems using sponge foam matrices or Millipore filter platforms up to 44 days. The light microscopic features throughout the period of observation were those of a relatively poorly differentiated small-cell glioma that did not form glial fibrils. Electron microscopy of the cell line originally obtained and of the same cells maintained in organ culture systems demonstrated only infrequent and sparse microfilaments and no glycogen rosettes. Immunofluorescence staining for glial fibrillary acidic (GFA) protein, a specific marker for fibrillary astrocytes, was positive in only an occasional cell in monolayer or suspension cell cultures, but was markedly positive in most cells maintained for 12–14 days on sponge foam matrices. These findings suggest that the production of GFA protein can occur in the absence of gliofibrillogenesis in an in vitro system that has been shown to favor differentiation, and that therefore the rat C-6 glioma cell line represents a still undifferentiated glial cell type with the potential to differentiate into fibrillary astrocytes.