Recent studies of gliosis in a variety of animal models are reviewed. The models include brain injury, neurotoxic damage, genetic diseases and inflammatory demyelination. These studies show that reactive gliosis is not a stereotypic response, but varies widely in duration, degree of hyperplasia, and time course of expression of GFAP immunostaining, content and mRNA. We conclude that there are different biological mechanisms for induction and maintenance of reactive gliosis, which, depending on the kind of tissue damage, result in different expressions of the gliotic response.

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20 -60 min at 43°C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFB p65 subunit, suggesting perturbation of NFB activation.A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development.In the preceding report (2) we have demonstrated by immunoelectron microscopy the intracellular (cytoplasmic) localization of a chondroitin sulfate proteoglycan in neurons and astrocytes of adult rat central nervous tissue. Its functional role in this location is still unknown, and although our immunocytochemical findings were not unexpected on the basis of previous biochemical studies of brain glycosaminoglycans, they are nevertheless quite different from the extracellular matrix or cell surface location of proteoglycans in other tissues (8,16,18,19).Since the possibility remained that during brain development this proteoglycan might be involved in extracellular processes associated with neural histogenesis and various types of cell-cell interactions, we also examined its localization in rat cerebellum during the period extending from 1 wk to l mo of postnatal age. These studies demonstrated that in contrast to the cytoplasmic localization observed in adult brain, the chondroitin sulfate proteoglycan is almost exclusively extraceUular in 7-d-old cerebellum, and progressively assumes its final intracellular location in neurons and astrocytes during the succeeding 3 wk. MATERIALS AND METHODSThe immunochemical and immunocytochcmical procedures employed were essentially as described in the preceding paper (2). However, for studies of immature brain (7-33 d postnatal), a two-step fixation procedure was used (3). After perfusion with 0.1 M sodium phosphate buffer (pH 7.4) containing 0.9% NaCl, rats were perfused with 4% freshly prepared formaldehyde and 0.1% glutaraldehyde in sodium phosphate buffer (pH 6.5), followed by the same aldehydes...

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and M~iller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.We have previously described the isolation and properties of a 6.5S chondroitin sulfate proteoglycan from a PBS extract of rat brain (19). This proteoglycan has a relatively high protein content (56% by weight), in addition to 24% glycosaminoglycans (predominantly chondroitin 4-sulfate) and 20% glycoprotein oligosaccharides, including a series of novel O-glycosidically linked oligosaccharides that contain mannose at their proximal ends (16, 28). The brain proteoglycan differs from the prototypical cartilage proteoglycans in a number of respects including its higher protein content, smaller monomer size, and its relatively limited ability to interact with hyaluronic acid to produce larger size aggregates.Biochemical analyses (for reviews, see references 27 and 28) demonstrated the presence of significant amounts of chondroitin sulfate in neuronal cell bodies, axons, and astrocytes (isolated in bulk from brain) as well as in purified nuclei, whereas there is little or no chondroitin sulfate in oligodendroglia, myelin, mitochondria, or nerve endings (synaptosomes). Approximately half of the chondroitin sulfate is found in the soluble fraction after high speed centrifugation of a brain homogenate, and much of the remainder is loosely associated with microsomal membranes from which it is easily extractable by mild washing procedures (19,29).When neurons are isolated in bulk from rat brain and lysed by a change in tonicity or pH, 82% of the chondroitin sulfate is released together with >90% of the lactate dehydrogenase (but only 20-25% of th...

Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densitometry of both stained gels and immunoblots of electrophoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinoculation (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton-insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one-and-a-half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton-insoluble protein (excluding histones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immunocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.