Cell reactions following acute brain injury: a review.

The proliferative behavior of glia following a cerebral stab wound in adult rats is reviewed. Proliferation was determined by both PCNA and [3H]thymidine labeling. Microglia were the first cells to divide and constituted the bulk of dividing cells. Both ramified and ameboid microglia divided. A smaller number of astrocytes entered the cell cycle a day later and were shown to derive from differentiated reactive cells. No differentiated oligodendroglia were labeled by thymidine, although a small number of dividing immature oligodendroglia could be detected in cultures of cells labeled in vivo. Recent studies of the properties of oligodendroglial precursors in brain repair mechanisms are discussed. The results so far support our conclusion that differentiated oligodendrocytes do not divide.

Cells of the oligodendrocyte lineage proliferate following cortical stab wounds: an in vitro analysis.

We have previously shown that a cortical stab wound induces the proliferation of microglia and astrocytes in situ, but no evidence was obtained for proliferation of cells of the oligodendrocyte lineage (Amat et al., 1996). To study further the properties of cells involved in repair following brain injury, groups of adult rats received either sham operations or bilateral stab wounds. Proliferating cells were labeled in vivo 3 days later with [3H]-thymidine (Thy) and sacrificed the same day. Oligodendrocyte-enriched preparations were isolated, cultured, and analyzed. The fate and antigenic phenotype of the proliferating cells was analyzed using three-color immunofluorescence combined with autoradiography at 1, 2, 3, 5, and 10 days in vitro (DIV). Cells were immunostained for ganglioside GD3 (glial stem cells), O4 antigen (cells of the oligodendrocyte lineage), galactosyl ceramide (GC, differentiated oligodendrocytes), and GFAP (astrocytes). Thymidine-labeled O4+/GC- cells were found only in cultures from wounded animals and most of them differentiated in vitro as mature oligodendrocytes, but no Thy+/O4+/GC+ oligodendrocytes were seen at 1, 2, or 3 DIV. There was also a marked increase in the number of Thy+/GD3+ cells in the experimental cultures. In both experimental and control groups the total number of Thy+ and Thy- GD3+ cells declined with time in culture concomitant with an increase in total number of both Thy+ and Thy- GFAP+ astrocytes, and without any significant change in the Thy+ cell fraction of O4+ oligodendrocytes in the experimental cultures. Therefore most of the GD3+/O4- cells apparently differentiated as GFAP+ astrocytes, not as oligodendrocytes. We conclude that O4+/GC- oligodendrocyte precursor cells, but not differentiated oligodendrocytes, proliferate in response to brain injury. These cells proliferate slowly or not at all in normal adult animals and constitute a phenotypically and kinetically distinct group from the GD3+ glial precursors. This result is consistent with the existence within the adult CNS of a quiescent premyelinating oligodendrocyte. We propose that these immature committed oligodendrocytes are induced to proliferate at the wound site and serve as a source of new oligodendrocytes.

Multiple sclerosis: altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin.

Recent studies have implicated heat shock proteins (HSP) in the pathogenesis of the multiple sclerosis (MS) lesion. Expression of the 73 kDa constitutive HSP (HSC70), the 72 kDa stress-inducible HSP (HSP70), and the 27 kDa small HSP (HSP27) was analyzed in white matter and myelin from central nervous system (CNS) tissue of MS and normal subjects using a combination of immunocytochemistry and quantitative immunoblotting. Plaques of all types were sharply defined by reduced immunostaining for HSC70, and shown by immunoblotting to contain 30 to 50% less HSC70 than surrounding white matter or normal tissue. In contrast, HSP27 was markedly enhanced 2.5- to 4-fold in plaque regions, especially in fibrous astrocytes and in hyperplastic interfascicular oligodendrocytes at the lesion edge. HSP70 was less abundant than HSC70, and no significant differences in HSP70 levels were noted between MS and normal white matter. Myelin isolated from active plaques contained 3- to 4-fold more HSC70 than normal myelin. Pronounced expression of HSP70 and HSP27 was also found in MS myelin, although neither protein was detected in normal myelin. Thus, white matter undergoing immune-mediated destruction in MS was associated with altered distribution and expression of HSC70 and HSP27. These changes may initially serve to protect myelin from further destruction and facilitate repair; however, enhanced expression of HSC70, HSP70, and HSP27 in myelin may subsequently present as additional immune targets involved in the progression of disease.

Induction of oligodendrocyte apoptosis by C2-ceramide.

Tumor necrosis factor-alpha induces oligodendrocytes apoptosis, and is known to stimulate the hydrolysis of sphingomyelin to form the lipid mediator, ceramide. These data encouraged us to determine whether ceramide itself is able to induce apoptosis in oligodendrocytes. For this purpose the cell-permeable ceramide analog, C2-ceramide was used. Treatment of bovine oligodendrocyte cell cultures with this compound induced cell death in a time- and concentration-dependent manner. The induction of cell death was specifically associated with the action of C2-ceramide and could not be elicited by dioctanoylglycerol (DC8) or phorbol 12-myristate 13-acetate (PMA). Treatment of the cultures with neutral sphingomyelinase, which increased the hydrolyses of endogenous sphingomyelin, resulted in oligodendrocyte death, whereas exposure of the cells to phospholipase C and A2 did not. C2-ceramide treatment caused DNA fragmentation. Morphologic analysis of the cells showed that C2-ceramide treatment resulted in a loss of their processes, reduction of cell volume, chromatin condensation, and formation of apoptotic bodies. These results indicate that ceramide can induce oligodendrocyte apoptosis, and suggest that sphingolipid metabolism plays a key role in the regulation of this process.

Study of the interaction of the myelin monomeric GTP-binding proteins with other brain proteins.

Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.

Nerve growth factor and neurotrophin-3 differentially regulate the proliferation and survival of developing rat brain oligodendrocytes.

There is increasing evidence that the neurotrophins, particularly nerve growth factor (NGF) and neurotrophin-3 (NT-3), play a role in the regulation of glial development in the CNS. Recent studies have shown that the proliferation of optic nerve-derived O2A progenitors (OLPs) is potentiated by NT-3 in combination with platelet-derived growth factor, whereas NT-3 alone supports the survival of their differentiated progeny (Barres et al., 1994). In this study, we have examined the expression of the high-affinity neurotrophin receptors (trks) and the low-affinity nerve growth factor receptor p75 in developing oligodendrocytes (OLs). In addition, we have examined the effects of NGF and NT-3 on proliferation and survival of OLPs and OLs, respectively. TrkC, the high-affinity NT-3 receptor, and trkA, the high-affinity NGF receptor, are both expressed from the early OLP through the mature OL stage. The truncated form of trkB, lacking the tyrosine kinase domain, and the low-affinity neurotrophin receptor p75 are expressed at low levels in OLPs and are upregulated in mature OLs. NGF and NT-3 both induced the phosphorylation of mitogen-activated protein kinase (MAPK) in OLPs and in OLs. In both OLPs and OLs, NT-3 sustained the activation of MAPK more than NGF. NT-3 enhanced the proliferation of OLPs and supported the survival of OLs. By contrast, unless coadministered with FGF-2, NGF did not exhibit mitogenic effects on OLPs but did enhance the survival of differentiated OLs. Our data demonstrate the presence of functional trkA and trkC in developing OLs and indicate that both NGF and NT-3 have a broad spectrum of developmental actions on cells of the OL lineage.

Biochemistry of demyelination.

The myelin sheath, a lipid-rich multilamellar membrane of relative stability, both insulates and enhances conduction in nerve axons. A notable feature of myelin-specific proteins, in particular myelin basic protein, is their susceptibility to proteolytic activity and their encephalitogenicity, which induces inflammatory demyelination in the CNS. The final common pathway of myelin breakdown in vivo is well documented and there is evidence that myelin disruption can be mediated directly by soluble (circulating) factors and for following receptor-driven phagocytosis by macrophages. However the exact mechanism(s) of demyelination in multiple sclerosis is still unresolved, both antigen-specific and--non-specific events having the potential to generate the myelinolytic process.

Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds.

Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocyte hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside GD3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [3H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for GD3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B4 binding (all microglia). Animals were labeled with thymidine at 1,2,3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of GD3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2-3 dpl and one third to one half were GD3+. Astrocytes proliferated maximally at 3-4 dpl, and some were also GD3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all GD3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither GD3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from GD3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and GD3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages.

Do oligodendrocytes divide?

Remyelination occurs in the adult central nervous system following a wide variety of experimental and naturally occurring demyelinating conditions, including multiple sclerosis. Remyelination is preceded by the appearance of new oligodendrocytes. These new cells may be generated from glial precursor cells, or from pre-existing differentiated oligodendrocytes that re-enter the cell cycle, which may first dedifferentiate, or both processes may occur. The evidence for the source of new oligodendrocytes following toxic or immune-mediated lesions is reviewed. Good evidence exists that fully differentiated oligodendrocytes can incorporate [3H]thymidine but this may be a rare event. Most of the evidence points towards glial precursor cells as the source of new oligodendrocytes in the adult, but definitive experiments have not yet been done. Research strategies, using our current knowledge and techniques, are outlined for solving this problem.

The 70-kDa heat shock cognate protein (HSC70) is a major constituent of the central nervous system and is up-regulated only at the mRNA level in acute experimental autoimmune encephalomyelitis.

The expression of the 70-kDa heat shock cognate (HSC70) and stress-inducible (HSP70) proteins, and their mRNAs, was examined in experimental autoimmune encephalomyelitis, a model of inflammatory demyelination in the CNS. This study was undertaken as an extension of previous work demonstrating an abrupt decline in mRNA levels of both glial fibrillary acidic protein and the low-molecular-weight neurofilament subunit in experimental autoimmune encephalomyelitis spinal cord at 12 days after inoculation, the height of inflammation and clinical signs. Using the same total RNA preparations as our previous study, we report here that mRNA levels for HSC70 increased approximately sixfold over control values at the same time that glial fibrillary acidic protein and low-molecular-weight neurofilament subunit messages decreased and were similar to controls by 21 days after inoculation. In situ hybridization experiments showed that HSC70 mRNA was predominantly expressed in neurons and that the influx of inflammatory cells into the CNS was not responsible for the large increase in HSC70 message. Despite this elevation in mRNA, only small (if any) increases in protein levels for HSC70 were detected by both western blotting and in vitro cell-free translation systems. However, by quantitative immunoblotting, we determined that constitutive levels of HSC70 comprised a substantial portion of CNS proteins, representing 2-3% of the total protein content of spinal cord. Immunohistochemical staining illustrated that the distribution of HSC70 was consistent with that of its message. In contrast, no HSP70 mRNA or protein was detected in either control or experimental animals.

Differentiation of glial precursor cells from developing rat brain in vitro.

We have previously shown that enriched preparations of oligodendrocytes from mature bovine brain or 30d rat brain contain 4-10% ganglioside-GD3+ glial precursor cells, which differentiate into astrocytes in culture. These findings are in contrast to those described by others in cultures of neonatal rat brain, which contain precursors that differentiate into both astrocytes and oligodendrocytes. We have extended this study to determine whether the properties of glial precursor cells vary during development. Cells isolated by the same technique from 5-, 10- and 20-day-old rats, were placed in culture and double-immunostained at 1,2,3,6 and 10 days in vitro (DIV) for GD3/glial fibrillary acidic protein (GFAP), galactosylceramide (GC)/GFAP, GD3/GC, GD3/antigen O4, GC/O4 and GFAP/O4. After 1 DIV the isolates from 5 day rats contained 30% GD3+ cells, 1% oligodendrocytes (GC+) and 3.5% astrocytes (GFAP+). The corresponding percentages from 10 day rats were: 35% GD3+, 3% GC+ and 3% GFAP+; and from 20 day rats: 28% GD3+, 35% GC+ and 1% GFAP+. Thus the number of oligodendrocytes in the initial isolate increased dramatically between 10 and 20 days. At all 3 ages immature cells were a major component of the total isolate. GFAP+ cells increased rapidly in all cultures. In all cultures the numbers of GFAP+/GD3+ cells reached a maximum and then declined coincident with the increase of GFAP+/GD3- cells, but there were many more of these double-stained cells in cultures from 20 day rats. Moreover, all cultures at 1 DIV contained some GFAP+/GD3- cells. Thus astrocytes appeared to derive both from pre-existing GFAP+/GD3- cells and from GD3+ cells, the latter pathway being more significant in the older rats. GC+ cells increased in cultures prepared from 5 day and 10 day rats, but remained relatively constant in cultures from 20 day rats. The number of GD3+/O4+ cells decreased coincident with an increase of GC+/O4+ cells (all GC+ cells were O4+), but the number of GC+/GD3+ cells was insignificant in any culture from any age animal. These findings in developing animals support the scheme shown by others in neonates that oligodendrocytes derive from GD3+ cells via O4+/GC-intermediate cells. These data show that more GD3+ cells differentiate into oligodendrocytes in 5- and 10-day-old animals than in 20-day-old animals, and a larger percentage differentiate into astrocytes in 20-day-old animals than in the younger animals. The most reasonable explanation of these results is that two committed populations of GD3+ precursor cells exist in the brain, and that the ratio of these populations changes during development. The implication of this conclusion is that bipotential progenitor cells do not persist beyond the neonatal period, but become committed to separate lineages during development.

Quantitative aspects of reactive gliosis: a review.

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.

Widespread differentiation stage-specific expression of the gene encoding phosphoprotein p19 (metablastin) in mammalian cells.

p19 is a highly conserved 19 kD cytosolic protein that undergoes phosphorylation in response to diverse extracellular factors in mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. To gain insights regarding its function, we analyzed the expression of p19 mRNA in a variety of cell types during induction of differentiation. Murine erythroleukemia cells showed a moderate increase followed by a marked decrease in the abundance of p19 mRNA during induction of differentiation. In murine C2 myoblasts and primary fetal rat osteoblasts, p19 mRNA was abundant in replicating cells and decreased to undetectable levels during differentiation. In resting human peripheral blood lymphocytes, p19 mRNA was virtually undetectable but was strongly induced during blast transformation of both B and T cells. In rat liver, p19 mRNA was abundant on embryonic day 17 and decreased during early postnatal development. Upon fractionation of adult rat liver cells by centrifugal elutriation, p19 mRNA was not detected in hepatocytes while a low level was observed in a fraction enriched in non-parenchymal epithelial cells. CCl4-induced liver regeneration resulted in induction of p19 mRNA in hepatocytes. Primary cultures of embryonic and neonatal rat brain were analyzed by indirect immunofluorescence using co-staining with stage-specific markers. p19 expression was restricted to immature neurons and oligodendrocyte precursors. In contrast to the other cell types examined, the neuronal and glial precursors that express p19 were shown, using BrdU labeling, to be postmitotic both in primary culture and in vivo. The data demonstrate widespread, stage-specific expression of p19 and suggest that the protein exerts a general, lineage-independent function during induction of differentiation of mammalian cells. In view of the available evidence on the stimulation of serine phosphorylation of p19 by several growth factors, our working hypothesis is that phosphorylation of p19 may be involved in the mechanism by which growth factors control cell differentiation.

Cytokine cytotoxicity against oligodendrocytes. Apoptosis induced by lymphotoxin.

The cytotoxic effect of recombinant human cytokines was tested on glial cells cultured from mature bovine brain. Lymphotoxin (LT) and TNF induced injury to oligodendrocytes in a time and dose-dependent fashion. The other cytokines tested, IFN-gamma, IL-6, and IL-2, did not affect oligodendrocytes in culture over a 72-h observation period. None of the cytokines injured astrocytes cultured from the same source. LT showed a much more potent cytotoxicity than TNF toward oligodendrocytes; cytotoxic changes were noted earlier (24 h) and at lower units of activity. Morphologic studies showed that the LT-mediated effects were associated with early retraction of cell processes, depolymerization of F-actin and subsequent nuclear degeneration. Lack of early cytoplasmic membrane injury as measured by 51Cr release and electron microscope studies demonstrating nuclear disintegration suggested an apoptotic mechanism of oligodendrocyte injury evoked by LT, which was supported by DNA integrity assay. These results demonstrate that LT possesses potent cytotoxic activity against oligodendrocytes and that the major mechanism involved in this process is DNA fragmentation.

Tumor necrosis factor-induced proliferation of astrocytes from mature brain is associated with down-regulation of glial fibrillary acidic protein mRNA.

Previous results from this laboratory have shown that tumor necrosis factor (TNF) is mitogenic for bovine astrocytes in chemically defined (CD) medium. The maximum mitogenic response was detected with 200 U/ml at 48 h. We have now extended these studies to assess the effect of TNF on message levels for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The results have shown that, whereas TNF had only a slight effect on vimentin mRNA, TNF induced a marked decrease to 4.3 +/- 2.0% of controls in GFAP mRNA which was both time and dose dependent. The lowest effective dose was 50 U/ml and the maximal effective dose was 200 U/ml. Kinetic analysis of this response demonstrated that a marked decrease in GFAP mRNA was present at 12 h and continued to decrease through 72 h. To determine the reversibility of the TNF effect, astrocyte cultures were exposed to 200 U/ml TNF for varying periods of time and then cultured in fresh CD medium. A 1-h pulse with TNF was sufficient to reduce GFAP mRNA levels when measured 24 h later. However, cultures incubated with 200 U/ml TNF for 48 h followed by incubation in CD medium without TNF for 7 days showed that GFAP mRNA levels had returned to 60% of the control values. Nuclear runoff assays showed that the effect of TNF on GFAP mRNA was at the posttranscriptional level. Polyacrylamide gel electrophoretic analysis of astrocyte cytoskeletal proteins demonstrated that GFAP levels were reduced after a 5-day incubation with 200 U/ml TNF whereas protein levels of vimentin and actin were not significantly changed.(ABSTRACT TRUNCATED AT 250 WORDS)

GFAP mRNA levels following stab wounds in rat brain.

We previously reported that glial fibrillary acidic protein (GFAP) levels increased significantly at 3 days after stab wounds, relative to sham-operated controls, reaching a maximum of 200% of control value at 5-7 days. They then fell to near-normal values by 21 days. To determine whether these protein changes correlated with changes in GFAP mRNA we performed Northern blot analyses. Total RNA, isolated from lesioned, sham-operated and intact rat forebrains, was hybridized with 32P-labeled mouse GFAP cDNA and quantified by densitometry. The maximum increase in total RNA content in lesioned animals was only 20% over controls at 12 h. GFAP mRNA levels increased to 2-fold control values at 6 h and reached 5-fold at 12 h. Thereafter they remained at 3.5- to 6-fold until 5 days and then declined to 1.5-fold by 21 days. The rapid increase of GFAP message at 12 h preceded a significant increase in GFAP by 2 days and the decrease of message after 5 days was more precipitate than the slow decrease in GFAP content. Sham-operated animals showed no significant changes in GFAP mRNA, compared to intact controls, during the period 3 h to 14 days postoperation. GFAP mRNA and GFAP in the stab-wound model reached levels similar to those found in the experimental autoimmune encephalomyelitis (EAE) model, but returned to normal much more rapidly.

Biochemical and immunocytochemical changes in glial fibrillary acidic protein after stab wounds.

Changes of glial fibrillary acidic protein (GFAP) in the forebrain of rats with stab wounds were determined by quantitative immunoblots and by immunohistochemistry. Bilateral stab wounds were made stereotaxically in the cortex and hippocampus. In control rats, the scalp was retracted and depressions were etched on the intact skull. At various times up to 21 days postoperation, one cerebral hemisphere was homogenized, proteins were separated by polyacrylamide gel electrophoresis and immunoblots were quantitated by densitometry. The contralateral hemisphere was immunostained for GFAP. Three hours postoperation GFAP+ cells were detected around the wound but there was no increase of total GFAP. At 6 h postoperation total GFAP in the forebrain decreased to 80% of the sham-operated control value and the number of GFAP+ cells was lower, compared to the controls, in layer 1 of the cortex, corpus callosum, cingulum, external capsule, internal capsule, hippocampus, optic tracts and around blood vessels. This early relative decrease in GFAP levels was actually due to an increase in GFAP in the sham-operated controls, which mounted a stronger gliotic response during the first 24 h. In neither group of animals did the GFAP levels drops below those of intact unoperated animals. At 24 h total GFAP began to increase. The number and intensity of reactive glia in the vicinity of the wound increased steadily, appearing to reach a maximum at about 7 days, then declining significantly by 21 days. The glial reaction was most pronounced in the hippocampus. Total GFAP reached 180% of the control value by 7 days and then declined to 117% by 21 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of glial fibrillary acidic protein and neurofilament mRNA in gliosis induced by experimental autoimmune encephalomyelitis.

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5-2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 +/- 35 micrograms (mean +/- SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11-14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11-14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to approximately 50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms and suppression of inflammatory demyelination.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system commonly used as a model for multiple sclerosis. In both of these diseases demyelination occurs association with perivascular infiltrates of T-cells and macrophages. The similarities in immunopathology suggest that these two diseases share common mechanisms of tissue destruction. We have proposed a general mechanism to explain the clinical and histopathological features of EAE. T-cells sensitized to the inducing antigen, myelin basic protein (MBP), react with antigen-presenting cells (possibly endothelial cells, microglia or astrocytes) in the central nervous system. As a consequence of this reaction, T-cells release lymphokines which activate macrophages, stimulate an augmenting inflammatory response, and, through the action of vasoactive amines, induce vasospasm and breakdown of the blood-brain barrier. The activated macrophages secrete inflammatory mediators, including plasminogen activator and other proteinases, which, in concert with serum plasminogen and complement, initiate myelin destruction. The macrophage products also serve to enhance the inflammatory response and vascular permeability. In support of this hypothesis we find that: (1) macrophage-secreted proteinases can degrade MBP in lyophilized myelin and that proteolysis is amplified in the presence of plasminogen; (2) proteolysis of proteins in fresh myelin by macrophage proteinases and plasminogen or by plasmin is potentiated by complement; (3) removal of macrophages from the circulation suppresses EAE; (4) proteinase inhibitors suppress EAE; and (5) prazosin, an alpha 1-adrenergic receptor antagonist, suppresses the clinical signs of EAE and the increased vascular permeability but only delays the inflammatory response. We believe that prazosin acts on the vascular alpha 1-adrenergic receptor to inhibit vasospasm and prevent opening of the blood-brain barrier. Thus it is possible to suppress both clinical signs and pathology by interceding at several steps of the cell-mediated immune reaction.

Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor.

The effect of cytokines on astrocytes cultured from mature bovine brain was determined both in a serum-containing medium and in a chemically-defined medium. The results showed that in serum-free medium, human TNF and, to a lesser degree, IL-6 and lymphotoxin, were mitogenic for astrocytes. Increased uptake of [3H]thymidine could be detected within 36 h in vitro and its presence in astrocytes was confirmed by autoradiography. In contrast, neither IL-1 alpha nor IL-1 beta induced astrocyte proliferation in serum-free medium but showed some synergistic effect with serum after 72 h. The proliferative effect of TNF and IL-6 was confirmed by cell counting. None of the cytokines tested was toxic for astrocytes as measured by 51Cr release. No mitogenic effect for oligodendroglia, purified from the same source, was detected. The results support a role for products of activated inflammatory cells in the development of astrocyte proliferation that may contribute to the reactive gliosis found in white matter diseases of the central nervous system such as multiple sclerosis.