Reactive gliosis in the brains of Lewis rats with experimental allergic encephalomyelitis

This paper assesses reactive gliosis in the optic tracts and other regions of brain in Lewis rats with experimental autoimmune encephalomyelitis (EAE). Enhanced immunostaining for glial fibrillary acidic protein (GFAP) in brains from rats with EAE occurred primarily in the white-matter tracts and was not restricted to sites of inflammation. Immunocytochemical staining for other putative astrocytic antigens demonstrated glutathione-S-transferase (Yb isoenzyme) to be localized extensively in GFAP-positive cells and vimentin to be present both in inflammatory cells and in some GFAP-positive astroglial cells. Positive staining for carbonic anhydrase and glutamine synthetase was observed in oligodendrocytes. In the optic tracts glutamine synthetase, but not carbonic anhydrase, was also observed in some astrocytes.     

The astrocyte as a locus of carbonic anhydrase in the brains of normal and dysmyelinating mutant mice

There is some controversy in the literature whether carbonic anhydrase occurs in astrocytes, as well as in oligodendrocytes and myelin, in the mammalian brain. In the present study this issue was addressed by double immunostaining for carbonic anhydrase and two astrocytic "markers" in the brains of normal mice and two dysmyelinating mutants, jimpy and shiverer. In the brains of young mice, carbonic anhydrase and glutamine synthetase were colocalized in astrocytes in the cortical gray matter. In gray matter of the adult mouse brain, it was possible to immunostain both carbonic anhydrase and glial fibrillary acidic protein (GFAP) in the same cells. However, in contrast to the findings in gray matter, in and near subcortical white matter carbonic anhydrase could be detected only in oligodendrocytes and myelinated fibers. In the brains of jimpy mice, virtually all the carbonic-anhydrase-positive cells were also GFAP positive, even in regions normally occupied by white matter. In the brains of young and adult shiverer mice, carbonic anhydrase was localized in astrocytes in the gray matter, but in and near the tracts normally occupied by white matter carbonic anhydrase could be detected only in oligodendrocytes and their abundant processes. The findings confirmed the oligodendrocyte-myelin unit to be the primary locus of carbonic anhydrase in the normal mouse brain and showed the astrocytes in gray matter normally to be a secondary locus of carbonic anhydrase. The immunostaining in the jimpy mouse brain suggested further that reactive astrocytes, in particular, might be rich in carbonic anhydrase.     

Astrocytic reactivity and intermediate filament metabolism in experimental autoimmune encephalomyelitis: The effect of suppression with prazosin

In either actively or passively transferred experimental autoimmune encephalomyelitis (EAE), increased immunocytochemical staining of glial fibrillary acidic protein (GFAP) in astrocytes was detected early in the disease process in both the gray and white matter of the spinal cord. Staining was not restricted to areas of perivascular mononuclear infiltration, and was observed at all levels of the cord. This enhanced staining pattern was delayed in rats in which clinical signs of EAE had been suppressed by treatment with the alpha 1-adrenoceptor antagonist prazosin. This glial reaction in EAE was not accompanied by increased GFAP synthesis, as measured by in vitro labeling of spinal cord slices, nor an increase in GFAP content, as measured by densitometry of intermediate filament fractions separated by polyacrylamide gel electrophoresis. Total protein synthesis was increased, with vimentin being labeled especially heavily; in prazosin-treated EAE animals, the increase in total protein synthesis was reduced and delayed.     

[H]Thymidine labeling of astrocytes in experimental allergic encephalomyelitis

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [³H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11–15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [³H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.     

Expression of 48-kilodalton intermediate filament-associated protein in differentiating and in mature astrocytes in various regions of the central nervous system

In this paper we present evidence that the 48-kD intermediate filament-associated protein (IFAP) is expressed relatively late in maturation of astrocytes, after they have acquired the glial fibrillary acidic protein (GFAP). In the astrocytes of white matter in the cerebellum the GFAP is detected at P3, whereas the 48-kD IFAP is detected only at P11. In the periventricular region and the hippocampus the 48-kD IFAP was detected at P6, long after the appearance of GFAP. In adult mice the 48-kD IFAP was observed in GFAP-positive astrocytes in the white matter of cerebellum, spinal cord, brainstem, and corpus callosum as well as in GFAP-positive cells in the grey matter of cerebral cortex and spinal cord. The 48-kD IFAP was not, however, detected in radial glia and their derivatives, in Bergmann glia or in Müller glia. Thus, not all the GFAP-positive astroglia express the 48-kD IFAP. Similarly, 48-kD IFAP was not detected in cells which were GFAP-negative.     

Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors

We have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound 1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; 2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and 3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP-containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase-positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogenesis.     

[3H]Thymidine labeling of astrocytes in experimental allergic encephalomyelitis

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [³H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11–15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [³H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.     

Appearance of GFAP-positive cells in adult human brain cultures spontaneously decelerated in growth

Cell cultures were derived from adult human brain biopsies [from cortical gray (cultures 9-HB-G and 33-HB-G) and white (culture 14-HB-W) and stroke-injured white matter (culture 33-HB-IW)]. The morphology and growth rate of cultured cells were examined and correlated with the presence of vimentin and glial fibrillary acidic protein (GFAP). The cultures from various brain matters differed in cell morphology and rate of growth but not in GFAP and vimentin staining. Cells of primary and rapidly proliferating cultures were GFAP-negative and vimentin-positive. Spontaneous growth deceleration occurred in culture 14-HB-W within passages 5 to 10 and in cultures 9-HB-G, 33-HB-G, and 33-HB-W within passages 17 to 20. This deceleration, as well as the successive complete growth arrest, were accompanied by an appearance of GFAP-positive cells and an elevated intensity for vimentin staining. We propose that GFAP-positive astrocytes originate from glial precursor cells that migrate from the explants and differentiate under prolonged subcultivation.     

Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was analyzed. Western blot analysis showed that three isotypes of caveolins including caveolin-1, -2 and -3 increased significantly in the spinal cords of rats during the early stage of EAE, as compared with the levels in control animals (p<0.05); the elevated level of each caveolin persisted during the peak and recovery stage of EAE. Immunohistochemistry demonstrated that caveolin-1 and -2 were expressed constitutively in the vascular endothelial cells and ependymal cells of the normal rat spinal cord, whereas caveolin-3 was almost exclusively localized in astrocytes. In EAE lesions, the immunoreactivity of caveolin-1 was increased in the ependymal cells, some astrocytes, and some inflammatory cells of the spinal cord, while that of caveolin-2 showed an intense immunoreactivity. Caveolin-3 was expressed constitutively in some astrocytes, but not in endothelial cells; its immunoreactivity was increased in reactive astrocytes in EAE lesions. The results of the Western blot analysis largely confirmed the observations obtained with immunohistochemistry. Taking all the findings into consideration, we postulate that the expression levels of each caveolin begin to increase when EAE is initiated, possibly contributing to the modulation of signal transduction pathways in the affected cells.     

实验性变态反应性脑脊髓炎大鼠星形胶质细胞的变化

目的观察实验性变态反应性脑脊髓炎(EAE)大鼠脊髓中星形胶质细胞的变化,探讨EAE大鼠的发病相关生物学机制。方法采用免疫组化法,对豚鼠全脊髓匀浆诱导的Wistar大鼠EAE的过程中,脊髓内星形胶质细胞变化情况进行研究。结果EAE大鼠症状高峰期时星形胶质细胞开始激活,恢复期时激活达到高峰,而且活化的星形胶质细胞未见表达主要组织相容性抗原(MHC)。结论活化的星形胶质细胞可能与EAE大鼠的恢复有关。     

Expression of citrullinated proteins in murine experimental autoimmune encephalomyelitis

In this study, we demonstrate for the first time the immunohistochemical expression of citrullinated proteins in the central nervous system (CNS) of mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). By using an established monoclonal antibody (F95) against natural and synthetic citrullinated proteins (Nicholas and Whitaker [2002] Glia 37:328-336), numerous, small, previously unrecognized "patches" of citrullinated proteins were discovered throughout EAE brains, whereas EAE spinal cords showed similar but much larger lesions. On dual color immunofluorescence, these lesions were found to contain citrullinated myelin basic protein (MBP) and were surrounded by astrocytes immunoreactive for both glial fibrillary acidic protein (GFAP) and F95. These lesions became evident about the time when EAE mice became symptomatic and increased in size and number with increasing disease severity. In some sections of spinal cord but not brains of severely debilitated EAE mice, a widespread gliotic response was seen, with astrocytes containing citrullinated GFAP spread throughout the gray and white matter. Western blot analysis of acidic proteins from the brains and spinal cords of EAE mice had higher levels of multiple citrullinated GFAP isoforms compared with controls, with more F95-positive bands in the EAE brains vs. spinal cords. These results raise the possibility that citrullination of both GFAP and MBP may contribute to the pathophysiology of EAE and that the brains of EAE mice may contain more pathology than previously realized.     

Localization of glial cell antigens in the brains of young normal mice and the dysmyelinating mutant mice, jimpy and shiverer

Tissue sections from the brains of normal, jimpy, and shiverer mice were immunostained by the peroxidase antiperoxidase method for carbonic anhydrase (CA) and the putative astrocytic "markers" glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The cells in normal gray matter that immunostained with anti-CA and anti-GS were similar to one another in size and process elaboration. In the normal gray matter there were relatively few GFAP-positive astrocytes. When present, these cells resembled the CA- and GS-positive cells; however, the GFAP appeared to be concentrated in the astroglial processes, as distinguished from the cell bodies. Glial cell processes, immunostained for CA or GS, surrounded blood vessels and unstained neurons in the normal gray matter. The glial cells in shiverer gray matter were similar to those in the normal gray matter. When stained for GS or GFAP, the glial cells in the jimpy gray matter appeared to be somewhat hypertrophied, and when the glial cells in this mutant were stained for CA, the nuclei appeared to be swollen. It was concluded that some of the CA-positive cells in the gray matter of the normal and of each mutant mouse brain could be astrocytes. The patterns of immunostaining in the white matter emphasized the different complements of glial cells in the mutants. In the normal and shiverer mouse corpus callosum, CA, in particular, was detected only in the oligodendrocytes, their processes, and myelin. However, the data concerning the jimpy mouse suggested that the few CA-positive cells in the corpus callosum of that mutant could be astrocytes.     

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.     

Transplant-derived astrocytes migrate into host lumbar and cervical spinal cord after implantation of E14 fetal cerebral cortex into adult thoracic spinal cord

Fourteen-day gestation fetal cerebral cortex homografts were transplanted into the thoracic (T6) spinal cord between the left dorsal column and dorsal horn of adult host rats. The transplants were soaked in 2.0 micrograms/ml of the lectin Phaseolus vulgaris leucoagglutinin (PHAL) prior to implantation. Transplanted host spinal cords were utilized at 7, 14, and 24 d and at 1 and 2 months postimplantation. Paraffin-sectioned spinal cords were double labeled for PHAL and glial fibrillary acidic protein (GFAP) by using FITC- and RITC-conjugated secondary antisera, respectively. Montages of FITC- and RITC-positive cells were analyzed for cells containing both fluorescences. Double-labeled cells (PHAL-GFAP) were transplant-derived astrocytes. Transplant-derived astrocytes were observed to initiate migration in the white matter columns of the host at approximately 14 d after transplantation. Double-labeled astrocytes were observed in cervical and lumbar spinal cord of the host (ca. 3.5 cm away from the center of the transplant) at 2 months postoperative. These astrocytes migrated at approximately 0.76 mm a day (after a 14-d delay). At 2 months, transplant-derived astrocytes composed as much as 50% of the astrocytes in the white matter of the host 2.0 mm from the transplant. The migrated astrocytes were hypertrophied and appeared reactive. Astrocytes in spinal gray matter only migrate about 1.0 mm from the graft-host interface. Transplant-derived astrocytes can migrate the entire length of the spinal cord white matter.     

Immunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

The appearance of reactive astrocytes following brain injury was investigated in 4-week-old rats with special reference to their proliferation and chronological changes in the cytoskeletal proteins. Two days after the injury, glial fibrillary acidic protein (GFAP)-positive cells had increased in number around the lesion and spread to the entire ipsilateral cortex by 3 days after the injury. To investigate the distribution of mitotic cells and its chronological change, immunohistochemical staining with monoclonal antibody to bromodeoxyuridine (BrdU) was performed. BrdU-positive cells began to appear around the lesion and spread to the entire ipsilateral cortex by 3 days and their distribution was the same as that of GFAP-positive cells. To investigate the association of GFAP-positive cells with cell division, double labeling experiments using [3H]thymidine autoradiography and immunohistochemical staining with antiserum to GFAP were performed. Cells doubly labeled with GFAP and [3H]thymidine were localized in the area adjacent to the lesion, in the molecular layer of the cortex and in the white matter. By contrast, none of the cells were doubly labeled in the IInd to VIth layers of the cortex. Furthermore, only astrocytes in the former areas expressed vimentin transiently from 2 to 10 days after the injury. In the rats administered vincristine, cells arrested during mitosis were found in the regions which express vimentin. From these results, it was suggested that astrocytes in the molecular layer of the cortex and the white matter adjacent to the lesion proliferated in response to the injury and expressed vimentin transiently, then acquired GFAP, and that astrocytes in the IInd to VIth layers of the cortex became reactive astrocytes without mitosis.     

Axonal and Nonneuronal Cell Responses to Spinal Cord Injury in Mice Lacking Glial Fibrillary Acidic Protein

We have examined the regeneration of corticospinal tract fibers and expression of various extracellular matrix (ECM) molecules and intermediate filaments [vimentin and glial fibrillary acidic protein (GFAP)] after dorsal hemisection of the spinal cord of adult GFAP-null and wild-type littermate control mice. The expression of these molecules was also examined in the uninjured spinal cord. There was no increase in axon sprouting or long distance regeneration in GFAP−/− mice compared to the wild type. In the uninjured spinal cord (i) GFAP was expressed in the wild type but not the mutant mice, while vimentin was expressed in astrocytes in the white matter of both types of mice; (ii) laminin and fibronectin immunoreactivity was localized to blood vessels and meninges; (iii) tenascin and chondroitin sulfate proteoglycan (CSPG) labeling was detected in astrocytes and the nodes of Ranvier in the white matter; and (iv) in addition, CSPG labeling which was generally less intense in the gray matter of mutant mice. Ten days after hemisection there was a large increase in vimentin⁺cells at the lesion site in both groups of mice. These include astrocytes as well as meningeal cells that migrate into the wound. The center of these lesions was filled by laminin⁺/fibronectin⁺cells. Discrete strands of tenascin-like immunoreactivity were seen in the core of the lesion and lining its walls. Marked increases in CSPG labeling was observed in the CNS parenchyma on either side of the lesion. These results indicate that the absence of GFAP in reactive astrocytes does not alter axonal sprouting or regeneration. In addition, except for CSPG, the expression of various ECM molecules appears unaltered in GFAP−/− mice.     

Tanycytes transplanted into the adult rat spinal cord support the regeneration of lesioned axons

During past years a number of therapeutic strategies have been developed in order to stimulate axonal regeneration after traumatic injuries of the spinal cord. Recently, encouraging data have been obtained by grafting specific glial cells such as Schwann cells or olfactory ensheathing glial cells, known to support the regeneration of peripheral or central axons, respectively. In a recent series of studies, we have shown that tanycytes, a particular glial cell type present in the mediobasal hypothalamus, were able to support the regeneration of a variety of axons innervating this region. The aim of the present study was to determine whether tanycytes could also support the regeneration of lesioned spinal axons. Cultured hypothalamic tanycytes and cortical astrocytes were prelabeled with Fast blue (FB) and grafted into the thoracic spinal cord of adult rats. Three weeks after the transplantation, the animals were fixed and spinal cord sections treated for multiple fluorescence detection of the FB-labeled transplanted cells on the one hand and of various glial and neuronal markers on the other hand. We show here that in all the spinal cords examined, transplanted tanycytes or astrocytes formed large spherical clusters of about 0.5 mm in diameter, located in the mediolateral spinal cord layer. The immunodetection of glial markers showed that transplanted astrocytes exhibited intense immunostaining for both glial fibrillary acidic protein (GFAP) and vimentin (VIM), whereas transplanted tanycytes were intensely immunostained for VIM, but GFAP negative. The immunodetection of axonal markers showed that contrasting with astrocyte transplants, tanycyte transplants were invaded by numerous axonal fibers. These data indicate that tanycyte transplants may represent a useful therapeutic tool for the reparation of the lesioned spinal axons.     

Glutamine synthetase in the central nervous system is not confined to astrocytes.

Glutamine synthetase (GS) immunoreactivity is frequently used as an astroglial 'marker'. However, when sections of adult rat spinal cords were immunostained with antibodies against sheep glutamine synthetase, intense immunofluorescence was observed in cells resembling oligodendrocytes. In white matter in the rat brain GS immunostaining was also found in ovoid oligodendrocyte-like cells, whereas in gray matter in the same tissue sections GS immunostaining was found in astrocytes. Like the antibodies against sheep GS, antibodies against rat GS also immunostained putative oligodendrocytes, and colocalization with 2',3'-cyclic nucleotide-3'-phosphohydrolase in spinal cord supported the designation of the GS-positive cells as oligodendrocytes.     

Axonal regeneration in old multiple sclerosis plaques

Summary Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225–232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.Supported by NIH grant NS 13034 and by the Veterans Administration     

Expression of osteopontin and its ligand, CD44, in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of osteopontin (OPN) and one of its ligands, CD44, was studied in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that osteopontin significantly increased at the early and peak stage of EAE and slightly declined thereafter. Osteopontin was constitutively expressed in some astrocytes adjacent to pia mater and neurons in normal rats, and was shown to be increased in the same cells and also in some inflammatory cells including macrophages at the early and peak stage of EAE. CD44, a ligand for osteopontin, was constitutively expressed in astrocytes in normal and control spinal cords and was also expressed in inflammatory cells, as well as increased expression in astrocytes in EAE. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of osteopontin in rat EAE, and osteopontin may interact with its ligand CD44 on astrocytes and inflammatory cells in EAE, possibly mediating autoimmune central nervous system (CNS) diseases in rats.