Immunohistochemical localization of citrullinated proteins in adult rat brain

By using hybridoma technology, an IgM monoclonal antibody (F95) against multiple citrullinated synthetic and natural peptides was recently developed and used to stain immunohistochemically subsets of astrocytes and myelin basic protein (MBP) from selected regions of human brain (Nicholas and Whitaker [2002] Glia 37:328-336). With this antibody, the present study provides a more detailed localization of citrullinated epitopes in the central nervous system (CNS) by examining immunohistochemical staining patterns for F95 in the normal adult rat brain. Thus, immunohistochemical labeling for citrullinated epitopes was seen in white matter areas consistent with myelin staining; however, in general, it was more prominent and uniform in the caudal CNS (spinal cord, medulla oblongata, pons, and cerebellum) than in more rostral areas. F95 staining was also seen in cells and fibers often intimately associated with blood vessels and/or ventricular surfaces. By using dual-color immunofluorescence, the vast majority of this latter staining was colocalized within a subset of astrocytes also immunoreactive for glial fibrillary acidic protein (GFAP). By using Western blot analysis of rat brain proteins, multiple GFAP- and MBP-immunoreactive proteins and peptide fragments were seen, and many of them were also reactive with the F95 antibody. Thus, the present study not only demonstrates that citrullinated epitopes in normal rat brain are most concentrated in subsets of myelin and astrocytes but also provides evidence that GFAP, like MBP, may be present as multiple citrullinated isoforms.     

Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis

In this study, we demonstrate that grossly unaffected white matter from secondary progressive multiple sclerosis (SP-MS) patients is heavily citrullinated, as compared to normal white matter from control patients. Citrullination was most pronounced at plaque interfaces and was shown to colocalize with glial fibrillary acidic protein (GFAP)-immunoreactivity using dual color immunofluorescence. In contrast, the plaques themselves weakly stained for citrullinated proteins compared to control white matter and usually contained a blood vessel with surrounding astrocytes that were positive both for citrullinated proteins and GFAP. In SP-MS brain samples, but not in normal brains, long fibers of colocalized GFAP- and citrullinated proteins extended into the gray matter. Increased numbers of astrocytes containing citrullinated proteins and GFAP were also present at the junction between the gray and white matter in SP-MS brains. Western blot analysis of acidic brain proteins from nonplaque-containing white matter showed upregulation of multiple citrullinated GFAP proteins in SP-MS brains as compared to controls. Our results demonstrate that increased amounts of citrullinated GFAP are present in SP-MS brains, but also shows that these proteins are present in areas of MS brains that were grossly normal appearing. These data raise the possibility that citrullination of GFAP contributes to the pathophysiology of MS.     

Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.     

Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis

Immunization of mammals with central nervous system (CNS)-derived proteins or peptides induces experimental autoimmune encephalomyelitis (EAE), a disease resembling the human autoimmune disease multiple sclerosis (MS). Both diseases are accompanied by destruction of a part of the of the myelin sheaths, which surround neurites in the CNS. Previous studies in MS have described alterations in the citrullination of myelin basic protein, one of the main protein constituents of the myelin sheath. Here, we show that, also during the development of EAE in mice, hypercitrullination occurs in the areas of the spinal cord that show the highest degree of inflammation and that myelin basic protein and glial fibrillary acidic protein are among the hypercitrullinated proteins. We conclude that hypercitrullination of myelin proteins in the CNS is a common phenomenon in demyelinating disease. Hypercitrullination may cause conformational changes in proteins, so the affected proteins may be involved in the pathogenesis of CNS autoimmune disease by acting as autoreactive T-cell epitopes. This is the first report in which hypercitrullination of CNS proteins in EAE is described and in which proteins other than myelin basic protein are reported to be citrullinated during autoimmune-mediated CNS inflammation.     

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.     

Developmental changes of glial fibrillary acidic protein and myelin basic protein in perinatal leukomalacia: relationship to a predisposing factor

Focal white matter necrosis is frequently seen in the brains of infants with perinatal cerebral hypoperfusion. Periventricular leukomalacia (PL) occurs in the deep white matter of premature and term neonates and subcortical leukomalacia (SL) in the subcortical white matter of young infants. Using immunoperoxidase methods in normal infants, glia positive for glial fibrillary acidic protein (GFAP) were found first in the deep zones of white matter and with increasing age they became more prominent in the subcortical zone. They increased diffusely in the deep or subcortical zones of the cases of PL or SL, respectively. The number of myelin basic protein-positive glia is much larger than that of GFAP-positive glia in the cases of old PL. These findings suggest that an increased number of positive glia may be a reaction to hypoxic, ischemic, or toxic insults, or this shifting, transient increase of positive glia in cerebral white matter may be one of several predisposing factors leading to perinatal leukomalacia. Furthermore, positive staining of GFAP and MBP for reactive astrocytes in old PL suggests that at a certain stage of gliogenesis both GFAP and myelin basic protein may be present within the same cell.     

Gliosis in the spinal cords of rats with experimental allergic encephalomyelitis: immunostaining of carbonic anhydrase and vimentin in reactive astrocytes

Spinal cord sections from rats sensitized to develop experimental allergic encephalomyelitis (EAE) were immunostained with antibodies against glial fibrillary acidic protein (GFAP), carbonic anhydrase, and vimentin, to see whether the latter two antigens could be detected in GFAP-positive reactive astrocytes. Sixteen days after sensitization (16 dpi) there was intense carbonic anhydrase immunostaining in GFAP-positive cells in the spinal cords of EAE rats, particularly in the white matter. At 13 and 20 dpi carbonic anhydrase immunostaining in astrocytes was less intense, and in the spinal cord white matter of control animals carbonic anhydrase was not detected in the few GFAP-positive cells. In the spinal cords of EAE rats vimentin immunostaining was observed in inflammatory cells and astrocytes. In the latter, GFAP and carbonic anhydrase were colocalized with vimentin. The data suggest that carbonic anhydrase expression in astrocytes is an acute response to injury and that vimentin can be detected in astrocytes, as well as inflammatory cells, as early as 16 dpi.     

[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.     

Immunohistochemical localization of the myelin basic protein and of the glial fibrillary acidic protein: comparative study in normal, quaking and jimpy mice

The topographical distribution of myelin basic protein (MBP) and of glial fibrillary acidic protein (GFA) have been compared in the CNS of 18-day-old normal, quaking, and jimpy mice using indirect immunofluorescence. Comparison was made in brain and spinal cord sections. MBP was strongly decreased in quaking and jimpy mice in all parts of the CNS. A pronounced gliosis was observed in jimpy mice with occurrence of numerous hypertrophied astrocytes. To a lesser extent, an astrocytic reaction was also observed in quaking CNS.     

GFAP mRNA fluctuates in synchrony with chronic relapsing EAE symptoms in SJL/J mice

Activation of astrocytes and hypertrophy of their processes is a result of a number of pathological conditions in the central nervous system. Astrocytic gliosis is especially prominent in multiple sclerosis (MS), where astrocytic fibers form a dense matrix around demyelinated axons. Experimental allergic encephalomyelitis (EAE), a laboratory model for MS, is also accompanied by astrocytic hyperactivity. We have previously shown the formation of plaque-like structures which stain heavily for glial fibrillary acidic protein (GFAP) in the brains and spinal cords of SJL/J mice after several episodes of chronic relapsing EAE (Smith and Eng: J Neurosci Res 18:203, 1987). To further investigate the mechanisms of this phenomenon, we have measured the levels of mRNA for GFAP throughout the course of three episodes and recoveries of EAE in the SJL/J mouse. Mice were immunized with spinal cord homogenate and subsequently developed EAE. After recovery they were again immunized at appropriate intervals, resulting in successive episodes of EAE, with partial or complete recovery between the paralytic stages. At appropriate times in the course of the different stages of EAE, spinal cords were dissected and RNA was prepared from each spinal cord. RNA Was analyzed by Northern blots to determine the levels of mRNA for GFAP and, as a control, for the 70 kDa neurofilament (NF-L). With the onset of the first EAE episode GFAP mRNA in spinal cords from animals with mild symptoms increased to sixfold the control level (P < 0.02) and to 20-fold in those with paralysis (P < 0.01). With recovery, the GFAP mRNA level decreased to twice the control. With each subsequent episodes, a chronic but stable neurological deficit was established, with GFAP mRNA at about eightfold the control levels (P < 0.01). Over the course of several episodes, the GFAP rose to about 2.8 times the control, while vimentin increased by a factor of 3.6. Thus multiple episodes of EAE resulted in upregulation of GFAP mRNA and accumulation of GFAP, which are associated with astrocyte activation and hypertrophy. Similar events may occur in the human demyelinative disease MS, where multiple episodes of inflammatory cell invasion occur, resulting in a neurological deficit. © 1995 Wiley-Liss, Inc.     

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。     

Experimental autoimmune encephalomyelitis: Antigen-induced inhibition of biochemical and immunohistological alterations

A comprehensive biochemical, immunological, and histological study was undertaken during suppression of experimental autoimmune encephalomyelitis (EAE) induced by antigen-specific inhibition of the immune response. Pretreatment of Wistar rats by intraperitoneal administration of low doses of saline-soluble bovine myelin or myelin basic protein (MBP) but not with ovalbumin suppresses the appearance of the clinical symptoms of EAE induced by sensitization with bovine myelin in complete Freund's adjuvant. Analysis of the central nervous system (CNS) of animals pretreated with MBP or whole myelin shows inhibition of the diminution of MBP and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) activity observed in the EAE animals or in rats pretreated with ovalbumin. With respect to the CNS lipid content, these suppressive treatments abolish the increase in esterified cholesterol and partially revert the diminution in the content of cerebrosides and total cholesterol characteristic of the acute stage of the disease. Concomitantly, meningeal and parenchymal infiltration with mononuclear cells and deposits of immunoglobulins in the infiltrated regions as well as in spinal cord motor neurons were reduced. Analysis of the humoral response to myelin antigens shows that all EAE as well as treated animals developed antibodies to MBP and other myelin proteins. However, a higher incidence and level of these antibodies was observed in nontreated EAE animals and MBP- and ovalbumin-treated rats, while rats treated with total bovine myelin showed a highly reduced humoral response. The present results indicate that intraperitoneal treatment with soluble forms of myelin antigens, concomitant with the suppression of the clinical symptoms of the disease, markedly reduces the biochemical and histological alterations occurring in EAE animals and produces changes in the autoimmune humoral response. © 1996 Wiley-Liss, Inc.     

Developmental expression of glial-specific mRNAs in primary cultures of rat brain visualized by in situ hybridization

The localization of mRNAs which encode the glial-specific marker proteins, glial fibrillary acidic protein (GFAP), glycerol phosphate dehydrogenase (GPDH, EC 1.1.1.8), and myelin basic protein (MBP), was mapped by in situ hybridization in primary cultures of 1-2-day-old rat brain in serum-supplemented medium. Developmental changes of these expressed mRNAs were examined after various times in culture ranging from 8 to 50 days and were correlated with the histological, morphological, and positional characteristics of the cells. By day 8, the culture stratified into a population of flat polygonal astrocytes covered by another population of phase-dark process-bearing cells. When counterstained with May-Grunwald histological stain, astrocytes appeared pale blue, whereas two subpopulations of phase-dark cells stained differentially; one was dark blue while the other was red and smaller. GFAP-specific sequences were abundant at day 8, increased in the astrocyte bedlayer as the culture became confluent, and plateaued at approximately day 16. A minor proportion of blue phase-dark cells contained GFAP mRNA although at a lower abundance. In contrast, GPDH mRNA positive blue phase-dark cells were seen scattered throughout the upper layer of the culture and also around the perimeter of large clumps of red phase-dark cells. These cells were infrequent at day 8 but increased in number at later time points. The expression of MBP mRNA differed from GPDH in that it was more abundant at early time points, plateaued between day 20 and day 24, and was predominantly localized in red phase-dark cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental autoimmune encephalomyelitis induction in peptidylarginine deiminase 2 knockout mice

During the development of multiple sclerosis the destruction of the myelin sheath surrounding the neurites is accompanied by citrullination of several central nervous system (CNS) proteins, including myelin basic protein and glial fibrillary acidic protein. In experimental autoimmune encephalomyelitis (EAE), a disease induced in animals by immunization with proteins or peptides from the CNS, the animals develop symptoms similar to multiple sclerosis (MS). The increased levels of citrullinated CNS proteins associated with MS are also observed during the development of EAE. To study the role of CNS protein citrullination in EAE development, we induced EAE with a peptide derived from myelin oligodendrocyte glycoprotein (MOG(35-55)) in mice lacking the peptidylarginine deiminase 2 (PAD2) protein, because this enzyme was the most likely candidate to be involved in catalyzing CNS protein citrullination in the diseased state. Even though the PAD2 knockout mice displayed a dramatic reduction in the amount of citrullination present in the CNS, indicating that PAD2 is indeed responsible for the majority of detectable citrullination observed in EAE, the development of EAE was not impaired by genetic deletion of PAD2, suggesting that PAD2 catalyzed citrullination is not essential to the development of EAE.     

Bone morphogenetic proteins 4, 6, and 7 are up-regulated in mouse spinal cord during experimental autoimmune encephalomyelitis

Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up-regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG-peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT-PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2- to 4-fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle-treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG-peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up-regulated during EAE. (c) 2007 Wiley-Liss, Inc.     

[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.     

Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co-localizes with tubulin

Differentiation of oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we have shown the presence of MAP1B in oligodendrocytes grown in primary glial cultures by double-label immunofluorescence using antibodies to galactocerebroside (GC) and MAP1B. The staining of the cultures showed that GC-positive oligodendrocytes were also stained with MAP1B antibodies. However, MAP1B stain was limited to cell bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta-tubulin in secondary glial cultures that were enriched for oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double-labeling of the cells showed that MAP1B was co-localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)-positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MAP1B (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.     

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.     

实验性自身免疫性脑脊髓炎的小胶质细胞和星形胶质细胞反应

目的确定小胶质细胞和星形胶质细胞在实验性自身免疫性脑脊髓炎（ＥＡＥ）发病及病变发展中的作用。方法用牛脊髓髓鞘碱性蛋白（ＭＢＰ）免疫豚鼠发生ＥＡＥ,用免疫组化法观察ＥＡＥ不同病期小胶质细胞和星形胶质细胞对炎性脱髓鞘病灶的反应。结果发生ＥＡＥ的前３天,小胶质细胞即开始激活,在临床症状出现时其数量及激活程度达高峰,并持续至高峰期。恢复期数量逐渐减少,激活程度逐渐减弱。星形胶质细胞在症状高峰期开始激活并围绕在浸润细胞和病变血管周围,似有隔离小胶质细胞与病灶接触的作用,至恢复期激活明显。结论小胶质细胞激活在ＥＡＥ的发病及进展中起重要作用,而星形胶质细胞主要与疾病的恢复有关。     

Induction of experimental autoimmune encephalomyelitis in guinea pigs using myelin basic protein and myelin glycolipids

Strain 13 guinea pigs were immunized with galactocerebroside, asialo-GM1 (GA1) or GM4 ganglioside in association with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) to produce experimental autoimmune encephalomyelitis (EAE). The clinical and pathological features, serum antibodies, and lipid compositions of affected brains and spinal cords were compared with those of guinea pigs immunized with MBP, in CFA, alone. Perivascular demyelination was seen in brains from all guinea pigs immunized with GA1/MBP. The incidence and degree of demyelination in this group were significantly higher than in the group immunized with only MBP. The onset of EAE was slightly, but significantly, retarded in groups of animals immunized with GM4/MBP and there was no detectable demyelination. Otherwise, no significant differences were detected between groups. Augmentation of EAE by myelin glycolipids may provide some important clues in understanding the mechanism of demyelinating diseases.