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.     

Upregulation of β-1,4-Galactosyltransferase I in Rat Spinal Cord with Experimental Autoimmune Encephalomyelitis

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. β-1,4-Galactosyltransferase I (β-1,4-GalT-I) is a major galactosyltransferase responsible for selectin–ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of β-1,4-GalT-I was low in normal or complete Freund’s adjuvant (CFA) control rats’ spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of β-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of β-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of β-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65–25 kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that β-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that β-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin–ligand biosynthesis and interaction with E-selectin.     

Activation of mitogen-activated protein kinases in experimental autoimmune encephalomyelitis

The expression of mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK), and p38, was analyzed in experimental autoimmune encephalomyelitis (EAE) in rats.

Western blot analysis showed that the three MAP kinases (phosphorylated ERK (p-ERK), p-JNK, and p-p38) were increased significantly in the spinal cords of rats with EAE at the peak stage as compared with the levels in controls (p<0.05), and both p-ERK and p-JNK declined slightly in the recovery stage of EAE. Immunohistochemistry showed that p-ERK was constitutively expressed in brain cells, including astroglial cells, and showed enhanced immunoreactivity in those cells in EAE, while some T cells and macrophages were weakly immunopositive for p-ERK in EAE lesions. Both p-JNK and p-p38 were intensely immunostained in T cells in EAE lesions, while a few glial cells and astrocytes were weakly positive for both.

Taking all these facts into consideration, we postulate that increased expression of the phosphorylated form of each MAP kinase plays an important role in the initiation of acute monophasic EAE. Differential expression of three MAP kinases was discerned in an animal model of human autoimmune central nervous system diseases, including multiple sclerosis.     

The subarachnoid space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis

To characterize the phenotype of inflammatory cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), Lewis rats were immunized with guinea pig myelin basic protein and frozen sections of the spinal cord with EAE were examined immunohistochemically using a panel of monoclonal antibodies against T cells and adhesion molecules. In addition, double immunostaining was performed with glial and T cells markers to examine the interaction between infiltrating T cells and reactive brain cells during the course of EAE. In the early stage of EAE, inflammatory cells first appeared in the subarachnoid space (SAS) and infiltrated the subpial region. The majority of inflammatory cells in SAS expressed TCRaβ and either CD4 or CD8 molecules. However, only CD4⁺ T cells infiltrated the parenchyma while the majority of CD8⁺ cells remained in SAS. A similar differential localization of T cells was observed with regard to CD45RC molecules. Inflammatory cells in SAS consisted of both CD45RC⁺ and CD45RC^- population, while those in the parenchyma were largely CD45RC˜. With regard to adhesion molecules, the leptomeninges constitutively expressed fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1). Most SAS inflammatory cells expressed very late activation antigen 4 (VLA-4) and, to lesser extent, lymphocyte function-associated antigen 1 (LFA-1) in the early stage of EAE. On the other hand, parenchyma! infiltrating cells expressed LFA-1 more strongly in the peak stage. Double staining for Vβ8.2 TCR and microglia demonstrated an increase in the number of microglia together with morphological changes into rod-shape cells in the vicinity of infiltrating T cells. Furthermore, these cells expressed adhesion molecules, such as LFA-1, ICAM-1 and CD4. These findings suggest that VLA-4/FN and LFA-1/ICAM-1 interactions between infiltrating cells and brain cells may be involved in the early and peak stages of EAE. Phenotype switching occurring in the process of inflammatory cell infiltration may be regulated by these adhesion molecules and factor(s) provided by the parenchyma, possibly by microglia.     

Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions

Expression of osteopontin and CD44 in the brain was studied after cryolesioning to understand how osteopontin and its receptor, CD44, are involved in processes in the brains of rats with cryolesions. Western blot analysis showed that osteopontin increased significantly at days 4 and 7 post-injury and declined slightly thereafter in cryolesioned brains in comparison with levels in sham-operated controls. An immunohistochemical study localized osteopontin in activated microglia/macrophages in the core lesions, where the majority of macrophages proliferate. Osteopontin was also detected temporarily in some neurons and a few astrocytes in the lesion periphery on days 4 and 7 post-injury, but the immunoreactivity in macrophages, neurons, and astrocytes disappeared by day 14 post-injury. There was some CD44, a receptor for osteopontin, in the brain cells of sham-operated rats. After injury, intense CD44 immunostaining was seen in the majority of macrophages and in reactive astrocytes, but not in neurons, in the ipsilateral lesions after day 4 post-injury, and this immunoreactivity remained on day 14 post-injury. These findings suggest that activated microglia/macrophages and some neurons are major sources of osteopontin during the early stage of brain damage induced by a cryolesion and that osteopontin interacts with CD44 expressed on astrocytes and activated microglia/macrophages in the damaged cerebral cortex, possibly mediating cell migration after cryolesioning in the rat brain.     

Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system

Gliosis is a repair process of lesions appearing in the central nervous system (CNS). Although gliosis by astrocytes (astrocytic gliosis) has been well documented, that by microglia (microglial gliosis) remains poorly understood. In the present study we induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats and examined microglial and astroglial reactions to EAE lesions at various stages of the disease by immunohistochemistry. For the demonstration of microglia and astrocytes, antibodies against complement receptor type 3 (OX42) and glial fibrillary acidic protein (GFAP) were used, respectively. It was revealed that the whole course of microglial and astroglial reactions to EAE lesions is divisible into three stages, i.e., initial, peak and recovery stages. Microglial and astroglial reactions to EAE lesions at each stage correspond well with the clinical and histological stages of EAE. At the initial stage, rats showed mild clinical signs and a few inflammatory foci were found in the CNS. Microglia were increased in number in close association with inflammatory cell aggregates, whereas astrocytes showed no significant reaction in spite of the presence of inflammatory cells. At the peak stage, rats showed full-blown EAE and the number of inflammatory cells reached maximum. The most characteristic finding at this stage was 'encasement' of inflammatory lesions by astrocytic fibers. Microglia were increased in number, but association of microglia with lesions was prevented by astrocytes. Interestingly, however, such characteristic distribution of microglia and astrocytes was not observed at the recovery stage. Residual inflammatory cell aggregates were intermingled with dense microglial and astrocytic gliosis, forming 'micro-astroglial scars'. Double immunofluorescence staining with anti-GFAP and anti-bromodeoxyuridine (BrdU), or with OX42 and anti-BrdU revealed that BrdU-incorporated microglia, but not astrocytes, were present mainly at the initial and peak stages, suggesting that microglia would proliferate by cell division to create gliosis, whereas astrocytic gliosis would be a result of migration of astrocytes and/or up-regulation of expression of GFAP molecule. Taken together with previous in vitro findings that microglia, but not astrocytes, stimulate encephalitogenic T cell proliferation, these in vivo findings suggest that microglia augment, whereas astrocytes suppress, inflammatory processes in the CNS.     

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.     

Fas system up-regulation in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated disorder characterized by infiltration of the central nervous system (CNS) by mononuclear cells and macrophages, and serves as a model for multiple sclerosis. In acute monophasic and relapsing remitting forms of EAE, the CNS inflammatory infiltrates are cleared within a few days and, simultaneously, animals recover from their clinical disability. The mechanisms for rapid disappearance of the inflammatory cells are not fully understood. Fas and Fas-ligand (Fas-L) molecules are thought to play an important role in the deletion of autoimmune reactive T cells through apoptosis. However, recent observations in transgenic lpr and gld mice show that mutations inactivating Fas and Fas-L respectively ameliorate signs of EAE despite persistence of immune cell infiltrates into the CNS. In the current study, the expression of Fas and Fas-L was investigated by immunochemistry and in situ hybridization during the course of EAE in DA rats that were actively immunized with syngenic spinal cord homogenate. CNS apoptotic cells were simultaneously examined using terminal transferase dUTP nick end-labeling techniques. During the acute phase of the disease, a significant proportion of CNS CD4+ cells (80%) and macrophages (50%) expressed Fas and Fas-L (80 and 60%, respectively). Simultaneously, about 20% of CD4+ cells and 30% of macrophages were found to be apoptotic. Some astrocytes and neurons also expressed Fas and Fas-L, although they did not appear to be apoptotic. These results further support a role for Fas-mediated lymphocyte and macrophage apoptosis in this model of CNS autoimmune disease but they also suggest a more complex role for Fas/Fas-L interactions in CNS autoimmunity, including resident cells.     

黏附分子CD44在实验性自身免疫性脑脊髓炎中的作用

目的 探讨黏附分子CD44在实验性自身免疫性脑脊髓炎（EAE）发病中的作用。方法 将20只大鼠随机分为正常对照组及EAE组,EAE组采用粗制髓鞘碱性蛋白（MBP）抗原注入大鼠后足掌皮下（0.2 ml/100 g）制作EAE模型,观察大鼠的发病情况及病理表现;并采用免疫组织化学法检测两组大鼠脑组织CD44的含量。结果 正常对照组大鼠未发病,EAE组大鼠均有不同程度的发病。HE染色后,光镜下观察,正常对照组大鼠脑和脊髓无异常;EAE组大鼠可见脑及脊髓实质内小血管充血,小静脉周围有大量炎性细胞浸润,血管周围白质脱髓鞘改变。免疫组化显示,正常对照组大鼠脑和脊髓组织未发现CD44阳性细胞;EAE组大鼠中枢神经系统（CNS）白质及灰白质交界处可见大量CD44阳性细胞。结论 EAE模型中存在黏附分子CD44的高表达,其对EAE的发病可能起到促进作用。     

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.     

黏附分子CD44在实验性自身免疫性脑脊髓炎中的作用

目的探讨黏附分子CD44在实验性自身免疫性脑脊髓炎(EAE)发病中的作用。方法将20只大鼠随机分为正常对照组及EAE组,EAE组采用粗制髓鞘碱性蛋白(MBP)抗原注入大鼠后足掌皮下(0. 2 ml/100 g)制作EAE模型,观察大鼠的发病情况及病理表现;并采用免疫组织化学法检测两组大鼠脑组织CD44的含量。结果正常对照组大鼠未发病,EAE组大鼠均有不同程度的发病。HE染色后,光镜下观察,正常对照组大鼠脑和脊髓无异常; EAE组大鼠可见脑及脊髓实质内小血管充血,小静脉周围有大量炎性细胞浸润,血管周围白质脱髓鞘改变。免疫组化显示,正常对照组大鼠脑和脊髓组织未发现CD44阳性细胞; EAE组大鼠中枢神经系统(CNS)白质及灰白质交界处可见大量CD44阳性细胞。结论 EAE模型中存在黏附分子CD44的高表达,其对EAE的发病可能起到促进作用。     

左旋咪唑对实验性自身免疫性脑脊髓炎大鼠脊髓内CD4 、CD8、CD28和CD152表达的影响

目的 探讨左旋咪唑（Levamisole,LMS）对实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis，EAE）大鼠脊髓内单个核细胞CD4、CD8、CD28及CD152表达的影响及其意义。方法 采用临床症状评分、病理学检查和流式细胞术，观察LMS处理和非处理条件下豚鼠全脊髓匀浆诱导的Wistar大鼠EAE临床表现、病理改变及脊髓内单个核细胞CD4、CD8、CD28及CD152分子表达水平。结果 LMS同时给药促发组和预处理组EAE大鼠脊髓内单个核细胞CD4、CD28表达水平明显高于非LMS处理的EAE模型组（P〈0．01），病理变化更加明显。而CD8、CD152表达水平差异无统计学意义。结论 LMS可促进EAE大鼠脊髓内单个核细胞CD4、CD28的表达，提示LMS上调CD4、CD28的表达可能与LMS诱发炎性脱髓鞘白质脑病有关。     

Upregulation of water channel aquaporin-4 in experimental autoimmune encephalomyeritis

Aquaporin-4 (AQP4) is a water channel protein that plays an important role in water movement in the central nervous system (CNS). Recently, presence of anti-AQP4 antibody has been reported in the sera from patients with neuromyelitis optica. AQP4 is therefore a possible target for inflammatory mechanisms in CNS. In the present investigation, we performed semi-quantitative analysis of AQP4-mRNA in brain and spinal cord from mice affected with experimental autoimmune encephalomyelitis (EAE) using real-time PCR. AQP4-mRNA expression was increased in EAE; reaching a peak in the spinal cord at 14 days, and in the brain at 21 days after first inoculation. Immunohistochemical analysis showed that AQP4 is expressed on astrocytes, indicating that the increase in AQP4 expression may correlate with astrocytic activation. This is the first study to demonstrate upregulation of AQP4 in EAE. The upregulation of AQP4 could be involved in the development of inflammation in the acute phase of EAE.     

Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS). EAE pathogenesis involves various cell types, cytokines, chemokines, and adhesion molecules. Given the complexity of the inflammatory response in EAE, it is likely that many immune mediators still remain to be discovered. To identify novel immune mediators of EAE pathogenesis, we performed an Affymetrix gene array screen on the spinal cords of mice at the onset stage of disease. This screening identified the gene encoding lipocalin 2 (Lcn2) as being significantly upregulated. Lcn2 is a multi-functional protein that plays a role in glial activation, matrix metalloproteinase (MMP) stabilization, and cellular iron flux. As many of these processes have been implicated in EAE, we characterized the expression and role of Lcn2 in this disease in C57BL/6 mice. We show that Lcn2 is significantly upregulated in the spinal cord throughout EAE and is expressed predominantly by monocytes and reactive astrocytes. The Lcn2 receptor, 24p3R, is also expressed on monocytes, macrophages/microglia, and astrocytes in EAE. In addition, we show that EAE severity is increased in Lcn2(-/-) mice as compared with wild-type controls. Finally, we demonstrate that elevated levels of Lcn2 are detected in the plasma and cerebrospinal fluid (CSF) in MS and in immune cells in CNS lesions in MS tissue sections. These data indicate that Lcn2 is a modulator of EAE pathogenesis and suggest that it may also play a role in MS.     

Expression of Erythropoietin in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

Background and Purpose

Erythropoietin (Epo), originally recognized for its central role in erythropoiesis, has been shown to improve the outcomes in patients with various neurological disorders. The aim of this study was to elucidate the Epo expression pattern in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) and to assess the systemic effect of Epo during the course of EAE.

Methods

We used an EAE model induced in Lewis rats by immunization with myelin basic protein. Immunized rats were given recombinant human Epo (rhEpo) intraperitoneally at a dose of 5,000 U/kg for 7 consecutive days, either starting on day 3 post-immunization (five rats) or on the day of clinical symptom onset (score ≥1, five rats). After immunization, the rats were observed daily for clinical signs of EAE. Epo expression was investigated by Western blot analysis and immunohistochemistry.

Results

Western blot analysis showed that, Epo expression was significantly elevated relative to control in the rat spinal cord during the peak stage of EAE (p<0.05), and then decreased thereafter. Immunohistochemistry demonstrated that Epo was expressed in some neurons and glial cells. Epo immunoreactivity was detected in ED1-positive macrophages and astrocytes in EAE lesions. Furthermore, we found that the intraperitoneal administration of rhEpo reduced both the disease severity and duration of paralysis in EAE rats, and reduced macrophage activity and increased Epo activity.

Conclusions

Based on these findings, we postulate that Epo expression begins to increase at the start of EAE and that rhEpo administration leads to functional recovery from EAE paralysis.     

实验性自身免疫性脑脊髓炎大鼠脑神经干细胞的增殖

目的通过建立实验性自身免疫性脑脊髓炎动物模型,检测EAE神经干细胞的增殖。方法以豚鼠脑脊髓匀浆加完全弗氏佐剂作为抗原．给予Wistar大鼠四足垫皮内注射,建立EAE模型,对正常组、免疫后7d组、EAE发病后3,7,14,21d组,分别进行5一溴脱氧尿嘧啶（BrdU）免疫组织化学染色,以观察正常大鼠脑内Brdu、EAE免疫损伤后BrdU的变化规律。结果在正常组大鼠,Brdu阳性细胞主要分布在室管膜下区（SVz）及海马齿状回的颗粒层（SGZ）;Brdu阳性细胞教在免疫后呈现先上升后下降的变化趋势,于发病后3d达高峰,21d恢复正常。结论EAE可以诱导大鼠脑内神经干细胞的增殖。     

Ceruloplasmin gene‐deficient mice with experimental autoimmune encephalomyelitis show attenuated early disease evolution

We conducted a microarray study to identify genes that are differentially regulated in the spinal cords of mice with the inflammatory disease experimental autoimmune encephalomyelitis (EAE) relative to healthy mice. In total 181 genes with at least a two‐fold increase in expression were identified, and most of these genes were associated with immune function. Unexpectedly, ceruloplasmin (Cp), a ferroxidase that converts toxic ferrous iron to its nontoxic ferric form and also promotes the efflux of iron from astrocytes in the CNS, was shown to be highly upregulated (13.2‐fold increase) in EAE spinal cord. Expression of Cp protein is known to be increased in several neurological conditions, but the role of Cp regulation in CNS autoimmune disease is not known. To investigate this, we induced EAE in Cp gene knockout, heterozygous, and wild‐type mice. Cp knockout mice were found to have slower disease evolution than wild‐type mice (EAE days 13–17; P = 0.05). Interestingly, Cp knockout mice also exhibited a significant increase in the number of astrocytes with reactive morphology in early EAE compared with wild‐type mice at the same stage of disease. CNS iron levels were not increased with EAE in these mice. Based on these observations, we propose that an increase in Cp expression could contribute to tissue damage in early EAE. In addition, endogenous CP either directly or indirectly inhibits astrocyte reactivity during early disease, which could also worsen early disease evolution. © 2014 Wiley Periodicals, Inc.     

实验性自身免疫性脑脊髓炎小鼠脑内主要组织相容性复合体Ⅱ类抗原和分化群3ε的变化

目的观察实验性自身免疫性脑脊髓炎(EAE)小鼠脑内主要组织相容性复合体Ⅱ类抗原(MHC-Ⅱ)和分化群3ε(CD3ε)的变化。方法 25只C57BL/6小鼠随机分为EAE组(n=13)和正常对照组(n=12)。应用髓鞘少突胶质细胞糖蛋白35-55抗原诱导小鼠EAE模型。观察记录小鼠行为学变化;采用常规及髓鞘染色方法观察脊髓损伤和炎症细胞浸润程度;荧光定量PCR检测脑MHC-Ⅱ和CD3εmRNA的表达。结果 EAE组小鼠发病后EAE症状评分逐渐增加;脊髓炎症细胞浸润明显;髓鞘脱失较多;脑组织MHC-Ⅱ和CD3εmRNA表达显著高于正常对照组(均P<0.01),并与EAE症状评分呈正相关。结论 EAE小鼠脑内MHC-Ⅱ及CD3εmRNA表达水平增高与其病情严重程度一致。     

Phagocytotic removal of apoptotic,inflammatory lymphocytes in the central nervous system by microglia and its functional implications

Calpain activity and expression at the protein level were examined in inflammatory cells, activated microglia, and astrocytes prior to or at onset of symptomatic experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). EAE was induced in Lewis rats by injection of guinea pig spinal cord homogenate and myelin basic protein (MBP) emulsified with Complete Freund's Adjuvant (CFA). Calpain translational expression, determined by Western blot and immunocytochemistry, was correlated with calpain activity, infiltration of inflammatory cells, and myelin loss at 2-11 days following challenge with antigen. Controls (CFA only) did not show any changes over time in these parameters and very few changes (CD11+ microglia/mononuclear phagocytes) were seen in either group from days 2 to 8 post-induction. In contrast, from days 9 to 11, the animals that developed the disease (at least grade 1) demonstrated extensive cellular infiltration (CD4+, CD25+, and CD11+ as well as increased calpain expression (content) and activity. This study demonstrates that cell infiltration and increased calpain activity do not begin in the CNS until the onset of clinical signs.