SD大鼠缓解-复发型实验性变态反应性脑脊髓炎模型的建立

目的:建立大鼠缓解-复发型实验性变态反应性脑脊髓炎(EAE)模型,进行病理学研究,为多发性硬化(MS)的研究提供据.方法:采用大鼠脊髓和弗氏完全佐剂混合乳剂一次性注入 SD 大鼠双足垫和尾部,1周后半剂量注射进行一次加强,诱导大鼠发生 EAE;观察发病情况,HE 染色观察病理变化,监牢兰染色观察脱髓鞘情况.结果:空白对照组大鼠均未出现症状,HE 染色小脑及脊髓无炎性细胞浸润,监牢兰染色未见髓鞘脱失;模型组临床症状发生率为90%以上,HE 染色可见小脑白质及脊髓血管周围有大量炎性细胞浸润,监牢兰染色发现有大片髓鞘脱落.结论:用 SD 大鼠脊髓髓鞘蛋白和弗氏完全佐剂混合乳剂可诱导同种 SD 大鼠发生缓解-复发型 EAE.     

复发缓解型实验性变态反应性脑脊髓炎动物模型的建立

目的:建立复发缓解型实验性变态反应性脑脊髓炎(EAE)动物模型,进行病理学研究,为多发性硬化(MS)的发病机制研究及治疗药物研发提供合适的动物模型。方法:选择不同品系动物(昆明种、BALB/c和C57BL/6小鼠,SD大鼠),采用各自品系来源的脊髓和弗氏完全佐剂混合乳剂一次性注入动物双足垫,1周后半剂量注射进行一次加强,诱导EAE;观察发病情况,HE染色观察病理变化,监牢兰染色观察脱髓鞘情况。结果:昆明种、BALB/c和C57BL/6小鼠均无明显症状,HE染色小脑的脊髓无炎性细胞浸润;SD大鼠对照组正常,模型组临床症状发生率为90%以上,HE染色可见小脑白质及脊髓血管周围有大量的炎性细胞浸润,监牢兰染色可见大片髓鞘脱落。结论:EAE模型在昆明种、BALB/c及C57BL/6小鼠中用脊髓匀浆不易于诱导,而用SD大鼠脊髓髓鞘蛋白和弗氏完全佐剂混合乳剂可高效率诱导出同种SD大鼠的复发缓解型EAE。     

髓鞘碱性蛋白片段69-85诱导建立大鼠自身免疫性脑脊髓炎模型

目的探索以大鼠髓鞘碱性蛋白片段69-85（MBP69-85）为单一抗原,建立Wistar大鼠实验性自身免疫性脑脊髓炎（EAE）模型,并观察其病理改变。方法MBP69-85以生理盐水溶解,与完全福氏佐剂（CFA）充分混匀制备免疫乳剂;雌性Wistar大鼠70只,留取10只作为正常对照组,余者根据免疫乳剂组分的不同随机分为A、B、C组（n=20）。A组：MBP69-85每只50μg＋CFA（含卡介苗6 mg）;B组：MBP69-85每只25μg＋CFA（含卡介苗6 mg）;C组：MBP69-85每只25μg＋CFA（含卡介苗12 mg）。脑和脊髓组织切片进行HE染色,MBP及神经微丝（neurofilament,NF）免疫组化检测,观察神经组织的病理改变。结果A组内部分大鼠在免疫后第12-16天发病,表现为尾部及四肢无力或麻痹、斜颈等,平均临床症状评分为2.38±1.89;B、C组均未见发病;HE染色可见神经组织内炎细胞浸润,血管＂袖套＂样病灶形成;MBP及NF免疫组化染色显示病变组织内白质脱髓鞘及轴突损伤明显。结论EAE模型建立与免疫抗原的剂量有一定的依赖关系;佐剂中的卡介苗含量不是诱导大鼠发病的主要原因;本模型具有多发性硬化的典型临床表现及病理改变,是研究多发性硬化发病机制及治疗的可靠的动物模型。     

实验性自身免疫性脑脊髓炎小鼠的视神经损害

目的:探讨实验性自身免疫性脑脊髓炎(EAE)的视神经损害以及将其作为视神经炎模型的可能性.方法:取MOG35-55多肽和完全弗氏佐剂制备成抗原乳剂免疫C57BL/6小鼠,并在腹腔内注射2次百日咳杆菌,建立EAE模型.在EAE疾病高峰时,观察小鼠视神经炎的发生率及病理学改变.结果:模型组12只小鼠中有10只从免疫后第12天开始陆续起病,约在第17～19天达到疾病高峰,发病率为83.3%;而对照组未出现任何神经功能受损的症状.HE染色结果显示模型组的视神经组织中有大量的炎症细胞浸润,动物视神经炎的发生率为83.3%,其炎症评分为2.1±0.8分,与对照组相比均有统计学意义.LFB染色可见模型组的视神经有明显的脱髓鞘改变,病灶内可见炎症细胞浸润;而对照组小鼠的视神经未见明显异常改变.结论:借助EAE建立视神经炎模型是可行的,这为深入探讨视神经炎的发病机制提供了理想的动物平台.     

MMP1 在炎症大鼠牙髓组织中表达

目的:探讨基质蛋白酶MMP-1在炎症性牙髓中的表达。方法:内毒素制备大鼠牙髓炎模型,取牙髓炎大鼠及健康大鼠牙髓组织行HE染色检测牙髓组织的组织形态学变化,MMP-1特异性免疫组织化学染色和Western blot等检测MMP-1在牙髓组织的表达,以明确MMP-1在大鼠急性牙髓炎期的空间分布改变及蛋白表达量变化。结果:大鼠牙髓组织急性炎症期牙髓炎模型中可见大量炎性细胞浸润,结缔组织有断裂破坏。免疫组织化学分析及Western Blot分析均显示牙髓炎组织MMP-1蛋白显著增高(P0.05)。结论:MMP-1在牙髓炎组织中高表达,可能参与调节牙髓炎症反应的疼痛机制调节。     

EAE大鼠SFO血红素氧合酶-1基因和蛋白表达的变化

目的：探讨实验性变态反应性脑脊髓炎（EAE）时,大鼠血红素氧合酶-1（HO-1）在穹隆下器（SF0）中的变化,为证明SFO是感受外周信息物质的早期位点之一提供依据。方法：分别用免疫组化和原位杂交方法,观察了完全福氏佐剂-豚鼠脊髓匀浆（CFA-GPSCH）诱导大鼠EAE1d、7d、14d、21d时SFO部位HO-1基因和蛋白表达的动态变化,并分析了与症状之间的相关性。结果：对照组大鼠脑仅有少量HO-1基因和蛋白表达;实验组大鼠诱导EAE后,伴随着大鼠EAE症状及脑组织病理损伤的出现和进行性加重,其HO-1基因和蛋白表达量逐渐增高;在诱导后1d,SFO部位即出现HO-1mRNA和蛋白表达,而其他脑区变化不明显;7d时进一步增多;14d时,H01蛋白表达至高峰。HO-1阳性细胞主要位于脉络丛、穹隆下器、血管“套袖样”病灶的周围,与EAE病变部位一致,此时大鼠EAE病情最重、体重减轻最显著、脑组织病理改变最明显;21d时脑组织HO-1基因和蛋白表达量逐渐下降,大鼠EAE症状也逐渐恢复。应用HO-1特异性抑制剂Snpp9后,大鼠EAE症状和脑组织病变明显减轻,说明脑组织HO-1的动态变化与EAE症状及脑组织损伤密切相关。结论：在EAE发病早期,SFO即已经感受到外界的氧化应激变化,SFO可能是外周免疫信息物质向中枢神经系统传递的重要而早期的通道之一;其次,应用HO-1抑制剂可能成为防治该病的有效方法之一。     

脑组织核因子-κB激活对实验性变态反应性脑脊髓炎的作用

为探讨脑组织核因子－κB（NF－κB）对实验性变态反应性脑脊髓炎（EAE）的作用,分别用凝胶电泳迁移分析和NF－κB p65免疫组化方法测定了CFA－GPSCH诱导大鼠EAE1、7、14和21d时脑组织NF－κB活性和蛋白表达的动态变化,并观察了这些变化与EAE症状之间的关系。结果表明;对照组大鼠脑组织仅有少量NF－κB蛋白表达,其活性也很低;诱导EAE后,伴随着大鼠EAE症状及脑组织病理损伤的出现和进行性加重,其NF－κB活性和蛋白表达量逐渐增高;在免疫后14d达到高峰,NF－κB阳性细胞主要位于脉络丛、穹隆下器、血管“套袖样”病灶的周围,与EAE病变部位一致,此时大鼠EAE发病率最高、病情最重、体重减轻最显著、脑组织病理改变也最明显;21h脑组织NF－κB活性和蛋白表达量逐渐下降,大鼠EAE症状也逐渐恢复。应用NF－κB特异性抑制剂PDTC以抑制脑内组织NF－κB活性和蛋白表达量逐渐下降,大鼠EAE症状也逐渐恢复。应用NF－κB特异性抑制剂PDTC以抑制脑内NF－κB蛋白表达后,大鼠EAE症状和脑组织损伤明显减轻,说明脑组织NF－κB的动态变化与EAE症状及脑组织损伤程度密切相关。结论：脑组织NF－κB的激活对EAE的发病起着关键的作用,应用NF－κB抑制剂可能是防治该病的有效方法之一。     

CT感染大鼠子宫内膜植入窗期基质金属蛋白酶的表达

目的 通过大鼠生殖道沙眼衣原体（chlamydia trachomatis,CT）感染模型研究CT初次感染后妊娠大鼠在子宫内膜植入窗期基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制物-1（TIMP-1）的表达对胚胎着床的影响。方法选择成年雌性SD大鼠30只,随机均分为对照组和感染组,感染组通过阴道接种CTD型株,而后在妊娠大鼠的植入窗期（即妊娠第5、6、7天）分别处死大鼠,采用链菌生物素蛋白一过氧化酶连接法和免疫组化测定两组子宫内膜种植窗期MMP-9及TIMP-1。采用HPIAS-1000高清晰度彩色病理图文小报告管理系统测定感染组和对照组中MMP-9＼T1MP-1在单位面积中各项免疫组织化学反应阳性颗粒的平均光密度值,并通过SPSS软件对数据差异显著性进行分析。结果在两组大鼠的子宫内膜中MMP-9均呈阳性表达。腺上皮细胞、腔上皮细胞和基质细胞胞浆内均可见棕黄色颗粒沉着,上皮细胞染色强度大于基质;感染组的表达较对照组弱,差异有显著性（P〈0．05）。TIMP-1表达特点和MMP-9相似,感染组的表达也较对照组弱,差异有显著性（P〈0．05）。结论生殖道感染CT后,妊娠大鼠子宫内膜植入窗期MMP-9及TIMP-1的低表达,可能是影响胚胎着床、导致不孕的重要因素。     

实验性变态反应性脑脊髓炎大鼠发病中脑组织血红素氧合酶-1基因和蛋白表达的动态变化

为探讨脑组织血红素氧合酶-1(hemeoxygenase-1,HO-1)对实验性变态反应性脑脊髓炎(experimental allergic encephalomyelitis,EAE)的作用,分别应用逆转录-聚合酶链反应(RT-PCR)和免疫组化技术测定了豚鼠脊髓生理盐水匀浆 完全福氏佐剂诱导EAE大鼠1、7、14、21d时,脑组织HO-1基因和蛋白表达的动态变化,并观察与症状之间的关系。结果显示：对照组大鼠脑组织仅有少量HO-1基因和蛋白表达：诱导EAE后,伴随着大鼠EAE症状及脑组织病理损伤的出现和进行性加重,脑组织HO-1基因和蛋白表达量逐渐增高。在豚鼠脊髓生理盐水匀浆 完全福氏佐剂诱导7d后,HO-1 mRNA上升至高峰。14d时,HO-1蛋白表达至高峰,HO-1阳性细胞主要位于脉络丛、穹隆下器、血管“套袖样”病灶的周围,与EAE病变部位一致。此时大鼠的病情最重、体重减轻最显著、脑组织病理改变最明显。21d时脑组织HO-1基因和蛋白表达量逐渐下降,大鼠EAE症状也逐渐减轻。应用HO-1特异性抑制剂锡原卟啉-9以抑制脑内HO-1蛋白表达后,大鼠EAE症状和脑组织损伤明显减轻,说明脑组织HO-1的动态变化与EAE症状及脑组织损伤密切相关。提示脑组织HO-1基因和蛋白过表达对EAE发病起着重要的作用,应用HO-1抑制剂可能是防治该病的有效方法之一。     

Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.     

Lipocalin-2 Protein Deficiency Ameliorates Experimental Autoimmune Encephalomyelitis: THE PATHOGENIC ROLE OF LIPOCALIN-2 IN THE CENTRAL NERVOUS SYSTEM AND PERIPHERAL LYMPHOID TISSUES*

Lipocalin-2 (LCN2) plays an important role in cellular processes as diverse as cell growth, migration/invasion, differentiation, and death/survival. Furthermore, recent studies indicate that LCN2 expression and secretion by glial cells are induced by inflammatory stimuli in the central nervous system. The present study was undertaken to examine the regulation of LCN2 expression in experimental autoimmune encephalomyelitis (EAE) and to determine the role of LCN2 in the disease process. LCN2 expression was found to be strongly increased in spinal cord and secondary lymphoid tissues after EAE induction. In spinal cords astrocytes and microglia were the major cell types expressing LCN2 and its receptor 24p3R, respectively, whereas in spleens, LCN2 and 24p3R were highly expressed in neutrophils and dendritic cells, respectively. Furthermore, disease severity, inflammatory infiltration, demyelination, glial activation, the expression of inflammatory mediators, and the proliferation of MOG-specific T cells were significantly attenuated in Lcn2-deficient mice as compared with wild-type animals. Myelin oligodendrocyte glycoprotein-specific T cells in culture exhibited an increased expression of Il17a, Ifng, Rorc, and Tbet after treatment with recombinant LCN2 protein. Moreover, LCN2-treated glial cells expressed higher levels of proinflammatory cytokines, chemokines, and MMP-9. Adoptive transfer and recombinant LCN2 protein injection experiments suggested that LCN2 expression in spinal cord and peripheral immune organs contributes to EAE development. Taken together, these results imply LCN2 is a critical mediator of autoimmune inflammation and disease development in EAE and suggest that LCN2 be regarded a potential therapeutic target in multiple sclerosis.     

Identification and quantitation of T lymphocyte subsets found in the spinal cord of the Lewis rat during acute experimental allergic encephalomyelitis

Monoclonal antibodies specific for different rat T cell subsets and Ia-positive cells were used in a quantitative morphologic study of the cellular infiltrates in the spinal cords of Lewis rats during acute, actively induced experimental allergic encephalomyelitis (EAE). The predominant cell types in the inflammatory spinal cord lesions are W3/25-positive and Ia-positive cells. The relative percentages represented by each cell type remain quite constant regardless of the degree of clinical illness exhibited by the rat. These data demonstrate a quantitative profile of the infiltrating cells in acute, active EAE, and suggest that the principal inflammatory cells in these lesions are T helper cells and Ia-bearing cells (macrophages, B cells, or activated T helper cells).     

Elevated ATG5 expression in autoimmune demyelination and multiple sclerosis

Multiple sclerosis (MS) is an inflammatory central nervous system (CNS) disorder characterized by T cell mediated demyelination. In MS, prolonged T cell survival and increased T cell proliferation have been linked to disease relapse and progression. Recently, the autophagy related gene 5 (Atg5) has been shown to modulate T cell survival. In this study, we examined the expression of Atg5 using both a mouse model of autoimmune demyelination as well as blood and brain tissues from MS cases. Quantitative real-time PCR analysis of RNA isolated from blood samples of experimental autoimmune encephalomyelitis (EAE) mice revealed a strong correlation between Atg5 expression and clinical disability. Analysis of protein extracted from these cells confirmed both upregulation and post-translational modification of Atg5 the latter of which was positively correlated with EAE severity. Analysis of RNA extracted from T cells isolated by negative selection, indicated that Atg5 expression was significantly elevated in individuals with active relapsing-remitting MS compared to non-diseased controls. Brain tissue sections from relapsing-remitting MS cases examined by immunofluorescent histochemistry suggested that encephalitogenic T cells are a source of Atg5 expression in MS brain samples. Together these data suggest that increased T cell expression of Atg5 may contribute to inflammatory demyelination in MS.     

The gelatinases,MMP-2 and MMP-9, as fine tuners of neuroinflammatory processes

This review focuses on the complementary roles of MMP-2 and MMP-9 in leukocyte migration into the brain in neuroinflammation, studied mainly in a murine model of experimental autoimmune encephalomyelitis (EAE) that has similarity to the human disease multiple sclerosis. We discuss the cellular sources of MMP-2/MMP-9 in EAE, their sites of activity, and how cleavage of the to-date identified MMP-2/MMP-9 substrates at the blood-brain barrier facilitate leukocyte filtration of the central nervous system (CNS). Where necessary, comparisons are made to inflammatory processes in the periphery and to other MMPs relevant to neuroinflammation. While the general principles concerning MMP-2 and MMP-9 function discussed here are relevant to all inflammatory situations, the details regarding substrates and molecular mechanisms of action are likely to be specific for neuroinflammation.     

Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade

In joint diseases of both the inflammatory (rheumatoid arthritis, or RA) or the degenerative variety (osteoarthritis, or OA), matrix metalloproteinases (MMPs) are essential mediators of irreversible tissue destruction. MMP-9 is secreted as a stable, inactive zymogen and is proteolytically converted to the active enzyme. To understand the activation mechanism of MMP-9 in joint diseases, the process was investigated in serum-free cocultures of human articular chondrocytes and macrophages. Macrophages extensively expressed and secreted pro-MMP-9 whereas chondrocytes failed to produce the enzyme. However, efficient activation of pro-MMP-9 required soluble and membrane-associated chondrocyte proteinases. Two alternative activation pathways mainly involved MMPs and, marginally, serine or cysteine proteinases. MT1-MMP (MMP-14), the only MT-MMP expressed in chondrocytes, converted pro-MMP-13 which, in turn, cleaved pro-MMP-9. Alternatively, pro-MMP-9 was activated less efficiently by MMP-3, which was converted by autocatalysis or by serine or cysteine proteinases. Both pathways were triggered by chondrocytes from OA, but not normal joints. Therefore, articular chondrocytes are not innocent bystanders in joint diseases. They not only produce destructive enzymes guided by environmental cues but also they can instruct inflammatory cells or cells from surrounding tissues to do so by converting in several ways zymogens produced but not activated by these cells themselves.     

The immunopathology of experimental allergic encephalomyelitis (EAE). III. Differential in situ expression of strain 13 Ia on endothelial and inflammatory cells of (strain 2 x strain 13)F1 guinea pigs with EAE

This study was undertaken to determine if there is differential expression of the relevant strain-specific Ia antigen on endothelial and parenchymal inflammatory cells in the central nervous systems (CNS) of guinea pigs (GP) with acute experimental allergic encephalomyelitis (EAE). Adult inbred GP were sensitized with GP spinal cord homogenate and complete Freund's adjuvant. Strain 13 GP and (2 x 13)F1 hybrids developed clinical disease within 2 to 3 wk after sensitization, whereas strain 2 GP did not, although all sensitized GP had CNS mononuclear inflammatory infiltrates. By using monoclonal antibodies to strain-specific and framework Ia epitopes with an immunoperoxidase technique, the distribution and amount of the strain 2 and strain 13 Ia were analyzed. Equivalent strain 2 and strain 13 Ia expression was found in normal tissues from F1 animals. Strain 2 and strain 13 GP sensitized for EAE had increased strain-specific Ia staining of CNS vessels and inflammatory cells over controls. However, F1 GP with EAE had markedly increased strain 13, but not strain 2, Ia on CNS parenchymal vessels and mononuclear inflammatory cells (p less than 0.001, for both). These results suggest for the first time that specific major histocompatibility complex gene products are selectively expressed on endothelial and inflammatory cells in situ in immune reactions in the target organ of individuals of heterogeneous immunogenetic composition.