Wistar大鼠实验性变态反应性脑脊髓炎病理探讨

目的研究缓解-复发型EAE大鼠的基本病理改变。方法对缓解-复发型EAE大鼠进行HE染色、Weil髓鞘染色和改良的Bielschowsky染色,以免疫组化方法标记GFAP和MMP-2、MMP-9。结果组织内可见多个血管周围淋巴细胞浸润呈袖套样分布,伴有大片状脱髓鞘,部分脱失的髓鞘内有炎细胞浸润,轴索呈空泡样缺失,GFAP染色证明在旧病灶的周围部分可见星形胶质细胞增生,MMP-2、MMP-9在血管内皮细胞、细胞外基质、炎细胞及脑(脊)膜内呈阳性表达。结论①缓解-复发型EAE大鼠的组织病理学改变与临床表现一致,有活动性和非活动性病灶并存的现象。②MMP-2、MMP-9在活动性病灶内表达,参与疾病的发生。③缓解-复发型EAE病理改变与MS相似,是理想的动物模型。     

Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ～70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.     

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。     

Neuroglial response after induction of experimental allergic encephalomyelitis in susceptible and resistant rat strains

Experimental allergic encephalomyelitis (EAE), the animal model for multiple sclerosis in humans, a T-cell mediated disease of the central nervous system is characterized by inflammatory infiltrates of myelin antigen(s)-specific T cells and consecutive demyelination. Spinal cord tissue emulsified in complete Freund's adjuvant clinical disease in the genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although similar inflammatory infiltrates in the CNS are observed in both strains 10-12 days after induction. We have shown that the resistance to clinical disease of AO rats is associated with rapid clearance of infiltrating mononuclear cells by a mechanism of apoptosis. Here, we demonstrate by immunohistochemical and FACS analyses of the expression of CD11b/c that microglial cells respond differently to disease induction in the two strains. Whereas microglial cells are activated throughout the period of day 10-28 days after EAE induction in AO rats they are only activated at the inception and resolution phases but not at the peak of clinical disease in DA rats when there is the highest level of CD4+ T cell infiltration. Our findings are compatible with the notion that microglia terminate effector T cells by apoptosis and that lack of this mechanism as evidenced by the lack of CD11b/c expression, support T cell survival and clinical expression of disease.     

Curcumin und Multiple Sklerose

Curcumin and Multiple Sclerosis In Multiple Sclerosis (MS), the inflammation caused by the patient's own immune system against the central nervous tissue leads to progressive loss of neurological functions. Reactive oxygenated species and nitric oxid, the latter produced by nitric oxid‐Synthases (NO) destroy axons and myelin sheets. In experimental autoimmune encephalomyelitis (EAE), an animal model for MS, arginase I expression is upregulated and an inhibition of arginase helps to delay the onset of EAE‐symptoms. Since arginases compete with NO for the substrate arginine, a complex network regulates NO activity. The prevalence of MS in societies, where curcumin is part of the daily diet, is lower than in the northern hemisphere. A specific inhibition of arginases by curcuma extract in vitro and evidence for a direct association of compounds therein with arginases suggests a perspective for the treatment of MS.     

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis

Current treatments and emerging oral therapies for multiple sclerosis (MS) are limited by effectiveness, cost, and/or toxicity. Genetic and environmental factors that alter the branching of Asn (N)-linked glycans result in T cell hyperactivity, promote spontaneous inflammatory demyelination and neurodegeneration in mice, and converge to regulate the risk of MS. The sugar N-acetylglucosamine (GlcNAc) enhances N-glycan branching and inhibits T cell activity and adoptive transfer experimental autoimmune encephalomyelitis (EAE). Here, we report that oral GlcNAc inhibits T-helper 1 (Th1) and T-helper 17 (Th17) responses and attenuates the clinical severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administered after disease onset. Oral GlcNAc increased expression of branched N-glycans in T cells in vivo as shown by high pH anion exchange chromatography, MALDI-TOF mass spectroscopy and FACS analysis with the plant lectin l-phytohemagglutinin. Initiating oral GlcNAc treatment on the second day of clinical disease inhibited MOG-induced EAE as well as secretion of interferon-γ, tumor necrosis factor-α, interleukin-17, and interleukin-22. In the more severe 2D2 T cell receptor transgenic EAE model, oral GlcNAc initiated after disease onset also inhibits clinical disease, except for those with rapid lethal progression. These data suggest that oral GlcNAc may provide an inexpensive and nontoxic oral therapeutic agent for MS that directly targets an underlying molecular mechanism causal of disease.     

Chaperone Activity of Small Heat Shock Proteins Underlies Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73–92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.     

Analysis of the Gadolinium retention in the Experimental Autoimmune Encephalomyelitis (EAE) murine model of Multiple Sclerosis

ObjectivesThe aim of this study is to quantitatively investigate, at the preclinical level, the extent of Gd retention in the CNS, and peripheral organs, of immune-mediated murine models (Experimental Autoimmune Encephalomyelitis –EAE) of Multiple Sclerosis, compared to control animals, upon the injection of gadodiamide. The influence of the Gadolinium Based Contrast Agent administration timing during the course of EAE development is also monitored.MethodsEAE mice were injected with three doses (1.2 mmol/kg each) of gadodiamide at three different time points during the EAE development and sacrificed after 21 or 39 days. Organs were collected and the amount of Gd was quantified through Inductively Coupled Plasma-Mass Spectrometry. Transmission electron microscopy (TEM) and MRI techniques were applied to add spatial and qualitative information to the obtained results.ResultsIn the spinal cord of EAE group, 21 days after gadodiamide administration, a significantly higher accumulation of Gd occurred. Conversely, in the encephalon, a lower amount of Gd retention was reached, even if differences emerged between EAE and controls mice. After 39 days, the amounts of retained Gd markedly decreased. TEM validated the presence of Gd in CNS. MRI of the encephalon at 7.1T did not highlight any hyper intense region.ConclusionIn the spinal cord of EAE mice, which is the mostly damaged region in this specific animal model, a preferential but transient accumulation of Gd is observed. In the encephalon, the Gd retention could be mostly related to inflammation occurring upon immunization rather than to demyelination.