实验性自身免疫性脑脊髓炎大鼠血-脑脊液屏障功能及动态变化

目的　探讨实验性自身免疫性脑脊髓炎 ( EAE)大鼠血 -脑脊液屏障功能的动态变化及其作用 ,以求进一步揭示 EAE发病机制。方法　检测 EAE大鼠免疫后第 4、6、8、1 0、1 2、1 4、1 6、1 8、2 0天血清和脑脊液 ( CSF)中清蛋白 ( ALB)含量 ,其 CSF与血清 ALB比值 ( QA)作为评价血 -脑脊液屏障损害的指标。结果　 QA值免疫后第 8天即出现显著性升高 ,早于临床症状出现 ;随免疫时间的延长 QA值呈逐渐增高后缓慢下降趋势 ,并且 QA值与EAE大鼠病情评分呈显著正相关 ( r =0 .81 ,P =0 .0 0 1 )。结论　血 -脑脊液屏障的早期损害在 EAE发病过程中具关键作用。     

雷公藤内酯醇对实验性自身免疫性脑脊髓炎大鼠血-脑脊液屏障的影响

目的 探讨雷公藤内酯醇(Tri)对实验性自身免疫性脑脊髓炎(EAE)大鼠血-脑脊液屏障(BCB)的免疫调节作用.方法 制备Wistar大鼠EAE模型,应用Tri治疗EAE,评估EAE组、Tri治疗组的临床症状,在不同时间点测定脑脊液(CSF)和外周血白蛋白(Alb)水平并计算二者比值(QA),通过QA值观察BCB变化.于免疫后(p.I.)6、8、10、12、14、16 d处死动物,用免疫组化技术检测脑和脊髓细胞间黏附分子-1(ICAM-1)和干扰素-γ(IFN-γ)表达.结果 免疫后14 d为EAE组发病高峰期,Tri治疗组临床症状评分(0.14±0.08)较EAE组(2.19±1.68)低(P<0.01).免疫后 8 d EAE组QA达最大值(0.30±0.05),较佐剂组(0.13±0.05)明显增高(P<0.01).免疫后6-14 d,EAE组与佐剂组、Tri治疗组比较QA值差异均有统计学意义(F=28.34,P<0.01).免疫后14 d Tri治疗组 QA达最大值(0.19±0.02),QA波峰时间较EAE组延迟且峰值明显较低.免疫后12-16 d,EAE组与佐剂组、Tri治疗组比较ICAM-1阳性表达高(F=17.11,F=29.87,均P<0.01);免疫后10-16 d,Tri治疗组较EAE组IFN-γ阳性表达低(F=447.92,P<0.01).结论 EAE中BCB损伤早于临床症状出现.Tri能有效抑制EAE进展,可能与其下调ICAM-1、IFN-γ表达,恢复BCB正常功能有关.     

阿托伐他汀对EAE大鼠脊髓ICAM-1和TGF-β1表达的影响

目的 探讨阿托伐他汀对实验性自身免疫性脑脊髓炎(EAE)大鼠脊髓细胞间黏附分子-1(ICAM-1)和转化生长因子-β1(TGF-β1)表达的影响. 方法 80只清洁级健康雌性Wistar大鼠按照随机数字表法分为4组:正常对照组、EAE模型组、低剂量治疗组、高剂量治疗组,每组20只.各组再分为第14天、21天两个亚组,每个亚组10只.利用新鲜豚鼠全脊髓匀浆及完全福氏佐剂免疫Wistar大鼠建立EAE模型.低、高剂量治疗组大鼠分别给予2 mg/(kg·d)、10 mg/(kg·d)阿托伐他汀灌胃,正常对照组及EAE模型组应用生理盐水灌胃.观察每组大鼠发病情况并进行神经功能障碍评分,采用HE染色方法观察病理改变,免疫组化方法检测ICAM-1及TGF-β1的表达. 结果 与EAE模型组及低剂量治疗组比较,阿托伐他汀高剂量治疗组发病率明显减轻,炎性病灶明显减少,组织中ICAM-1表达明显减少,TGF-β1表达明显增多,差异均有统计学意义(P＜0.05).结论 阿托伐他汀可改善EAE大鼠的临床症状及病理改变,并且与剂量有关,其作用推测与减少ICAM-1表达、提高TGF-β1表达有关.     

α-硫辛酸对实验性自身免疫性脑脊髓炎大鼠脑和脊髓内MMP-9表达的影响

目的通过α—硫辛酸(α—LA)对实验性自身免疫性脑脊髓炎(EAE)大鼠脑和脊髓内MMP—9表达水平的影响,探讨α—LA是否具有血—脑脊液屏障(BBB)的保护作用。方法选取雌性Wistar大鼠45只,随机分为3个组,对照组、EAE组、α—LA组(免疫后第6天开始给予腹腔注射30mg·kg-1),分别于免疫后8d、12d、20d断头取脑与脊髓,取大鼠脑和脊髓腰膨大处组织进行免疫组织化学染色检测脑组织和脊髓中MMP—9的表达。结果 EAE组12d组与对照组12d组比较,大鼠脑组织视交叉和脊髓腰膨大处MMP—9阳性细胞数明显增多;α—LA 12d组与EAE组12d组相比较,大鼠脑组织视交叉处和脊髓腰膨大处MMP—9阳性细胞表达数明显减少(P0.05)。结论α—LA可以降低EAE大鼠的发病率,能够降低BBB通透性的标志物MMP—9的水平,具有稳定BBB的作用,提示其在防治多发性硬化(MS)方面,具有很好的应用前景。     

苦参素对实验性自身免疫性脑脊髓炎大鼠脑内神经干细胞增值的影响

目的观察苦参素对实验性自身免疫性脑脊髓炎(EAE)大鼠的治疗作用及对脑内神经干细胞标志物-巢蛋白Nestin、转录因子Sox-2表达的影响,探讨苦参素对EAE大鼠的治疗机制。方法将60只雌性Wistar大鼠随机分为正常组、模型组和治疗组,每组20只。用新鲜豚鼠全脊髓匀浆免疫诱导建立EAE模型,自免疫后第11天(发病初期)起,治疗组大鼠每日腹腔注射苦参素6.70 ml/kg(250 mg/kg),同时正常组与模型组注射等量生理盐水。每天观察并记录大鼠的体重变化及神经功能学评分。给药1 w后将大鼠处死,取脊髓和大脑,免疫组织化学法检测脑内Nestin的表达,RT-PCR法测定脑内SOX-2含量的变化。结果与模型组相比,治疗组大鼠神经功能评分明显降低,Nestin阳性细胞数和SOX-2的表达均显著增加(P0.05)。结论苦参素对EAE大鼠有治疗作用,其机制可能与增加中枢神经系统中神经干细胞的数量有关。     

AMD3100、CXCR7抗体对自身免疫性脑脊髓炎大鼠脊髓和脾细胞SDF1α、CXCR4、CXCR7 mRNA表达的影响

目的研究AMD3100、CXCR7抗体对实验性自身免疫性脑脊髓炎(experimental autoimmune en-cephalomyelitis,EAE)大鼠脊髓和脾脏中趋化因子SDF1α及其受体CXCR4、CXCR7表达的影响。方法采用MBP68-86和完全福氏佐剂配置的完全抗原免疫Lewis大鼠制备EAE模型。将大鼠随机分为对照组、AMD3100组、CXCR7抗体组和EAE组,于免疫后第0、2、4、6天给予相应药物干预:AMD3100组按体质量2.5mg/kg腹腔注射AMD3100,CXCR7抗体组腹腔注射CXCR7抗体20μg/只,EAE组腹腔注射等量生理盐水。每天对大鼠进行神经功能评分,免疫后第15天行RT-PCR检测各组脊髓及脾脏中SDF1α、CXCR4、CXCR7mRNA表达,行HE染色观察大鼠脊髓组织病理变化。结果 (1)与对照组相比,EAE组大鼠脊髓血管周围有大量性炎细胞浸润,神经功能评分明显增加,AMD3100组、CXCR7抗体组和EAE组脊髓SDF1α、CXCR4、CXCR7 mRNA表达增加(P<0.01),脾细胞SDF1α、CXCR7 mRNA表达减少(P<0.01);(2)与EAE组相比,AMD3100组大鼠神经功能评分增加,脾细胞CXCR4、CXCR7 mRNA表达增加(P<0.01),脊髓SDF1α、CXCR4、CXCR7表达无统计学差异;(3)与EAE组相比,CXCR7抗体组大鼠神经功能评分增加,脾细胞SDF1α、CXCR4、CXCR7 mRNA表达增加(P<0.01),脊髓SDF1α、CXCR4、CXCR7 mRNA表达无统计学差异。结论 AMD3100和CXCR7抗体均能加重EAE病情,上调EAE脾细胞CXCR4和CXCR7 mRNA的表达。     

实验性自身免疫性脑脊髓炎大鼠脑组织Nogo-A和NgR的变化

背景：Nogo-A蛋白是一种中枢神经系统髓鞘相关轴突再生抑制因子,与其受体作用可抑制轴突再生。 目的：观察实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis,EAE）大鼠侧脑室周围Nogo-A和NgR蛋白的动态变化,探讨Nogo-A和NgR蛋白在EAE发生发展中的可能作用机制。 设计、单位、时间：随机对照动物研究。试验于2008年9月至11月在湖南省人民医院临床研究所进行。 材料：6~8周龄清洁级雌性Wistar大鼠60只,体重（180±10）g,由湖南农业大学动物科技学院实验动物养殖场提供。 方法：60只大鼠随机分为EAE组和对照组各30只,各组再分为6个亚组,每组5只大鼠,分别于免疫后11天、13天、15天、18天、24天、30天处死各亚组大鼠取脑。采用免疫组化方法测定2组大鼠侧脑室周围白质Nogo-A和NgR蛋白在不同时间点表达情况。 主要观察指标：通过免疫组化后阳性细胞数计数观察侧脑室周围Nogo-A和NgR表达水平。 结果：EAE组在免疫第11天Nogo-A蛋白表达升高,第13天下降,后又升高,第30天仍高于对照组;EAE组NgR蛋白在第13、15、18表达升高。 结论：Nogo-A和NgR蛋白可能在EAE的发生发展中起重要作用     

基质金属蛋白酶9在实验性自身免疫性脑脊髓炎大鼠发病过程中的动态表达

目的通过检测基质金属蛋白酶-9(MMP-9)在EAE大鼠发病过程中不同阶段外周血和中枢神经系统中的表达水平及动态变化,以探讨其在多发性硬化发病过程中的作用及机制。方法 Wistar雌性大鼠80只,分为模型组(EAE组)、完全福氏佐剂组(CFA组),分别于免疫后6天、8天、10天、12天、14天、16天、18天及20天取视交叉处脑组织和脊髓腰膨大段行HE染色观察炎性细胞的浸润状况;免疫组化法检测脑和脊髓组织中MMP-9的表达,酶联免疫吸附实验测定血清中MMP-9的含量。结果 EAE组大鼠脊髓和脑组织中MMP-9阳性细胞数及血清中MMP-9水平均显著高于同期CFA组(P<0.05);EAE 10天组与EAE 6天、14天、18天及20天组比较,大鼠脊髓腰膨大处MMP-9阳性细胞数较多(P<0.05);EAE 12天组与EAE 6天组、8天组、14天组、16天组、18天组及20天组比较,大鼠脑组织视交叉处MMP-9阳性细胞数较多(P<0.05);EAE 12天组与EAE 6天组、8天组、10天组、14天组、16天组、18天组及20天组相比较,大鼠血清中MMP-9含量较高(P<0.05)。结论在EAE大鼠发病前期即有中枢组织内MMP-9的高表达,且EAE大鼠脊髓中MMP-9的高表达要早于脑组织和外周血,但MMP-9表达的高峰在第12天与病理变化和疾病进展是同步的。     

雷公藤内酯醇对实验性自身免疫性脑脊髓炎大鼠脑和脊髓ICAM-1表达的影响

目的探讨雷公藤内酯醇(Triptolide,Tri)对实验性自身免疫性脑脊髓炎(EAE)大鼠脑和脊髓细胞间黏附分子-1(ICAM-1)表达的影响。方法模型组采用豚鼠髓鞘蛋白匀浆和福(氏)完全佐剂诱发大鼠急性EAE,治疗组在此基础上给予不同剂量Tri[0.2 mg/(kg.d),0.4 mg/(kg.d)],观察其临床表现并进行评分。采用Loyez(氏)髓鞘染色法观察髓鞘病理改变,免疫组化法检测脑和脊髓ICAM-1表达。结果高剂量Tri治疗组大鼠未出现临床症状,其髓鞘结构完整;低剂量Tri治疗组临床症状较模型组轻且发病时间延迟,其髓鞘部分脱失。高剂量Tri治疗组ICAM-1阳性血管数为2.31±1.23,与模型组和低剂量Tri治疗组比较差异有统计学意义(P<0.01)。结论Tri对EAE的治疗作用与其抑制ICAM-1表达有关。     

左旋咪唑通过促进抗原呈递细胞MHC-Ⅱ表达而加重实验性自身免疫性脑脊髓炎

目的：探讨左旋咪唑加重大鼠实验性自身免疫性脑脊髓炎（EAE）是否通过上调抗原呈递细胞（APC）主要组织相容性复合体Ⅱ类抗原（MHC-Ⅱ）表达而实现的。方法：SD大鼠28只随机分为完全弗氏佐剂（CFA）组（n=8）,EAE组（n=10）,左旋咪唑组（n=10）。用豚鼠脊髓匀浆和CFA的混匀乳剂免疫SD大鼠制作EAE模型。在造模前3d至造模后7d腹腔注射左旋咪唑10mg·kg-1（左旋咪唑组）。分析各组行为学变化及神经组织病理学改变,用流式细胞术检测脾脏APC表面分子MHC-Ⅱ的表达。结果：左旋咪唑加速了大鼠EAE的病程,加重了神经缺损症状与中枢神经病理学损害;左旋咪唑上调了脾脏APC表面分子MHC-Ⅱ的表达,且与EAE神经缺损症状程度呈正相关。结论：左旋咪唑上调了APC表面分子MHC-Ⅱ的表达可能为左旋咪唑促发EAE的作用机制。     

Mast cell activity in experimental allergic encephalomyelitis

The number and functional reactivity of peritoneal mast cells (MCs) were evaluated in rats with experimental allergic encephalomyelitis (EAE). Cells were counted following staining with toluidine blue and activation was measured by B-hexosaminidase (B-hex) release. The number of detectable MCs and their capacity to release B-hex decreased significantly by 40 and 65%, respectively, as compared with normal controls just prior to the onset of clinical signs. These values returned to normal on clinical recovery. Preliminary data on MC counts performed on histological sections of rat brains with EAE suggested a similar pattern of response, i.e., an early decrease prior to disease onset with subsequent normalization on recovery. In an attempt to modify the course of EAE, rats were treated with the MC stabilizing agent nedocromil or with the MC activating agent, compound 48/80. Nedocromil induced a slight delay in the onset of EAE, but only when administered at the time of EAE induction. Compound 48/80 did not seem to affect the clinical course of the disease. Our results suggest that MCs are involved in the pathogenesis of EAE and may contribute to the induction of the disease rather than to the effector phase and its clinical expression.     

Metallothionein I+II expression and their role in experimental autoimmune encephalomyelitis

We examined the expression and roles of neuroprotective metallothionein-I+II (MT-I+II) in the rat CNS in experimental autoimmune encephalomyelitis (EAE), an animal model for the human autoimmune disease, multiple sclerosis (MS). EAE caused significant macrophage activation, T-lymphocyte infiltration, and astrogliosis in spinal cord, brain stem, and cerebellum, which peaked 14-18 days after immunization. The remission of symptoms and histopathological changes began at days 19-21 and were completed by days 30-40. MT-I+II expression was increased significantly in EAE infiltrates. In order to study the effects of increased MT levels, we administered Zn-MT-II intraperitoneally (i.p.) to rats during EAE. Clinically, Zn-MT-II treatment reduced the severity of EAE symptoms and mortality in a time- and dose-dependent manner. Histopathologically, Zn-MT-II increased reactive astrogliosis and decreased macrophages and T lymphocytes significantly in the CNS. In spleen sections, the number of macrophages both in control and EAE-sensitized rats was reduced by Zn-MT-II, while the number of lymphocytes remained unaltered by Zn-MT-II. Therefore, we suggest that MT-II has peripheral mechanisms of action on macrophages, while T lymphocytes are affected locally in the CNS. During EAE, oxidative stress was decreased by Zn-MT-II, which could contribute to the diminished clinical scores observed. None of the effects caused by Zn-MT-II could be attributable to the zinc content. These results suggest MT-I+II as potentially useful factors for the treatment of EAE/MS.     

Induction of experimental allergic encephalomyelitis in a low-susceptible Albino Oxford rat strain by somatostatin analogue SMS 201-995

OBJECTIVE: The effect of the somatostatin analogue SMS 201-995 (octreotide; OCT) on the course of experimental allergic encephalomyelitis (EAE) in the relatively resistant Albino Oxford (AO) strain of rats was studied. METHODS: Animals were actively immunized with bovine brain homogenate in complete Freund's adjuvant. OCT was given subcutaneously in the hind legs on days 7, 8 and 9 after immunization, at a dose of 3 x 5 microg/kg/day. Rats in control groups were treated with saline or were left untreated. EAE was scored clinically and immunophenotypically, estimating by flow cytometry the changes in the popliteal lymph nodes (PLN) and spleen and monitoring immunohistologically the brain sections of rats recovered from disease. RESULTS: In control AO rats, EAE was induced in only 2 of 22 rats (9%). In OCT-treated rats, however, EAE developed in 11 of 20 rats (55%), in comparison with 3 of 17 saline-treated animals (17%) (p <0.05). In PLN of OCT-treated rats during the clinical course of EAE, a decreased proportion of OX8+ cells was seen, followed by increases in OX39+ and W3/25+ cells on days 17 and 26. In spleen, OCT decreased the proportion of OX1+, OX39+ and OX8+ cells (on days 12 and/or 17), and increased the proportion of OX39+ cells on days 26 and 31. In the brain sections of saline-treated rats recovered from EAE, numerous Mac-1+, Mac-3+ and OX8+ cells were found. These cells were, however, absent in OCT-treated rats; instead, several W3/25+ cells were noticed. CONCLUSIONS: These data imply that OCT increases the susceptibility of AO rats to EAE, interfering with specific and/or nonspecific defense mechanisms operating in both the initial and recovery phase of EAE.     

Type IV phosphodiesterase inhibition in experimental allergic encephalomyelitis of Lewis rats: sequential gene expression analysis of cytokines, adhesion molecules and the inducible nitric oxide synthase.

Type IV phosphodiesterase inhibitors are able to suppress EAE. To investigate the effects of this therapy in the central nervous system, we serially analyzed from days 7 to 17 postinoculation the gene expression pattern of tumor necrosis factor (TNF), lymphotoxin, interferon-gamma, interleukin-1beta, the inducible nitric oxide synthase (iNOs), interleukin-10, the vascular cell adhesion molecule-1 (VCAM-1) and the intercellular adhesion molecule-1 (ICAM-1) in the spinal cord of Lewis rats with actively induced EAE, treated with Rolipram. Treated rats had a delayed and milder disease, and reduced numbers of infiltrates in the nervous tissue. The gene expression profile was similar to that of untreated rats, although delayed, with no evidence of IL-10 upregulation during the observation period. The delayed inflammation was not associated with changes in the expression of VCAM-1 and ICAM-1. In peripheral blood mononuclear cells, TNF mRNA levels were decreased and interleukin-10 was unchanged. This therapy did not alter the proliferative ability of T lymphocytes against myelin basic protein. The encephalitogenic potential of splenocytes from treated animals was also unaffected. The high levels of both iNOs mRNA and nitric oxide (NO) found before the appearance of clinical signs, suggests that NO generation might be a contributing factor to the therapeutic benefit achieved by Rolipram in the rat.     

Recovery phase of acute experimental autoimmune encephalomyelitis in rats corresponds to development of endothelial cell unresponsiveness to interferon gamma activation

Activation of the vascular endothelium is important in the development of inflammation. Activated endothelial cells (EC) express surface markers not expressed by quiescent EC. These surface markers augment adhesion reactions and leukocyte migration. We examined microvessel EC activation longitudinally in experimental autoimmune encephalomyelitis (EAE) in Lewis rats. CNS microvessels were isolated at 0, 3, 7, 12, 20, and 30 days post-inoculation (PI). Normal and CFA-injected rat microvessels do not express activation antigens (Ag). Increased expression of major histocompatibility complex (MHC) class II molecule and intercellular adhesion molecule-1 (ICAM-1) were detected on CNS microvessels from immunized rats at 7 days PI, prior to development of clinical signs, and at 12 days PI. Enhanced MHC class I molecule was seen only at 12 days. MHC class II molecule expression was focally expressed along microvessel fragments. By 20 days PI, EC did not exhibit increased levels of any of the markers tested. Perivascular cells (possibly pericytes), however, were found to express MHC class II molecule and ICAM-1 up to 30 days PI. During the recovery phase isolated CNS microvessels from MBP-immunized rats were unresponsive to IFNγ-mediated endothelial activation. Unresponsiveness was independent of IFNγ concentration. These results suggest that the endothelium is restored to functional quiescence during the recovery phase of acute EAE. © 1996 Wiley-Liss, Inc.     

小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的建立

目的探索以小剂量豚鼠脊髓匀浆为抗原,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。方法按脊髓湿重与冰盐水体积为1:10的比例制备豚鼠脊髓匀浆抗原,免疫Wistar大鼠,建立EAE模型;留取脑和脊髓进行HE染色及三色染色,光镜下观察病理改变。结果Wistar大鼠免疫后第17.20±6.48天第一次发病,发病形式多样,除表现EAE常见症状外,尚出现痉挛状态、斜颈等特殊症状。HE染色发现神经组织内炎细胞浸润,血管"袖套"样病灶形成;三色染色可见轴突肿胀,呈串珠状,且不连续,着色不均匀;髓鞘结构层次紊乱,疏松,崩解。结论小剂量脊髓匀浆诱导的EAE模型,病程及发病表现更接近于人类多发性硬化的表现,且经济实用,是研究多发性硬化的理想动物模型。     

特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值

目的特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值,并观察其病理的改变。方法按脊髓重量与冰盐水体积之比为1:5的比例制备豚鼠脊髓匀浆抗原,免疫Wistar大鼠,建立EAE模型;组织切片进行HE染色,三色染色,髓鞘碱性蛋白(MBP)及神经微丝(NF)免疫组化,光镜下观察病理改变。结果Wistar大鼠免疫后第16.07±4.25天发病,发病形式多样,除表现EAE经典症状外,尚出现痉挛状态、斜颈等特殊症状。HE染色发现神经组织内炎细胞浸润,血管"袖套"样病灶形成;三色染色可见轴突肿胀,呈串珠状,且不连续,着色不均匀;髓鞘结构层次紊乱,疏松,崩解;MBP及NF免疫组化研究发现病变组织内白质脱髓鞘及轴突损伤。结论三色染色结合MBP、NF免疫组化检测,较常规HE染色更能直接地、准确地显示EAE大鼠的病理变化,可推广应用。