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。     

Wistar大鼠实验性变态反应性脑脊髓炎的模型建立

目的:建立Wistar大鼠多病程实验性变态反应性脑脊髓炎(EAE)的动物模型,并进行病理学研究,为多发性硬化(MS)的研究提供实验依据。方法:以豚鼠全脊髓匀浆(GPSCH)为抗原免疫Wistar大鼠建立EAE的动物模型,并在光镜下观察不同发病类型EAE的病理改变。结果:根据病理和临床表现可将Wistar大鼠EAE模型分为5种发病形式:急性型、缓解-复发型、持续进展型、良性型和隐匿型。光镜下可见不同发病时期的EAE的病理改变有所不同,但都以血管"袖套"状改变、脑室周围及白质脱髓鞘改变为主,伴有神经元肿胀变性。结论:首次建立了Wistar大鼠多病程EAE,是研究多发性硬化的理想动物模型。     

Wistar大鼠实验性自身免疫性脑脊髓炎模型的建立

目的:建立Wistar大鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalo-myelitis,EAE)模型,并检测可溶性血管细胞黏附分子-1(soluble vascular cell adhesion molecule-l,sVCAM-1)在EAE大鼠中的动态表达。方法:将豚鼠麻醉后,不用磷酸盐缓冲液(PBS)灌注,直接取新鲜豚鼠全脊髓制备匀浆(Guinea Pig Spinal Cord Homoge-nate,GPSCH)为抗原,免疫Wistar大鼠建立EAE的动物模型。观察其体重及神经功能评分的变化;取脑、脊髓组织石腊包埋,病理切片,光镜观察;采用双抗体夹心法酶联免疫吸附实验(ELISA)检测血清中sVCAM-1的表达。结果:Wist-ar大鼠在免疫后10天出现明显的神经系统体征,发病率达100%;脑脊髓组织可见血管周围炎细胞呈袖套样浸润;sV-CAM-1的表达与对照组比较有显著性升高(P<0.01)。结论:采用非灌注法制备的豚鼠全脊髓匀浆作为抗原,能够成功诱发Wistar大鼠的EAE模型,为研究多发性硬化(multiple sclerosis,MS)的发病机制及治疗奠定了一定的基础。     

实验性自身免疫性脑脊髓炎模型的建立和长期观察研究

目的 建立实验性自身免疫性脑脊髓炎小鼠模型(EAE)并长期观察研究.方法 C57BL/6小鼠30只,随机分为EAE模型组、PBS对照组和正常对照组.应用神经功能评分进行临床评估,通过HE和髓鞘染色观察组织病理变化.结果 小鼠在诱导后的12±3d急性起病,16±2d内达到高峰,严重度评分为3.2±0.6分.半年观察期内复发1次,复发率为25%.光镜下可见EAE组以脊髓组织病变为主,表现为大量炎性细胞浸润和白质脱髓鞘.结论 采用MOG_(35-55)诱导C57BL/6小鼠建立的模型既往被认为是一种慢性迁延EAE模型,本研究通过长期观察发现其存在缓解复发现象.

Abstract：

Objective To establish a mice model of experimental autoimmune encephalomyelitis (EAE) and perform a long term study. Methods C57BL/6 mice were immunized with 300μg MOG_(35-55) in complete Freund' s adjuvant (CFA) to establish EAE model in EAE group (n = 10). Mice in adjuvant group( n = 10)were treated with CFA without MOG_(35-55) and control group( n = 10)were treated with normal saline. The pathologic changes of the central nervous system were studied by HE staining and myelin staining. Results The clinic symptoms of EAE were present in the 12±3th day post-immunization,and went to the peek in the 16±2 th day post-immunization. The severity score was 3.2±0.6. One relapse was observed in the term of 6 months, and the rate was 25%. Light microscopy showed there were abundant inflammatory cells infiltrated especially in spinal cord tissues in EAE mice,with evident demyelination in white matter. Conclusion The relapse of this EAE model was observed in the study, though it was believed to be a chronic persistent model without relapse.     

实验性自身免疫性脑脊髓炎大鼠模型脑组织硫化氢的变化

目的 观察实验性自身免疫性脑脊髓炎(EAE)模型大鼠脑组织中硫化氢(H2S)动态变化,探讨H2S与多发性硬化(MS)的关系. 方法 按随机区组设计方法将SD大鼠分成对照组及模型组,每组78只.模型组制备EAE模型,对照组用生理盐水代替诱导乳剂,余措施相同.采用HE染色、尼氏染色观察2组大鼠脑组织病理变化;比较不同时间点(建模后5、10、15、20、25、30、35、40、45、50、55、60 d和65 d)2组大鼠平均临床评分;采用比色法检测各时间点H2S和丙二醛(MDA)水平. 结果 (1)HE和尼氏染色显示模型组中可见明显的炎性细胞浸润现象及血管袖套形成,对照组未发现明显改变.(2)模型组于建模后第20天及第50天出现2次发病高峰,临床评分分别为(4.0±0.55)分和(3.5±0.25)分;对照组的评分为0分.(3)模型组大鼠各时间点脑组织中H2S表达量随着发病呈逐渐下降趋势,从第10天开始低于对照组,差异有统计学意义(P＜0.05).相反,模型组大鼠各时间点脑组织中MDA呈逐渐增加趋势,从第10天开始高于对照组,差异有统计学意义(P＜0.05).对照组大鼠H2S和MDA水平无明显变化.(4)模型组大鼠脑组织中H2S水平的变化与临床评分、MDA水平呈负相关(r=0.960,P=0.000;r=-0.920,P=0.000);临床症状评分和MDA水平呈正相关(r=0.910,P=0.000). 结论 H2S在EAE模型中表达明显降低,在MS中的作用机制有待进一步研究.     

多病程Wistar大鼠实验性变态反应性脑脊髓炎的病理研究

目的:在建立wistar大鼠多病程实验性变态反应性脑脊髓炎(EAE)的动物模型的基础上,进行病理学研究,探讨不同发病类型EAE的基本病理改变如炎细胞浸润、脱髓鞘和轴索损伤等方面的差别,为多发性硬化(MS)的研究提供实验依据。方法:以豚鼠全脊髓匀浆(GPSCH)为抗原免疫Wistar大鼠建立EAE的动物模型,进行常规HE染色、Weil髓鞘染色和改良的Bielschowsky,并行GFAP免疫组化染色,观察不同发病类型EAE的病理政变。结果:根据病理和临床表现可将Wistar大鼠EAE模型分为5种发病形式:急性型、缓解-复发型、持续进展型、良性型和隐匿型。光镜下可见不同发病时期的EAE的病理改变有所不同,急性型EAE炎症浸润明显,尤脱髓鞘改变,缓解-复发型和持续进展型EAE髓鞘脱失和轴索损伤更明显,且有陈旧病灶周围的星型胶质细胞增生,而新发病灶无此表现,良性型EAE则改变接近正常。结论:首次建立了Wistar大鼠多病程EAE,且病理证实不同类型EAE的炎细胞分布、髓鞘脱失及轴索损伤等基本病理改变是不同的,它具有人类MS的许多发病特点,其中多病程的发病形式和主要病理特点与MS极其相似,是理想的MS动物模型。     

实验性自身免疫性脑脊髓炎大鼠中1,25二羟基维生素D3的免疫调节作用

目的探讨1,25二羟基维生素D3[1,25(OH)2D3]对实验性自身免疫性脑脊髓炎(EAE)的免疫调节作用。方法建立Lewis大鼠主动免疫EAE实验动物模型,分别于致敏当天(预防组)及EAE症状出现当天给药(治疗组),并设立相应对照组。动态观察各组大鼠的临床评分,于致敏第13天处死,测定其引流淋巴结中细胞总数,单个核细胞(MNC)中CD 4CD 25T细胞含量、CD 86干细胞含量及MNC培养上清中干扰素(IFN)γ和白细胞介素(IL)4含量。结果预防组1,25(OH)2D3使EAE发病高峰延迟,治疗组显示1,25(OH)2D3能减轻EAE的病情,使高峰期评分[(3.3±0.6)分]比对照组[(4.0±0.3)分]降低(P<0.05);1,25(OH)2D3干预后EAE大鼠高峰期发现淋巴结中CD 4CD 25T细胞、引流淋巴结的细胞总数与对照组差异无统计学意义;而预防组CD 4CD 25/CD 4T细胞比值(15.1±3.3)高于其对照组(12.3±2.6,P<0.05);预防组、治疗组CD 86细胞明显降低(P<0.05)。1,25(OH)2D3干预后预防组及治疗组中淋巴结MNC培养上清中IFNγ无明显改变,而IL4显著增多(P<0.01)。结论应用1,25(OH)2D3治疗EAE大鼠,可通过改变共刺激分子表达、调节性T细胞比例以及不同细胞因子的分泌能力使EAE症状缓解。     

实验性自身免疫性脑脊髓炎的历史和现状

<正> 多发性硬化(multiple sclerosis,MS)是一种中枢神经系统(central nervous system,CNS)炎性脱髓鞘性自身免疫性疾病,其确切的发病机制仍不清楚,研究MS的实验动物模型成为探索该病病因学的最佳途径和方法。MS的实验动物模型主要分为免疫介导模型和病毒诱导模型两种,其中以免疫介导模型多用,该模型主要是通过注射CNS髓鞘蛋白(或分子肽段)与抗原佐剂的乳化复合物诱导动物对抗原反     

实验性自身免疫性脑脊髓炎动物模型早期轴索损伤

目的 研究实验性自身免疫性脑脊髓炎(EAE)动物模型发病早期的轴索损伤.方法 采用髓鞘蛋白脂蛋白(PLP139-151)多肽作为抗原诱发EAE小鼠模型.小鼠经免疫后每天进行神经功能评分及称体质量,于发病后剥离脑及脊髓组织进行病理学研究.结果 7只(23.3%)小鼠于免疫后15～22 d内发病,平均免疫后发病时间为(19.00±2.58)d,平均神经功能评分为(2.14±0.69)分.免疫前小鼠平均体质量[(21.85±0.94)g]与发病后体质量[(23.24±1.55)g]比较差异无统计学意义.HE染色可见发病小鼠软脊膜下脊髓组织炎性细胞浸润明显,以小血管周围为主的血管袖套形成.病变累及脊髓多见,且较大脑病变重.LFB染色可见炎细胞浸润处髓鞘有不同程度脱失,同时Bodian银染可见轴索肿胀、横断.免疫组织化学染色可见髓鞘碱性蛋白着色的相同区域淀粉样前体蛋白浓染,而星形胶质细胞GFAP染色增生不明显.结论 PLP多肽诱发SJL/J小鼠的EAE模型临床症状较轻,病变主要累及脊髓,而在大脑未发现与MS类似的典型病灶.在EAE发病早期,髓鞘还未明显脱失时即可见轴索损伤,且早期轴索损伤与临床症状并不平行.     

一氧化氮供体对实验性自身免疫性脑脊髓炎的作用

目的探讨一氧化氮供体3-吗啉-斯德酮亚胺(3-morpholinosydnonimine,SIN-1)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)大鼠的作用。方法应用豚鼠髓鞘碱性蛋白68-86(myelin basic protein 68-86,MBP68-86)主动免疫制作EAE实验动物模型。将大鼠随机分为SIN-1组和对照组,SIN-1组大鼠于致敏后第0~7天给予SIN-1药物干预,动态观察两组大鼠的临床症状及体质量变化,致敏后第14天采用ELISA方法检测各组大鼠单个核细胞(mononuclear cells,MNC)培养上清中γ干扰素(IFN-γ)和白细胞介素-4(IL-4)水平,并观察大鼠脑组织病理变化。结果与对照组比较,SIN-1组大鼠发病时间延迟,恢复时间提前,体质量明显增加,临床症状明显减轻;疾病症状最高评分明显降低。SIN-1组大鼠MNC培养上清中IFN-γ水平为(90.29±9.07)pg/mL,较对照组的(121.57±10.44)pg/mL明显降低(P<0.05);IL-4水平为(18.14±3.98)pg/mL,较对照组的(8.14±1.95)pg/mL明显增加(P<0.05)。SIN-1组大鼠组织病理损伤较对照组明显减轻。结论一氧化氮供体SIN-1可抑制EAE大鼠病情发展,对EAE具有保护作用。     

Gonadotropin-releasing hormone reduces the severity of experimental autoimmune encephalomyelitis, a model of multiple sclerosis

It has been reported that the spinal cord possesses Gonadotropin-releasing hormone (GnRH) receptor and that GnRH has neurotrophic properties. Experimental autoimmune encephalomyelitis (EAE) causes neurodegeneration in spinal cord. Thus, the present study was designed to determine whether administration of GnRH reduces the severity of EAE. The clinical signs of locomotion, axonal morphometry and neurofilaments (NFs) expression were evaluated. Clinical signs remained significantly lower in EAE rats with GnRH administration compared to animals without treatment. Morphometric analysis, there were more axons of larger areas in the spinal cord of EAE + GnRH group compared to EAE animals. Western blot analysis demonstrated that GnRH administration significantly increased the expression of NFs of 68, 160 and 200 kDa in the spinal cord of EAE animals. Our results indicate that GnRH administration reduces the severity of EAE in the rat.     

辛伐他汀抑制大鼠实验性自身免疫性脑脊髓炎的实验研究

目的 探讨辛伐他汀(STATINS)对实验性自身免疫性脑脊髓炎IEAE)的作用及机理.方法 Wistar大鼠55只采用随机数字表法分为EAE组(15只)、STATINS组(15只)、雷公藤(TP)组(15只)、正常对照组(10只).使用STATINS干预EAE大鼠,以TP为阳性对照,观察大鼠P53蛋白、IL-6、TNF-α、TGF-β的表达变化.结果 与EAE组相比,STATINS组发病率降低、临床症状减轻、体质量下降减少、病灶数减少、潜伏期延长,而TP组临床症状减轻、病灶数减少,差异均有统计学意义(P＜0.05),TP组体质量变化、潜伏期、发病率无显变化,差异无统计学意义(P＞0.05);STATINS组IL-6和TNF-α的表达降低,P53蛋白和TGF-β的表达增高;TP组仅TNF-α的表达降低,差异均有统计学意义(P＜0.05).与TP组相比较,STATINS组P53蛋白与TGF-β的表达增加,差异有统计学意义(P＜0.05),而TNF-α及Ⅱ-6的表达差异无统计学意义(P＞0.05).结论 STATINS可以有效抑制EAE.其可能的机制是抑制IL-6和TNF-α等促炎症因子的表达,促进P53蛋白与TGF-β的表达,效果可能优于TP.     

猴急慢性实验性变应性脑脊髓炎的病理研究

目的　探讨急、慢性猴 (猕猴类 )实验性变应性脑脊髓炎 (EAE)的病理特点。方法　从EAE猴病程不同阶段进行病理取材 ,制成切片 ,用HE、Bielschowsky改良法、Page法、Holzer法染色及间接免疫荧光法观察CD4、CD8和B淋巴细胞。结果　 ( 1)猴急性EAE脑组织炎症反应重 ,分布在小静脉周围形成血管套 ,以CD4淋巴细胞浸入为主。脱髓鞘病灶微小 ,分布弥散 ,轴突改变轻 ,无出血 ,类似急性播散性脑脊髓炎 (ADEM)。 ( 2 )猴慢性EAE脑组织炎症反应轻 ,分布在血管外周 ,以CD8和B细胞为主。脱髓鞘病灶较大 ,界线清晰 ,呈局灶性分布。病变部位轴突变性严重及轴突丧失 ,有胶质斑块形成 ,与慢性多发性硬化 (MS)的活动期病理改变有惊人的相似。结论　支持猴慢性EAE是MS的优秀模型。另外 ,用相同抗原诱导出不同病程的EAE提示ADEM和MS可能是同一疾病的不同病程 ,或可能是同一种病因 ,而致病程不同。     

Protracted, relapsing and demyelinating experimental autoimmune encephalomyelitis in DA rats immunized with syngeneic spinal cord and incomplete Freund's adjuvant

Experimental autoimmune encephalomyelitis (EAE) is a model for multiple sclerosis (MS). However, MS is a chronic, relapsing and demyelinating disease, whereas EAE in rats is typically a brief and monophasic disorder showing little demyelination. We demonstrate here that DA rats develop severe, protracted and relapsing EAE (SPR-EAE) after a subcutaneous immunization at the tail base with syngeneic spinal cord and incomplete Freund's adjuvant (IFA). The neurological deficits were accompanied by demyelinating inflammatory lesions in the spinal cord, with infiltrating T lymphocytes and perivascular deposition of immunoglobulins and complement. The induction of SPR-EAE was associated with humoral autoreactivity to myelin oligodendrocyte glycoprotein (MOG) and cellular autoreactivity to the rat myelin basic protein (MBP) peptides 69–87 and 87–101. These two peptides, as well as whole rat MBP, were encephalitogenic. In conclusion, we believe that the presently described demyelinating SPR-EAE represents a useful model for MS.     

大鼠实验性变态反应性脑脊髓炎模型研究

目的 用豚鼠脊髓匀浆诱发实验性变态反应性脑脊髓炎(EAE)大鼠模型.方法 应用豚鼠脊髓匀浆加完全弗氏佐剂和百日咳杆菌免疫Wistar大鼠.结果 免疫后8～14d大鼠发病,发病率为67%,光镜下病理学证实发病大鼠脑和脊髓内具有典型的脱髓鞘改变和炎性细胞浸润.电镜下也可见到髓鞘结构破坏和神经元轻微改变.结论 以豚鼠脊髓匀浆为抗原免疫Wistar大鼠可诱发EAE,具有发病率较高、费用低廉、模型稳定的特点,可以作为研究多发性硬化的动物模型.     

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。     

Variation in experimental autoimmune encephalomyelitis scores in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a common demyelinating central nervous system disease associated with progressive physical impairment. To study the mechanism underlying disease pathogenesis and develop potential treatments, experimental autoimmune encephalomyelitis (EAE) is often used as an animal model. EAE can be induced in various species by introducing specific antigens, which ultimately result in motor dysfunction. Although the severity of the paralysis is indicated using the EAE score, there is no standard scoring system for EAE signs, and there is variability between research groups with regard to the exact EAE scoring system utilized. Here, we describe the criteria used for EAE scoring systems in various laboratories and suggest combining EAE score with another quantitative index to evaluate paralysis, such as the traveled distance, with the goal of facilitating the study of the mechanisms and treatment of MS.     

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.