A neuropathological analysis of experimental autoimmune encephalomyelitis with predominant brain stem and cerebellar involvement and differences between active and passive induction

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune demyelinating disease that can be induced in a variety of animal species and which is commonly used as an animal model of multiple sclerosis. In rodent EAE models, the clinical disease is typified by ascending paralysis; however, other clinical patterns can also be observed by inducing disease with particular peptides of myelin proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein. Here we describe EAE induced in C3H/HeJ mice by inoculation with residues 190–209 of PLP. This form of EAE is manifested clinically by a movement disorder, with axial rotation of the head and trunk. Histologically, this form of EAE is characterized by predominant cerebellar or brain stem involvement, depending on whether EAE is induced by active immunization with the PLP peptide, or by passive transfer of T cells specific for the peptide. The inflammatory cell infiltrate is composed of polymorphonuclear cells and mononuclear cells. This rotatory form of EAE may be a useful model for studying the neuropathological characteristics of multiple sclerosis affecting the brain stem and cerebellum. Received: 23 June 1999 / Revised, accepted: 25 October 1999     

雌激素在实验性自身免疫性脑脊髓炎中的保护作用

多发性硬化(multiple sclerosis,MS)与其他多种自身免疫性疾病一样存在着性别差异,表现为:与男性相比,女性发病率较高,且初发时间也较早。女性患者在妊娠期伴随着体内雌激素水平的波动,MS患者复发次数和严重程度也出现明显变化[1]。在MS动物模型即实验性自身免疫性脑脊髓炎(exp     

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.     

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.     

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.     

Th17细胞与多发性硬化/实验性自身免疫性脑脊髓炎

多发性硬化(multiple sclerosis,MS)及其理想动物模型实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是主要累及中枢神经系统的自身免疫性疾病.     

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.     

Dysregulation of the hypothalamic-pituitary-gonadal axis in experimental autoimmune encephalomyelitis and multiple sclerosis

The ability of sex hormones to regulate cytokine production is well established, but the ability of cytokines to regulate sex hormone production has only begun to be investigated. We measured sex hormones in mice with passive experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS) patients with sexual dysfunction. Abnormally low serum testosterone levels were found in male mice with EAE and in male MS patients, while serum estrogen levels in female mice with EAE were normal. An inverse relationship between cytokine and testosterone levels in male mice with EAE, coupled with an increase in serum luteinizing hormone (LH) levels, suggests that inflammatory cytokines suppress testosterone production by a direct effect on testicular Leydig cells. Gender differences in the sensitivity of the hypothalamic–pituitary–gonadal (HPG) axis to inflammation may be an important factor regulating the duration and severity of central nervous system (CNS) autoimmunity.     

Prevention of chronic relapsing experimental autoimmune encephalomyelitis by soluble tumor necrosis factor receptor I

We have evaluated the effect of the type I (p-55, type β) soluble tumor necrosis factor receptor (sTNFrI) in an animal model of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) was induced in SJL/J mice by adoptive transfer of T lymphocytes sensitized to myelin basic protein (MBP). sTNFrI completely blocked both clinical signs of disease and pathological changes that included CNS demyelination and inflammatory cell infiltration. Effective inhibition of disease expression was obtained using several different regimens of subcutaneous (s.c.) injection. These included daily doses starting at day 0, every other day injections starting at day 0, daily doses starting on day 4, and two doses separated by 12 h on day 1 and 2. Furthermore, treatment with sTNFrI for 15 days completely protected these animals from the recurrent episodes of disease normally associated with adoptively transferred EAE. These findings suggest that TNF plays a major causative role in EAE and that the sTNFrI may prove to be a useful therapeutic approach in multiple sclerosis.     

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。     

B cells from glatiramer acetate-treated mice suppress experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) thought to be primarily mediated by T cells. However, emerging evidence supports an important role for B cells in the pathogenesis and inhibition of MS. Glatiramer acetate (GA), a Food and Drug Administration-approved drug for the treatment of MS, has a good safety profile. But GA's mechanism of action in MS is still elusive. In this study, we showed that B cells from GA-treated mice increased production of IL-10 and reduced expression of co-stimulatory molecules viz.: CD80 and CD86. B cells from GA-treated mice also diminished proliferation of myelin oligodendrocyte glycoprotein (MOG_35-55) specific T cells. Purified B cells transferred from GA-treated mice suppressed experimental autoimmune encephalomyelitis (EAE) in recipient mice compared with B cells transferred from mice treated with PBS or ovalbumin. The treatment effect of GA in EAE was abrogated in B cell-deficient mice. Transfer of B cells from GA-treated mice inhibited the proliferation of autoreactive T cells as well as the development of Th1 and Th17 cells but promoted IL-10 production in recipient mice. The number of peripheral CD11b⁺ macrophages in recipient mice also decreased after transfer of B cells from GA-treated mice; however, the number of dendritic cells and regulatory T cells remained unaltered. These results suggest that B cells are important to the protective effects of GA in EAE.     

Spatial diversity of blood–brain barrier alteration and macrophage invasion in experimental autoimmune encephalomyelitis: A comparative MRI study

Inflammation plays a central role in the development of numerous disorders of the central nervous system (CNS) such as multiple sclerosis (MS). For a long time it was assumed that recruitment of macrophages into the CNS and breakdown of the blood–brain barrier (BBB) are closely linked. In the present study we challenge this concept. We used small superparamagnetic iron oxide particles (SPIO)-enhanced T2-weighted (T2-w) magnetic resonance imaging (MRI) on a routine 1.5 T MRI unit to follow macrophage infiltration in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. After an initial SPIO-enhanced MRI, gadofluorine M (Gf), an experimental contrast agent which proved to be more sensitive in detecting BBB leakage than gadolinium (Gd)–DTPA (Bendszus, M., Ladewig, G., Jestaedt, L., Misselwitz, B., Solymosi, L., Toyka, K.V., Stoll, G., Gadofluorine-M enhancement allows more sensitive detection of inflammatory CNS lesions than T2-w imaging: a quantitative MRI study. Brain 2008; 1-12), was applied to the same animals followed by a second scan. Areas with SPIO-induced signal loss on T2-w MRI indicative of recent macrophage infiltration were matched with areas showing Gf enhancement as a measure of BBB disturbance. Overall 87 EAE lesions showed iron-related signal loss, while 57 lesions showed Gf enhancement. By direct comparison we could detect concomitant SPIO-induced signal loss and Gf enhancement only in a small minority of lesions. In conclusion, our findings show macrophage infiltration in the CNS during EAE in areas with a closed BBB for humoral factors. This holds true despite the use of a more sensitive MR contrast agent for BBB disruption than Gd-DTPA. Our experimental observations may have implications for disease monitoring in MS patients by MRI which guides treatment decisions.     

小鼠实验性自身免疫性脑脊髓炎的病理变化

目的用髓鞘少突胶质细胞糖蛋白多肽(MOG35-55)诱发实验性自身免疫性脑脊髓炎(experi-m ental autoimmune encephalomyelitis,EAE)小鼠模型。方法应用MOG35-55抗原加完全弗氏佐剂免疫C57BL/6小鼠,利用光镜、电镜观察小鼠组织学改变。结果光镜下可见小血管周围炎细胞浸润,呈袖套状改变、血管周围明显脱髓鞘及神经元变性,B ieschowsky银染显示大量轴索肿胀和轴索卵形体的形成,电镜下可见髓鞘结构松散、断裂或融合,包括不同程度的髓鞘重建,脊髓病变广泛,程度重于脑部。结论EAE的病理改变为血管周围炎性细胞浸润、白质脱髓鞘及髓鞘重建。     

复发缓解型EAE大鼠长期、自然病程的视神经病理改变

目的建立小剂量复合抗原成分诱导的EAE长病程动物模型,通过多种方法观察EAE大鼠的视神经病理改变。方法雌性Wistar大鼠72只,6~8周龄,体重180~200g,其中12只为对照组,余60只诱导EAE模型,进行EAE动物的神经功能评分(按照Kono五级评分法[1]进行),观察大鼠每次发病时的病情、病程变化。于第一次发病后第7d和半年时行眼底照相检查,并取视神经行HE染色光镜观察和透射电镜观察其病理改变。结果 EAE组大鼠视神经光镜主要表现为有不同程度的炎症反应;电镜主要表现为髓鞘稀疏,其髓鞘板层松解,轴突萎缩、变性,结构模糊不清。EAE组第一次发病后第7d及半年时眼底照相提示视乳头萎缩,边界不清楚。半年时视乳头萎缩更明显。结论 EAE大鼠存在明显的视神经病变,主要表现为视神经炎症反应、脱髓鞘改变和轴索损伤。     

Survival of oligodendrocytes in chronic relapsing experimental autoimmune encephalomyelitis

Demyelinated plaques of chronic relapsing experimental autoimmune encephalomyelitis (EAE) have been examined in Strain 13 guinea pigs. Oligodendrocytes could be identified within these lesions adjacent to naked axons and astrocytic processes. Oligodendrocytes were identified both ultrastructurally and immunocytochemically. Many of these cells showed bizarre shapes and myelin within their cytoplasm. The survival of oligodendrocytes within these lesions suggests that the myelin sheath, not the oligodendrocyte, is the primary target in autoimmune demyelination. A similar sequence of events has been proposed in multiple sclerosis, for which chronic relapsing EAE serves as a laboratory model. The persistence of myelinating cells in areas of chronic demyelination and gliosis might have significant reparatory implications.     

Preventing the BDNF and NGF loss involved in the effects of cornel iridoid glycoside on attenuation of experimental autoimmune encephalomyelitis in mice

The present study was designed to investigate the beneficial effects of cornel iridoid glycoside (CIG), a main component extract from Cornus officinalis, on neurotrophin expression in mouse experimental autoimmune encephalomyelitis (EAE), a classical model of multiple sclerosis (MS). After EAE initiation, CIG was intragastrically administered daily for 32 days and reduced disease severity. Histopathological staining and western blotting both showed that CIG could prevent brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) loss in the spinal cord of EAE mice. In conclusion, our findings indicated that CIG treatment suppressed disease severity of EAE partially through blocking downregulation of neurotrophic factor expression such as BDNF and NGF, suggesting that CIG may have beneficial effects for the treatment of demyelinating diseases such as MS.     

Stress and hypothalamic-pituitary-adrenal axis function in experimental autoimmune encephalomyelitis and multiple sclerosis - a review

Multiple sclerosis (MS) is an inflammatory and degenerative disease of the CNS with an assumed autoimmune-mediated pathogenesis. Stressful life events have been hypothesized as potential triggers of disease exacerbation. Animal studies using experimental autoimmune encephalomyelitis (EAE), as a model for MS, suggest that decreased hypothalamic-pituitary-adrenal (HPA) function may play a role in the increased susceptibility and severity of the disease. Histopathological studies of the hypothalamus point to disturbances in corticotropin-releasing hormone (CRH) regulation as a result of MS lesions in this area. Functional endocrine tests (e.g., the combined Dexamethasone-CRH test) showed a disturbed negative feedback after steroid application in MS patients. Hyper- and hypoactivity of the HPA axis, have been described to be associated with more severe courses. This paper presents an overview of the evidence for a role of HPA dysfunction in EAE and MS based on stress-experimental studies.     

Blood-brain barrier integrity is unaltered in human brain cortex with diabetes mellitus

Diabetes-related cognitive dysfunction has been recognized for many years in humans, but the pathogenesis of this condition is poorly understood. Evidence from animal studies suggests that altered function of the blood-brain barrier (BBB) could be a potential cause contributing to this disease. This study aimed to investigate whether the permeability of the BBB is affected in the brains of persons with diabetes mellitus (DM). On postmortem prefrontal and temporal cortex of diabetic patients and controls, immunohistochemical stainings were carried out using specific antibodies against three proteins (PAL-E, IgG and albumin), which are considered as markers for the vascular permeability status of the BBB. Rare or no PAL-E staining was found in the capillaries of the prefrontal and temporal cortex parenchyma, in both DM and control materials. IgG and albumin were localized in and directly around blood vessel walls in the prefrontal and temporal cortex. No obvious differences in the staining pattern of IgG and albumin were observed between brain samples of persons with DM and controls. This study suggests that the BBB in diabetic patients is well maintained.     

Blood-brain barrier alterations in both focal and diffuse abnormalities on postmortem MRI in multiple sclerosis

Postmortem MRI-guided tissue sampling significantly enhances the yield of MS lesions in autopsy material, but so far it is unknown whether abnormalities concur with blood-brain barrier alterations. Here we sampled MS lesions with focal and diffuse abnormalities (diffusely abnormal white matter; DAWM) on MRI; both were coupled to the presence of MS lesions upon neuropathological examination. Extravascular distribution of fibrinogen, indicating BBB disturbance, was observed in so-called (p)reactive lesions that reflect discrete areas of microglial activation without demyelination within an otherwise normal appearing white matter. Leakage became more extensive in active demyelinating MS lesions to chronic inactive lesions. An enlargement of the perivascular (Virchow-Robin) space containing infiltrated leukocytes was associated with both DAWM and focal abnormalities on postmortem MRI. This study shows for the first time that in MS brain changes in the vasculature take place not only in focal lesions but also in DAWM as detected by postmortem MRI.     

Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation

Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG_35-55/CFA and 100–600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.