ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis

Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the na?ve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.     

CD24 and myosin light polypeptide 2 are involved in prevention of experimental autoimmune encephalomyelitis by myelin basic protein-pulsed dendritic cells

We previously demonstrated that injection of myelin basic protein-pulsed (MBP-pulsed)--but not of unpulsed--autologous bone marrow-derived dendritic cells (DC) efficiently prevents experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To define the molecules involved, we used 3 groups of rats pretreated subcutaneously with MBP-DC, or unpulsed DC, or PBS (control EAE). Four weeks later, all rats were immunized with encephalitogenic MBP peptide and adjuvant. Microarray analyses were done to screen for genes that differ among the 3 groups. Based on microarray analysis data, we used real-time PCR to measure expression of six probably involved genes in draining lymph node cells obtained on day 0, day 7 and day 14 post immunization (p.i.). Two of these 6 genes were consistently altered in both microarray analyses and RT-PCR. They are CD24 antigen being persistently low, and myosin light polypeptide 2 (Myl2) being high in the acute immune response in MBP-DC pretreated rats that develop resistance to EAE. These two genes could be targeted to treat EAE and, possibly, multiple sclerosis.     

Interferon γ, interleukin 4 and transforming growth factor β in experimental autoimmune encephalomyelitis in lewis rats: Dynamics of cellular mrna expression in the central nervous system and lymphoid cells

The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon -γ (IFN-γ), interleukin 4 (IL-4) and transforming growth factor β (TGF-β) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63–88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-γ, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.), and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-β did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-γ, while TGF-β may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63–88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63–88-induced production of both IFN-γ and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63–88 induced lymphoid cells to TGF-β production. Thus, an autoantigen-specific production of TGF-β occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically. © 1995 Wiley-Liss, Inc.     

细胞间黏附分子-1在实验性自身免疫性脑脊髓炎大鼠中表达的动态变化及作用

目的探讨细胞间黏附分子-1(ICAM-1)在实验性自身免疫性脑脊髓炎(EAE)大鼠中表达的动态变化及其作用。方法分别取免疫后第4、6、8、10、12、14、16、18、20天EAE大鼠脑和脊髓制成石蜡切片,行HE染色和ICAM-1半定量免疫组化分析。结果免疫后第8天ICAM-1表达即出现明显上调,早于临床症状的发生;随免疫后时间的延长,ICAM-1表达呈逐渐增高后缓慢下降的变化趋势,并且与EAE大鼠病情评分呈显著正相关(r=0.57,P=0.003)。结论ICAM-1的表达上调可能在EAE发病中具重要作用。     

Prazosin suppresses development of axonal damage in rats inoculated for experimental allergic encephalomyelitis

The effectiveness of the alpha 1-adrenergic antagonist prazosin for preventing monoaminergic axonal damage in the spinal cords of rats that were inoculated for experimental allergic encephalomyelitis (EAE) was assessed using immunohistochemistry. Prazosin injections (2 mg, i.p.) given twice daily from day 7 to day 15 postinoculation significantly reduced paralysis, spinal cord inflammation and monoaminergic axonal damage compared to saline injections. A close positive correlation between severity of inflammation and severity of axonal damage was found for both prazosin- and saline-treated rats that were inoculated for EAE. These findings confirmed previous observations of suppression of the development of clinical signs of EAE by prazosin treatment and supported the hypothesis that some factor associated with spinal cord inflammation may be responsible for the bulbospinal monoaminergic axonal damage that occurs during EAE.     

盐酸法舒地尔治疗实验性自身免疫性脑脊髓炎的潜能与抗炎作用

目的：探讨盐酸法舒地尔对实验性自身免疫性脑脊髓炎（EAE）的治疗效果及机制。方法：雌性C57BL/6小鼠,随机分为EAE对照组、盐酸法舒地尔干预组和盐酸法舒地尔治疗组。采用髓鞘少突胶质细胞糖蛋白多肽诱导慢性EAE模型。干预和治疗分别在免疫后第3天和症状出现时予以腹腔注射盐酸法舒地尔,观察EAE模型小鼠体重变化和临床症状,进行苏木精-伊红和CD4＋T细胞染色,同时检测磷酸化肌球蛋白磷酸酶（p-MYPT1）和核因子（NF-κB）。结果：盐酸法舒地尔可推迟并改善EAE小鼠症状,减轻中枢神经系统炎细胞浸润,抑制脊髓和脑p-MYPT1及脊髓NF-κB的表达。     

Competitive dissociation of encephalitogenic complexes between antigen presenting cells and myelin basic protein

Splenic T cells from myelin basic protein (MBP)-immunised Lewis rats were activated to transfer experimental autoimmune encephalomyelitis (EAE) by co-culture with MBP-pulsed lymphoid dendritic cells (DC). MBP-pulsed DC could be kept for at least 24 h at 37 degrees C in antigen-free medium without affecting their ability subsequently to activate encephalitogenic T cells. However, MBP-pulsed DC were rendered much less stimulatory after a 6 h, but not 2 h, secondary incubation with ovalbumin. Thus, although encephalitogenic complexes between MBP and DC appear very stable in the absence of competing antigens, in their presence, antigen exchange can take place over a period of a few hours; this has positive implications for therapy of EAE by antigen competition.     

Basic and acidic fibroblast growth factors protect spinal motor neurones in vivo after experimental spinal cord injury

We studied the effect of a single focal injection of recombinant basic (FGF2) or acidic (FGF1) fibroblast growth factor on the survival of spinal motor neurones at 24 h after a standardized spinal cord contusion injury (SCI) in the rat. Both FGF2 and FGF1 (3 μg), microinjected into the injury site at 5 min post-injury (p.i.), protected at least two functionally important classes of spinal motor neurones, autonomic preganglionic neurones in the intermediolateral (IML) column and somatic motor neurones in the ventral horn (VH). Moreover, there was enhanced choline acetyltransferase (ChAT) immunoreactivity in surviving VH and IML neurones, suggesting an improved functional status. Thus, neurotrophic factors such as FGF2 and FGF1 may contribute to an overall strategy to treat acute SCI and improve recovery of function.     

Down regulation of trophic factors in neonatal rat spinal cord after administration of cerebrospinal fluid from sporadic amyotrophic lateral sclerosis patients

Accumulating evidence supports neuroprotective role of trophic factors in amyotrophic lateral sclerosis (ALS). Previous studies from our laboratory report that the CSF of patients with sporadic ALS (ALS-CSF) induces degenerative changes in the rat spinal motor neurons and reactive astrogliosis in the surrounding gray matter. The present study was aimed to investigate if the ALS-CSF affected the expression of trophic factors namely, brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) in the newborn rat spinal cords. ALS-CSF was intrathecally injected into the neonatal rats and the mRNA levels of the trophic factors were determined by quantitative real-time polymerase chain reaction. Here, we report significant down regulation in the gene expression of trophic factors for BDNF, FGF2 and IGF1. BDNF mRNA levels were found to be reduced by 6.8-fold in the ALS-CSF injected group compared to control groups. The levels of IGF1 and FGF2 mRNA were also decreased by 3.91- and 2.13-fold, respectively, in the ALS group. We further found that exogenous supplementation of BDNF considerably reduced the aberrant phosphorylation of neurofilaments, complementing our earlier findings of restored expression of voltage gated sodium channel. Reduced expression of trophic factors indicates an altered microenvironment of the motor neurons and could possibly be one of the contributing factors in the degeneration process.     

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.     

Immunoregulation of experimental autoimmune encephalomyelitis by the selective CB1 receptor antagonist

During immune-mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here we explored the cellular distribution of the CB1 receptor (CB1R) in the central nervous system (CNS) and detected the level of CB1R expression during experimental autoimmune encephalomyelitis (EAE) by RT-qPCR, Western blotting, and immunostaining. Expression of CB1R was observed in neurons and microglia/macrophages but was barely detected in astrocytes. During EAE, the expression of CB1R in spinal cords was reduced at days 9, 17, and 28 postimmunization (p.i.), but the level of CB1R expression in spleens did not show a significant difference compared with complete Freund's adjuvant (CFA)-immunized mice. A selective CB1R antagonist (SR141716A) increased EAE clinical score, accompanied by weight loss. Unexpectedly, SR141716A inhibited the expression of CB1R but increased the expression of CB2R in brains, spinal cords, and spleens simultaneously. The administration of SR141716A increased interferon-γ, interleukin-17 (IL-17), and inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α in brains and/or spinal cords. A similar increase was observed in spontaneous and specific antigen-stimulated splenic mononuclear cells compared with vehicle controls. Interestingly, the expression of CX3CL1 was increased in brains and spinal cords but declined in spleens of EAE mice treated with SR141716A. These results indicate that manipulation of the CB1R may have therapeutic value in MS, but its complexity remains to be carefully considered and studied in further clinical application.     

Mechanisms of recovery from experimental allergic encephalomyelitis induced with myelin basic protein peptide 68-86 in Lewis rats: a role for dendritic cells in inducing apoptosis of CD4+ T cells.

Spontaneous remission of experimental allergic encephalomyelitis (EAE) is usually associated with prominent apoptosis. The mechanisms behind apoptosis are unknown. We examined the functions of dendritic cells (DC) from Lewis rats with EAE induced by immunization with myelin basic protein peptide 68-86 (MBP68 - - 86). Recovery from EAE was associated with three major functional changes of freshly prepared DC: (1) elevated proliferation, (2) increased nitric oxide (NO) production, and (3) augmented IFN-gamma secretion. In Freund's complete adjuvant (FCA)-immunized control rats, no increase of proliferation, NO production or IFN-gamma secretion was observed on day 21 post-immunization (p.i.), i.e., recovery from EAE. In vitro effects of IFN-gamma, TNF-alpha, TGF-beta1, IL-4 and IL-10 on DC were examined. IFN-gamma enhanced proliferation and NO production by DC, while TNF-alpha and IL-4 induced only slight DC proliferation. DC from recovering EAE rats (day 21 p.i.) suppressed MBP68 - - 86-induced T cell proliferation compared to DC obtained at other time points in EAE and FCA-immunized rats. DC-derived NO induced apoptosis of CD4+ T cells, thereby inhibiting autoreactive T cell responses. Besides IFN-gamma stimulation, NO production by DC was mainly induced in an antigen-dependent manner when DC were co-cultured with T cells. The results suggest that spontaneous recovery from EAE is associated with augmented DC functions. Overproduction of NO by DC results in apoptosis of autoreactive CD4+ T cells, thereby decreasing autoreactive T cell reactivities. The existence of such a NO negative feedback loop may contribute to remission of EAE.     

Restoration of axon conduction and motor deficits by therapeutic treatment with glatiramer acetate

Glatiramer acetate (GA; Copaxone) is an approved drug for the treatment of multiple sclerosis (MS). The underlying multifactorial anti‐inflammatory, neuroprotective effect of GA is in the induction of reactive T cells that release immunomodulatory cytokines and neurotrophic factors at the injury site. These GA‐induced cytokines and growth factors may have a direct effect on axon function. Building on previous findings that suggest a neuroprotective effect of GA, we assessed the therapeutic effects of GA on brain and spinal cord pathology and functional correlates using the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Therapeutic regimens were utilized based on promising prophylactic efficacy. More specifically, C57BL/6 mice were treated with 2 mg/mouse/day GA for 8 days beginning at various time points after EAE post‐induction day 15, yielding a thorough, clinically relevant assessment of GA efficacy within the context of severe progressive disease. Therapeutic treatment with GA significantly decreased clinical scores and improved rotorod motor performance in EAE mice. These functional improvements were supported by an increase in myelinated axons and fewer amyloid precursor protein‐positive axons in the spinal cords of GA‐treated EAE mice. Furthermore, therapeutic GA decreased microglia/macrophage and T cell infiltrates and increased oligodendrocyte numbers in both the spinal cord and corpus callosum of EAE mice. Finally, GA improved callosal axon conduction and nodal protein organization in EAE. Our results demonstrate that therapeutic GA treatment has significant beneficial effects in a chronic mouse model of MS, in which its positive effects on both myelinated and non‐myelinated axons results in improved axon function. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

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。     

Dendritic cells exposed to estrogen in vitro exhibit therapeutic effects in ongoing experimental allergic encephalomyelitis

Immunomodulatory effects of estrogen have been demonstrated by clinical and experimental observations, but the mechanisms by which estrogen exhibits the effects remain to be defined. One possible mechanism by which estrogen inhibits the development of experimental allergic encephalomyelitis (EAE), a commonly used model of multiple sclerosis (MS) in humans, is over the functions of dendritic cells (DC). Here, we describe that splenic DC from Lewis rats obtained on day 12 post-immunization (p.i.) with myelin basic protein (MBP) encephalitogenic peptide 68-86+Freund's complete adjuvant (FCA), after being exposed in vitro 17beta-estradiol, exhibited therapeutic effects on acute EAE when injected subcutaneously on day 5 p.i. Blood mononuclear cells (MNC) were isolated from thus treated rats on day 12 p.i. Administration of estrogen-exposed DC prevented the expansion of CD4+ T cells and increased proportions of regulatory T cells producing IL-10 and CD4+CD28- suppressor T cells, accompanied with increased IL-10 and IFN-gamma, and reduced TNF-alpha production. Infiltrates of CD68+ macrophages within the central nervous system and MBP 68-86-induced T cell proliferation were inhibited in rats injected with estrogen-exposed DC compared to rats injected with naive DC. Estrogen up-regulated the expression of indoleamine 2,3-dioxygenase, which promotes tolerogenic properties of DC. The results suggest that in vitro exposure of DC to estrogen modulates DC functions and results in a therapeutic effect of DC.     

Lipoic acid inhibits expression of ICAM-1 and VCAM-1 by CNS endothelial cells and T cell migration into the spinal cord in experimental autoimmune encephalomyelitis

Lipoic acid (LA) suppresses and treats murine experimental autoimmune encephalomyelitis (EAE), which models multiple sclerosis. However, the mechanisms by which LA mediates its effects in EAE are only partially known. In the present study, LA (25, 50 and 100 microg/ml) inhibited upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-alpha (TNF-alpha) stimulated cultured brain endothelial cells. Immunohistochemical analysis of spinal cords from SJL mice that had received LA (100 mg/kg/day) following immunization to induce EAE exhibited markedly reduced expression of ICAM-1 and VCAM-1 compared with that of EAE mice receiving saline. Co-localization analysis showed that ICAM-1 and VCAM-1 expression increased over endothelial cells (staining positive for von Willebrand factor, vWF) in EAE and that LA decreased the expression levels to that observed in na?ve mice. Spinal cords from mice receiving LA had significantly reduced inflammation (decreased CD4 and CD11b staining) as compared to EAE mice that received saline. Overall, our data suggest that the anti-inflammatory effects of LA in EAE may be partly due to inhibition of ICAM-1 and VCAM-1 expression by central nervous system (CNS) endothelial cells.