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.     

Hypertension and neuronal degeneration in excised rat spinal cord studied by high-b value q-space diffusion magnetic resonance imaging

Hypertension is one of the major risk factors of stroke and vascular dementia (VaD). We used stroke prone spontaneous hypertensive rats (SPSHRs) as a model for neuronal degeneration frequently occurring in humans with vascular disease. Recently, high b value q-space diffusion-weighted imaging (DWI) was shown to be very sensitive to the pathophysiological state of the white matter. We studied the spinal cords of SPSHR rats ex vivo after the appearance of motor impairments using diffusion anisotropy and q-space diffusion imaging (measured at a high b value of up to 1 x 10(5) s/mm(2)). The diffusion anisotropy images computed from low b value data set (b(max) approximately 2500 s/mm(2)) showed a small but statistically significant decrease (approximately 12%, P < 0.05) in the diffusion anisotropy in the spinal cords of the SPSHR group as compared to control rats. However, more significant changes were found in the high b value q-space diffusion images. The q-space displacement values in the white matter of the SPSHR group were found to be higher by more than 70% (P < 0.002) than that of the control group. These observations concurred with electron microscopy (EM) that showed significant demyelination in the spinal cords of the SPSHR group. These results seem to indicate that high b value q-space DWI might be a sensitive method for following demyelination and axonal loss associated with vascular insults.     

左旋咪唑诱导大鼠急性EAE外周血CD4+/CD8+和IL-6的检测及意义

目的 探讨急性实验性变态反应性脑脊髓炎(EAE)大鼠外周血CD4 /CD8 及IL-6的改变及意义.方法 采用豚鼠髓鞘蛋白匀浆和福氏完全佐剂注射并加用左旋咪唑诱发大鼠EAE模型,分别注射生理盐水/福氏佐剂、左旋咪唑作为对照,注射后16d采用Loyez氏髓鞘染色观察大鼠脊髓髓鞘改变、流式细胞仪检测外周血CD4 /CD8 比值和采用ELISA法检测IL-6水平,并与对照组比较.结果 模型组发病率87.5%,正常组、NS/CFA组和LMS对照组未见异常.模型组髓鞘染色见髓鞘结构疏松、部分髓鞘脱失;外周血CD4 /CD8 比值为2.33±1.04,及IL-6为194.92±56.85,较其余各组明显增高(P＜0.01,P＜0.01).结论 外周血CD4 /CD8 比值及IL-6水平增高在大鼠EAE的发生过程中发挥重要作用.     

实验性变态反应性脑脊髓炎的病理学研究

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%,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。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,可作为研究多发性硬化的动物模型。     

Magnetic resonance imaging of antibody-mediated demyelinating experimental allergic encephalomyelitis

Two models of demyelinating experimental allergic encephalomyelitis (EAE) were studied on Lewis rats in whom the disease was induced by injections of either (i) lentil-lectine binding myelin glycoproteins plus myelin basic protein (MBP)-specific T cells (36 rats), or (ii) myelin/oligodendrocyte glycoprotein-specific monoclonal antibody plus MBP-specific T cells (16 rats). In our 24 control rats, 20 received MBP-specific T cells only, and four received myelin glycoproteins plus purified protein derivative-specific T cells. The extent of the resulting blood-brain barrier (BBB) permeability, vasogenic oedema and/or demyelination was assessed in vivo using magnetic resonance imaging (MRI) techniques. The results show that in both demyelinating EAE models the disease appeared more quickly, progressed very rapidly and was more severe than when induced with a similar number of MBP-specific T cells alone. Almost all animals developed hypercute EAE, with a very high mortality rate. MRI showed a very intense BBB breakdown and vasogenic oedema in all the normally ‘leaky’ areas of the central nervous system, and focal lesions corresponding to plaque formation in the brain stem or spinal cord near the ‘leaky’ areas. During the 40-day observation period, the rare survivors of this hyperacute form of EAE presented a chronic form of EAE with serious sequelae. Our results demonstrate that the synergistic effect observed between MBP-specific T cells and antibodies to myelin glycoproteins, especially to myelin/oligodendrocyte glycoprotein, does not only induced demyelinating lesions and chronic clinical signs, but is further responsible, via the normally ‘leaky areas’, for the fatal increase of the BBB breakdown and vasogenic oedema of which there are ample acute clinical signs.     

Inhibition of chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse

Chronic relapsing experimental allergic encephalomyelitis (CREAE) can be reproducibly induced in Biozzi AB/H mice following injection of spinal cord homogenate (SCH) emulsified in complete Freund's adjuvant (CFA). Active clinical disease is associated with mononuclear cell infiltration of the central nervous system (CNS), mainly the spinal cord. Whole brain homogenate (BH), however, failed to induce clinical or histological disease. In contrast, substituting sciatic nerve homogenate in the inoculum induced experimental allergic neuritis (EAN). Clinical disease was manifest earlier (13.1 +/- 0.3 days) than CREAE (16.2 +/- 1.4) and was accompanied by mononuclear infiltration of the peripheral nervous system (PNS). In comparison to CREAE induction, pretreating mice with SCH or BH in incomplete Freund's adjuvant (IFA) suppressed the development of SCH-induced disease. The BH was more tolerogenic than the SCH and this hyporesponsiveness was CNS antigen-specific as PNS tissue failed to inhibit the course of CREAE. Tolerance induced by pretreatment with SCH or BH in IFA was reversed by a single injection of 200 mg/kg cyclophosphamide, 2 days prior to CREAE induction. This suggests that IFA-induced hyporesponsiveness is actively regulated, possibly via the action of suppressor cells. In addition, treatment with neuroantigens in IFA appears to be mainly afferent acting as it serves to prevent initial disease induction. This treatment after immunization for CREAE, however, fails to prevent disease progression. Furthermore, treatment with CNS antigens emulsified in IFA during the post-acute remission stage appeared to synchronize and induce (32 +/- 1 days) the onset of clinical relapse, compared with untreated controls (41 +/- 5 days). This indicates that such IFA treatment has minimal value in controlling an ongoing immune disease of the CNS.     

Adoptive transfer of experimental allergic encephalomyelitis from immature guinea pig donors

In immature Strain 13 guinea pigs sensitized to syngeneic spinal cord, a chronic allergic encephalomyelitis is elicited reminiscent of demyelinating diseases of man and which features relapses or progressive downhill course and extensive areas of demyelination in the central nervous system. However, juvenile recipients of syngeneic lymphocytes from similarly sensitized juveniles show only the acute form of experimental allergic encephalomyelitis. Neuropathologically, the CNS of affected animals displayed mild changes only and minimal demyelination. These observations indicate that the age-dependent differences seen between the acute disease of adults and the chronic disease of juveniles may be due to differences in availability of modulating or reparatory factors, rather than differences in the central nervous system organ or in the immune response itself.     

Abrogation of induced resistance to experimental allergic encephalomyelitis in guinea pigs by host-versus-graft reaction

Experimental allergic encephalomyelitis (EAE) effector cells known to exist in guinea pigs with myelin basic protein-incomplete Freund's adjuvant (MBP-IFA)-induced resistance to EAE could be activated in vivo by means of allogeneic confrontation (local host-versus-graft reaction (HVGR)). The abrogation of the resistance was observed only when HVGR was combined with encephalitogenic challenge (myelin basic protein-complete Freund's adjuvant (MBP-CFA)) in a certain order and at certain time intervals. The injection of 20 x 10(7) gamma-irradiated allogeneic lymphoid cells 7 or 4 days prior to or along with MBP-CFA treatment resulted in development of EAE with delayed onset and protracted course. The effect was most prominent when HVGR was induced on day -4. Histological examination revealed inflammatory lymphoid cell infiltrations in spinal cord. Serum level of total and anaphylactic anti-MBP antibodies correlated with the clinical picture.     

新西兰兔和SD大鼠实验性变态反应性脑脊髓炎模型的比较

目的比较两种不同种属的动物及不同免疫原诱发的实验性变态反应性脑脊髓炎（EAE）模型的各自特点及其适用的优缺点。方法应用兔脊髓匀浆（R-SCH）、SD大鼠脊髓匀浆（M-SCH）、异种牛大脑白质匀浆（C-SCH）为免疫原，分别免疫新西兰兔和SD大鼠制备EAE动物模型。观察比较模型的发病过程、临床评分、病理变化和病理组织学评分。结果新西兰兔EAE模型呈单相发病过程，发病时间长，发病率较低，但其病灶大且集中，MRI更易观察和定位。SD大鼠EAE模型则呈典型的双相病程，发病时间短，发病率高，复发率高，其发病更像人类多发性硬化的发病过程。同时发现同种免疫原诱发的EAE模型其发病过程更稳定，发病率也较异种免疫原高．其导致的模型病理变化也更明显，尤其是脱髓鞘更明显。结论不同的动物物种和不同的免疫原制备的EAE模型具有各自适用的优缺点和特点，可根据研究目的的不同选取所需的模型。     

Lymphokine mRNA expression in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis is associated with a host recruited CD45R hi/CD4 population during recovery

To evaluate CD4⁺ T cell subpopulations involved in the induction and recovery from experimental autoimmune encephalomyelitis (EAE), the CD45R phenotype and lymphokine mRNA profile was evaluated for encephalitogenic CD4⁺ T cell lines in vitro and compared to CD4^* T cells islated from the spinal cord of Lewis rats with EAE were > 90% of the myelin basic protein (MBP)-specific T cell lines and clones that adoptively transferred EAE were > 90% CD4⁺ and > 90% CD45R lo. A time course of EAE disease progression was monitored as a function of the percentage of CD45R hi/CD4⁺ T cells isolated from the spinal cords of diseased animals. The majority of CD4⁺ T cells found in the central nervous system during the early phase of passive EAE were CD45R lo (the same as the encephalitogenic lines/clones). A large increase of the CD45R hi/CD4⁺ T cells (up to 45%) was observed during the peak and recovery phases of EAE. Lymphokine mRNA production was analyzed from antigen-stimulated MBP-specific lines, and from spinal cord lymphocytes isolated from rats with EAE. The BP-specific lines produced Th1 lymphokines (IL-2, IFN-γ, and TNF-α), while the spinal cord lymphocytes produced the same Th1 lymphokines as well as IL-4 and IL-10. The CD45R hi/CD4⁺ T cells isolated from the spinal cords were larger and expressed more lymphokine RNA per cell than the CD45R lo/CD4⁺ T cells. The encephalitogenic cells (CD45R hi/CD4⁺ T detected in the spinal cords of rats with a fluorescent dye and by allelic transfers and all of the CD45R hi/CD4⁺ lymphocytes found in the spinal cells were found to be host recruited. Thus it appears that the CD45R hi/CD4⁺ lymphocytes found in the spinal cord represent a host-recruited, activated cellular infiltrate that increased in number in the recovery phase of EAE and synthesized both Th1 and Th2 lymphokines.     

Myelin-induced experimental allergic encephalomyelitis in Lewis rats: tumor necrosis factor α levels in serum and cerebrospinal fluid Immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord

Tumor necrosis factor a (TNFa) activity was measured in serum and cerebrospinal fluid (CSF) of Lewis rats after experimental allergic encephalomyelitis (EAE) induction and during the clinical course of acute disease. TNFa bioactivity expression preceded the clinical symptoms and paralleled the severity of disease. We further investigated the identity of the central nervous system (CNS) cells involved in TNFa expression and their regional localization during EAE. Tissue sections of brain, cerebellum, dorsal spinal cord and optic nerve were studied by indirect double labelling immunofluorescence. Spinal cord white matter and optic nerve showed a widespread TNFa immunoreactivity at critical stages of EAE in macrophages/microglia and astrocytes. We have shown changes in CSF/serum albumin ratio and immunoglobulin G index during EAE. Our results confirm the very important role of TNFa in EAE.     

Antibodies to P2 and P1 myelin antigens in experimental allergic neuritis and allergic encephalomyelitis

We confirmed earlier observations that experimental allergic neuritis (EAN) in Lewis rats induced by injection of bovine peripheral nerve myelin in complete Freund's adjuvant is not accompanied by development of experimental allergic encephalomyelitis. However, sera of these animals contained elevated titers of antibodies against central nervous system myelin basic protein (BP), likely induced by peripheral nerve P1 protein. Anti-BP antibodies were not seen in sera of rats with EAN induced by peripheral nerve P2 protein. Lack of encephalogenicity of bovine myelin in Lewis rats does not reflect simply lack of immune responses induced against BP.     

[H]Thymidine labeling of astrocytes in experimental allergic encephalomyelitis

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [³H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11–15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [³H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.     

High resolution nuclear magnetic resonance imaging of the spinal cord in experimental demyelinating disease

Summary Chronic recurrent experimental allergic encephalomyelitis was induced in a strain 13 guinea pig by inoculation of isologous spinal cord homogenate. The spinal cord was obtained after perfusion with 4% paraformaldehyde and examined with nuclear magnetic resonance (NMR) imaging. Proton NMR spin echo images (repetition time: 3 s; echo times: 20 and 60 ms) were obtained from intact, isolated spinal cord in a 4.7 Tesla, 50 mm bore magnet. The slice thickness of the images was 380 m and the inplane resolution was 40×40 m. The images showed superficial areas of low signal intensity in the lateroventral regions of the white matter, in some instances with a seam of higher signal intensity. Neuropathologically, these abnormalities corresponded exactly to areas of demyelination. Control images did not show these abnormalities. The present high resolution imaging allowed a correlation between demyelination and abnormal NMR signals in a small laboratory animal with an inflammatory demyelinating disease.Supported by the Belgian Foundation of Medical Scientific Research (FGWO, grant 3.0096.86 and grant 3.0019.86), by the Institute for the promotion of Scientific Research in Industry and Agriculture (IWONL) and by the Scientic Research Planning Office of the Belgian Government (DPWB), contract no. 87/92-120     

Suppression of experimental allergic encephalomyelitis by the encephalitogenic peptide, in solution or bound to liposomes

We have investigated the ability of liposome-bound encephalitogenic peptide to suppress experimental allergic encephalomyelitis (EAE) in the guinea pig. EAE was induced by challenge with the encephalitogenic peptide, residues 113-122 of human myelin basic protein (MBP) in complete Freund's adjuvant. The peptide was acylated with stearic acid in order to anchor it to the lipid bilayer. The liposomal-bound peptide effectively suppressed clinical signs of EAE at relatively low doses, when given subcutaneously or intraperitoneally without incomplete Freund's adjuvant, several days after challenge. In vitro proliferation of lymphocytes from treated, protected animals in response to the peptide was greatly decreased but that to the purified protein derivative of tuberculin antigen was not, indicating an antigen-specific effect. However, histological signs of EAE were not reduced. The free peptide in solution was somewhat less effective when given intraperitoneally but was as or nearly as effective as liposome-bound peptide when given subcutaneously. Binding to liposomes may decrease the rate of clearance or degradation of the peptide when given intraperitoneally.     

The neuropathology of chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rat by inoculation with whole spinal cord and treatment with cyclosporin A

Summary Chronic relapsing experimental allergic encephalomyelitis was induced in Lewis rats by inoculation with guinea-pig spinal cord and complete Freund's adjuvant followed by treatment with low-dose cyclosporin A. In most animals, tail and limb weakness developed in a relapsing remitting pattern but in some these signs were persistent or progressive from onset. Histological studies during the early stages of clinically active disease (< 25 days after inoculation) revealed inflammation and primary demyelination in the central nervous system (CNS), particularly the spinal cord, and in the peripheral nervous system (PNS), specifically the ventral and dorsal roots and dorsal root ganglia. Animals studied in the later stages of clinically active disease (> 28 days after inoculation) had extensive spinal cord demyelination but minimal PNS demyelination. In these animals, large plaques of demyelination with gliosis and prominent plasma cells occurred particularly in the thoracic spinal cord, and lesions of different ages were present within the spinal cord. CNS and PNS remyelination by oligodendrocytes and Schwann cells, respectively, was present in all animals studied later than 18 days after inoculation (the time of the first remission, if it occurred). In both early and late clinically active disease electron microscopy revealed macrophages invading and destroying CNS myelin sheaths. Active demyelination was sometimes found in regions of CNS remyelination, suggesting that remyelinated fibres were being attacked. Axonal degeneration occurred in the spinal cord. During clinical remission there was CNS and PNS remyelination and much less inflammation; however, active demyelination still occurred to a limited degree.Supported by the National Health and Medical Research Council of Australia, University of Queensland Foundation and Clive and Vera Ramaciotti Foundations is gratefully acknowledged. During this work Glenn Stanley was a recipient of a Research Training Fellowship of the National Multiple Sclerosis Society of Australia     

Regional suppression, therapy after onset and prevention of relapses in experimental allergic encephalomyelitis by mitoxantrone

Mitoxantrone, a new antineoplastic anthracenedione drug, suppressed the development of experimental allergic encephalomyelitis (EAE) in rats when administered during the incubation period. One dose of mitoxantrone had a favorable effect on EAE even when withheld until after the onset of clinical signs. In a model of relapsing EAE, a single dose during a remission prevented the expected relapse. Mitoxantrone had a particularly favorable effect when administered at the site of inoculation of the encephalitogenic antigen and adjuvant, indicating the importance of its interaction with the regional draining lymph nodes. These results support the possible clinical use of mitoxantrone for immunosuppression.     

雌激素对实验性变态反应性脑脊髓炎大鼠发病影响的研究

目的探讨雌激素对实验性变态反应性脑脊髓炎(EAE)的影响。方法将30只大鼠随机分为EAE组及大、小剂量雌激素干预组,每组10只,制作EAE模型,大、小剂量雌激素干预组分别给予皮下注射苯甲酸雌二醇1 mg/kg/d、250μg/kg/d,连续10 d,观察各组的发病情况并采用HE染色观察脑和脊髓组织病理变化。结果大、小剂量雌激素干预组的临床症状均较EAE组轻,表现为发病率减少、潜伏期延长、进展期缩短、高峰期神经功能损害较轻。病理切片提示雌激素干预组脑和脊髓炎症细胞浸润明显减少,其中以大剂量组更明显。结论雌激素对多发性硬化动物模型EAE具有保护作用,且与剂量相关。     

Visualisation of the kinetics of macrophage infiltration during experimental autoimmune encephalomyelitis by magnetic resonance imaging

Macrophages are considered to be the predominant effector cells in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Ultra small particles of iron oxide (USPIO) can be used to detect macrophage infiltrates in the CNS with magnetic resonance imaging (MRI). Here, we investigated whether the kinetics of lesion formation in EAE can be visualised by altering the time point of USPIO injection and the time interval between particle injection and MRI. When USPIO are systemically injected 24 h before MRI, hypo intense regions are detected in different brain regions depending on the disease stage. These regions correspond to sites of macrophage infiltration. A more complete visualisation of sites of inflammation is accomplished by USPIO injection at disease onset and postponing MRI to top of disease. This study demonstrates that the distribution pattern and amount of inflammatory lesions detected with USPIO, depends on timing of USPIO administration and subsequent MRI. These findings are important for a correct application and interpretation of USPIO dependent contrast imaging of CNS inflammation.