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Multiple sclerosis (MS) is a chronic autoimmune disease with demyelination and neurodegeneration of the central nervous system. It has been shown that the regulatory T (Treg) cells are responsible for maintaining tolerance to self-antigens and can suppress the autoimmune process in several animal models such as experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Recent basic studies have demonstrated that forkhead box P (FOXP3) and BTB domain and CNC homolog 2 (BACH2) are the master transcription factors of these cells playing a pivotal role in the polarization of naïve T cells into Treg cells. In the current study, the expression of FOXP3 and BACH2 genes and FOXP3 promoter methylation were evaluated in T cells of the EAE-induced mice. The results of this study showed a prominent and significant hypermethylation of the FOXP3 gene promoter in the EAE-induced mice compared to the sham and control groups. The expression of FOXP3 and BACH2 genes was significantly decreased in the EAE group in comparison with the sham and control groups. This study suggests that the epigenetic modification of FOXP3 gene is involved in the pathogenesis of EAE and this could be important in therapy in an appropriate and logical statement., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Astroglia, the most abundant cells in the human CNS, and even more prominent in multiple sclerosis patients, participate in CNS innate and adaptive immunity, and have been hypothesized to play an important role in multiple sclerosis progression. Experimental autoimmune encephalomyelitis elicited in mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 provides a means by which to explore the genesis and disease significance of astrogliosis during a chronic immune-mediated CNS inflammatory/demyelinative disorder that, in its' pathological features, strongly resembles multiple sclerosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Current concepts attribute an early and central role for auto-aggressive, myelin-specific T-lymphocytes in the pathogenesis of multiple sclerosis. This view emerged from immunological and pathological findings in experimental autoimmune encephalitis, an animal model characterised by pathological lesions closely resembling the ones found in multiple sclerosis. Furthermore, therapeutic strategies targeting the functions of these encephalitogenic T cells which attenuate their pathogenicity such as glatiramer acetate or anti-VLA4 antibody treatments represent proven approaches in multiple sclerosis. Nonetheless, all therapies evaluated to date either insufficiently dampen down inflammation or completely block immune processes. For this reason, there is a need to identify new therapeutic targets. We have employed live intravital two-photon microscopy to learn more about the behaviour of T cells during the preclinical phase of EAE, when T cells acquire the properties required to invade their target organ. Furthermore, we were able to identify an unexpected locomotive behaviour of T cells at the blood-brain barrier, which occurs immediately before diapedesis and the induction of paralytic disease. Such studies might open new avenues for the treatment of CNS autoimmune diseases. Multiple sclerosis is considered to be an autoimmune disease in which self-reactive T cells enter the central nervous system (CNS) and create an inflammatory milieu that destroys myelin and neurons. Immunomodulatory strategies for the treatment of multiple sclerosis target this process by attempting to inactivate these auto-aggressive T cells. However, so far, these strategies have failed to extinguish disease activity completely. For this reason, there is a need to understand in more detail the mechanisms by which T cells become encephalitogenic, how they enter the nervous system, and what the signals are that guide them along this path. If these processes could be better understood, it may be possible to design more effective and specific therapies for multiple sclerosis. This article will give a brief overview about our recent findings obtained using intravital imaging of autoaggressive effector T cells in an experimental model of multiple sclerosis. This new technological approach might help to fill some gaps in the understanding of autoimmune pathogenesis of multiple sclerosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The interaction between the immune and nervous systems can be both detrimental and beneficial. Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune demyelination that histologically and clinically mimics multiple sclerosis (MS). Myelin-reactive T cells produce and release brain-derived neurotrophic factor (BDNF) directly in the central nervous system, which stimulates tissue repair after traumatic injury. In EAE and MS, T cells in the vicinity of actively demyelinating lesions express BDNF, suggesting a neuroinflammatory reaction that is designed to limit brain damage and contribute to the repair process. Despite some evidence supporting MS therapies that enhance BDNF production by immune cells, no published reports have actually demonstrated that increased BDNF production can substantially ameliorate the clinical symptoms of MS. BDNF binds to a small subset of peripheral T cells that express TrkB, which is the BDNF receptor. This binding confers a partial resistance to apoptosis upon T cell activation, which could underlie the chronic nature of the inflammatory process. Here we will review the main aspects of BDNF and TrkB receptor involvement in neuroprotective autoimmunity in both EAE and MS. We will also discuss the latest findings with respect to the role of the BDNF/TrkB axis in regulating the survival of autoreactive T cells, with a focus on potential selectively immunomodulating strategies that may favor neuroprotection in MS.

Although it was originally synthesised to induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, glatiramer acetate (GA) is actually used in the treatment of this human disease. Serendipity thus was responsible for the discovery of the therapeutic properties of what has become one of the only two first-line therapies currently approved for relapsing-remitting multiple sclerosis. Despite being discovered over forty years ago, novel aspects of the mechanism of action of GA are still being uncovered today. Initially, the immunomodulatory effects of GA were believed to involve high-affinity binding of the polypeptide to MHC Class II molecules on antigen-presenting cells. Subsequently, it was demonstrated that GA activated a specific population of GA-reactive T cells of a type-2 helper (Th2) phenotype, promoting an antiinflammatory environment and the preferential migration of GA-specific Th2 cells into the central nervous system, leading to decreased local inflammation through 'bystander suppression'. More recently, it has been shown that GA-reactive Th2 cells will secrete neurotrophins, important factors for neuronal survival and for axonal protection, in the central nervous system. Moreover, perhaps by this mechanism, GA increases proliferation, differentiation and survival of oligodendrocyte precursor cells; potentially enhancing myelin repair processes in situ. In parallel to this work, light has been shed on immunomodulatory effects of GA on other immune cell types. These findings were stimulated by the observation that adoptive transfer of GA-specific T cells alone had a limited capacity to suppress experimental autoimmune encephalomyelitis compared to injection of GA itself, suggesting that other cell types such as monocytes also played a role. It has now been documented that GA treatment can also modulate antigen-presenting cells such as monocytes, dendritic cells, and also additional adaptive immune system cell types such as CD8+ T cells and Treg cells. In this respect, it is important to note that the interplay between such antigen-presenting cells and T cells is fundamental given the coordinated and bidirectional interactions between these two cell types in the immune network., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Myelin oligodendrocyte glycoprotein (MOG) is commonly used as an immunogen to induce an immune response against endogenous myelin, thereby modeling multiple sclerosis in rodents. When MOG is combined with complete Freund's adjuvant (CFA), multifocal, multiphasic disease ensues; whereas when MOG is combined with incomplete Freund's adjuvant (IFA), clinical disease is usually absent. MOG-IFA immunized animals can be induced to have neurological disease after intraspinal injections of cytokines and ethidium bromide (EtBr). In this study, we investigated whether MOG-IFA immunized rats exhibited subclinical injury as defined by somatosensory evoked potential (SEP) recordings. The titration of anti-MOG-125 antibodies showed robust peripheral mounting of immune response against myelin in MOG-immunized rats. However the SEP measures showed no significant change over time. Upon injecting cytokine-EtBr in the spinal cord after MOG sensitization, the SEP recordings showed reduced amplitude and prolonged latency, suggestive of axonal injury and demyelination in the dorsal column, respectively. These findings were later confirmed using T2-weighted MRI and histological hematoxylin-eosin stain of the spinal cord. This report establishes that MOG-IFA immunization alone does not alter neuronal conduction in SEP-related neural-pathways and that longitudinal in-vivo SEP recordings provide a sensitive read-out for focal myelitis (MOG-IFA and intraspinal cytokine-EtBr) in rats.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the highly specialized endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). It is well established that leukocyte recruitment across this vascular bed is unique due to the predominant involvement of alpha4-integrins in mediating the initial contact to as well as firm adhesion with the endothelium. In contrast, the involvement of the selectins, L-selectin, E- and P-selectin and their respective carbohydrate ligands such as P-selectin glycoprotein (PSGL)-1 in this process has been controversially discussed. Intravital microscopic analysis of immune cell interaction with superficial brain vessels demonstrates a role for E- and P-selectin and their common ligand PSGL-1 in lymphocyte rolling. However, E- and P-selectin-deficient SJL- or C57Bl/6 mice or PSGL-1-deficient C57Bl/6 mice develop EAE indistinguishable from wild-type mice. Considering these apparently discrepant observations, it needs to be discussed whether the molecular mechanisms involved in leukocyte trafficking across superficial brain vessels are irrelevant for EAE pathogenesis or whether the therapeutic efficacy of targeting alpha4-integrins in EAE is truly dependent on the inhibition of leukocyte trafficking across the BBB.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) and the helpful tool in preclinical testing of various substances considered for treatment of this human CNS disease. Ribavirin (R) and tiazofurin (T) are purine nucleoside analogues, with the broad spectrum of anti-viral, anti-tumoral and anti-inflammatory properties. We proposed that combined treatment with RT, administrated during the effector phase of EAE, would attenuate disease severity, both clinically and pathologically. Ribavirin was given daily at a dosage of 30 mg/kg and tiazofurin was given at a dosage of 10 mg/kg every other day for 15 days. We detected amelioration of clinical signs and faster recovery in the RT group compared to the control group. Immunohistochemical analyses revealed that RT treatment decrease the number of T cells, macrophages and microglia. In the controls, we detected reactive type of microglia, while in the RT group we noticed ramified/resting form. Demyelination areas and axonal damage were not recorded in the RT group, in contrast to the control group where multiple areas of demyelination zones and axonal loss were found. RT combination treatment suppresses ongoing EAE, prevents demyelination and axonal loss, and therefore may well be the potential therapy for the treatment of MS.

It is widely accepted that chronic disabilities in multiple sclerosis (MS) patients are due in part to neuronal damage. The central aim of this study was to characterize axonal disruption in the spinal cord of mice with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), a model of progressive MS. To accomplish this goal, we induced MOG-EAE in thy1-yellow fluorescent (thy-YFP)-transgenic mice in which all spinal motorneurons express the YFP reporter protein. We demonstrate that a build-up of YFP fluorescence occurs in profiles reminiscent of tortuous fragmented axons and axonal spheroids/globules as seen in various neurodegenerative/neuroinflammatory diseases. Approximately two-thirds of these damaged axons were decorated by the monoclonal antibody SMI 32, which recognizes hypophosphorylated neurofilament-H (hypoP-NF-H), an established marker of CNS axonal pathology. Unexpectedly, one third of damaged axons were hypoP-NF-H negative but could be visualized by their expression of the YFP transgene, whilst the remaining profiles were hypoP-NF-H positive but did not exhibit YFP fluorescence. Thus, using YFP transgenic mice in conjunction with hypoP-NF-H immunoreactivity provides a more comprehensive depiction of axonopathy in the ventral-lateral aspect of lumbosacral spinal cord in MOG-EAE. When YFP fluorescence was used in conjunction with a monoclonal antibody that recognizes CD11b; a marker of subsets of inflammatory cells, we were able to discern evidence of an early inflammatory attack on white matter axons. Finally, we show the accumulation of hyperphosphorylated neurofilament-H (hyperP-NF-H) expression in YFP+, lesioned WM areas and in a subpopulation of neuronal perikarya in the lumbar spinal cords of EAE mice.

Standard CD44 (CD44s) and its alternatively spliced variants (CD44v) were found to be associated with the metastatic potential of tumor cells, and with cell migration of autoimmune inflammatory cells, including cells involved in experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to evaluate whether induction of anti-CD44 immune reactivity, through cDNA vaccination could down-regulate EAE. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of SJL/J mice immunized with myelin antigens for EAE induction. Animals vaccinated with CD44v cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. The in vitro proliferation of lymphocytes was preserved regarding myelin antigens and mitogens. Histopathological examinations revealed a significant reduction of EAE lesions and enhanced apoptosis in central nervous system (CNS)-infiltrating cells of the successfully vaccinated animals. Such methods of cDNA vaccination with CD44 could be applicable in inflammatory CNS diseases, like multiple sclerosis.

Acute immunosuppression with lymphocytic agents given at maximally tolerated doses, followed by hematopoietic stem cell rescue achieved by autologous bone marrow or peripheral blood stem cell transplantation (BMT), has proved effective in various experimental models of autoimmunity. The rationale for such an approach in autoimmune diseases is based on the concept of lymphoablation of self-reactive lymphocytes followed by de novo immune system reconstitution, which, in the presence of the autoantigens in the thymus, may reinduce self-tolerance. Our previous work shows that in experimental autoimmune encephalomyelitis (EAE), autologous/syngeneic BMT not only prevents the appearance of paralytic signs, but can also partially reverse chronic disease and induce long-term, antigen-specific tolerance. However, there are serious reservations to be considered when interpreting these data and before applying similar protocols in patients with multiple sclerosis. (1) The model of EAE is not a completely reliable model of multiple sclerosis. (2) In animals with chronic EAE, although further relapses were prevented, the established paralysis was usually not reversible. According to recent data, in chronic multiple sclerosis (MS) lesions, damage caused by axonal loss/transection and cortical/spinal cord atrophy is irreversible and probably amenable to immunotherapy. (3) Long-term, antigen-specific tolerance may be induced with BMT, but not in all cases; in passively induced CR-EAE, many of the mice relapsed upon challenge with myelin antigens, which may indicate that the presence of the immunizing, myelin antigens (on the site of immunization) during the process of immune reconstitution is critical for induction of tolerance. Finally, one should weigh the procedure-related risks (including mortality of up to 5%) of bone marrow or peripheral stem cell transplantation (SCT). A more radical solution for autoimmunity may involve the use of non-myeloablative allogeneic transplantation., (Copyright 2004 Elsevier B.V.)

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2+ cells. In the present study, T cell, macrophage (CD11b/c+) and B cell (CD45RA+) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 microg nor 2 x 100 microg doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5+ and CD11b/c+ cells. Treatment with 2 x 100 microg/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5+, CD11b/c+ and CD45RA+ cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5+ T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking.

Axonal degeneration has been identified as the major determinant of irreversible neurological disability in patients with multiple sclerosis (MS). Axonal injury begins at disease onset and correlates with the degree of inflammation within lesions, indicating that inflammatory demyelination influences axon pathology during relapsing-remitting MS (RR-MS). This axonal loss remains clinically silent for many years, and irreversible neurological disability develops when a threshold of axonal loss is reached and compensatory CNS resources are exhausted. Experimental support for this view-the axonal hypothesis-is provided by data from various animal models with primary myelin or axonal pathology, and from pathological or magnetic resonance studies on MS patients. In mice with experimental autoimmune encephalomyelitis (EAE), 15-30% of spinal cord axons can be lost before permanent ambulatory impairment occurs. During secondary progressive MS (SP-MS), chronically demyelinated axons may degenerate due to lack of myelin-derived trophic support. In addition, we hypothesize that reduced trophic support from damaged targets or degeneration of efferent fibers may trigger preprogrammed neurodegenerative mechanisms. The concept of MS as an inflammatory neurodegenerative disease has important clinical implications regarding therapeutic approaches, monitoring of patients, and the development of neuroprotective treatment strategies.

The immunomodulating capacity of heparin led us to test the effect of the synthetic heparin-mimicking and low anticoagulant compound RG-13577 on the course of experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation. EAE was induced in SJL mice by inoculation with whole mouse spinal cord homogenate. RG-13577, delivered intraperitoneally, inhibited the clinical signs of acute EAE and markedly ameliorated inflammation in the spinal cord, primarily by inhibiting heparanase activity in lymphocytes and astrocytes and thus impairing lymphocyte traffic. RG-13577 treatment was effective when started on day of disease induction or day 7 after induction. The low molecular weight heparin, enoxaparin, tested under the same conditions, exerted only a minor insignificant inhibitory effect. RG-13577 also inhibited the tyrosine phosphorylation of several proteins, particularly Erk1 and Erk2 of the MAP kinase signaling pathways associated with inflammation and cell proliferation. RG-13577 blocked the activity of sPLA(2) and inhibited CNS PGE(2) production both in vivo and in vitro.

In Lewis rats, treatment with high doses of cyclosporin A (CsA) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE), although disease occurs when treatment is ceased. Treatment with low doses of CsA causes EAE to take a chronic relapsing course. We have previously shown that CsA treatment causes a decline in the number of T cells and increased inflammatory cell apoptosis in the spinal cord. The present study was undertaken to assess whether CsA therapy also modulates cytokine mRNA expression by inflammatory cells in the spinal cord of rats with EAE, looking for changes that might contribute to the observed effects of CsA on the course of EAE. EAE was induced in Lewis rats by inoculation with myelin basic protein and adjuvants. At the peak of neurological signs, on day 14 after inoculation, rats were given a single intraperitoneal injection of saline, or CsA at a dose of 8, 16, 32 or 64 mg/kg. The next day, rats were sacrificed, the spinal cords removed, inflammatory cells were extracted from the cords, and mRNA isolated from these cells. Expression of cytokine mRNA was assessed by semi-quantitative reverse transcription polymerase chain reaction (PCR) and by quantitative real-time PCR. With both techniques, we found that CsA suppressed the expression of interferon-gamma mRNA and interleukin-2 (IL-2) mRNA. With real-time PCR, we found that CsA caused significantly increased expression of transforming growth factor-beta mRNA. With the different techniques, we observed no consistent pattern of alteration of expression of interleukin-10 or interleukin-4 mRNA. It is possible that these changes in cytokine mRNA expression contribute to the modulation of the clinical course of EAE that is produced by CsA treatment.

Previous studies have shown that activation of blood coagulation and fibrin depositions around CNS vessels are observed in animals with experimental autoimmune encephalomyelitis (EAE), which provides an animal model for human autoimmune demyelinating disorders. We examined the values of peripheral blood fibrinogen, thrombin-antithrombin III complex (TAT), fibrinolytic activity, and fibrin degradation products in Lewis rats with EAE to elucidate the role of the blood coagulation-fibrinolysis system in EAE. Plasma TAT values increased immediately prior to development of symptoms, and decreased according to the improvement of symptoms. There was significant correlation between TAT values and clinical scores of EAE; other markers were not correlated with the symptoms of EAE. These results suggest that plasma TAT levels are sensitive markers of the severity of EAE, and may be useful clinical indicators for the severity of human autoimmune demyelinating disorders.

L-selectin is an adhesion molecule expressed on T cells and monocytes. It mediates rolling--the initial step of transendothelial migration. In this study, we investigated the role of L-selectin in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. EAE was induced in Lewis rats by active sensitization with myelin basic protein (MBP-EAE), or by adoptive transfer using MBP specific T cells (AT-EAE). Treatment with HRL3, a monoclonal antibody to L-selectin, and its F(ab')2 fragments efficiently suppressed MBP-EAE, and had a mild inhibitory effect on AT-EAE. Histological examination revealed a marked reduction of inflammatory infiltrates after treatment with HRL3. Administration of the control antibody HRL4 did not significantly alter the course of the disease. HRL3 caused T-cell depletion in the draining lymph nodes and spleen and a downregulation of L-selectin expression on T cells. We conclude that L-selectin-dependent mechanisms are involved in the pathogenesis of EAE. Modulation of L-selectin in vivo by antibodies or by competitive antagonists could be a novel therapeutic approach to autoimmune diseases of the central nervous system.

To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE.

We have compared the clinical signs, brain pathology and visually evoked responses (VEP) of guinea pigs with experimental allergic encephalomyelitis (EAE). Animals immunized with myelin basic protein had a milder disease, both from the clinical and histological points of views, compared to those immunized with crude white matter extract. However, VEP findings were quite similar in both groups. The VEP of the majority of animals from both groups showed changes before or at the same time that neurological signs appeared. Electrophysiological responses were usually characterized by abnormal wave shapes and prolonged latencies. Recovery of the VEP usually preceded the recovery from clinical signs. In contrast, the severity and incidence of brain tissue pathology was not correlated to either clinical signs or VEP changes. Possible explanations of the electrophysiological, clinical and histopathological changes and their time-course are discussed.

Histological and electrophysiological studies were performed on Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with guinea-pig spinal cord and Freund's adjuvants, in order to determine the cause of the neurological signs. These studies demonstrated demyelination-induced nerve conduction block in the large and also the smaller diameter fibres at the ventral root exit zone (VREZ) of the lumbar spinal cord. The demyelination at the VREZ affected both centrally and peripherally myelinated internodes, but predominantly the former. Studies on the H reflex recorded from a hindfoot muscle indicated normal peripheral nerve motor conduction but interruption of the monosynaptic reflex arc, as would be anticipated from this efferent conduction block and previously reported afferent conduction abnormalities. It is concluded that conduction block in alpha, beta and gamma motor fibres at the VREZ is an important cause of hindlimb weakness in whole spinal cord-induced acute EAE.

Experimental allergic encephalomyelitis (EAE) was induced In Lewis rats by the intradermal inoculation of an homogenate of guinea pig spinal cord in complete Freund's adjuvant. In these animals the effectiveness with which the capillary barrier excluded mannitol (a substance which normally only crosses this barrier very slowly) from the brain and spinal cord involvement were at their height, 14 days after the inoculation, the effectiveness of the barrier was reduced since the rate of diffusion of the mannitol out of the blood into the brain-stem and spinal cord was approximately doubled. Even as early as 7 days after inoculation, before any clinical signs had appeared, the rate of diffusion was significantly increased in the region of the lumbo-sacral cord. These changes roughly paralleled the histological changes seen in many of the small vessels. We believe that the changes are due to an increase in the permeability of the vessels to mannitol. The bearing of these findings on multiple sclerosis is discussed.

Lewis rats develop experimental allergic neuritis (EAN) when injected with bovine dorsal root (BDR) emulsified in complete Freund's adjuvant (CFA). In this study the susceptibility to EAN and subsequent relapse was studied in animals ranging from 4 to 25 weeks of age. Older animals exhibited a severe acute illness which was monophasic over the period of observation, whereas younger animals developed a less severe and frequently relapsing illness. Very young animals often relapsed more than once and sometimes to a more severe degree than shown in their first attack. Older animals which were in the late recovery stage of EAN (44 days after injection with BDR/CFA) were completely resistant to a second challenge with antigen. Young adult animals also developed resistance but not as strongly as the older animals. Animals first injected at weaning developed resistance as in the adults, but the analysis is complicated by the occurrence of spontaneous relapses. Because of differences in disease pattern between EAN and acute inflammatory polyneuropathy (Guillain-Barré syndrome) in man, caution should be exercised in drawing too close a parallel between the animal model and the human disease. The affinities may be closer to chronic relapsing inflammatory polyradiculopathy in man.

Histological and electrophysiological studies were performed in Lewis rats with acute experimental allergic encephalomyelitis (EAE) in order to determine the extent of dorsal root ganglion (DRG) involvement. Histological studies showed inflammation and demyelination in both the central nervous system (CNS) and peripheral nervous system (PNS). The DRG was the most affected region of the PNS and its involvement increased caudally. Nerve conduction abnormalities were demonstrated in the regions of the lumbar, sacral or coccygeal DRGs in some of the rats with EAE. However, the overall DRG involvement was much less severe, both histologically and functionally, than what we recently found in rabbits with EAE. Conduction through the lumbar dorsal root entry zone was normal. We conclude that lesions of the afferent pathway to the spinal cord do not contribute significantly to the disturbances of hindlimb motor function in Lewis rats with EAE.

An ascending impairment of tail nociception is a previously undescribed clinical sign of acute experimental allergic encephalomyelitis (EAE) in the rat. It occurs in EAE induced by inoculation with purified central nervous system (CNS) myelin basic protein (MBP) as well as with whole spinal cord. It is invariably present and consists of an absence of the vocalization response to noxious mechanical stimulation of the tail. This impairment of nociception evolves over 1-3 days, simultaneously with the development of tail weakness, and resolves more rapidly than the tail weakness. Light-microscopic, electron-microscopic and electrophysiological studies indicate that it is due to demyelination-induced conduction block in the small diameter myelinated afferent (A delta) fibres in the sacral and coccygeal dorsal root ganglia, dorsal roots and dorsal root entry zones. Unmyelinated fibres appear to be largely spared.

Experimental allergic encephalomyelitis (EAE) in Lewis rats is an acute, monophasic autoimmune disease, and rats recovering from EAE are generally resistant to active reinduction. However, following immunosuppression with a high dose (100-200 mg/kg) of cyclophosphamide (CY), EAE was reinduced in convalescent rats, irrespective of whether or not they were rechallenged with the encephalitogenic inoculum. Reinduction of EAE was not observed in rats treated with low doses (20 mg/kg or less) of CY. A selective inhibition of suppressor cells during the convalescent stage may be responsible for the reinduction of EAE in Lewis rats.

Adult inbred Strain 13 guinea pigs develop an acute, fatal form of experimental allergic encephalomyelitis (EAE) about 2 weeks after a single injection of isologous spinal cord in complete Freund's adjuvant (CFA), but similarly injected juveniles develop a delayed, rarely fatal chronic form. Thirty-seven sensitised adult Strain 13 animals were separated into 2 groups. One group was permitted to develop acute EAE. The other group was injected intramuscularly with 1 mg of guinea pig or bovine myelin basic protein (MBP) in incomplete Freund's adjuvant (IFA) on day 2, 7 or 10 post-inoculation (PI) followed by 0.2 mg in IFA every third day for a total of 10 doses. Animals in the unsuppressed group succumbed to acute EAE 13-16 days post-sensitisation. No animal in the suppressed group died during this period. Animals treated with MBP beginning 2 days PI showed no clinical signs, but mild clinical manifestations occurred in animals suppressed from days 7 and 10 PI. These signs remitted by 21 days post-sensitisation. One suppressed animal (out of 21) died during the fourth week postsensitisation. The other 20 suppressed animals appeared clinically normal towards the end of the course of MBP injections and remained so for the 6 months of study. Morphological examination revealed that CNS lesions occurred in all animals. In animals suppressed with MBP beginning on day 2 PI, lesions consisted only of a few meningeal inflammatory cells. Animals given MBP beginning on day 7 or 10 PI and sampled 1-2 weeks later, had lesions which could not be distinguished from those occurring in the non-suppressed acute EAE group. In time, the suppressed animals developed lesions which were typical of chronic EAE with remyelination as a predominant feature. Preliminary experiments on the suppression of chronic EAE in 5 juvenile Strain 13 guinea pigs have revealed that 3 MBP-injected animals failed to develop clinical disease over a 28-week period of study although lesions typical of chronic EAE were present. Simultaneously, 2 non-suppressed juvenile animals developed clinical signs by 12 weeks. These were associated with both acute inflammation and demyelination superimposed upon regions of chronic demyelinative activity.

Severe hypothermia and an ascending impairment of shivering are previously undescribed clinical signs in hyperacute experimental allergic encephalomyelitis (EAE) in the Lewis rat. These occurred in hyperacute EAE induced by inoculation with guinea pig spinal cord homogenate and heat-killed Bordetella pertussis. Hypothermia was first detected on day 6-7 post-inoculation, within 12-24 h of the onset of neurological signs, and became more severe as the disease progressed. Rectal temperatures less than or equal to 30 degrees C were common at ambient temperatures of 19-22 degrees C. Shivering was assessed by palpation and by cold tremor electromyography. Shivering was absent in the tail by day 6-7 post-inoculation. The impairment then progressed to affect the hindlimbs, thorax and occasionally the forelimbs. Shivering was absent in hindlimbs with only mild or moderate weakness. Histological studies revealed perivascular inflammation with polymorphonuclear and mononuclear cells, oedema, fibrin deposition, haemorrhage, primary demyelination and axonal degeneration in the spinal cord, dorsal root ganglia and spinal roots. The brainstem was also involved but the cerebral hemispheres, including the hypothalamus, were spared. The close relationship between the severity of hypothermia and the extent of shivering impairment indicates that reduced shivering is an important cause of hypothermia in hyperacute EAE. It is concluded that this impairment of shivering is due not to hypothalamic damage but to lesions elsewhere in the central and peripheral nervous systems.

Histological and electrophysiological studies were performed on Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with guinea-pig myelin basic protein (MBP) and Freund's adjuvant. The histological studies showed demyelination in the lumbar, sacral and coccygeal dorsal and ventral spinal roots and to a lesser extent in the spinal cord, including the dorsal root entry and ventral root exit zones. The electrophysiological studies demonstrated reduced conduction velocities between the lumbar ventral roots and sciatic nerve. Conduction block was demonstrated at the ventral root exit zone of the lumbar spinal cord but was less severe than in rats with whole spinal cord-induced acute EAE. Recordings of the M wave and H reflex elicited in a hindfoot muscle by sciatic nerve stimulation showed a normal M wave, indicating normal peripheral nerve motor conduction, but a markedly reduced H reflex. The reduction in the H reflex is accounted for by demyelination-induced nerve conduction block in the dorsal and ventral spinal roots, intramedullary ventral roots and at the dorsal root entry and ventral root exit zones of the spinal cord. Demyelination and nerve conduction abnormalities were well established in the relevant lumbar segments on the day of onset of hindlimb weakness. It is concluded that demyelination in the lumbar ventral roots and to a lesser extent in the lumbar spinal cord, including the ventral root exit zone, is an important cause of hindlimb weakness in myelin basic protein-induced acute EAE in the Lewis rat.

The reduction in the effectiveness of the blood-brain and blood-spinal cord barriers, previously seen in rats at the height of the acute episode of experimental allergic encephalomyelitis, has now been measured at various stages in the development of the disease up to 60 days after inoculation with guinea pig spinal cord in complete Freund's adjuvants. The marker of extracellular space, radioactively labelled mannitol, only crosses the blood-central nervous system barriers very slowly by passive diffusion in normal rats. An abnormal penetration of this marker into the central nervous system began to develop during the second week after inoculation, appearing first in the lower spinal cord, where it also reaches the highest level during the acute phase of the attack. The leak begins before either the clinical signs become evident or cuffing is seen around blood vessels in stained sections. As the clinical signs are disappearing, from about 15 days onwards, the permeability of the barrier returns steadily to its normal low value, starting in the spinal cord, especially the caudal part. The timing of the reduction in the effectiveness of the blood-central nervous system barrier in relation to other clinical and histological changes suggests that it may play a part in the development of the lesion. The relation between the timing of these changes in EAE and that in the development of a new lesion in (exacerbation of) multiple sclerosis is discussed.

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs with bovine myelin basic protein (BP) with adjuvant of either synthetic muramyl dipeptide (Mdp) or Mycobacterium tuberculosis (Tbc). The following results were obtained: (1) The body temperature of the animals was studied serially after sensitization and its elevation was shown to be an early sign of EAE. (2) Several animals developed the clinical and histological signs of hyperacute EAE. (3) An optimal combined dosage of BP and adjuvant was found for induction of clinical EAE and for the the production of complement fixing (CF) antibodies. (4) Little passive hemagglutinating (PH) antibody was produced by single immunization. These results displayed no essential difference in EAE induced by either adjuvant. (5) Detectable PH antibodies developed later in addition to CF antibodies in a few animals immunized with Tbc adjuvant. These animals were skin-tested to BP, and had recovered from body weight loss or limb weakness. The results suggest that humoral antibodies play a role in modifying the disease process, even if they are not essential in production of EAE.

Relapse of experimental allergic encephalomyelitis (EAE) was achieved in Lewis rats by cyclophosphamide (CY). All rats, immunized with an emulsion of guinea pig spinal cord homogenate in complete Freund's adjuvant and treated with 100 mg/kg of CY 21 days postimmunization (pi), developed moderate to severe paralysis 9-14 days following CY injection. A second relapse was observed in 4 of 11 rats reinjected with CY 49 days pi. Histologically, focal mononuclear cell infiltration with or without demyelination of the white matter of the central nervous system was observed. Cyclophosphamide administration caused transient leukopenia and T-cell defect, the resolution of which coincided with relapse of clinical EAE. Lymphocyte proliferative responses to myelin basic protein (BP) and concanavalin A (Con A) and antibody titers to BP were preserved in CY-treated rats. Adoptive transfer of EAE to naive recipients with Con A-activated spleen cells from donors with CY-induced relapse was unsuccessful.

By combining tissue pO2 measurements with morphological visualization and counting of perfused capillaries, early microvascular changes were investigated in paralysis of skeletal muscle of the rat. The models included experimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN), 6-aminonicotinamide intoxication, and peripheral denervation. The results showed that in all models tissue oxygenation was either normal or elevated. In most cases this was accompanied by a reduced perfusion of the capillary bed, supporting the hypothesis of a dysfunction in oxidative metabolism. The effects of paralysis on capillary perfusion were more marked in red than in white muscles, except in spastic paresis. Flaccid paralysis had more deleterious effects on muscle microcirculation than spastic paralysis. Our data suggest that: (1) microvascular changes occur very early after the onset of paralysis and (2) later developing changes in the skeletal muscle such as atrophy cannot be ascribed to a lack of oxygen supply but rather, at least in part, to a dysfunction in oxidative metabolism.