Experimental allergic encephalomyelitis: Modification of optic nerve pathology by antecedent virus infection

Summary Strain 13 guinea pigs, sensitised with spinal cord and Freund's complete adjuvant at 18–21 days of age, developed an experimental allergic encephalomyelitis (EAE) characterised by a non-fatal first attack followed by recovery, then remissions and relapses in the majority of animals. Pretreatment with an i.p. injection of an avirulent strain (A 774) of Semliki forest virus at 1 day of age resulted in an EAE with an accelerated onset and enhanced mortality during the first attack. Unlike the acute phase pathology in unpretreated animals, optic nerve demyelination was observed at 14 days after neuroantigen sensitisation. In sections of optic nerve from virus-treated animals, endothelial cell vesicularisation was seen and the role of this change in disease exacerbation is discussed. Virus treatments of animals older than 1 day of age resulted in a failure to potentiate the disease. Virus inoculated simultaneously with spinal cord alone failed to act as an adjuvent. The mechanism of virus action in modifying the immunopathogenesis of EAE and its relevance to multiple sclerosis is discussed.We thank Multiple Sclerosis Society of Great Britain and Northern Ireland for the financial support of this work     

Effect of viral infection on experimental allergic encephalomyelitis in mice

BALB/c mice were irradiated with 350 R and injected with mouse spinal cord homogenate (MSCH) in complete Freund's adjuvant. Only 15-30% of these animals developed signs of experimental allergic encephalomyelitis (EAE) at 21-28 days after inoculation. Intraperitoneal infection with the non-lethal A7 strain of Semliki forest virus (SFV) 7 days after sensitization reduced the mean appearance time of the EAE symptoms to 14 days and the number of animals with clinical EAE increased up to 70%. In contrast, virus inoculation 10 days before induction of EAE decreased significantly the incidence of clinical EAE in both BALB/c and SJL mice. Demyelination with increased cellularity, presence of macrophages, stripping of myelin from the axons and sparing of oligodendrocytes was observed in spinal cords of animals at days 13-16 after induction of EAE and subsequent virus infection. No demyelination was seen in specimens taken at the same time from mice inoculated with MSCH or SFV alone. Combined MSCH and virus inoculations induced changes in the general immune response which may be one of the major reasons for the increase or decrease in demyelination in this model.     

Serum and cells from Theiler's virus-infected mice fail to injure myelinating cultures or to produce in vivo transfer of disease. The pathogenesis of Theiler's virus-induced demyelination appears to differ from that of EAE

Intracerebral inoculation of SJL mice with Theiler's Murine Encephalomyelitis virus (TMEV) results in a biphasic disease characterized by early grey matter involvement followed by late, chronic white matter inflammation and demyelination. Morphological parameters of TMEV-induced demyelination are essentially identical to those of experimental allergic encephalomyelitis (EAE) and immunosuppression has been shown to prevent demyelination. To test whether the pathogenesis of demyelination in TMEV infection is based on an autoimmune attack on myelin as in EAE, we tested sera and cells from infected animals for their ability to produce in vitro demyelination and cells for their ability to transfer disease in vivo. Isogeneic organotypic cultures were exposed to either serum or splenocytes from diseased animals. Neither serum nor splenocytes demyelinated or prevented myelination in these cultures. Splenocytes from diseased animals were also incubated with basic protein or whole spinal cord and assayed for their proliferative response or their ability to transfer disease to naive recipients. Neither proliferation nor transfer of disease was observed. These results show that the immunopathology of demyelination in the Theiler's model differs from that of EAE in a number of important parameters and support the contention that demyelination in this viral infection is produced by immunological mechanisms different from those operating in EAE.     

Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss

Autoimmune injury to oligodendrocytes evokes an endogenous response in the central nervous system, which initially limits the acute injury to oligodendrocytes and myelin, and subsequently promotes remyelination. The key molecular and cellular events responsible for this beneficial outcome are incompletely understood. In this article, we utilize murine autoimmune encephalomyelitis (EAE) to focus on the effect of endogenously produced leukemia inhibitory factor (LIF) upon mature oligodendrocyte survival after demyelinating injury. We show that the mRNA for LIF is markedly upregulated in the spinal cord in the context of acute inflammatory demyelination. After clinical disease onset, administration of neutralizing anti-LIF antibodies over a four day period significantly worsens disease severity in two different murine EAE models. We also show that administration of neutralizing antibodies results in reduced activation of the cognate LIF receptor components in the spinal cord. Histologically, anti-LIF antibody administration increases the extent of acute demyelination (P < 0.01) and doubles the oligodendrocyte loss already induced by EAE (P < 0.05), without altering the extent of inflammatory infiltration into the spinal cord. Although acute EAE induces a rapid, three-fold increase in the proliferation of NG2 positive oligodendrocyte progenitors (P < 0.001), this response is not diminished by antagonism of endogenous LIF. We conclude that endogenous LIF is induced in response to autoimmune demyelination in the spinal cord and protects mature oligodendrocytes from demyelinating injury and cell death, thereby resulting in attenuation of clinical disease severity.     

Concomitant detection of changes in myelin basic protein and permeability of blood-spinal cord barrier in acute experimental autoimmune encephalomyelitis by electroimmunoblotting

An electroimmunoblotting technique was used with a monoclonal antibody to myelin basic protein (MBP) to assess demyelination in 3 defined regions of the spinal cord in rats with acute experimental autoimmune encephalomyelitis (EAE). A slight loss in MBP was detected only in the sacrococcygeal region of the spinal cord after the onset of clinical signs. In all 3 spinal cord regions studied, significantly elevated levels of albumin and IgG were detected during the course of EAE by the same technique. At the onset of clinical signs, the levels of IgG and albumin were highest in the more caudal regions of the spinal cord. As the clinical signs became more severe, IgG and albumin levels increased in the more cranial regions of the spinal cord. These changes thus correlated with the ascending progression of clinical signs typical of EAE in rats. These results provided added evidence that in rats affected with acute EAE, the clinical signs occur independently of demyelination and coincide with vasogenic edema.     

Demyelinative myelopathy in mice induced by the DA virus.

An attenuated tissue culture adapted strain of DA virus, an agent related to the Theiler murine encephalomyelitis viruses (TMEV), was used to induce a chronic myelopathy in mice. Spastic paraparesis first appeared 5 months after weanings were inoculated intracerebrally with the virus. None died as a direct result of the infection, and none improved once paretic. The major pathological change in these mice was demyelination of thoracic segments of spinal cord. No clinical illness or demyelinative pathology were detected during the first 4 months after inoculation. Encephalitic virus was present in brain and spinal cord as late as 10 months after inoculation. No neutralization antibody activity to DA virus was present in sera from 10 patients with amyotrophic lateral sclerosis, 10 with multiple sclerosis, or in 10 controls.     

Infection of mice with lactic dehydrogenase virus prevents development of experimental allergic encephalomyelitis

A significant reduction in the incidence of experimental allergic encephalomyelitis (EAE) in SJL/J mice was observed when mice were infected with lactic dehydrogenase virus (LDV) 14 days before, on day 0, or 3 days after immunization with spinal cord homogenate. These results are discussed in terms of the selective infection by LDV of I-region-associated antigen- (Ia) positive macrophages.     

Spinal motoneurone distress during experimental allergic encephalomyelitis

The main pathophysiological feature characterizing multiple sclerosis (MS) is demyelination. However, the possibility of neural damage has recently been proposed as a mechanism in chronic disease. Experimental allergic encephalomyelitis (EAE) is the most widely used experimental model for MS. We investigated occurrences of microglial activation and astrocytosis in the spinal cord, choline acetyl-transferase (ChAT) and calcitonin gene-related peptide (CGRP) mRNA regulation in spinal motoneurones during EAE. EAE was induced in female Lewis rats by injecting guinea pig spinal cord tissue in complete Freund's adjuvant (CFA) to which heat-inactivated Mycobacterium had been added. Rats injected with CFA and uninjected rats were used as controls. ChAT and CGRP mRNAs were studied by in situ hybridization in the lumbar spinal cord and a computerized grain counting procedure was used for quantification. No differences in ChAT mRNA level were found between control and CFA-injected rats. ChAT mRNA level was strongly reduced in EAE 14 days after immunization and then recovered (29 days after immunization). CGRP mRNA increased 14 days after immunization, and then recovered to control level. Extensive long-lasting gliosis developed in the spinal cord and around motoneurones and a transient expression of p75LNGFR in motoneurones was also found. These data suggest that during EAE, gliosis induces distress in spinal cord neurones involving the synthesis enzyme for the main transmitter.     

实验性自身免疫性脑脊髓炎的视神经病理改变

目的 研究实验性自身免疫性脑脊髓炎(EAE)的视神经病理改变.方法 足垫皮下注射豚鼠脊髓匀浆和完全弗氏佐剂(CFA)混合物制作Wismr大鼠EAE模型,于发病后第6d将大鼠处死,取视神经、脑和脊髓,行HE和LFB染色,光镜和电镜下观察其病理改变.结果 病理检查发现EAE模型组大鼠脑、脊髓有不同程度的炎症反应和脱髓鞘改变;均有视神经病变,光镜主要表现为炎症反应和脱髓鞘,视神经髓鞘脱失重于炎症反应;电镜主要表现为髓鞘稀疏,少突胶质细胞数量减少、胞核固缩,其周围包裹的髓鞘板层松解,轴突髓鞘分离.结论 EAE大鼠存在明显的视神经病变,主要为视神经炎症反应和脱髓鞘改变.     

The Distribution of Inflammatory Demyelinated Lesions in the Central Nervous System of Rats with Antibody-Augmented Demyelinating Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) has long been studied as an animal model of the human demyelinating disease Multiple Sclerosis. However, EAE induced in the Lewis rat by injection of myelin basic protein (MBP), or MBP-specific T-lymphocytes, is primarily an inflammatory condition of the central nervous system (CNS) with little or no demyelination. In EAE models in which demyelination does result, it is either not very widespread or is unpredictable in its degree and location. In this study we have produced antibody-augmented demyelinating EAE (ADEAE) in the Lewis rat by injection of activated MBP-specific T-lymphoblasts, followed by injection 4 days later of a monoclonal antibody against myelin/oligodendrocyte glycoprotein, an extrinsic protein of myelin. We have documented the extent and location of inflammatory cell infiltrates and demyelination throughout the CNS using histochemistry, immunofluorescence, and image analysis. Perivascular inflammatory infiltrates were seen in the deep cerebellar white matter and in the folia. Perivascular, periventricular, and subpial inflammation was widespread throughout the pons/medulla and at all levels of the spinal cord. Very little inflammation was apparent in the forebrain. MBP immunofluorescence demonstrated extensive areas of periventricular demyelination in the forebrain around the third ventricle. Both periventricular and perivascular lesions were commonly observed in the cerebellum and pons/medulla. The extent of demyelination in the spinal cord increased caudally with large confluent areas of subpial demyelination seen throughout the lumbar cord. The extensive and reproducible distribution of inflammatory demyelinating lesions in ADEAE provide the possibility to select areas of the CNS for more detailed analysis of the cellular changes that accompany demyelination and remyelination.     

Chronic neurologic disease in Theiler's virus infection of SJL/J mice

This study demonstrates that most SJL/J mice inoculated intracerebrally (IC) with 1000 suckling mouse 50% mean lethal doses of Theiler's encephalomyelitis virus (TMEV) develop flaccid paralysis 10–21 days after infection when there is acute spinal cord gray matter involvement (early disease). Surviving mice later develop a distinctive chronic neurologic disorder which is associated with marked mononuclear cell infiltrates and active demyelination in spinal cord white matter (late disease). Moreover, about one-fourth of infected animals only develop signs of late disease which may begin after an incubation period as long as 2 and a half months. Affected mice are less active, incontinent, and have a waddling, spastic gait. Minimal stimulation induces prolonged extensor spasms of all limbs. These late-developing manifestations of chronic TMEV infection are progressive and clinical remissions have not been observed. The effect of persistent CNS infection on general development was monitored by weekly measurement of body weight; however, the growth of chronically-infected mice was found to parallel that of control animals.     

Persistence of Theiler's virus infection following promotion of central nervous system remyelination

Chronic infection of SJL/J mice with the Daniel's strain of Theiler's virus develop primary demyelination, viral persistence but minimal central nervous system (CNS)-type remyelination. In contrast, treatment of virus-infected mice with sera or immunoglobulin G (IgG) from mice immunized with homogenized spinal cord (SCH) emulsified in incomplete Freund's adjuvant promotes CNS remyelination. We measured levels of infectious virus, virus antigen and virus-specific antibody to determine if treatments which promote CNS remyelination are able to modulate infection. Levels of virus-specific antibody were higher in mice treated with SCH/IgG than control treatment groups and correlated positively with extent of remyelination. Although number of virus antigen-positive cells in spinal cord was less in mice treated with SCH/IgG than mice treated with phosphate buffered saline (PBS)/IgG, there was only a slight negative correlation with extent of remyelination by regression analysis. Titers of infectious virus isolated three to six months following infection were not different among treatment groups. Even though treatment of mice with SCH/IgG reduced number of virus antigen-positive cells and enhanced levels of virus-specific antibody, CNS remyelination can occur despite presence of infectious virus.     

Chronic experimental allergic encephalomyelitis in rhesus monkeys and its modification by treatment.

Rhesus monkeys were immunized with low doses of encephalitogenic mixture. Twenty two of 24 monkeys developed experimental allergic encephalomyelitis (EAE) lasting from 3 to 252 days. Fifteen of 22 monkeys developed chronic progressive EAE with remissions and relapses. In the early stages of EAE multiple perivascular foci of demyelination and lymphoid histiocytic infiltration were found within the central nervous system (CNS). In advanced disease these foci became confluent, developing foci of plaque type with demyelination and gliofibrosis. Eight affected monkeys received an emulsion of spinal cord with incomplete adjuvant; 6 of them showed a good therapeutic response. In the CNS of those monkeys 514 to 667 days later plaques of demyelination and gliofibrosis and minimal inflammatory lesions were detected. Two monkeys without clinical evidence of EAE had plaques of demyelination and gliofibrosis in the CNS 2 years after immunization. It is suggested that chronic EAE in monkeys may be considered an adequate model for multiple sclerosis (MS).     

Human mesenchymal stem cells infiltrate the spinal cord, reduce demyelination, and localize to white matter lesions in experimental autoimmune encephalomyelitis

Mesenchymal stem cells (MSCs) can abrogate the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), but whether this therapeutic effect occurs entirely through systemic immune modulation and whether CNS infiltration occurs after peripheral delivery are uncertain. We studied the clinical and neuropathologic effects of intravenously administered human MSCs (hMSCs) in C57BL/6 mice with EAE. Human MSCs significantly reduced the clinical disease severity, particularly in later disease. Large numbers of hMSCs migrated into gray and white matter at all levels of the spinal cord in both naive mice and mice with EAE. In the latter, hMSCs accumulated over time in demyelinated areas. There were 2 distinct morphological appearances of the hMSCs in the tissue, that is, rounded and less numerous process-bearing forms; very few expressed neural markers. The number of spinal cord white matter lesions and areas of white matter demyelination were reduced after hMSC treatment compared with control treatment. These findings show that central nervous system infiltration occurs after peripheral delivery of hMSCs, that they accumulate where there is myelin damage, and that they are associated with a reduced extent of demyelination. These data support a potential role for hMSCs in autologous cell therapy in multiple sclerosis.     

A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis viruses (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain produces acute polioencephalomyelitis in mice, whereas the DA strain produces demyelination with virus persistence in the spinal cord. A comparative study of GDVII and DA strains suggested that low host immune responses are responsible for the development of acute GDVII infection and that the persistence of infected macrophages plays a crucial role in the development of chronic white matter lesions in DA infection. All 78 mice infected with GDVII died or became moribund by day 13, while none of 54 mice infected with DA died. In the acute stage, the distribution of viral antigens in the central nervous system (CNS) tissue was similar in both GDVII and DA infections, although the virus titer was higher in GDVII infection. In DA infection, a substantial number of T cells were recruited to the CNS on day 6 when they were virtually absent in GDVII infection. The titer of neutralizing antibody was already high on day 6 in DA infection but was negligible in GDVII infection. Development of chronic paralytic disease from day 35 of the DA infection was accompanied by focal accumulation of viral antigen-positive macrophages in the spinal white matter. In addition, white matter lesions comparable to those in chronic DA infection were induced in the spinal cord within 7 days after intracerebral injection of DA-infected murine macrophages. Received: 26 June 1995 / Revised, accepted: 27 December 1995     

Effect of cyclosporin A, silica quartz dust, and protease inhibitors on virus-induced demyelination

Treatment with cyclosporin A, from the time of virus infection, suppressed inflammation and demyelination in the spinal cord of SJL/J or ASW(H-2s) mice persistently infected with Theiler's murine encephalomyelitis virus. Demyelination was not decreased if treatment was given after inflammation was established. The decrease was independent of serum titers of immunoglobulin G to purified viral antigen but did correlate with decreased proliferation of T lymphocytes to virus and myelin antigens. Silica quartz dust, a direct toxin of macrophages, suppressed demyelination and inflammation if begun at time of virus infection. No therapeutic effect was seen with inhibitors of plasminogen activators or other neutral proteases found primarily in macrophages.     

Intra-axonal virus in demyelinative lesions of experimental herpes simplex type 2 infection

Three-week-old mice which had been infected intracerebrally with herpes simplex virus type 2 (HSV-2) were examined electron-microscopically for the presence of intra-axonal virus in or near optic nerve and spinal cord demyelinative lesions. Acute lesions and their margins frequently contained a very small proportion of abnormal axons, and in a few of these mature virus particles, nucleocapsids, or other incomplete forms were found. A similar range of particle morphology was present in the cytoplasm of infected and degenerating glia. Axons containing similar particles were not identified in fibers in normal white matter surrounding demyelinative lesions. It is proposed that neuronal infection and axonal transport of virus may lead to foci of oligodendroglial infection, destruction and central nervous system (CNS) demyelination near to or remote from the cell bodies of infected neurons. In some instances, the topography of lesions could reflect a tract association. Anatomical features of nervous tissue could favor amplification of demyelination from a relatively minimal neuronal infection, with little evidence of tract degeneration. This hypothesis is consistent with the great predominance of demyelination relative to gray matter disease seen experimentally in non-fatal CNS infections with HSV-2. It would also explain the marked tendency for demyelinative lesions in at least certain CNS locations to be greatly elongated in the long axis of fiber tracts. This mechanism could be of importance in other animal models of virus-induced demyelination, and perhaps also in multiple sclerosis.     

Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.     

Abrogation of resistance to Theiler's virus-induced demyelination in C57BL mice by total body irradiation

Theiler's murine encephalitis virus (TMEV) produces an unusual biphasic disease in susceptible mice characterized by poliomyelitis with early viral replication in neurons, followed by chronic demyelination with viral antigen expression in spinal cord white matter. In addition, infectious virus persists in the central nervous system (CNS) throughout the chronic phase of disease. Previous studies have indicated an important role for major histocompatibility complex (MHC)-gene products in determining resistance/susceptibility to disease. In particular, certain class I gene products of the D region of the H-2 gene complex render mice of the C57BL lineage resistant to induction of demyelination. Intracerebral infection of B10.S(DS) mice results in demyelination in the spinal cord while infection of C57BL/10(Db) or B10.S(9R)(Dd) fails to produce white matter destruction. In this study we showed that immunosuppression with gamma irradiation renders normally resistant B10.S(9R) and C57BL/10 mice susceptible to TMEV-induced demyelination and allowed for increased viral replication. In addition, the majority of irradiated C57BL/10 mice infected with virus showed extensive areas of CNS remyelination by oligodendrocytes beginning at 63 days post-infection. In contrast, immunosuppression of normally susceptible B10.S mice resulted in acute disease and high mortality accompanied by overwhelming destruction of neurons. The study supports the hypothesis that MHC-conferred resistance in C57BL mice is associated with MHC D region products and indicate an important active role for the immune system early in infection in limiting vital infection during disease induction in nonimmunosuppressed mice.     

Induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in Lewis rats

Intracerebral inoculation of weanling Lewis rats with measles virus led to the development of subacute measles encephalomyelitis (SAME) 4-8 weeks after infection. The disease is characterized pathologically by an intense inflammatory infiltration within both the white and grey matter of the central nervous system (CNS) without apparent demyelination. Both during and after SAME splenic lymphocytes from these animals could be restimulated in vitro to proliferate in the presence of myelin base protein (MBP). MBP-specific class II MHC-restricted T cell lines were isolated from this cell population. They were shown to exhibit no cross-reactivity with measles virus and to induce experimental allergic encephalitis (EAE) in naive syngeneic recipients following adoptive transfer. The clinical and histopathological signs of this T cell-mediated disease were identical to that seen in classical T cell-mediated EAE. A humoral immune response to MBP was only detected in a limited number of those rats with SAME. These results indicate that autoimmune reactions to brain antigen can arise during measles virus infection which may contribute to the pathogenesis of measles virus-associated encephalomyelitis.