Brain Advance Access originally published online on July 28, 2004
Brain 2004 127(10):2201-2213; doi:10.1093/brain/awh260
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Brain Vol. 127 No. 10 © Guarantors of Brain 2004; all rights reserved
Clinicopathological study of a myelin oligodendrocyte glycoprotein-induced demyelinating disease in LEW.1AV1 rats
1 Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience and 2 Department of Paediatrics and Developmental Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
Correspondence to: Yoh Matsumoto, Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2–6 Fuchu, Tokyo 183-8526, Japan E-mail: matyoh{at}tmin.ac.jp
Received April 16, 2004. Revised June 3, 2004. Accepted June 4, 2004.
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Although multiple sclerosis is considered to be an autoimmune disease in the CNS, the immune responses that take place in the CNS and lymphoid organs remain to be elucidated. Here, we have successfully induced various subtypes of experimental autoimmune encephalitis (EAE) in LEW.1AV1 rats carrying RT1av1 on the Lewis background genes by immunization with recombinant rat myelin oligodendrocyte glycoprotein (MOG) in various solutions with adjuvants. The purpose of the present study was to analyse in more detail the clinical and immunopathological features of MOG-induced EAE in LEW.1AV1 rats. Immunization with high doses of soluble MOG with pertussis toxin induced acute, frequently fatal EAE, whereas medium doses of partially aggregated MOG without pertussis toxin produced relapsing and remitting EAE. Secondary progressive EAE was induced in some rats by immunization with the immunization protocol having an intermediate nature between the above two. The optic nerve (
60% of the immunized rats) and spinal cord (100%) were frequently involved and detectable both clinically and pathologically, while there was no lesion in the cerebrum. Histological examination revealed that, despite variety in the clinical subtypes, progression of the pathological processes was strikingly uniform, i.e. initial inflammation with minimal demyelination followed by predominant demyelination with minimal lymphocyte infiltration. These findings suggest that the lesion during the later stage is maintained by humoral factors. Taken together, this experimental system can serve as a model of neuromyelitis optica. Further analysis will provide useful information to elucidate the pathogenesis and to develop immunotherapy for neuromyelitis optica and multiple sclerosis.
Key Words: neuromyelitis optica; experimental autoimmune encephalomyelitis; myelin oligodendrocyte glycoprotein; LEW.1AV1 rat
Abbreviations: BS = brain stem; CP = chronic persistent; EAE = experimental autoimmune encephalomyelitis; H & E = haematoxylin and eosin; IgG = immunoglobulin G; MBP = myelin basic protein; MHC = major histocompatibility complex; MOG = myelin oligodendrocyte glycoprotein; NMO = neuromyelitis optica; PI = post-immunization; PT = pertussis toxin; RR = relapsing-remitting; SP = secondary progressive
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Introduction |
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The pathogenesis of neuroimmunological disorders including multiple sclerosis, neuromyelitis optica (NMO), acute disseminated encephalomyelitis (ADEM) and their variants is still poorly understood. One of the reasons for this is that there are many variants in terms of the clinical course (Lublin and Reingold, 1996
) and pathology (Lucchinetti et al., 2000). For example, there have been controversies with regard to the relationship between multiple sclerosis and NMO (or opticospinal multiple sclerosis). While some neurologists think that these two disease conditions belong to the different entities (Lucchinetti et al., 2002; Weinshenker, 2003), others believe that these exist at both ends of a wide disease spectrum (Kira, 2003). However, such discussions will not be fruitful without sufficient knowledge of the immune responses that take place in patients with the diseases. Analysis of animal models that mimic multiple sclerosis and NMO could provide clues to elucidate their pathomechanisms and to develop effective immunotherapy.
Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) has been extensively investigated for several reasons (Iglesias et al., 2001; von Budingen et al., 2001). First, immunization of rats and mice with MOG reproduce well the clinical course found in multiple sclerosis, i.e. relapsing and remitting (RR), primary progressive (PP) and secondary progressive (SP) multiple sclerosis (Lublin and Reingold, 1996; Storch et al., 1998; Tsunoda et al., 2000; Iglesias et al., 2001). Secondly and more importantly, anti-MOG antibodies are elevated in the sera of multiple sclerosis patients (Reindl et al., 1999) and bind specifically to disintegrating myelin around axons in multiple sclerosis lesions (Genain et al., 1999). Furthermore, it has been reported that patients with anti-MOG and anti-myelin basic protein (MBP) antibodies had relapses more often and earlier than patients without these antibodies (Berger et al., 2003). Together, these findings strongly suggest that MOG and anti-MOG antibodies play an essential role in the development of multiple sclerosis. In this regard, analysis of MOG-induced EAE could provide useful information to elucidate the pathogenesis of multiple sclerosis. However, due to varieties in the clinical course and neurological deficits, clinical and pathological features of the disease have not been well described.
The purpose of the present study was to delineate the clinicopathogy of MOG-induced EAE in LEW.1AV1 rats and to establish the most appropriate immunization protocol to induce a particular subtype of EAE. Through this investigation we found that, despite variety in the clinical subtypes, progression of the pathological processes was strikingly uniform, i.e. initial inflammation with minimal demyelination followed by predominant demyelination with minimal lymphocyte infiltration. We also found that the histopathological characteristics are similar to those of NMO (Lucchinetti et al., 2002). Further analysis will provide useful information to elucidate the pathogenesis and to develop the immunotherapy for NMO and multiple sclerosis.
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Animals
LEW.1AV1 rats were kindly provided by Dr R. Gold, Department of Neurology, Würzburg University, Germany, and maintained in our animal facility. Lewis (LEW) and DA rats were purchased from Japan SLC Inc. (Shizuoka, Japan). All rats used were 8–12 weeks old.
Reagents
Recombinant rat MOG was prepared as follows. The gene coding the extracellular domain (amino acid 1–125) of MOG was amplified using primers specific for the corresponding MOG sequence. The PCR products were then digested with SphI and HindIII and sub-cloned into pQE30 (QIAGEN, Tokyo, Japan) for large-scale preparation. The sequence of the construct was confirmed by sequencing. Recombinant MOG produced in transformed E.coli were isolated under denaturing conditions and purified using Ni-NTA Agarose (QIAGEN). Then, purified MOG was diluted and refolded in one of the following solutions: (i) 25 mM glycine-HCl (pH 3.0); (ii) phosphate-buffered saline (PBS) containing 1 M L-arginine, 2 mM glutathione (reduced form), 0.2 mM glutathione (oxidized form); or (iii) PBS, pH7.4. Solutions (i) and (ii) produced the soluble form and solution (iii) produced the insoluble form. In some experiments, PBS containing 0.6 M and 0.3 M arginine was used as solution. As a final step, recombinant protein was incubated with Detoxi-Gel (PIERCE, Funakoshi, Tokyo, Japan) overnight to remove endotoxins. The protein obtained contained <10 EU endotoxins per mg protein as determined with a Toxinometer ET-2000 (Wako, Tokyo, Japan).
Overlapping 18–23 mer peptides were prepared using a peptide synthesizer, PSSM-8 (Shimadzu Biotech, Kyoto, Japan). The purity of each peptide was determined by high pressure liquid chromatography (HPLC) (Waters 486, Waters 600 and Bondasphere C18 column, Waters, Tokyo, Japan) and all peptides were >90% pure.
The myelin fraction was extracted from the bovine spinal cord as described previously but with a few modifications (Agrawal et al., 1972; Casado et al., 1988). Briefly, the spinal cord tissue was homogenized and washed in 0.32 M sucrose before the suspension was overlaid on 0.84 M sucrose. After centrifugation, the interface was collected, washed with Mili Q (Millipore, Tokyo, Japan) water and homogenized. Using fractions other than the interface, this process was repeated and the interface was collected. These preparations were lyophilized and kept at –80°C until use. Western blot analysis revealed that the purified myelin preparation contained MBP, proteolipid protein and MOG (data not shown). Guinea pig, bovine and rat MBP was prepared as described previously (Deibler et al., 1972).
EAE induction and clinical evaluation
LEW.1AV1 rats were immunized in the tail base with recombinant MOG in the indicated solution and at the indicated dose in complete Freud's adjuvant (CFA) containing the indicated amount M. tuberlosis along with or without intraperitoneal injection of pertussis toxin (PT) (2 µg). Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail (partial paresis: 0.5; complete paresis: 1.0). Thus, the clinical score of complete paralysis of four limbs plus tail or the moribund conditions was 5. The majority of rats reaching a score of 5 died or were killed under ether anaesthesia for histological examination. Disease remission was defined as an improvement by 2 points, which was maintained for at least 2 days. Relapse was defined as an increase in clinical signs of at least 2 points that lasted for 2 days as described previously (Weissert et al., 1998). In addition to this paralysis score, the optic and brain stem signs were recoded separately. The presence or absence of the optic sign (either visual disturbance or blindness) was examined as follows. First, examiners shook a hand in front of rats' eyes. Rats with normal visual acuity stared at the hand. When there was moderate or severe visual disturbance, the rats did not stare at the examiner. Rats that did not exhibit escape activity when a sharp-pointed object was held in front of each eye were judged to be blind. The presence of rotation of rats in one direction was defined as the brain stem (BS) sign in this study. Some rats showed ataxia, but this was not included in the BS signs because it was occasionally difficult to estimate due to paraparesis of multiple limbs.
Histological and immunohistochemical examination
The optic nerve and the cervical, thoracic and lumbar spinal cord were examined routinely. The cerebrum, brain stem and cerebellum were also examined in some cases. The tissues were fixed in 4% paraformaldehyde and processed for paraffin embedding. Six micrometre sections were cut and stained with haematoxylin and eosin (H & E) and with Kruever and Barrera's (K–B) method. Inflammatory lesions were graded using sections stained with H & E and W3/13 for T cells into four categories (grade 1: leptomeningeal and adjacent subpial cell infiltration; grade 2: mild perivascular cuffing; grade 3: extensive perivascular cuffing; grade 4: extensive perivascular cuffing and severe parenchymal cell infiltration). Demyelinating lesions were graded using sections stained with the K–B method and ED1 for macrophages into five categories (grade 1: trace of perivascular or subpial demyelination; grade 2: focal demyelination; grade 3: demyelination involving a quarter of tissues examined, i.e. the spinal tract, brain stem, cerebellar white matter or optic tract; grade 4: massive confluent demyelination involving half of the tissue; grade 5: extensive demyelination involving the entire tissues) according to Storch et al. (1998) with a few modifications.
Single immunoperoxidase staining was performed as described previously (Matsumoto and Fujiwara, 1987; Ohmori et al., 1992). Briefly, paraffin-embedded sections were deparaffinized and rehydrated. After blocking the endogenous peroxidase activity with methanol containing 0.3% hydrogen peroxide, sections were incubated with mAb W3/13 (Dainippon Pharm, Osaka, Japan) for T cell-staining, ED1 (purified from the hybridoma supernatant) for macrophages, anti-MBP (DAKO Japan, Kyoto, Japan), anti-glial fibrillary acidic protein (GFAP) (DAKO Japan) or anti-neurofilament (Nichirei, Tokyo, Japan) antibodies. After washing, sections were incubated with biotinylated anti-mouse or rabbit immunoglobulin G (IgG) (Vector, Burlingame, CA, USA) followed by horseradish peroxidase (HRP)-labeled VECTSTAIN Elite ABC Kit (Vector). IgG deposition was detected using biotinylated anti-rat IgG (Vector). HRP-binding sites were detected in 0.005% diaminobenzidine and 0.01% hydrogen peroxide. To confirm the specificity of the staining, the primary antibodies were omitted or replaced with normal mouse IgG. The controls did not show any specific staining.
Enzyme-linked immunosorbent assay (ELISA) and western blot analysis
The level of anti-MOG and anti-MBP antibodies was measured by the standard ELISA test. Recombinant rat MOG or purified rat MBP (10 µg/ml) was coated onto microtitre plates and serially diluted sera from normal and immunized animals were applied. After washing, appropriately diluted horseradish-conjugated anti-rat IgG, IgG1 or IgG2a was applied. The reaction products were then visualized after incubation with the substrate. The absorbance was read at 450 nm.
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Results |
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We immunized both DA and LEW.1AV1 rats with MOG, but finally decided to focus on EAE induced in LEW.1AV1 rats because some DA rats developed severe arthritis, making it difficult to estimate the degree of paralysis. Furthermore, the incidence of the disease seemed to be slightly lower in DA than in LEW.1AV1 rats. A total number of 93 LEW.1AV1 rats were immunized with recombinant rat MOG using various protocols. Due to initial inappropriate procedures in preparing the antigen and immunization, 24 rats were excluded from the analysis.
Clinical course of MOG EAE in LEW.1AVI rats and its relationship to the immunization protocol
As reported previously (Storch et al., 1998; Tsunoda et al., 2000), MOG-induced EAE in mice and rats showed a variety of clinical courses depending on the differences in the solubility of MOG, the immunization protocol and the strain used. LEW.1AV1 rats used in the present study also showed acute RR, SP, CP and monophasic EAE. The representative clinical courses are shown in Fig. 1 and the results are summarized in Table 1. We categorized the clinical course into six groups. In ‘early-onset acute’ EAE, rats developed the clinical signs on days 9–13 post-immunization (PI) and developed moribund conditions or died within 1–6 days (Fig. 1A). ‘Late-onset acute’ EAE showed the essentially the same course, except that rats developed EAE on days 20–27 PI (Fig. 1B). RR and SP types of EAE were almost the same as those seen in multiple sclerosis (Figs. 1C and 1D). CP-EAE was rare in our series (two out of 69), but a definite subtype, in which paralysis of the limbs persisted for more than two weeks without amelioration or exacerbation of the disease (Fig. 1E). Monophasic EAE was also found rarely, as shown in Fig. 1F.
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We next examined the relationship between the subtype of EAE and the immunization protocol. A previous study suggested that the soluble form of MOG tends to induce acute EAE, whereas insoluble MOG induces the chronic form (Storch et al., 1998
). However, precise information was not available. Here, we performed the overall study and confirmed that soluble MOG in the glycine buffer pH3 [solution (i)], induced acute EAE (Table 1, rows 1 and 2). In our series, it was shown that the use of PT heavily influenced the induction of the acute form because MOG dissolved in the solution containing 1 M arginine with PT (Table 1, rows 3–5 and 7–9), but not without PT (rows 10–14), induced acute EAE. SP-EAE was mainly induced by immunization with high to moderate doses of MOG in 1 M Arg solution with PT. In contrast, moderate to low dose of the same antigen solution without PT generally induced RR-EAE (Table 1, rows 10–14). In our study, slightly aggregated MOG induced SP- or RR-EAE (rows 17 and 18), but the completely aggregated form did not induce clinical EAE (row 18). Although the number of rats used for each immunization was not large, the results obtained were judged reliable. This was because similar protocols (e.g. 50 µg MOG/2.5CFA and 100 µg MOG/2.5CFA) produced similar subtypes of EAE (Table 1, rows 10–13). The findings presented in Table 1 clearly indicate that the immunization protocol should be chosen carefully for the induction of a particular subtype of EAE. During the course of the experiments, we noticed that in addition to the differences in clinical course, there were subtypes of the disease with regard to paralysis pattern (Fig. 2). One is the hind-limb-dominant type as shown in Fig. 2B. In this case, the rat showed SP-EAE (Fig. 2A) and the paralysis score in the hind limbs was much higher than that in the fore limbs (Fig. 2B). The difference became much greater by the later stage of the disease. In contrast, in a rat with RR-EAE (Fig. 2C), the paralysis score of the hind limbs was almost the same as that of the fore limbs throughout the observation period (Fig. 2D) (the quadroparesis type). A few rats (six out of 48 in Table 2) showed a combination of the above two types (data not shown). In addition, it was difficult to determine the paralysis subtype in some rats due to the acute fatal clinical course. These paralysis types were found to be closely associated with the presence or absence of the optic sign (see below).
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Optic and BS signs and the EAE subtype
Although previous investigators noticed the presence of optic and BS signs during the course of MOG-induced EAE, it remained unknown when and in what kind of EAE subtype these signs appeared. We have addressed this question by examining each rat and recording the presence or absence of the signs daily. Representative results are illustrated in Fig. 3 and all the results are summarized in Table 2. As shown in Fig. 2 and Table 3, optic and BS signs appeared for all the subtypes—acute EAE (Fig. 2A), SP-EAE (Fig. 2B and C) and RR-EAE (Fig. 2D). Interestingly, these signs were present only during the active stage of the disease as shown in Fig. 3B and C. The rat featured in Fig. 3C showed only the optic sign.
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We then examined the relationship between optic and brain stem involvement and the EAE subtype (Table 2). With regard to the clinical course subtype, the optic signs were observed significantly less frequently in the acute type compared with other subtypes. The paralysis subtypes were found to be closely associated with the optic involvement. While
30% of rats with lower limb dominant paralysis showed optic signs and pathology (representative figures are shown in Fig. 4), all rats with quadroparesis and combined types showed optic signs, and 75–80% were confirmed pathologically. The brain stem involvement showed essentially the same tendency as optic involvement, but the inflammation and/or demyelination was found in all rats examined (Table 2).
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Pathological features were relatively uniform despite the variety of clinical courses
We routinely examined the cervical, thoracic and lumbar spinal cords and optic nerves. In addition, the cerebrum, cerebellum and brain stem were examined in some cases. Our pathological findings are summarized in Table 3 (in order of the day PI at sampling) and Fig. 5. Strikingly, progression of pathological processes of the CNS lesion was rather uniform regardless of the difference in the clinical course. At the early stage (day 14 PI), there was severe lymphocyte infiltration (Fig. 5A and C), whereas demyelination was minimal (Fig. 5D). Granulocyte infiltration was found in some rats at this stage (Fig. 5B). From day 17 to day 27 PI, there was co-localization of inflammation and demyelination (Table 3). From day 31 to day 77 PI (the end point of examination), marked demyelination with minimal inflammation became the main pathological feature. The representative pathology around day 40 PI is shown in Fig. 4E–H. The main feature of the lesion was demyelination as shown in Fig. 5E, whereas axons were relatively preserved (Fig. 5F). In accordance with this finding, a large number of macrophages (Fig. 5C) and very few T cells (Fig. 5H) infiltrated the demyelinating lesion. Unlike infiltrating macrophages appearing during the early stage, the majority at this stage were ‘foamy’ macrophages, suggesting that they phagocytized debris formed by demyelination (not shown). However, in the most severe lesions, there were very few remaining axons with the feature of necrosis. Infiltrating macrophages gradually decreased in number, and astrocytosis revealed by immunohistochemical staining for GFAP became predominant around day 40 PI (data not shown). At the latest stage (days 63 and 77 PI), demyelinating lesions in the spinal cord improved slightly, but most of the rats showed severe optic nerve involvement (Table 3). There was no inflammatory and/or demyelinating lesions in the cerebrum of all the rats examined (Table 3).
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Anti-MOG, but not anti-MBP, antibodies are elevated throughout the course of EAE
We finally evaluated B cell activities in MOG-induced EAE in LEW.1AV1 rats. At various time points, sera were taken and the titres of anti-MOG and anti-MBP antibodies were measured by ELISA. As shown in Fig. 6A, several interesting findings were obtained. First, the anti-MOG titres during the early and intermediate stages (between days 14 and 21) were relatively low compared with the later stage (between days 32 and 77). Secondly and more importantly, the titres at the later stage did not correlate with the clinical severity. The titre of sera taken from rats during remission (numbers 4630, 4631, 4633 and 4695) was not significantly different from that from rats with the active disease (numbers 4721, 4338 and 4339) (see also Table 3). Finally, the anti-MBP antibody titre remained low thought the disease course, demonstrating that intermolecular epitope spreading at the B cell level did not occur in this type of MOG-induced EAE.
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Immunohistochemical staining for IgG revealed that IgG deposition was limited on the luminal surface of the blood vessels in the intact spinal cord. However, diffuse staining of the parenchyma was detected in the spinal cord with inflammation and/or demyelination throughout the observation period (data not shown). Interestingly, such parenchymal staining was more marked in the less demyelinated area (Fig. 6B and, arrows) than in the severely demyelinated area (arrowheads).
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Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
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Multiple sclerosis is thought to be an autoimmune disease characterized by the presence of multiple demyelinating lesions in the CNS (Bar-Or et al., 1999
; Noseworthy et al., 2000). Furthermore, the variation in the clinical course is one of the characteristic features of this disease. To elucidate the pathological mechanisms of multiple sclerosis, it is essential to establish a good disease model that fulfils these features. In the present study, we immunized LEW.1AV1 rats with MOG and successfully produced EAE resembling multiple sclerosis and NMO in terms of the presence of multiple demyelinating lesions and RR or SP clinical course. Using this animal model, we performed detailed clinicopathological studies to clarify the relationship among the subtype of the clinical course, the immunization protocol, the presence or absence of optic involvement and the histological nature of the lesion.
In the present study, we have demonstrated for the first time that the clinical subtype of EAE is closely associated with the immunization protocol. Immunization with relatively high doses of soluble MOG in glycine-HCl buffer, pH3, or in 1 M arginine buffer with PT administration induced acute, frequently fatal EAE, whereas immunization with moderate doses without PT induced RR or CP EAE (Table 1). The SP form was produced by either of the above two immunization protocols, suggesting an intermediate nature between acute and RR-EAE. Despite variety in the clinical course, the progression of pathological processes in the spinal cord was surprisingly uniform. During the early stage (day 14 PI), lymphocyte—and sometimes granulocyte—infiltration was the main feature in the lesion as reported previously (Storch et al., 1998; Stefferl et al., 1999). Demyelination was minimal or absent in most cases at this stage. On the other hand, the main pathology of the later stage (day 31 PI and thereafter) was demyelination with extensive macrophage, but very mild lymphocyte, infiltration. Between the two stages (from day 17 to day 27 PI), there was co-localization of inflammation and demyelination. Interestingly, late-onset acute EAE examined on days 32 and 34 PI mainly showed demyelination, implying that this EAE subtype should be interpreted as chronic EAE that is latent during the early inflammatory stage. Optic nerve involvement, one of the characteristic features of this type of EAE, was almost the same as that found in the spinal cord, i.e. early inflammation followed by later demyelination.
A characteristic finding was that a single immunization with the same antigen (i.e. MOG) produced various clinical subtypes of EAE. In particular, we were interested in the development of the relapsing and remitting form and examined its association with our published and unpublished data. Table 4 summarizes the clinical subtypes of EAE in LEW, DA and LEW.1AV1 rats induced by immunization with MOG, MBP or purified myelin. RR-EAE or SP-EAE was induced only by immunization with MOG in rats carrying RT1av1. All other combinations resulted in monophasic or biphasic EAE. It should be noted that immunization of LEW.1AV1 rats with MBP induced acute monophasic EAE. In addition to RT1av1, RT1n and RT1u have been reported to be associated with chronic EAE (Stefferl et al., 1999; Weissert et al., 1998, 2001). Thus, the clinical subtype of EAE is highly regulated by both immunizing antigens and major histocompatibility complex (MHC) haplotype. The previous study suggested that optic nerve involvement is dependent on non-MHC genes (Storch et al., 1998). However, this was not the case in our study because both LEW.1AV1 and DA rats, which carry the same MHC gene with different background genes, developed optic and spinal cord lesions.
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Little is known about what kind of factors are involved in the relapse in multiple sclerosis and EAE. Using B cell-deficient mice, it was initially reported that B cells and antibodies are not necessary for the development of inflammation and demyelination (Hjelmstrom et al., 1998
). Later studies demonstrated that MOG-specific antibodies are critical (Lyons et al., 1999, 2002) or at least contribute to the severity of MOG-induced EAE (Svensson et al., 2002). In this study, we examined the titre of anti-MOG antibodies in the sera and the location of immunoglobulins in the spinal cord. Immunohistochemical localization revealed that immunoglobulins were mainly deposited in less damaged areas rather than in severe necrotic lesions, suggesting that immunoglobulins are involved in ongoing lesion formation. However, the titre of anti-MOG antibodies was always high and was not associated with relapse or remission. Previously suggested substances including compliments (Lucchinetti et al., 2002) and chemokines (Jee et al., 2002) should be examined in further studies.
The optic nerve and spinal cord involvement in this MOG-induced EAE suggest that this experimental system could be a model for NMO. There have been several controversies regarding the criteria for diagnosis (Baudoin et al., 1998; Wingerchuk et al., 1999); according to Wingerchuk et al. (1999), absolute criteria for NMO include ‘no evidence of clinical disease outside the optic nerve or spinal cord’, while a review article by Baudouin et al. (1998) includes cases with brain stem lesions. It is also unclear whether NMO is identical to opticospinal multiple sclerosis, which is frequently found in Japan (Kira et al., 1996; Yamasaki et al., 1999; Misu et al., 2002). Such discrepancies appear to be mainly based on the paucity of information regarding the pathological mechanisms of NMO. Recently, the precise pathological features of NMO were reported and strongly suggested that the humoral mechanism is involved in lesion formation (Lucchinetti et al., 2002). As well as previous reports (Storch et al., 1998, 2002; Stefferl et al., 1999; Tsunoda et al., 2000), we have shown in our system that inflammation characterized by T and granulocyte infiltration was limited only to the early stage. Furthermore, we have demonstrated that demyelination with extensive macrophage and minimal T cell infiltration is the main feature of lesions in the optic nerve and spinal cord in all subtypes of MOG-induced EAE. These findings suggest that demyelinating lesions in MOG-induced EAE are produced by humoral factors including anti-MOG antibodies. Information obtained by analysis of MOG-induced EAE could be helpful to resolve the discrepancies mentioned above.
In summary, we have successfully induced various types of EAE in LEW.1AV1 rats by immunization with recombinant rat MOG in various solutions with adjuvants. Histopathological examination revealed that optic nerve and spinal cord lesions mimic those of NMO. Further analysis will provide useful information to help elucidate the pathogenesis of NMO and multiple sclerosis.
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Acknowledgements |
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We wish to thank: Dr K. Fujihara, Department of Neurology, Tohoku University, Sendai, Japan, for critical reading of this manuscript; Dr R. Gold, Department of Neurology, Würzburg University, Würzburg, Germany for providing LEW.1AV1 rats; and Y. Kawazoe for technical assistance. H.S. is grateful to Professor S. Mizutani, Department of Paediatrics and Developmental Biology, Graduate School, Tokyo Medical and Dental University, Tokyo for his continuous support. This study was supported in part by grants-in aid from the Ministry of Education, Japan.
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