Histopathological Characterization of Magnetic Resonance Imaging-Detectable Brain White Matter Lesions in a Primate Model of Multiple Sclerosis
1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65606-4
ISSN1525-2191
AutoresBert A. ‘t Hart, Jan Bauer, H Müller, Bert P.C. Melchers, Klaas Nicolay, Herbert Brok, Ronald E. Bontrop, Hans Lassmann, Luca Massacesi,
Tópico(s)Herpesvirus Infections and Treatments
ResumoExperimental autoimmune encephalomyelitis in the common marmoset, a nonhuman primate species (Callithrix jacchus), is a new model for multiple sclerosis. Given the close immunological relationship between marmosets and humans, it is an attractive model for investigating immunopathological pathways relevant to multiple sclerosis and to evaluate new treatments for the disease. Unlike in the originally documented model, experimental autoimmune encephalomyelitis induced without the use of Bordetella pertussis led to a chronic disease of moderate severity. The clinical course of experimental autoimmune encephalomyelitis in the present model was mainly chronic and progressive, but periods of incomplete remission did occur. At the chronic stage of the disease, actively demyelinating lesions were found together with inactive demyelinated and remyelinated (shadow) plaques. Before immunization and during clinically active experimental autoimmune encephalomyelitis, T1- and T2-weighted magnetic resonance brain images were obtained. Correlation of the data from the magnetic resonance images and the neuropathology analysis revealed that the hyperintense regions in T2-weighted images represented both active and inactive remyelinating lesions. Quantification showed that the number of lesions in T2-weighted magnetic resonance images equalled those found by pathological examination, and thus T2-weighted magnetic resonance imaging can be used to discern the total lesion load. Extravasation of gadolinium-diethylenetriamine-penta-acetic acid (triple dose) was found only in lesions, which by histopathology were shown to be engaged in the process of active demyelination. Experimental autoimmune encephalomyelitis in the common marmoset, a nonhuman primate species (Callithrix jacchus), is a new model for multiple sclerosis. Given the close immunological relationship between marmosets and humans, it is an attractive model for investigating immunopathological pathways relevant to multiple sclerosis and to evaluate new treatments for the disease. Unlike in the originally documented model, experimental autoimmune encephalomyelitis induced without the use of Bordetella pertussis led to a chronic disease of moderate severity. The clinical course of experimental autoimmune encephalomyelitis in the present model was mainly chronic and progressive, but periods of incomplete remission did occur. At the chronic stage of the disease, actively demyelinating lesions were found together with inactive demyelinated and remyelinated (shadow) plaques. Before immunization and during clinically active experimental autoimmune encephalomyelitis, T1- and T2-weighted magnetic resonance brain images were obtained. Correlation of the data from the magnetic resonance images and the neuropathology analysis revealed that the hyperintense regions in T2-weighted images represented both active and inactive remyelinating lesions. Quantification showed that the number of lesions in T2-weighted magnetic resonance images equalled those found by pathological examination, and thus T2-weighted magnetic resonance imaging can be used to discern the total lesion load. Extravasation of gadolinium-diethylenetriamine-penta-acetic acid (triple dose) was found only in lesions, which by histopathology were shown to be engaged in the process of active demyelination. Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS). Although there is a large variability among individual patients, MS typically follows a chronic-progressive or relapsing-remitting course. In the latter case, disease episodes with neurological abnormalities (relapse/exacerbation) alternate with periods of partial or complete recovery (remission). The typical histopathological hallmark of MS is the presence of inflammatory, demyelinating lesions within the CNS. Most of these lesions contain mononuclear cells such as lymphocytes and macrophages, whereas granulocytes are not typically found. Demyelinated lesions are found throughout the CNS in a perivascular or periventricular location.1Adams CWM The general pathology of multiple sclerosis: morphological and chemical aspects of the lesions.in: Hallpike JF Adams CWM Toutelotte WW Multiple Sclerosis: Diagnosis and Management. Chapman and Hall, London1983: 83-97Google Scholar Magnetic resonance imaging (MRI) is presently the diagnostic imaging modality of choice in MS.2Thorpe JW Miller DH MRI: its implication and impact.Int Mult Scler J. 1994; 1: 7-15Google Scholar, 3Young IR Hall AS Pallis CA Nuclear magnetic resonance imaging of the brain in multiple sclerosis.Lancet. 1981; 2: 1063-1066Abstract PubMed Scopus (399) Google Scholar, 4McFarland HF Clinical trials in multiple sclerosis.in: Porter RJ Schoenberg BS Controlled Clinical Trials in Neurological Disease. Kluwer Academic, Boston1990: 321-341Crossref Google Scholar T2-weighted sequences are especially sensitive to structural alterations of CNS tissue. All lesion stages, from early inflammation to late chronic stages, are visualized as hyperintense regions. Therefore, T2-weighted MRI is usually applied to assess the spatial distribution of plaques and to determine the total lesion load.5Harris JO Frank JA Patronas NJ McFarlin DE McFarland HF Serial Gd-enhanced magnetic resonance imaging scans in patients with early, relapsing-remitting multiple sclerosis: implications for clinical trials and natural history.Ann Neurol. 1991; 29: 548-555Crossref PubMed Scopus (289) Google Scholar Active lesions can be distinguished from inactive ones by increased permeability of the blood-brain barrier (BBB) for intravenously injected gadolinium. This results in local enhancement of the signal intensity of T1-weighted images. Lesions with contrast-induced regional signal enhancement correlate well with disease activity both in MS6Thompson AJ Kermode AG Wicks D MacManus DG Kendall BE Kingsley DPE McDonald WI Major differences in the dynamics of primary and secondary progressive multiple sclerosis.Ann Neurol. 1991; 29: 53-62Crossref PubMed Scopus (433) Google Scholar, 7Barkhof F Scheltens P Frequin STFM Nauta JJP Tas MW Valk J Hommes OR Relapsing-remitting multiple sclerosis: sequential enhanced MR imaging vs. clinical findings in determining disease activity.Am J Roentgenol. 1992; 159: 1041-1047Crossref PubMed Scopus (143) Google Scholar, 8Van Walderveen MAA Barkhof F Hommes OR Polman CH Tobi H Frequin STFM Valk J Correlating MRI, and clinical disease activity in multiple sclerosis: relevance of hypointense lesions on short-TR/short TE (T1-weighed) spin-echo images.Neurology. 1995; 45: 1684-1690Crossref PubMed Scopus (307) Google Scholar, 9Hawkins CP Munro PMG MacKenzie F Kesselring J Tofts PS du Boulay EPGH Landon DN McDonald WI Duration and selectivity of blood-brain-barrier breakdown in chronic relapsing experimental allergic encephalomyelitis studied by Gd-DTPA and protein markers.Brain. 1990; 13: 365-378Crossref Scopus (206) Google Scholar and in the experimental model experimental autoimmune encephalomyelitis (EAE).10Morrissey SP Stodal H Zettl U Simonis C Jung S Kiefer R Lassmann H Hartung HP Haase A Toyka KV In vivo MRI, and its histological correlates in acute adoptive transfer experimental allergic encephalomyelitis: quantitation of inflammation and oedema.Brain. 1996; 119: 239-248Crossref PubMed Scopus (88) Google Scholar However, limited information is available on pathological characteristics of MRI-detectable lesions. The objectives of this study were to investigate the histopathological basis of MRI-detectable lesions in the EAE model in marmoset monkeys. The obvious advantage of any EAE model is that MR images can be recorded before and at defined time points after disease onset. When regions of interest appear in MRI, animals can be sacrificed immediately after in vivo MRI analysis to characterize regions of interest by immunohistopathological techniques. The EAE model in marmosets is of particular interest in view of the close similarity between the human and marmoset immune systems.11Quint DJ Buckham SP Bolton EJ Solari R Champion BR Zanders ED Immunoregulation in the common marmoset, Callithrix jacchus: functional properties of T and B lymphocytes and their response to human interleukins 2 and 4.Immunology. 1990; 69: 616-621PubMed Google Scholar, 12Neubert R Nogueira AC Neubert D Thalidomide derivatives and the immune system. 1. Changes in the pattern of integrin receptors and other surface markers on T lymphocyte subpopulations of marmoset blood.Arch Toxicol. 1993; 67: 1-17Crossref PubMed Scopus (56) Google Scholar, 13Neubert R Foerster M Nogueira AC Helge H Cross-reactivity of antihuman monoclonal antibodies with cell surface receptors in the common marmoset.Life Sci. 1995; 15: 317-324Crossref Scopus (38) Google Scholar, 14Bontrop RE Otting N Slierendregt BL Lanchbury JS Evolution of major histocompatibility complex polymorphisms and T cell receptor diversity in primates.Immunol Rev. 1995; 143: 33-62Crossref PubMed Scopus (125) Google Scholar, 15Uccelli A Oksenberg JR Jeong M Genain CP Rombos T Jaeger EEM Giunti D Lanchbury JS Hauser SL Characterization of the TCRB chain repertoire in the New World monkey Callithrix jacchus.J Immunol. 1997; 158: 1201-1207PubMed Google Scholar The present study shows that the development of lesions in the marmoset brain white matter can be very well visualized with MRI techniques similar to those used for diagnosis of MS. Moreover, for histopathological characterization of MRI-detectable lesions, the same reagents could be used as those applied for the stage characterization of lesions in the CNS of MS patients. Our present pathological investigation reveals the presence of both active and inactive, as well as remyelinating, plaques in the CNS of EAE-affected marmosets that strongly resemble the plaques in the CNS of MS patients. Correlation of MRI and pathology revealed that the number of lesions in tissue sections equalled those found by T2-weighted MRI. Furthermore, none of the pathologically defined inactive lesions displayed gadolinium enhancement. Nearly all lesions that did show gadolinium enhancement of the T1-weighted nuclear magnetic resonance (NMR) signal could be classified as early active. In summary, we demonstrate here that the present EAE model resembles MS not only in its clinical expression, but also in radiological and histopathological aspects of the lesions that are found in the CNS white matter. Eleven marmosets (Callithrix jacchus) were used for this study. The sex and birth dates (month/year) of the monkeys were: EB: male, 4/1992; EC: male, 8/1990; ED: female, 6/1992; EE: male, 4/1992; EG: female, 8/1992; EH: male, 6/1992; EI: male, 8/1992; EJ: male, 4/1993; EK: male, 4/1993; EL: male, 4/1993; and GY: male, 7/1994. The body weight of the monkeys at the start of the experiment ranged between 295 and 320 g. The monkeys were bred and raised at the Biomedical Primate Research Centre (Rijswijk, The Netherlands). Marmosets of either sex can be used, because there is no sex-linked difference in susceptibility to EAE. During the experiments, the monkeys were individually housed in spacious cages with padded shelter on the bottom. The daily diet consisted of commercial food pellets for nonhuman primates (Hope Farms, Woerden, The Netherlands), supplemented with rice and fresh fruit. Drinking water was providedad libitum. Myelin was isolated as described16Van Noort JM El Ouagmir M Boon J Van Seghel AC Fractionation of central nervous system myelin proteins by reversed phase high performance liquid chromatography.J Chromatogr. 1994; 653: 155-161Crossref Scopus (14) Google Scholar from human brain white matter, which was kindly provided by Dr. Rivka Ravid of the Dutch Brain Bank (Amsterdam, The Netherlands). The myelin concentration in the stock solution was 30 mg/ml on a dry-weight basis and 1.3 mg/ml protein on a protein basis as measured according to Bradford.17Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar In a first group of five monkeys (EB, EC, ED, EE, and EG), EAE was induced essentially as described earlier.18Massacesi L Genain CP Lee-Parritz D Letvin NL Canfield D Hauser SL Actively and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis.Ann Neurol. 1995; 37: 519-530Crossref PubMed Scopus (123) Google Scholar, 19Genain CP Lee-Parritz D Nguyen MH Massacesi L Joshi N Ferrante R Hoffman K Moseley M Letvin NL Hauser SL In healthy primates, circulating autoreactive T cells mediate autoimmune disease.J Clin Invest. 1994; 94: 1339-1345Crossref PubMed Scopus (91) Google Scholar The myelin stock solution was emulsified in an equal volume of enriched complete adjuvant. Enriched complete adjuvant was prepared by mixing incomplete adjuvant (DIFCO Laboratories, Detroit, MI) with 6 mg/ml desiccated mycobacteria (Mycobacterium tuberculosis, H37A, DIFCO) followed by brief sonication. Under ketamine anesthesia, each monkey was injected intradermally on the back with 600 μl of emulsion divided over four spots, two in the inguinal and two in the axillary region. In addition, 1 ml phosphate-buffered saline (pH 7.4) containing 1010 heat-inactivated B. pertussisparticles was injected immediately after immunization and 48 hours later. In a second group of six monkeys, the original immunization protocol was modified in two respects. First, the Mycobacteriumconcentration in the antigen-adjuvant emulsion was reduced from 1.5 to 0.5 mg/ml to diminish the severity of ulceration around the injection sites. Secondly, B. pertussis administration as second adjuvant was omitted to obtain a milder EAE in which patterns of EAE reactivity can be associated with the presence of certain mhc alleles and cytokine profiles.20Van Lambalgen R Jonker M Experimental allergic encephalomyelitis in rhesus monkeys: 1. Immunological parameters in EAE-resistant and susceptible rhesus monkeys.Clin Exp Immunol. 1985; 68: 100-107Google Scholar, 21Slierendregt BL Hall M 't Hart BA Otting N Anholts J Verduyn W Lanchbury JS Bontrop RE Identification of an Mhc-DPB1 allele involved in susceptibility to experimental autoimmune encephalomyelitis in rhesus macaques.Int Immunol. 1995; 7: 1671-1679Crossref PubMed Scopus (44) Google Scholar The clinical course of EAE was recorded daily by a trained observer using semiquantitative scoring: 0 = no clinical signs; 0.5 = apathy, loss of appetite, altered walking pattern without ataxia; 1 = lethargy and/or anorexia; 2 = ataxia; 2.5 = para- or monoparesis and/or sensory loss and/or brain stem syndrome; 3 = para- or hemiplegia; 4 = quadriplegia; 5 = spontaneous death attributable to EAE. The highest per-day scores were averaged over 1 week. Moreover, each monkey was weighed at least three times per week to obtain a more objective score of the clinical wellbeing. MRI was performed at the Bijvoet Center of Utrecht University, The Netherlands. For each monkey, T2- and T1-weighted magnetic resonance (MR) images were recorded, the latter also with contrast enhancement after intravenous injection of gadolinium-diethylenetriamine-penta-acetic acid (DTPA) (triple dose). The time points for performing MRI after immunization were chosen during periods of clinically active EAE. In preparation for the experiment, the monkeys were anesthetized with ketamine/Vetranquil (9/1 v/v). During scanning, each monkey was placed on a 37°C water-filled heating pad (Granulab International BV, Amersfoort, The Netherlands) to prevent hypothermia. The head of the monkey was fixed in a custom-built stereotactic apparatus made of metal-free plastics to ensure reproducible positioning in the magnetic field and to minimize movement artifacts. The stereotactic apparatus was placed inside a saddle-type radiofrequency coil. MRI was performed on a SISCO 200-MHz spectrometer (Varian, Palo Alto, CA) equipped with an actively shielded gradient (maximum gradient 3.2 G/cm, 33 cm inner diameter). T2-weighted MR images were recorded from formalin-fixed brains to enable the determination of the exact localization of the lesions that were detected in vivo. In both scannings the same orientation points were chosen for slice localization (see below). Because movement artifacts were absent and long acquisition times can be used, images of very high contrast can be obtained. First, a sagittal scout scan was made. The posterior and anterior positions of the corpus callosum were chosen as orientation markers for precise localization of the slices for the in vivo and postmortem MRI. A T2 (echotime (TE)/repetition time (TR), 60/2500 ms)-weighted multislice scan (20 slices of 1 mm thickness) was obtained followed by a T1-weighted scan (TE/TR, 25/1000 ms) with the same spatial prescription. Gadolinium-DTPA (Magnevist; Schering AG, Berlin, Germany) was injected intravenously (0.3 mmol/kg) and allowed to circulate for 10 minutes to ensure adequate distribution. Next, the T1-weighted MRI was repeated to attain a postcontrast data set. Each slice was recorded with a matrix of 256 × 128 data points and a field of view of 5 × 5 cm. The data set was analyzed on an Apple Macintosh Performa 630 using the public domain NIH Image program. The T1-weighted MR images had an unexpected gray-white matter contrast. In images recorded with the NMR machines that are now used in clinical settings, which are 1.5 Tesla or less, a contrast conversion of the white and gray matter is normally seen on T1-weighted versusT2-weighted images. In T1-weighted images the NMR signal of the white matter is hyperintense in comparison with that of the gray matter, whereas in T2-weighted images the white matter signal is hypointense compared with that of the gray matter. In both the T1- and T2-weighted images recorded on our 4.7-Tesla machine, white matter is hypointense as compared with gray matter, and lesions are visible as hyperintensities. This is not a unique feature of the marmoset brain, given that it was also observed in cat brains (unpublished observation). The most likely explanation for this discrepancy is that with the high magnetic field used, T1 values of gray and white matter converge, whereas T2 values are unaffected by the strength of the field. In our T1-weighted images, the intensities of white and gray matter are most likely determined by the longer T2 value of gray matter and the different proton densities of both tissues. This phenomenon did not affect the detection of gadolinium enhancement, because even long TR/short TE images ("proton-density" weighted) contrast enhancement can be observed.22Barkhof F Valk J Hommes OR Scheltens P Nauta JJP Gadopentetate diglumine enhancement of multiple sclerosis lesions on long TR spin-echo images at 0.6 T.Am J Neuroradiol. 1992; 13: 1257-1259PubMed Google Scholar The brain and spinal cord of sacrificed monkeys were excised and fixed in toto for 3 days in 4% buffered formalin and subsequently stored at 4°C in phosphate-buffered saline, pH 7.4, containing sodium azide until analysis. In some experiments the fresh brain hemispheres were separated with a surgical blade, after which one hemisphere was fixed in buffered formalin and the other snap frozen in liquid nitrogen for immunohistological analysis. The analysis of the frozen brains has been published separately.23Laman JD Van Meurs M Schellekens MM De Boer M Massacesi L Melchers B Lassmann H Claassen E 't Hart BA Expression of accessory molecules and cytokines in acute experimental autoimmune encephalomyelitis (EAE) in marmoset monkeys (Callithrix jacchus).J Neuroimmunol. 1998; 86: 30-45Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Formalin-fixed brains were embedded in paraffin. For the exact localization of lesions that were MRI detectable in vivo, use was made of the postmortem images. Because high-contrast images were made in the coronal and axial directions using the same orientation parameters as for the in vivo images, this enabled an accurate three-dimensional positioning of each lesion. From this material, coronal sections with a thickness of 3 to 5 μm were cut. The extent of inflammation, demyelination, and axonal pathology was evaluated on tissue sections stained for hematoxylin and eosin to visualize infiltrated cells, with Klüver Barrera stain (Luxol Fast Blue (LFB) combined with periodic acid-Schiff (PAS)) for staining myelin and myelin degradation products and with Bielschowsky silver impregnation for staining axons. Immunocytochemistry was performed with a biotin-avidin system as described earlier.24Vass K Lassmann H Wekerle H Wisniewski HM The distribution of Ia-antigen in the lesions of rat acute experimental allergic encephalomyelitis.Acta Neuropathol. 1996; 70: 149-160Crossref Scopus (193) Google Scholar T cells were stained with anti-human CD3 antibodies (Dakopatts, Glostrup, Denmark). Astrocytes were stained with polyclonal anti-glial fibrillary acidic protein (Dakopatts). Macrophages were detected by staining with the antibodies 27E10 and MRP14, which are both commercially available from BMA Biomedicals (Augst, Switzerland).25Lucchinetti CF Brück W Rodriguez M Lassmann H Distinct patterns of multiple sclerosis pathology indicates heterogeneity in pathogenesis.Brain Pathol. 1996; 6: 259-274Crossref PubMed Scopus (683) Google Scholar Myelin and oligodendrocytes were stained with antibodies recognizing 2′-3′-cyclic nucleotide-3′-phosphodiesterase (CNP; Affinity Research, Nottingham, UK), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) which were kindly provided by Dr. S. Piddlesden (University of Cardiff, UK). Immunoglobulin (Ig) depositions in the lesions were detected using biotinylated anti-(human) Ig (Amersham, Buckinghamshire, UK) as a primary antibody. Depositions of complement factor C9 were detected by a polyclonal antibody as described earlier.26Piddlesden SJ Lassmann H Zimprich F Morgan BP Linington C The demyelinating potential of antibodies to myelin oligodendrocyte glycoprotein is related to their ability to fix complement.Am J Pathol. 1993; 143: 555-564PubMed Google Scholar The maturation stage of a certain lesion was assessed using previously published criteria for the classification of MS lesions.27Brück W Porada P Poser S Rieckmann P Hanefeld F Kretzschmar A Lassmann H Monocyte/Macrophage differentiation in early multiple sclerosis lesions.Ann Neurol. 1995; 38: 788-796Crossref PubMed Scopus (405) Google Scholar In this classification scheme, lesions are staged as follows: 1) early active lesions, presence of LFB and MOG myelin degradation products in macrophages and/or the presence of macrophages stained for macrophage antigen MRP14; 2) late active lesions, presence of myelin degradation products LFB and PLP but not MOG in macrophages and/or the presence of macrophages stained for macrophage antigens by 27E10 but not MRP14; 3) inactive lesions, presence of PAS-positive myelin degradation products but without LFB, PLP, or MOG degradation products in macrophages; and 4) remyelinated lesions, presence of thin LFB-positive myelin. According to Dutch law on animal experimentation, the experimental procedures of this study have been reviewed and approved by the Institute's Animal Care and Use Committee. In a first set of experiments, the originally documented EAE model in marmoset monkeys18Massacesi L Genain CP Lee-Parritz D Letvin NL Canfield D Hauser SL Actively and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis.Ann Neurol. 1995; 37: 519-530Crossref PubMed Scopus (123) Google Scholar, 19Genain CP Lee-Parritz D Nguyen MH Massacesi L Joshi N Ferrante R Hoffman K Moseley M Letvin NL Hauser SL In healthy primates, circulating autoreactive T cells mediate autoimmune disease.J Clin Invest. 1994; 94: 1339-1345Crossref PubMed Scopus (91) Google Scholar was reproduced, namely by immunization with human myelin in complete adjuvant (with 3 mg M. tuberculosis per ml oil) and additionally intravenous administration of B. pertussis. As described in the original reports, this procedure induced a severe relapsing-remitting EAE. In our experience, however, the high Mycobacterium dose in the inoculum was found to cause large ulcerative skin lesions at the injection sites. This not only meant discomfort for the monkeys but also obscured early symptoms of neurological disorder in the lower part of the body. Furthermore, histopathological examination of CNS lesions showed that selective demyelination occurred only in small perivenous lesions. In the larger demyelinated lesions that were found around the ventricles, but only in one of the five monkeys (Table 1), the axons were completely disrupted, suggesting that the lesions were formed by a destructive inflammatory process rather than by selective demyelination. For these reasons we chose to further investigate EAE induced without administration ofB. pertussis and using a reduced Mycobacteriumdose in the inoculum.Table 1Histopathology of EAE in Marmosets Induced With and Without BordetellaAnimalBordetella*+, with Bordetella; −, without Bordetella.Time point†Time point (weeks after immunization) at which each animal was sacrificed.Clinical score‡Clinical score at time of sacrifice.Inflammation§0, no inflammation; +, rare (1 to 3 perivascular cuffs/section); ++, moderate numbers (3 to 10) of perivascular cuffs/section; +++, widespread perivascular cuffing and parenchymal infiltration.Infiltrate compositionDEM¶Demyelination: +/−, single demyelinated fibers; +, perivenous demyelination; ++, perivenous demyelination and some confluent plaques; +++, multiple confluent plaques.Lesion activity∥A, active lesions; I, inactive lesions (<, less than; >, more than).Brain lesion topographySpinal cord**−, no involvement; +, limited involvement; ++, moderate involvement; +++, extensive involvement.EB+213.0++Ly, MΦ++AOpt, Fo+++EC+133.0++Ly, MΦ+ACSO, Opt++ED+63.0+Ly, MΦ+/−ACSO, CWM+EE+133.0+Ly, MΦ−ICSO, Opt−EG+114.0+++Gr, Ly, MΦ++ACSO, Thal+++EH−761.0++Ly, MΦ++A < IOpt, CC, CI, CSO++EI−352.0+++Ly, MΦ+++A < IOpt, CC, CI, CSO, Thal++EJ−352.5+/−Ly, MΦ++A < ICC, Cer, Med Obl+EK−242.5++Ly, MΦ, Gr++ICC, CSO+GY−122.5+++Ly, MΦ+++A > IOpt, CC, CI, CSO, Thal++Ly, lymphocytes; MΦ, macrophages; Gr, granulocytes; Opt, optic system; Fo, fornix; CSO, centrum semiovale (periventricular white matter); CWM, cerebellar white matter; Thal, thalamus; CC, corpus callosum; CI, capsula interna; Med Obl, medulla oblongata; Cer, cerebellum.* +, with Bordetella; −, without Bordetella.† Time point (weeks after immunization) at which each animal was sacrificed.‡ Clinical score at time of sacrifice.§ 0, no inflammation; +, rare (1 to 3 perivascular cuffs/section); ++, moderate numbers (3 to 10) of perivascular cuffs/section; +++, widespread perivascular cuffing and parenchymal infiltration.¶ Demyelination: +/−, single demyelinated fibers; +, perivenous demyelination; ++, perivenous demyelination and some confluent plaques; +++, multiple confluent plaques.∥ A, active lesions; I, inactive lesions (<, less than; >, more than).** −, no involvement; +, limited involvement; ++, moderate involvement; +++, extensive involvement. Open table in a new tab Ly, lymphocytes; MΦ, macrophages; Gr, granulocytes; Opt, optic system; Fo, fornix; CSO, centrum semiovale (periventricular white matter); CWM, cerebellar white matter; Thal, thalamus; CC, corpus callosum; CI, capsula interna; Med Obl, medulla oblongata; Cer, cerebellum. The clinical course of EAE was monitored using semiquantitative criteria (Figure 1a) and body weight measurement as a surrogate disease parameter (Figure 1b). All six monkeys developed a protracted form of EAE, but substantial interindividual variation of disease courses was observed. Monkey EH experienced several relatively mild attacks, maximally reaching score 2.0 (ataxia), with episodes of complete remission in between. This monkey went into remission in the 49th week and remained free of clinical signs up to week 76, when it was sacrificed for pathological examination. In the five other monkeys (EI, EJ, EK, EL, and GY), disease episodes of higher severity alternated with episodes of incomplete remission, as was particularly apparent from body weight measurements (Figure 1b). In the advanced stages of EAE, clinical remission in these monkeys was incomplete. Clinical signs progressed to paraparesis (EI) and hemiplegia (EJ) or paraplegia (EK, EL, and GY). Monkey EI was sacrificed after a long period of chronic ataxia, monkey EJ with one paralyzed leg and monkeys GY, EK, and EL with both legs paralyzed. Importantly, none of the monkeys in this experimental group died spontaneously. To visualize abnormalities in the brain white matter, T1- and T2-weighted MR data sets were recorded during episodes of active EAE. The first MR images were made of all monkeys in the 17th week after immunization. At that stage, regions with hyperintense signal intensity were found around the ventricles on T2-weighted images. Comparison with the T1-weighted images at that stage with those recorded before EAE induction, showed that the ventricles themselves were not enlarged. This observation points to periventricular edema (not shown). MRI recorded at later disease stages showed a number of circumscribed hyperintense regions within white matter tracts such as the corpus callosum, but also in cerebral gray matter ((Figure 2, c and d). To assess the total lesion load in the brain, a T2-weighted data set from all fixed brains was attained. These postmortem MR images were compared with the data from in vivo MR images, which were recorded 1 hour before the monkey was sacrificed. Because of t
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