MOG35-55 Peptide Induced RR-EAE And Its Pathology

Multiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system (CNS). It causes relapsing-remitting and progressive neurological impairment. There are many factors that are associated with the pathogenesis of MS which is still unclear at present. Its typical pathology is the infiltration of inflammatory cells, demyelination, the degeneration of the axon and the proliferation of gliocyte, etc. It has the characteristics of relapsing-remitting and high morbidity. The uneasily available of specimen of MS has limited the research of the disease, so it is very important to establish a proper animal model to study the pathogenesis, pathology and treatment of MS. Experimental autoimmune encephalomyelitis (EAE) is acknowledged to be the ideal model of MS. Nowadays the inducement of EAE by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 is considered to mimic MS mostly in the pathogenesis. What is more, the inducement of EAE of mice or rats by MOG can mimic many types of MS such as: relapsing-remitting (RR), primary progress (PP), secondary progress (SP), Marburg and Devic, etc. The strain of C57BL/6 which is sensitive to MOG35-55 is pure and we have known more about their background of heritage, genetics and immunology, etc. The strain is cheaper and more available than strains of PL/J and SJL, and so on. So the inducement of EAE in C57BL / 6 mice by MOG35-55 can work better to study the pathogenesis of MS/EAE.Objective: In our study we want to induce an EAE model by MOG35-55 in C57BL/6 mice and to observe the whole course of EAE and its pathology which can make a basis for the further study of the pathogenesis, pathology and treatment of MS.Methods: 24 female C57BL/6 mice (inbred strain, aged 8-10 weeks) wheighted 18-20g were induced by MOG35-55 to make EAE model. They were randomly divided into an EAE group and a healthy control group. Each group was divided into two subgroups: an acute phase group and a chronic group. There were separately 9 and 6 in the two subgroups of EAE; 3 and 6 in the two subgroups of healthy control. All the experimental mice were immunized subcutaneously in the four points of back with 0.1ml emulsion, which including myelin oligodendrocyte glycoprotein (MOG) as antigen, emulsified with an equal volume of complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis 4mg/ml (final concentration). Pertussis toxin (PTX) was given intraperitoneal 0 hour and 48 hour. The healthy control group left untreated. Clinical signs of EAE were assessed twice a day by two investigators. Mice were examined daily for clinical signs and scored as follows: 0, no paralysis; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, moribund or dead. During the experiment, the mean maximal score and weight of animals at different phages were observed and scored.Mice of EAE (9) and control (3) of acute phase and chronic phase (separately 3) of two groups were sacrificed after anesthesia with Hydrated chlorine aldehyde. Tissues of the brain and spinal cord were fixed with 4% formalin, then the tissues were embedded in paraffin and sectioned at 5μm or 8μm thickness. The pathological changes of tissue sections were observed separately stained by Haematoxylin & Eosin (HE), Mallory, LFB and Bielschowsky's. There mice of two groups of chronic phase were fixed with 4% glutaraldehyde for electric microscope observation.Results:1 At the end of the study, the total morbidity was 100% .The mean time of onset was at 14.00±2.45d, the mean time of the peak neurological deficits score was at 18.75±4.10d and the mean peak score was 2.42±0.66.There had relapsing and remitting in all mice. At the end of the course 66.7% of the mice recovered to 0.5-1; while 33.3% did not remit, instead they occurred continuously neurological deficits;2 The mean clinical score of EAE and healthy control respectively was:1.28±0.70,0.00±0.00, and the differences of the two groups were statistically significant(P <0.05);3 The mean bodyweight of the two groups respectively was:19.27±0.87,20.99±1.55, and the differences between the two groups were statistically significant(P <0.05);4 Haematoxylin & Eosin (HE) staining showed an infiltration of inflammation at different stages of EAE. The mean score of inflammation in the two subgroups respectively was: 17.33±4.63,28.50±8.09; while the healthy control group was 5.00±0.00. The differences between groups were all statistically significant(P <0.05);5 There was varying demyelination in each group showed by Luxol Fast Blue staining .The mean score was: 31.33±3.31,18.83±5.00 and the control was: 5.00±0.00. The differences between the stage acute and chronic were statistically significant (P <0.05);6 Bielschowsky's silver stain revealed varying axonal welling and degeneration and the formation of spheroids;7 Electron microscopy showed the loose,broken and collapsion inward of the myelin sheaths; and some remyelination in different degrees. We also saw the swelling of the chondriosome and the degeneration and missing of the axonal.Conclusions: We have induced chronic EAE model successfully by mixed artificially synthesized MOG35-55 peptide and complete Freund's adjuvant in the C57BL/6 mice. It has been reported that the course of EAE induced by MOG35-55 in the C57BL/6 mice was chronic progression or chronic non-remitting. But we have seen relapsing and remitting in the whole course of our experiment in this model. We have described in detail the different pathological characteristics of different stages which will make a basis for the future study of the pathogenesis, pathology and treatment of MS.