The Establishment And Pathologic Study Of Experimental Autoimmune Encephalomyelitis In Rats Induced By Simplex/complex Antigen

Objective:Multiple sclerosis(MS) is an inflammatory demyelination disease of the central nervous system(CNS),with the feature of recurrent attacks. It is one of the momentous diseases of nervous system for its high incidence, puber-attaching and chronic course. As the short of data from MS patients,the establishment of an ideal animal model becomes the foundation for further pathogenetic and therapeutic investigations.Experimental autoimmune encephalomyelitis(EAE),an autoimmune disease, characterised by myelinolysis and perivascular cuffings,is an ideal animal model for MS.The orthodox EAE model was induced by guinea pig spinal cord homogenate(GPSCH) but the complexity of antigenic components limited the studys on molecular level. To explore the relationship between EAE and the dose and composition of antigen,we established an EAE model with a low-dose guinea pig spinal cord homogenate(GPSCH), For further study of the encephalitogenic role of specific antigenic component of spinal cord and its quantitative effects on EAE,we induced another EAE model by MBP69-85. Methods:1 Preparation for immunizing antigen:1.1 GPSCH-immunizing antigen Weigh the whole spinal cord, mix spinal cord and icy salt water separately with the proportion W/V 1:1,1:5 and 1:10 to produce homogenate,then a 1:1 mixture of the homogenate and complete Freund's adjuvant (CFA) resulted in the encephalitogenic emulsion.1.2 MBP69-85-immunizing antigen MBP69-85 dissolved in normal saline was mixed with complete Freund's adjuvant to prepare the encephalitogenic emulsion.2 The rats grouping and EAE induction2.1 GPSCH-EAE EAE group: 60Wistar rats were randomly and equally divided into 3 groups, separately immunized with that three kinds of homogenate antigen,0.5ml/rat,hypodermic injection in five points of foot pads and the back. Control group:10Wistar rats were immunized in the same way only with CFA。2.2 MBP69-85-EAE EAE group: 60Wistar rats were divided randomly and equally into 3 groups, separately immunized with MBP69-85 25μg,50μg,100μg per rat(0.5mL encephalitogenic emulsion), hypodermic injection in five points of foot pads and the back. Control group:10Wistar rats, immunized in the same way only with CFA。3 Neural function assessment: Scores were assigned on the basis of the following symptoms: 1, tail weakness; 2, tail weakness plus limb asthenia; 3, mild limb paralysis; 4, severe limb paralysis; 5,moribund/dead.4 Histopathology: Rats in each group were sacrificed after anesthesia with intraperitoneal injection of 10% chloralhydrate at the crest-time,paracmasis and relapse of the disease within 50 days after immunization. Tissues of the brain and spinal cord were fixed within 4% paraform after left ventricle perfusion with normal saline and 4% paraform, then the tissues were embedded in paraffin and sectioned at 6μm thickness.The sections were stained with HE staining,trichrome staining and immunohistochemistry of MBP(myelin basic protein) and NF(Neurofilament). The ultrathin sections were observed under electron microscope for pathological changes of myelin sheath and axon.Results:1 GPSCH-EAE1.1 Homogenate antigen 1:1 group1.1.1:The morbidity and clinical manifestation of EAE rats The incidence of the rats was 60% with a relapse rate 33.3%(1/3). The mean time of morbility was 14.25±1.66 days and mean clinical symptom score was 3.25±0.63.Rats in EAE group showed disorders 12 days after immunization,including tail hypomyotonia or acratia,hind limbs asthenia or general fatigue or paralysis and gatism.The minority rats showed the symptom of acratia of anterior limbs or mortuus of the disease. There was no morbidity in the co ntrol g roup.1.1.2 Histopathologic examination 1.1.2.1 HE staining: There were infiltration of inflammatory cells inside nerve tissue and perivascular cuffings in the junctional zone of gray and white matter at the crest-time of the disease. Perivascular cells decreased or vanished at paracmasis of the disease,and increased with conspicuous infiltration of cells in white and grey matter at relapse of the disease.1.1.2.2 Trichrome staining: The axon with inhomogeneous chromatosis was swollen and beaded-discontinued;The myelin was porous and partly disaggregated.1.1.2.3 Immunohistochemistry: The immunohistochemistry of MBP and NF showed demyelination in the white matter and the injury of axon.1.1.2.4 Electron microscope: The main pathological change was myelinoclasis with the swell of axon at the crest-time of the disease;There was concomitance of myelinoclasis and serva with indefinite construction inside the axon at paracmasis of the disease;The destruction of axon was obvious with the vacuolization or the absence of axon at relapse of the disease.1.2 Homogenate antigen 1:5 group1.2.1 The morbidity and clinical manifestation of EAE rats The incidence of the rats was 70% with a relapse rate 35.71%. The mean time of morbility was 16.07±4.25 days and mean clinical symptom score was 1.68±0.93.Rats in EAE group showed disorders 12 days after immunization,including tail hypomyotonia or acratia, hind limbs asthenia or general fatigue or paralysis and gatism.The minority rats showed the symptom of anterior limbs acratia,paralysis, spasticity,head tilt or mortuus of the disease. There was no morbidity in the control group.1.2.2 Histopathologic examination1.2.2.1 HE staining: There were infiltration of inflammatory cells inside nerve tissue and perivascular cuffings in the junctional zone of gray and white matter at the crest-time of the disease. Perivascular cells decreased or vanished at paracmasis of the disease,and increased with conspicuous infiltration of cells in white and grey matter at relapse of the disease.1.2.2.2 Trichrome staining: The axon with inhomogeneous chromatosis was swollen and beaded-discontinued;The myelin was porous and partly disaggregated.1.2.2.3 Immunohistochemistry: The immunohistochemistry of MBP and NF showed demyelination in the white matter and the injury of axon.1.2.2.4 Electron microscope: The main pathological change was myelinoclasis with the swell of axon at the crest-time of the disease;There was concomitance of myelinoclasis and serva with indefinite construction inside the axon at paracmasis of the disease.The destruction of axon was obvious,vacuolization or absence of axon at relapse of the disease.1.3 Homogenate antigen 1:10 group1.3.1 The morbidity and clinical manifestation of EAE rats The incidence of the rats was 50% with a relapse rate 50%. The mean time of morbility was 19.20±6.48 days and mean clinical symptom score was 2.45±1.23. Rats in EAE group showed disorders 13 days after immunization,including tail hypomyotonia or acratia, hind limbs asthenia or general fatigue or paralysis, gatism.The minority rats showed the symptom of anterior limbs acratia,paralysis, spasticity,head tilt or mortuus of the disease. There was no morbidity in the control group.1.3.2 Histopathologic examination1.3.2.1 HE staining: There were infiltration of inflammatory cells inside nerve tissue and perivascular cuffings in the junctional zone of gray and white matter at the crest-time of the disease.Perivascular cells decreased or vanished at paracmasis of the disease,and increased with conspicuous infiltration of cells in white and grey matter at relapse of the disease.1.3.2.2 Trichrome staining:The axon with inhomogeneous chromatosis was swollen and beaded-discontinued;The myelin was porous and partly disaggregated.1.3.2.3 Immunohistochemistry:The immunohistochemistry of MBP and NF showed demyelination in the white matter and the injury of axon.1.3.2.4 Electron microscope:The main pathological change was myelinoclasis with the swell of axon at the crest-time of the disease;There was concomitance of myelinoclasis and serva with indefinite construction inside the axon at paracmasis of the disease.The destruction of axon was obvious,vacuolization or the absence of axon at relapse of the disease.2 MBP69-85-EAE2.1 The morbidity and clinical manifestation of EAE rats The rats ,immunized with MBP69-85 50μg,showed a incidence 30% with the mean time of morbility 13.83±1.47 days and mean clinical symptom score 2.38±1.89 but no relapse. Rats in EAE group showed disorders 12 days after immunization,including tail catatonosis or acratia, hind limbs or general fatigue or paralysis, gatism.The minority rats showed the symptoms of head tilt or mortuus of the disease. There was no morbidity in other groups.2.2 Histopathologic examination:2.2.1 HE staining: There were infiltration of inflammatory cells inside nerve tissue and perivascular cuffings in the junctional zone of gray and white matter in HE staining.2.2.2 trichrome staining: In trichrome staining,the axon with inhomogeneous chromatosis was swollen and beaded -discontinued;The myelin was porous and partly disaggregated.2.2.3 Immunohistochemistry: The immunohistochemistry of MBP and NF showed demyelination in the white matter and injury of axon.Conclusions:1 To some extent, the morbility of EAE rats had no dependence on the dose of immunizing antigen.The clinical manifestations,with the feature of diversity and specificity, altered with the different location of the neural damage.2 The discrepancy between the clinical manifestation and the pathological changes indicated that there minght be compensatory mechanisms for the neural function.Tere was a certain relationship between the myelinoclasis and the dose of homogenate antigen at the first attack of the disease.Tere were myelinoclasis and remyelination in the pathological changes of EAE,but the pathological changes of axon was progressive.3 MBP69-85,containing special phlogogenous determiners, had a role to cause encephalomyelitis.The morbility of EAE need autoimmune responses that triggered by a suitable dose of immunizing antigen and with adequate intensity.4 The concordance of the pathological changes between rat EAE and MS indicated that these EAE models we established were reliable and could be used for further study on the pathogenesis and therapeutics of EAE and MS on the molecular level.