Axonal Damage Mechanism Of EAE And Therapeutic Effects Of F₀₅

Multiple sclerosis is an inflammatory and demyelinatting disease in C.N.S, which is characterized by the multiple focuses, relapsing-remitting and aggravating gradually. Up to now, most of the studies about this disease were on the mechanisms of demyelination. There were still little documents about the significance, the mechanisms, the treatment and the rehabilitation for the axonal damage in this disease. In this research, the EAE model that had the similar clinical feature and pathology with MS was used to study the mechanism of axonal damage in EAE in vivo. On the other hand, technique of culture for nerve cell is used to investigate the potential mechanism of axonal damage in EAE in vitro. The last and the most important is that the extractive F₀₅ of Chinese traditional medicine was studied on its potential therapeutic effect for axonal damage in MS/EAE.Objectives:1. (1) To establish the RR-EAE (Relapse Remitting Experimental Autoimmune Encephalomyelitis, RR-EAE) mouse model by proteolipid protein (139-151 and 178-191), which had the similar clinical situation, course and histological alterations to MS. To confirm that it could be used for the research on the demyelination and the axonal damage mechanism of EAE. (2)To explore the mechanism about the expression of the hyperphosphorylated p38 MAPK in the central nervous system (CNS) of EAE mice and its relationship to the axonal damage. (3) To investigate the potential regulation of F₀₅ to p38 MAPK and its protection for the damaged axons in the CNS of EAE mice.2. (1) To establish the axonal damage model in vitro. (2) To explore the mechanism of axonal damage induced by γδT cell and the regulation of y8T cell to cytokine and the intervention of F₀₅. (3) To explore the therapeutic effect and its mechanism of F₀₅ for the axonal damage of EAE mice induced by y5T cell.Material and methods:1. Each SJL mouse was injected s.c. over the abdominal wall with PLP_139-151 and PLP_178-191, Mycobacteria H37RA and IFA, and Bordetella pertussis bacilli.2. HE and the Luxol Fast Blue were used to observe the demyelination in EAE mice. Fluorescence immunity and image analysis were used to explore the axonal loss marked by the expression of neurofilament protein (NF) . The axonal damage in the earlier period of EAE was observed.3. Immunohistochemistry was used to explore the mechanism of the activated kinases, the hyperphosphorylated p38 MAPK. And the relationship between the expression of p38 MAPK and APP expression was studied. Meanwhile, effect of the stress-activated kinases on axonal damage and the therapeutic effect and mechanism of F₀₅ for the axonal damage of EAE mice caused by hyperphosphorylated p38 MAPK were also observed.4. Fluorescence immunity marking flow cytometry was used to detect the change of y8T cell extracted from the ilEL and LNC.5. MTT and ELISA were used to study the regulation of y8T cell to cytokine (IFN-γ, IL-4) and the effect of F₀₅.6. Cultured neurons were detected by the enzyme terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling (TUNEL) assay and immunohistochemistry of NF to explore the therapeutic effect of F05 for the axonal damage of EAE mice caused by γδT cell.Result:1. The model of EAE mouse induced by both two PLP peptides manifested significant neurological symptoms, signs and features of relapse and remitting. In spinal and cerebral tissue, obvious phenomena of perivascular inflammatory cuffing, satellitism, predominant perivascular inflammatory cell infiltration and demyelinated lesion could be found. NF loss was observed even at the earlier 7th day after immunization and it got less with the development of EAE. But there was no evidence for recovery.2. Activated p38 MAPK was observed even at the earlier 7th day after immunization in the CNS of EAE mice. Also, the expression of APP could be found in the same area at the same period, and either of them could express in neurons. However, activated p38 MAPK was also observed in the area of both sides of lateral cerebral ventricle. The expression of APP can also restrained and the clinical score was improved after given F05 (p<0.05).3. The percentage of γδT cell in CD3⁺ cells went up obviously in the PLP_178-191group compared to the normal group( P<0.01). The number of CD3⁺ cells from LNC was significantly lower in the PLP_178-191 group (p<0.01) . However, the percentage of γδT cell in the CD3+ cells was similar to that in ilEL. The density of IFN-γ in PLP_178-191 group coated by y8TCR antibody was higher than that in the group without yγδTCR antibody (p<0.01) and this could be down regulated by F₀₅(p<0.01) . Meanwhile, the density of IL-4 in PLPm-191 group coated by ySTCR antibody was lower than that of group without γδTCR antibody (p<0.01) . The percentage of CD3⁺ cells from LNC in the F₀₅ group was also lower than that in the normal group( P<0.01).4. Compared to the normal group, the cultured neurons were damaged obviously after the stimulus of lympho- supernatant in the yd T group (p<0.01) , which showed decrease of the survival rate(p<0.01); increase in leakage of LDH, the decrease of NF number, the increase of the gray scale and increase number of neuron apoptosis(p<0.01). These could all be improved by F₀₅ (p<0.01). Conclusion:1. RR-EAE model induced by both two PLP peptides had more similarity to the relapse-remitting of MS. Meanwhile, earlier axonal damage could be reliably observed in EAE model. It is an ideal EAE model for the research on axonal damage in MS/EAE.2. The expression of APP could mark the recent axon transsection. The quantity of APP-positive axons in multiple sclerosis lesions was correlated with the disease duration and course. APP-positive axons were detected even before the onset in EAE. Activated p38 MAPK might be one of the reason of axonal damage in EAE. F05 might down regulate the expression of APP and activate p38 MAPK, improved clinical outcome compared with control group. This might be helpful to the treatment of axonal damage in MS.3. The percentage of γδT cell in iIEL and LNC was up-regulated after immunization by PLP in the early period. They might be the initial source of the γδT cell in the periphery of the earlier focus of EAE mice. γδT cell might play a important role in the mechanism of axonal damage in MS. PLP can hasten the secretion of Th1 cell through activating y8T cell. However, F₀₅ can inhibit this process. Therefore, γδT cell might play a key role in the polarization...