Objectives The aim of our study is to investigate the changes of peripheral blood lymphocytes in Experimental Autoimmune Encephalomyelitis (EAE) and their possible mechanism.
Methods 1. EAE animal models were established by injecting MOG33-35 (Myelin Oligodendrocyte Glycoprotein) with CFA (Complete Freunds’ Adjuvant) subcutaneously into C57BL/6 female mice’s axillae and boosting with PT (Pertussis Toxin) injection into the tail on day 0 and the second day. 2. Collecting the peripheral blood from the orbital sinus or plexus of the mouse dealt with heparin sodium on the fifth, eighth, eleventh, thirteenth, twenty-firth day after immunisation respectively.
3. Culturing and stimulating the lymphocytes for 6 hours with MOG, anti-mouse IL-2 and DMEM. 4. Using flow cytometry to determine the distribution of lymphocyte subtypes. 5. Using immunofluorescence cytometry to monitor the expression of tissue lymphocytes of the fiozen section of spinal.
Results 1. EAE animal models were successfully established. 2 Mean clinic scores between CFA and EAE models had significant difference Since the twenty-first day after immunisation (***P < 0.05). 3. On the fifth, eighth, eleventh, thirteenth and twenty-first day after immunisation, the result of peripheral blood lymphocytes flow cytometry showed: compared with CFA, CD45R+ B cells quantity had no statistically significant difference, however, at the initial stage, the fifth day after immunisation, and twenty-first day after immunisation, CD45R+ B cells in EAE group expressed higher. EAE group expressed CD4+CD44+T cells higher on the fifth day after immunisation. Since then EAE group began to decline. Compared with CFA group, CD4+TCD44+T cells quantity had statistically significant difference (***p <0.001) on the eleventh day after immunisation. CD8+CD44+Tcells of EAE group expressed higher than the CFA group at the initial stage, the fifth day after immunisation, the rest days were lower. The quantity of γδT cells had statistically significant difference (***p <0.001) on the twenty-first day after immunisation. 4. The result of immunofluorescence showed that: 14 days, 18 days and 21 days after immunisation, compared with CFA group, EAE group had significantly higher CD4+T cell and CD45R+ B cell expression during these periods.
Conclusions 1. In experimental autoimmune encephalomyelitis, B, γδT cells CD8+CD44+T cells, CD4+CD44+T cells all have an effect on the in the pathogenesis of the mice. And CD4+CD44+T plays an important pathogenic role.