Establishment of experimental allergic encephalomyelitis in C57BL/6 mice and testing of expressions of Foxp3 and quantifies of CD4~+ CD25~+ regulatory T cells / 中华微生物学和免疫学杂志

ABSTRACT

Objective To induce experimental allergic encephalomyelitis (EAE) in female C57BL/6 mice with the extracellular domain of myelin oligedendroglia glycoprotein(MOG~(Igd)). Percentages of CD4~+ CD25~+ T cell (Tr) were tested , and also normalized expressions of Foxp3. Methods Molecular cloning technology was used to produce MOG~(Igd) fusion protein. The MOG~(Igd)-TrxA fusion protein and TrxA protein were purified by metal chelate affinity chromatography (MCAC). Mice were injected s. c. in the flank with 300 μg MOG~(Igd) in complete Frcund's adjuvant (CFA) supplemented with 4 μg/μl Mycobacterium tuberculosis. H37Rv. Mice received 0.4 ml emulsion of spinal cord homogenate of guinea pigs (GPSCH) in positive control group, and the same volume emulsiom of TrxA in negative control group, while mice served as normal control received only saline/adjuvant. Mice were monitored two times a day for continuously 30 days by double bind. Clinical scores and histopathology were evaluated. Then, mice were sacrificed. The spinal cord and brain were removed and fixed in buffered formalin. Horizontal sections taken from the central nervous system(CNS) were stained with haematoxylin and eosin (HE), and Kluver-Barrera staining. Also, immunohistochemistry was performed. Percentages of CD4~+ CD25~+ T cells were tested through flow cytometric analysis, and real-time PCR was performed to test normalized expressions of Foxp3 mRNA. Then, correlations between the two were performanced. Results Mice in both MOG group and GPSCH group shew chronic non-remitting course. The onset of disease, time when the most severe clinical symptoms happened and the clinical score between the two groups shew no significant differnces (P>0.05). However, neither in TrxA treated group nor in normal control group did animals exhibit clinical signs of EAE. Histologic sections of the brain and spinal cord taken from affected animals shew perivascular infiltration of mononuclear cells, gliosis, and multifocal demyelination. Lesions scattered throughout the CNS including brainstem, spinal cord, cerebellum, and penventricular white matter. There were significant differences between MOG group and TrxA group in the level of lesion-ceutric AQP-4 expression showing up by immunohistochemistry (P<0.05). Percentages of CD4~+ CD25~+ T cells in MOG group and GPSCH group were (4.71±1.61) % and (1.44±0.65) %, respectively, both of which were significantly lower than those in the normal control group or TrxA treated group (P<0.01). And the difference between MOG group and GPSCH group also reached statistics meaning (P<0.01). Normalized expression of Foxp3 mRNA in MOG group was 2.26± 1.97, and was not significantly higher than the 1.44±1.20 level in GPSCH group (P>0.05). However, they beth were statistically lower than that in the negative control group, namely 8.58±3.34 (P<0.01). Percentages of CD4~+ CD25~+ T cells was statistically correlated with expressions of Foxp3 mRNA (P< 0.05). Conclusion EAE induced in C57BL/6 mice with MOG~(Igd) is reproduceable. It shares the similar clinial signs and pathologic features with human multiple sclerosis(MS). Thus, we find a good way to further study the immune mechanisms of MS and also to search for the effective treatments.