ABSTRACT
Objective To explore the appropriate dosage of drugs inducing experimental allergic en cephalomyelitis (EAE) in mice,and evaluate the modified model mice.Methods Different doses of myelin oligodendrocyte glycoprotein (MOG35-55:200 μg,100 μg,50 μg,25 μg),together with different doses of inactivation of mycobacterium tuberculosis (H37RA:800 μg,250 μg,100 μg) and pertussis toxin (500 ng,200 ng),were used to induce the EAE model.After immunized,the clinical disease severity of EAE mice was measured by the standard EAE grading clinical score daily.The open field test was used to detect the locomotion of mice.The Western blot and immunofluorescence were used to detect the level of myelin basic proteins (MBP) in different brain regions of mice.Results Compared with the EAE mice induced with high-dose drugs,the mice with low-dose drugs (25 μg MOG35-55,100 μg H37RA,200 ng pertussis toxin) had low neu rological scores.And they displayed normal locomotion compared with the control mice (day 16:group EAE (8.885±0.772) cm/s vs control group (8.933±0.567) cm/s,P＞0.05;day 31:group EAE (11.130±0.630) cm/s vs control group (10.670±0.959) cm/s,P＞0.05;day 55:group EAE (7.686±0.428) cm/s vs control group (8.313±0.918) cm/s,P＞0.05).Moreover,there was a significant decrease of MBP in the parahippocampal cortex (PHC) and fimbria-fornix of EAE mice induced with low-dose of drugs (PHC:group EAE (0.369±0.096) vs control group (1.000±0.163),P＜0.05;fimbria-fomix:group EAE (0.494±0.071) vs control group (1.000±0.143),P＜0.05).Conclusion The EAE mice induced with low-dose drugs(25 μg MOG35-55,100 μg H37RA,200 ng pertussis toxin) have low neurological scores,normal locomotion,and myelin impairment in the central neuronal system.And it can be used in the cognitive behavioral research of demyelination disease,such as multiple sclerosis.
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.
ABSTRACT
Experimental allergic encephalomyelitis (EAE) lesions by autoimmune inflammatory mechanism are characterized by the activation of microglia and astrocytes during the peak symptomatic stage of the disease. Besides it is well known that ROS and nitric oxide (NO), which is come out from activated inflammatory cells, play important role in the pathogenesis of EAE lesions. And vitamin C (L-ascorbic acid), which may protect from the deleterious effect by reducing iron (Fe2+) or copper (Cu2+) ions and maintain tissue homeostasis by removing of oxygen free radical, is inevitable to help many enzymatic reactions of cells. Previous report already investigated expression and functional analysis on various vitamin C transporters of vitamin C in many cell types. However, the researches for the vitamin C transporters are mostly performed in the normal state but not disease model yet. Therefore, for the first time, we investigated to know whether the SVCT1, 2 immunoreactivity may be observed in the astrocyte of EAE rat spinal cord. In the comparison of control and peak time group, the number of SVCT1, 2 immunoreactive cell was inclined to increase (P<0.05) as respectively 100+/-29.93, 135+/-34.62 in the control group, and 179+/-54.29, 349+/-73.56 in the peak time group. SVCT2 immunoreactivity was not doubly colocalized with GFAP antibody in the control group. In contrast, the astrocytes of the peak time group showed SVCT2 immunoreactivity in the perivascualr region and the cell number of doubly (SVCT2, GFAP) colocalized was 15+/-5.67 (P<0.05). We are firstly demonstrated that, in the evolving processes of EAE, astrocytes are able to use the vitamin C via the SVCT2. Taken all findings into consideration, the present data on the typical anti-oxidant vitamin C and its transporters, which may play a role in removing ROS, could be considered as a target to the therapeutic strategy of EAE and is also very useful to identify the characterization of vitamin C in the biological organism.