| [Objective]CNS demyelination in white matter lesions of multiple sclerosis(MS) is the main pathological features, astrocytes abnormal and axonal damage is being paid more and more attention in recent years. In our previous study, we confirmed that icariin(ICA) can be affected by inflammatory cytokines and HPA axis way to regulate inflammatory immune mechanisms exert on experimental autoimmune encephalomyelitis(EAE) mice symptoms relief and the protective effect of myelin. This study intends to in based on the previous research, continue to use female C57BL/6 EAE mice as the research object, then investigate the level changes of weight, clinical symptoms scoring, immunofluorescence staining of GFAP, NF200+β-APP in corpus callosum. To investigate the effects of ICA on the abnormal astrocytes and axonal injury in EAE mice, and to reveal whether ICA has a protective effect on EAE.[Methods]Part one Select 65 female C57BL/6 mice, 15 mice as normal control group, 50 mice were used to make model. The mice were anesthetized then subcutaneous injected MOG35-55 antigen emulsion oil in water state on both sides of the spine(four parts injection) by dose of each mouse in 300 ug. Intraperitoneal injection of pertussis toxin 500 ng on the same day and 48 hours after immunization. Seventh days after the first immunization use MOG35-55 to strengthen the immunity. Dynamic observation of mice in neurological symptoms and score of neurological impairment after immunization. Randomly selected three EAE mice and three normal mice at the peak of the disease to sacrifice, take the lumbar enlargement of spinal cord for HE staining, observe the histological changes of spinal cord in the microscope to determine the success of modeling.The second part According to the principle of the same grade EAE mice were randomly assigned to three groups. Including the estrogen group(positive control group),ICA group and model control group, with the normal control group, a total of four groups.Given diethylstilbestrol 0.2mg/kg.d, ICA300mg/kg.d, the equal volume of 0.5% sodium carboxymethyl cellulose, the equal volume of 0.5% sodium carboxymethyl cellulose respectively. Continuous administration for five days, The mice were sacrificed sixth days after the administration. In paraformaldehyde perfusion samples of brain tissues, frozen section after fixing and sugar dehydration, and then fluorescence staining for GFAP and NF200 + β-APP respectively. Firstly, GFAP(astrocyte marker) single fluorescent staining:according to immunofluorescence staining steps were added first antibody GFAP, second antibody Cy3 with red fluorescence light, GFAP emits red fluorescence in the fluorescence microscope.Secondly, NF200(axon marker) + β-APP(axonal damage marker)immunofluorescence double staining: according to immunofluorescence staining steps were added first antibody NF200 and β-APP, second antibody Cy3 with red fluorescence light and Alexa Fluor488 with green fluorescence light. In the fluorescence microscope NF200 emits green fluorescence and β-APP emits red fluorescence. The dyed section in the 40 x lens were observed under fluorescence microscope and photographed. Using Image J software to analysis of the fluorescence intensity of GFAP, NF200 + β-APP. Using SPSS Statistics 17 software for statistical analysis, using Graph Pad Prism software for drawing.Measurement data using(x±s). One way ANOVA was used to compare between groups was statistically significant difference for P<0.05, P<0.01 had significant difference. Related variables using Pearson correlation analysis.[result]Part one Mice got sick 13-15 days after immunization, 2-3 days of disease peak.The tail weakness as the first symptom accounted for the majority of mice. There are a few mouse to walk the balance disorder, gradually increase to limb weakness, paralysis of limbs.The hightest nerve function score was 5 points. The normal control group mice had no obvious abnormalities. The EAE mice HE staining of spinal cord showed a large amount of lymphocyte infiltration around the vessels was a "cuff" change.The normal control group had no pathological changes. According to the symptoms of mice, the neurological score and HE staining histology shows that constructing EAE mouse model successfully.The second part(1)The changes of weight in mice: The mice,s weight began to decline after antigen stimulation, with the passage of time, weight loss is more and more obvious.After treatment, the weight of estrogen group and ICA group of EAE mice increased significantly(P<0.01), compared with the model group after treatment, the estrogen group and ICA group have significantly improved in weight(P<0.01), no significant difference was found between the estrogen group and ICA group(P>0.05).(2)The changes of nerve function in mice: After treatment,the estrogen group and ICA group of EAE mice,s neurological function score improved significantly(P<0.05),compared with the model group after treatment, the neurological function score in the estrogen group and ICA group also significantly improved(P<0.05), no significant difference was found between the estrogen group and ICA group(P>0.05).(3)EAE mice have abnormal astrocytes and axonal damage: The model control group GFAP(astrocyte marker) fluorescence intensity was significantly higher than the normal group, and the cell morphological abnormalities, the increase in the number of instructions,indicating that have astrocyte activation and proliferation in EAE mice; model control group(NF200 mark) axon density than normal group significantly decreased, indicating that EAE mice have axonal damage.(4)Effect of ICA in astrocytes on EAE mouse: Astrocytes were smaller in normal group,such as protrusions to extend the peripheral filaments, no abnormal shape, GFAP(astrocyte marker) fluorescence intensity is low; the model control group was significantly higher than the normal fluorescence intensity(P<0.05), and in the active state, abnormal cell morphology, cell body size and less the rules, processes short and thick; compared with the model group, estrogen group and ICA group significantly decreased the fluorescence intensity(P<0.05), form close to normal. No significant difference was found between the estrogen group and ICA group(P>0.05).(5)Effect of ICA in axons on mice of EAE : Model control group(NF200 mark) axon density than normal group decreased significantly(P<0.01); estrogen, ICA treatment group and axonal density than the model control group was significantly increased, which was statistically significant, ICA group increased more significantly(P<0.01), between the estrogen group and ICA group showed no significant difference(P>0.05). While no damage the normal group of axons(beta-APP markers), β-APP fluorescence intensity is highest in model control group, showed the most serious axonal damage, compared with the model group, after treatment group and estrogen group ICA β-APP fluorescence intensity decreased significantly(P<0.01), between the estrogen group and ICA group showed no significant difference(P>0.05).[Conclusion]( 1) There were abnormal astrocytes in the corpus callosum of EAE mice, which showed the proliferation and activation state, the abnormal cell morphology, the increase of the number, and the presence of axonal damage;(2)After ICA treatment, the activation of astrocytes in the corpus callosum of EAE mice decreased, and ICA could improve the neural function damage in model rats by inhibiting the proliferation of astrocytes;(3)ICA has a non inflammatory neuroprotective effect on EAE mice, which may be closely related to the regulation of axonal regeneration and reduce axonal damage. |
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