| Objective:In this study,CPZ was used to induce C57BL/6 mice to establish the demyelination model of central nervous system(CNS)at different time.The demyelination,immune response and glial cell response of mice fed with different CPZ were observed.We try to find the ideal target of drug intervention,therefore providing theoretical and experimental basis for the remyelination and neuroprotection in the demyelinating diseases.Methods:The demyelination model of C57BL/6 male mice were established by feeding CPZ.The mice were divided into control group,2 weeks group,4 weeks group and6 weeks group,8 mice in each group.The control group was fed with the normal rodent standard diet for 6 consecutive weeks;in the 2 weeks group,mice were fed with normal diet for 4 weeks,and then the mixed feed containing 0.2%(w/w)CPZ for 2 consecutive weeks;in the 4 weeks group,mice were fed with normal diet for 2 weeks,and then mixed feed containing 0.2%(w/w)CPZ for 4 consecutive weeks.The 6 weeks group was fed with mixed feed containing 0.2%(w/w)CPZ for 6 weeks.The weight of the animals were recorded from the beginning of modeling.At the end of the sixth week,the mice were sacrificed,the spleen and brain tissues were taken,the spleen mononuclear cells(MNCs)were prepared.Brain tissues were divided into two parts:protein extraction and brain fixation.Luxol Fast Blue staining,Black Gold II staining,MBP immunofluorescence staining were used to observe the damage and loss of myelin sheath;Western blot was used to detect the expression of MBP;Elisa was used to detect the release of related cytokines in the supernatant of cultured splenocytes and extract of brain tissues;Immunofluorescence staining was used to observe the changes of immune cells and glial cells.The experimental data of each group was analyzed by GraphPad Prism 7.0.Results:(1)The results of myelination staining were as follows:Luxol Fast Blue staining of the corpus callosum in mice(the average optical density of the corpus callosum in the control group was 0.4855±0.0006766;the average optical density in the 2 weeks group was 0.3964±0.006159,p<0.0001;the average optical density in the 4 weeks group was 0.1146±0.007348,p<0.0001;the average optical density in the 6 weeks group was0.07621±0.00228,p<0.01,with statistical significance between the two adjacent groups).Black Gold II staining(the average light density of the corpus callosum in the control group was 0.459±0.002406;2 weeks group was 0.4242±0.004816,p<0.05;4 weeks group was 0.2057±0.01395,p<0.0001;6 weeks group was 0.1676±0.002224,p<0.01,there was statistical significance between the two adjacent groups).MBP immunofluorescence staining(the average light density of the corpus callosum in the control group was 0.1228±0.006121;2 weeks group was 0.1076±0.003484,p<0.01;4weeks group was 0.09098±0.0003428,p<0.01;6 weeks group was 0.07569±0.001767,p<0.01,there was statistical significance between the two adjacent groups).With the prolongation of CPZ feeding time,the demyelination gradually increased,and a stable demyelination model was established in 4-6 weeks of CPZ feeding.(2)Immunofluorescence staining was used to detect the expression of ZO-1~+vascular endothelial cells of in the brain.The results showed that compared with the control group,the expression of ZO-1~+on vascular endothelial cells in CPZ fed group decreased gradually,while the vascular endothelial cells also lacked vascular characteristics.The numbers of ZO-1~+cells in the control group were(43.33±0.8819),in the 2 weeks group were(30±1.528,p<0.001),in the 4 weeks group were(19.67±0.8819,p<0.01),in the 6 weeks group were(15.67±1.202,p<0.01).There was statistical significance between the two adjacent groups.(3)The expression and status of CD4~+T cells in the brain were detected by immunofluorescence staining.The results showed that compared with the control group,the infiltration of CD4~+T cells in the dorsolateral septal nucleus of mice fed with CPZ increased significantly,and increased with the prolongation of CPZ feeding time.The numbers of CD4~+T cells in the control group were(0.3333±0.3333),in the 2 weeks group were(4.333±0.3333,p<0.001),in the 4 weeks group were(8.333±0.3333,p<0.001),in the 6 weeks group were(11.33±0.3333,p<0.001).In addition,the numbers of CD4~+IL-17~+and CD4~+IFN-γ~+T cells increased with the increase of CPZ feeding time.The numbers of CD4~+IL-17~+cells in the control group were(0.3333±0.3333),in the 2 weeks group were(2.667±0.3333,p<0.05),in the 4 weeks group were(6±0.5774,p<0.01),in the 6 weeks group were(9±0.5774,p<0.01).The numbers of CD4~+IFN-γ~+cells in the control group were(0.3333±0.3333),in the 2 weeks group were(3.333±0.3333,p<0.01),in the 4 weeks group were(5.333±0.3333,p<0.05),and in the 6 weeks group were(7±0.5774).There was statistical significance between the two adjacent groups.At the same time,the levels of IL-17 and IFN-γin the extracts of brain extracts detected by Elisa were also matched with the results of immunofluorescence.The concentration of IL-17 and IFN-γin the brain increased after CPZ feeding,and reached the peak at 4-6 weeks.There was no significant difference after CPZ feeding for 2 weeks,which was basically consistent with the results of immunofluorescence.The level of IL-17 in the control group was(1435±301.3),and that in the 6 weeks group was(3762±746.7,p<0.05).The level of IFN-γwas(1398±343.7)in the control group and(4526±1097,p<0.05)in the 4weeks group,which was statistically significant compared with the control group.(4)The concentrations of IFN-γ,IL-17,TNF-α,IL-6,IL-1βand IL-10 in the supernatant of spleen culture were detected by Elisa.The results showed that the levels of the IFN-γand IL-17 contents in the supernatant of cultured spleen cells increased slightly after CPZ feeding for 2 weeks,but there was no statistical significance.Unexpectedly,compared with the mice fed CPZ for 2 weeks,IFN-γand IL-17 were significantly lower after 4 and 6 weeks(p<0.01 and p<0.0001,respectively).In addition,IL-1βincreased slightly after CPZ feeding for 2 weeks,while TNF-αand IL-6 increased significantly(p<0.01 and p<0.0001,respectively)after CPZ exposure for 4 and 6 weeks in the supernatant of cultured spleen cells,and CPZ feeding did not change the concentration of IL-10.(5)The numbers of Iba1~+microglia and expression of ICAM-1 in Iba1~+microglia were detected by immunofluorescence staining.The results showed that compared with the control group,the numbers of Iba1~+microglia and expression of ICAM-1 in Iba1~+microglia were enhanced by CPZ feeding,which further increased with the increase of CPZ feeding time.(6)The numbers of GFAP~+astrocytes and expression of SOX2 in GFAP~+astrocytes were detected by immunofluorescence staining.Compared with the control group,GFAP~+astrocytes and SOX2~+expression were elevated obviously,which further increased with the prolongation of CPZ feeding time.In addition,the expression of GFAP~+BDNF~+,,GFAP~+CNTF~+and GFAP~+IGF-II~+were detected by immunofluorescence staining.Compared with the control group,CPZ feeding increased the expression of BDNF,CNTF and IGF-II by astrocytes,which further increased the expression with the prolongation of CPZ feeding time.(7)The expression of NG2~+,PDGF-Rα~+,Olig2~+OPCs and PDGF-Rα~+SOX2~+were detected by immunofluorescence staining.The results showed that NG2~+,PDGF-Rα~+and Olig2~+OPCs were enhanced in CPZ group,and Olig2~+OPCs gradually migrated to the corpus callosum.Compared with the control group,PDGF-Rα~+SOX2~+OPCs decreased significantly,indicating that the spontaneous formation of OPCs in demyelinating model is insufficient to differentiate into mature oligodendrocytes(p<0.001 and p<0.0001,respectively).Conclusion:(1)After 2 weeks of feeding,CPZ did not cause obvious microglia reaction,but after 4 weeks of feeding,CPZ caused obvious microglia migration and accumulation to myelin sheath.The increased production of inflammatory cytokines IL-1βand IL-6 in the extracts of brain indicates that there is an inflammatory microenvironment mediated by microglia in the brain,which should contribute to BBB destruction and T cell infiltration.(2)CPZ can activate astrocytes and up-regulate the expression of BDNF,CNTF and IGF-II,thus providing neurotrophic microenvironment for the remyelination.(3)At the 4th and 6th week of CPZ feeding,microglia-mediated inflammatory microenvironment coexist with astrocyte-driven neurotrophic microenvironment.We speculate that BDNF,CNTF and IGF-II from astrocytes can induce the formation of NG2~+and PDGF-Rα~+OPCs,but the neuroinflammatory microenvironment mediated by microglia should affect the differentiation of OPCs into mature oligodendrocytes. |
PDF Full Text Request