小胶质细胞在双环己酮草酰二腙诱导的急性脱髓鞘小鼠模型中的动态变化及意义

The changes and significance of microglia in acute demyelination mice model mediated by cuprizone

目的探讨小胶质细胞在双环己酮草酰二腙(cuprizone,CPZ)诱导的急性脱髓鞘小鼠模型中的时空演变及其意义。方法选取C57BL/6雄性小鼠32只,随机分为对照组以及髓鞘脱失2(2周组)、4(4周组)、6周组(6周组),每组8只。对照组给予正常饲料饲喂,另3组分别给予含0.2%(质量分数)CPZ的混合饲料饲喂2周、4周和6周。采用ELISA法检测各组脑组织干扰素γ(IFN-γ)、白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平,采用蛋白印迹实验检测脑组织髓鞘碱性蛋白(MBP)表达水平,采用免疫荧光染色观察MBP、Iba-1、Iba-1+/诱导型一氧化氮合酶(iNOS+)、Iba-1+/精氨酸酶1(Arg1+)和Iba-1+/MBP+表达。结果与对照组比较,CPZ喂食2周后,小鼠胼胝体区髓鞘开始脱失,并随喂食时间延长而加重(P<0.01)。喂食4周后,髓鞘损害区小胶质细胞明显活化,并向损害区迁移和聚集(P<0.01),表达iNOS的M1型小胶质细胞和表达Arg1的M2型小胶质细胞增加。与对照组比较,CPZ喂食4~6周小鼠脑组织小胶质细胞与MBP共定位。ELISA结果显示,喂食2周后,小鼠脑组织IFN-γ水平升高(P<0.01);喂食4周后,脑组织IL-1β水平表达升高(P<0.01);而TNF-α水平无统计学变化(P>0.05)。结论CPZ喂食可导致严重的髓鞘脱失,并触发小胶质细胞活化;小胶质细胞的炎性作用和吞噬作用共存,但髓鞘仍然脱失严重。改变小胶质细胞极化表型,增加神经营养因子的释放,促进其有效吞噬髓鞘碎片,改善局部炎症环境,可能对减轻髓鞘脱失、促进轴突再生具有重要意义。

Objective To investigate the temporal and spatial evolution and meaning of microglia in the acute demyelination mice model induced by cuprizone(CPZ).Methods Thirty-two male C57BL/6 mice were selected and randomly divided into a normal control group,a demyelination 2 weeks group(2 weeks),a demyelination 4 weeks group(4 weeks),and a demyelination 6 weeks group(6 weeks),with 8 mice in each group.The control group was fed normal diet,while the other three groups were fed with a mixed diet containing 0.2%CPZ for 2,4 and 6 weeks respectively.Interferonγ(IFN-γ),interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)in the brain were detected by ELISA.Myelin basic protein(MBP)was detected by western blot.Expressions of MBP,IBA-1,IBA-1+/iNOS+,IBA-1+/Arg1+and IBa-1+/MBP+were observed by immunofluorescence staining.Results Compared with the controls,after CPZ feeding for 2 weeks,the myelin sheath in the corpus callosum began to lose,and increased with the feeding time(P<0.01).After 4 weeks of CPZ feeding,microglia in the myelin damaged area were activated significantly;they migrated and gathered to the damaged area(P<0.01).At the same time,the polarization of M1 microglia expressing iNOS and M2 microglia expressing Arg1 were significantly increased.Compared with the control group,the microglia co-located with MBP at 4-6 weeks of CPZ feeding.The results of ELISA showed that the level of IFN-γincreased 2 weeks after CPZ feeding(P<0.01),IL-1βincreased significantly 4 weeks after CPZ feeding(P<0.01),and TNF-αdid not change significantly after CPZ feeding.Conclusions CPZ led to severe demyelination and triggered the activation of microglia.Microglia existed in both inflammatory and phagocytosis states,but the demyelination was still serious.Therefore,with regard to microglia,to change its polarized phenotype,increase its release of neurotrophic factors,promote its effective phagocytosis of myelin fragments,and improve the local inflammatory environment may be of great significance for the protection of myelin sheath loss and the promotion of axon regeneration.