Effects Of Gut Microbiota On Microglia Phenotype In Substantia Nigra Of Mice Model Of Parkinson’s Disease

Objective:To investigate the effects of gut microbiota on microglia phenotype in the substantia nigra region of the brain in MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)model of chronic Parkinson disease(PD)mice.Methods:Eight-week-old C57BL/6 male mice weighed about 20 g.After feeding for 7 days,they were randomly divided into two groups: normal control group and PD model group.In PD model group,mice were intraperitoneally injected with MPTP(25mg/kg)+ promethanoxide(250mg/kg)for 10 times,3.5 days per time,to establish chronic PD model.Normal control group mice were given peritoneal injection of the same volume of normal saline.During feeding,movement disorders(tremor,autonomous activity,etc.)of mice were observed,body weight and stool characteristics of mice were recorded,and disease activity index(DAI)was measured according to the records.On the 36 th day of the experiment,the success of the PD model was evaluated by animal behavior test(rod climbing test,rotating rod test)and immunofluorescence detection of Tyrosine hydroxylase(TH)expression in the substantia nigra of mice.After the validation of the model,the following indexes were detected:(1)The diversity and composition of gut microbiota of mice were analyzed by 16 Sr DNA gene sequencing technology;(2)The histological changes of cecal mucosa of mice were detected by HE staining.(3)The expressions of inducible nitric oxide synthase(i NOS)and ionized calcium binding adaptor molecule-1(Iba-1)in the substantia nigra of mice were detected by immunofluorescence staining.(4)The expression of IBA-1,i NOS and Arginase in the substantia nigra of mice midbrain was detected by Western Blot.(5)The interleukin-1β(IL-1β)and interleukin-10(IL-10)in the substantia nigra region of mouse midbrain were detected by real-time quantitative PCR.Then,PD model group mice were randomly divided into three groups: normal control group,PD model group,and PD + faecal bacteria transplantation group.From day 36,PD model and faecal bacteria transplantation group were given 200 μL fresh faecal bacteria suspension(108 CFU/ m L)by gavage once a day for 14 days.Mice in normal control group and PD model group were given the same volume of normal saline by gavage every day.On the 50 th day of the experiment,animal behavior experiment(pole climbing experiment,rotating rod experiment)and immunofluorescence were conducted to detect the expression of Th in the substantia nigra of mice,and related indexes were detected(the detection indexes were the same as those on the 36 th day of the experiment).Results:1.The mice model of chronic PD induced by intraperitoneal injection of MPTP showed PD symptoms and positive behavioral evaluation after modeling,accompanied by the down-regulation of Th expression in the substantia nigra in the brain of mice,which was an effective mouse model of chronic PD.2.Compared with normal control mice,the diversity of gut microbiota of chronic PD model mice was increased and the bacterial composition was changed.3.Fecal traits,DAI score and cecal pathology of mice from childhood showed intestinal inflammation in chronic PD model mice.4.The microglia in the substantia nigra region of the brain of mice with chronic PD model were activated and mainly showed pro-inflammatory M1 phenotype.5.After faecal bacteria transplantation,the movement disorder symptoms and animal behavior tests of chronic PD model mice were improved,and the expression of TH in the substantia nigra was increased.6.After faecal bacteria transplantation,the gut microbiota diversity of chronic PD model mice decreased,and the intestinal flora structural composition basically recovered,and there was no significant difference compared with the normal control group.7.After faecal bacteria transplantation,fecal traits,DAI score and cecal pathology of mice showed that intestinal inflammation was improved in chronic PD model mice.8.After faecal bacteria transplantation,the activation of microglia cells in the substantia nigra of the chronic PD model mice was mainly transformed from M1 phenotype to M2 phenotype,and the secretion of inflammatory factors decreased while the secretion of anti-inflammatory factors increased.Conclusion:Compared with normal control mice,chronic PD model mice showed increased diversity of gut microbiota,altered bacterial composition and accompanied by intestinal inflammation.Microglial cells in the substantia nigra region were activated and dominated by pro-inflammatory M1 phenotype,which was divided into pro-inflammatory factors.After faecal bacteria transplantation,the gut microbiota diversity of PD model mice decreased and the bacterial composition basically recovered.The activation of microglia in the substantia nigra region was mainly M2 phenotype,the secretion of pro-inflammatory factors decreased,the secretion of anti-inflammatory factors increased,the Parkinson’s symptoms and animal behavioral performance of mice were improved,and the expression of Th was increased.The changes of gut microbiota affect the activation and phenotypic differentiation of microglia in the substantia nigra of chronic PD mice,and regulating gut microbiota may be one of the means to regulate the phenotype of microglia.