| Background and objectivesSpontaneous intracerebral hemorrhage is a non traumatic intracerebral hemorrhage with high disability rate and high mortality rate.Some studies have shown that the immune response assoicated with intestinal flora is related to the prognosis of cerebrovascular diseases,and trimethylamine oxide(TMAO),the metabolite of intestinal flora,is considered to be one of the new risk factors independent of the traditional risk factors related to the occurrence of ischemic stroke.It has also been suggested that TMAO may aggravate the cognitive dysfunction of Alzheimer's disease.The specific role of TMAO in the pathophysiological process of brain diseases is currently unclear.In order to further explore the pathophysiological mechanism of acute ICH and find the effective therapeutic target of ICH,this study observed the effect of TMAO on brain inflammation,brain injury and neurological function of mice with ICH by giving choline diet to increase the concentration of TMAO in the serum of mice,and demonstrated whether TMAO can promote the inflammatory response and aggravate brain injury and neurological dyfunction after ICH.The results may provide some theoretical support for the prevention and treatment ofICH.Materials and methods1.Animals and experimental groupsOne hundred and forty-four male C57BL/6 mice(10-12 months old,weighing about 25-35 g,SPF)were randomly divided into 4 groups according to random number table:Sham+Control group(general diet for 6 weeks before operation),sham+Choline group(general diet+1.2%choline diet for 6 weeks before operation),ICH+Control group(general diet for 6 weeks before operation),ICH+Choline group(general diet+1.2%choline diet for 6 weeks).After the operation,the mice in each group were given the corresponding diet according to their groups until the end of the experiment.2.Establish the model of ICH in miceWe injected 0.075 U VII collagenase in 0.5 ul sterile saline to induce hemorrhage at the following stereotactic coordinates:0.6 mm anterior and 2.00 mm lateral of the bregma and 3.2 mm in depth.Postoperative NDS below 4 was identified as model failure,then exculded from the experment.Mice in sham group only received injection of 0.5 ul sterile saline in the same place.3.Experimental assessment(1)The concentration of TMAO in the serum of mice in each group was detected by UPLC-MS/MS at 24 h and 72 h after ICH.(2)The expression of p38 Mitogen-Activated Protein Kinase(p3 8 MAPK),Myeloid differentiation facter 88(MyD88),High mobility group boxl protein(HMGB1)and Interleukin-1?(IL)around hematoma was detected by Western blot at 24 h after ICH.(3)the volume of hematoma,brain swelling,the activation of microglia and astrocytes around hematoma,and the infiltration of neutrophils were detected by Luxol fast blue/cresyl violet staining and immunofluorescence staining at 72 h after ICH;Brain water content was detected by dry wet weight method at 72 h after ICH;and the residual lesion volume,brain atrophy and white matter damage were detected by Luxol fast blue/cresyl violet staining on day 28 after ICH.(4)neurological function score(NDS)and corner turn test were used to evaluate the neurological function on day 1,3,7,14 and 28 post-ICH,and the body weight,anal temperature and survival of mice were recorded at the same time.4.Statistical analysisSPSS 23.0 and graphpad prism 7 were used for statistical analysis.The quantitative data were expressed by mean ±standard deviation(x±s).Student t test was used for the comparision of the differences between the two group,one-way analysis of variance or nonparametric test followed by Bonferroni were used for the difference among multiple groups.Chi square was used for the comparations of mortality of ICH groups.P<0.05 was statistically significant.Result1.Effect of choline diet on the concentration of TMAO in serum of mice.The results of UHPLC-MS/MS showed that the choline diet significantly increased the concentration of TMAO in the serum of mice treated with sham operation compared with the general diet group(n?6 mice/group,P=0.01).The concentration of TMAO in the serum of mice with the choline diet decreased compared with that of the sham group at 24 h and 72 h after ICH.The concentration of TMAO in the serum of mice with choline diet was still significantly higher than that of mice with general diet at 24 h after ICH(n=6 mice/group,P=0.03).Although the concentration of TMAO was still higher in the serum of mice with choline diet than that of general diet at 72 h after ICH,the difference was not statistically significant(n=6 mice/group,P>0.05).2.Effect of TMAO on the histomorphology and brain water content of mice with ICHWe detected brain injury,brain swelling and brain water content in the ipsilateral cerebral hemisphere of mice treated with general diet and choline diet at 72 h after ICH,and there were no statistical significance in the hematoma volume size,brain swelling and brain water content in this two groups(n=8 mice/group,All P>0.05).3.The effect of TMAO on brain inflammation in mice with ICHThe expression of MyD88 around the hematoma in the choline diet group was significantly higher than that in the general diet group at 24 h after ICH(n=6 mice/group,P=0.01);there was no significant difference in p38 MAPK,HMGB1 and IL-1 ? in two groups at 24 h after ICH(n=6 mice/group,P>0.05).There were activation of microglia and astrocytes and infiltration of neutrophils around the hematoma at 72 h after ICH both in the general diet group and the choline diet group.The results revealed that the number of microglia and astrocytes around the hematoma in the choline diet group increased significantly when compared with the general diet group at 72h after ICH(n=6 mice/group,P=0.001).4.Effects of TMAO on the long-term brain damage and neurological function in mice with ICHWe detected the residual lesion volume,white matter injury,and brain atrophy in ICH mice treated with general diet and choline diet on day 28 after ICH,and no statistical significance were found between this two groups(n=12 mice/group,All P>0.05).Additionally,Neurological function analysis revealed that there were also no statistical significance in corner turn test,NDS,and the individual tests of NDS(including the gait test,body symmetry test,circle test,compulsory circling,climbing test and forelimb symmetry test)between these two groups at different time points after ICH(the F values of corner turn test,NDS,gait test,body symmetry test,circle test,compulsory circling,climbing test and forelimb symmetry test were 1.730,3.458,1.878,2.271,1.347,3.337,0.214,1.843,respectively;all P values were greater than 0.05).The results revealed that TMAO had no effect on neurological deficit after ICH.Conclusion1.Choline diet increased the concentration of TMAO in the serum of mice in the sham group.Although the concentration of TMAO in the serum of the choline diet group after ICH showed a decreasing trend compared with that of the sham group,the concentration of TMAO was still higher than that of the general diet group after ICH.2.Although high serum TMAO concentration in the serum of mice induced by dietary pathway increased the inflammatory response to some extent in the acute stage of ICH,it had no influence on brain injury and long term neurological function. |
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