Fecal Microbiota Transplantation From Normal Healthy Donors Alter Intestinal Microorganisms To Promote Neurological Recovery In The Mice Of Traumatic Brain Injury

Traumatic brain injury(TBI)is defined as damage to normal structures of brain tissue and brain dysfunction caused by external forces.In addition,TBI is the leading cause of death in trauma patients,accounting for approximately 30-40% of all trauma-related mortality.However,current therapeutic measures for TBI are rather limited and often unsatisfactory.Therefore,more researches on the diagnosis and treatment of TBI are urgently needed.Previous studies have shown that post-cranial trauma in rats can lead to extensive changes in intestinal structure and function,affecting intestinal permeability.Previous human studies have also found corresponding changes in intestinal flora after craniocerebral injury in patients.In addition,TBI leaded to changes in intestinal flora,and that changes in intestinal flora may lead to intracranial microglia activation and brain tissue inflammation in mice,thereby exacerbating brain injury.It has also been reported that changes in the gut microbiota can alter the intestinal mucosal barrier and trigger a peripheral gut neurogenic or inflammatory response,which in turn may lead to neuroinflammation and neurodegeneration in the central nervous system(CNS).Studies have confirmed that the CNS,including Alzheimer’s Disease(AD),Parkinson’s Disease(PD),and many other CNS diseases lead to dysbiosis of the gut flora,and correcting dysbiosis of the gut flora can alleviate these diseases.It has also been shown that altering gut bacteria can modulate peripheral inflammatory pathways through inflammatory vesicle signaling,which in turn can help influence brain homeostasis.The gut microbiota can regulate the development and function of the brain,and the brain in turn can influence the gut microbiota through neuroimmune and neuroendocrine pathways,a two-way communication system is summarized as the Microbiome-Gut-Brain Axis(MGBA).This axis regulates homeostatic functions related to behavior,cognition,and mental health.Thus,disease states in either system may affect both systems.The microbiota-gut-brain axis is a complex bidirectional network that ensures balanced in vivo control of gastrointestinal function and is influenced by the intestinal microbiome.TBI can lead to changes in the intestinal microbiota,alter the intestinal mucosal barrier and cause an inflammatory response,which in turn may lead to neuroinflammation and neurodegeneration in CNS.Many metabolites of the gut microbiota such as short chain fatty acids,can enter the circulating blood,further regulating brain function.Furthermore,some of the metabolites from probiotics were reported to regulate the function of mitochondria and this may help in controlling secondary injury and promote repair.It was found that FMT(fecal microbiota transplantation from normal healthy donors)may have effects on the intestinal microenvironment,affecting the structure and function of the intestinal microbiota,as well as the signal exchange between microbes and the host.FMT may increase the proportion of beneficial intestinal bacteria and flora diversity,making the composition of the intestinal flora in the host similar to that in the donor.In addition,studies have shown that FMT can improve the intestinal inflammatory response,the amount and activity of neurotransmitters and vasoactive substances,and energy metabolism to rebuild the intestinal microbiota and thus treat disease.In addition to acting on the enteric nervous system,FMT may also act on the CNS.FMT may not only modulate intestinal flora composition and intestinal barrier function,but may also affect CNS function by modulating MGBA.Recent studies have found that FMT improves neurological deficits in TBI rats.In a previous study conducted by our group,we found that the intestinal flora and its metabolites can change accordingly in mice with different levels of TBI by 16 s RNA and non-targeted mass spectrometry analysis.However,the relationship between the alteration of gut microbiota after TBI and the recovery of neurological function after TBI is still inconclusive.Based on this,our group proposed the hypothesis that FMT could not only change the abundance and diversity of intestinal flora,but also change the intestinal flora and its corresponding metabolites in TBI mice,thus promoting the recovery of neurological function in mice.In this study,we established a mouse TBI model using C57BL/6 male mice as the target,and then the normal fecal microbiota from healthy donors was transplantated into them before examining the neurobehavior of TBI mice and brain water content,and analyzing the intestinal flora and its metabolites by 16 s RNA,LC-MS/MS high throughput targeted absolute quantification method.Part I FMT promotes neurological recovery in TBI miceObjective:To investigate the effect of FMT on the recovery of neurological function after TBI by establishing mouse controlled cortical impact(CCI)models..Methods:48 C57BL/6 male mice were randomly divided into three groups:(1)sham-operated group(Sham)(n=16);(2)the group of TBI(n=16);(3)the group of FMT+TBI(n=16);CCI model was used to establish the TBI mouse model,and the effects of FMT on the neurological function of TBI mice were used HE staining,NSS,rotation experiment,and brain water content measurement.Results :1.HE staining showed that 3 days after TBI,TBI mice had severe brain injury with massive and disorganized cortical and brain tissue loss,while mice in the FMT+TBI group had partial brain cortical and brain tissue loss and a significantly smaller injured area than the TBI group(P < 0.05).2.Compared with the Sham group,the brain water content of the TBI mice increased(P < 0.05),while the brain water content of the mice in the FMT+TBI group decreased significantly(P < 0.05)compared with the TBI group.3.On days 3,5 and 7 after TBI,FMT treatment significantly reduced the NSS scores of TBI mice compared with TBI(P < 0.05).On days 1,3,5 and 7 after TBI,TBI mice had a shorter latency to fall from the bar in the rotation test than the Sham group,while mice in the FMT+TBI group had a higher latency to fall on days 3,5 and 7 than TBI mice(P <0.05).Conclusion:FMT can promote neurological recovery in TBI mice by reducing brain water content and improving neurological deficits and motor coordination in TBI mice.Part II Study on the effect of FMT on the changes of intestinal flora in TBI miceObjective:To explore the changes of intestinal flora in mice after TBI by 16 s RNA sequencing,and to explore the effect of FMT on the changes of intestinal flora.Methods:The animals were grouped and molded as in the first part,and the mice were executed on the third day after TBI,and the cecum contents of each group were collected,and the specimens were divided into two parts,one for detecting the difference of intestinal flora in each group by 16 s RNA method,and one for LC-MS/MS mass spectrometry analysis in the third part.Results:1.In the alpha diversity analysis,Ace and Chao1 indices were increased and shannon index wasdecreased in the TBI group,and intestinal flora abundance index was high and diversity decreased in the TBI group;compared with the TBI group,Ace,Chao1 and shannon indices aredecreaseing in the FMT+TBI group,and intestinal flora abundance and diversity weredecreased in the FMT+TBI group,and the differences were statistically significant.2.From the change of β diversity,it can be seen that the difference between groups is greater than the difference within groups,shown that the grouping is reasonable and the difference is statistically significant.3.In view of the analysis of intestinal flora structure,it was shown that at the phylum level,compared with the Sham group,Firmicutes and Bacteroidetes decreased significantly in the TBI group,while Proteobacteria and Actinobacteria increased significantly in the TBI group;and compared with the TBI group,in the FMT+TBI group: Bacteroidetes and Verrucomicrobia increased,while Firmicutes,Proteobacteria,and Actinobacteria decreased.At the family level,compared with the Sham group,the TBI group showed thatdecreases in Lachnospiraceae and Ruminococcaceae,and increases in Enterobacteriaceae;compared with the TBI group,the FMT+TBI group:Lachnospiraceae,Akkermansiaceae,and Ruminococcaceae increased,and Enterobacteriaceae decreased.At the genus level,compared with the Sham group,the TBI group showed a significant decrease in Parabacteroidesand Alistipes,while Bacteroidetes and Parasutterella increased significantly;compared with the TBI group,the FMT+TBI group : Akkermansia increased significantly,and Alistipes and Parasutterella decreased significantly.Conclusion:1.Dysbiosis occurred in the intestinal flora of TBI mice,which resulted in a increase in the abundance of intestinal flora and a decrease in the diversity.Meanwhile,the abundance of intestinal beneficial bacteria decreased and the abundance of pathogenic bacteria increased relatively.2.FMT can change the abundance and diversity of intestinal flora in TBI mice,increasing the number of beneficial bacteria and decreasing the number of pathogenic bacteria,and the protective effect of FMT on TBI may be achieved by correcting the disorder of intestinal flora after TBI.Part III Study on the effect of FMT on the changes of intestinal metabolites in TBI miceObjective:To perform absolute quantitative targeted detection of intestinal metabolites in TBI mice by LC-MS/MS mass spectrometry,to explore the changes of intestinal metabolites in mice after TBI,and to explore the effect of FMT on the changes of metabolites.Methods:Absolute quantitative targeted detection of changes in intestinal metabolites by LC-MS/MS mass spectrometry.Results:1.260 statistically significant metabolites were identified by mass spectrometry analysis(P<0.05),among which the top 10 were 57 amino acids,48 fatty acids,33 bile acids,18 carbohydrates,16 benzoic acids,12 indoles,10 dicarboxylic acids,10 hydroxy acids,9 purine nucleotides,and 6 phenols.2.The OPLS-DAshowed that the differences between groups were greater than the differences within groups,so the grouping was reasonableand the model was robust.3.The analysis of the differences between groups showed that compared with the Sham group,the TBI group had significantly higher metabolites of UCA,Nor DCA,UDCA,CDCA,HDCA,DCA,Iso DCA,and GCDCA;while the metabolites of tauro-a-muricholic acid,tauro-w-muricholic acid,b-TMCA,and glutaric acid were significantly decreased.4.Analysis of the differences between groups showed that metabolites in FMT group such as THDCA,GCA,TDCA,TCDCA,HCA,CA,w-MCA increased significantly,while metabolites weredecreased significantly in TBI group,such as Nor DCA,UCA,LCA,b HDCA,and DCA,.5.It is evident from the KEGG pathway enrichment analysis that the FMT+TBI group,compared with the TBI group,showed significant differences in MTOR signaling pathway,insulin secretion,aminoacyl-t RNA biosynthesis,regulation of adipocyte lipolysis,CAMP signaling pathway,biosynthesis of phenylalanine,tyrosine and tryptophan,amino acid biosynthesis,glycine,unsaturated fatty acid.Conclusion:The results of this part in the experiment indicated that the intestinal metabolites of TBI mice changed significantly,and the bile acid profile changed significantly,with an increase in toxic bile acids and a decrease in beneficial bile acids;and FMT may have been used to reduce the amount of toxic bile acids and increase the amount of beneficial bile acids to protect against neurological impairment after TBI.