The Influence Of Gut Microbiome On The Secondary Brain Injury In Rats After Traumatic Brain Injury

Backgroud:Traumatic brain injury（TBI）is a commom disease in neurosurgery deparement,which bring serious threat to human health.TBI can not only lead to dysfunction of the brain tissue itself,but also cause other organs function damage.Recently,an increasing number of the research were focused on the Microbiome-Gut-Brain Axis（MGBA）,and the focus were focused on microbime colonized the gut.It have reported that the gut microbiome structure and composition were altered in the neurological diseases,such as Alzheimer’s disease,Parkinson’s disease,and the method to rescue the the gut microbiome dysbiosis could bring some therapeutic effect.In the present study,Sprague Dawley male rats（SD rats）were used to esatablishe TBI model,and then the 16S ribosomal RNA（r RNA）sequencing,neurobehavioral test and metabolomics was used to explore the role and mechanism of gut microbiome in the pathophysiological process of TBI.PART Ⅰ:THE NEUROPROTECTIVE EFFECT OF HEALTH FECAL MICROBILME TRANSPLANTATION IN RATS AFTER TRAUMATIC BRAIN INJURYObjective:To detect the changes of gut microbiome in rats after TBI and the effect of health fecal microbilme transplantation on the neurological function in rats after TBI,and explore the possibility of health fecal microbilme transplantation in the treatment of TBIMethodsExperiment 1:The changes of gut microbiome in rats after TBIA total of 50 male SD rats were divided into five groups randomly:Sham group（n=10）,TBI group（n=10）,TBI+saline group（n=10）,TBI+FMT group（n=10）and 10 SD rats were selected as donor rats.The controlled cortical impact was performed to established TBI model,and then health fecal microbiota transplantation（h FMT）were performed via enema for 7 consecutive days.The fecal samples from the sham,TBI,TBI+saline,and TBI+FMT groups were collected 1 day before TBI（pre-TBI）,and on 8 days after injury.The fecal samples from donor rats were also collected.And then the 16S RNA sequencing of fecal samples was performed to analyze the microbiome change.Experiment 2:The influence of gut microbiome induced by TBI on neurobehavior in rat.A total of 52 male SD rats were divided into four groups randomly:TBI group（n=10）,Normal group（n=14）,ABX group（n=14）,ABX+FMT_（TBI）group（n=10）.The broad-spectrum antibiotics was used to deplete gut microbiota and the rats received the fecal microbiota induced by TBI transplantation for 3 consecutive days.And the rats was used to explore influence of the gut microbiome induced by TBI on behavior through Morris water maze（MWM）,Elevated plus maze（EPM）,Forced swim test（FST）,open field test（OFT）.Experiment 3:Effect of health fecal microbiota transplantation（h FMT）on the recovery of neural function in rats after TBI.A total of 84 male SD rats were divided into four groups randomly:Sham group（n=21）,TBI group（n=21）,TBI+saline group（n=21）,TBI+FMT group（n=21）.The controlled cortical impact was performed to established TBI model,and then health fecal microbiota transplantation（h FMT）were performed via enema for 7 consecutive days.The modified neurological severity scores（mNSS）,Morris water maze（MWM）,Elevated plus maze（EPM）,Forced swim test（FST）,open field test（OFT）were conducted to explore the effect of health FMT on the neurobehavioral functions.Experiment 4:Effect of health fecal microbiota transplantation on neurons and blood brain barrier（BBB）in rats after TBI.A total of 80 male SD rats were divided into four groups randomly:Sham group（n=20）,TBI group（n=20）,TBI+saline group（n=20）,TBI+FMT group（n=20）.The controlled cortical impact was performed to established TBI model,and then brain tissue samples were collected after health fecal microbiota transplantation（h FMT）continuing for 7 consecutive days.The brain water content and Evans Blue（EB）permeability in brain were measured and the Western blot was used to detect the expression of apoptosis related proteins,namely Cleaved caspase 3,Bax,Bcl-2 and tight junction proteins Zo-1,Occlaudin,Claudin-5,Tunel was used to test the number of apoptotic cells and immunofluorescence was used to test the effect of h FMT on neuron and BBB（blood brain barrier）in the rat after TBI.ResultExperiment 1:There was no difference in the alpha and beta diversity and structure of the gut microbiome in Sham,TBI,TBI+saline and TBI+FMT group before injury,which indicated that there were no biological gut microbiome differences among these groups before surgery.When compared with Sham group,there were significance changes in the gut microbiome,including the alpha-and beta-bacterial diversity,as well as the microbiome composition in TBI group at 8 days after TBI,while there was no difference of gut microbiome among TBI and TBI+saline group.Health FMT could rescue these gut microbiome changes after TBI.Experiment 2:In MWM test,when compared with Nromal and ABX groups,there were no difference in the time and distance to find the platform with the ABX+FMT_（TBI）group.In the probe test,there was a shorter time in the platform quadrant where the platform was originally located in ABX+FMT_（TBI）group comapred with Nromal and ABX groups.The time and number of entries into open arm entry in the EPM test and the time entry into the central area of OFT test was decreased in ABX+FMT_（TBI）group when compared with Nromal and ABX groups.In FST test,the ABX and ABX+FMT_（TBI）group have a longer immobility time compared with Normal group,but there was no difference in immobility time between ABX and ABX+FMT_（TBI）groups.Experiment 3:The mNSS score was higher in rats from TBI,TBI+saline and TBI+FMT group on 1,3,7,14,21 days after TBI compared with Sham group,while there was no difference among TBI and TBI+saline group.The mNSS score of TBI+FMT group was significantly lower than that of TBI and TBI+saline group on days 3,7,14,21 after TBI.In the MWM test,the time and distance to find the hidden platform were no difference between the TBI and TBI+saline group but significantly reduced in TBI+FMT group when compared with TBI and TBI+saline group.In the probe test,the TBI+FMT group spent more time in the quadrant where the platform was originally located compared to the TBI and TBI+saline groups,but there was no difference between TBI and TBI+saline group.The time and number of entries into open arm in the EPM test and the time entry into the central area of OFT test was increase in TBI+FMT group when compared with TBI and TBI+saline groups,but there was no difference between TBI and TBI+saline group.In FST test,the TBI,TBI+saline and TBI+FMT group have a longer immobility time compared with Sham group,but there was no difference in immobility time between TBI,TBI and TBI+saline groups.Experiment 4:The expression of Cleaved-caspase3 and Bax protein in the ipsilateral brain were increased and the number of apoptosis cell around the peri-contusion in the brain was increased in TBI and TBI+saline group,the number of neurons in cortex and hippocampus was decreased in TBI and TBI+saline group when compared with Sham group,but there was no difference between the TBI+saline and TBI groups.When compared with the TBI and TBI+saline groups,the expression of Cleaved caspase3 and Bax was decreased and the expression of Bcl-2 was increased in the ipsilateral brain,the number of apoptosis cell around the peri-contusion in the brain was decreased,the number of surviving neurons in hippocampal and cortical were increased in TBI+FMT group.When compared with the Sham group,the brain water and EB permeability in ipsilateral hemisphere from TBI and TBI+saline groups was increased and the expression of Zo-1,Occlaudin,and Claudin-5 in ipsilateral brain were decreased,but there was no difference between the TBI and TBI+saline groups,when compared with the TBI and TBI+saline groups,the brain water and EB permeability in ipsilateral hemisphere from TBI+FMT group was decreased,and the expression of Zo-1,Occlaudin,and Claudin-5 were increased.Conclusion:TBI can lead to gut microbiota dysbiosis,which can influence the memory and lead to anxiety-like behavior in rats.Health FMT can restore gut microbiota dysbiosis,relieve neurological deficits,promote spatial learning and memory functions recovery,reduce anxiety-like behaviors,preserved neuronal survival,protect BBB and decrease apoptosis around the injured tissue after TBI.PART Ⅱ:THE MECHANISM OF THE INFLUENCE OF GUT MICROBIOME ON THE SECONDARY BRAIN INJURY IN RATS AFTER TRAUMATIC BRAIN INJURYObjective:To explore the effect of the intestinal flora in the pathophysiological process after TBIMethod:Expeiment1:The serum and brain tissue metabolites change after h FMTA total of 45 male SD rats were divided into three groups randomly:Sham group（n=15）,TBI+saline group（n=15）,TBI+FMT group（n=15）,The controlled cortical impact was performed to established TBI model,and then health FMT were performed via enema for consecutive 7days and the feces,serum and ipsilateral brian were collected on 8 days after TBI.Then untargeted and targeted Metabolomics profiling and ELISA were used to screen the molecule change due to h FMT after TBI.Experiment 2:The molecular mechanism of the influence of the gut microbiome induced by TBI on the brainThe male SD rats were divided into two parts:,the first part was divided into as follow:Sham（n=25）group,TBI（n=10）group,TBI+saline（n=25）group,TBI+FMT（n=25）group,TBI+DMB(（n=25）group;the rats in the second part were received the treatment of broad-spectrum antibiotics and one group recrive the fecal from TBI rats microbiota transplantation and divided into as follow:Noraml（n=18）group,ABX（n=18）group,ABX+FMT_（TBI）group（n=18）and ABX+FMT_（TBI）+DMB group（n=18）.We used the mNSS scores,MWM,EPM and OFT test to further explore whether the gut microbiome after TBI can affect the behavior of rats through screening metabolic molecules,and the oxidative stress indicators,the expression of FoxO3a and MsrA were also tested.Result:Experiment1:When compared with the Sham group,trimethylamine（TMA）level in feces,the trimethylamine oxide（TMAO）level in the serum and ipsilateral brain in the TBI+saline group were increased,but trimethylamine（TMA）level in feces,the trimethylamine oxide（TMAO）level in the serum and ipsilateral brain were decreased in the TBI+FMT group when compared with TBI+saline group.There was no difference in the concentrations of betaine,creatinine,choline,and carnitine in the feces of the three groups.Experiment2:When compared with the TBI+saline group,mNSS scores in TBI+FMT and TBI+DMB group were significantly reduced;In the MWM test,the time and distance to find the hidden platform was significantly reduced in TBI+FMT and TBI+DMB group whe compared with TBI+saline group.In the probe test,the TBI+FMT and TBI+DMB group spent more time in the quadrant where the platform was originally located compared to TBI+saline group,but TBI+FMT group spent more time in the quadrant compared to TBI+DMB group.The time and number of entries into open arm in the EPM test and the time entry into the central area of OFT test was increase in TBI+FMT and TBI+DMB group when compared with TBI+saline groups.Compared with the TBI+saline group,the TMAO concentration in the brain was decreased,the expression of FoxO3a and MsrA were increased,and the oxidative stress damage was decreased in TBI+FMT and TBI+DMB group.There were no difference in the time and distance to find the platform in MWM test in ABX+FMT_（TBI）,and ABX+FMT_（TBI）+DMB group.In the probe test,there was a shorter time in the platform quadrant where the platform was originally located in ABX+FMT_（TBI）group comapred with ABX+FMT_（TBI）+DMB groups.The time and number of entries into open arm in the EPM test and the time entry into the central area of OFT test was decrease in ABX+FMT_（TBI）group when compared with ABX+FMT_（TBI）+DMB groups.The concentration of TMAO in brain was increased,and the expression of FoxO3a and MsrA were decreased and the oxidative stress damage was increased in ABX+FMT_（TBI）group when compared with the ABX+FMT_（TBI）+DMB groups.Conclusion:The gut microbiome dysbiosis induced by TBI can lead to anxiety-like behavior and influence the memory of rats throug increasing the content of TMAO,a gut-derived metabolite,inhibiting the expression of Fox03a and MsrA,aggravateing oxidative stress damage in brain.Health fecal microbiota transplantation can relieve neurological deficits and decrease anxiety-like behavior through reducing the level of TMAO,increaseing the expression of Fox03a and MsrA and reduceing the oxidative stress damage in ipsilateral brain after TBI.