| BackgroundWith the development of society and the advancement of medical and health technology,the life expectancy of human beings is constantly increasing,and the aging of the population is the inevitable result of the progress and development of human civilization.It is estimated that by 2050,the number of people over the age of 65 in the world will reach 1.5 billion,and China’s elderly population will be close to 400 million.Aging is a natural process that cannot be reversed.It is not a disease in itself,but it will decrease the body’s physical and psychological adaptation to the environment,weaken resistance,and increase susceptibility to diseases.It is reported that aging is a high-risk factor of many central nervous system diseases,including AD and POCD,and cognitive decline is one of the most important features of brain aging.China has included the issue of population aging in the "Healthy China" national strategy.Therefore,exploring the underlying mechanism of aging and seeking effective prevention and intervention measures to improve aging-related cognitive impairment and achieve healthy aging is not only an important scientific issue in the field of life sciences,but also a global social issue with important strategic significance.The number of microbes living in the human intestine is as high as one hundred billion,which is more than 10 times than the total number of human cells,and its genome is more than a hundred times than the human genome.For hundreds of thousands of years,the gut microbes have formed the most complex and huge symbiosis with the human body.The intestine is called the "second brain" of the human body,and the intestinal flora is also called a forgotten "organ".A large number of studies have confirmed that the intestinal flora may regulate brain function and behavior through the "microbe-gut-brain" axis,and there is evidence that changes in the intestinal flora may be related to Alzheimer’s disease,Parkinson’s disease,depression,autism and other neuropsychiatric diseases.However,there are few studies on the role of gut microbiota in aging-related learning and memory impairment,and the role of fecal microbiota transplantation therapy in it has not been reported,and further research is needed.POCD is a serious central nervous system complication that occurs after anesthesia and surgery.It is mainly manifested as impaired learning and memory function,impaired executive ability,confusion,and anxiety.At present,clinical studies have shown that POCD has a higher incidence in the elderly.The clinical diagnosis is mainly based on the individualized evaluation of the learning and memory cognitive scale,while in animal studies,the diagnosis and evaluation are performed on a group basis.There are no objective diagnostic markers.Therefore,it is necessary to perform individualized diagnosis and treatment research.Hence,this study intends to explore the alterations of cognitive behavior and gut microbiota in SAMP8 mice,and to further explore the role of gut microbiota in it through FMT.In addition,to explore the alteration of gut microbiota in the physiological aging process of mice and the effects of physiological aging on the incidence of POCD.Moreover,we set out to investigate the possibility of gut microbiota as an individualized diagnostic marker of susceptibility to POCD in mice.Objective,Methods and Results Part ⅠAlteration of cognitive function and gut microbiota in SAMP8 miceObjective:To detect the cognitive function and the alteration of gut microbiota in SAMP8 mice.Methods:7-month-old male SAMP8 mice and SAMR1 mice were included in the experiment.After seven days of accommodation,the Morris water maze test was used to assess the spatial learning and memory function of the mice.Then,fecal samples were collected for 16S rRNA high-throughput sequencing to detect the richness and diversity of the gut microbiota.Results:After seven days of accommodation,there was no significant difference in body weight between the two groups.Compared with SAMR1 mice,SAMP8 mice demonstrated a significantly increased escape latency and path length.In the probe trial,platform crossing and time spent in each quadrant were both significantly lower for SAMP8 mice than for SAMR1 mice.16S rRNA high-throughput sequencing results showed that the Chao 1 and Shannon indices were significantly lower in fecal samples from SAMP8 mice than from SAMR1 mice,although there was no significant difference in the Simpson index between the two groups.Regarding β-diversity,principal coordinates analysis(PCoA)plots of BrayCurtis dissimilarity and principal component analysis(PCA)between the two groups showed that the dots of SAMP8 mice were not close to the dots of SAMR1 mice.Statistical analysis at two different levels of phyla and genus showed that the relative abundance of 12 bacteria was significantly decreased in SAMP8 mice compared with SAMR1 mice.Part ⅡThe effect of FMT on cognitive function and the gut microbiota in pseudo germ-free miceObjective:To explore whether FMT can improve the cognitive function of pseudo germ-free mice by regulating the gut microbiota.Methods:8-week-old male C57BL/6J mice were assigned randomly into four groups:CONT group,normal drinking for 14 days and gavage with sterile PBS solution for 14 days;Vehicle group,14 days of antibiotic "cocktail"(Ampicillin,Neomycin sulphate,and Metronidazole)to construct pseudo-sterile mice,followed by gavage with sterile PBS solution for 14 days;SAMR1 group,14 days of antibiotic "cocktail" to construct pseudo germ-free mice,followed by fecal microbiota transplantation of the SAMR1 mice;SAMP8 group,14 days of antibiotic "cocktail" to construct pseudo germ-free mice,followed by fecal microbiota transplantation of the SAMP8 mice.After FMT,the Morris water maze test was used to assess the spatial learning and memory function,and 16S rRNA high-throughput sequencing was performed to detect the effect of FMT on the gut microbiota of the pseudo germ-free miceResults:The body weights of mice in the four groups showed no significant changes on days 1,15and 28.We found a slight increase in escape latency and path length in the vehicle-treated group compared with the control group,although there was no significant change between the two groups.In the probe trial,platform crossing and time spent in each quadrant were both significantly lower for vehicle-treated mice than for control group mice.Compared with the Vehicle group,the SAMR1 group mice have a slight decrease in escape latency and path length,and platform crossing and time spent in each quadrant increased significantly,while the SAMP8 group showed no significant changes.The 16S rRNA high-throughput sequencing results showed that compared with the control group,Vehicle-treated pseudo germ-free mice had significantly decreased α-diversity indices,including the Chao 1 index,observed species index,PD whole tree,and Shannon index.Interestingly,Gut microbiota transplantation from SAMR1 mice,but not from SAMP8 mice,significantly improved abnormality in Chao 1 index,observed species index,PD whole tree,and Shannon index.In addition,PCoA analysis plots for the four groups showed that the dots of vehicle-treated mice were not close to the dots of control mice and that the dots of SAMR1 mice were close to the dots of control mice but not SAMP8 mice.Moreover,it was found that a total of 6 bacteria were significant altered at the phylum and genus level.Compared with the CONT group,the vehicle-treated group showed significant changes at the level of Lachnospiraceae NK4A136,Moryella,Peptococcus,and Prevotellaceae NK3B31,but SAMP8 or SAMR1 fecal microbiota transplants failed to elicit any changes.Interestingly,SAMP8 or SAMR1 fecal microbiota transplants significantly improved or further aggravated changes in another two bacteria in pseudo germ-free mice.Compared with the vehicle group,the relative abundance of Firmicutes was significantly reduced in SAMP8 group,and the relative abundance of Parasutterella was significantly increased in SAMP8 group;Compared with the vehicle group,the relative abundance of Parasutterella was significantly reduced in SAMR1 group,and there was no significant difference in the relative abundance of Firmicutes in SAMR1 group.Part ⅢAlteration of gut microbiota in C57BL/6J mice at different ages and the effects on postoperative cognitive dysfunctionExperiment 1Objective:To explore the changes of gut microbiota at different ages of C57BL/6J miceMethods:Normal male C57BL/6J mice aged 2,9,18 months,were included in the following three groups:M2 group(2 months old mice),M9 group(9 months old mice),M18 group(18 months old mice).The fecal samples of the three groups of mice were collected,and the differences in gut microbiota were explored through 16S rRNA gene sequencing.Results:16S rRNA gene sequencing results showed that there was no significant difference in the Chao 1 index,Shannon index and Simpson index of the gut microbiota among the three groups of mice at the age of 2 months,9 months and 18 months.However,the results of principal component analysis and principal coordinate analysis suggest that there are significant differences in the composition of the gut microbiota of the three groups of mice.At the phylum level,the relative abundance of Proteobacteria,Deferribacteres,Spirochaetes,Tenericutes,Actinobacteria and Saccharibacteria significantly increased,while the relative abundance of Cyanobacteria decreased with the progress of physiological aging.At the genus level,we found that a total of 29 bacterial genera changed significantly among the three groups of mice.Experiment 2Objective:To explore the incidence of POCD susceptibility in different ages of C57BL/6J miceMethods:Normal 2-month-old,9-month-old,and 18-month-old male C57BL/6J mice were anesthetized and operated(intramedullary fixation of tibial fractures)to construct postoperative cognitive dysfunction model.The Open field test was used to assess the motor function of mice undergone anesthesia and surgery,and the Morris water maze test was used to measure the spatial learning and memory function.The mice were divided into POCD group(POCD susceptible group),Non-POCD group(POCD resilient group)and Undetermined group(uncertain group)by the results of by hierarchical cluster analysis of the MWMT.Followed by the hierarchical cluster analysis,the incidence of POCD among 2,9,and 18-month-old mice were compared.Results:The results of the open field test showed that there was no significant difference in the total distance travelled among the POCD group and the Non-POCD group with the corresponding Control group mice.The Morris water maze test showed that compared with the corresponding control group mice,the 2,9,and 18-month-old POCD group mice required longer escape latency to locate the platform on the fifth day in Place navigation test,they crossed the platform crossing fewer times and spent less time in the target quadrant.The Non-POCD group mice at the age of 2,9,and 18 months shortened the escape latency,increased the platform crossing and the time spent in the target quadrant compared with the POCD group.There were no statistically significant differences in the escape latency,the number of crossing platforms and the time spent in the target quadrant between the Non-POCD group and the corresponding control group.The results of hierarchical cluster analysis showed that the proportions of POCD-susceptible mice at the age of 2,9 and 18 months after anesthesia and surgery were 31.58%,38.10%and 57.45%,respectively.Experiment 3Objective:To explore the differences of individualized gut microbiota between POCD susceptible and non-susceptible mice in elderly miceMethods:Anesthesia and surgery(intramedullary fixation of tibial fractures)were performed in normal 18-month-old male C57BL/6J mice.The mice were divided into POCD group and Non-POCD group by the results of by hierarchical cluster analysis of MWMT.Then,16S rRNA gene sequencing was used to detect the differences in the gut microbiota of the two groups of mice,and the ROC curve was used to evaluate the individualized diagnostic efficacy of the gut microbiota relative abundance in POCD mice.Results:There was a significant decrease in the Shannon index but a significant increase in the Simpson index in POCD mice compared with Non-POCD mice.The PLS-DA analysis revealed that there was a significant difference in the composition of the gut microbiota between the groups.16S rRNA gene sequencing results showed that a total of 24 gut bacteria at six phylogenetic levels(phylum,class,order,family,genus,and species)were significantly altered in fecal samples of mice between the groups.The relative abundance of 10 bacteria was significantly increased in the POCD group compared with the Non-POCD group.In contrast,the relative abundance of 14 bacteria was significantly decreased in the POCD group compared with the Non-POCD group.ROC curves were constructed to indicate the diagnostic ability of the gut bacteria in POCD,the results indicated that the area under the ROC curve of family Dehalobacteriaceae and genus Dehalobacterium was 1,and the sensitivity,specificity,positive and negative predictive values,and accuracy were 100%.Conclusions1.Abnormalities in the gut microbiota may be involved in the pathogenesis of aging-related cognitive impairment in SAMP8 mice;2.Fecal microbiota transplantation is a promising intervention treatment for the richness,diversity and composition of gut microbiota,thereby improving the spatial learning and memory function of pseudo germ-free mice;3.Alteration of the gut microbiota may be involved in the process of physiological aging,and may play an important role in POCD susceptible mice;4.Family Dehalobacteriaceae and Genus Dehalobacterium can be used as individualized diagnostic markers for POCD susceptible mice. |
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