Correlation Between Dysregulation Of Intestinal Flora And Postoperative Cognitive Dysfunction

ObjectiveTo explore the potential mechanism of anesthesia and surgery induced dysregulation of intestinal flora and its metabolites in aging mice,promoting the release of inflammatory cytokines,triggering the neuroinflammatory response in the peripheral and central nervous system,and driving the postoperative cognitive dysfunction related to aging.MethodsA total of 40 SPF male C57BL/6J mice,weighing 27-35g and aged 16-17 months,were divided into two groups according to random number table method:surgery group(A+S,n=20)and control group(Sham,n=20).Isoflurane and exploratory laparotomy were used to construct the model of postoperative cognitive dysfunction.Mice in the operation group were anesthetised with 1.4%isoflurane and 100%oxygen for 15 min,and then continued to be anesthetised with 1.4%isoflurane and 100% oxygen for explorative laparotomy.The operation lasted for 10 min.After surgery,mice were put back into a transparent anesthetic box composed of 1.4% isoflurane and 100% oxygen for 2 hours.The mice in the control group could not perform exploratory laparotomy.The feces of mice in the surgery group(A+S)and Sham control group(SHAM)were collected 48 hours after surgery,and the preoperative baseline feces of mice in the A+S group were collected.The DNA extracted from the feces was detected by 16S rDNA high-throughput sequencing,and the changes in intestinal bacterial richness,composition and diversity of the feces of mice were analyzed.The changes in fecal metabolic profiles of mice in the A+S operation group and the SHAM control group were detected by liquid chromatography-mass spectrometry.Blood samples of both groups were collected 48 hours after operation,and the levels of IL-1β,IL-6 and TNF-α in hippocampus and peripheral blood were determined by ELISA.The open field test and Morris water maze were used to test the memory and cognition function of the mice 2 days after operation.Finally,fecal bacteria from Sham group and A+S group were transplanted into antibiotic-induced pseudo sterile mice.After continuous intragastric treatment for 2 weeks,the effects of fecal bacteria transplantation on learning and memory behavior of mice in each group were detected by Morris water maze.Results(1)Behavioral results:In open field experiment,there were no significant differences in dwell time and exploration distance between the two groups(P BBB 00.05).Water maze test:localization and navigation test:Compared with Sham group,the escape latency of mice in A+S group was significantly prolonged(P<0.05),and there was no statistical significance in the average movement velocity between the two groups(P>0.05).Space exploration test:the time spent in the target quadrant of mice in the operation group decreased,and the difference was statistically significant(P<0.05).(2)Intestinal microecological results:α-diversity and β-diversity index analysis showed that the richness and diversity of fecal intestinal flora of mice in A+S group were different after the operation(Post＿A+S)and before the operation(Base＿A+S).Compared with Base＿A+S group,In the Post＿A+S group,the relative abundance of Bacteroidales＿unclassified,Mucispirillum and Clostridiales unclassified decreased.However,Escherichia-Shigella,Actinomyces,Ruminococcus＿gnavus＿group and Lachnospiraceae＿FCS020＿group were all classified into three groups:Escherichia-Shigella,Actinomyces,Ruminococcus and Lachnospiraceae＿FCS020＿groupThe relative abundance of).In addition,compared with Sham group,the relative abundance of intestinal microflora(6 species)and genera(15 species)in postoperative feces of mice in A+S group was significantly different(P<0.05).Among them,the proportion of relative abundance of Fusobacteria,Escherichia-Shigella,Fusobacterium and Proteobacteria in A+S group increased significantly.(3)Change of metabolic profile detected by LC/MS:There were significant differences in fecal metabolic profiles between Sham group and Post＿A+S group(P<0.05),which involved the synthesis and degradation of amino acids,neurotransmitters and fatty acids of some metabolites.In the Post＿A+S group,tryptophan,kynurenic acid,N-oleoyl γ-aminobutyric acid(GABA),The contents of 2-indolecarboxylic acid and glutamic acid were significantly increased.Two groups of 35 different metabolites were identified by MS/MS and online database comparison.At the same time,16S and metabonomics combined analysis showed that some different metabolites were associated with the changes in the abundance of specific bacteria.(4)Expression of pro-inflammatory cytokines:Compared with Sham group,the expression of IL-1β and IL-6 in the hippocampus of A+S group was significantly increased,while the expression of IL-1β,IL-6 and TNF-α in serum of A+S group had no statistical significance(P>0.05).(5)Fecal bacteria transplantation experiment:The results of water maze navigation showed that compared with the pseudo sterile mice transplanted in Sham group,the escape latency of pseudo sterile mice transplanted in A+S group was longer(P<0.05),and there was no significant difference in the average velocity of mice among all groups(P>0.05).The results of space exploration test showed that the target quadrant residence time of pseudo sterile mice in postoperative feces of transplanted A+S group was shortened,and the difference was statistically significant compared with other control groups(P<0.05).ConclusionIn this present study,we preliminarily confirmed that,compared with group Sham,the number of conditioned pathogenic bacteria in group A+S was increased,suggesting that it is conducive to the occurrence and development of intestinal inflammation.Compared to the Sham group,the pathways involved in synthesis and degradation of amino acids,fatty acids,and organic acids were interfered with,thus affecting their important roles in maintaining intestinal barrier function and inflammation-related immunosuppression.The results of intestinal microbiota taxonomy and metabonomic showed that these metabolites were strongly associated with disease-related microorganisms,and the altered intestinal flora was highly likely to be associated with the pathogenesis of postoperative cognitive dysfunction in elderly mice.