In this study, we found that anesthesia/surgery caused intestinal flora imbalance and induced neurocognitive impairment in aged rats. Intestinal flora may act on the central nervous system by activating intestinal Th17 cells and increase the secretion of IL17, leading to cognitive impairment, which may be a potential mechanism of PND in aged rats.
There are a huge number of microorganisms on the surface and in the body of our human body, about 10 ^ 14 bacteria, which contain 100 times the number of genomes of humans . The intestinal flora plays an important role in maintaining human health and can affect the CNS in many ways, such as changing the composition of the intestinal flora , activating the immune system , the vagus nerve , the tryptophan metabolites , the secretion of intestinal hormones , the bacterial metabolites  and others. In view of the important role of intestinal flora in maintaining human health, the intestinal flora was even called "neglected human organ" .
A large number of studies have confirmed that intestinal flora played an important role in the pathophysiology of neurodegenerative diseases. In the animal model of POCD caused by abdominal exploratory surgery, using the method of cluster analysis, it was found that the abundance and diversity of the intestinal flora in the POCD group was significantly reduced . Similar to the results of the above study, we found that anesthesia/surgery caused intestinal flora imbalance and cognitive impairment in aged rats. When compound antibiotics were given 4 weeks before anesthesia/surgery to relatively eliminate the intestinal flora, the memory ability of old rats was improved. This result is consistent with the study of others . Through 16S rRNA sequencing, we found that the abundance of five family-level intestinal flora changed significantly, including Lactobacillaceae, Lachnospiraceae, Turicibacteraceae, Erysipelotrichaceae and Enterobacteriaceae. At the genus level, the abundance of Lactobacillus, Blautia, Turicibacter and Dorea changed significantly. In patients with depression, multiple sclerosis (MS) and Hashimoto’s thyroiditis, the abundance of Blautia and Dorea was increased [33–36]. Lactobacillus regulated emotional behavior and the expression of central gamma-aminobutyric acid (GABA) receptor through the vagus nerve . Lactobacillus reuteri reversed the autism-like behavior of various types of autistic mice . Studies have confirmed that Ruminococcaceae and Lachnospiraceae were related to learning and memory. In the model of memory impairment caused by high-fat diet, the increased abundance of Ruminococcaceae and Lachnospiraceae was related to the increase of inflammation-related genes in the hippocampus . In our study, we found that anesthesia/surgery reduced the abundance of Lactobacillus, and increased the abundance of Lachnospiraceae and Ruminococcaceae in the intestinal flora. Compared with the CON group, the compound antibiotics had no obvious effect on the memory ability of aged rats; Compared with the SUR group, the compound antibiotics improved the memory ability of aged rats. It suggested that the occurrence of PND may correlate to the increase of “harmful” bacteria in the intestine, for example Lachnospiraceae and Ruminococcaceae, and elimination of the “harmful” bacteria through compound antibiotics helped to improve the cognitive function.
Intestinal flora played a decisive role in the maturation of the immune system by stimulating local and systemic immune responses in the intestine , especially the maturation of intestinal-associated lymphoid tissues . The lamina propria of the small intestine is rich in Th17 cells, and the changes in the local microenvironment are the main factors for Th17 cells activation. In 2015, Koji Atarashiit et al. discovered that the adhesion of microorganisms to intestinal epithelial cells was a crucial condition for inducing the production of Th17 cells . The level of intestinal Th17 cells could be changed by regulating the intestinal flora through diet [42, 43]. Some researchers pointed out that intestinal microbes were an important factor affecting the proliferation and differentiation of Th17 cells . The phenotype of differentiation of intestinal CD4 + T cell was determined by microorganisms rather than cytokines . The above studies suggested that the intestinal flora, especially the interaction of intestinal flora with the local microenvironment, was a key factor in the production of Th17 cells.
Anesthetic drugs and surgical trauma may affect the intestinal homeostasis. In our study, we found that anesthesia/surgery caused intestinal flora imbalance, and simultaneously, Th17 cells increased significantly in the PPs, MLN and brain. When compound antibiotics were given before anesthesia/surgery to eliminate the intestinal flora, the number of Th17 cells reduced simultaneously in above tissues. The compound antibiotics treatment significantly reduced intestinal Th17 cells [45, 46] and IL-17 cytokines .
Studies have shown that IL-17A was involved in the pathophysiological process of neurodegenerative diseases. Researchers found that the level of IL-17A was significantly increased in the cerebrospinal fluid and plasma of patients with MS, AD, and PD, and was related to the severity of these diseases [48, 49]. However, how IL17A causes neurodegenerative diseases is less known yet and controversies remain. Most people believe that IL-17A caused the occurrence and development of diseases by activating glial cells (especially microglia) [48, 50]. For example, bone cancer pain leaded to the activation of Th17 cells, which promoted the activation of microglia and further aggravated the pain of bone cancer patients. Intrathecal injection of IL17 antibody could relieve pain in such patients . Through immunofluorescence, we found that anesthesia/surgery significantly increased the fluorescence intensity of IL17 and IL17R in the hippocampus of rats. When compound antibiotics were given before anesthesia/surgery, the fluorescence intensity of IL17 and IL17R in the hippocampus was significantly reduced. We also found that most of the IBA1 that specific in the microglia could co-label with IL17R in the hippocampus. These results indicated that anesthesia/surgery caused a significantly increase in the number of activated microglia in the hippocampal of aged rats, while the administration of compound antibiotics before anesthesia/surgery could evidently reduce the number of activated microglia.
Microglia are macrophages of the CNS and have multiple functions . Sequencing technology revealed that microglia were the determinants of central nervous system diseases. Through Genome-Wide Association Studies (GWAS), many risk genes for central neurodegenerative diseases (including AD, PD, schizophrenia, autism, and MS) that have been discovered were expressed by microglia . Studies have confirmed that microglia also played an important role in the occurrence of POCD. Study has shown that through inhibiting the colony stimulating factor 1 receptor (CSF1R) in adult mice, it effectively depleted the microglia of the CNS, effectively prevented inflammation infiltration in the hippocampus, and improved cognitive function after tibial fracture .
In summary, the intestinal homeostasis of old rats was relatively fragile. Under the combined action of anesthesia and surgery, the intestinal flora was prone to imbalance, harmful flora increased, beneficial flora decreased. Intestinal flora imbalance caused activation and increase of intestinal Th17 cells, which increased the secretion of IL17 and other pro-inflammatory cytokines to aggravate system inflammation response. Increased Th17 cells and IL17 damaged BBB (confirmed in the previous study ), entered the CNS and acted on microglia, and consequently caused central immune inflammation and cognitive impairment (Fig. 7).
There are some controversies about the effects of compound antibiotics on cognitive function. Some researchers have found that the use of compound antibiotics to clear the intestinal flora from the weaning of mice affected the new objects recognition memory . Short-term (11 days) use of compound antibiotics affected the new objects recognition memory, but spatial memory was not affected . In our study, we found that the administration of 4-week compound antibiotics has no effect on space and working memory, which is consistent with the previous study . For the effects of compound antibiotics on cognitive function, more research is needed to further confirm.
Our study has some limitations. We did not identify which intestinal flora was involved in Th17 cells activation or cognitive impairment caused by anesthesia/surgery. Further research is needed to identify specific gut bacteria involved in PND, such as transplanting feces or specific gut bacteria into germ-free mice. Besides, in our experiment, we did not set up a simple anesthesia group, because in the process of modeling, our anesthesia time was short to 30 minutes, and a large number of studies also confirmed that sevoflurane has little effect on cognitive function. In primate experiments, it was found that sevoflurane exposure had little effect on the cognitive function of adult monkeys and did not cause the activation of microglia . According to reports, 2.5% sevoflurane exposure for 1 hour had little effect on the cognitive function in adult rodents . There were even studies reported that sevoflurane could alleviate the cognitive impairment induced by lipopolysaccharide (LPS) . On the other hand, anesthesia and surgery cannot be completely separated. So, we did not set up a simple anesthesia group.