Effects of repeated sevoflurane inhalation on cognitive deficits mediated by the gut-brain axis

Effects of repeated sevoflurane inhalation on cognitive deficits mediated by the gut-brain axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of repeated sevoflurane inhalation on cognitive deficits mediated by the gut-brain axis Mengzhu Wang, Chunchun Tang, Yuanping Zhong, Fan Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6843275/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study was designed to determine the effects of repeated sevoflurane (Sev) exposure on children and the possible role of the gut-brain axis. Thirty 28-day Sprague-Dawley rats were randomly divided into five experimental groups. The Sev group was exposed to 3.2% Sev for 2 h on postnatal day (P) 35, P42, and P49. The Sev-Con group only inhaled carrier gas for 2 h at the same time points. The ANT group received antibiotic pre-treatment only since P28 for 3 weeks. The Sev + ANT group was treated with antibiotics for 3 weeks and then exposed to 3.2% Sev for 2 h at each time point. Control group (Con group): animals were freely raised without special treatment. Behavioral tests performance significantly decreased in the Sev group compared to the other groups. Sev exposure promoted serum levels of LPS and TMAO, as well as levels of IL-1β, IL-6, TNF-α, LPS, and TMAO in hippocampal tissue, reduced the expression of ZO-1 in colon tissue and hippocampal vascular tissue, and activated the TLR4/NF-κB/NLRP3 inflammatory pathway in hippocampal tissue. After ANT treatment, the expression of related proteins improved. There were no significant changes in levels of short-chain fatty acids (SCFAs) between groups. Finally, the 16S rRNA sequencing indicated that at the genus level, Ligilactobacillus , Faecousia , and Alloprevotella showed an increasing trend, while Lactobacillus showed a decreasing trend. The results may reflect that repeated Sev anesthesia may induce learning and memory deficits through the gut-brain axis at the developmental stage. sevoflurane gut-brain axis Intestinal flora developmental phase postoperative cognitive impairment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Sevoflurane (Sev) is the mostly used inhalational general anesthetic for pediatric patients due to its unique pharmacological advantages [ 1 ]. One study shows that by the time children reach the age of 3, one seventh of them have experienced general anesthesia at least once, and most of these cases involved the use of Sev inhalation for general anesthesia [ 2 ]. However, postoperative learning and memory deficits related to Sev are gradually being reported [ 3 , 4 ], but the relevant mechanisms are still unclear, causing poor treatment effectiveness. Recent studies suggested that the gut-brain axis pathway may also be an important reason for postoperative cognitive dysfunction (POCD) caused by repeated Sev anesthesia in early life, which may provide new ideas for the prevention and control of POCD [ 5 – 7 ]. Sophisticated glucose metabolism processes are essential to dysfunction in mild cognitive impairment. The SCFAs produced by bacterial fermentation have been recognized as the pivot molecules regulating several neurodegenerative diseases [ 8 , 9 ]. These findings indicate that studies about how the gut-brain axis and microbiota factors impact POCD are urgently needed. The present study aimed to establish a model of intermittent and repeated inhalation of Sev at developmental phase, simulating the situation where clinical children require multiple anesthesia to cooperate with diagnosis and surgical treatment procedures. Our goal was to explore whether Sev anesthesia had a potential effect on learning and cognitive function through the gut-brain axis, to provide new ideas for the prevention and treatment of POCD after anesthesia for pediatric patients. Materials and Methods Animals. In this study, 30 neonatal male SD rats were sourced from Chengdu Dashuo Experimental Animal Co., Ltd (SCXK 2020-0030). These animals were maintained in cages with a standard temperature control system, exposed to a 12-hour light/dark cycle, and provided with food and water and libitum. The research (project no. zyfy-an-2023-0071) was granted approval by the Animal Ethics Committee of Zunyi Medical University and followed the guidelines for the care and use of laboratory animals set forth by the International Institute of Health. Anesthesia management and study design. All subjects were situated in a transparent simulated clinical inhalation anesthesia chamber [ 10 ] that was linked to an anesthesia machine. (Fabius Anesthetic Apparatus, Drägerwerk AG & Co, Lübeck, Germany). The inhalation anesthesia concentration detectors were connected to both sides of the anesthesia chamber to ensure that the inhalation concentration accurately matched clinical requirements. (Vamos Variable Anesthetic Gas Monitor, Drägerwerk AG & Co). On postnatal day (P) 35, P42, and P49, the Sev group was subjected to 2 hours of 3.2% Sev anesthesia. The carrier gas used consisted of 1L/min oxygen and 1L/min air. In contrast, the Sev-Con group was only given the carrier gas for 2 hours at the same time points above. The ANT group received antibiotic pre-treatment since P28 for 3 weeks. The Sev + ANT group was treated with antibiotics for 3 weeks, and exposed to 3.2% Sev for 2 h on P35, P42, and P49. While the animals were under anesthesia, they were maintained at a warm temperature on a heating pad set to 26–28°C. In the chamber, soda lime (Intersurgical, UK, 1181619) was placed at the bottom to absorb carbon dioxide. During the inhalation process, the rats' skin color was monitored, and their oxygen saturation was measured at intervals of every half an hour using a pulse oximeter (Nonin Medical INC, 2500A). Neurobehavioral tests. The Morris water maze test (MWM) mainly comprised three distinct tests: adaptive training, place navigation, and spatial probe tests. The apparatus was a circular black pool measuring 120 cm in diameter, 60 cm in height, and 32 cm in depth, filled with water at a temperature of 24–26℃. To obscure the platform's visual location, 0.5 kg of skim milk was added to the water. The pool is divided into four equal quadrants, with a removable platform (15 cm in diameter and 30 cm in height) placed underwater at the center of the fourth quadrant. A camera and lighting system are installed above the pool to collect data. During the place navigation test, the rats were introduced into the pool from different quadrants for each trial. The time it took for the animal to locate the hidden platform from the moment it entered the water was noted as the escape latency. In the spatial probe test, the platform was removed, and the frequency with which the rat entered the original platform area was documented. Following the swimming tests, the rats were promptly returned to their thermal cages. Western blotting. Hippocampal tissue samples of identical quantity were completely lysed on ice utilizing RIPA lysis buffer (provided by Biosharp Technology Co., Ltd., based in Beijing, China) for a duration of 30 minutes to effectively extract hippocampal proteins. Subsequently, the concentration of the extracted proteins was ascertained via the BCA method (reagents sourced from Biyuntian Biomedical Technology Co., Ltd., located in Shanghai, China). To standardize the protein concentration, a 5 × loading buffer was incorporated and the proteins were subjected to boiling to induce denaturation. Following electrophoresis, the protein samples were electrotransferred from the gel onto a PVDF membrane (acquired from Sigma-Aldrich, Shanghai, China). The membrane was then blocked with a 5% skimmed milk solution at room temperature for 2 hours. Primary antibodies (from Bioss, Beijing, China) were subsequently applied and the membrane was incubated at 4℃ overnight. After thorough washing with TBST three times, each for 5 minutes, a secondary antibody (obtained from affbiotech, Jiangsu, China) was added and the membrane was incubated at room temperature for 2 hours. Further washing with TBST was conducted three times, each for 10 minutes, followed by ECL color development. The resulting image was captured using a gel imaging system (Tanon, Shanghai, China). The Gel-Pro analyzer 4 software was employed for gray value analysis. The expression level of the target protein was quantified by calculating the ratio of the gray value of the target protein band to that of the internal reference protein. ELISA. Rat kits for IL-1β/IL-6/TNF-α detection were sourced from Shanghai Zhuocai Biotechnology Company. Rat hippocampal tissue and serum samples were collected and placed into chilled EP tubes. A 1:9 ratio of PBS was added to the samples, which were then homogenized until clear. Following this, the samples were centrifuged at 4℃ for a duration of 10 minutes. After centrifugation, the pellet was discarded, and the supernatant was carefully pipetted out to be used as the sample for subsequent measurements. For the enzyme-linked immunosorbent assay (ELISA), 50µL of standard samples with varying concentrations and the test samples were pipetted into the enzyme plate wells. Subsequently, 100µL of detection antibodies conjugated with horseradish peroxidase were added to each well. The plate was then incubated at 37℃ for 1 hour. After incubation, the liquid was removed, and the plate was washed thoroughly five times. Next, 50µL of the substrate solution was added to each well, followed by incubation at 37℃ in the dark for 15 minutes. Following this, 50µL of stop solution was added to each well, and the optical density (OD) values of the samples were promptly measured at a wavelength of 450nm. Finally, a linear regression curve was plotted based on the standard samples, and the concentration of each test sample was determined by applying the curve's equation. Immunofluorescence. After each Morris Water Maze test, rats from each group were euthanized via intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg). Their brains were then dissected out and placed into a 4% paraformaldehyde solution. Following 24 hours of fixation, the brains were embedded in paraffin. Subsequently, 2-µm-thick paraffin sections were obtained from each animal using a microtome (Leica RM 2016, Shanghai, China). The expression of ZO-1 and CD31 in both colonic and hippocampal tissues was assessed through immunohistochemistry with streptomycin–peroxidase. In this study, CD31 antibodies (abcam, Shanghai, ab182981), ZO-1 antibodies (Servicebio, Wuhan, GB111402), HRP labeled goat anti rabbit IgG (Servicebio, Wuhan, GB23303) antibodies, and CY3 labeled goat anti rabbit (Servicebio, Wuhan, GB21303) were utilized. The areas and integral optical densities of the hippocampal CA1, CA3, and dentate gyrus (DG) regions in serial sections taken from the same area were measured in microns using image analysis software (Image-Pro plus 6.0; Media Cybernetics Inc., Rockville, MD). The average optical densities of ZO-1, CD31, and other proteins in these regions were used to reflect their expression levels. Gas chromatography testing. Gas chromatographic analysis was conducted utilizing a Trace 1310 gas chromatograph from Thermo Fisher Scientific (USA). Mixed standard stock solutions containing 10 mg/mL of seven short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and caproic acid) were prepared. Calibration curves were then generated by plotting the concentration of the standard solutions on the x-axis and the ratio of the peak area of each standard to that of the internal standard on the y-axis, following the determination of peak areas at various concentrations of the standard solutions. 16S rDNA gene amplicon and transcriptome sequencing. When sequencing the full length 16S rRNA of microbial diversity, high-throughput sequencing technology was used. We entrusted Chengdu Lilai Biotechnology Co., Ltd. to conduct this test. Statistical analysis. The data were analyzed using SPSS 22.0 (SPSS, Chicago, IL). Shapiro-Wilk tests and Levene’s tests were conducted to assess normality and homogeneity of variance, respectively. The escape latency and the crossing of platform times, the level of pro-inflammatory cytokines and immunofluorescence are expressed as mean ± standard error of the mean (SEM). Intergroup comparisons of measured data were performed via one-way analysis of variance. Statistical significance was defined as P < 0.05. Results The impact of multiple exposure to sevoflurane on behaviors During the process, all rats maintained steady breathing. While inhaling Sev, their oxygen saturation remained stable within the range of 95–100%. Throughout the MWM tests, none of the rats were unable to swim or died. In the tests (Fig. 1 ), the escape latency and the crossing of platform times were significantly prolonged and decreased in the Sev group when compared with the Sev-Con group ( P < 0.05 ). However, after treatment with antibiotics, the escape latency of ANT groups showed a decreasing trend, and the number of crossings the platform times had been improved, thus there were no significant differences between the experimental groups ( P > 0.05 ). The effect of multiple sevoflurane exposure on inflammatory damage and the blood-brain barrier of developing rat brain tissue Study results revealed that Sev exposure increased the levels for the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and decreased the levels for the BDNF protein expression compared with Sev-con group ( P < 0.05 ). However, the inflammatory factors IL-1 β, IL-6, and TNF - α in the hippocampal tissue of Sev + ANT group were significantly reduced, and the relative protein expression level of BDNF was significantly increased compared to ANT group ( P < 0.05 ), shown in Fig. 2 A and 2 B. Next, we collected hippocampal samples from all groups of rats. Histomorphological changes of mice were observed by HE staining (Fig. 2 C). Our HE results showed no significant changes in the hippocampal tissue of experimental groups ( P > 0.05 ). This may be due to the short modeling time, and currently molecular level changes have been observed only. The metabolic products of gut microbiota, LPS and TMAO, may enter brain tissue through the intestinal barrier and blood-brain barrier, inducing inflammatory damage to brain tissue. Therefore, we first examined the changes in the blood-brain barrier of the hippocampal tissue (Fig. 3 ). Compared with the Sev-Con group, the expression level of ZO-1 in the colon tissue of Sev group was significantly reduced ( P < 0.01 ). After treatment with ANT, the expression level of ZO-1 in ANT group increased again compared to Sev + ANT group ( P < 0.05 ). The above results further indicate that the gut microbiota metabolites LPS and TMAO may cause brain damage and cognitive impairment through damaged blood-brain barrier. We also investigated the potential role of the inflammatory pathway of the process. Compared with the Sev-Con group, the TLR4, p-p65 and NLRP3 proteins significantly increased in Sev group ( P < 0.01 ), however, compared with the Sev + ANT group, the TLR4, p-p65 and NLRP3 proteins significantly decreased in ANT group ( P < 0.05 ). The above results indicate that the metabolites LPS and TMAO from the gut microbiota may enter the brain tissue through the damaged intestinal barrier and blood-brain barrier, thereby activating the TLR4/NF-κB/NLRP3 inflammatory pathway in rat brain tissue and then induce occurrence finally (Fig. 4 ). Sevoflurane exposure altered intestinal barrier structure Furthermore, we tested the relevant indicators of the intestinal barrier to verify the hypothesis above. As shown in Fig. 5 , there were no significant changes in the colon tissue of experimental groups yet, however, we found significant changes at the molecular level. Compared with the Sev-Con group, the expression level of ZO-1 in the colon tissue of the Sev group was significantly reduced ( P < 0.01 ). After treatment with ANT, the expression level of ZO-1 in the colon connective tissue of Sev group increased gradually ( P < 0.01 ). Sevoflurane exposure led to changes in intestinal metabolites and microbial richness Fecal samples of rats were collected for targeted SCFAs analysis by gas chromatography-mass spectrometry (GC-MS). The research results demonstrate changes in the levels of SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid) in the colon contents and serum (Fig. 6 ). The levels of SCFAs in the colon contents of the Con group, Sev-Con group, and Sev group showed no significant changes. The levels of SCFA in the colon contents of rats were significantly reduced after treatment with ANT ( P < 0.05 ). In addition, the serum results showed no statistically significant difference in the levels of SCFAs. To further investigate the role of microbiota, the composition of intestinal microflora of mice in different groups was analyzed by 16S rDNA sequencing technology. As shown in Fig. 7 , at the phylum level, there were no significant changes in the composition and structure of gut microbiota between the groups. At the genus level, Ligilactobacillus , Faecousia and Alloprevotella showed an increasing trend, while Lactobacillus showed a decreasing trend. Lefse was used to search for differential species between groups, the results are shown in Fig. 8 , Actinobacteriota Coriobacteriia is the dominant family of bacteria in the Sev group. Discussion The widespread presence of POCD brings a heavy burden to families and society, especially when patients and their families pay more attention to whether anesthesia affects intelligence than the disease itself in China now. At present, the trend of young patients receiving general anesthesia is prominent. The perinatal period and early infancy are crucial periods for the development of the body's brain. Epidemiological research [ 11 ] indicates that more than 2 million children under the age of 5 are administered general anesthesia every year, including 1.5 million infants under 12 months of age. It is of great importance to study and prevent the effects of narcotics on children's learning and memory and its pathogenesis. Sev has been extensively applied in pediatric anesthesia because of its rapid induction, stable anesthesia maintains, and minimal respiratory tract irritation. However, many reports confirmed that inhalation of sevoflurane can lead to negative neurocognitive changes [ 12 , 13 ]. Moreover, studies suggest that Sev may be an independent risk factor contributing to the emergence of behavioral disorders and learning difficulties in children who are under the age of 3 [ 14 ]. Nevertheless, the specific mechanism by which Sev causes neurotoxicity remains unclear, thus leads to poor treatment outcomes. Recently, there was growing evidence that gut microbiota and metabolites are almost involved in the entire process of central nervous system development and functional maintenance [ 15 , 16 ]. Sev may cause disruption of the intestinal microenvironment, resulting in structural changes and metabolic abnormalities in the gut microbiota [ 17 ]. Additionally, dysregulation of the gut microbiota may activate NLRP3 inflammasomes in the gut and brain, disrupt the gut/blood-brain barrier, and impair cognitive function [ 18 ]. The above research has inspired us that the gut-brain axis pathway may also be an important cause of postoperative cognitive impairment caused by Sev. The importance of the microbiota gut-brain axis in maintaining internal balance has been recognized [ 19 ]. The gut and brain are associated through various pathways, including the immune system, tryptophan metabolism, vagus nerve, and intestinal nervous system, involving microbial metabolites such as SCFAs [ 20 ]. However, there are few reports on the relationship between gut microbiota and cognitive learning impairment caused by anesthesia [ 21 ]. The existing treatment of POCD mainly focuses on cognitive function therapy, drug therapy, exercise therapy, etc., but the results are very limited, and new ideas are urgently needed for the treatment targets of cognitive impairment [ 22 ]. Our research therefore highlights the role of gut-brain axis in Sev-related neurotoxicity during developmental period and searches for effective prevention measures. According to our study, the repeated Sev exposure across postnatal development affected exploring and learning ability in the young significantly regarding the behavior tests. Reducing the gut microbiota of Sev rats through ANT treatment alleviated cognitive impairment caused by sevoflurane, indicating that Sev induced cognitive impairment in rats is related to gut microbiota. Furthermore, our results indicate that the levels of inflammatory cytokines and BDNF relative protein expression in rat hippocampal tissue induced by Sev are related to gut microbiota, and regulating gut microbiota may be a therapeutic target for cognitive impairment. However, our HE results showed that there were no significant changes in the hippocampal tissues of rats between groups, which may be due to the short modeling time and only molecular level changes currently observed. The metabolic products of gut microbiota, LPS and TMAO, may enter brain tissue through the intestinal barrier and blood-brain barrier, inducing inflammatory damage to brain tissue. The above results indicate that the blood-brain barriers in the hippocampus of Sev group rats were damaged, and ANT treatment can improve the blood-brain barrier in the hippocampus of Sev induced cognitive impairment rats, indicating that the blood-brain barrier in the hippocampus of Sev induced cognitive impairment rats is related to gut microbiota. Next, we detected the expression levels of LPS and TMAO in serum and hippocampal tissue, as well as further validating the pertinent TLR4/NF-κB/NLRP3 inflammatory pathway that LPS and TMAO may activate. The above results indicate that the metabolites LPS and TMAO from the gut microbiota of rats with Sev induced cognitive impairment may enter the brain tissue through the intestinal barrier and blood-brain barrier, thereby activating the TLR4/NF-κB/NLRP3 inflammatory pathway in rat brain tissue. Finally, we observed changes in the levels of SCFAs and found that the effect of sevoflurane on cognitive impairment in developing rats was likely unrelated to the metabolism of SCFAs in the gut microbiota, but additional evidence is required to verify this hypothesis. This study also has certain limitations, for example, our research on animals did not receive real surgical treatment after anesthesia as animals were too small. Furthermore, The relatively brief duration of the modeling in this study might introduce certain interferences to the research outcomes. Further basic and clinical research is needed before applying modulation to prevent or treat Sev-related neurotoxicity. In summary, similarly to previous studies, our study found that Sev affected gut microbiota composition significantly. The results of this study suggest that gut microbiota may be associated with postoperative cognitive impairment caused by Sev. Declarations Data availability statement The 16s rRNA sequencing data reported in this paper has been deposited at NBCI BioSample database. The accession numbers for the datasets are BioProject ID: PRJNA1280557. Data is publicly available as of the date of publication. Ethics statement The animal study was approved by the Animal Ethics Committee of Zunyi Medical University (approval no. zyfy-an-2023-0071). The study was conducted in accordance with the local legislation and institutional requirements. Author contributions FZ conceived and performed the study idea, gathered and analyzed the data, and wrote the manuscript. CT conducted the data analysis of sc-RNA seq. YZ contributed to the LC-MS of glutamine. MW contributed to the final manuscript, conceived and supervised the project, and wrote the manuscript. All authors read and approved of the final manuscript. Funding This research was supported by Natural Science Project of Guizhou Provincial Department of Science and Technology [project No. Qiankehe-zk (2024) General 298]. Consent for publication Not applicable Acknowledgements The present authors would like to acknowledge all the technician included in the study. The authors are grateful to professor Zebing Zheng from the Pediatric Surgery Department for the surgical modeling guidance for animals. Conflict of interest The authors declare no competing interests. References Zhong Y, Zhang C, Wang Y, et al. Multiple exposures to sevoflurane across postnatal development may cause cognitive deficits in older age[J]. Pediatr Res. 2023;93(4):838–44. 10.1038/s41390-022-01943-x . Shi Y, Hu D, Rodgers EL, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6843275","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476734000,"identity":"8ea0179e-e7c2-4754-90c2-fae936750a9e","order_by":0,"name":"Mengzhu Wang","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengzhu","middleName":"","lastName":"Wang","suffix":""},{"id":476734004,"identity":"a97032fb-32cb-48c3-8ac6-91965abcc71f","order_by":1,"name":"Chunchun Tang","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunchun","middleName":"","lastName":"Tang","suffix":""},{"id":476734005,"identity":"979a135d-35d6-4077-a438-52c06fb69b93","order_by":2,"name":"Yuanping Zhong","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanping","middleName":"","lastName":"Zhong","suffix":""},{"id":476734007,"identity":"8d010355-9e83-4cef-9c4b-be9aaf8b5c4e","order_by":3,"name":"Fan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYFACHjYgIcfAwN7AcIAULcZA+gDJWiQSiNQg33/22IMfFQby5pLPHx4uqGGQ5xcjYBljw7l0w54zBoY7Z+cYHJ5xjMFw5mwC1jEz9phJ8Lb9YdxwO4fhMNCRCQa3CWhhY+Yxk/zbZmC/4ebxB4d5/hGhhYeNx0yat80gccMNBoPDvG1EaJHg4UuTljljkLzhDNAvvH0ShP0CCjHJNxUGthuOH3/8meebjTy/NAEtGLaSpnwUjIJRMApGAXYAAJmuP3XVttL0AAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Fan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-06-07 14:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6843275/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6843275/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85819287,"identity":"188e94e1-38b9-4360-9674-be0a615d783c","added_by":"auto","created_at":"2025-07-02 06:13:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6634280,"visible":true,"origin":"","legend":"\u003cp\u003eThe behavioral test results of the experiment. A: Typical trajectory diagram of positioning navigation experiment. B: MWM space exploration experiment. Data are expressed as mean ± SD. Error bars are standard deviation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/9937e68297c562b5956b2209.png"},{"id":85819288,"identity":"c959e6e3-b0da-4e97-8724-f6345a462f6d","added_by":"auto","created_at":"2025-07-02 06:13:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18526530,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression of pro-inflammatory cytokines. A: ELISA analysis of IL-1β, IL-6 and TNF-α. B: WB analysis of BDNF expression. C: mice hippocampal samples observed by HE staining. (Data are expressed as mean ± SD; *\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e: different from Comparison with the ANT group; ** \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e: significantly different from Comparison with the Sev-Con group.)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/c95bc85f5ee6aa57ed9417d4.png"},{"id":85819298,"identity":"c344c2d3-54e6-423a-af70-a8ae45ae3b34","added_by":"auto","created_at":"2025-07-02 06:13:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9593731,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes of blood-brain barrier of developing rat brain tissues. WB analysis of expression level of ZO-1. Expression values of ZO-1 and CD31 analyzed by immunohistochemistry. (Data are expressed as mean ± SD; * \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e: different from Comparison with the ANT group; ** \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e: significantly different from Comparison with the Sev-Con group.)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/33d8d1881ee7ccac2d018210.png"},{"id":85819627,"identity":"1a6575ae-e8bc-42cb-a175-ae9b016ca361","added_by":"auto","created_at":"2025-07-02 06:21:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2681891,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of TLR4/NF - κB/NLRP3 inflammatory pathway expressions. (Data are expressed as mean ± SD; * \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e: different from Comparison with the groups; **\u003cem\u003e P \u0026lt; 0.01\u003c/em\u003e: significantly different from Comparison with groups.)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/bc9721933ae71f255963c626.png"},{"id":85819315,"identity":"2056a11f-70d1-4ee2-8b11-a37a83c2e729","added_by":"auto","created_at":"2025-07-02 06:13:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29504631,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes of intestinal barrier of developing rat brain tissue. (Data are expressed as mean ± SD; ** \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e: significantly different from Comparison with groups.)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/f0b14a57c95ea189aa9164b2.png"},{"id":85819310,"identity":"22ec6905-1789-4ea4-9677-ab605ce0953a","added_by":"auto","created_at":"2025-07-02 06:13:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1445538,"visible":true,"origin":"","legend":"\u003cp\u003eBar plot showed the metabolite enrichment. (Data are expressed as mean ± SD; * P \u0026lt; 0.05: different from Comparison with the groups; ** \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e: significantly different from Comparison with groups.)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/6c63e9471ad439f1eba57e12.png"},{"id":85819293,"identity":"f4581d50-6f50-48d5-9599-c1dfce97f994","added_by":"auto","created_at":"2025-07-02 06:13:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1025360,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in the microbiota composition at the phylum and genus levels in rats.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/4eb7ff5b71830e0d45095bf0.png"},{"id":85819635,"identity":"53055ee3-aec6-4bf3-99c4-a0e9a52123a3","added_by":"auto","created_at":"2025-07-02 06:21:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3034486,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in intestinal microbial richness by Lefse tests.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/a12e7cebbc9969d56eab820b.png"},{"id":101880505,"identity":"abe830f3-465d-46b8-bfaf-ba17baee6cf7","added_by":"auto","created_at":"2026-02-04 15:03:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":63412229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/66621b38-fb18-441d-93dd-cf5f993cea86.pdf"},{"id":85819633,"identity":"ae9b6ae8-a0bd-4446-a029-225ac7f580b8","added_by":"auto","created_at":"2025-07-02 06:21:35","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14765159,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedGelsandBlotsimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-6843275/v1/7388944a3d1423908f63dc86.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of repeated sevoflurane inhalation on cognitive deficits mediated by the gut-brain axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevoflurane (Sev) is the mostly used inhalational general anesthetic for pediatric patients due to its unique pharmacological advantages [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One study shows that by the time children reach the age of 3, one seventh of them have experienced general anesthesia at least once, and most of these cases involved the use of Sev inhalation for general anesthesia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, postoperative learning and memory deficits related to Sev are gradually being reported [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], but the relevant mechanisms are still unclear, causing poor treatment effectiveness.\u003c/p\u003e \u003cp\u003eRecent studies suggested that the gut-brain axis pathway may also be an important reason for postoperative cognitive dysfunction (POCD) caused by repeated Sev anesthesia in early life, which may provide new ideas for the prevention and control of POCD [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Sophisticated glucose metabolism processes are essential to dysfunction in mild cognitive impairment. The SCFAs produced by bacterial fermentation have been recognized as the pivot molecules regulating several neurodegenerative diseases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings indicate that studies about how the gut-brain axis and microbiota factors impact POCD are urgently needed.\u003c/p\u003e \u003cp\u003eThe present study aimed to establish a model of intermittent and repeated inhalation of Sev at developmental phase, simulating the situation where clinical children require multiple anesthesia to cooperate with diagnosis and surgical treatment procedures. Our goal was to explore whether Sev anesthesia had a potential effect on learning and cognitive function through the gut-brain axis, to provide new ideas for the prevention and treatment of POCD after anesthesia for pediatric patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eAnimals.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, 30 neonatal male SD rats were sourced from Chengdu Dashuo Experimental Animal Co., Ltd (SCXK 2020-0030). These animals were maintained in cages with a standard temperature control system, exposed to a 12-hour light/dark cycle, and provided with food and water and libitum. The research (project no. zyfy-an-2023-0071) was granted approval by the Animal Ethics Committee of Zunyi Medical University and followed the guidelines for the care and use of laboratory animals set forth by the International Institute of Health.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnesthesia management and study design.\u003c/b\u003e All subjects were situated in a transparent simulated clinical inhalation anesthesia chamber [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] that was linked to an anesthesia machine. (Fabius Anesthetic Apparatus, Dr\u0026auml;gerwerk AG \u0026amp; Co, L\u0026uuml;beck, Germany). The inhalation anesthesia concentration detectors were connected to both sides of the anesthesia chamber to ensure that the inhalation concentration accurately matched clinical requirements. (Vamos Variable Anesthetic Gas Monitor, Dr\u0026auml;gerwerk AG \u0026amp; Co).\u003c/p\u003e \u003cp\u003eOn postnatal day (P) 35, P42, and P49, the Sev group was subjected to 2 hours of 3.2% Sev anesthesia. The carrier gas used consisted of 1L/min oxygen and 1L/min air. In contrast, the Sev-Con group was only given the carrier gas for 2 hours at the same time points above. The ANT group received antibiotic pre-treatment since P28 for 3 weeks. The Sev\u0026thinsp;+\u0026thinsp;ANT group was treated with antibiotics for 3 weeks, and exposed to 3.2% Sev for 2 h on P35, P42, and P49.\u003c/p\u003e \u003cp\u003eWhile the animals were under anesthesia, they were maintained at a warm temperature on a heating pad set to 26\u0026ndash;28\u0026deg;C. In the chamber, soda lime (Intersurgical, UK, 1181619) was placed at the bottom to absorb carbon dioxide. During the inhalation process, the rats' skin color was monitored, and their oxygen saturation was measured at intervals of every half an hour using a pulse oximeter (Nonin Medical INC, 2500A).\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeurobehavioral tests.\u003c/b\u003e The Morris water maze test (MWM) mainly comprised three distinct tests: adaptive training, place navigation, and spatial probe tests. The apparatus was a circular black pool measuring 120 cm in diameter, 60 cm in height, and 32 cm in depth, filled with water at a temperature of 24\u0026ndash;26℃. To obscure the platform's visual location, 0.5 kg of skim milk was added to the water. The pool is divided into four equal quadrants, with a removable platform (15 cm in diameter and 30 cm in height) placed underwater at the center of the fourth quadrant. A camera and lighting system are installed above the pool to collect data.\u003c/p\u003e \u003cp\u003eDuring the place navigation test, the rats were introduced into the pool from different quadrants for each trial. The time it took for the animal to locate the hidden platform from the moment it entered the water was noted as the escape latency. In the spatial probe test, the platform was removed, and the frequency with which the rat entered the original platform area was documented. Following the swimming tests, the rats were promptly returned to their thermal cages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting.\u003c/b\u003e Hippocampal tissue samples of identical quantity were completely lysed on ice utilizing RIPA lysis buffer (provided by Biosharp Technology Co., Ltd., based in Beijing, China) for a duration of 30 minutes to effectively extract hippocampal proteins. Subsequently, the concentration of the extracted proteins was ascertained via the BCA method (reagents sourced from Biyuntian Biomedical Technology Co., Ltd., located in Shanghai, China). To standardize the protein concentration, a 5 \u0026times; loading buffer was incorporated and the proteins were subjected to boiling to induce denaturation. Following electrophoresis, the protein samples were electrotransferred from the gel onto a PVDF membrane (acquired from Sigma-Aldrich, Shanghai, China). The membrane was then blocked with a 5% skimmed milk solution at room temperature for 2 hours. Primary antibodies (from Bioss, Beijing, China) were subsequently applied and the membrane was incubated at 4℃ overnight. After thorough washing with TBST three times, each for 5 minutes, a secondary antibody (obtained from affbiotech, Jiangsu, China) was added and the membrane was incubated at room temperature for 2 hours. Further washing with TBST was conducted three times, each for 10 minutes, followed by ECL color development. The resulting image was captured using a gel imaging system (Tanon, Shanghai, China). The Gel-Pro analyzer 4 software was employed for gray value analysis. The expression level of the target protein was quantified by calculating the ratio of the gray value of the target protein band to that of the internal reference protein.\u003c/p\u003e \u003cp\u003e\u003cb\u003eELISA.\u003c/b\u003e Rat kits for IL-1β/IL-6/TNF-α detection were sourced from Shanghai Zhuocai Biotechnology Company. Rat hippocampal tissue and serum samples were collected and placed into chilled EP tubes. A 1:9 ratio of PBS was added to the samples, which were then homogenized until clear. Following this, the samples were centrifuged at 4℃ for a duration of 10 minutes. After centrifugation, the pellet was discarded, and the supernatant was carefully pipetted out to be used as the sample for subsequent measurements.\u003c/p\u003e \u003cp\u003eFor the enzyme-linked immunosorbent assay (ELISA), 50\u0026micro;L of standard samples with varying concentrations and the test samples were pipetted into the enzyme plate wells. Subsequently, 100\u0026micro;L of detection antibodies conjugated with horseradish peroxidase were added to each well. The plate was then incubated at 37℃ for 1 hour. After incubation, the liquid was removed, and the plate was washed thoroughly five times. Next, 50\u0026micro;L of the substrate solution was added to each well, followed by incubation at 37℃ in the dark for 15 minutes. Following this, 50\u0026micro;L of stop solution was added to each well, and the optical density (OD) values of the samples were promptly measured at a wavelength of 450nm. Finally, a linear regression curve was plotted based on the standard samples, and the concentration of each test sample was determined by applying the curve's equation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence.\u003c/b\u003e After each Morris Water Maze test, rats from each group were euthanized via intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg). Their brains were then dissected out and placed into a 4% paraformaldehyde solution. Following 24 hours of fixation, the brains were embedded in paraffin. Subsequently, 2-\u0026micro;m-thick paraffin sections were obtained from each animal using a microtome (Leica RM 2016, Shanghai, China). The expression of ZO-1 and CD31 in both colonic and hippocampal tissues was assessed through immunohistochemistry with streptomycin\u0026ndash;peroxidase. In this study, CD31 antibodies (abcam, Shanghai, ab182981), ZO-1 antibodies (Servicebio, Wuhan, GB111402), HRP labeled goat anti rabbit IgG (Servicebio, Wuhan, GB23303) antibodies, and CY3 labeled goat anti rabbit (Servicebio, Wuhan, GB21303) were utilized. The areas and integral optical densities of the hippocampal CA1, CA3, and dentate gyrus (DG) regions in serial sections taken from the same area were measured in microns using image analysis software (Image-Pro plus 6.0; Media Cybernetics Inc., Rockville, MD). The average optical densities of ZO-1, CD31, and other proteins in these regions were used to reflect their expression levels.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGas chromatography testing.\u003c/b\u003e Gas chromatographic analysis was conducted utilizing a Trace 1310 gas chromatograph from Thermo Fisher Scientific (USA). Mixed standard stock solutions containing 10 mg/mL of seven short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and caproic acid) were prepared. Calibration curves were then generated by plotting the concentration of the standard solutions on the x-axis and the ratio of the peak area of each standard to that of the internal standard on the y-axis, following the determination of peak areas at various concentrations of the standard solutions.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rDNA gene amplicon and transcriptome sequencing.\u003c/b\u003e When sequencing the full length 16S rRNA of microbial diversity, high-throughput sequencing technology was used. We entrusted Chengdu Lilai Biotechnology Co., Ltd. to conduct this test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e The data were analyzed using SPSS 22.0 (SPSS, Chicago, IL). Shapiro-Wilk tests and Levene\u0026rsquo;s tests were conducted to assess normality and homogeneity of variance, respectively. The escape latency and the crossing of platform times, the level of pro-inflammatory cytokines and immunofluorescence are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Intergroup comparisons of measured data were performed via one-way analysis of variance. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of multiple exposure to sevoflurane on behaviors\u003c/h2\u003e \u003cp\u003eDuring the process, all rats maintained steady breathing. While inhaling Sev, their oxygen saturation remained stable within the range of 95\u0026ndash;100%. Throughout the MWM tests, none of the rats were unable to swim or died. In the tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the escape latency and the crossing of platform times were significantly prolonged and decreased in the Sev group when compared with the Sev-Con group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). However, after treatment with antibiotics, the escape latency of ANT groups showed a decreasing trend, and the number of crossings the platform times had been improved, thus there were no significant differences between the experimental groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of multiple sevoflurane exposure on inflammatory damage and the blood-brain barrier of developing rat brain tissue\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStudy results revealed that Sev exposure increased the levels for the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and decreased the levels for the BDNF protein expression compared with Sev-con group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). However, the inflammatory factors IL-1 β, IL-6, and TNF - α in the hippocampal tissue of Sev\u0026thinsp;+\u0026thinsp;ANT group were significantly reduced, and the relative protein expression level of BDNF was significantly increased compared to ANT group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we collected hippocampal samples from all groups of rats. Histomorphological changes of mice were observed by HE staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Our HE results showed no significant changes in the hippocampal tissue of experimental groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). This may be due to the short modeling time, and currently molecular level changes have been observed only.\u003c/p\u003e \u003cp\u003eThe metabolic products of gut microbiota, LPS and TMAO, may enter brain tissue through the intestinal barrier and blood-brain barrier, inducing inflammatory damage to brain tissue. Therefore, we first examined the changes in the blood-brain barrier of the hippocampal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Compared with the Sev-Con group, the expression level of ZO-1 in the colon tissue of Sev group was significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). After treatment with ANT, the expression level of ZO-1 in ANT group increased again compared to Sev\u0026thinsp;+\u0026thinsp;ANT group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The above results further indicate that the gut microbiota metabolites LPS and TMAO may cause brain damage and cognitive impairment through damaged blood-brain barrier.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also investigated the potential role of the inflammatory pathway of the process. Compared with the Sev-Con group, the TLR4, p-p65 and NLRP3 proteins significantly increased in Sev group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), however, compared with the Sev\u0026thinsp;+\u0026thinsp;ANT group, the TLR4, p-p65 and NLRP3 proteins significantly decreased in ANT group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The above results indicate that the metabolites LPS and TMAO from the gut microbiota may enter the brain tissue through the damaged intestinal barrier and blood-brain barrier, thereby activating the TLR4/NF-κB/NLRP3 inflammatory pathway in rat brain tissue and then induce occurrence finally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSevoflurane exposure altered intestinal barrier structure\u003c/h3\u003e\n\u003cp\u003eFurthermore, we tested the relevant indicators of the intestinal barrier to verify the hypothesis above. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, there were no significant changes in the colon tissue of experimental groups yet, however, we found significant changes at the molecular level. Compared with the Sev-Con group, the expression level of ZO-1 in the colon tissue of the Sev group was significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). After treatment with ANT, the expression level of ZO-1 in the colon connective tissue of Sev group increased gradually (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSevoflurane exposure led to changes in intestinal metabolites and microbial richness\u003c/h3\u003e\n\u003cp\u003eFecal samples of rats were collected for targeted SCFAs analysis by gas chromatography-mass spectrometry (GC-MS). The research results demonstrate changes in the levels of SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid) in the colon contents and serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The levels of SCFAs in the colon contents of the Con group, Sev-Con group, and Sev group showed no significant changes. The levels of SCFA in the colon contents of rats were significantly reduced after treatment with ANT (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In addition, the serum results showed no statistically significant difference in the levels of SCFAs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of microbiota, the composition of intestinal microflora of mice in different groups was analyzed by 16S rDNA sequencing technology. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, at the phylum level, there were no significant changes in the composition and structure of gut microbiota between the groups. At the genus level, \u003cem\u003eLigilactobacillus\u003c/em\u003e, \u003cem\u003eFaecousia\u003c/em\u003e and \u003cem\u003eAlloprevotella\u003c/em\u003e showed an increasing trend, while \u003cem\u003eLactobacillus\u003c/em\u003e showed a decreasing trend. Lefse was used to search for differential species between groups, the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cem\u003eActinobacteriota Coriobacteriia\u003c/em\u003e is the dominant family of bacteria in the Sev group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe widespread presence of POCD brings a heavy burden to families and society, especially when patients and their families pay more attention to whether anesthesia affects intelligence than the disease itself in China now. At present, the trend of young patients receiving general anesthesia is prominent. The perinatal period and early infancy are crucial periods for the development of the body's brain. Epidemiological research [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] indicates that more than 2\u0026nbsp;million children under the age of 5 are administered general anesthesia every year, including 1.5\u0026nbsp;million infants under 12 months of age. It is of great importance to study and prevent the effects of narcotics on children's learning and memory and its pathogenesis.\u003c/p\u003e \u003cp\u003eSev has been extensively applied in pediatric anesthesia because of its rapid induction, stable anesthesia maintains, and minimal respiratory tract irritation. However, many reports confirmed that inhalation of sevoflurane can lead to negative neurocognitive changes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, studies suggest that Sev may be an independent risk factor contributing to the emergence of behavioral disorders and learning difficulties in children who are under the age of 3 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, the specific mechanism by which Sev causes neurotoxicity remains unclear, thus leads to poor treatment outcomes.\u003c/p\u003e \u003cp\u003eRecently, there was growing evidence that gut microbiota and metabolites are almost involved in the entire process of central nervous system development and functional maintenance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Sev may cause disruption of the intestinal microenvironment, resulting in structural changes and metabolic abnormalities in the gut microbiota [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, dysregulation of the gut microbiota may activate NLRP3 inflammasomes in the gut and brain, disrupt the gut/blood-brain barrier, and impair cognitive function [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The above research has inspired us that the gut-brain axis pathway may also be an important cause of postoperative cognitive impairment caused by Sev.\u003c/p\u003e \u003cp\u003eThe importance of the microbiota gut-brain axis in maintaining internal balance has been recognized [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The gut and brain are associated through various pathways, including the immune system, tryptophan metabolism, vagus nerve, and intestinal nervous system, involving microbial metabolites such as SCFAs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, there are few reports on the relationship between gut microbiota and cognitive learning impairment caused by anesthesia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The existing treatment of POCD mainly focuses on cognitive function therapy, drug therapy, exercise therapy, etc., but the results are very limited, and new ideas are urgently needed for the treatment targets of cognitive impairment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our research therefore highlights the role of gut-brain axis in Sev-related neurotoxicity during developmental period and searches for effective prevention measures.\u003c/p\u003e \u003cp\u003e According to our study, the repeated Sev exposure across postnatal development affected exploring and learning ability in the young significantly regarding the behavior tests. Reducing the gut microbiota of Sev rats through ANT treatment alleviated cognitive impairment caused by sevoflurane, indicating that Sev induced cognitive impairment in rats is related to gut microbiota.\u003c/p\u003e \u003cp\u003eFurthermore, our results indicate that the levels of inflammatory cytokines and BDNF relative protein expression in rat hippocampal tissue induced by Sev are related to gut microbiota, and regulating gut microbiota may be a therapeutic target for cognitive impairment. However, our HE results showed that there were no significant changes in the hippocampal tissues of rats between groups, which may be due to the short modeling time and only molecular level changes currently observed.\u003c/p\u003e \u003cp\u003eThe metabolic products of gut microbiota, LPS and TMAO, may enter brain tissue through the intestinal barrier and blood-brain barrier, inducing inflammatory damage to brain tissue. The above results indicate that the blood-brain barriers in the hippocampus of Sev group rats were damaged, and ANT treatment can improve the blood-brain barrier in the hippocampus of Sev induced cognitive impairment rats, indicating that the blood-brain barrier in the hippocampus of Sev induced cognitive impairment rats is related to gut microbiota.\u003c/p\u003e \u003cp\u003eNext, we detected the expression levels of LPS and TMAO in serum and hippocampal tissue, as well as further validating the pertinent TLR4/NF-κB/NLRP3 inflammatory pathway that LPS and TMAO may activate. The above results indicate that the metabolites LPS and TMAO from the gut microbiota of rats with Sev induced cognitive impairment may enter the brain tissue through the intestinal barrier and blood-brain barrier, thereby activating the TLR4/NF-κB/NLRP3 inflammatory pathway in rat brain tissue.\u003c/p\u003e \u003cp\u003eFinally, we observed changes in the levels of SCFAs and found that the effect of sevoflurane on cognitive impairment in developing rats was likely unrelated to the metabolism of SCFAs in the gut microbiota, but additional evidence is required to verify this hypothesis.\u003c/p\u003e \u003cp\u003eThis study also has certain limitations, for example, our research on animals did not receive real surgical treatment after anesthesia as animals were too small. Furthermore, The relatively brief duration of the modeling in this study might introduce certain interferences to the research outcomes. Further basic and clinical research is needed before applying modulation to prevent or treat Sev-related neurotoxicity.\u003c/p\u003e \u003cp\u003eIn summary, similarly to previous studies, our study found that Sev affected gut microbiota composition significantly. The results of this study suggest that gut microbiota may be associated with postoperative cognitive impairment caused by Sev.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16s rRNA sequencing data reported in this paper has been deposited at NBCI BioSample database. The accession numbers for the datasets are\u0026nbsp;BioProject ID: PRJNA1280557. Data is publicly available as of the date of publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the Animal Ethics Committee of Zunyi Medical University (approval no. zyfy-an-2023-0071). The study was conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFZ conceived and performed the study idea, gathered and analyzed the data, and wrote the manuscript. CT conducted the data analysis of sc-RNA seq. YZ contributed to the LC-MS of glutamine. MW contributed to the final manuscript, conceived and supervised the project, and wrote the manuscript. All authors read and approved of the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Natural Science Project of Guizhou Provincial Department of Science and Technology [project No. Qiankehe-zk (2024) General 298].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present authors would like to acknowledge all the technician included in the study. The authors are grateful to professor Zebing Zheng from the Pediatric Surgery Department for the surgical modeling guidance for animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhong Y, Zhang C, Wang Y, et al. Multiple exposures to sevoflurane across postnatal development may cause cognitive deficits in older age[J]. Pediatr Res. 2023;93(4):838\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41390-022-01943-x\u003c/span\u003e\u003cspan address=\"10.1038/s41390-022-01943-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Y, Hu D, Rodgers EL, et al. 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Neurobiol Dis. 2024;200:106627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nbd.2024.106627\u003c/span\u003e\u003cspan address=\"10.1016/j.nbd.2024.106627\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sevoflurane, gut-brain axis, Intestinal flora, developmental phase, postoperative cognitive impairment","lastPublishedDoi":"10.21203/rs.3.rs-6843275/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6843275/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was designed to determine the effects of repeated sevoflurane (Sev) exposure on children and the possible role of the gut-brain axis. Thirty 28-day Sprague-Dawley rats were randomly divided into five experimental groups. The Sev group was exposed to 3.2% Sev for 2 h on postnatal day (P) 35, P42, and P49. The Sev-Con group only inhaled carrier gas for 2 h at the same time points. The ANT group received antibiotic pre-treatment only since P28 for 3 weeks. The Sev\u0026thinsp;+\u0026thinsp;ANT group was treated with antibiotics for 3 weeks and then exposed to 3.2% Sev for 2 h at each time point. Control group (Con group): animals were freely raised without special treatment. Behavioral tests performance significantly decreased in the Sev group compared to the other groups. Sev exposure promoted serum levels of LPS and TMAO, as well as levels of IL-1β, IL-6, TNF-α, LPS, and TMAO in hippocampal tissue, reduced the expression of ZO-1 in colon tissue and hippocampal vascular tissue, and activated the TLR4/NF-κB/NLRP3 inflammatory pathway in hippocampal tissue. After ANT treatment, the expression of related proteins improved. There were no significant changes in levels of short-chain fatty acids (SCFAs) between groups. Finally, the 16S rRNA sequencing indicated that at the genus level, \u003cem\u003eLigilactobacillus\u003c/em\u003e, \u003cem\u003eFaecousia\u003c/em\u003e, and \u003cem\u003eAlloprevotella\u003c/em\u003e showed an increasing trend, while \u003cem\u003eLactobacillus\u003c/em\u003e showed a decreasing trend. The results may reflect that repeated Sev anesthesia may induce learning and memory deficits through the gut-brain axis at the developmental stage.\u003c/p\u003e","manuscriptTitle":"Effects of repeated sevoflurane inhalation on cognitive deficits mediated by the gut-brain axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 06:13:29","doi":"10.21203/rs.3.rs-6843275/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c606f194-1c19-4464-b11e-6e49499e55db","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T08:57:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 06:13:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6843275","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6843275","identity":"rs-6843275","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}