Accepted Manuscript Title: Intestinal microbiota impact sepsis associated encephalopathy via the vagus nerve Authors: Suyan Li, Jian Lv, Jianguo Li, Zhaolong Zhao, Hui Guo, Yanni Zhang, Shichao Cheng, Jianbin Sun, Hongming Pan, Shaopeng Fan, Zhongxin Li PII: DOI: Reference:
S0304-3940(17)30826-1 https://doi.org/10.1016/j.neulet.2017.10.008 NSL 33152
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23-5-2017 4-8-2017 5-10-2017
Please cite this article as: Suyan Li, Jian Lv, Jianguo Li, Zhaolong Zhao, Hui Guo, Yanni Zhang, Shichao Cheng, Jianbin Sun, Hongming Pan, Shaopeng Fan, Zhongxin Li, Intestinal microbiota impact sepsis associated encephalopathy via the vagus nerve, Neuroscience Letters https://doi.org/10.1016/j.neulet.2017.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Intestinal microbiota impact sepsis associated encephalopathy via the vagus nerve Running title: Intestinal microbiota and SAE
Suyan Li1 , Jian Lv1, Jianguo Li2,Zhaolong Zhao1, Hui Guo2, Yanni Zhang1,Shichao Cheng1, Jianbin Sun1, Hongming Pan2 , Shaopeng Fan1, Zhongxin Li1. 1. Second Department of Surgy, The Fourth Hospital of Hebei Medical University, 12 Health Road, Shijiazhuang, Hebei 050011, P. R. China 2. Department of Emergency, Hebei General Hospital , 348 Heping Road, Shijiazhuang, Hebei 050011, P. R. China *Correspondence to: Professor Zhongxin Li, Tel: 86-0311-66696447; Fax: 86-0311-86077634; E-mail: [email protected]
Sepsis was induced by intravenous administration of lipopolysaccharide (LPS; 10 mg/kg). Fecal microbiota transplantation was performed with fresh feces from the healthy donor rats. Animals received vagotomy at the left cervical level.
Intestinal microbiota was confirmed by 16S rDNA-based molecular analysis of microbiota composition in fecal samples. Behavioral tests and EEG were measured at 7th day as indicators of brain function. In addition, hypothalamus were studied using immunohistochemistry. Moreover, cytokine levels in hypothalamus were analyzed by ELISA. Iba-1, a7nAchR levels in hypothalamus were analyzed by Western-blot at day 7th.
LPS induced a decline of both neurophysiological parameters, which was prevented by FMT,but more importantly, VGX can reversed this benefit of FMT.
Our results suggested that the gut-microbiota-brain axis was vagally mediated, the vagus nerve might be individually sufficient to transmit gut flora information to the central nervous system during sepsis-associated encephalopathy.
Abstract Objective The pathogenesis of sepsis associated encephalopathy (SAE) remains poorly understood. Vagus nerve plays an important role in gut-microbiota-brain axis. This study aimed
to investigate whether vague nerve is a key mediator of the impact of intestinal microbiota on SAE. Methods Male rats were randomly divided into four groups (n=20): SHAM (SH) group, lipopolysaccharide (LPS) group, fecal microbiota transplantation (FMT) +LPS group, and vagotomy (VGX)+ LPS+FMT group. The left cervical vagotomy was performed 30 min before LPS administration in LPS+FMT+VGX group. LPS+ FMT and LPS+FMT+VGX groups received nasogastric infusion of feces from healthy donor three times a day. Fecal samples were collected every two days to monitor changes in microbiota composition by 16S rDNA analysis. Brain function was evaluated by behavioral tests and EEG. The levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-10 in brain cortex were detected by ELISA. The expression of Iba-1 in brain cortex was assessed by immunohistochemistry and Western blot analysis. Results Significant modification of microbiota composition, characterized by a profound increase of commensals in the Firmicutes phylum and depletion of opportunistic organisms in the Proteobacteria phylum, was observed in FMT groups compared to LPS group. Furthermore, we identified a reconstituted bacterial community enriched in Firmicutes and depleted of Proteobacteria. In both FMT groups the diversity of the fecal microbiota and the microbiota composition were similar to SH group. LPS mice treated with FMT demonstrated a better spatial memory and less EEG abnormalities, significantly attenuated levels of IL-1β, IL-6, TNF-α, and decreased number of Iba-1 positive microglia in the cortex, but these beneficial effects of FMT were reversed by VGX. Conclusions FMT can change intestinal microbiota in sepsis patients, and vagus nerve is a key mediator between intestinal microbiota and SAE. These findings suggest that FMT and vagus nerve are potential therapy targets for treating SAE.
Key words: intestinal Microbiota; sepsis associated encephalopathy; sepsis; vagus nerve; fecal microbiota; vagotomy; gut-microbiota-brain axis
Key words：The intestinal microbiota; Sepsis associated encephalopathy; The vagus nerve; The gutmicrobiota-brain axis.
Introduction Sepsis remains a major clinical challenge in modern medicine and the quality of life among sepsis survivors has now been focused on their brain function. In spite of the absence of direct infection of the central nervous system (CNS), sepsis survivors frequently experience significant neurological morbidity[2,3]. Brain dysfunction is a major complication of sepsis and is called sepsis-associated encephalopathy (SAE), including delirium, coma, seizure, and focal neurological signs[4-6]. Despite the high mortality rate and poor prognosis associated with SAE, effective therapy for SAE is still lacking. Currently, SAE treatments focus on controlling the systemic spread of infection and providing supportive therapy[7,8]. The pathophysiology of SAE is multifactorial and related to the effects of systemic inflammation on cerebral perfusion and neuronal activity, including inflammatory cytokines, microscopic brain injury, blood-brain barrier (BBB) compromise, and altered cerebral metabolism, neurotransmission and cerebral microcirculation[9-11]. Neuroinflammation is the main mechanisms underlying the development of SAE[12,13]. Therefore, novel therapeutic strategies for SAE aim to reduce brain inflammation. The nervous system, via an inflammatory reflex of the vagus nerve, can inhibit cytokine release and prevent tissue injury and death[14, 15]. When pathogens invade the body, inflammatory cytokines are produced and released to solitary the afferent sensory nerve, which in turn activates the efferent vagus nerve, promoting its terminus to release acetylcholine (Ach). Ach then stimulates Ach receptor on the surface of inflammatory cells to suppress the synthesis and release of proinflammatory cytokines, inhibiting local and systemic inflammatory responses. The cholinergic anti-inflammatory pathway is involved in the regulation of inflammation in experimental sepsis, and higher levels of vagal activity are associated with lower systemic levels of proinﬂammatory cytokines. Vagal nerve stimulation decreased
lipopolysaccharide (LPS) induced systemic TNF-α release in adult rats. The role of cholinergic anti-inflammatory pathway in SAE is increasingly appreciated. The dysbiosis of intestinal microbiota plays an important role in the dysfunction of the brain . The anti-inflammatory vagus nerve is involved in the gut-microbiota-brain axis. However, the role of vagus nerve in SAE remains largely unknown. In the present study, we aimed to examine whether the vagus nerve is a key mediator of the impact of intestinal microbiota on SAE.
Material and Methods Animals Eighty adult male Sprague-Dawley (SD) rats were purchased from Hebei Medical University, Shijiazhuang, China. The protocol was approved by the Ethics Committee of Hebei Medical University, and all procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The animals were housed under a 12-h light/dark cycle in a temperature-controlled room at 24 ± 1°C with free access to food and water.
Experimental procedures SD rats were randomly divided into SH group, LPS group, LPS+FMT group, and LPS+FMT+VGX group (n=20). Rats in all groups except SH group received intravenous injection of 10 mg/kg body weight LPS (LPS from Escherichia coli, O111:B4; Sigma-Aldrich Chemie GmbH, Germany) through femoral vein, while rats in SH group were given the same volume of saline. Severn days later, fecal samples were obtained, and the brains were removed and stored at −80°C. For the rats in LPS+FMT+VGX groups, left vagotomy were performed 7 days before LPS administration. The left vagus nerves were exposed at the cervical level and carefully dissected from the common carotid artery. For the rats in LPS+FMT group and LPS+FMT+VGX group, fecal microbiota transplantation was performed with fresh feces from the healthy donor rats three times a day, until 7th days. The feces (3-5 g) freshly collected were
diluted with sterile saline (5 ml). The homogenized solution was filtered twice through a sterilized metal sieve. The filtrates (2 ml) were infused into the rats via gavage administration on the day after LPS administration. The rat’s stool was collected at 1, 3, 5, 7 days, and an aliquot (1 g) of each sample was immediately stored at −80°C until DNA extraction.
Morris water maze test Morris water maze test was performed in a circular pool with a diameter of 100 cm and a height of 50 cm, in an isolated environment (Jiliang Software, Shanghai, China). Different shapes were marked on the inner walls of the pool to recognize the relative position of the mouse. Water (21.5 ± 0.5℃) containing food-grade titanium dioxide (JianghuTaibai, Shanghai, China) was filled to a height of the three quarters of the wall. The pool was divided into four quadrants and monitored with a video camera on the top. A platform with a diameter of 7 cm was place into one of the four quadrants, 1 cm below the water surface. Four group rats were trained for Morris water maze task from the 4th day to 7th day after LPS or saline administration. The rats were place on the platform for a total of 10 seconds and were then removed from the pool. In the subsequent training session, the rats were individually placed into each quadrant and were allowed to search the platform for a period of 60 seconds. If the rats were unable to reach the platform within 60 seconds, they would be placed on the platform for an additional 10 seconds. The escape latency, distance of swimming, and time spent in the target quadrant were recorded for each training process. Half an hour after the last training session on the 7th day, the rats were subjected to the probe trial with the platform removed from the pool. All rats were monitored for 60 seconds to observe the distance of swimming, time spent in the target quadrant, and frequency of crossing the platform.
Enzyme-linked immunosorbant (ELISA) assay The hippocampus samples were collected for TNF-α, IL-6, and IL-1β detection at 7 days. The concentrations of TNF-α, IL-6, IL-10 and IL-1β were detected by ELISA kits according to the manufacturer’s instructions. A standard curve was constructed using various dilutions of TNF-
α, IL-6, IL-10 and IL-1β standard preparation. The levels of the cytokines were calculated according to standard curves.
Western blot analysis The mice were killed by decapitation and the brains were removed for the determination of Iba-1 levels in the hippocampus at the indicated time points. Briefly, the hippocampus was homogenized on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH=7.4, 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). The lysates were collected, centrifuged at 10,000 g for 10 min at 4°C. The supernatant was removed, and protein concentration was determined using the Pierce bicinchoninic acid Protein Assay kit (Pierce, Iselin, NJ, USA). Equal amounts of protein were separated on 4-12% NuPAGE Novex Bis-Tris gradient gels (Invitrogen, NY, USA) and transferred to the nitrocellulose membranes. After blocking with 5% non-fat milk for 1 h at room temperature, membranes were incubated with anti- Iba-1 antibody (1:500; Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Pittsburgh, PA, USA) for 2 h at room temperature. The protein bands were detected by enhanced chemiluminescence and the quantitation of bands was performed using the Image J software.
Immunohistochemistry The brains were paraffin-embedded and cut into 4 µm sections. The sections were incubated in 0.1% H2O2 for 30 min to block endogenous peroxidase, followed by incubation with 1.5% normal goat serum for 30 min. The sections were then incubated overnight at 4°C with Iba-1 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA), and then washed and incubated with peroxidase conjugated secondary antibody (Dako EnVision system; Dako) for 1 h at room temperature. The sections were then washed and observed under microscope.
PCR and sequence analysis
Fecal samples were harvested and used for bacterial DNA extraction and sequencing of the V4 hypervariable region in the 16S rRNA gene.
EEG Recordings and analysis EEG was recorded at 7th days after LPS or saline administration. Standard EEG was performed using a Nihon Kohden manufactured EEG-9100J/K portable digital EEG system. EEG recordings and analysis followed the guidelines of the International Federation of Clinical Neurophysiology.
Statistics analysis All data are presented as means ± standard error of the mean (SEM) or standard deviation (SD). Statistical analyses were performed using a one-way analysis of variance. In cases of significance, a Fisher post hoc test was applied (Statview, SAS, Cary, NA, USA). Correlation between two variances was estimated using linear regression analysis with a Pearson test. The significance level was set to P < 0.05.
Results The diversity of the fecal microbiota The diversity of the fecal microbiota in the four groups at 7th days was assessed based on the Chao1 index and observed species richness. LPS group had signiﬁcantly lower phylogenetic diversity compared to SH group, indicating a less diverse fecal microbial composition. LPS+FMT group and LPS+FMT+VGX group had approximately equal phylogenetic diversity compared to SHAM group at 7 days post FMT (Fig. 1A,B). These data indicate that LPS has significant impact on the richness of the gut microbiota, a change that is often related to pathological conditions such as hepatic encephalopathy, obesity, diabetes and inflammatory bowel disease, while FMT can improve the richness of the gut microbiota.
Fecal microbiota transplantation changed gut microbiome in sepsis rats
At the phylum level, the most abundant microbes in the gut of sepsis rats were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Microbiome composition was altered in LPS group compared to SH group, in which significantly greater abundance of Proteobacteria and significantly lower abundance of Firmicutes were observed. Meanwhile, the abundance of Bacteroidetes was decreased whereas Actinobacteria was almost unchanged (Fig. 2A). Bacterial community composition was similar in LPS +FMT group and LPS+FMT+VGX group compared to SH group. The ratio of Firmicutes to Proteobacteria was significantly lower in LPS group compared to SH group (Fig. 2B), but it was similar in LPS +FMT group and LPS+FMT+VGX group compared to SH group. Firmicutes to Bacteroidetes ratio was significantly decreased in LPS group compared to SH group, but it was similar in LPS +FMT group and LPS+FMT+VGX group compared to SH group (Fig. 2C). At the genus level, 2 genus were found only in LPS group (Fig. 2D-G). Among all genus, the genus from the phylum of Firmicutes and Proteobacteria were dominant. Analysis of fecal flora confirmed that LPS group had significantly lower ratios of Bifidobacterium, lactobacillus, Bacteroides, Clostridium, Enterobacter, Enterococcus, especially fewer Bifidobacterium and Lactobacillus compared to SH group, but had significantly higher ratio of Campylobacter, Staphylococcus, Pseudomonas. Meanwhile, LPS+FMT group and LPS+FMT+VGX group had similar ratios of Bifidobacterium, lactobacillus, Bacteroides, Clostridium, Enterobacter, Enterococcus compared to SH group following FMT administration, and the new gut flora was stable for up to 7 days.
Fecal microbiota transplantation improved spatial learning and memory of septic mice Morris water maze training was performed for a total of 4 days, after LPS or Sham administration. After the water maze training, the platform was removed to test the distance, time spent in the target quadrant, and frequency of crossing. The distance travelled was similar among the four groups (P > 0.05) (Fig. 3A). The mice in LPS group spent the shortest amount of time in the target quadrant (6.47 ± 4.63s), which was significantly less than that of SH group
(41.89 ± 7.53s, P < 0.05) and LPS+FMT group (16.35 ± 6.19s, P < 0.05). The time spent in the target quadrant in LPS+FMT group was shorter than in Sham group, but was longer than in LPS group and LPS+FMT+VGX group (P < 0.05, Fig. 3B). The frequency of crossing the platform was significantly reduced in LPS and LPS+FMT+VGX groups (0.17 ± 0.41, 0.16 ± 0.38) compared to SH group (2.67 ± 0.82, P < 0.05), but was significantly increased in LPS+FMT group compared to LPS and LPS+FMT+VGX groups (Fig. 3C). These data indicate that FMT could improve spatial learning and memory of septic mice, and this is mediated by the vagus nerve because VGX disrupted the improvement of spatial learning and memory by FMT.
Fecal microbiota transplantation inhibited microglial activation in the cortex of septic mice Iba-1 is a marker of the activation for microglia. Western blot and Immunohistochemistry analysis showed that Iba-1 expression in the cortex was significantly increased in LPS group, while FMT reduced Iba-1 expression significantly (P< 0.05). In addition, VGX led to increased Iba-1 expression after FMT (Fig. 4). Collectively, these data indicate that FMT could inhibit microglial activation in the cortex of septic mice, and this is mediated by the vagus nerve.
Fecal microbiota transplantation inhibited the release of cytokines in the hippocampus of septic mice ELISA showed that on day 7, the levels of TNF-α, IL-6 and IL-1β were significantly higher in LPS group than in SH group, but were lower in LPS+FMT group compared with LPS group (P