The Effects of Vagus Nerve Stimulation Mediated Protection of Intestinal Epithelial Glycocalyx on Acute Lung Injury Associated With Traumatic Hemorrhagic Shock/Resuscitation

Background Acute lung injury (ALI) is a common complication after THS/R, and vagus nerve stimulation (VNS) could alleviate lung injury by activating cholinergic anti-inammatory pathway (CAP) during traumatic hemorrhagic shock/resuscitation (THS/R). The purpose of this study was to explore the effects of VNS on intestinal epithelial glycocalyx and acute lung injury associated with THS/R. Methods cannulated PE-50 tubing blood anticoagulated infusion BP-100 blood pressure monitor. internal jugular vein dissected cannulated with PE-50 tubing, the vagus was identied carefully along the carotid midline suture. carotid


Background
Traumatic Hemorrhagic shock (THS) is the most common type of shock. Although signi cant advances in treatment of THS, the one of most effective therapy is volume resuscitation. The process of traumatic hemorrhagic shock/resuscitation (THS/R) could lead to intestinal ischemia-reperfusion injury , disrupting the gut barrier and increasing intestinal permeability, then the cytokines generated by gut could across to sterile internal milieu and induce systematic in ammatory response (1,2). Overwhelming gut-derived factors could cause pulmonary in ammatory reaction, increasing lung vascular permeability causing tissue edema and eventually leading to acute lung injury(ALI) (3) or acute respiratory distress syndrome (ARDS). ALI/ARDS after THS/R might trigger the process of multiple organ dysfunction failure and increase disability rate and mortality rate (4).
Cholinergic anti-in ammatory pathway (CAP) is a kind of neuroimmunomodulation mechanism to regulate in ammation reaction (5), which processed by the end of vagus nerve releasing acetylcholine (Ach) to combine with the alpha 7 nicotinic acetylcholine receptor(α7nAchR) on the surface of in ammatory cells to regulate the level of in ammatory cytokines, such as tumor necrosis factor-α(TNFα), nuclear transcription factor-κB(NF-κB) and interleukin-6(IL-6) (6). Many studies show that vagus nerve stimulation (VNS) could alleviate the damage of gut barrier associated with THS/R by the activation of CAP but the speci c mechanism is still unknown (7,8).
Glycocalyx is a kind of complex compound located at the surface of endothelial cells and epithelial cells, consisting mostly of glycosaminoglycans(GAGs), including heparan sulfate(HS) (9), hyaluronic acid(HA), and chondroitin sulfate(CS). Hyaluronidase could diminish glycocalyx layers by cleavage HA. Although many studies showed that endothelial glycocalyx is the rst barrier to maintain vascular endothelial permeability (10,11), the function of glycocalyx on intestinal epithelial cells (IECs) is still unclear. Thus, the aim of this study is to explore the effects of VNS on glycocalyx located on IECs and acute lung injury associated with THS/R.

Animals
Male Sprague-Dawley rats (SD rats, weighing 250 ± 20 g) were purchased from Wuhan Center for Disease Control and Prevention, China. All rats were kept in air-conditioned room with 12hours (h) light and 12 h dark cycle, free access to water, but 12-hour fast before experiment. All experiments were approved by the Ethics Investigation Board of the Central Theater Command General Hospital of the Chinese People's Liberation Army, Wuhan, China.

Animal model constructing
This experiment consisted of two parts. In rst part, twelve SD rats (n=12) were randomly divided into 2 groups(n=6), Control group and glycocalyx inhibitor group(GI group). Rats were anesthetized with 6% chloral hydrate (5ml/kg) intraperitoneally. We incised the skin along the midline of the animals' neck and separated the trachea and performed the tracheotomy, then imbedded 14G catheter into the trachea, retaining spontaneous respiration. Temperature measurement probe was inserted into anus to monitor temperature. A midline laparotomy length 5cm was performed, and the cecum was identi ed and protected with moist gauze. A 10-cm segment of ileum was identi ed at 20-30cm from cecum, ligated both ends of the ileum, and incised distally and ushed with 2.0 mL of 0.9% normal saline to remove feces. Once ushed, the incision was closed. 12mg/kg Hyaluronidase and 20mg/ml Fluorescein isothiocyanate dextran (FD4) were prepared. A total of 1-mL Hyaluronidase or 0.9% normal saline was injected into the 10-cm segment of ileum in GI or Control group respectively, then the abdominal incision is closed using 4-0 suture.30 minutes later, midline laparotomy incision was released, and 1ml 20mg/ml FD4 was injected into the 10-cm segment of ileum carefully preventing any spillage onto the external bowel allowing for a circulation of 30 minutes. Then 1 mL of venous blood was taken from the right ventricle into a heparinized syringe, protected from light and placed on ice, then the rats were sacri ced.
During the whole procedure, we used thermal equipment to maintain rats' body temperature between 36.0℃ and 38.0℃.
In second part, twenty-four (n=24) SD rats were randomly divided in 4 groups (n =6), Sham surgery group(SS group), traumatic hemorrhagic shock/resuscitation (THS/R group), Vagus nerve stimulation group(VNS group) and Vagus nerve stimulation-methyllycaconitine group(VSM group). SD rats were anesthetized, intubated and monitored temperature as above. Right carotid artery was dissected and cannulated with PE-50 tubing for blood withdraw, anticoagulated with heparin(5U/mL) infusion and attached to a BP-100 blood pressure monitor. Left internal jugular vein was dissected and cannulated with PE-50 tubing, and the right vagus nerve was identi ed and separated carefully along the right carotid artery. THS/R group, VNS group and VSM group received abdominal trauma and traumatic hemorrhagic shock/ resuscitation. A midline laparotomy length 5cm was performed to simulate trauma, then the incision was closed using 4-0 suture. Blood was withdrawn through the right carotid artery into an anticoagulation solution syringe until the mean arterial pressure (MAP) reached 30 to 35 mmHg at the rate of 1 mL per minute and maintained for 60 minutes. Before the resuscitation, VNS group and VSM group received VNS, VSM group also received methyllycaconitine(MLA,10mg/Kg) intraperitoneally before VNS, and THS/R group only separated the right vagus nerve. The process of VNS was as following: a platinum electrode was placed across the nerve, attached to the neurostimulator, and the VNS was applied for 15 minutes at 1.0mA 0.1ms 1Hz. Then the rats were resuscitated with shed blood and 0.9% normal saline at the rate of 1 mL per minute through internal jugular vein to make MAP reach the 90% of baseline, and maintained for 2.0 hours. 20 mg/kg Evans Blue dye (EBD) and 25 mg/mL FD4 were prepared. Then, midline laparotomy incision was released, and the cecum was identi ed and disposed as above. Then a total of 1mL FD4 was injected into the 10-cm segment, carefully preventing any spillage onto the external bowel. Meanwhile, EBD (20mg/kg) was administered via the internal jugular vein. After circulating for 30min, 2mL of venous blood was removed from the internal jugular catheter into a heparinized syringe, the lungs were perfused with 0.9% normal saline through ventriculus dexter, then the rats were sacri ced to obtain lung tissue and gut tissue. During the whole procedure, we used thermal equipment to maintain temperature between 36.0℃ and 38.0℃.

Lung vascular permeability assay
Lung vascular permeability was measured as Huang. et al described (12).We used EBD extravasation to assess the pulmonary vascular leakage. The lungs were excised and imaged, then the lungs were homogenized in formamide. Following overnight extraction, the tissue uid was centrifuged at 12000g for 10 min. The EBD concentration of the supernatant was obtained by comparing with a standard curve measured on Microplate Reader at 620 nm absorption.

Gut Permeability Assay
Gut permeability was measured as Levy. et al described (13). 1 ml venous blood was protected from light and placed on ice. Plasma FD4 was obtained by centrifuging 3000 rpm for 10 minutes at 4°C and the concentration was obtained by comparing with a standard curve measured on Microplate Fluorescence reader at excitation 485/20, emission 528/20, and a sensitivity of 40.

ELISA
ELISA assays were used to measure the levels of cytokines in lung tissues and Ach in plasma. The TNF-a, IL-6, NF-κB, and Ach ELISA kits were purchased from Coolaber(Shanghai, China). All procedures were performed according to the manufacturers' protocols.

Immuno uorescence
After rats sacri ced, gut tissues were collected and xed with 4% paraformaldehyde, para n embedded, and cut into 4 μm slices. Those slices were depara nized with xylene, and then dehydrated in gradient ethanol. Subsequently, antigen retrieval was performed, and slices were washed three times in PBS (5 min/wash). After blocking, the slices were incubated with primary antibodies to SDC-1 and FITCconjugated secondary antibody. After washing, the slices were stained with DAPI and then sealed with an anti-fade uorescence medium. The slices were observed with a uorescence microscope and analyzed by ImageJ software.

H&E staining
The lung tissues and gut tissues were collected and xed with 4% paraformaldehyde, para n embedded, and cut into 5 μm slices. Those slices were depara nized with xylene, and dehydrated in gradient ethanol, then stained with hematoxylin and eosin and observed under an optical microscope. Five random elds were identi ed in a blinded fashion at 100x magni cation to quantify morphologic damage. The lung injury scores (14) were obtained by assessments of in ammatory cell in ltration in the airspace or vessel wall, alveolar congestion, hemorrhage, alveolar wall thickness, and hyaline membrane formation. The degree of gut injury was identi ed if any one of the following were present(15): a subepithelial space, moderate lifting of the epithelial layer from the lamina propria, massive epithelial lifting down the sides of the villi, denuded villi with lamina propria and dilated capillaries and/or digestion and disintegration of the lamina propria with hemorrhage, and ulceration.

Western blot analysis
The lung tissues and gut tissues were frozen at -80℃. The extraction of proteins was performed using protein extraction kit (Coolaber, China) in accordance with the manufacturer's instructions. The lung tissues and gut tissues were homogenized respectively on ice with RIPA buffer, and the protein concentrations were determined by BCA protein assay kit (Coolaber, China). The protein samples were boiled in sample buffer and loaded into each lane, separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were washed three times in Tris-buffered saline with Tween 20 (TBST) and blocked with 5% nonfat milk for 2 h at room temperature. Subsequently, the samples were incubated with primary antibodies (MPO TNF-α IL-6 IL-10 NF-κB) overnight at 4 °C, followed by membrane washing three times for 10 min. Then, the samples were incubated with the second antibody at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit. The images were quantitatively analyzed by using the Image J analysis software.
2.9 Quantitative real time polymerase chain reaction (RT-qPCR) Total RNA was isolated from lung tissues and gut tissues respectively using TRIzol reagent (Simgen, China), according to the manufacturer's instructions. cDNA was synthesized using a ReverTra Ace qPCR RT kit (Simgen China), according to the manufacturer's protocol. RT-PCR was performed using SYBR Premix Ex Taq (Simgen China). The levels of mRNA expression of target gene were measured using primers purchased from TSangon Biotech (Shanghai, China); these primer sequences have been shown in Table 1 Data were presented as Mean ± SD and compared by independent Sample T test and one-way analysis of variance (ANOVA), and the Student-Newman-Keuls (SNK) post hoc test was used for statistical analysis to compare the data among all groups. A significant difference was presumed when p<0.05.

Hyaluronidase damaged glycocalyx on IECs and increased intestinal barrier permeability
Syndecan-1(SDC-1) is the main component of the core and side-chain structures of the glycocalyx, and is often used as an indicator of the integrity of the glycocalyx. In this study, compared with Control group, the results showed that pretreatment with hyaluronidase could damage the glycocalyx on IECs in GI group ( Figure.1A-B, P <0.0001). The gut permeability was re ected by FD4 concentration in plasma, and higher FD4 concentration meant higher gut permeability. The results shown that, compared with Control group, pretreatment with hyaluronidase in GI group could increase FD4 concentration in plasma P=0.0043 ( Figure.1C).

Hyaluronidase induced intestinal and lung histology injury
In our study, the results showed that, compared with Control group, pretreatment hyaluronidase could induce slight intestinal and lung histology injury, such as the formation of some subepithelial spaces ( Figure.2A) and in ammatory cells in ltrating into pulmonary interstitium ( Figure.2B). In addition, the results showed that, in GI group, the concentration of TNF-α and NF-κB in lung tissues were signi cantly increased versu Conrol group, but the level of IL-6 didn't show statistically signi cant difference ( Figure.2C-2E). Figure

VNS decreased the expression of TNF-α, IL-6 and MPO, and increased the expression of IL-10 in lung tissue
Neutrophil-derived in ammation plays an important role in the development of ALI, and the expression of IL-10, TNF-α and IL-6 could be the biomarkers of in ammation and the level of MPO could re ect the degree of neutrophil activation (16). As shown by the results (Figure 4A-E), compared with SS group, the mRNA and protein level of IL-10 was decreased, and the expression of TNF-α, IL-6 and MPO were increased in THS/R group; after treatment with VNS, the expression of IL-10 was increased, and the level of TNF-α, IL-6 and MPO were decreased, however, after administrating MLA, the effect of VNS was reversed remarkably.

VNS attenuated glycocalyx damage induced by THS/R and decreased intestinal barrier permeability.
Previous studies suggested that THS/R could damage gut barrier and increase intestinal barrier permeability (17). Our results showed that, compared with SS group, THS/R could damage the glycocalyx on IECs and increase gut permeability, but the treatment of VNS before resuscitation could mitigate the damage of glycocalyx and alleviate the elevation of gut permeability. However, the intraperitoneal injection of MLA before VNS could inhibit the effect of VNS on alleviating the glycocalyx damage and relieving the increase of gut permeability (Figure.5).
3.6 VNS reduced the gut histology injury induced by THS/R During THS/R, gut tissue could be damaged because of ischemia-reperfusion (18). As the results shown, compared with SS group, the gut tissue was markedly damaged after THS/R, showing the information of subepithelial spaces, massive epithelial lifting down the sides of the villi, denuded villi with lamina propria as well as dilated capillaries, but treated with VNS before liquid resuscitation could relieve the gut damage. However, the intraperitoneal MLA could partly offset the effect of VNS ( Figure.6).
3.7 VNS increased the level of Ach in plasma and IL-10 in gut, and decreased the expression of TNF-α, IL-6, NF-κB and MPO in gut As the results shown that, compared with THS/R group, the level of acetylcholine in plasma increased in VNS group ( Figure.7A). Compared with THS/R group, the mRNA and protein expression of TNF-α, IL-6, NF-κB and MPO decreased signi cantly in VNS group, and the expression of IL-10 mRNA and protein upregulated. However, intraperitoneal MLA before VNS could reverse the anti-in ammatory effect of VNS( Figure.7B-7G).

Discussion
To the best of our knowledge, this study rstly demonstrated that the effect of glycocalyx on IECs on intestinal barrier permeability, and VNS could alleviate the lung injury after THS/R by relieving the damage of glycocalyx on IECs.
ALI which is characterized by increased pulmonary vascular permeability, in ammatory cell in ltration and pulmonary edema is a common complication in patients after THS/R, and could develop into ARDS which presents as persistent hypoxemia, eventually resulting in respiratory failure (19,20). Previous studies suggested the gut injury induced by THS/R plays a crucial role in the development of ALI, which means that THS/R could damage gut barrier and increase intestinal barrier permeability, so that lots of gut-derived cytokines could be carried in the mesenteric lymph where they reach the pulmonary system and cause lung injury (21)(22)(23). Our study also suggested that increased intestinal barrier permeability could cause in ammatory cell in ltration and increased cytokines in lung tissue, such as TNF-α and NF-κB. Many studies showed that kinds of treatments, including vagus nerve stimulation(VNS), could alleviate in ammation reaction and lung injury by protecting intestinal barrier, but the speci c mechanisms were still unclear (13,21,24). In this study, we showed that THS/R could cause evident gut injury and markedly increased the intestinal barrier permeability, as well as cause in ammatory cell in ltration and interstitial edema and increase the level of IL-6, MPO and TNF-α in lung tissue, however, after administrating VNS, the intestinal barrier permeability was decreased, then lung injury and pulmonary vascular permeability were alleviated.
The intestinal barrier consists of mucous layer, gut microbiota, intestinal immune system and the integrity of intestinal epithelial cells. Glycocalyx is a kind of complex compound located at the surface of epithelial cells and endothelial cells, consisting mostly of glycosaminoglycans (GAGs) including heparan sulfate (25), hyaluronic acid (HA), and chondroitin sulfate (CS) (26), and the destruction of any element would lead to glycocalyx shedding. The function of endothelial glycocalyx had been studied in previous researches, which is to constitute the rst barrier to maintain vascular endothelial permeability (11,27). However, the function of epithelial glycocalyx was still unclear, and some scholars proposed that mucosal epithelial glycocalyx could maintain physical barrier (28). In our study, we injected hyaluronidase, an enzyme for cleavage HA, into the ileum to nd out the effect of intestinal epithelial glycocalyx on intestinal permeability for the rst time, and used syndecan-1 as an indicator to detect the content of glycocalyx (12,29). Our results showed that intestinal barrier permeability increased after intestinal epithelial glycocalyx was damaged by hyaluronidase.
Glycocalyx could be damaged by in ammation factors and ischemia-reperfusion injury, the change of wall shear stress , the increase of cholesterol and decrease of high-density lipoprotein (27,(30)(31)(32).Previous studies showed that in ammation factors, such as matrix metalloproteinase-1 (MMP-1), MMP-7, MMP-15 and MMP-9, are capable of cleaving GCX components (9,30,(33)(34)(35)(36). Recently, many clinical trials demonstrated that the increase of other in ammatory factors, like IL-6, TNF-α, and the decrease of antiin ammatory, such as IL-10, also could damage glycocalyx (37,38).In this study, we found that there were extensive damage of glycocalyx on IECs after THS/R, and the level of IL-6, TNF-α and NF-κB in gut were increased. After VNS treatment, the level of IL-6, TNF-α and NF-κB were decreased and the level of IL-10 was increased, then the damage of glycocalyx on IECs was alleviated, which meant that VNS could alleviate the shedding of glycocalyx by suppressing in ammatory reaction in gut. Vagus nerve is an important part of CAP, which is a kind of neuroimmunomodulation mechanism to regulate in ammation reaction, and VNS could activate CAP by releasing Ach to combine with α7nAchR which located on the surface of in ammatory cells or nerve cells (5). Many research have revealed that VNS is an effective way to reduce the level of in ammatory factors, like IL-1, IL-6, TNF-α and NF-κB as well as alleviate tissue damage following THS/R by activating CAP (7,21,25). In this study, when we pretreated with MLA, a selective α7nAchR antagonist, the above protective effect of VNS were reversed, which suggest that the glycocalyx protective effect of VNS might be mediated by activating CAP.

Conclusion
Our study rstly demonstrated that the shedding of glycocalyx on IECs could increase intestinal barrier permeability, and that the intestinal epithelial glycocalyx was damaged after THS/R, and VNS could relieve lung injury via alleviating the damage of glycocalyx on IECs by activating CAP. The results could provide a potential mechanism for the protective effect of VNS on lung injury after THS/R.   and NF-κB in lung tissues were measured by ELISA. GI represent glycocalyx inhibitor group, ****p<0.0001, ***p<0.001, ns p>0.05.