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.
2.2 Animal model constructing
This experiment consisted of two parts. In first 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 identified and protected with moist gauze. A 10-cm segment of ileum was identified at 20-30cm from cecum, ligated both ends of the ileum, and incised distally and flushed with 2.0 mL of 0.9% normal saline to remove feces. Once flushed, 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 sacrificed. 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 identified 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 identified 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 sacrificed to obtain lung tissue and gut tissue. During the whole procedure, we used thermal equipment to maintain temperature between 36.0℃ and 38.0℃.
2.3 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 fluid 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.
2.4 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 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.
After rats sacrificed, gut tissues were collected and fixed with 4% paraformaldehyde, paraffin embedded, and cut into 4 μm slices. Those slices were deparaffinized 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 FITC-conjugated secondary antibody. After washing, the slices were stained with DAPI and then sealed with an anti-fade fluorescence medium. The slices were observed with a fluorescence microscope and analyzed by ImageJ software.
2.7 H&E staining
The lung tissues and gut tissues were collected and fixed with 4% paraformaldehyde, paraffin embedded, and cut into 5 μm slices. Those slices were deparaffinized with xylene, and dehydrated in gradient ethanol, then stained with hematoxylin and eosin and observed under an optical microscope. Five random fields were identified in a blinded fashion at 100x magnification to quantify morphologic damage. The lung injury scores (14) were obtained by assessments of inflammatory cell infiltration in the airspace or vessel wall, alveolar congestion, hemorrhage, alveolar wall thickness, and hyaline membrane formation. The degree of gut injury was identified 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.
2.8 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. Target gene expression was quantified as the average of triplicate samples using the △△CT equation and normalized to glyceraldehyde 3-phosphate dehydrogenase gene expression.
2.10 Statistical Analysis
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.