Experimental Animals
Fourteen male C57BL/6 mice were purchased from SLAC Laboratory Animal Inc. (Shanghai, China) at seven-weeks-old. All animals received water and normal chow (SLAC, Laboratory Animal Inc., Shanghai, China) ad libitum after birth. At the age of 8 weeks, all animals were fed a high-fat diet (HFD) (60 kcal% fat, #D12492; Research Diets Inc., New Brunswick, NJ, USA) for 12 weeks and subsequently allocated to two body weight-matched groups (sham and RYGB) before undergoing surgery. All mice were housed under a 12-h light/12-h dark cycle at room temperature (21±2°C). The procedures of all animal investigations were performed according to Central South University guidelines for the use of animals, with the approval of the Institutional Animal Care and Use Committee in Central South University. This study was carried out in compliance with the ARRIVE guidelines.
Preoperative Preparation
Fourteen mice were randomly allocated into two groups: the RYGB group (RYGB surgery, n=7), and the sham group (sham operation, n=7). All animals fasted overnight presurgery. The surgical procedures were performed under anesthesia with isoflurane (3-4% for induction and 1-2% for maintenance).
Surgery
All animal surgeries were performed under an approved surgical model protocol14. In brief, a 1.0-cm midline incision was made on the upper abdomen. For mice in the RYGB group, the jejunum was transected 4.0 cm distal to the Treitz ligament, and the proximal part was the biliopancreatic limb. Four centimeters of the distal part was measured and designated as the alimentary limb and then anastomosed to the biliopancreatic limb in an end-to-side fashion by interrupted suture. The gastroesophageal junction was ligated to exclude the stomach, and then a longitudinal incision of 0.2 cm was made on the distal esophagus. The alimentary limb was anastomosed to the distal esophagus in side-to-side pattern by running sutures. The abdominal incision was closed followed by postoperative care. Mice in the sham group underwent jejunal transection and reanastomosis in situ, and the operating time was extended to be the same as that of the RYGB group.
Postoperative Care
After surgery, all animals were placed on a heating pad after injection of 1.0 ml 0.9% of saline and 1.0 mg/kg buprenorphine. Mice received a liquid diet (ENSURE, Abbott, Zwolle, Netherlands) for 24 hours after surgery and then were switched to HFD.
Body Weight, IPGTT, and ITT
Mouse body weight was measured weekly at 11:00-12:00 a.m to the nearest 0.1 g. After 16 hours of fasting at postoperative week 7, an intraperitoneal glucose tolerance test (IPGTT) was performed after administering glucose solution (1.5 g/kg body weight) by intraperitoneal injection. The insulin tolerance test (ITT) was conducted with intraperitoneal injection of insulin at 0.75 u/kg body weight after 4 hours of fasting. At 0, 15, 30, 60, 90 and 120 min after injection, mouse blood was retrieved from the tail vein for blood glucose measurement using an ACCU-CHEK Performa glucometer (Roche, Mannheim, Germany).
Plasma IL-6, TNFα and LPS
Mice were euthanized at 8 weeks postsurgery, followed by the collection of blood and tissue samples. Plasma IL6 and TNFα levels were measured with the relevant enzyme-linked immunosorbent assay kits (BiomeTech, Greifswald, Germany) and plasma LPS concentrations were determined using the limulus amebocyte lysate (LAL) test (Associates of Cape Cod, East Falmouth, MA, USA).
RNA Extraction and Quantitative Real-time PCR
The intestine of RYGB mice was divided into four segments after RYGB, including the alimentary limb, biliopancreatic limb, common limb and colon, which were rapidly frozen in liquid nitrogen after sampling and then stored at -80°C for further analysis. The common limb of RYGB mice is equal to the lower small bowel of Sham mice. RNA was extracted from snap-frozen specimens with TRIzol reagent (Invitrogen, CA, USA). Reverse transcription of RNA was performed using cDNA synthesis kits (Thermo Fisher Scientific, NY, USA). SYBR green dye was used as a fluorochrome forquantitative real-time PCR (qPCR) in the ViiA7 System (Life Technology). qPCR was conducted to detect the gene expression levels in the intestine.
Ussing Chamber Experiments and Intestinal Permeability Assay of the Common Limb and Colon
Intestinal permeability was examined 8 weeks after surgery. For the Ussing chamber experiment, in short, it is essential to keep excised intestinal tissue fresh. The intestinal muscle layer was stripped-off and the mucosa was mounted in a Ussing chamber (WPI Instruments, Sarasota, FL, USA) containing 5 ml of circulating oxygenated Krebs buffer (136 mM NaCl, 4.3 mM KCl, 1.5 mM CaCl2, 1.4 mM MgSO4, 27 mM NaHCO3, 1.6 mM KH2PO4, pH 7.30)15. The mucosal-to-serosal flux level of dextran conjugated to fluorescein isothiocyanate (dextran-FITC, Sigma, MO, USA) was expressed as the intestinal permeability. After the dextran probe was added, specimens of serosal buffer were retrieved at 0, 30, 60, 90 and 120 min and then was added with Krebs buffer/glucose. With respect to data analysis, the concentration was estimated in accordance with a standard curve.
Statistical Analysis
SPSS 21.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze all results. Statistical analysis of the data was performed by Student’s t-test or ANOVA. All data are shown as the mean ± SEM. P ≤ 0.05 was considered to be statistically significant.