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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Duodenal-jejunal bypass (DJB) surgery can improve glucose metabolism and reduce insulin resistance. Here, we present a protocol to establish a stable and reliable mouse model of DJB.

Abstract

The prevalence of obesity and type 2 diabetes is a serious global health concern. Obesity is a major pathogenic factor in type 2 diabetes, cardiovascular disease, and some cancers. Bariatric surgery offers a long-term and effective treatment option for both obesity and diabetes. Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) are widely recognized as the most popular bariatric surgeries. Additionally, several exploratory bariatric surgeries have demonstrated promising therapeutic effects. Duodenal-jejunal bypass (DJB), specifically tailored for diabetics with low body mass index, has shown beneficial metabolic outcomes. However, its weight-independent metabolic benefits are not fully understood due to limited animal models. In this article, we describe the optimized care protocols and surgical techniques for performing DJB surgery in diet-induced obese (DIO) diabetic mice. Using a mouse model contributes to a better understanding of the nature of changes induced by DJB surgery while facilitating related clinical practice.

Introduction

Obesity and type 2 diabetes are the most common chronic diseases in the world, and their prevalence is increasing among young individuals1. Bariatric surgery is the most effective treatment for obesity and diabetes, facilitating long-term blood glucose stabilitywhile also improving the complications associated with obesity2,3. There are several types of bariatric surgery, classified by whether they reduce gastric volume or intestinal absorption; these include restrictive, malabsorptive, and combination4,5.

Duodenal-jejunal bypass (DJB) was first developed by Rubino and Marescaux, who demonstrated that type 2 diabetes could be alleviated by connecting the duodenum and jejunum rather than by reducing the gastric volume6,7. DJB preserves the entire stomach and bypasses the entire duodenum and proximal jejunum. The bowel is divided into biliopancreatic, digestive, and common limbs6,8. DJB shares some similarities with bariatric surgeries, including Roux-en-Y gastric bypass (RYGB), mini-gastric bypass, biliopancreatic bypass, duodenal diversion, and DJB plus sleeve gastrectomy9. Compared to RYGB, DJB does not require a gastrointestinal anastomosis, which reduces the operative time and improves the success rate of the procedure. DJB is similar to RYGB in improving glucose metabolism but does not affect body weight10. After DJB surgery, rapid food delivery to the distal small intestine stimulates the secretion of glucagon-like peptide-1 (GLP-1), resulting in improved glucose metabolism11,12.

The use of animal models is essential for understanding the metabolic, cellular, and molecular pathways. Animal models of bariatric surgery have contributed to our understanding of potential mechanisms underlying obesity and diabetes13,14. However, due to the physiological differences between species, it is impossible to perfectly replicate human diseases in animal models15. Among various animal models available for research purposes, the diet-induced obesity (DIO) mouse model most closely resembles human obesity and metabolic syndrome16. Mice were selected for DJB surgery to determine the feasibility of the surgery and to provide techniques for further research. This manuscript provides a comprehensive summary of both technical aspects and experimental details of DJB surgery.

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Protocol

All protocol steps described below follow the guidelines of the Animal Care and Use Committee of the General Hospital of the Southern Theater Command under the approval number 2020112501.

1. General preoperative preparation

NOTE: Thirty 6-week-old male C57BL/6 mice were purchased. Mice were housed in a Specific Pathogen-Free (SPF) laboratory under a 12 h light/dark cycle; temperature, 22 ± 2 °C; and humidity, 55-65%. Mice were given free access to water and fed a 60% kcal fat diet for 6 weeks to induce obesity. An intraperitoneal injection of 40 mg/kg streptozotocin was administered for 5 days to induce diabetes17. Among the thirty mice, twenty-two mice were screened for random blood glucose > 300 mg/dL and randomly assigned to the DJB (n=15) and sham surgery (n=7) groups. Eight mice were excluded from the experiment due to substandard blood glucose.

  1. Fast the mice for 8 h prior to the surgery. Withdraw water 2 h before the surgery.
  2. Administer 1% sodium pentobarbital solution (6 mL/kg) and buprenorphine (1 mg/kg) intraperitoneally. Touch the toes or tails of the mice with forceps and ensure that the mice do not show any obvious twitching or shaking. Under adequate anesthesia, the mice can breathe freely without supplemental oxygen.
  3. Place the mice in the supine position on a sterileboardand under a stereomicroscope. Apply eye ointment to the eyes. Use an electric blanket to keep the mice warm throughout the procedure. Use sterile procedures, including surgical gowns, sterile gloves, and autoclaved instruments.
  4. In sham control mice, make the two incisions respectively in the duodenum 1 cm below the pylorus and in the jejunum 5 cm below the Treitz and then suture the incisions.

2. Duodenal jejunal bypass: Surgical Procedure

  1. Apply the depilatory paste from the xiphoid to the abdomen to remove hair from this region. Wipe off the cream and ensure that the skin is clean. Scrub the area three times with alternating scrub of iodine and alcohol solutions.
  2. Cover the mouse with a sterile drape, leaving the operational area exposed, and make a 2 cm incision from the xiphoid to the abdomen.
  3. Use an abdominal retractor to expose the abdominal cavity. Push away the abdominal fat using a wet cotton swab, and move the liver to the cephalic side to fully expose the stomach and bowels.
  4. The stomach and pylorus are below the liver, and the ligament of Treitz is at the distal duodenum. Double ligate the jejunum, 5 cm distal to the ligament of Treitz with a 6-0 silk suture (Figure 1A). Cut the jejunum at the midpoint of the two ligations and suture the jejunal stump with a 10-0 silk suture (Figure 1B).
    NOTE: Avoid twisting the mesenteric blood vessels.
  5. Pull the proximal jejunal incision 5 cm along the bowel to the jejunum to create a jejunal-jejunal anastomosis. Align the two bowels horizontally, and then use a 10-0 silk suture to create the lateral anastomosis (Figure 1C).
    NOTE: During surgery, keep the bowel moist with saline to reduce water loss. This will prevent the bowel from curling up and will make suturing easier.
  6. Secure both ends of the bowel and cut the incision to equal length (Figure 1D). Suture the second layer of the posterior intestinal wall with a full-thickness continuous suture.
    NOTE: The incision length is approximately 0.5-0.6 cm, and the needle distance is ~0.5 mm.
  7. Secure both ends of the bowel and suture the anterior wall of the bowel. Suture the first layer of the anterior wall with a simple continuous suture and the second layer with a horizontal varus suture (Figure 1E-F).
    NOTE: Swab the intestinal contents with cotton swabs to prevent infection of the abdominal cavity.
  8. Pull the distal jejunal incision into the duodenum 1 cm below the pylorus to create a duodenal-jejunal anastomosis (Figure 1G). Suture it using the same method as in steps 2.6-2.7 (Figure 1H-K).
    NOTE: Check the suture at the anastomotic corner to reduce anastomotic leakage.
  9. Ligate the bowel with the micro forceps and cut the bowel with the micro scissors. Double ligate the duodenum with a 6-0 silk suture, 2 mm from the distal end of the duodenal-jejunal anastomosis. Cut at the midpoint with micro scissors and suture the stump with a 10-0 silk suture (Figure 1L).
    NOTE: The gastroduodenal vessels branch perpendicular to the head of the pancreas and are adjacent to the pylorus, requiring careful exploration of the vessels in the transverse section. The confluence of the common bile duct and duodenum was located, taking care not to damage the pancreas or the common bile duct.
  10. Rinse the abdominal cavity with 30 °C saline. Return the bowel to the abdominal cavity. Suture the muscle and the skin separately with a 6-0 silk. Then, disinfect the skin with iodophor.
  11. After surgery, inject 30 °C saline (30 mL/kg) subcutaneously in the back to prevent dehydration. Inject penicillin (10 mg/kg) intramuscularly to prevent infection.

figure-protocol-5629
Figure 1: Duodenal-jejunal bypass procedure. (A) Location of the jejunum 5 cm distal to the ligament of Treitz. (B) Double ligate the jejunum with 6-0 silk, cut through the middle of the ligature. (C) Jejunal-jejunal anastomosis. (D) Make a 0.5-0.6 cm incision, and suture the posterior wall. (E-F) Complete Jejunal-jejunal anastomosis. (G) Pull the distal jejunal to the duodenum 1 cm below the pylorus. (H) Duodenal-jejunal anastomosis. (I) Cut the duodenal-jejunal anastomosis with a 0.5-0.6 cm incision and suture the posterior wall with a simple continuous suture. (J-K) Complete duodenal-jejunal anastomosis. (L) At 2 mm from the distal to the duodenal-jejunal anastomosis, double ligate the duodenum with 6-0 silk and cut through the middle of the ligature. Please click here to view a larger version of this figure.

3. General postoperative care

  1. After surgery, place the mice on an electric blanket to prevent hypothermia. Allow the mice to crawl freely until fully awake before returning to their cages.
  2. On the postoperative day, restrict the food and water and inject 2 mL of saline subcutaneously into the back of the mice. On the first postoperative day, give 10 mL of 10% glucose and a functional drink (1:1 ratio) without food, and inject 1 mL of saline subcutaneously.
  3. On the second and third postoperative days, feed the mice with a mixture of 20 mL of 10% glucose and a functional drink. After the fourth day, give the mice pure water and a high-fat diet. Transitional feeding should be performed according to the postoperative recovery status in the following order: solution, semi-liquid, or solid food.
  4. Postoperative analgesia: inject buprenorphine (0.1 mg/kg) every 12 h from Days 1 to 3, and then once daily until Day 5.
  5. After surgery, observe the feeding conditions, activity, feces, and wound healing of the mice.

figure-protocol-8078
Figure 2: The diagram of DJB surgery. (A) Duodenal-jejunal anastomosis. (B) Jejunal-jejunal anastomosis. (C) Biliopancreatic limb. (D) Digestive limb. (E) Common limb. Please click here to view a larger version of this figure.

figure-protocol-8717
Figure 3: Anatomy of DJB surgery. (A) Duodenal-jejunal anastomosis. (B) Jejunal-jejunal anastomosis. (C) Biliopancreatic limb. (D) Digestive limb. (E) Common limb. Please click here to view a larger version of this figure.

4. Postoperative evaluation of metabolic parameters

  1. Body weight measurement
    1. Fast the mice for 8 h prior to the measurement. Weigh the mice weekly on Monday before and after surgery for 8 weeks.
  2. Food intake measurement
    1. House one mouse in each cage. Measure the amount of solid food in each cage before and after 24 h. The difference between the two values represents daily food intake.
  3. Random blood glucose measurement
    1. At 8:00 a.m. on the weekly Monday of measurement, collect a drop of blood from the tip of the mouse tail and apply it to a glucose strip inserted into the glucometer.
  4. Oral glucose tolerance test
    1. Eight weeks after the DJB surgery, fast the mice for 8 h before the oral glucose tolerance test. Collect a drop of blood from the tip of the mouse tail and place it on a glucose strip inserted into the glucometer. Administer an oral dose of 20% D-glucose (2 g/kg). Measure the blood glucose levels at 0, 15, 30, 60, 90, 120, 150, and 180 mins after gavage.

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Results

General conditions
The mean operative time for the DJB procedure was 84.5 ± 2.6 min. Fifteen mice underwent DJB surgery, nine mice survived. As shown in Table 1, the majority of deaths occurred during surgery or in the following 7 days. The causes of postoperative death were bleeding (n=2) on postoperative day 1, anastomotic leakage (n=1) on postoperative day 4, anastomotic obstruction (n=2) on postoperative day 7, and unknown cause (n=1) three weeks after surgery.

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Discussion

In 1953, Varco et al.18 performed the first jejuno-ileal bypass as the beginning of bariatric surgery. Since then, numerous bariatric surgeries have been performed by surgeons. These surgeries have resulted in weight loss and improved metabolic complications4,19,20. Furthermore, in 1967, Mason and Ito21 performed the first Roux-en-Y gastric bypass (RYGB), which can effectively redu...

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by a grant from the Science and Technology Planning Project of Guangdong Province of China (No. 202002020069).

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Materials

NameCompanyCatalog NumberComments
Abdominal retractorF.S.T17000-03Colibri Retractor -3cm,retractor range 1.5cm/3cm long
1% sodium pentobarbital solutionGuangzhou Chemical Reagent Factory/ Dissolved 500mg of pentobarbital sodium powder in 50ml of normal saline to obtain 1% pentobarbital sodium solution.
Benzylpenicillin sodium for InjectionNorth China Pharmaceutical Company Ltd.F2062121Penicillin
BuprenorphineGuangzhou Chemical Reagent Factory/Analgesia
BuprenorphineGuangzhou Otsuka Pharmaceutical Co., Ltd/Analgesic
Citric acid-sodium citrate bufferLEAGENER00522Buffer solution
Cotton budsHaoZheng Medical60220610Cotton swabs
Depilatory pasteVeetAAPR-S222Hair removal cream
Ear tagZEYASUS304Ear-mark
Electric blanketZOSENZS-CWDRTHeat pad
Electronic scaleWETTLER TOLEDO20060902-6Measure the weight
Enteral nutritional powderAbbott Laboratories/Nutrition powder
Eye ointmentGuangzhou Otsuka Pharmaceutical Co., Ltd/Protect the eyes
GlucometerRoche6993788001Assess blood glucose
Graphpad Prism version 9.4.1GraphPad Softwareversion 9.4.1Software for statistical analysis
High-fat diet (High Fat [60FDC] Purified Rodent Diet)Dyets11225260kcal% High Fat Diet
Micro ForcepsJinzhong Medical18-1140Micro forceps
Micro needle holderJinzhong MedicalEMT-160-ZNeedle holder
Micro ScissorsJinzhong MedicalYBC010Micro scissors
Microscope cameraLAPSUNE 2000Video
Ophthalmic scissorsJinzhong MedicalY00030Surgical scissors
PentobarbitalGuangzhou Chemical Reagent Factory/Narcosis
Sodium chloride InjectionGuangzhou Otsuka Pharmaceutical Co., LtdB21L0301NaCl 0.9%
Stereo microscopeZEISSStemi 305Binocular stereomicroscope
StreptozotocinSigmaS110910-1gSTZ
Suture lineLINGQIAO SUTUREZS-LQPMRZ5/0Prolene 6/0,Prolene 10/0
Tissue forcepsJinzhong MedicalH1701Surgical forceps

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