Roux-en-Y Gastric Bypass Procedure: A Bariatric Surgical Procedure for Gastric Resectioning in Murine Model

протокол

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. General Preoperative Preparation

1. Fast the mice for 6 h before the surgery. Gel diet food was given 3 days before surgery and exclusively one day before surgery. Induce anaesthesia in a chamber with 5% isoflurane (0.4 L/min) and O₂ (dioxygen; 0.4 L/min). Perform a toe-pinch test to confirm that the anesthetization is effective. Administer analgesics (0.1 mg/kg buprenorphine), antibiotics (10 mg/kg marbofloxacin), and pro-kinetics (1 mg/kg metoclopramide) via subcutaneous injections.
2. Allocate a specific work area for surgical procedures (distant from laboratory traffic and ventilation fans). Clean the surgical area with dedicated hard surface disinfectants. Place a heating pad on the working space area to maintain mouse homeothermy during the surgery. Apply a clean, absorbent under pad over the heating pad.
3. Shave the abdomen from sternum to pelvis using an electric razor. Wrap the mouse abdomen in sterilized plastic wrap (Figure 1A) and place the mouse in supine position on the heating pad.
4. Apply vet ointment on the eyes to prevent dryness while under anaesthesia. Place the mouse in the nose cone and maintain anaesthesia with 2% isoflurane (0.4 L/min) and O₂ (0.4 L/min).
5. Before beginning the surgery, put on a sterile examination gown, a disposable scrubs cap, a facemask, and sterile gloves. Open an autoclaved package containing a sterilized surgical instrument set, which is required for all steps of the surgery. Cut a window in the sterilized plastic wrap and disinfect the skin with 2 successive povidone-iodine solutions.

2. Roux-en-Y Gastric Bypass: Surgical Procedure

1. Median laparotomy
   1. Under a binocular microscope and with a scalpel, perform a midline incision from the sternum to the middle of the abdomen to open the abdominal cavity (do not damage the abdominal muscles). Protect the skin with a sterile compress soaked with 37 °C saline solution (Figure 1B).
2. Biliopancreatic limb and alimentary limb
   1. Externalize the intestine (Figure 1C). Measure 8 cm from the pylorus and perform two ligatures of the intestine with 5.0 non-absorbable sutures (Figure 1D).
   2. Cut the intestine between the two ligatures (Figure 1E). Place the proximal limb of the two ends in the upper-left quadrant of the abdomen. Note: This will be used as the alimentary limb (Figure 1F).
   3. Place the distal limb of the two ends facing the alimentary limb 6 cm below the proximal limb (Figure 1F).
      NOTE: This will be used as the biliary limb.
3. Jejuno-jejunostomy
   1. Cut both the proximal limb and the intestine loop using micro scissors and perform two antimesenteric incisions of the same length (Figure 1G and 1H).
   2. Perform side-to-side anastomosis with two 8.0 non-absorbable sutures. Perform the dorsal side anastomosis first (Figure 1I), followed by the ventral side anastomosis (Figure 1J).
   3. Gently roll 2 moistened cotton swabs towards each side of the anastomosis to ensure that the suture is leak-proof. Complete with 8.0 non-absorbable sutures in case of leakage.
4. Gastro-jejunostomy
   1. Gently mobilize the stomach using moistened cotton swabs. Free the stomach from its lateral close connective tissue attachments using moistened cotton swabs or micro scissors.
   2. Gently, externalize the stomach fully, placing a re-absorbable hemostatic collagen compress behind the stomach. Perform a ligature of the pylorus using a 5.0 non-absorbable suture passed through the omentum using curved micro forceps (Figure 1K and 1L).
   3. Cut both the ventral side of the stomach, 1.5 cm from the pylorus, and the distal limb using micro scissors, creating two incisions of the same length (Figure 1M).
   4. Perform side-to-side anastomosis with two 8.0 non-absorbable sutures. Start with the dorsal side anastomosis (Figure 1N), and then perform the ventral side anastomosis (Figure 1O). Gently roll 2 moistened-cotton swabs towards each side of the anastomosis to ensure that the suture is leak-proof. Complete with 8.0 non-absorbable sutures in case of leakage.
5. Abdominal closure
   1. Close the muscle layer of the abdominal wall using 5.0 non-absorbable sutures (Figure 1P). Reduce the anaesthesia by reducing the isoflurane concentration to 1%. Close the skin using 5.0 non-absorbable sutures (Figure 1Q). Administer 25 mL/kg of warm saline solution via subcutaneous injection.

3. General Postoperative Care

1. Stop isoflurane and continue with an O₂ flow of 0.8 L/min until the mouse is fully awake. Do not leave the mouse unattended until it regains motor control, indicated by the animal starting to move around the cage and being able to stand and walk without falling.
2. Place the mouse (only one mouse per cage) in an incubator under 30 °C temperature condition for 5 days.
3. Maintain iron (0.5 mg/kg/day; subcutaneous injection) and vitamin (800 mg/180 mL in water) supplementations in the RYGB mice until the end of the protocol.
4. Return free access to gel diet food (high-fat gel diet: 10% lard, 10% liquid sugar, 57% water) for 5 days after surgery. Reintroduce a solid diet 3 days after surgery.
5. Subcutaneously inject buprenorphine (0.1 mg/kg, twice daily, from day 0 to day 3 after surgery), meloxicam (1 mg/kg, from day 0 to day 3 after surgery), metoclopramide (1 mg/kg, from day 0 to day 5 after surgery), and marbofloxacin (10 mg/kg, from day 0 to day 3 after surgery)

Результаты

Figure 1: Roux-en-Y bypass Procedure. (A) Preoperative preparation. (B) Median laparotomy. (C) Externalization of the small intestine. (D) Biliopancreatic and alimentary limbs ligatures. (E) Small intestine section between the ligatures. (F) Intestinal limb positioning for the jejuno-jejunos...

Материалы

Name	Company	Catalog Number	Comments
Drugs
High Fat diet	DIO diet	Safe
Isoflurane	Forane	Baxter
Buprenorphin	Buprecare	Animalcare
Marbofloxacine	Marbocyl	Vetoquinol
Ammonium iron citrate, vitamins PP-B12	Fercobsang	Vetoquinol
Vitamins A-D3-E-K-B	Vita Rongeur	Virbac
NaCl 0.9%	NaCl 0,9%
Povidone solution	Betadine Scrub	Betadine
Povidone solution	Betadine Solution	Betadine
Carboptol 980 NF	Ocrygel	TVM
Sutures
Prolene®	8.0, 6,5 mm	Ethicon
Prolene®	5.0, 13 mm	Ethicon
Surgical equipments
Scissors		FST
Needle holder	Olsen-Hegar	FST
Micro scissors	Vannas	FST
Micro forceps	Graefe	FST
Micro forceps curved	Graefe	FST
Curved micro needle holder	Castroviejo	FST
Hemostatic collagen compress	Pangen	Urgo
Absorbent underpads		VWR
Specific equipments
Stereo microscope	MZ6	Leica