Murine Ileal Resection Model: Studying Ileectomy-Induced Bile Metabolism in Murine Model

프로토콜

1. Ileectomy and Anastomosis

1. Anesthetize mice with isoflurane (2 - 3%) in a small animal incubation chamber. Determine adequate anesthetization using the toe pinch technique while the animal is on isoflurane.
2. Remove abdominal hair by applying hair removal products and wipe hair away using surgical sponges while maintaining anesthesia. Place the mouse on a temperature-controlled small animal surgical table (Figure 1A) to maintain body temperature at 37 °C. Maintain anesthesia with isoflurane (1 - 2%) through a facemask. Treat the mouse eyes with ocular ointment.
3. Clean the skin using povidone-iodine and 70% alcohol and cover the surgical area of the abdomen with sterile surgical gauze (Figure 1B).
4. Make a midline abdominal incision using a surgical scalpel once anesthesia is in effect. Use a cotton-tipped applicator to protect the intestine and pull mouse abdominal muscle with retractors to fully open and expose the abdominal cavity (Figure 1C).
5. Locate the cecum. Starting from the cecum, carefully move the connected ileum and part of the jejunum out of the abdominal cavity (Figure 1D).
   NOTE: The cecum can be easily identified due to its large size even after fasting.
6. Ligate the upper branch of the superior mesenteric artery with a 7-0 silk suture to occlude the blood supplying the ileal segment that is to be excised. The ileal color changes from pink to dark purple after ligation. (Figure 1E - F).
7. Depending on the purpose of the experiment, using scissors, excise and remove 50% or 90% of the ileum.
   NOTE: For sham surgery, do not perform the superior mesenteric artery ligation and do not remove the ileum.
8. Flush the lumen of both ileal ends with 0.9% saline.
   NOTE: As the remaining intact ileum is still receiving a normal blood supply from the superior mesenteric artery, a small amount of blood will be flushed out during the process. This also indicates that the blood supply to the ileal ends is normal and ensures no ischemia during the anastomosis procedure (Figure 1G).
9. Locate the mesenteries on the side of both ileal ends. Align the mesenteries and suture the ileal ends together using 8-0 suture (Figure 1G - H).
10. Suture the contralateral side of the ileum to keep the ileum anastomosed in a natural manner (Figure 1I).
11. Suture the upper and lower sides between the two original sutures to thoroughly join the two ileal ends together (Figure 1J).
12. Confirm that there is no leakage from the anastomosis site after finishing the three-step suturing procedure (Figure 1G-I). Return the cecum and the small intestine into the abdominal cavity to the original anatomical location. Wash the surgery area with warm 0.9% saline using a blunt needle. (Figure 1K).
13. Close the incision of the abdominal muscle layer with a 6-0 suture. Align the abdominal skin incision using forceps and suture the abdominal skin to facilitate optimal wound healing (Figure 1L).

2. Post-operative Care

1. Transfer the post-surgery mice to an intensive care unit for recovery. House them in a paper-bedding cage on a temperature-controlled heating pad to continue the post-surgery recovery overnight. Supply mice with soft food in addition to regular food and water.
2. Administer buprenorphine (0.05 - 0.1 mg/kg) with subcutaneous injection every 8-12 h for analgesia.
   NOTE: Euthanize mice by CO₂ if severely sick.
3. At the end-point, sacrifice mice using overdosed isoflurane and harvest samples as needed (section 3).

3. Evaluation of Ileectomy-induced Bile Overaccumulation

1. Weigh and dissect the mice one day after resection of 0% (sham), 50%, or 90% of the ileum.
2. Remove the GI tract and weigh it. Calculate the GI weight to body weight ratio to evaluate the severity of bile salt malabsorption and overaccumulation.
3. Transfer the GI tracts into 15 mL conical tubes and cut them into short segments using scissors. After cutting, centrifuge at 3,000 x g for 10 min. Transfer the supernatant (GI fluid containing bile salts) to a clean tube.
4. Measure the total volume and weight of GI fluid and calculate the fluid weight to GI tract weight ratio to further assess bile overaccumulation in the GI tract.
5. Determine the total bile amount in the supernatant by bile acid assay.

결과

Figure 1: Surgery Procedures of Ileectomy. (A) Maintain body temperature using a temperature-controlled small animal surgical table. (B) Cover the surgery area with sterile gauzes after abdominal skin preparation. (C) Expose the small intestine through a midline abdominal incision. (D) Cecum (highlighted by the white dashed line) is marked to locate the ileum. Expose the ileum (yellow...

자료

Name	Company	Catalog Number	Comments
C57BL/6J	The Jackson Laboratory
Dissection microscope	Olympus	SZ61	For surgery
Animal temperature controller	Physitemp Instruments, Inc	TCAT-2LV	For body temperature control
Isoflurane anesthetic vaporizer	VetEquip	911104	For anesthesia
Dissection forceps	Fine Science Tools, Inc.	11274-20	For surgery
Scissors	Fine Science Tools, Inc	14084-08	For surgery
Needle holder	Roboz Surgical Instrument Co.	RS-7882	For surgery
Micro knives-needle blade	Fisher Scientific	10318-14	For surgery
6-0 monofilament suture	Ethicon	1698G	For abdominal skin closure
7-0 silk suture	Ethicon	766G	For ligation
8-0 monofilament suture	Ethicon	1714G	For anastomosis
Surgical sponges	Dynarex Corp.	3333	For surgery
Small cotton-tipped applicators	Fisher Scientific	23-400-118	For surgery
Isoflurane	Piramal Healthcare Limited	66794-013-25	For anesthesia
Buprenorphine hydrochloride	Reckitt-Benckiser Pharmaceuticals	12496-0757-1	For analgesia
0.9% sodium chloride Injection	B. Braun Medical Inc.	0264-7800-10	For washing/injection
Povidone iodine prep solution	Dynarex Corp.	1413	For skin preparation
Puralube vet ointment	Dechra Veterinary Products	17033-211-38	For eye protection
Hair remover lotion	Church & Dwight Co., Inc.		For skin preparation
Intensive care unit	ThermoCare	FW-1	For post-surgery recovery
Total bile acid assay kit	Genzyme Diagnostic	DZ042A-K01	For bile acid assay