Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Summary

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Abstract

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Introduction

In modern biomedical research, genetically manipulated animal models are widely utilized to glean insights into human diseases. In particular, tissue or cell-specific gain-and-loss functions of genes have been used to study molecular regulation as well as induced biological effects. Despite the advancements in manipulating target genes in vivo, there are lingering limitations. First, many cell or tissue specific deletions will affect multiple organs. For example, epithelial gene deletion will eliminate expression in epithelia of multiple tissues. Further, even if deletion is restricted to a specific tissue, spatial control is rarely feasible. For example, in a tissue like the intestine, distinct segments carry out very specific functions that cannot be manipulated with precision in vivo. In these situations, resection of the gene-containing tissues is considered to be a more efficient studying approach to determine the mechanistic and functional significance of tissue communication.

Ileectomy is mostly used in patients with Crohn's and inflammatory diseases involving the distal ileum ^1,2,3. The ileum typically produces several energy storage hormones like fibroblast growth factor 15/19 (FGF15/19), peptide YY (PYY), and glucagon-like peptide 1/2 (GLP1/2); these hormones play important local and endocrine roles in many biological functions^4,5,6. Among these hormones, FGF15 has been identified as a robust endocrine inhibitor of bile acid synthesis in the liver. Once reabsorbed into ileal enterocytes, bile acids activate the nuclear receptor farnesoid X receptor (FXR) to stimulate Fgf15 expression, which subsequently leads to feedback inhibition of hepatic bile acid synthesis ⁷. In a recent study, we introduced the mouse ileectomy model in order to study the ileal kruppel-like factor 15 (KLF15)-Fgf15 signaling axis that regulates circadian bile acid production in the liver ⁸. Most importantly, we introduced a novel family, the kruppel-like factors, particularly KLF15, into bile acid biology. Based on functional studies including ileectomy surgery, we determined that KLF15 upregulates bile acid synthesis via an indirect non-hepatic mechanism. Finally, ileal KLF15 is also identified as the first endogenous negative regulator of Fgf15.

The intestinal segments descending from proximal to distal regions are responsible for absorption of different nutrients. The ileum is the major segment responsible for bile acid and vitamin B₁₂ (V_B12) absorption ⁹. An earlier study employed a mouse model of proximal gut resection to study short bowel syndrome; various resection lengths, diets, and suture types were proposed to maintain an optimal post-surgery survival rate ¹⁰. Furthermore, a more recent review indicates that ileal resection typically results in more severe disease than other gastrointestinal (GI) segment resections because of the decreased adaptive capacity of the remaining tract ¹¹. This topic has gained intensive interests of basic and clinical research groups, whereas the understanding of recovery and the effective therapeutic approaches are still limited.

Bile acid diarrhea results from imbalances in bile acid homeostasis in the enterohepatic circulation ^12,13. It can be a consequence of ileal resection, gastrointestinal disease, or a result of idiopathic bile acid malabsorption. More than 80% of patients have been found to present with diarrhea after undergoing ileal resection ¹⁴. Ileectomy has the potential to be an important surgery model for the investigation of bile acid diarrhea. In this study, a series of ileal resections provide a gradient assessment of FGF15 deficiency as well as intestinal bile salt malabsorption, overaccumulation, and toxic damages.

Protocol

Animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Case Western Reserve University School of Medicine and was conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). Mice were euthanized by methods compliant with the American Veterinary Medical Association (AVMA) Guidelines for Euthanasia of Animals (2013 Edition). C57BL/6J, male, 8 - 16 week old mice were used in this protocol. Mice were housed in a 12 h dark/light cycle environment.

1. Pre-operative Preparation

1. Transfer mice to a clean cage 24 h before the procedure. Allow animals free access to water. Replace chow food with soft food (gel dietary supplement) 4 - 6 h before the surgery to reduce the contents of the small intestine for surgery.
2. Sterilize all surgical instruments and prepare the single-use sterile surgical materials. Clean the surgery area and the anesthetic nose cone with 70% ethanol.
3. Set up a dissection microscope, isoflurane anesthetic vaporizer, instruments, and a temperature-controlled small animal surgical table for body temperature maintenance. Organize instruments, sutures, and syringes in a location for free access during the surgery.
4. Set up a light source to provide enough light for the surgical area.
5. Prepare sterile 0.9% saline in 5 mL syringe for intestinal and abdominal cleaning.

2. 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 product 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 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 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 6-0 suture. Align the abdominal skin incision using forceps and suture the abdominal skin to facilitate optimal wound healing (Figure 1L).

3. 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. Administrate 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 4).

4. Evaluation of Ileectomy-induced Bile Overaccumulation

1. Weigh and dissect the mice one day after a resection of 0% (sham), 50%, or 90% of 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 as described in reference ⁸.

Results

The procedures for ileectomy are shown in Figure 1. The first step includes preparing mouse abdominal skin, making an abdominal incision, and using retractors to fully expose the intestine (Figure 1A-C). Next, the mouse cecum was located (Figure 1D); given that its size and shape make it an easily identifiable landmark. The cecum, ileum, and part of the distal jejunu...

Discussion

In order to perform a successful ileectomy, the superior mesenteric artery must be ligated in advance to block the blood supply to the resecting segment. The ischemic ileal segment will turn dark purple after ligation. The ileal segment must then be completely resected. A normal blood supply must be ensured for the retained ends. This is essential to avoid bleeding and improper removal, which can easily cause surgical failure due to ischemic necrosis after suturing. During the anastomosis, it is important to join the ile...

Materials

Name	Company	Catalog Number	Comments
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 anasthesia
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 pretection
Hair remover lotion	Church & Dwight Co., Inc.		For skin preparation
Intensive care unit	ThermoCare	FW-1	For post-surgery recovery
DietGel recovery	ClearH2O	72-06-5022	For post-surgery recovery
Aurum total RNA fatty and fibrous tissue kit	Bio-Rad	7326830	For RNA isolation
iScript reverse transcription supermix for RT-qPCR	Bio-Rad	1708841	For reverse transcription assay
TaqMan fast advanced master mix	Applied Biosystems/Life Technologies	4444965	For QPCR analysis
Total bile acid assay kit	Genzyme Diagnostic	DZ042A-K01	For bile acid assay
C57BL/6J	The Jackson Laboratory