This research is supported by Tom Peterson Foundation and NIH grant R01-HL119780 (Jain, MK).

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...

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The authors have nothing to disclose.

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...

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&#39;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 functions4,5,6. 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 (VB12) 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.

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			<td height="21" style="width:248px;height:21px;">Dissection microscope</td>
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			<td height="21" style="width:248px;height:21px;">8-0 monofilament suture</td>
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ileectomy-induced bile overaccumulation in mouse intestine

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.

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...

Watch this Scientific Journal Video about Ileectomy-induced Bile Overaccumulation in Mouse Intestine at JoVE.com

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

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.

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

The overall goal of this surgery is to evaluate ileectomy-induced bile malabsorption, overaccumulation and toxicity in the mouse intestine. Diseases of the ileum as manifesting conditions such as Crohn's, cancer, autoimmune disorders or infection are important sources of morbidity and mortality. The development of this model that involves segmental removal of the ileum, we believe, can be leveraged for insights into biology as well as therapy.The implications of this technique extend toward the therapy of bile acid diarrhea as ileectomy potentially induce bile acid overproduction by the liver and the malabsorption and overaccumulation by the intestine. Generally, individuals new to this method will struggle with the intestinal anastomosis which is the most critical and difficult portion of this surgery. Begin by confirming the appropriate level of sedation by toe pinch and applying depilatory cream to the mouse's abdomen.Remove the hair with surgical sponges and place the animal on a temperature-controlled small-animal surgical table set to 37 degrees Celsius. Apply ointment to the animal's eyes and disinfect the exposed skin with a sequential povidone-iodine and 70%ethanol scrub. Cover the surgical area of the abdomen with sterile surgical gauze and use a scalpel to make a midline incision in the abdomen and cut through the muscle layer.Using a cotton-tipped applicator to protect the intestines, fully open the mouse abdominal muscles with retractors to expose the abdominal cavity. Starting from the cecum, carefully move the connected ileum and part of the jejunum out of the abdominal cavity and use a 7-0 silk suture to ligate the upper branch of the superior mesenteric artery. When the tissue color changes from pink to dark indicating a full occlusion, use scissors to excise 50 or 90%of the ileum depending on the parameters of the experiment.Flush the lumen of both ileal ends with 0.9%saline. After locating the mesentery on the side of both ileal ends, align the mesenteries on both ends of the excised tissue and use an 8-0 suture to join the ends together. Suture the contralateral side of the ileum to keep the ileum anastomosed in a natural manner and suture the upper and lower sides between the two original sutures to thoroughly join the two ileal ends.After confirming the lack of leakage from the anastomosis site, return the cecum and small intestine to their original anatomical locations and use a blunt needle to wash the surgical area with warm 0.9%saline. Close the abdominal muscle layer with a 6-0 suture and align the abdominal skin incision to close the skin and to facilitate optimal wound healing. Then, transfer the mouse to an intensive care unit in a paper bedding cage on a temperature-controlled heating pad for overnight recovery with soft food in addition to regular food and water.One day after the surgery, weigh the whole animal. Then, harvest the gastrointestinal or GI tract and weight the intestinal tissue. Transfer the GI tissue into a 15-milliliter conical tube and use scissors to cut the tissue into short segments.Then, pellet the tissue pieces by centrifugation and transfer the bile salt-containing supernatant into a new tube. Quantitative real-time polymerase chain reaction analysis reveals an abundant expression of fibroblast growth factor 15, apical sodium-bile acid transporter, ileal bile acid-binding protein and organic solute transporter beta mRNA within the ileum with less to extremely low expression within the jejunum and the rest of the intestine. Adenomatous polyposis coli mRNA levels which are measured as a negative control remain similar throughout the entire intestine.After 24 hours, the intestine from the sham surgery is normal demonstrating a similar size and morphology compared to that of non-surgery animals. The fluid volumes exhibit a gradient enhancement from 0%to 90%ileectomy corresponding to the significantly increased GI to body weight ratio observed after a 90%resection. The GI fluid collected from the post-surgery GI tracts and the GI fluid weight to GI tract weight ratio also demonstrate a gradient increase after resection.Further, the bile acid assay confirms that the amount of bile acids detected in the supernatant increases as the size of ileal resection increases suggesting that the 50%ileum resection is a more applicable mouse ileectomy model due to its relatively more moderate adverse effects. Once mastered, this technique can be completed in 30 minutes if it's performed properly. After its development, this technique pave the way for researchers in the field of microsurgery to explore ileum biology and also the bile syndrome in small animals.After watching this video, you should have a good understanding of how to properly identify and remove the ileal segment and how to perform an intestinal anastomosis.

ileectomy-induced-bile-overaccumulation-in-mouse-intestine

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JoVE Journal

Medicine

9.4K vistas.  Case Western Reserve University. The overall goal of this surgery is to evaluate ileectomy-induced bile malabsorption, overaccumulation and toxicity in the mouse intestine. Diseases of the ileum as manifesting conditions such as Crohn's, cancer, autoimmune disorders or infection are important sources of morbidity and mortality. The development of this model that involves segmental removal of the ileum, we believe, can be leveraged for insights into biology as well as therapy.The implications of this technique extend t...