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.
