The overall goal of this surgical procedure is to reliably and consistently produce intestinal obstructions in mice that facilitate the study of the molecular and phenotypic changes in gastrointestinal tissue that occur in response to the obstructions. This method can help answer key questions in the gastrointestinal field surrounding the cellular mechanisms underlying the phenotypic changes that result from intestinal obstruction. The main advantages of this technique is that it's reliable, replicable, and cost effective.
The implication of this technique extend toward the therapy of intestinal obstruction as they mimic phenotypic conditions observed in human pathologies caused by bowel obstruction. Demonstrating this procedure will be Se Eun Ha, a post doc, and Lai Wei, a graduate student, both from our laboratory. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and place the mouse onto a warming pad.
Remove the hair from the abdomen with depilatory cream and clean the exposed skin with 70%ethanol. Disinfect the surgical site with povidone-iodine solution and drape the animal with a 25 by 50 centimeter sterile paper with a 2.5 by 2.5 centimeter opening for the surgical area. Secure the drape with sterile strips at the boundaries of the opening and the skin and use a number 15 scalpel blade to make a three centimeter longitudinal skin incision.
Using forceps and surgical scissors, carefully separate the skin from the musculoperitoneal layer without incising the tissue and locate the linea alba. Then use micro forceps and scissors to cut two centimeters along the linea alba to expose the intraperitoneal cavity and carefully locate the cecum. Use the micro forceps to slowly and gently move the cecum, proximal colon, and ileum from within the intraperitoneal cavity onto the sterile drape and immediately moisten the intestinal tissue with 0.9%sterile saline soaked gauze.
Locate the mesentery between the ileum and proximal colon and make a one centimeter incision in the mesentery parallel to and just below the ileum taking care to avoid cutting any vasculature. Cut longitudinally to open the mesenteric vessel and use micro forceps to open an appropriately sized autoclaved sterilized silicone ring. Insert one end of the ring through the incision and bring the end of the ring into contact with the first end to return the instrument to its original shape.
Confirm that the ring is completely surrounding the ileum and close the ring with a suture. The incisions in the mesentery should be large enough for the ring to fit, but small enough that no blood vessels are incised. Also make sure the ring is placed on the ileum, not on the colon.
Carefully return the intestines to their original anatomical location and use an absorbable suture in a simple continuous stitch to close the musculoperitoneal layer along the linea alba. Clean any bleeding with 0.9%sterile saline soaked gauze and use a new nylon suture and a simple continuous stitch to close the skin. Then disinfect the wound site with povidone-iodine and inject antibiotics before placing the animal in a warming gate with monitoring until full recumbency.
In this experiment, the small intestine was partially filled and distended at eight days post partial obstruction surgery and fully filled and distended at 13 days post partial obstruction surgery compared to control sham operated animals. Further, fecal pellet formation in the colon of day eight and 13 post partial obstruction surgery mice was reduced compared to that in control animals. The smooth muscle layer of the ileum just upstream of the ring was hypertrophied at eight days post partial obstruction surgery with further hypertrophy observed at day 13.
The smooth muscle cells grew at progressively rapid rates in all three layers of tissue at eight and 13 days post partial obstruction surgery as assessed by immunochemical analysis while the interstitial cells of Cajal degenerated within the intra and intermuscular layers. Platelet-Derived Growth Factor Receptor alpha positive cell subsets also known as PDGFR alpha cells were dynamically remodeled within the intermuscular layers of partial obstruction mice with growth observed at eight days post surgery that had degenerated by day 13. In addition, neuronal cells were also significantly lost in the inter and intramuscular regions of partial obstruction mice at both days eight and 13 post surgery.
Once mastered, this technique should be completed within 30 minutes to ensure the survival of the mouse. While attempting this procedure, it is important to treat the tissue quickly and delicately. Following this procedure, other methods like gastrointestinal trend testing can be performed to answer additional questions about how obstructions alter intestinal peristalsis.
After its development, this technique paved the way for researchers in the field of gastrointestinal research to explore the consequences of intestinal obstruction in mice. After watching this video, you should have a good understanding of how to quickly and properly place a ring around the mouse ileum for the creation of intestinal partial obstruction. Don't forget that working with surgical tools and needles can be extremely hazardous so take the proper precautions.
Also, be sure to wear sterile gloves and practice aseptic techniques while performing this procedure.
