The overall goal of the following experiments is to perform an intestinal manipulation to study the development and dissolution of the postoperative ileus in a rodent model, this is achieved by standardized manipulation of the small bowel between two moist cotton applicators. Next, gastrointestinal and colonic transit are analyzed to measure postoperative motility. Then histochemical analysis and organ culture of the small bowel muscularis, externa, or me is performed to examine levels of inflammation.
Results are obtained that show a strong infiltration of neutrophil granulocytes into the me and excess production of nitric oxide in me cultures resulting in delays in gastrointestinal and colonic transit times. This method can help answer key questions concerning the development and the solution of postoperative ilios, and in investigating potential prophylaxis or therapy, Demonstrating the procedure will be Mario Lewison, a technician from our lab After administering 0.8 to 1%isoflurane in oxygen to a male mouse. Place the animal in a supine position and use adhesive tape to fix the extremities.
Use a slight pain stimulus to test for deep sedation. Then perform a median laparotomy, two centimeters in length. Next, insert two sterile retractors to keep the abdomen open and carefully eventuate the small bowel to the left and place it on wet cotton gauze.
Using two moist cotton applicators, evacuate the luminal contents of the entire small bowel by simultaneously rolling the applicators from the pori to the cecum. Put the gut back into the peritoneal cavity without twisting to prevent ischemia or mechanical obstruction or, and using two layers of continuous 5.0 polyamide non-absorbable sutures. Close the abdominal incision.
Place the animal under a heating lamp and monitor it until it recovers from anesthesia. Then place the mouse back in its cage and allow it access to food and water. The next day.
After fully anesthetizing the mouse, carefully hyper extend its head and use blunt forceps To pull out the tongue, fill a one milliliter syringe with at least 100 microliters of fluorescein labeled dextran. Connect it to an arterial catheter and carefully insert the catheter as a gastric tube into the stomach. Quickly inject 100 microliters of FITC dextran and remove the catheter.
Allow the animal to regain consciousness and wait 90 minutes. After the 90 minutes, euthanize the mouse and remove the entire gastrointestinal tract from stomach to rectum. Place the intestine onto a polystyrene pad and avoid stretching.
Next, measure the full length of the small bowel and the colon. Use cannulas to pin down the bowel to the polystyrene pad. Divide the small bowel into 10 equal segments and the colon into three equal segments.
After transecting the intestine at the march positions, flush the sections once with one milliliter of Krebs heli, bicarbonate buffer, or KHB. Transfer the sections into consecutively labeled two milliliter tubes and vortex vigorously centrifuge the probes. Then transfer the supernat into clean, numbered 1.5 milliliter tubes.
Store at four degrees Celsius in the dark until analysis. Next pipette. Duplicate a hundred microliter aliquots of each supernatant into a black 96 well plate and use a fluorescence reader to read the plate to check colonic transit time After weekly anesthetizing the animal.
Carefully examine colonic patency by inserting a fistula. Probe through the anus into the colon using the probe. Insert a two millimeter glass ball, three centimeters into the colon, and remove the probe.
Immediately start a timer. Place the mouse in a transparent cage and monitor it for the length of time it takes for the ball to be excreted. For histochemical analysis of mid toal samples.
After cutting tissue segments from the bowel and immersing them in cold KHB in a silicone filled dish, use insect needles to pin the mesentery to the dish. Cut open the segments along its mesenteric border and stretch the tissue to 120%of its length and width. Secure the tissue with needles starting at the mesentery.Mechanically.
Separate the muscularis externa or me from the tunica mucosa. Fix and stain the samples according to the written portion of this protocol to culture. The me.
24 hours after the intestinal manipulation procedure, resect the entire small intestine and place it in cold, KHB containing penicillin, G and streptomycin. Cut the gut into three centimeter lengths and pin each segment down on a silicone coated glass dish using fine scissors. Remove the mesentery and slide the intestine onto a nodding needle with sharp forceps.
Gently inci the me and using a moist cotton applicator. Strip it off from the submucosa and place it in cold, KHB plus Ps.Next, cut the ME strips into two to four millimeter length pieces and incubate it in ice cold KHB plus PS for 30 minutes. Rinsing the specimen several times.
Then transfer approximately 50 to 60 milligrams of me to a sterile 24 well plate containing 500 microliters of DMEM. Incubate the tissue at 37 degrees Celsius and 5%CO2 for 24 hours. Collect the supernatant and centrifuge at 1000 RCF for five minutes.
Then snap, freeze it in liquid nitrogen for cytokine or nitric oxide measurements. Finally, using a clean paper tissue blot, dry the muscle tissue for 30 seconds and then weigh it. The most important parameter for evaluating the severity of postoperative ileus is the examination of gastrointestinal transit or GIT in vivo.
This figure demonstrates a typical distribution of FITC dextran along the gastrointestinal tract in untreated controls and intestinally manipulated mice 24 hours after surgery. As shown here, sham operated animals showed normal GIT with a calculated geometric center in the cecum. While Im led to a significant delay in GIT in the proximal jejunum, in the colonic motility analysis using a glass ball, sham operated mice excreted the ball within 48 to 200 seconds, and Im prolonged the excretion time to 470 to 775 seconds to measure inflammation.
Within the ME leukocyte levels were analyzed using histo chemistry or immunohistochemistry as seen here. Few polymorphonuclear neutrophils were observed in control mice, however, I am of the small bowel led to their strong infiltration. Many inflammatory mediators liberated from cells are difficult to detect in tissue.
Lysates organ cultures allow detection of inflammatory markers in the culture. Supernatant, a representative mediator in POI is nitric oxide, or no, which is a major inhibitory neurotransmitter in the gastrointestinal tract. This figure shows that nno could not be detected in me.
Organ culture, supernatants of control, animals in sham operated mice, basal nno levels were observed. However, me cultures of intestinally manipulated mice produce significantly more nno after a 24 hour incubation Once mastered. This technique of intestinal manipulation can be done within 15 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to induce postoperative ileus in mice and how to analyze its severity.
