Our protocol allows us to directly measure postprandial metabolism in flowing mesenteric lymph. This is powerful because we can quantitatively determine the movement of nutrients from the small intestine into the lymph. And we can measure cytokines, dietary lipids, other dietary components and lymphocytes.
This technique can help us dissect the dynamics of dietary lipids moving into lymph, the synthesis and secretion of chylomicrons. The one day model presented here has several advantages. First, it reduces experimental time.
Second, it reduces animal stress. It increases animal survival. And overall, decreases experimental number.
Demonstrating our technique today will be our Senior Lab Surgeon, Mr.Nick Dedousis. And our Research Scientist, Dr.Lihong Teng. After preparing the mouse for surgery, grasp the skin with tweezers and make a midline abdominal incision with small scissors.
Then, cut toward the sternum and down to the inguinal fat. Using the retractor, move the peritoneal viscera out of the way until the lymph duct is visible. Using a saline soaked Q-tip, move the liver toward the top right side of the body and the intestines and stomach to the animal's left.
Then expose the superior mesenteric artery and the intestinal lymph duct by stretching the duodenum transversely toward the left. Prepare a 30 to 40 centimeter length of cannula tubing by inserting a blunt needle. Then, using a one milliliter syringe, flush a small amount of heparin through the tube and cut a bevel at the tip of the cannula tubing using scissors.
Then, make a shallow incision and lymph duct to using iris scissors as described in the manuscript. Hold the cannula tubing with a pair of fine forceps and gently insert the tip bevel into the duct. As the lymph duct is fragile, use a drop of cyanoacrylate to glue the lymph cannula into the mesenteric lymph instead of tying the catheter with a suture.
Now, using an 18 gauge needle, puncture a hole through the pyloric region of the stomach posterior to the pyloric sphincter, and insert the duodenal infusion tube in the stomach one to two millimeters beyond the pyloric sphincter into the duodenum. Then, secure the tube to the stomach with a purse-string ligature using a silk 5/0 suture and sealed with a drop of cyanoacrylate glue to prevent leakage. Start the intraduodenal infusion of 5%glucose and 0.9%saline.
Replace the organs in the body cavity and suture the midline incision. Place the mouse gently in a snuggle restraint. Provide the mice with a continuous intraduodenal infusion of saline and glucose for one hour.
Check the cannula for lymph flow and start milking the lymph cannula to keep lymph flowing. Perform the classic lipid infusion of a 0.3 milliliter lipid emulsion bolus via the intraduodenal infusion tube. Then, switch the infusion back to 5%glucose and 0.9%saline at 0.3 milliliters per hour continuously until the end of the experiment.
Collect the lymph samples and pre-weigh micro centrifuge tubes for 60 minutes and keep the lymph on ice. Weigh the tubes the second time to establish the weight of lymph secreted every 60 minutes. Collect the stomach, cecum and colon and place each organ into a 15 milliliter glass tube.
Divide the small intestine into three or four segments. Cut it open longitudinally and collect the luminal contents by rinsing the tissue with two to three milliliters of cold PBS. Remove the intestinal mucosa comprising epithelial cells and associated lamina propria from the muscular layer by scraping each section in two to three milliliters of cold PBS with a curved tweezer.
Place all tissues, the luminal and mucosal isolates, and the muscular layer in glass tubes. The triglyceride concentration in lymph increased in response to an intraduodenal bolus of 300 microliters of SMOFlipid. Peak triglyceride concentration is reached at two to three hours post bolus and decreases steadily through the six hour time.
The lymph flow rate increases from time zero bolus infusion through the end of the experiment. A major goal of this protocol was to reduce animal stress increase survival rate, and decrease anesthesia time. In order to accomplish this, we switched Bollman restraint cages for snuggles, and we added humidity, warmth, and fluid replenishment.
Our one day limp fistula protocol allows us to measure in real time, in vivo, lipid absorption dynamics. It also allows us to study gut organ crosstalk, metabolism, dietary nutrient absorption, and immune phenotypes.
