On the day before the surgery, cut the cannulas to the appropriate lengths, and use a sterile surgical blade to make a bevel at the tip of each piece of tubing. Next, to prepare the intraduodenal cannula, loop the cannula back onto itself, and then, heat the cannula to create an anchor point in the tubing.
On the day of the surgery, shave the fur from the right side of the abdomen and neck and left clavicle regions of an anesthetized adult rat, and then, clean the surgical regions aseptically with three successive povidone-iodine solution and 70% ethanol scrubs.
To cannulate the mesenteric lymph duct, place the animal with its right side facing up and use a sterile scalpel blade to open the top layer of the abdominal muscle wall with a straight four-centimeter incision extending from the xiphoid process to the right flank, approximately two centimeters below the costal margin.
Retract the small intestine under the left abdominal wall, and use two to three pieces of sterile gauze saturated with normal saline to keep it in place. Then, bridge the rat over a 10-milliliter plastic syringe placed horizontally under the animal's back at the level of the right kidney to assist in the visualization of the mesenteric lymph duct.
Locate the superior mesenteric lymph duct, which is approximately 0.5 to 1 millimeter in diameter, perpendicular to the right kidney, and immediately rostral and parallel to the dark red pulsating mesenteric artery. Note that in non-fasted rats, as shown here, the superior mesenteric lymph duct is white, opaque, and easier to visualize than in fasted rats.
Next, pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney parallel to the superior mesenteric lymph duct, and through the connective tissue layers, immediately below the vena cava. Then, using the tip of the forceps, pull the lymph cannula through the peri-renal fat bed with one end immediately adjacent to the mesenteric lymph duct, and the other exteriorized from the animal at the level of the right kidney.
Then, using a surgical microscope and jeweler's forceps, isolate the mesenteric lymph duct through the overlying layers of connective and fat tissue by blunt dissection, starting at the lower end and taking care not to damage the lymph duct. When the duct has been isolated, use the tip of the fine-tip forceps to make a small hole in the lymph duct. After ensuring that the lymph cannula is completely filled with anticoagulant solution and no air gaps, use a small pair of forceps to insert the lymph cannula approximately two to four millimeters inside the mesenteric lymph duct via the puncture.
If the cannulation is successful, a gradual flow of intestinal lymph will be observed from the free-collecting end of the cannula. When this occurs, place a small drop of cyanoacrylate glue over the entrance hole into the lymph duct to secure the cannula, taking care not to occlude the vessel with excess glue.
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