This protocol makes it possible to perform in vivo observation of lymphocyte dynamics in rat Peyer's patches for long periods of time. It can be used to explore the effects of various tracks on lymphocytes in vivo. Demonstrating the procedure will be Kazuki Horiuchi, a medical doctor from my laboratory.
Begin by forming a cannulation tube and dipping it in hot water, then bend and fix it to a plastic board with adhesive tape. After anesthetizing a male Wistar rat and removing the hair on its abdomen, make a horizontal incision with surgical scissors from the midline to the left subcostal area, taking care not to damage the blood vessels in the parietal peritoneum. Wrap the intestine with wet gauze and move it gently to the outside of the body to reveal the retroperitoneal organs.
Insert a notch between the erector muscles of the spine and the adipose tissue with surgical scissors, then bluntly separate them with fingers. Carefully strip off the connective tissue around the thoracic duct. Expose the thoracic duct from just beneath the left crus of the diaphragm caudally until 20 millimeters of the duct is visible.
Gently separate adhesions between the thoracic duct and aorta using precision tweezers or wet cotton swabs. Apply a ligature on the thoracic duct just beneath the left crus of the diaphragm causing the caudal thoracic duct to become distended. Make a five millimeter hole in the abdominal wall by stabbing it with the surgical scissors, pass the hairpin-curved cannulation tube into the hole and ligate the tube on the iliopsoas muscle.
Fill the cannulation tube with normal saline containing 10 units per milliliter heparin. After placing a string beneath the distended thoracic duct, stab the duct with a sharply cut edge of the cannulation tube, cannulate it approximately five millimeters toward the tail and ligate the thoracic duct with the cannulation tube. To prevent dehydration, create a three millimeter hole in the anterior wall of the antrum of the stomach using precision tweezers and pass a two millimeter silicon tube to the duodenum through the pyloric ring.
After closing the wound, maintain the animals in Bollman's cages and start to infuse sugar-laden saline into the rat's duodenum from the silicon tube at a flow rate of three milliliters per hour. Cover the entire cage with a paper towel to keep it warm. To collect the thoracic duct lymphocytes, fix the cannulation tube to the hole in the center of the lid of a 15 milliliter conical tube on ice containing six units per milliliter heparin, 10%fetal bovine serum and RPMI 1640 medium.
Dissolve CFDSE and dimethyl sulfoxide to 15.6 millimolar and incubate one times 10 to the ninth lymphocytes in 15 milliliters of RPMI 1640 with 50 microliters of CFDSE solution for 30 minutes at 37 degrees Celsius. Under continuous anesthesia with 2%isoflurane, placed the Wistar rat on a portable stainless steel plate with a rectangle hole around the center covered with a glass slide and open the abdomen of the recipient Wistar rat via a midline incision. Select approximately 10 centimeters of the ileal segment with Peyer's patches for observation.
Keep the intestine moist and as warm as possible by soaking the gauze that is used to cover the small intestine with PBS warmed to 37 degrees Celsius. Put the slide on the stage of the microscope and choose suitable areas for observation of the microcirculation in Peyer's patches where some high endothelial venules are running through the serosa. Cover the adjacent intestinal segment and mesentery with absorbent cotton soaked with PBS.
Place the intestine segment between two rolled cotton balls and position it as far as possible from the rat's body to prevent it from being vibrated by the rat's heartbeat and breathing. Use a one milliliter syringe to slowly inject the CFSE-labeled thoracic duct lymphocytes into the jugular vein of the recipient rats from the venous catheter. Continuously monitor thoracic duct lymphocytes in the microvasculature of the Peyer's patches using CLSM and perform time-lapse imaging for three hours at 32nd intervals.
Inject 25 milligrams per kilogram Texas Red-dextran into the jugular vein of the recipient rat to stain the bloodstream and five milligrams per kilogram Hoechest 33342 to stain the cell nuclei. To quantify the lymphocyte dynamics, define lymphocytes adhering to the high endothelial venules for more than 30 seconds as adhesive lymphocytes and those immigrating from the high endothelial venules to stroma as migrating lymphocytes. Then calculate the average percentage of migration in each field of view.
This protocol was used to collect naive gut tropic lymphocytes and observe their migration in rat Peyer's patches. Microscopic images of the Peyer's patches are shown here. The nuclei of vascular endothelial cells are stained blue.
The blood plasma is red, and the blood flow is detected by the flow of unstained cells. Gut tropic lymphocytes stained green can be seen adhering to high endothelial venules. When performing this protocol, keep in mind that the preparation to create the optimal environment for cannulation is important, including the exposure of the thoracic duct and adjustment of the positional relationship between the thoracic duct and the catheter running parallel.
Observing the lymphocytes dynamics in the Peyer's patches under several conditions may be are foundations for future research of gut immunity.
