Rat Pancreas Isolation

The following procedure is used for isolating pancreata from rats weighing 150-300 g. For optimal success, the procedure should be performed as quickly as possible to avoid tissue degradation. This protocol can be used to isolate pan-creata from one to five rats during a single isolation. It is not recommended to use more than five rats per isolation because of the extended time for pancreas removal. The protocol anticipates that the total surgery time will be no longer than 30 min for a five-rat isolation. The surgery should be treated as a sterile procedure, although it is acceptable to perform the procedure on a bench top with care.

1. Prepare a minimum of 200 mL HBSS and fill each 20-cm3 syringe with cold HBSS (use one 20-cm3 syringe per rat). Attach the cannulas to each 20-cm3 syringe.

2. Anesthetize rats using approved institutional animal care guidelines. Once anesthetized, wet the abdominal fur with a 4 X 4 gauze pad soaked in 70% ethanol. Place the rat on its back with the head toward the surgeon. Make a midline incision of the skin down the abdomen using the large forceps and operating scissors. The incision should begin at the sternum and end at the level of the symphisis pubis. Wipe off the blades of the scissors with an ethanol-soaked gauze pad after the first incision to remove any fur. Make a second midline incision following the linea alba, from the sternum to the symphisis pubis, through the abdominal musculature and peritoneum to expose the internal organs.

3. Lay the edge of an unfolded gauze pad at the sternal edge of the incision. Using both hands, gently apply pressure at the edge of the gauze using a downward motion to flip all of the lobes of the liver cephalad. Secure the lobes with the free unfolded flap of the gauze. This will expose the common bile duct.

4. Locate the point at which the common bile duct enters the duodenum. Using the Halsted hemostat, clamp off the duct at the point where it enters the duodenum. Gently lay the hemostat in a position parallel with the animal's body. This will create tension on the duct and will slightly raise it for easier cannulation.

5. Locate the area where the common bile duct bifurcates into the dorsal lobes of the liver (see Fig. 1A). Using the small curved eye dressing forceps, make a small hole in the connective tissue just under and caudal to the bifurcation. Thread a piece of ligature through the hole and under the common bile duct with the small curved forceps. Tie a loose single knot just above the bifurcation. This will hold the can-nula in the duct, once in place.

6. Using the small curved iris scissors, make a small cut on the top of the widest part of the bifurcation. Be careful not to cut through the duct. Insert the cannula into the common bile duct through the hole at the bifurcation, with the bevel facing downward. Gently tighten the ligature around the cannula to secure it in the duct.

7. Inject HBSS into the pancreas at a rate of approx 6 mL/min. By injecting too quickly, the increased pressure can cause the outer capsule of the pancreas to burst and full inflation will not be achieved.

Bile Duct Cannulation Rat

Fig. 1. Cannulation of the common bile duct. These figures shows the cannulation point in a rat (A) and mouse (B) common bile duct. Note that the ligature knot placement is caudal to the insertion point of the cannula in the rat and that the clamp is placed at the juncture of the duct entering the duodenum in the mouse.

Fig. 1. Cannulation of the common bile duct. These figures shows the cannulation point in a rat (A) and mouse (B) common bile duct. Note that the ligature knot placement is caudal to the insertion point of the cannula in the rat and that the clamp is placed at the juncture of the duct entering the duodenum in the mouse.

8. Once the pancreas is inflated, remove the cannula, hemostat, and ligature. Using the small curved eye dressing and straight dressing forceps, gently tease the inflated pancreas away from the small intestine, the spleen, and the stomach. The remaining attachments will be near the great vessels deep in the abdominal cavity. Remove the remaining tissue by placing the forceps underneath the tissue and lifting upward. To prevent excessive tissue degradation, the pancreas should be removed in one piece.

9. Place the pancreas in a small beaker containing approx 10 mL cold HBSS and keep on ice. Once all the pancreata are excised, remove any fatty tissue, visible lymph nodes, and blood clots from the pancreas by moving it to a Petri dish and cutting away the unwanted tissues with the small curved iris scissors and curved forceps. This cleaning procedure should be completed as quickly as possible. The pancre-ata are now ready for digestion and isolation.

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  • mia
    How to remove pancreas in a rat?
    7 years ago
  • Claudia Moench
    How to remove pancrease from a rat?
    6 years ago

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