The overall goal of this video is to establish a mouse model of acute pancreatitis through retrograde injection of sodium taurocholate into biliopancreatic duct. All experiments involving animals were approved by the Animal Ethics Committee of Soochow University. All surgical procedures were performed under full anesthesia.
Analgesics were not used in order not to interfere with the natural course of the disease, which was evidenced by previous literatures and approved by the Animal Ethics Committee of Soochow University. Fast the mouse eight to 12 hours before the surgery. Prepare 5%sodium taurocholate solution filters through a 0.22 microfilter.
Autoclave the surgical instruments and materials, including forceps, scissors, blade holder, needle holder, hemostatic clip, vascular clamps, and the sterile drapes. The micro-syringe was sterilized through irradiation. With the mouse, induce a general anesthesia with intraperitoneal injection of 1%pentobarbital sodium solution at a dosage of 50 milligram per kilogram.
Fix the mouse in the supine position on an irradiated foam flat plate with the tape. Remove fur of a mouse with a depilation cream from the xiphoid process level down to the connecting line of the bilateral anterior superior iliac spine, with the left and right sides parallel to the front axillary line. Wipe the skin two to three times with 0.5%iodophor and let it dry for one to two minutes each time.
Cover the mouse with sterile drapes. Cut open skin layer and then open subcutaneously. Make an approximate two to three centimeters long median abdominal incision.
Locate the duodenum from left to right along the stomach. Gently pull the duodenum out and to the left side using surgical forceps and a disinfectant-soaked cotton swab to expose the pancreas, the biliopancreatic duct, and the duodenal papilla. Use 8-0 sutures to pass through the connection between the duodenum and pancreas around the papilla, pull the suture to the left side of the mouse body to fix the duodenum.
Gently pull the skin and the lower edge of the liver aside. Clamp the common hepatic duct. Pierce the microinjector into the distal biliopancreatic duct retrogradely.
Fix the needle tip near the duodenal papilla with a clamp. Infuse 10 microliters of 5%sodium taurocholate into the biliopancreatic duct at a rate of 5 microliters per minute. Keep the biliopancreatic duct closed for 5 min to achieve a stable pressure.
Remove the clamps. Pull out the microinjector, remove the other clamp and fix the suture. Restore the duodenum to the original anatomical position and check the bleeding.
Close the wound with 6-0 sutures. Remove the sterile drape. Disinfect the incision with 0.5%iodophor.
Recover the animals from anesthesia on a 37 degree Celsius thermal pad. Administer 0.8 milliliter of 0.9%saline subcutaneously for fluid supplementation. At 24 hour post-operation, anesthetize the mouse with pentobarbital sodium using the same dose and injection route.
Collect the whole blood from the abdominal aorta and spin at 1, 500 x g for 15 minutes to isolate the serum for serological testing. Collect the pancreas and the lung tissues for histology evaluation. Immerse the pancreas into 4%paraformaldehyde solution for 24 hours.
Dehydrate in a series of alcohol concentrations and embed in paraffin. Use a microtome to prepare 5 micro thick pancreas tissue sections. Perform H&E staining for the histological evaluation of the pancreas.
The mice were slightly inactive and had weight lost at 24 hours post-operation compared to pre-operation. The survival rates of the control and SAP mice were 100%and 72.7%respectively, at 72 hours post-operation. According to the H&E staining results from pancreas sections, pulpal necrosis of the pancreas tissue could be observed in SAP mice while no significant changes could be found in control mice.
Moreover, significantly increased levels of amylase, lipase, pancreas, and lung MPO activity were found in SAP mice compared to control mice. In addition, significantly elevated levels of the liver and kidney indexes including ALT, AST, BUN, and creatinine were also found. Using retrograde injection of sodium taurocholate into biliopancreatic duct, severe acute pancreatitis could be induced in mice.
Well exposure of the biliopancreatic duct to ensure the successful puncture. During the puncture, ensure the appropriate angle and force to avoid puncturing of the biliopancreatic duct. TC induced SAP model descried here is a suitable model to study the mechanisms involved in severe acute pancreatitis.
It is an economic model using less expensive reagents and this video helps training the novice surgeon faster.
