This method can be used for reproducing the inflammatory events that occur in acute biliary pancreatitis. It is possible to study changes in pancreatic tissue in its early stages, which is generally not possible in the clinic. The main advantages of this technique are that it is quick and easy to reproduce for a trained researcher, and it offers similar outcomes to acute pancreatitis in humans.
The method can be used to study pharmacological immune responses of drug molecules and other therapeutic interventions, as it allows the study of inflammatory-mediating cells in the pancreas and adjacent organs. When attempting this protocol, locating the organs and establishing the cannulation region is crucial. Memorize the location of the organs and exercise the injection application first using a colored solution such as methylene blue.
After checking the depth of anesthesia with a toe pinch, clean the abdominal area of an anesthetized mouse with a 5%povidone iodine solution. Using a trimmer, remove hair between the chest and lower abdomen and clean an approximately two square centimeter surgical area with 70%alcohol. Immobilize the animal on the surgical board and use scissors to cut five millimeters of the skin horizontally on the upper part of the abdomen and one centimeter below the xiphoid process.
Repeat the cut on the peritoneum to perform the laparotomy with minimal exposure of the cavity. Using a retractor, pull the liver towards the head at approximately one centimeter from the intestine and locate the region of the pancreas and the duodenum. Using forceps, lift the liver towards the animal head.
By gently pulling the portion of the small intestine, fix the two lateral ends with a 6-0 polypropylene suture for a better view of the distal portion of the common bile duct too. Using a micro vessel clip, occlude the proximal common bile duct temporarily. Expose the organ out of the abdominal cavity.
Then make a temporary occlusion of the distal common bile duct with an 8-0 suture. To access the common bile duct, puncture the periampullary region, which appears as a whitish part of the small intestine's wall. With a 0.4 millimeter needle connected to a 0.54 millimeter polyethylene tube, start the infusion pump for 2.5%sodium taurocholate solution infusion at a constant speed of 10 microliters per 10 grams of body weight for three minutes.
After the infusion, remove the micro vessel clip, the temporary 8-0 suture, and the injection needle from the pancreatic bile duct. Suture the abdomen with a 6-0 nonabsorbent monofilament polypropylene suture. The time between the laparotomy and the end suture should not be more than 30 minutes.
After the surgery, house the animals in polyethylene boxes lined with wood shavings and water and food ad libitum. Gently hold the animal with skin pulled back, promoting a slight protrusion of the eyeball, and position the animal with the eye facing upwards. Instill a drop of eye ointment containing local anesthetic in the animal's eye.
Position the end of a capillary tube in the medial corner of the eye and insert it gently under the eyeball at an angle of approximately 30 to 45 degrees. Rotate the capillary tube until blood flow begins. Once the collection is over, ensure homeostasis by keeping the eyelids closed with light compression.
Later, using a 27 gauge needle, inject four milliliters of ice cold PBS into the peritoneal cavity of the mouse. After the injection, gently massage the peritoneum for 10 seconds to remove adhered cells. Using scissors and tweezers, make a small cut of 0.5 centimeters on the inner skin and musculature to expose the abdominal cavity.
Insert a bulb pipette in the peritoneum and collect as much fluid as possible, taking care not to aspirate fatty tissue. Collect a part of the pancreas from the head region for further processing. Deposit the collected fluid into tubes kept on ice.
Then centrifuge the peritoneal fluid at 250 times G for five minutes and stock the supernatant for interleukin-6 dosing. To obtain serum, centrifuge the collected blood samples at 700 times G for 15 minutes and stock the supernatant for amylase and interleukin-6 dosing. Analysis of pancreatic tissue 12 hours after induction of acute pancreatitis showed intense inflammation in the affected organ when compared to tissue samples from the sham group.
Similar results were represented by histological examination after hematoxylin eosin staining of tissue samples from sham and APTA. Analysis of blood serum presented a significant increase in the serum amylase and interleukin-6 cytokine concentration in the AP group compared to the sham group. At the same time, interleukin-6 concentration was significantly higher in peritoneal cavity fluid in the AP group.
The severity of pancreatitis in this model is proportionally dependent on the concentration, the volume and pressure of the infusion, and the time of acute pancreatitis induction. With the help of this technique, the effects of continuous peritoneal lavage on severe acute pancreatitis can be studied.
