Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

Treating severe acute pancreatitis is difficult due to high mortality rates using animal models, specifically mice with C57BL/6J selected background. We are investigating SAP as pathologic to identify therapeutic targets and develop new treatments, providing polymice method at the one SAP research.

This research uncovered high expression of HMGB1 genes injured pancreatitis, suggesting its association with the inflammatory stone of severe acute pancreatitis. Our protocol involves Caerulein to induce gall bladder contraction and disrupt fasting in mice, leading to pancreatic tissue damage by blocking digestive fluid secretion. Combined with LPS injection, we induced severe pancreatitis with organ failure in mice. This approach is non-invasive and easy to implement with short cycles and high repeatability.

[Demonstrator] To begin, measure and record the weight of the experimental mouse. For intraperitoneal injection of Caerulein, grab and hold the mouse with the belly facing up and the head positioned lower than the tail. Hold the syringe in the right hand and insert the needle into the subcutaneous skin on the left side of the abdominal white line. Move the needle forward approximately three to five millimeters and insert it into the abdominal cavity at a 45-degree angle. Keep the needle stationary and slowly inject the Caerulein. Pull out the needle, then gently massage and press the injection site with a sterile cotton swab to fully diffuse the drug into the abdominal cavity. 12 hours after injection, place the mouse in the middle of the grid in the experimental box facing away from the experimenter. Turn on the video tracking software and set up the monitoring for four simultaneous fields of view. Mark the length and width of the experimental object that can move within the box in the real-time monitoring screen. Record the mouse activity for 15 minutes. Macroscopic changes measured in the pancreatic injury mice model during the five cycles demonstrated that mice in the Pancreatic Injury Group 5 maintained a low level of movement distance within three minutes, while the immobility ratio within three minutes increased with each subsequent cycle. Pancreatic Injury Group 5 showed the smallest total movement distance compared to the other experimental groups, and the difference was statistically significant. Except for the Control and Pancreatic Injury Group 1, all groups showed negative D-value weight growth, with the Pancreatic Injury Group 5 showing the greatest change. The survival rate analysis indicated an 80% mortality rate in the Pancreatic Injury Group 5 by day five, unlike the other groups which showed no significant difference from the Control. After 36 hours of intraperitoneal injection of Caerulein, euthanize and secure the mouse in a supine position on a foam plate. Make an abdominal median incision and flip the small intestine tube to the right to expose the pancreas. Disconnect the duodenum and pyloric duct. Locate the small intestine beneath the pancreas, then entirely free the pancreatic tissue along the intestinal duct. Using toothless forceps, clamp the spleen and gently pull upwards. Sharply dissect the posterior pancreatic ligament tissue up to the head of the pancreas. Then disconnect the bile duct and blood vessels. Remove the pancreatic tissue from the abdominal cavity and pat dry the surface moisture with absorbent paper. Measure and record the weight of the pancreatic tissue. After preparing paraffin sections, de-wax the pancreatic tissues twice in xylene for five minutes each. Treat each section with 100 microliters of proteinase K and incubate at 37 degrees Celsius for 20 minutes. Add the required amount of 3% hydrogen peroxide to the tissue and incubate for 20 minutes to fully infiltrate it. Cover the entire area of the sample with 50 microliters of equilibration buffer and incubate for 10 minutes. Then add 56 microliters of TDT incubation buffer to each tissue sample and incubate for one hour. Wash the tissue sections four times with PBS for five minutes each before incubating for 30 minutes in 100 microliters of Streptavidin-HRP reaction solution. For DAB staining, add 50 microliters of DAB to each tissue section. Immerse the sections in hematoxylin staining solution for three to five minutes before rinsing with pure water. Dehydrate the samples with four rounds of fresh anhydrous ethanol for five minutes each. Perform a histological examination under a white light microscope. TUNEL staining on the pancreatic tissues of mice revealed an increase in cellular necrosis in the Pancreatic Injury Group 5, with significantly higher gray scale values and positive necrosis rates compared to other groups. Protein immunoblotting assessed Caspase-3 expression, a marker of cellular necrosis, and showed a significant rise in the Pancreatic Injury Group 5 tissues. Flow cytometry of pancreatic acinar cell suspensions, labeled with a Nexin 5 fit CPI, indicated a notably higher cell death rate in the Pancreatic Injury Group 5 compared to others, with statistical significance.