Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice
June 28th, 2021
Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice.
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.
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Overview
This article presents a protocol for inducing severe acute biliary pancreatitis in C57BL/6 mice, allowing for the study of early inflammatory events and tissue changes. The method enables researchers to observe the pathophysiological alterations that occur during the initial stages of pancreatitis.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Pathophysiology
Background
- Animal models are essential for studying severe acute pancreatitis.
- Understanding early inflammatory responses can inform therapeutic strategies.
- Current clinical methods do not allow for early-stage observation of pancreatic changes.
- This protocol aims to replicate human acute pancreatitis conditions in a controlled environment.
Purpose of Study
- To provide a reproducible method for inducing acute biliary pancreatitis in mice.
- To facilitate the study of pharmacological responses and therapeutic interventions.
- To analyze inflammatory mediators in pancreatic tissue and adjacent organs.
Methods Used
- Retrograde infusion of sodium taurocholate into the pancreatic duct.
- Preparation of the surgical area and anesthesia of the mice.
- Monitoring and maintaining anesthesia during the procedure.
- Collection and analysis of pancreatic tissue and serum samples post-surgery.
Main Results
- Significant inflammation observed in pancreatic tissue 12 hours post-induction.
- Increased serum amylase and interleukin-6 levels in the acute pancreatitis group.
- Histological examination confirmed the severity of inflammation.
- Results indicate the method's effectiveness in mimicking human acute pancreatitis.
Conclusions
- The protocol is quick and reproducible for trained researchers.
- It allows for detailed study of inflammatory processes in pancreatitis.
- Future studies can explore the effects of treatments on acute pancreatitis.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma researchers to model early inflammatory events in acute biliary pancreatitis, supporting target validation and mechanistic de-risking in preclinical studies. By replicating human-relevant pathophysiological responses in C57BL/6 mice, the method facilitates pharmacological screening and biomarker analysis in a genetically tractable system. The approach enhances predictive confidence in evaluating therapeutic interventions targeting inflammatory pathways.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of inflammatory mediators and pathway activation in pancreatic tissue.
- Operational Value: Provides a reproducible model for assessing target engagement of drug candidates.
- Strategic Value: Supports hypothesis-driven target validation through measurable inflammatory outputs.
Screening & Assay Development
- Scientific Value: Generates quantifiable biomarkers such as serum amylase and interleukin-6 for assay readouts.
- Operational Value: Standardizes surgical and infusion parameters for consistent phenotype induction.
- Strategic Value: Enables high-fidelity screening of immunomodulatory compounds in a disease-relevant system.
Translational & Preclinical Research
- Scientific Value: Mirrors human acute pancreatitis inflammatory cascades for translational biomarker alignment.
- Operational Value: Utilizes C57BL/6 strain compatibility with antibody and reagent arsenals for downstream analysis.
- Strategic Value: Facilitates preclinical evaluation of therapeutic candidates with genetic modification feasibility.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by providing a validated inflammatory phenotype for target confirmation and lead optimization stages.
- Discovery Biology: Supports mechanistic interrogation of inflammatory pathways and cellular responses in pancreatitis.
- Screening: Delivers standardized biochemical readouts (amylase, IL-6) for compound effect assessment.
- Analytics: Enables quantitative comparison of biomarker levels across treatment and control groups.
- Translational Research: Aligns with preclinical validation through histopathological and cytokine profiling.
- Enterprise Reuse: Establishes a reusable surgical induction platform for multiple therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in inflammatory pathway modulation studies.
- Operational Value: Ensures reproducibility through defined infusion concentration, volume, and timing parameters.
- Strategic Value: Improves go/no-go decisions by linking target inhibition to phenotypic rescue.
- Portfolio Impact: Enables risk-adjusted prioritization based on target-mediated pancreatitis amelioration.
Implementation Considerations
- Requires expertise in murine surgical techniques and vascular cannulation.
- Dependent on precision infusion pumps and microsurgical instrumentation.
- Necessitates standardized anesthesia and postoperative monitoring protocols.
- Involves adaptation considerations for different mouse strains or genetic backgrounds.
- Limited by procedural complexity and animal welfare constraints in high-throughput settings.
