Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Summary

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.

Abstract

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 µL/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Introduction

In humans, the presence of gallstones is the most common cause of pancreatitis due to the obstruction of the terminal portion of the choledochal, interrupting the flow of pancreatic secretions and causing an intense inflammatory process in the pancreas, with an increase in the concentration of digestive enzymes in the serum and inflammatory mediators^1,2.

Two different theories have been proposed to explain the development of acute pancreatitis (AP). The "common channel" theory suggests that the stones present in the gallbladder obstruct the distal common bile duct system, al....

Protocol

This protocol was approved by the Ethics Committee for the use of animals of the USP Medicine School, Num. Project: 1343/2019-CEUA: FMUSP. For this protocol, C57BL/6 mice, aged 6 weeks, weighing 20 ± 2 g were used (n = 9/group).

1. Laparotomy

1. Anesthetize animals with xylazine (10 mg/kg) and ketamine solution (80 mg/kg), subcutaneously (0.1 mL/10g of body weight) using a 1 mL syringe and 13x0.45mm needle 26G ½. Check for sufficient anesthesia depth by pinching the toe. Con.......

Discussion

The method of inducing acute pancreatitis by retrograde sodium taurocholate infusion has already been shown in rats^22,23,24. Three similar works, published in 2008, 2010 and 2015, served as a reference for the protocol^3,14,15. In this work, we list all the critical steps for reproducing this method in C57BL/6 mice and some possibilities.......

Materials

Name	Company	Catalog Number	Comments
0.4 mm needle	INTRAG MEDICAL TECH	90183210	30G
0.54 mm polyethylene tube	Tygon	730010	-
Styrofoam block	-	-	-
masking tape for mounting the mouse	Missner	1236	-
Infusion pump scheduled to 10µL / min.	Havard aparatus-Peristaltic Pump Series	MA1 55-7766	Model 66 Small Peristaltic
Scissors and forceps
Antiseptic providine iodine	Pfizer	12086OR	antisepsis
70% ethanol	SIGMA	459836	Mix 700 mL 100% ethanol with 300 mL dH2O
Razor blade	Lord	bdk9a1ghk6	For trichotomy
Sodium taurocholate	Sigma-Aldrich	86339- 1G	CAS NUMBER- 345909-26-4
microvessel clip	Medicon Surgical	56.87.35	Approximator, opening 4.0 mm, closing pressure 30 - 40 g
6-0 prolene	Bioline	5162	Suture line
Ketamin NP (cloridrato de dextrocetamina) 50mg/mL	Cristália
Xilazine 2%	Syntec
Sterile saline solution (0.9% (wt/vol) saline)	Farmace	105851
Methyl Blue	Sigma-Aldrich Chemicals	M5528
MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay	MERCK	MCYTOMAG-70K	Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.
Amylase Assay	Labtest	11
Desmarres retractor 13-mm width	ROBOZ	RS-6672