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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 mediators1,2.

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.......

Representative Results

Pancreatitis severity was scored between 0-3 according to the Schmidt's scale21 where zero corresponds to the absence, 1 corresponds to a mild presence (<25%), 2 corresponds to a moderate presence (between 25 and 50%) and 3 corresponds to an intense presence (> 50%) (Table 1). The measurements performed were plasma amylase activity, pancreatic edema, acinar cell, injury/necrosis, pancreatic inflammation (by histology analysis of H&E.......

Discussion

The method of inducing acute pancreatitis by retrograde sodium taurocholate infusion has already been shown in rats22,23,24. Three similar works, published in 2008, 2010 and 2015, served as a reference for the protocol3,14,15. In this work, we list all the critical steps for reproducing this method in C57BL/6 mice and some possibilities.......

Acknowledgements

The authors thanks to Post Graduation Program in Medical Clinic of University of São Paulo; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and University of São Paulo Medical School (FMUSP).

....

Materials

NameCompanyCatalog NumberComments
0.4 mm needleINTRAG MEDICAL TECH9018321030G
 0.54 mm polyethylene tubeTygon730010-
Styrofoam block---
masking tape for mounting the mouseMissner1236-
Infusion pump scheduled to 10µL / min.Havard aparatus-Peristaltic Pump SeriesMA1 55-7766 Model 66 Small Peristaltic
Scissors and forceps
Antiseptic providine iodinePfizer12086ORantisepsis
70% ethanolSIGMA459836Mix 700 mL 100% ethanol with 300 mL dH2O
Razor bladeLordbdk9a1ghk6For trichotomy
Sodium taurocholateSigma-Aldrich86339- 1GCAS NUMBER- 345909-26-4
microvessel clipMedicon Surgical56.87.35Approximator, opening 4.0 mm, closing pressure 30 - 40 g
6-0 proleneBioline5162Suture line
Ketamin NP (cloridrato de dextrocetamina) 50mg/mLCristália
Xilazine 2%Syntec
Sterile saline solution (0.9% (wt/vol) saline)Farmace105851
Methyl BlueSigma-Aldrich ChemicalsM5528
MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex AssayMERCKMCYTOMAG-70KSimultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.
Amylase AssayLabtest11
Desmarres retractor 13-mm
width
ROBOZRS-6672

References

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Acute PancreatitisC57BL 6 MiceSodium TaurocholateBiliary PancreatitisInflammatory ResponsePharmacological InterventionLaparotomyCommon Bile DuctDuodenumInfusionAnimal Model

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