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Abstract

Introduction

Protocol

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Medicine

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Published: February 28th, 2021

DOI:

10.3791/62080

1Department of Surgery, Medical University of South Carolina, 2Ralph H. Johnson Veterans Affairs Medical Center
* These authors contributed equally

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a chronic inflammatory disease characterized by the atrophy of the pancreas, fibrosis, abdominal pain, and eventual loss of both exocrine and endocrine functions1. Current medical and surgical treatments are not curative but are undertaken to relieve symptoms that are the consequence of the disease: refractory abdominal pain, endocrine and exocrine dysfunction. Therefore, more effective treatments are urgently needed2. Animal models provide an essential tool for developing a better understanding of the disease and investigating potential therapeutics3. Multiple mouse m....

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All procedures were conducted with the approval of the Institutional Animal Care and Use Committees at the Medical University of South Carolina and the Ralph H. Johnson Medical Center. C57BL/6J male mice between 8-10 weeks of age were used in this study. Mice were housed under a standard 12 light/ 12 dark cycle with ad libitumaccess to food and water.

1. Preparation of TNBS solution for injection

  1. Prepare 10% ethanol in 0.9% saline. Dissolve stock TNBS (see Table of Material.......

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The bile duct infusion procedures were optimized to reduce mouse mortality associated with this procedure10. TNBS was first given in a total volume of 35 μL or 50 μL. Injection of TNBS in a volume of 50 μL could reach the whole pancreas and induce a more homogeneous disease phenotype (Figure 1B). In addition, injection of TNBS using insulin syringe with 31G needle could better control infusion speed relative to regular syringes and needle sizes. Freshly.......

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Bile duct infusion of TNBS to induce chronic pancreatitis is technically challenging in mice, as up to 22.5% of mice can die within 3-4 days of drug infusion10. Here, this report refined the procedure based on previous studies and reduced early mouse mortality to <10%. For example, the increased drug volume (from 35 μL to 50μL) can ensure the drugs reach the whole pancreas. Using an insulin syringe and a smaller needle size (31G) reduces potential damage to the pancreatic duct a.......

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This study was supported by the Department of Veterans Affairs (VA-ORD BLR&D Merit I01BX004536), and the National Institute of Health grants # 1R01DK105183, DK120394, and DK118529 to HW. We thank Dr. Hongju Wu for sharing technical experience.

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Name Company Catalog Number Comments
10% Neutral buffered formalin v/v Fisher Scientific 23426796
Alcohol prep pads, sterile Fisher Scientific 22-363-750
Animal Anesthesia system VetEquip, Inc. 901806
Buprenorphine hydrochloride, injection Par Sterile Products, LLC NDC 42023-179-05
Centrifuge tubes, 15 mL Fisher Scientific 0553859A
Ethanol, absolute (200 proof), molecular biology grade Fisher Scientific BP2818500
Extra fine Micro Dissecting scissors 4” straight sharp Roboz Surgical Instrument Co. RS-5882
Graefe forceps 4” extra delicate tip Roboz Surgical Instrument Co. RS-5136
Heated pad Amazon B07HMKMBKM
Hegar-Baumgartner Needle Holder 5.25” Roboz Surgical Instrument Co. RS-7850
Insulin syringe with 31-gauge needle BD 324909
Iodine prep pads Fisher Scientific 19-027048
Isoflurane Piramal Critical Care NDC 66794-017-25
Micro clip applying forceps 5.5” Roboz Surgical Instrument Co. RS-5410
Micro clip, straight strong curved 1x6mm Roboz Surgical Instrument Co. RS-5433
Micro clip, straight, 0.75mm clip width Roboz Surgical Instrument Co. RS-5420
Picrylsulfonic acid solution, TNBS, 1M in H2O Millipore Sigma 92822-1ML
Polypropylene Suture 4-0 Med-Vet International MV-8683
Polypropylene Suture 5-0 Med-Vet International MV-8661
Sodium chloride, 0.9% intravenous solution VWR 2B1322Q
Surgical drape, sterile Med-Vet International DR1826
Tissue Cassette Fisher Scientific 22-272416
Von Frey filaments Bioseb EB2-VFF

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