Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

Summary

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

Abstract

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Introduction

Surgical models of acute liver injury and repair, such as partial hepatectomy or common bile duct ligation (cBDL), have been used for decades in rodents to study liver regeneration and pathobiology^1,2. In cBDL, the common bile duct (which drains all bile produced in the liver) is ligated, resulting in obstructive cholestasis, inflammation, and fibrosis in the first 2-3 weeks following surgery, with eventual progression to cirrhosis³. Bile duct proliferation, transdifferentiation, and induction of resident liver progenitor cells are also features of cBDL^4,

Protocol

All procedures were conducted in accordance with ethical guidelines of the Institutional Animal Care and Use Committee of the University of Pittsburgh.

1. Basic Techniques and Common Procedures

1. Autoclave all microsurgical equipment before use.
2. To avoid killing the mouse by anesthesia, check its breathing pattern carefully.
3. Touch organs and intestines as little as possible to avoid damaging organs and causing bleeding. Press bleeding points with wet cotton swabs or gauze sponges.
4. Use wet cotton swabs for gentle manipulation of organs. Wet, non-woven gauze sponges are used in two sizes, 3.5 × 3.5 cm and 6.0 × 6.....

Results

For this typical experiment, pBDL was performed in triplicate PiZ transgenic mice, which recapitulate the human liver disease found in alpha-1 antitrypsin (A1AT) deficiency. The PiZ mouse overexpresses a transgene consisting of multiple genomic fragments of DNA that contain the coding regions of the mutant human A1AT gene and promoter, together with introns and ~2 kilobases of upstream and downstream flanking regions^14,15. Normal .......

Discussion

Although cBDL is the most common experimental technique for obstructive cholestasis, it can present multiple challenges in rodents. Depending on the number of post-operative days required to achieve an endpoint, the animals can experience significant morbidity and mortality as time progresses. The degree of tissue injury and biliary necrosis is further exacerbated when working with some transgenic mouse models of liver disease, which demonstrate abnormal liver parenchyma and function at baseline. Compared to cBDL, report.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope-Leica Wild M650, 6–40× magnification	Leica Microsystems	n/a
Tec 3 isoflurane funnel fill vaporizer	General Anesthetic Services	n/a
Stevens Tenotomy Scissors	Accurate Surgical & Scientific Instruments Corporation	ASSI.9193
Adson Forceps	Fine science tools	11006-12
Mosquito classic delicate hemostatic forceps	Codman	30-4472
Micro-retractor	Murdock; Roboz Surgical Instrument	RS-6550
Nonwoven gauze sponges	Fisherband	870-PC-DBL
Puritan 6” Small Cotton Swab w/Wooden Handle	Puritan Medical Products Company	868-WCS
Dumont SS Forceps - Standard Tips/Straight/Inox/13.5cm	Fine science tools	11203-23
Dumont SS Forceps - Standard Tips/Angled 45°/Inox/13.5cm	Fine science tools	11203-25
Vannas Spring Scissors - 3mm Cutting Edge	Fine science tools	15000-00
Microneedle holder	Aesculap Surgical Instruments	FD231R
Micro AROSuture, sterile 10-0 nylon suture, 70 µm, TAP points	AROSurgical Instruments Corporation	T4A10N07
4-0 Vicryl	Ethicon	J662H
5-ml Syringe	BD	309646
Falcon tissue culture dish, 60 × 15 mm	Corning	353002
Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml	Piramal Healthcare	NDC 66794-017-25
Buprenex injectable (buprenorphine hydrochloride)	Reckitt Benckiser Pharmaceuticals	NDC 12496-0757-1
Cefazolin for injection , USP	APP Pharmaceuticals	NDC 63323-238-61
PVP scrub solution (povidone–iodine 7.5%)	Medline	NDC 12496-0757-1
70% Ethanol	Decon Labs	2716
Phosphate Buffered Saline Without Calcium or Magnesium	LONZA	17-516F
Sirius Red	Sigma	365548
Hematolxylin	Fisher (Richard-Allan Scientific)	22-050-111 (7211)
Eosin	Anatech (Fisher)	832 (NC9686037)
Formalin	Fisher	SF100-20