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^4,5. Although cBDL elucidates specific mechanisms of obstructive cholangiopathy, which are well-characterized with reproducible outcomes in wild-type mice and in those strains with normal livers^6,7,8, the procedure can be technically demanding in some transgenic mouse models of liver diseases, requiring more animals than anticipated due to the higher morbidity and mortality of biliary obstruction over time. This technique is also beneficial for studying transgenic mice with underlying liver disease, which might not otherwise tolerate the stress of prolonged time course experiments following cBDL.

pBDL can provide a reasonable alternative to help overcome some of these challenges. Initial pBDL studies in wild-type mice aimed to distinguish locally acting and systemic mediators of inflammation⁹. In pBDL, the main biliary confluence of the right and left hepatic bile ducts above the common bile duct is carefully visualized at the hilum, and only the left hepatic bile duct is ligated to generate a localized obstructive cholestasis. pBDL offers several advantages to cBDL. In pBDL, the unligated right lobe serves as an identically matched internal control for the ligated left lobe, subjected to the same systemic effects, such as nutritional status or circulating factors. Although systemic pro-inflammatory mediators were present, Osawa et al. observed less fibrosis and necrosis in the unligated lobes, but increased inflammatory cells and transforming growth factor-β expression in the ligated lobe⁹.

More recently, pBDL has been redefined to study liver parenchymal effects of retained bile¹⁰. By using the same animal for internal control (unligated "sham") and experimental groups, pBDL effectively halves the number of animals needed per experiment, which in turn is more cost-effective. The mice tolerate the effects of pBDL much better, so that the ideal time course experiment (≥2 weeks) can be achieved in the ligated lobe. Most importantly, pBDL can distinguish direct intrahepatic effects of obstructive cholestasis and retained bile components in the ligated lobe, compared to the unligated control lobe. Overall, pBDL is an attractive method to study lobe-specific effects of localized cholestasis in the liver.

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 × 6.0 cm. Use the smaller one to retract whole liver. Use the larger one to wrap the small and large intestines.

2. Presurgical Preparation

1. Use male c57/b6 mice, 8-12 weeks of age (when body weight is approximately 20-35 g) for partial bile duct ligation.
2. Allow the mice free access to water and food until induction of anesthesia.

3. Partial Bile Duct Ligation Operation

1. Anesthetize the mouse by putting the animal in an induction chamber with a mix of 1.5-2.0% isoflurane and 1 L/min oxygen. Monitor the animal carefully by observing the respiration rate. When the respiration rate of the animal is one breath per second, the mouse is well-anesthetized. Since front limb withdrawal reflex disappears more rapidly, pinch the hind limb foot as well to confirm complete absence of the withdrawal reflex. Eyes should be lubricated with an ophthalmic lubricating agent to prevent corneal ulcers. Note that first dose of analgesic can be given pre-operatively to prevent stimulation of pain receptors.
2. Shave the abdominal wall with hair clippers, and place the mouse on the surgical table under the surgical microscope. Fix all limbs to the table with tape. Use isoflurane inhalation to maintain the mouse under general anesthesia.
   1. Adjust the isoflurane vaporizer and use 2.0-3.0% isoflurane for induction.
3. Using sterile swabs, disinfect the abdominal wall first with 0.3-0.5 mL povidone-iodine applied topically, followed by 70% ethanol. Animal should be covered with a sterile drape.
4. Make a long midline abdominal incision from the xiphoid to the pubis using the scissors and Adson Forceps. After making the abdominal incision, reduce isoflurane to 0.8-1.0% for maintenance.
5. Expose the abdominal cavity by using a Micro-retractor. Hold the xiphoid with mosquito forceps and retract toward the head of the mouse. Fix the mosquito forceps in place using soft clay.
6. Instill 1-2 mL of warmed (37 °C) sterilized phosphate-buffered saline (PBS) into the abdominal cavity to avoid injuring abdominal organs. Hold the small intestines with wet cotton swabs and gently retract them toward the head of the mouse. Cut the ligaments between the small intestines and the retroperitoneum to avoid injuring the small intestines (Figure 1A).
7. Wrap the small and large intestines with a wet, non-woven gauze sponge. Place them on the caudal side of the abdominal cavity. Intestines wrapped in sterile gauze can be placed on the sterile draped field to avoid contamination.
8. Gently retract the whole liver toward the xiphoid by using a small wet non-woven gauze sponge to expose the hepatic hilum and hepato-duodenal ligament (Figure 1B).
9. Check to confirm the anatomy of the hepatic hilum. The common bile duct is located on ventral side of the portal vein. Confirm locations of the right and the left hepatic bile duct, which drain into the main biliary confluence at the hepatic hilum.
10. Identify the left hepatic bile duct which drains the left lobe, and encircle it with a 10-0 nylon suture using the blunt side of a suture needle (Figure 2A). Avoid injuring the portal vein, hepatic artery, and liver surface.
11. Ligate the left bile duct with 10-0 nylon. (Figure 2B).
12. Place the small and large intestines in the same position as before the operation by using wet cotton swabs.
13. Confirm there is no intestinal torsion and intra-abdominal bleeding, then close the muscle and the skin in two layers with 4-0 vicryl. Braided suture (Vicryl) was used for skin closure, but monofilament is preferred to reduce likelihood of infection.
    NOTE: Close fascia by continued suture from xiphoid to pubis, and close the skin by continued suture from pubis to xiphoid.
14. Stop anesthesia after closing the incision.

4. Postoperative Treatment and Follow-Up

1. Just after the surgery, pinch the dorsal skin of the mouse and inject Buprenex (0.1 mg/kg) and Cefazolin (100 mg/kg) subcutaneously, followed by Buprenex injection every 12 h for 3 days, and Cefazolin every 24 h for 3 days respectively^11,12,13. Note that first dose of analgesic can be given pre-operatively to prevent stimulation of pain receptors. Cefazolin and other peri-operative antibiotics are only necessary for immune-suppressed animals, such as those used in transplantation studies.
   ​NOTE: If the surgery is successfully performed, the mouse usually recovers within 30 min. Activity of the mice will be almost the same as the preoperative condition.
2. Check the mice daily for any signs of distress.
   1. Sacrifice the mice by the cervical dislocation under general anesthesia to procure livers and other tissue samples at defined time points (i.e., 7, 14, 21 days) after pBDL.

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