Determining Bile Duct Density in the Mouse Liver

Summary

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Abstract

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

Introduction

The biliary tree is a critical part of the mammalian liver, allowing the passage of bile from hepatocytes into the gut. Intrahepatic bile ducts (BDs) are formed by cholangiocytes, which differentiate from bipotential hepatoblasts through Notch and TGFβ signaling^1,2. Proper specification and commitment of cholangiocytes and their assembly into mature BDs are critical for the development of the intrahepatic biliary tree. As the liver grows during development or upon organ regeneration, the biliary system needs to develop along the liver to ensure proper bile drainage. Moreover, a number of syndromic and non....

Protocol

All animals were housed in a barrier animal facility at Baylor College of Medicine per Institutional Animal Care and Use Committee guidelines and under approved animal protocols.

1. Collection of Mouse Liver Tissue

1. Preparation of mouse for liver harvest
   1. Euthanize the mouse using isoflurane.
   2. Perform cervical dislocation of the mouse to ensure death.
   3. Make a transverse incision approximately one inch below the rib cage.
   4. Expose the entire ventral surface of the liver.
2. Collection of the mouse liver
   1. Carefully, with small ....

Results

We previously documented biliary defects in Jag1+/– animals, a mouse model of ALGS⁸. To determine the BD to PV ratio, we sectioned P30 mouse livers and co-stained them for CK8 and CK19 (wsCK) along with the vascular marker αSMA. We then imaged all the PVs in each of the liver lobes. As shown in Figure 2A, we defined PVs as αSMA-stained vessels that have adjacent wsCK staining (arrowheads). The αSMA-stained structures without w.......

Discussion

Analysis of BD development and repair in mice is an important tool in studying the pathogenesis and mechanism of cholestatic disorders. In addition, development of new therapies is in part dependent upon establishing a reproducible and preferably quantifiable phenotype. Current phenotyping in mouse models usually involves serum chemistry, liver histology and immunostaining for cell-type specific markers. Although these techniques generate valuable information about the structure and function of the biliary system, they d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isothesia (Isoflurane)	Henry Schein	11695-6776-2
Desiccator	Bel-Art	16-800-552
10% PFA	Electron Microscopy Sciences	15712
50 mL tube	ThermoScientific	339653
70% Ethanol	Decon Laboratories	2401
95% Ethanol	Decon Laboratories	2801
100% Ethanol	Decon Laboratories	2701
HistoChoice	VWR Life Sciences	H103-4L	clearing agent
Omnisette Tissue Cassette	Fisher HealthCare	15-197-710E
Macrosette	Simport	M512
Paraplast X-TRA	McCormick Scientific	39503002	Parrafin
Tissue Mold	Fisher Scientific	62528-32
Microtome	Microm	HM 325
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Xylene	Fisher Scientific	C8H10
Tris-Based Antigen Retrieval	Vector Laboratories	H-3301
Pressure Cooker	Instant Pot	Lux Mini
Mini Pap Pen	Life Technologies	8877
Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)	J.T. Baker	X251-07
Octyl Phenol Ethoxylate (Triton-X-100)	J.T. Baker	X198-07
Normal Goat Serum	Jackson Immunoresearch	005-000-121
anti-CK8	Developmental Studies Hybridoma Bank	TROMA-I	Antibody Registry ID AB531826
anti-CK19	Developmental Studies Hybridoma Bank	TROMA-III	Antibody Registry ID AB2133570
anti-αSMA	Sigma Aldrich	A2547, Clone 1A4
anti-rat-Alexa488	ThermoFisher	A21208
anti-mouse-Cy5	Jackson Immunoresearch	715-175-151
DAPI	Vector Laboratories	H-1000
22 x 50 mm2 micro cover glass	VWR Life Sciences	48393 059
Fluorescence Microscope	Leica	DMI6000 B
Kimwipes	Kimtech Science	05511
VECTASHIELD	Vector Laboratories	H-1000	Antifade Mounting Medium