Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

Summary

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

Abstract

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

Introduction

The genesis and progression of cholangiopathies such as biliary atresia, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC) are either unknown or incomplete^1,2. The limited understanding of the origin and progression of those diseases leads to a paucity of therapy options³. The most difficult obstacle in studying neonatal bile duct disorders is gaining a molecular understanding of the pathophysiology. One of the essential keys to a better understanding of molecular pathology is the best possible observation of affected tissue. To avoid reduced comparability and ....

Protocol

Following ethical approval (N045/2021), male and female C57BL/6 mice neonates were observed until 9 days old. The animals were born and provided for experimental purposes by the animal facility of the University Medical Center Hamburg-Eppendorf, Hamburg, Germany. The neonates were housed in a cage together with their parent animals. The environmental conditions were controlled in temperature (20-24 °C), 12:12 h light-dark cycle, and relative humidity of 40%-70%.

1. Experimental preparation

1. Prepare the required equipment for surgical operation, including scissors, forceps, etc. (see Table of Material....

Results

Figure 1A shows the EBDS of a murine neonate, which was dissected with the described technique. Microscopically, no further hepatic tissue is visible. The hepatic tissue has been removed during the final isolation steps of the protocol and could easily be distinguished from bile duct tissue regarding color and consistency. Figure 1B displays the isolated sample compared to a millimeter scale. The EBD's length (measured from gall bladder to duodenal papilla) is l.......

Discussion

This article reported and discussed the creation and validation of a new surgical approach for removing the EBDS of euthanized neonatal mice. Microscopical and histological findings reveal that the approach quickly detects EBDs and dissects them near the duct's margins, even in neonatal mice. Only surgical instruments and a microscope with a 20x magnification are required for the described protocol. Furthermore, the approach allows the isolation of the entire EBDS. The technique is highly efficient, straightforward, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Propanol	CHEMSOLUTE	11365000	used as a dehydrating agent
30 G canula	B Braun/Sterican, Melsungen Germany	4656300	canula for hydration of the sample
Air vent	C + P Möbelsysteme GmbH & Co. KG, Breidenbach, Germany	Tec-Ononmic AZ 1200	the use of an air vent helps to avoid inhalation of formalin-containing fixatives
Aqua ad injectabilia Braun	B Braun, Melsungen, Germany	2351744	saline; Container: Mini-Plasco connect, 20 x 10 mL, sterile
Bigger microsurgical Forceps	DIADUST von Aesculap, Trossingen Deutschland	FD253R	straight, 180 mm (7"), platform tip, round handle, width: 0,800 mm, diamond dust coated, non-sterile, reusable optional tool for observation and every step of preparation except very final preparation; Dividing skin of the peritoneum
Camera “SmartCAM 5”	Basler and Vision Engineering, Send, United Kingdom	EVC131A	optional Lynx Exo camera modul: sensortype: CMOS, resolution 2560 x 1920 pixels, sensor size: 1/2"; Used for videoproduction and technical evaluation
Dehydration machine/Citadel 2000 Tissue Processor	Fisher Scientific GmbH, Schwerte, Germany	12612613	used for automatic dehydration, short program (approx. 4.8 h)
Dehydration sponge	Carl Roth, Karlsruhe, Germany	TT56.1	sponge for final dissection step, other sponges/foam pads with a minimum pore size of 60 pores per inch are also suitable, the use of  two foam pads per embedding cassette is recomended to cover the sample from below and above to prevent sliding through the perforation of the embedding cassettes
Dulbecco´s Phosphat Buffered Saline (PBS)	Gibco	14190-144	Doesn´t contain Calzium or Magnesium, 500 mL
Embedding cassettes	Engelbrecht GmbH, Edermünde, Germany	17990
Eosin	MEDITE Medical GmbH, Burgdorf, Germany	41-6660-00	staining solution, ready to use
Fine Scissors CeramaCut	FST, Heidelberg Germany	14959-09	Tips: Sharp-Sharp, Alloy / Material: Ceramic Coated Stainless Steel, Serrated:, Yes; Feature: CeramaCut, Tip Shape: Straight, Cutting Edge: 22 mm, Length: 9 cm; Skin incision, incision of the peritoneal window
Graefe Forceps	FST, Heidelberg Germany	11051-10	Length: 10 cm, Tip Shape: curved, serrated, Tip width: 0.8 mm, Tip Dimensions: 0.8 x 0.7 mm, Alloy /Material: Stainless Steel
Hematoxylin	MEDITE Medical GmbH, Burgdorf, Germany	41-5130-00	staining solution, ready to use
Highresolotion microscope	Vision Engineering, Send United Kingdom	EVO503	Capable of enlargement up to 60x magnification, only 6x to 20x magnification were used
Microscope	Olympus Optical CO, Ltd., Hamburg, Germany	BX60F5
Microscope Cover Glases	Marienfeld, Lauda-Königshofen, Germany	101244	60 mm broad, made of SCHOTT D 263 glass
Microscope Slides	R. Langenbrinck GmbH, Emmendingen, Germany	03-0060
Microtome	Leica, Nußloch, Germany	SM2010R	Tool for sectioning (2 µm-slices)
Omnifix-F 1 mL syringe	B Braun, Melsungen, Germany	9161406V	syringe without canula
Paraffin	Sakura Finetec, Torrance, USA	4511	Tissue-Tek Paraffin Wax Tek III, without DMSO
Paraffin embedding machine	MEDITE Medical GmbH, Burgdorf, Germany	TES 99	The embedding machine used in this study contained the following three individual modules: TES 99.420, TES 99.250, TES 99.600. The sample should be embedded in Paraffin directly after the dehydration, no interim storage in a fridge should be performed due to possible shrinking and moisture in the fridge
Paraformaldehyde (PFA)	Morphisto	1176201000	Prepare 1 mL Aliquots in 2 mL Eppendorf conical Tubes for liver samples and 0.5 mL Aliquots in 1 mL Eppendorf conical Tubes for extrahepatic bile duct samples, 4% in PBS ph 7.4
Small Microsurgical Forceps	EPM (Erich Pfitzer Medizintechnik), Bütthard, Bayern, Germany	(00)165	Round handle, straight, 0.3 mm tip, tool for observation and every step of preparation, especially useful in final preparation
Stainless Steel Ruler	Agntho's AB, Lidingö, Sweden	30085-15	150mm With Metric & Inch Graduations
Surgical Scissors – Sharp-blunt for decapitation	FST, Heidelberg Germany	14001-14	Device for decapitation
Warming cabinet	Haraeus, Hanau, Germany	T 6060	the sliced samples should be kept in the warming cabinet to ensure the attachement of the sample on the microscope slides