The authors acknowledge Johanna Hagens, Pauline Schuppert, Clara Philippi, PD Dr. med Christian Tomuschat, Svenja Warnke, PD Dr. Diana Lindner, Prof. Dr. Dirk Westermann, Miriam Tomczak, Nicole L&#252;der, Nadine Kurzawa, Dr. rer nat. Laia Pagerols Raluy, Birgit Appl, and Magdalena Trochimiuk for their contributions.&#160;Hans Christian Schmidt was financially supported by the Else Kr&#246;ner-Fresenius-Stiftung iPRIME Scholarship (2021_EKPK.10), UKE, Hamburg.

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 &#176;C), 12:12 h light-dark cycle, and relative humidity of 40%-70%.
1. Experimental preparation
<ol>
	<li>Prepare the required equipment for surgical operation, including scissors, forceps, etc. (see Table of Materials).</li>
	<li>Place disinfected and autoclaved instruments on a sterile surface next to the operation table.</li>
	<li>Euthanize a P9-aged neonatal mouse quickly by decapitation with an operating scissor. Place the body of the euthanized neonatal mouse on a sterile operation field. Dissect the EBDSs in 9-day-old mouse neonates (step 5). 
	&#8203;NOTE: The described dissection procedure gives any scientist the proper tools to remove the EBDS from neonatal and older mice. The older the mouse, the easier the preparation.</li>
</ol>
2. Access to the peritoneal cavity
<ol>
	<li>Grasp the skin above the location of the urinary bladder with forceps. Incise a 2 mm diameter hole into the skin using scissors, without damaging the peritoneum and underlying structures. Expand the cut to the location of the decapitation, following the left front axillar line. Remove the skin from the left to the right side with the atraumatic forceps.</li>
	<li>Take hold of the peritoneum surrounding the spleen. Gently lift it up until the peritoneum resembles a tent-like structure and cut a 1 mm diameter hole in the center. Wait for the &#34;peritoneal tent&#34; to fill up with air. Use a 10x microscopic magnification for this and the following steps.</li>
	<li>Cut off the peritoneum in a window framed by the lower ribs, both lateral abdominal regions, and the lower bladder area to ensure full access to the liver, bile duct system, stomach, small intestines, and colon. 
	&#8203;NOTE: To improve access to the liver, an additional incision can be performed, removing the three lowest ribs while leaving the xiphoid process, falciform ligament, liver, and bile duct structures intact. The view of the liver is easy to obtain.</li>
</ol>
3. Examination of the gall bladder and bile ducts
NOTE: Ensure to keep the sample wet on a regular basis during all of the following steps.
<ol>
	<li>Carefully pull the xiphoid process in a cranioventral position to examine the gall bladder. 
	NOTE: The tension on the falciform ligament increases with this motion, and the attached gall bladder becomes visible.
	<ol>
		<li>Perform only a slight pull to avoid uncontrollable tearing of the falciform ligament, which could lead to the gall bladder tearing from the bile duct system. Release the pull of the xiphoid process before the next step.</li>
	</ol>
	</li>
	<li>Gently pull down the duodenum to free the bile duct system. 
	&#8203;NOTE: As the tension on the hepatoduodenal ligament increases, the bile duct tissue becomes visible.</li>
</ol>
4. En-bloc-resection
<ol>
	<li>Perform the lower en-bloc-mobilization following the steps below.
	<ol>
		<li>Identify the duodenal papilla, which connects the bile duct system to the duodenum.</li>
		<li>Cut through the duodenum about 2 cm from to the right lateral side&#160;of the papilla.</li>
		<li>Cut through the pyloric area. Ensure that the stomach contents are present in the pyloric area between the cutting location and the duodenal papilla. 
		NOTE: This is an important step for later security of orientation for the correct location of oral and aboral duodenal parts.</li>
	</ol>
	</li>
	<li>Perform the upper en-bloc-mobilization.
	<ol>
		<li>Gently pull the xiphoid process and get access to the falciform ligament.</li>
		<li>Perform a 1 cm long cut through the falciform ligament, as close as possible to the xiphoid process, between the gall bladder and the xiphoid process. Ensure not to damage the gall bladder.</li>
		<li>Cut through the following connecting structures between the liver and the thorax: esophagus, inferior vena cava, thoracic aorta, all ligaments that surround the bare area of the liver, and all dorsally remaining tissue connections. 
		&#8203;NOTE: The en-bloc sample is completely dissected. It contains the liver, bile duct system, and the duodenal corpus, which is connected with the pyloric region of the stomach.</li>
	</ol>
	</li>
</ol>
5. Final gall bladder and bile duct dissection
<ol>
	<li>Perform the gross preparation following the steps below.
	<ol>
		<li>Place the en-bloc sample on a foam pad usually used for dehydration. Use a 20x microscopic magnification and two microsurgical atraumatic forceps with a maximum tip size of 6 mm for this and the following steps.</li>
		<li>Assemble the sample on the foam pad. Reorganize the sample in the correct anatomical position. Flatten the oral and aboral part of the duodenum, performing a gentle movement.</li>
		<li>Start the movement at the duodenal papilla and continue to the cutting edges using atraumatic forceps. Smooth out the white pulpy contents of the stomach, which will only occur in the oral part of the duodenum. Ensure to identify the oral and the aboral part of the duodenum to rule out probable bile duct rotation.</li>
		<li>Cut away large remnants of hepatic tissue to begin dissection of the bile duct.</li>
	</ol>
	</li>
	<li>Perform the final isolation.
	<ol>
		<li>Gently press the remaining hepatic tissue into the pores of the foam mat. Ensure that the scraping movements start from the bile duct system and lead to the liver boundaries.</li>
		<li>Transfer the sample to a cleaner position on the foam mat after a few scraping movements in various directions. Use the advantage of less squeezed liver tissue in the background to optimize the view for the best possible differentiation between EBDS and unwanted cells. Scrape the hepatic tissue from the bile duct until nothing, or as little as possible, of the hepatic tissue remains.</li>
		<li>Process the hepatoduodenal ligament until the isolated EBDS remains. Remove intraligamentous blood vessels like the hepatic artery, portal vein, and small remnants. Remove this delicate filament, with a gentle pull and high care, to the left lateral side. This dissection step might have already started unintentionally or partly completed during the prior removal of hepatic tissue, which in the end leads to the same result. 
		NOTE: The blood vessels are emerging as a white and very delicate filament approximately 3-5 mm orally of the duodenal papilla and join the hepatoduodenal ligament from the left lateral side, accumulating with the bile duct to the glissonian triad. With the completion of this step, the final sample is completely isolated. If there is a need for a record, images can be captured after organizing the bile duct structures into their anatomical position (Figure 1). Doing the last preparation steps on a foam mat is recommended because the sample will not stick as much to the surface of the operation field. If the pores are wet, the bile duct levitates or floats. When moving the sample, it will not stick as firmly as it would with preparation on the operation cloth, resulting in no ripping while moving the sample.</li>
	</ol>
	</li>
</ol>
6. Preparation for histological analysis
<ol>
	<li>Put the isolated sample in a buffered solution, special medium, or formalin-containing fixatives (see Table of Materials) as soon as possible after the dissection.</li>
	<li>Chose a suitable storage solution depending on further planned processing steps. 
	CAUTION: Use formalin-containing fixatives only under an air vent because of acute toxicity, corrosivity, and diverse health hazards. 
	NOTE: In the presented study, the EBDS samples have been inserted in paraformaldehyde, dehydrated, and embedded in paraffin. They have been stored at room temperature and cooled down prior to sectioning. In a warming cabinet, 2 &#181;m slices were kept overnight and stained using conventional hematoxylin and eosin4 (see Table of Materials).</li>
</ol>

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	<li>Liwinski, T., Schramm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prim&#228;r+sklerosierende+Cholangitis.">Prim&#228;r sklerosierende Cholangitis.</a> Der Internist. 59 (6), 551-559 (2018).</li><li>Kobayashi, H., Stringer, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia.">Biliary atresia.</a> Seminars in Neonatology. 8 (5), 383-391 (2003).</li><li>Patman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+tract:+Newly+identified+biliatresone+causes+biliary+atresia.">Biliary tract: Newly identified biliatresone causes biliary atresia.</a> Nature Reviews Gastroenterology &amp; Hepatology. 12 (7), 369 (2015).</li><li>Mack, C. L., Sokol, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+pathogenesis+and+etiology+of+biliary+atresia.">Unraveling the pathogenesis and etiology of biliary atresia.</a> Pediatric Research. 57 (5), 87-94 (2005).</li><li>Karjoo, S., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neonatal+extrahepatic+cholangiocytes.">Isolation of neonatal extrahepatic cholangiocytes.</a> Journal of Visualized Experiments. (88), e51621 (2014).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+normal+bile+ducts.">Functional anatomy of normal bile ducts.</a> The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology. 291 (6), 653-660 (2008).</li><li>Nakanuma, Y., Hoso, M., Sanzen, T., Sasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+and+development+of+the+normal+and+pathologic+biliary+tract+in+humans,+including+blood+supply.">Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply.</a> Microscopy Research and Technique. 38 (6), 552-570 (1997).</li><li>Banales, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholangiocyte+pathobiology.">Cholangiocyte pathobiology.</a> Nature Reviews Gastroenterology &amp; Hepatology. 16 (5), 269-281 (2019).</li><li>de Buy Wenniger, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cholangiocyte+glycocalyx+stabilizes+the+'biliary+HCO3-+umbrella':+an+integrated+line+of+defense+against+toxic+bile+acids.">The cholangiocyte glycocalyx stabilizes the 'biliary HCO3- umbrella': an integrated line of defense against toxic bile acids.</a> Digestive Diseases. 33 (3), 397-407 (2015).</li><li>Pinto, C., Giordano, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-purity+myenteric+plexus+from+adult+human+and+mouse+gastrointestinal+tract.">Isolation of high-purity myenteric plexus from adult human and mouse gastrointestinal tract.</a> Scientific Reports. 5 (1), 9226 (2015).</li><li>Ishii, M., Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Laboratory Investigation. 74 (1), 303-313 (1996).</li><li>Paradis, K., Sharp, H. 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The authors declare no conflict of interest.

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&#39;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, ...

The genesis and progression of cholangiopathies such as biliary atresia, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC) are either unknown or incomplete1,2. 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 discrepancies between research, such as observing potential viral etiology of biliary atresia4,&#160;the need for the best possible preparation and sharing of the performed dissection techniques arise. A pure preparation of the target tissue is necessary for later microscopical investigations or breeding cell- and 3D-organoid cultures. However, in murine neonatal disorders, tissue samples are rare and only occur in a small amount due to the very small size. Regarding bile duct disorders, difficulties in a clean preparation of bile ducts in murine neonates have been described5. Due to the neonatal stage of development, tissue differentiation is not overly advanced, which complicates preparation and increases the difficulty compared to the preparation of adult samples. Therefore, the operating workgroup investigated a novel strategy for preparing the EBDS in a neonatal mouse model. In the present study, the technique allows an efficientdissection of each sample.
The bile duct system is intraperitoneally placed in the right upper abdomen, arising from the liver. The gall bladder is located underneath the visceral surface of the liver&#39;s right lobe. The bile duct, together with the portal vein and hepatic artery, is embedded in the hepatoduodenal ligament. It joins the liver and the duodenum directly and drains bile fluid into the duodenum6. Anatomically, the bile duct is divided into the right and left hepatic ducts, the common hepatic duct, the cystic duct, and the Ductus choledochus, which is formed by the confluence of the cystic duct and the common hepatic duct7. This one eventually empties bile fluid and saliva from the pancreatic duct into the duodenum via the Ampulla of Vater.
Cholangiocytes line the bile duct intra- and extrahepatically, dwelling in a complicated anatomic niche where they assist in bile production and homeostasis8. Bile fluid passes these specialized epithelial cells in high concentrations daily. In particular, the HCO3- umbrella maintenance is very important for protecting against bile acid toxicity9. Cholangiocytes are the first line of defense in the hepatobiliary system against, for example, luminal microorganisms10. The cholangiocytes&#39; defense efficacy against toxic assaults may be weakened by genetic predisposition. A toxic overload causes damage and destruction and can therefore lead to cholangiopathies. Furthermore, the developing bile duct is not completely capable of all self-protective mechanisms, leading to a higher susceptibility to environmental toxins in neonatal bile ducts11.

<table><tbody><tr><td>2-Propanol</td><td>CHEMSOLUTE</td><td>11365000</td><td>used as a dehydrating agent</td></tr><tr><td>30 G canula</td><td>B Braun/Sterican, Melsungen Germany</td><td>4656300</td><td>canula for hydration of the sample</td></tr><tr><td>Air vent</td><td>C + P M&amp;ouml;belsysteme GmbH &amp;amp; Co. KG, Breidenbach, Germany</td><td>Tec-Ononmic AZ 1200</td><td>the use of an air vent helps to avoid inhalation of formalin-containing fixatives</td></tr><tr><td>Aqua ad injectabilia Braun</td><td>B Braun, Melsungen, Germany</td><td>2351744</td><td>saline; Container: Mini-Plasco connect, 20 x 10 mL, sterile</td></tr><tr><td>Bigger microsurgical Forceps</td><td>DIADUST von Aesculap, Trossingen Deutschland</td><td>FD253R</td><td>straight, 180 mm (7&quot;), 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</td></tr><tr><td>Camera &amp;ldquo;SmartCAM 5&amp;rdquo;&amp;nbsp;</td><td>Basler and Vision Engineering, Send, United Kingdom</td><td>EVC131A</td><td>optional Lynx Exo camera modul: sensortype: CMOS, resolution 2560 x 1920 pixels, sensor size: 1/2&quot;; Used for videoproduction and technical evaluation</td></tr><tr><td>Dehydration machine/Citadel 2000 Tissue Processor</td><td>Fisher Scientific GmbH, Schwerte, Germany</td><td>12612613</td><td>used for automatic dehydration, short program (approx. 4.8 h)</td></tr><tr><td>Dehydration sponge&amp;nbsp;</td><td>Carl Roth, Karlsruhe, Germany</td><td>TT56.1</td><td>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&amp;nbsp; 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</td></tr><tr><td>Dulbecco&amp;acute;s Phosphat Buffered Saline (PBS)</td><td>Gibco</td><td>14190-144</td><td>Doesn&amp;acute;t contain Calzium or Magnesium, 500 mL</td></tr><tr><td>Embedding cassettes</td><td>Engelbrecht GmbH, Ederm&amp;uuml;nde, Germany</td><td>17990</td><td></td></tr><tr><td>Eosin</td><td>MEDITE Medical GmbH, Burgdorf, Germany</td><td>41-6660-00</td><td>staining solution, ready to use</td></tr><tr><td>Fine Scissors CeramaCut</td><td>FST, Heidelberg Germany</td><td>14959-09</td><td>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</td></tr><tr><td>Graefe Forceps</td><td>FST, Heidelberg Germany</td><td>11051-10</td><td>Length: 10 cm, Tip Shape: curved, serrated, Tip width: 0.8 mm, Tip Dimensions: 0.8 x 0.7 mm, Alloy /Material: Stainless Steel</td></tr><tr><td>Hematoxylin</td><td>MEDITE Medical GmbH, Burgdorf, Germany</td><td>41-5130-00</td><td>staining solution, ready to use</td></tr><tr><td>Highresolotion microscope</td><td>Vision Engineering, Send United Kingdom</td><td>EVO503&amp;nbsp;</td><td>Capable of enlargement up to 60x magnification, only 6x to 20x magnification were used&amp;nbsp;</td></tr><tr><td>Microscope</td><td>Olympus Optical CO, Ltd., Hamburg, Germany</td><td>BX60F5</td><td></td></tr><tr><td>Microscope Cover Glases</td><td>Marienfeld, Lauda-K&amp;ouml;nigshofen, Germany</td><td>101244</td><td>60 mm broad, made of SCHOTT D 263 glass</td></tr><tr><td>Microscope Slides</td><td>R. Langenbrinck GmbH, Emmendingen, Germany</td><td>03-0060</td><td></td></tr><tr><td>Microtome</td><td>Leica, Nu&amp;szlig;loch, Germany</td><td>SM2010R</td><td>Tool for sectioning (2 &amp;micro;m-slices)&amp;nbsp;</td></tr><tr><td>Omnifix-F 1 mL syringe</td><td>B Braun, Melsungen, Germany</td><td>9161406V</td><td>syringe without canula</td></tr><tr><td>Paraffin</td><td>Sakura Finetec, Torrance, USA</td><td>4511</td><td>Tissue-Tek Paraffin Wax Tek III, without DMSO</td></tr><tr><td>Paraffin embedding machine</td><td>MEDITE Medical GmbH, Burgdorf, Germany</td><td>TES 99</td><td>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</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Morphisto</td><td>1176201000</td><td>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&amp;nbsp;</td></tr><tr><td>Small Microsurgical Forceps&amp;nbsp;</td><td>EPM (Erich Pfitzer Medizintechnik), B&amp;uuml;tthard, Bayern, Germany</td><td>(00)165</td><td>Round handle, straight, 0.3 mm tip, tool for observation and every step of preparation, especially useful in final preparation</td></tr><tr><td>Stainless Steel Ruler</td><td>Agntho&#039;s AB, Liding&amp;ouml;, Sweden</td><td>30085-15</td><td>150mm With Metric &amp;amp; Inch Graduations</td></tr><tr><td>Surgical Scissors &amp;ndash; Sharp-blunt for decapitation</td><td>FST, Heidelberg Germany</td><td>14001-14</td><td>Device for decapitation</td></tr><tr><td>Warming cabinet</td><td>Haraeus, Hanau, Germany</td><td>T 6060</td><td>the sliced samples should be kept in the warming cabinet to ensure the attachement of the sample on the microscope slides</td></tr></tbody></table>

extrahepatic bile duct and gall bladder dissection in nine-day-old mouse neonates

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.

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&#39;s length (measured from gall bladder to duodenal papilla) ...

Watch this Scientific Journal Video about Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates at JoVE.com

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

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.

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

extrahepatic-bile-duct-gall-bladder-dissection-nine-day-old-mouse

Research

JoVE Journal

Medicine

1.8K Views.  University Medical Center Hamburg-Eppendorf. 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.

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

Determining Bile Duct Density in the Mouse Liver

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.

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.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Developmental Biology

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

Engineering

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Immunology and Infection

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Biology

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

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

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.

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

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

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

Summary

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Chapters in this video

Determining Bile Duct Density in the Mouse Liver

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A Neonatal Mouse Spinal Cord Compression Injury Model

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Isolation of Neonatal Extrahepatic Cholangiocytes

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

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