Name: determining bile duct density in the mouse liver
Uploaded: 2019-04-30T14:45:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

The authors acknowledge support from the National Institutes of Health (NIH) (R01 GM084135 and R01 DK109982), a Pilot/Feasibility Award from the Texas Medical Center Digestive Disease Center under NIH P30 DK56338, and an Alagille Syndrome Accelerator Award from The Medical Foundation.

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
<ol>
	<li>Preparation of mouse for liver harvest
	<ol>
		<li>Euthanize the mouse using isoflurane.</li>
		<li>Perform cervical dislocation of the mouse to ensure death.</li>
		<li>Make a transverse incision approximately one inch below the rib cage.</li>
		<li>Expose the entire ventral surface of the liver.</li>
	</ol>
	</li>
	<li>Collection of the mouse liver
	<ol>
		<li>Carefully, with small scissors, cut through the ligaments connecting the liver to other organs in the abdomen.</li>
		<li>Cut through the common BD to detach the liver from the intestine.</li>
		<li>Carefully remove the liver by holding onto the gallbladder and immediately place in a 50 mL tube filled to three-quarters by 4% paraformaldehyde (PFA).</li>
	</ol>
	</li>
</ol>
2. Fixation and Embedding the Liver in Paraffin
<ol>
	<li>Fixation
	<ol>
		<li>Fix the liver tissue for 48 h in 4% PFA at 4 &#176;C.</li>
		<li>Wash the tissue with 70% EtOH for 1 h at 4 &#176;C.</li>
		<li>Wash the tissue twice with 95% EtOH for 1 h each at 4 &#176;C.</li>
		<li>Wash the tissue twice with 100% EtOH for 1 h each at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Clearing
	<ol>
		<li>Wash the liver tissue with clearing agent (Table of Materials) three times for 30 min each at room temperature. 
		NOTE: The liver should feel rigid following the third wash.</li>
	</ol>
	</li>
	<li>Embedding in paraffin
	<ol>
		<li>Place the tissue cassette in a tissue mold in paraffin wax for 3 washes, 30 min each. Wax should be preheated to 60 &#176;C.</li>
		<li>Fill the tissue mold with paraffin wax to three-quarters height and keep on a heating block at 60 &#176;C.</li>
		<li>Place the liver in the mold with the ventral side facing up.</li>
		<li>Carefully remove the mold from the heating block.</li>
		<li>Place the top of the cassette on the mold and top off with hot liquid paraffin.</li>
		<li>Allow the mold and block to cool to room temperature overnight. 
		NOTE: Tissue blocks can now be stored at room temperature.</li>
	</ol>
	</li>
</ol>
3. Sectioning liver tissue
<ol>
	<li>Preparation of the block for sectioning
	<ol>
		<li>Place the mold on ice for 5 min before removing the block from the mold.</li>
		<li>Place the block on ice with a lab tissue paper present between block and ice.</li>
		<li>Keep the block on ice when not sectioning for best tissue slicing results.</li>
	</ol>
	</li>
	<li>Sectioning the liver blocks
	<ol>
		<li>Using a microtome, begin by sectioning through the superficial, dorsal side of the liver. Sections should be 5 &#181;m.</li>
		<li>Check the superficial sections under a dissection microscope to ensure sections are not sheared or folded.</li>
		<li>Take a section of the liver that includes the caudate lobe. 
		NOTE: For some blocks, you will have the left, medial, right and caudate lobes on the same tissue slice.</li>
		<li>For those blocks where all four lobes are not present on the same slide, continue to slice until the left, medial and right lobes are present on the same slide.</li>
	</ol>
	</li>
</ol>
4. Immunohistochemistry for wsCK and &#945;SMA
<ol>
	<li>Processing of slides for immunohistochemistry
	<ol>
		<li>Select one slide per genotype to be analyzed.</li>
		<li>Wash the slide for 15 min in Xylene, 100% EtOH, 95% EtOH and finally 70% EtOH (3 x 5 min in each solution).</li>
		<li>Wash the slide for 5 min in deionized H2O.</li>
		<li>Immerse the slide in the antigen retrieval solution (Tris-based, high pH).</li>
		<li>Heat under pressure in a pressure cooker for 3 min at 10 psi.</li>
		<li>Allow the slide to cool to room temperature (approximately 35 min).</li>
	</ol>
	</li>
	<li>Blocking the tissue sections
	<ol>
		<li>Using a Pap Pen, outline the sections on the slide.</li>
		<li>Apply phosphate-buffered saline (PBS) + 0.1% Tween to cover the section twice, 5 min each.</li>
		<li>Make blocking buffer by mixing Normal Goat Serum (NGS) at 1:50 in PBS + 0.3% Triton. To have enough buffer for both blocking and primary antibody application, 100 &#181;L per section is sufficient.</li>
		<li>Apply 100 &#181;L of blocking solution per section.</li>
		<li>Incubate the slides covered with the blocking solution at 4 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Staining for wsCK and &#945;SMA 
	<ol>
		<li>Dilute anti-CK8 and anti-CK19 antibodies16 (Developmental Studies Hybridoma Bank, TROMA-I and TROMA-III, respectively) 1:20 in blocking buffer to stain for wsCK. Dilute the anti-&#945;SMA antibody17 (Table of Materials) to 1:200 in the same buffer.</li>
		<li>Apply 100 &#181;L of the diluted antibody solution containing all three antibodies to each section.</li>
		<li>Incubate the slides covered with the antibody solution at 4 &#176;C overnight.</li>
		<li>Wash the slides with PBS + 0.1% Triton three times, 5 min each.</li>
		<li>Dilute secondary antibodies (anti-rat-Alexa488 and anti-mouse-Cy5) 1:200 in PBS + 0.3% Triton.</li>
		<li>Apply 100 &#181;L of the secondary antibody solution containing both secondary antibodies to the slides.</li>
		<li>Incubate at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>DAPI nuclear staining and mounting
	<ol>
		<li>Wash the slides three times, 5 min each.</li>
		<li>Apply 100 &#181;L of DAPI (1:3000) to each section for 10 min.</li>
		<li>Apply Antifade Mounting Medium (Table of Materials) to the slides and place a glass coverslip on top of the tissue sections. Leave the slides at 4 &#176;C overnight. Seal the slides the next day.</li>
		<li>Store the slides at 4 &#176;C and image within 1 week of mounting.</li>
	</ol>
	</li>
</ol>
5. Imaging and Quantification of BDs
<ol>
	<li>Imaging liver sections
	<ol>
		<li>Prior to imaging, blind yourself to the genotype of the sample with help from a lab member. Ensure all imaging files are devoid of genotype or other specific identifying information besides an animal/sample number.</li>
		<li>Using a fluorescent microscope, take 20x images at 1x zoom of each section and ensure that every PV&#160;across the liver is imaged. Include the left, medial, right and caudate lobes. 
		NOTE: We usually find 60-90 portal tracts per animal depending on the size of the liver.</li>
		<li>To identity the PVs, look for &#945;SMA plus wsCK staining. Structures that are &#945;SMA positive but lack wsCK staining are not portal structures.</li>
	</ol>
	</li>
	<li>Identification and counting of BDs
	<ol>
		<li>Create a spreadsheet with the following columns: Animal/Sample Number, Image Number, Number of PVs, and Number of BDs.</li>
		<li>Going through each image, identify and record the number of PVs per image.</li>
		<li>Identify patent BDs in each image by the presence of cholangiocytes (wsCK+) surrounding a definable lumen. Structures should be distinct and separated by mesenchyme from other wsCK+ cells.</li>
		<li>Count each patent BD and place in the same column as the image number.</li>
		<li>Do this for each image taken of a PV.</li>
		<li>Calculate the sum of all PVs and all BDs in the liver sample.</li>
		<li>Calculate the BD to PV ratio for the liver sample.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zong, Y., 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=Notch+signaling+controls+liver+development+by+regulating+biliary+differentiation.">Notch signaling controls liver development by regulating biliary differentiation.</a> Development. 136 (10), 1727-1739 (2009).</li><li>Clotman, F., 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=Control+of+liver+cell+fate+decision+by+a+gradient+of+TGF&#946;+signaling+modulated+by+Onecut+transcription+factors.">Control of liver cell fate decision by a gradient of TGF&#946; signaling modulated by Onecut transcription factors.</a> Genes &amp; Development. 19 (16), 1849-1854 (2005).</li><li>Karpen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+etiologies+and+management+of+neonatal+cholestasis.">Update on the etiologies and management of neonatal cholestasis.</a> Clin Perinatol. 29 (1), 159-180 (2002).</li><li>Roskams, T. A., 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=Nomenclature+of+the+finer+branches+of+the+biliary+tree:+canals,+ductules,+and+ductular+reactions+in+human+livers.">Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.</a> Hepatology. 39 (6), 1739-1745 (2004).</li><li>Oda, T., 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=Mutations+in+the+human+Jagged1+gene+are+responsible+for+Alagille+syndrome.">Mutations in the human Jagged1 gene are responsible for Alagille syndrome.</a> Nature Genetics. 16 (3), 235 (1997).</li><li>Li, L., 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=Alagille+syndrome+is+caused+by+mutations+in+human+Jagged1,+which+encodes+a+ligand+for+Notch1.">Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.</a> Nature Genetics. 16 (3), 243 (1997).</li><li>Xue, Y., 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=Embryonic+lethality+and+vascular+defects+in+mice+lacking+the+Notch+ligand+Jagged1.">Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1.</a> Human Molecular Genetics. 8 (5), 723-730 (1999).</li><li>Thakurdas, S. 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=Jagged1+heterozygosity+in+mice+results+in+a+congenital+cholangiopathy+which+is+reversed+by+concomitant+deletion+of+one+copy+of+Poglut1+(Rumi).">Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).</a> Hepatology. 63 (2), 550-565 (2016).</li><li>Hadchouel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paucity+of+interlobular+bile+ducts.">Paucity of interlobular bile ducts.</a> Seminars in Diagnostic Pathology. 9 (1), 24-30 (1992).</li><li>Emerick, K. 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=Features+of+Alagille+syndrome+in+92+patients:+frequency+and+relation+to+prognosis.">Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.</a> Hepatology. 29 (3), 822-829 (1999).</li><li>Poncy, A., 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=Transcription+factors+SOX4+and+SOX9+cooperatively+control+development+of+bile+ducts.">Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.</a> Dev Biol. 404 (2), 136-148 (2015).</li><li>Hofmann, J. 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=Jagged1+in+the+portal+vein+mesenchyme+regulates+intrahepatic+bile+duct+development:+insights+into+Alagille+syndrome.">Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.</a> Development. 137 (23), 4061-4072 (2010).</li><li>McCright, B., Lozier, J., Gridley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Alagille+syndrome:+Notch2+as+a+genetic+modifier+of+Jag1+haploinsufficiency.">A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency.</a> Development. 129 (4), 1075-1082 (2002).</li><li>Andersson, E. R., 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=Mouse+Model+of+Alagille+Syndrome+and+Mechanisms+of+Jagged1+Missense+Mutations.">Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.</a> Gastroenterology. 154 (4), 1080-1095 (2018).</li><li>Loomes, K. 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=Bile+duct+proliferation+in+liver-specific+Jag1+conditional+knockout+mice:+effects+of+gene+dosage.">Bile duct proliferation in liver-specific Jag1 conditional knockout mice: effects of gene dosage.</a> Hepatology. 45 (2), 323-330 (2007).</li><li>Brulet, P., Babinet, C., Kemler, R., Jacob, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+trophectoderm-specific+markers+during+mouse+blastocyst+formation.">Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.</a> Proc Natl Acad Sci U S A. 77 (7), 4113-4117 (1980).</li><li>Skalli, O., 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=A+monoclonal+antibody+against+alpha-smooth+muscle+actin:+a+new+probe+for+smooth+muscle+differentiation.">A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.</a> J Cell Biol. 103 (6 Pt 2), 2787-2796 (1986).</li><li>Kamath, B. 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=A+longitudinal+study+to+identify+laboratory+predictors+of+liver+disease+outcome+in+Alagille+syndrome.">A longitudinal study to identify laboratory predictors of liver disease outcome in Alagille syndrome.</a> Journal of Pediatric Gastroenterology and Nutrition. 50 (5), 526 (2010).</li><li>Mouzaki, 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=Early+life+predictive+markers+of+liver+disease+outcome+in+an+International,+Multicentre+Cohort+of+children+with+Alagille+syndrome.">Early life predictive markers of liver disease outcome in an International, Multicentre Cohort of children with Alagille syndrome.</a> Liver International. 36 (5), 755-760 (2016).</li><li>Shiojiri, N. <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+differentiation+of+bile+ducts+in+the+mammalian+liver.">Development and differentiation of bile ducts in the mammalian liver.</a> Microsc Res Tech. 39 (4), 328-335 (1997).</li><li>Crawford, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+intrahepatic+biliary+tree.">Development of the intrahepatic biliary tree.</a> Semin Liver Dis. 22 (3), 213-226 (2002).</li><li>Kaneko, K., Kamimoto, K., Miyajima, A., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+remodeling+of+the+biliary+architecture+underlies+liver+homeostasis.">Adaptive remodeling of the biliary architecture underlies liver homeostasis.</a> Hepatology. 61 (6), 2056-2066 (2015).</li><li>Schaub, J. R., 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=De+novo+formation+of+the+biliary+system+by+TGFbeta-mediated+hepatocyte+transdifferentiation.">De novo formation of the biliary system by TGFbeta-mediated hepatocyte transdifferentiation.</a> Nature. 557 (7704), 247-251 (2018).</li><li>Sparks, E. E., Huppert, K. A., Brown, M. A., Washington, M. K., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51 (4), 1391-1400 (2010).</li><li>Tanimizu, N., 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=Intrahepatic+bile+ducts+are+developed+through+formation+of+homogeneous+continuous+luminal+network+and+its+dynamic+rearrangement+in+mice.">Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.</a> Hepatology. 64 (1), 175-188 (2016).</li><li>Walter, T. J., Sparks, E. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-dimensional+resin+casting+and+imaging+of+mouse+portal+vein+or+intrahepatic+bile+duct+system.">3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system.</a> J Vis Exp. (68), e4272 (2012).</li><li>Popper, H., Kent, G., Stein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductular+cell+reaction+in+the+liver+in+hepatic+injury.">Ductular cell reaction in the liver in hepatic injury.</a> J Mt Sinai Hosp N Y. 24 (5), 551-556 (1957).</li><li>Yimlamai, D., 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=Hippo+pathway+activity+influences+liver+cell+fate.">Hippo pathway activity influences liver cell fate.</a> Cell. 157 (6), 1324-1338 (2014).</li></ol>

The authors have no conflict of interest.

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...

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 TGF&#946; signaling1,2. 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-syndromic diseases result in the paucity of intrahepatic BDs3. In addition, a number of acute and chronic liver diseases give rise to so-called ductular reactions in the liver, which are defined as the presence of a significant number of cells that express biliary markers but do not necessarily arise from biliary cells or form patent BDs4. In the multisystem disorder Alagille syndrome (ALGS), haploinsufficiency of the Notch ligand jagged1 (JAG1) results in poor BD formation and cholestasis5,6. Our lab recently demonstrated that a previously generated Jag1 heterozygous mouse line7 is an animal model of BD paucity in ALGS8. In this mouse model of ALGS, cholangiocytes are still present. However, they fail to commit to incorporation into mature, patent BDs8. Therefore, analysis of the liver in a model of BD paucity requires more than the apparent presence or absence of cholangiocytes. It is important to accurately assess the degree to which mature BDs are present in the liver.
In anatomic pathology, there are accepted quantitative methods for assessing whether BD paucity exists9. For example, studies on ALGS in human patients often quantify the BD to portal vein (PV) ratio by analyzing at least 10 portal vessels per liver biopsy9,10. Analysis of the shape and overall presence or absence of patent BDs, combined with serum chemistry, can provide valuable information about BD development in mice11,12,13. However, mice can lose a significant number of BDs with only a modest increase in serum bilirubin level8. Accordingly, a quantitative method that evaluates the number of BDs present per PV can provide a more direct measure of the degree of BD paucity in mice. In a recent report, we quantified the number of BDs per PV across all liver lobes and reported a significant decrease in the BD to PV ratio in Jag1+/&#8211; animals8. During the course of our analysis, we noticed that despite the significant variation in the degree of inflammatory response and ductular reactions, the BD to PV ratio does not show much variability8. Moreover, quantification of the BD to PV ratio allowed us to demonstrate that removing one copy of the glycosyltransferase gene Poglut1 in Jag1+/&#8211; animals can significantly improve their BD paucity8. In a Jag1+/+ background, conditional loss of Poglut1 in vascular smooth muscle cells results in a progressive increase in BD numbers, which is modest (20-30%) at P7 but becomes prominent in adults8. Again, this technique allowed us to show that even at P7, the increase in BD density in these animals is statistically significant. Of note, the increased BD density in this genotype at four months of age was validated through resin cast analysis as well.8 These observations and other reports which measured BD density in different ALGS mouse models14,15 prompted us to incorporate this method into our overall strategy to analyze biliary defects in various mutant and transgenic mice.
Here, we detail a straightforward technique which can be used to examine the degree of BD paucity in mouse models of liver disease (Figure 1). In this method, co-staining with cholangiocyte markers cytokeratin (CK) 8 and CK19 (hereafter wide-spectrum CK, wsCK) is used to visualize BDs and unincorporated cholangiocytes in the mouse liver. An antibody against alpha-smooth muscle actin (&#945;SMA) is added to the staining to label vessels. Systematic analysis of the BD to PV ratio in a section covering all liver lobes ensures that a large number of PVs are analyzed for each genotype. Since our method relies on quantifying BDs and PVs in 2D images, it is not suitable for studying the effects of a given mutation on the 3D structure of the biliary tree or the integrity of the small biliary conduits. Nevertheless, it provides a simple and objective strategy for investigators to assess biliary development in the mouse.

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

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 previously documented biliary defects in Jag1+/&#8211; 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 &#945;SMA. We then imaged all the PVs in each of the liver lobes. As shown in Figure 2A, we defined PVs as &#945;SMA-stained vessels that have adjacent wsCK staining (arrowheads). The &#945;SMA-stained...

Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Determining Bile Duct Density in the Mouse Liver

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 ...

determining-bile-duct-density-in-the-mouse-liver

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.8K Views.  Baylor College of Medicine. 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.

Video: Determining Bile Duct Density in the Mouse Liver

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Cancer Research

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 ...

Medicine

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

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

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 ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

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.

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 ...

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 ...

Biology

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...