Pipe-3D: A Pipeline Based on Immunofluorescence, 3D Confocal Imaging, Reconstructions, and Morphometry for Biliary Network Analysis in Cholestasis

  • Amruta Damle-Vartak
  • Brigitte Begher-Tibbe
  • Georgia Gunther
  • Fabian Geisler
  • Nachiket Vartak
  • Jan G. HengstlerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1981)


Cholestasis, the impairment of bile flux out of the liver, is a common complication of many pathological liver disorders, such as cholangiopathies, primary biliary sclerosis, and primary biliary cirrhosis. Besides accumulation of bile acids in the liver and blood, it leads to a proliferative response of the biliary tree termed as a ductular reaction. The ductular reaction is characterized by enhanced proliferation of cholangiocytes, which form the epithelial lining of bile ducts. This strong reaction of the biliary tree has been reported to generate a source of progenitor cells that can differentiate to hepatocytes or cholangiocytes during regeneration. On the other hand, it can cause periportal fibrosis eventually progressing to cirrhosis and death. In 2D histology, this leads to the appearance of an increased number of duct lumina per area of tissue. Yet, the biliary tree is a 3D vstructure and the appearance of lumina in thin slices may be explained by the appearance of novel ducts or by ramification or convolution of existing ducts in 3D. In many such aspects, traditional 2D histology on thin slices limits our understanding of the response of the biliary tree. A comprehensive understanding of architecture remodeling of the biliary network in cholestasis depends on robust 3D sample preparation and analysis methods. To that end, we describe pipe-3D, a processing and analysis pipeline visualization based on immunofluorescence, confocal imaging, surface reconstructions, and automated morphometry of the biliary network in 3D at subcellular resolution. This pipeline has been used to discover extensive remodeling of interlobular bile ducts in cholestasis, wherein elongation, branching, and looping create a dense ductular mesh around the portal vein branch. Surface reconstructions generated by Pipe-3D from confocal data also show an approximately fivefold enhancement of the luminal duct surface through corrugation of the epithelial lamina, which may increase bile reabsorption and alleviate cholestasis. The response of interlobular ducts in cholestasis was shown to be in sharp contrast to that of large bile ducts, de novo duct formation during embryogenesis. It is also distinct from ductular response in other models of hepatic injury such as choline-deficient, ethionine-supplemented diet, where parenchymal tissue invasion by ducts and their branches is observed. Pipe-3D is applicable to any model of liver injury, and optionally integrates tissue clearing techniques for 3D analysis of thick (>500 μm) tissue sections.

Key words

Bile duct ligation (BDL) Ductular reaction Cholestasis Confocal imaging Surface reconstructions Automated morphometry Passive tissue clearing technique (PTCT) 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) Choline-deficient ethionine diet (CDE) Hepatocyte nuclear factor 1 homeobox B (HNF1B) Aspartate aminotransferase (ASAT) Alanine aminotransferase (ALAT) Gamma-glutamyl transferase (GGT) 



This work was financially supported by the Virtual Liver Network, its successor Liver Systems Medicine, and Lebersimulator projects funded by the German Federal Ministry of Education and Research (BMBF). Special acknowledgments to Dr. Fabian Geisler for providing the HNF1beta_CRetdTom transgenic mice.

Supplementary material

Video 1

3D architecture visualization of the murine liver domains (MP4 63322 kb)

Video 2

Ductular mesh around a portal vein of a clarified liver tissue section (AVI 85277 kb)


  1. 1.
    Roskams TA, Theise ND, Balabaud C et al (2004) Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology 39:739–1745CrossRefGoogle Scholar
  2. 2.
    Esteller A (2008) Physiology of bile secretion. World J Gastroenterol 14:5641–5649CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Sellinger M, Boyer JL (1990) Physiology of bile secretion and cholestasis. Prog Liver Dis 9:237–259PubMedGoogle Scholar
  4. 4.
    Guicciardi ME, Gores GJ (2002) Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease. Dig Liver Dis 34:387–392CrossRefPubMedCentralGoogle Scholar
  5. 5.
    Vartak N, Damle-Vartak A, Richter B et al (2016) Cholestasis-induced adaptive remodeling of interlobular bile ducts. Hepatology 63:951–964CrossRefPubMedCentralGoogle Scholar
  6. 6.
    Kaneko K, Kamimoto K, Miyajima A et al (2015) Adaptive remodeling of the biliary architecture underlies liver homeostasis. Hepatology 61:2056–2066CrossRefGoogle Scholar
  7. 7.
    Roskams T, Desmet V (1998) Ductular reaction and its diagnostic significance. Semin Diagn Pathol 15:259–269PubMedPubMedCentralGoogle Scholar
  8. 8.
    Aller M-A, Arias J-L, García-Domínguez J et al (2008) Experimental obstructive cholestasis: the wound-like inflammatory liver response. Fibrogenesis Tissue Repair 1:6CrossRefPubMedCentralGoogle Scholar
  9. 9.
    Tanaka M, Itoh T, Tanimizu N et al (2011) Liver stem/progenitor cells: their characteristics and regulatory mechanisms. J Biochem 149:231–239CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Rókusz A, Veres D, Szücs A et al (2017) Ductular reaction correlates with fibrogenesis but does not contribute to liver regeneration in experimental fibrosis models. PLoS One 12:e0176518CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Ye F, Jing Y-Y, Guo S-W et al (2014) Proliferative ductular reactions correlate with hepatic progenitor cell and predict recurrence in HCC patients after curative resection. Cell Biosci 4:50CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Lee S-J, Park J-B, Kim K-H et al (2014) Immunohistochemical study for the origin of ductular reaction in chronic liver disease. Int J Clin Exp Pathol 7:4076–4085PubMedPubMedCentralGoogle Scholar
  13. 13.
    Ben-Ari Z, Weiss-Schmilovitz H, Sulkes J et al (2004) Serum cholestasis markers as predictors of early outcome after liver transplantation. Clin Transpl 18:130–136CrossRefGoogle Scholar
  14. 14.
    Li B, Wang Z, Fang J-J et al (2007) Evaluation of prognostic markers in severe drug-induced liver disease. World J Gastroenterol 13:628–632CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Abshagen K, König M, Hoppe A et al (2015) Pathobiochemical signatures of cholestatic liver disease in bile duct ligated mice. BMC Syst Biol 9:83CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Gouw ASH, Clouston AD, Theise ND (2011) Ductular reactions in human liver: diversity at the interface. Hepatology 54:1853–1863CrossRefPubMedCentralGoogle Scholar
  17. 17.
    Jörs S, Jeliazkova P, Ringelhan M et al (2015) Lineage fate of ductular reactions in liver injury and carcinogenesis. J Clin Invest 125:2445–2457CrossRefPubMedCentralGoogle Scholar
  18. 18.
    Renier N, Wu Z, Simon DJ et al (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159:896–910CrossRefGoogle Scholar
  19. 19.
    Tomer R, Ye L, Hsueh B et al (2014) Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9:1682–1697CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Marx V (2016) Optimizing probes to image cleared tissue. Nat Methods 13:205–209CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Hammad S, Hoehme S, Friebel A et al (2014) Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis. Arch Toxicol 88:1161–1183CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Amruta Damle-Vartak
    • 1
  • Brigitte Begher-Tibbe
    • 1
  • Georgia Gunther
    • 1
  • Fabian Geisler
    • 2
  • Nachiket Vartak
    • 1
  • Jan G. Hengstler
    • 1
    Email author
  1. 1.Department of Systems ToxicologyLeibniz Research Centre for Working Environment and Human Factors (IfADo) at TU DortmundDortmundGermany
  2. 2.Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar der Technische Universitaet MuenchenMunichGermany

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