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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
Protocol
  • 525 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1981)

Abstract

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) 

Notes

Acknowledgments

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)

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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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