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

• Published in: Archives of toxicology 2014-05, Vol.88 (5), p.1161-1183
• Main Authors: Hammad, Seddik, Hoehme, Stefan, Friebel, Adrian, von Recklinghausen, Iris, Othman, Amnah, Begher-Tibbe, Brigitte, Reif, Raymond, Godoy, Patricio, Johann, Tim, Vartak, Amruta, Golka, Klaus, Bucur, Petru O., Vibert, Eric, Marchan, Rosemarie, Christ, Bruno, Dooley, Steven, Meyer, Christoph, Ilkavets, Iryna, Dahmen, Uta, Dirsch, Olaf, Böttger, Jan, Gebhardt, Rolf, Drasdo, Dirk, Hengstler, Jan G.
• Format: Article
• Language: English
• Subjects: Animals; Antibody Specificity; Bile Canaliculi - cytology; Bioinformatics; Biomedical and Life Sciences; Biomedicine; Computer Science; Dipeptidyl Peptidase 4 - immunology; Environmental Health; Hepatocytes - cytology; Humans; Image Processing; Image Processing, Computer-Assisted - instrumentation; Image Processing, Computer-Assisted - methods; Life Sciences; Liver - blood supply; Liver - ultrastructure; Liver diseases; Male; Medical Imaging; Mice, Inbred C57BL; Microcirculation; Microscopy; Modeling and Simulation; Occupational Medicine/Industrial Medicine; Paraffin Embedding; Pharmacology/Toxicology; Protocols; Quality Control; Quantitative Methods; Reproducibility of Results; Staining and Labeling - methods; Swine; Systematic biology; Toxicity
• ISSN: 0340-5761; 1432-0738
• DOI: 10.1007/s00204-014-1243-5

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-μm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed ‘liver architectural staining’ for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled ‘S-phase staining’, where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 μm 3 ) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 μm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 μm), the number of intersection nodes per mm 3 (1