title: 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.
abstract: 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) (120.3 × 103 ± 42.1 × 10(3)), the average length of sinusoidal vessel per mm(3) (5.4 × 10(3) ± 1.4 × 10(3)mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 μm), length of the second-order branches (10.9 ± 1.8 μm), length of the dead-end branches (5.9 ± 0.7 μm), the number of intersection nodes per mm(3) (819.1 × 10(3) ± 180.7 × 10(3)), the number of dead-end branches per mm(3) (409.9 × 10(3) ± 95.6 × 10(3)), the length of the bile canalicular network per mm(3) (9.4 × 10(3) ± 0.7 × 10(3) mm) and the percentage of the bile canalicular volume with respect to the total liver volume (3.4 ± 0.005). A particular strength of our technique is that quantitative parameters of hepatocytes and bile canalicular as well as sinusoidal networks can be extracted from the same tissue block. Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms. Furthermore, protocols are presented for both human and pig livers. The technique is also applicable for both vibratome blocks and conventional paraffin slices.
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU Dortmund University, Dortmund, Germany, Seddik.hammad@vet.svu.edu.eg.
authors.forename: Seddik
authors.initials: S
authors.lastname: Hammad
authors.forename: Stefan
authors.initials: S
authors.lastname: Hoehme
authors.forename: Adrian
authors.initials: A
authors.lastname: Friebel
authors.forename: Iris
authors.initials: I
authors.lastname: von Recklinghausen
authors.forename: Amnah
authors.initials: A
authors.lastname: Othman
authors.forename: Brigitte
authors.initials: B
authors.lastname: Begher-Tibbe
authors.forename: Raymond
authors.initials: R
authors.lastname: Reif
authors.forename: Patricio
authors.initials: P
authors.lastname: Godoy
authors.forename: Tim
authors.initials: T
authors.lastname: Johann
authors.forename: Amruta
authors.initials: A
authors.lastname: Vartak
authors.forename: Klaus
authors.initials: K
authors.lastname: Golka
authors.forename: Petru O
authors.initials: PO
authors.lastname: Bucur
authors.forename: Eric
authors.initials: E
authors.lastname: Vibert
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Marchan
authors.forename: Bruno
authors.initials: B
authors.lastname: Christ
authors.forename: Steven
authors.initials: S
authors.lastname: Dooley
authors.forename: Christoph
authors.initials: C
authors.lastname: Meyer
authors.forename: Iryna
authors.initials: I
authors.lastname: Ilkavets
authors.forename: Uta
authors.initials: U
authors.lastname: Dahmen
authors.forename: Olaf
authors.initials: O
authors.lastname: Dirsch
authors.forename: Jan
authors.initials: J
authors.lastname: Böttger
authors.forename: Rolf
authors.initials: R
authors.lastname: Gebhardt
authors.forename: Dirk
authors.initials: D
authors.lastname: Drasdo
authors.forename: Jan G
authors.initials: JG
authors.lastname: Hengstler
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D001648:Bile Canaliculi / Q000166:cytology*; D018819:Dipeptidyl Peptidase 4 / Q000276:immunology; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D008099:Liver / Q000098:blood supply* / Q000648:ultrastructure; D008297:Male; D008810:Mice, Inbred C57BL; D008833:Microcirculation; D016612:Paraffin Embedding; D011786:Quality Control; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*; D013552:Swine
chemical_list: C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4
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