This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
SummaryWe investigated the expression and distribution of keratinocyte growth factor (KGF) (FGF-7) and its receptor (KGFR.) during reepithelialization of human skin. KGF mR.NA levels increased rapidly by 8-10-fold and remained elevated for several days. In contrast, KGFP,. transcript levels decreased early but were significantly elevated by 8-9 d. A KGF-immunoglobulin G fusion protein (KGF-HFc), which specifically and sensitively detects the KGFR., localized the receptor to differentiating keratinocytes of control epidermis, but revealed a striking decrease in receptor protein expression during the intermediate period of reepithelization. Suramin, which blocked KGF binding and stripped already bound KGF from its receptor, failed to unmask KGFR.s in tissue sections from the intermediate phase of wound repair. The absence of KGFP,. protein despite increased KGFR. transcript levels implies functional receptor downregulation in the presence of increased KGF. This temporal modulation of KGF and KGFR.s provides strong evidence for the functional involvement of KGF in human skin reepithelialization.
Using a anatomically based, vessel-oriented, parenchyma-preserving surgical technique in 95% liver resections led to long-term survival. This represents the maximal reduction of liver mass compatible with survival.
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 μm3) 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 mm3 (120.3 × 103 ± 42.1 × 103), the average length of sinusoidal vessel per mm3 (5.4 × 103 ± 1.4 × 103mm) 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 mm3 (819.1 × 103 ± 180.7 × 103), the number of dead-end branches per mm3 (409.9 × 103 ± 95.6 ×...
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