Letter to the editor

Quantification of three-dimensional structures in liver tissue: bile canalicular and sinusoidal networks

Hassan Ahmed1[*], Mosaab A. Omar2, Ahmed M. Abdou3

1Department of Physiology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

2Department of Parasitology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

3Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

EXCLI J 2014;13:Doc548

 

Dear Editor,

Unpredicted hepatotoxicity represents the most frequent reason for withdrawal of drugs from the market (Godoy et al., 2013[5]). Therefore, large efforts are currently undertaken to establish improved techniques to predict hepatotoxicity (Shimizu et al., 2009[25]; O’Brien et al., 2006[20]; Okwa et al., 2013[21]; Oloyede et al., 2013[22]; Lu et al., 2013[18]; Godoy et al., 2009[4]). The perhaps most intensive activities in this field of research can be observed in establishment of hepatocyte in vitro systems (Godoy et al., 2013[5]; Hewitt et al., 2007[13]; Ilkavets et al., 2013[16]; Hengstler et al., 2012[12], 2014[11]; Hammad et al., 2013[10]; Hammad, 2013[8]). One of the concepts of this type of in vitro research is that stress responses to chemicals are similar in vitro and in vivo. Therefore, activation of stress response pathways in vitro may indicate a potential hazard (Jennings, 2013[17]; Vinken et al., 2014[27]). Although hepatocyte in vitro systems have promoted our understanding of mechanisms (Meyer et al., 2011[19]; Schug et al., 2013[24]; Godoy et al., 2010[6], 2012[7]; Zellmer et al., 2010[28]; Hengstler et al., 2014[11]) it has also become clear that a full replacement of animal experiments for toxicity testing will not be possible within the next one or two decades (Adler et al., 2011[1]; Hammad, 2013[8]). Reasons are difficulties to include xenobiotic metabolism into in vitro tests, to model interactions between cell types, to extrapolate from in vivo doses to in vitro concentrations and to simulate the consequences of long term exposure in vitro (Tice et al., 2013[26]; Ghallab, 2013[3]). However, one additional aspect may currently be underestimated: we still know too little about mechanisms of in vivo toxicity to establish in vitro systems in a way that the most critical in vivo processes are recapitulated. Although this may seem paradoxical, progress in replacement of animal experiments currently depends on progress in understanding the mechanisms of toxicity in vivo.

Histological alterations often constitute a fingerprint of toxic mechanisms (Hammad et al., 2014[10]). Particularly, liver toxicity often leads to altered tissue microarchitecture (Höhme et al., 2007[15]; Hoehme et al., 2010[14]; Braeuning et al., 2010[2]; Schreiber et al., 2011[23]). Moreover, current studies on liver histology largely rely on image analysis of two-dimensional pictures. However, many aspects of liver tissue architecture such as the complex bile canalicular and sinusoidal networks can be much better quantified using three-dimensional reconstructions. In this context, it represents an important step forward that Dr. Hammad and his team have established a technique for staining, three-dimensional reconstruction and quantification of liver microarchitecture (Hammad et al., 2014[10]). These protocols which can be applied under routine conditions allow: (1) the simultaneous staining of bile canalicular and sinusoidal networks in approximately 100 µm thick liver tissue blocks; (2) identification of S-phase positive cells whereby the position, size and shape of all individual cells is robustly captured; (3) quantification of key parameters such as hepatocyte volume, the fraction of hepatocytes in contact with neighboring hepatocytes, sinusoids and bile canaliculi. Parameters of the bile canalicular network include the length and branching angles of individual canaliculi, the number of dead end branches and the length of canaliculi per tissue volume. Further, parameters that can be quantified are the radius of the sinusoids, percentage of vessel volume in relation to tissue volume, the branching angle and the length of intersection branches of sinusoids.

Briefly, key parameters of liver tissue microarchitecture can now be quantified routinely. It can be expected that the novel technique will lead to a more precise and robust characterization of hepatotoxicity.

 

References

1. Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, et al. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol. 2011;85:367-485.
2. Braeuning A, Singh Y, Rignall B, Buchmann A, Hammad S, Othman A, et al. Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice. Histochem Cell Biol. 2010;134:469-81.
3. Ghallab A. In vitro test systems and their limitations EXCLI J. 2013;12:1024-6.
4. Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Müller A, et al. Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology. 2009;49:2031-43.
5. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87:1315-530.
6. Godoy P, Lakkapamu S, Schug M, Bauer A, Stewart JD, Bedawi E, et al. Dexamethasone-dependent versus independent markers of epithelial to mesenchymal transition in primary hepatocytes. Biol Chem. 2010;391:73-83.
7. Godoy P, Reif R, Bolt HM. Alcohol hepatotoxicity: Kupffer cells surface to the top. Arch Toxicol. 2012;869:1331-2.
8. Hammad S. Advances in 2D and 3D in vitro systems for hepatotoxicity testing. EXCLI J. 2013;12:993-6.
9. Hammad S, Hoehme S, Friebel A, von Recklinghausen I, Othman A, Begher-Tibbe B, et al. 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. 2014;88:1161-83.
10. Hammad S, Marchan R, Hengstler JG. Cutting-edge topics in research on animal sciences. J Exp Appl Anim Sci. 2013;1:1-3.
11. Hengstler JG, Hammad S, Ghallab A, Reif R, Godoy P. In vitro systems for hepatotoxicity testing. In: Bal-Price A, Jennings P (eds.): In vitro toxicology systems. Methods in pharmacology and toxicology (pp 27-44). New York: Springer Science+Business Media, 2014.
12. Hengstler JG, Marchan R, Leist M. Towards the replacement of in vivo repeated dose systemic toxicity testing (Highlight report). Arch Toxicol. 2012;86:13-5.
13. Hewitt NJ, Lechón MJ, Houston JB, Hallifax D, Brown HS, Maurel P, et al. Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev. 2007;39:159-234.
14. Hoehme S, Brulport M, Bauer A, Bedawy E, Schormann W, Hermes M, et al. Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration. Proc Natl Acad Sci USA. 2010;107:10371-6.
15. Höhme S, Hengstler JG, Brulport M, Schäfer M, Bauer A, Gebhardt R, et al. Mathematical modelling of liver regeneration after intoxication with CCl(4). Chem Biol Interact. 2007;168:74-93.
16. Ilkavets I. A special issue about hepatotoxicity and hepatocyte in vitro systems. Arch Toxicol. 2013;87:1313-4.
17. Jennings P. Stress response pathways, toxicity pathways and adverse outcome pathways. Arch Toxicol. 2013;87:13-4.
18. Lu Y, Pei Y, Shao Y, Yan S, Ma L, Fang F, et al. Hepatotoxicity induced by zoledronic acid in an aged woman with primary osteoporosis. EXCLI J. 2013;12:115-7.
19. Meyer C, Godoy P, Bachmann A, Liu Y, Barzan D, Ilkavets I, et al. Distinct role of endocytosis for Smad and non-Smad TGF-β signaling regulation in hepatocytes. J Hepatol. 2011;55:369-78.
20. O'Brien PJ, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, et al. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch Toxicol. 2006;80:580-604.
21. Okwa IB, Akindele AJ, Agbaje EO, Oshinuga OT, Anunobi CC, Ademeyi OO. Effect of subclinical, clinical and supraclinical doses of calcium channel blockers on models of drug-induced hepatotoxicity in rats. EXCLI J. 2013;12:231-50.
22. Oloyede GK, Adaramoye OA, Oguntokun OJ. Phytochemical and hepatotoxicity studies on Adansonia digitata leaf extracts. J Exp Appl Anim Sci. 2013;1:25-34.
23. Schreiber S, Rignall B, Braeuning A, Marx-Stoelting P, Ott T, Buchmann A, et al. Phenotype of single hepatocytes expressing an activated version of β-catenin in liver of transgenic mice. J Mol Histol. 2011;42:393-400.
24. Schug M, Stöber R, Heise T, Mielke H, Gundert-Remy U, Godoy P, et al. Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes. Arch Toxicol. 2013;87:337-45.
25. Shimizu S, Atsumi R, Itokawa K, Iwasaki M, Aoki T, Ono C, et al. Metabolism-dependent hepatotoxicity of amodiaquine in glutathione-depleted mice. Arch Toxicol. 2009;83:701-7.
26. Tice RR, Austin CP, Kavlock RJ, Bucher JR. Improving the human hazard characterization of chemicals: a Tox21 update. Environ Health Perspect. 2013;121:756-65.
27. Vinken M, Whelan M, Rogiers V. Adverse outcome pathways: hype or hope? Arch Toxicol. 2014;88:1-2.
28. Zellmer S, Schmidt-Heck W, Godoy P, Weng H, Meyer C, Lehmann T, et al. Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes. Hepatology. 2010;52:2127-36.
 
 
 

[*] Corresponding Author:

Hassan Ahmed, Department of Physiology, Faculty of Veterinary Medicine, South Valley University, 83523 Qena, Egypt, eMail: hassan-younes@vet.svu.edu.eg