Solutions to the discrepancies in the extent of liver damage following ischemia/reperfusion in standard mouse models
Authors: Rowan F. van Golen, Megan J. Reiniers, Michal Heger, Joanne Verheij
Journal: Journal of Hepatology (2014)

Due to their experimental versatility, mouse models are commonly used to study hepatic ischemia/reperfusion (I/R) injury in relation to (I) the efficacy of surgical interventions [[1]Oberkofler C.E. Limani P. Jang J.H. Rickenbacher A. Lehmann K. Raptis D.A. et al.Systemic protection through remote ischemic preconditioning is spread via platelet-dependent signaling.Hepatology. 2014; 60: 1409-1417Crossref PubMed Scopus (36) Google Scholar], (II) the pathophysiological role of biomolecules [[2]Hou J. Xia Y. Jiang R. Chen D. Xu J. Deng L. et al.PTPRO plays a dual role in hepatic ischemia reperfusion injury through feedback activation of nf-κb.J Hepatol. 2013; 60: 306-312Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar] as well as cells [[3]Stewart R.K. Dangi A. Huang C. Murase N. Kimura S. Stolz D.B. et al.A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury.J Hepatol. 2014; 60: 298-305Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar], and (III) the interplay with parenchymal liver disease [[4]Kato H. Kuriyama N. Duarte S. Clavien P.-A. Busuttil R.W. Coito A.J. MMP-9 deficiency shelters endothelial pecam-1 expression and enhances regeneration of steatotic livers after ischemia and reperfusion injury.J Hepatol. 2014; 60: 1032-1039Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. For the assessment of post-ischemic liver damage, plasma alanine transaminase (ALT) and the extent of necrotic cell death, determined by routine clinical chemistry and histology, respectively, are commonly used as gold standard parameters. A strong correlation exists between hepatocellular necrosis and plasma ALT levels (Fig. 1K and L). However, during the development of an I/R mouse model for pharmacological intervention studies, we noticed that the reported damage profiles vary considerably amongst similar I/R models, even between studies from groups with extensive expertise in hepatic I/R research [1Oberkofler C.E. Limani P. Jang J.H. Rickenbacher A. Lehmann K. Raptis D.A. et al.Systemic protection through remote ischemic preconditioning is spread via platelet-dependent signaling.Hepatology. 2014; 60: 1409-1417Crossref PubMed Scopus (36) Google Scholar, 3Stewart R.K. Dangi A. Huang C. Murase N. Kimura S. Stolz D.B. et al.A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury.J Hepatol. 2014; 60: 298-305Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 4Kato H. Kuriyama N. Duarte S. Clavien P.-A. Busuttil R.W. Coito A.J. MMP-9 deficiency shelters endothelial pecam-1 expression and enhances regeneration of steatotic livers after ischemia and reperfusion injury.J Hepatol. 2014; 60: 1032-1039Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. This has several important implications for the use of mouse I/R models for the abovementioned research purposes, especially for laboratories with limited experience with these models. Our letter therefore serves to provide empirical evidence for the experimental discrepancies, address their implications, and furnish recommendations on how to manage the implications. The first implication is that mouse models should always be optimized and attuned to the desired set of outcome parameters per laboratory rather than on the basis of literature. This is supported by the fact that liver injury parameters (ALT) routinely vary >10-fold for the same ischemia and reperfusion times amongst similar mouse models (Fig. 1C and D; Supplementary Table 1). The observed interstudy differences most likely stem from variables related to animal selection (e.g., age, weight, or strain variations), the surgical procedure (e.g., injection or inhalation anesthesia, portal triad clamping technique), and the acclimatization conditions, all of which vary considerably in hepatic I/R literature (Supplementary Table 1). Consequently, the duration of ischemia should always be attuned to the ALT and necrosis values per model and not dogmatically adopted from literature, as the full spectrum of experimental details is not always reported. In support of this premise, we have profiled hepatocellular damage at 6 h (Fig. 1A; Supplementary Fig. 2) and 24 h of reperfusion (Fig. 1B and E–J) as a function of ischemia time, using a standardized mouse I/R model (technically detailed in the Supplementary material). Our experiments with C57BL/6J mice revealed a sigmoidal relationship between liver ischemia time and hepatocellular injury at both 6 h (Fig. 1A) and 24 h (Fig. 1B) of reperfusion. At 6 h of reperfusion, ALT plasma levels increased after an ischemic hit of ⩾20 min and peaked after ⩾45 min of liver ischemia (Fig. 1A and B), indicating that only a small bandwidth of ischemia times separates minor I/R injury from maximal I/R injury. Considering that ischemia intervals of ⩾60 min are used in the majority of hepatic I/R studies published in the last 5 years (Supplementary Table 1), it should be noted that extending the ischemia time from 60 to 90 min did not aggravate I/R injury after 24 h of reperfusion, as both ischemia times produced a maximum degree of liver damage (Fig. 1A, B, I, and J). Evidently, there is no gain in using 30 min extra ischemia time with no differences in liver damage. A ⩾30 min reduction in ischemia time can significantly decrease the total length of the experiments, particularly in larger studies. More importantly, performing studies on livers that are >75% necrotic is unsuitable for two reasons. Firstly, such damage profiles are not clinically representative, given that peak postoperative ALT values in patients subjected to I/R rarely exceed ±1000–1500 U/L [5van den Broek M.A. Bloemen J.G. Dello S.A. van de Poll M.C. Olde Damink S.W. Dejong C.H. Randomized controlled trial analyzing the effect of 15 or 30 min intermittent pringle maneuver on hepatocellular damage during liver surgery.J Hepatol. 2011; 55: 337-345Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 6Reiniers M.J. van Golen R.F. Heger M. Mearadji B. Bennink R.J. Verheij J. et al.In situ hypothermic perfusion with retrograde outflow during right hemihepatectomy: first experiences with a new technique.J Am Coll Surg. 2013; 218: e7-e16Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]; i.e., >10-fold lower than in most mouse models (Fig. 1A–D). Given this discrepancy between mouse models and the clinical situation, we strongly support the bench-to-bedside-to-bench approach recently reported by Hou et al. [[2]Hou J. Xia Y. Jiang R. Chen D. Xu J. Deng L. et al.PTPRO plays a dual role in hepatic ischemia reperfusion injury through feedback activation of nf-κb.J Hepatol. 2013; 60: 306-312Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar] where applicable, whereby experimental hypotheses and/or results are validated on/with patient material obtained in a trial with minimal patient burden. Such an approach could prevent unnecessary animal experiments and gauge the clinical relevance and, by extension, the therapeutic potential of a novel intervention modality. Better alignment of preclinical research with the clinical situation is crucial to expedite the clinical implementation of new treatments for hepatic I/R injury, which has proven problematic hitherto. Secondly, the (patho)physiological mechanisms in considerably damaged livers may differ from that in livers that are less extensively injured by I/R (i.e., in a clinically more representative model). This could be attributed to e.g., the allocation of residual intracellular energy by cells towards survival mechanisms (in case of moderate hepatocellular damage) versus cell death pathways (in case of damage beyond repair), which in turn dictates the type of cell death (e.g., necrosis, autophagy) and the severity of the consequent immunological responses [[7]van Golen R.F. van Gulik T.M. Heger M. The sterile immune response during hepatic ischemia/reperfusion.Cytokine Growth Factor Rev. 2012; 23: 69-84Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar]. In light of these differences in pathophysiology, the steep slopes of the ALT versus ischemia time graphs (Fig. 1A and B, linear segments) imply that even slight experimental variations have a disproportionally large effect on the type and extent of liver injury. As it is impossible to eliminate differences between laboratories altogether, it is recommended to test novel hypotheses or interventions in mild and severe mouse liver I/R models. Reporting results generated with different experimental regimens per research group circumvents the aforementioned comparability issues and automatically benefits the external validity of published liver I/R data. In conclusion, hepatic I/R experiments in mice, especially when diseased livers are concerned [[8]Reiniers M.J. van Golen R.F. van Gulik T.M. Heger M. Reactive oxygen and nitrogen species in steatotic hepatocytes: a molecular perspective on the pathophysiology of ischemia-reperfusion injury in the fatty liver.Antioxid Redox Signal. 2013; 21: 1119-1142Crossref Scopus (85) Google Scholar], are technically very difficult, even when conducted properly. To maximize outcomes with respect to hepatocellular (patho)physiology, model reproducibility, and clinical translatability, hepatic I/R models in mice should be optimized on the basis of experimental data rather than literature alone. In addition, it is advised to perform studies on mild and severe mouse models of I/R injury and to validate results generated with these models on clinical material. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Download .pdf (.34 MB) Help with pdf files Supplementary data