Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Jan 27, 2018; 10(1): 8-21
Published online Jan 27, 2018. doi: 10.4254/wjh.v10.i1.8
Preserved liver regeneration capacity after partial hepatectomy in rats with non-alcoholic steatohepatitis
David Haldrup, Sara Heebøll, Karen Louise Thomsen, Kasper Jarlhelt Andersen, Michelle Meier, Frank Viborg Mortensen, Jens Randel Nyengaard, Stephen Hamilton-Dutoit, Henning Grønbæk
David Haldrup, Sara Heebøll, Karen Louise Thomsen, Henning Grønbæk, Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus C DK-8000, Denmark
David Haldrup, Department of Internal Medicine, Randers Regional Hospital, Randers NØ DK-8930, Denmark
Kasper Jarlhelt Andersen, Michelle Meier, Frank Viborg Mortensen, Department of Surgical Gastroenterology, Aarhus University Hospital, Aarhus C DK-8000, Denmark
Jens Randel Nyengaard, Stereology and Electron Microscopy Laboratory, Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University Hospital, Aarhus C DK-8000, Denmark
Stephen Hamilton-Dutoit, Institute of Pathology, Aarhus University Hospital, Aarhus C DK-8000, Denmark
Author contributions: Haldrup D and Heebøll S performed the majority of the experiments with assistance and supervision by Thomsen KL, Andersen KJ, Meier M and Nyengaard JR; Thomsen KL, Andersen KJ, Meier M, Mortensen FV, Nyengaard JR and Grønbæk H concived and designed the study; Thomsen KL performed the statistical analysis; Hamilton-Dutoit S performed the histological evaluation; all authors contributed to the analysis and interpretation of data; Haldrup D wrote the original manuscript draft; Heebøll S, Thomsen KL, Andersen KJ, Meier M, Nyengaard JR, Mortensen FV, Nyengaard JR, Hamilton-Dutiot S and Grønbæk H critically revised the manuscript for important intellectual content; all authors saw and approved the final manuscript.
Supported by NOVO Nordisk Foundation (grant number 1013267) and Savværksejer Jeppe Juhl og Hustru Ovita Juhls Mindelegat to Grønbæk H; Villum Fonden to Nyengaard JR.
Institutional animal care and use committee statement: All animal work was conducted according to the national guidelines. The Danish Animal Experiments Inspectorate approved the study (2014-15-2934-00997).
Conflict-of-interest statement: To the best of our knowledge there are no competing interests.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: David Haldrup, MD, Department of Hepatology and Gastroenterology, Aarhus University Hospital, Nørrebrogade 44, Aarhus C DK-8000, Denmark.
Telephone: +45-20912152
Received: October 12, 2017
Peer-review started: October 13, 2017
First decision: November 7, 2017
Revised: November 20, 2017
Accepted: December 6, 2017
Article in press: December 6, 2017
Published online: January 27, 2018
Research background

Epidemiological studies showed that liver resections are associated with increased morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD)/ non-alcoholic steatohepatitis (NASH). It has been suggested that NASH livers are more vulnerable to surgical interventions because of decreased liver regeneration capacity (LRC). LRC has been studied in different animal models of NAFLD/NASH. However, these models may have significant limitations. Some models induce NASH but with severe weight loss, while other models induce simple steatosis only, further, genetic modified models may not reflect the etiological features of human NASH. In the present study we used a high fat high cholesterol diet (HFCD) rat model, which mimic human NASH better than previous models.

Research motivation

This is the first study of LRC in rats with NASH induced by a HFCD. Previous experimental NAFLD/NASH studies showed contradictory findings with decreased LRC or unchanged LRC, even when the same animal models were used. Clearly, the model and methods of evaluation may significantly influence the results and conclusions. For future treatment strategies of liver resections, it is important to understand whether the LRC of NAFLD/NASH livers is compromised.

Research objectives

The aim of the present study was to evaluate LRC in rats with NASH induced by a HFCD. Authors the methods of evaluation and the chosen model of NAFLD/NASH significantly influences the results and further research on the subject should be aware of this.

Research methods

Rats were fed a high-fat, high-cholesterol diet (65% fat, 1% cholesterol) or standard diet (STD) for 16 wk. After the feeding phase 1/3 of the animals were euthanised immediately and served as a baseline reference. The remaining 2/3 of the animals underwent 70% partial hepatectomy (PH) and the hepatectomized animals were euthanised either 2 or 5 d post-PH. The degree of steatosis and the presence of NASH were evaluated by an expert liver pathologist using both the Kleiner and Bedossa criteria. LRC was evaluated using: the total number of Ki-67 positive hepatocytes in the caudate lobe, N(Ki-67, lobe) evaluated in a stereology-based design, the regenerated protein ratio (RPR), prothrombin-proconvertin ratio (PP), and mRNA expression of genes related to regeneration. The study is the first to use a stereology based design to evaluate cell proliferation. The authors believe this design superior to former methods of evaluation.

The study is also the first to address that future research should be cautious using the regenerated liver weight only to evaluate LRC. The NASH liver weight is biased by fat accumulation and when using the liver weight only one cannot account for whether the NASH liver regenerates fat- or liver tissue. Thus, we estimated the total protein concentration in the livers and used this to describe the regenerated liver mass. Biochemical tests were used as markers of liver injury. The data was analyzed using STATA. Normality of data was checked by qq-plots. For continuous variables, comparisons were made using the ANOVA test for significance. Post-hoc comparisons were performed by Student’s t-test. Categorical data were analyzed using Fisher’s exact test. The qPCR data exhibited skewed distributions with variance heterogeneity. Therefore, these data were analysed using the non-parametric Kruskal-Wallis one-way analysis of variance on ranks test; when significant, post-hoc tests were performed using the Mann-Whitney rank sum test.

Research results

The HFCD NASH model showed significant steatosis with ballooning and inflammation, while no fibrosis was present. Mortality was similar in HFCD and STD animals following PH. Further, HFCD animals had significantly elevated markers of liver injury after PH. HFCD animals had a higher N(Ki-67, lobe) at baseline, day 2 after PH and day 5 after PH. However, we found no significant difference in RPR or PP neither 2 or 5 d post-PH. Expression of liver regeneration genes was higher at both day 2 and 5 post-PH in HFCD groups.Authors evaluated LRC at day 2 and 5 after PH; however, it would have been interesting also to evaluate the very early stages of liver regeneration including time points as early as a few hours after PH and at day 1 after PH. Further, it would be of interest to investigate this rat model after more prolonged HFCD diet treatment when fibrosis may be more pronounced and if this decreases LRC. In addition, finding and identifying relevant new and better methods of LRC evaluation may ease the interpretation of the results.

Research conclusions

The novel finding is that in a HFCD NASH model without fibrosis authors observed preserved LRC. The etiology and methods of evaluation is of great importance when evaluating LRC in animal models. Further, the fat accumulation in the NAFLD/NASH liver is a bias when estimating LRC and it needs to be addressed in future studies. In animal models the etiology of NAFLD/NASH and methods of evaluation is of significant importance in understanding LRC. Seemingly, NASH without fibrosis induced by a HFCD does not decrease LRC. HFCD induced NASH without fibrosis does not compromise LRC in rats following hepatectomy. HFCD induced NASH without fibrosis does not compromise LRC in rats following hepatectomy. When evaluating LRC the fat accumulation of the liver must be addressed, thus we have used both a stereological design to evaluate cell proliferation and measured the total protein concentration in the liver as a marker of regenerated liver mass. Prior to the study hypothesized LRC to be decreased, but in contrast we found a preserved LRC. It is too early to draw conclusions for clinical practice, but this study adds insight to the subject. Speculating, the reasons for increased morbidity and mortality in patients with NAFLD/ NASH following liver resections should be sought elsewhere than in decreased LRC.

Research perspectives

Identifying and/or optimising relevant animal models of NAFLD/NASH as well as methods of evaluation for LRC. Using a pure stereological design for evaluation of cell proliferation, as this is perfectly unbiased. Using different markers LRC and being aware of the potential bias fat accumulation brings when evaluating LRC based on liver weight alone.