Published online Jan 27, 2018. doi: 10.4254/wjh.v10.i1.8
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
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.
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.
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.
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.
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.
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.
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.