Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan;243(1):1-12.
doi: 10.1177/1535370217738730. Epub 2017 Oct 24.

Microcirculatory disturbances and cellular changes during progression of hepatic steatosis to liver tumors

Marie Liebig  1 , Alireza Hassanzada  1 , Malte Kämmerling  1 , Berit Genz  1   2 , Brigitte Vollmar  1 , Kerstin Abshagen  1
Affiliations

Microcirculatory disturbances and cellular changes during progression of hepatic steatosis to liver tumors

Marie Liebig et al. Exp Biol Med (Maywood). 2018 Jan.

Abstract

Non-alcoholic fatty liver disease is closely associated with metabolic syndrome and comprises a pathological spectrum of liver disease ranging from steatosis to steatohepatitis and can progress to fibrosis/cirrhosis and hepatocellular carcinoma. In 2013, a mouse model was described that mimics non-alcoholic fatty liver disease progression from steatohepatitis to tumors in a short time span and with high incidence. As microcirculatory disturbances play a crucial role in liver disease, the suitability of the steatosis-inflammation-tumor model for microcirculatory studies was assessed. Herein, we present a comprehensive view on morphological, microvascular, cellular, and functional aspects of non-alcoholic fatty liver disease progression in the steatosis-inflammation-tumor model using intravital microscopy, biochemical, and histological techniques. Mice develop steatohepatitis, mild fibrosis, and liver tumors at ages of 6, 12, and 20 weeks, respectively. Non-alcoholic fatty liver disease progression was accompanied by several general aspects of disease severity like increasing liver/body weight index, non-alcoholic fatty liver disease activity score, and hepatocellular apoptosis. Intravital microscopic analysis revealed significant changes in hepatic microcirculation with increasing structural alterations, elevated leukocyte adherence, and impaired nutritive perfusion. Non-alcoholic fatty liver disease was further characterized by a lower sinusoidal density with a striking rise at 20 weeks. The characteristic microcirculatory changes make the model a convenient tool for analysis of microcirculation during progression from steatosis to liver tumor. Impact statement Significant alterations of microcirculation contribute to progression of NAFLD, a chronic liver disease with increasing medical and socio-economic impact. Characterization of microcirculation in a NAFLD model reflecting all relevant stages of disease progression was still missing. Thus, we evaluated microcirculatory and cellular changes in a steatosis-inflammation-tumor model using in vivo microscopy. Analyses revealed increasing structural alterations, elevated leukocyte-endothelial interaction, and impaired nutritive perfusion. Thus, this model is suitable for further studies investigating therapeutic approaches _targeting these progressive microcirculatory disturbances.

Keywords: Non-alcoholic fatty liver disease; hepatic microcirculation; hepatocellular carcinoma; intravital microscopy; steatohepatitis.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
General characteristics during progression of NAFLD. (a) Non-fasting blood glucose levels of all analyzed STZ/HFD-treated mice at the ages from 4 to 20 weeks given as mean±s.e.m. (n=12–74). The red line represents the mean blood glucose value of healthy mice. (b) Non-fasting plasma insulin concentration of untreated (control) and STZ/HFD-treated mice. Values are given as mean±s.e.m. (control: n=4, 6 weeks: n=5, 8 weeks: n=5, 12 weeks: n=5, 20 weeks: n=6). Significance of differences between the groups was tested by one-way ANOVA (Holm-Sidak method), *P<0.05 vs. control. (c) Body weight of all analyzed STZ/HFD-treated mice at the ages from 4 to 20 weeks compared to healthy controls given as mean±s.e.m. (n=12–74). (d) Calculation of the liver to body weight ratio. Data (control: n=13, 6 weeks: n=17, 8 weeks: n=17, 12 weeks: n=19, 20 weeks: n=23) are presented as box plots indicating the median, the interquartile range in form of a box, and the minimum and maximum as whiskers. Significance of differences between the groups was tested by one-way ANOVA on Ranks (Kruskal–Wallis); *P<0.05 versus control. (e) Macroscopic images of livers of mice treated with STZ/HFD at an age of 6, 8, 12, and 20 weeks. (A color version of this figure is available in the online journal.)
Figure 2.
Figure 2.
Impaired nutritive perfusion in NAFLD. Quantitative in vivo microscopic analysis of sinusoidal perfusion (a) and density (b) of control mice and mice treated with STZ/HFD at ages of 6, 8, 12, and 20 weeks. (a) Percentage of perfused sinusoids (control: n=10, 6 weeks: n=11, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=11) and appropriate representative images (200× magnification) for control and six weeks old STZ/HFD-treated mouse showing decreased nutritive perfusion and tortuous sinusoids. Values are given as mean±s.e.m. (b) Quantification of sinusoidal length per area regardless of perfusion. Values are given as mean±s.e.m. (control: n=10, 6 weeks: n=10, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=10). Depicted are representative images (400× magnification) of a control mouse and a eight weeks old mouse treated with STZ/HFD. Significance of differences between the groups was tested by one-way ANOVA (Holm-Sidak method; sinusoidal length per area) and one-way ANOVA on Ranks (Kruskal–Wallis; perfused sinusoids), *P<0.05 vs. control; ***P<0.001 vs. control; +P<0.05 vs. 20 wks; +++P<0.01 vs. 20 weeks.
Figure 3.
Figure 3.
Raised hepatic lipid content and systemic lipid peroxidation in NAFLD. (a) Quantitative analysis of lipids by Oil Red O staining in livers of 6, 8, 12, and 20 weeks old mice treated with STZ/HFD and appropriate representative images of Oil Red O stained liver sections. Values are given as mean±s.e.m. (6 wks: n=7, 8 wks: n=7, 12 wks: n=6, 20 wks: n=7). The red line indicates basal level of hepatic fat content in healthy mice (∼8%). (b) Quantitative measurement of malondialdehyd (MDA) in plasma as a biomarker of lipid peroxidation. Values are given as mean±s.e.m. (control: n=7, 6 weeks: n=7, 8 weeks: n=7, 12 weeks: n=4, 20 weeks: n=6). Significance of differences between the groups was tested by one-way ANOVA on Ranks (Kruskal–Wallis); *P<0.05 vs. control. (A color version of this figure is available in the online journal.)
Figure 4.
Figure 4.
Increased hepatic inflammation during progression of NAFLD. (a) Intravital microscopic analysis of the number of adherent leukocytes in postsinusoidal venules of healthy livers (control) and livers of STZ/HFD-treated mice at ages of 6, 8, 12, and 20 weeks. Values are given as mean±s.e.m. (control: n=9, 6 weeks: n=10, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=11). Significance of differences between the groups was tested by one-way ANOVA (Bonferroni t-test), *P<0.05 vs. control. (b) Quantitative analysis of CAE-positive cells in liver sections of STZ/HFD-treated mice (6, 8, 12, and 20 weeks). Data are presented as box plots indicating the median, the interquartile range in form of a box, and the minimum and maximum as whiskers (6 weeks: n=7, 8 weeks: n=7, 12 weeks: n=9, 20 weeks: n=7). (c) Upper panel: Representative in vivo images of the liver of a control and a 20 weeks old STZ/HFD-treated mouse showing numerous leukocytes (arrows) adhering at the endothelium of the venule in the diseased liver (12 weeks) (400× magnification). Lower panel: The images depict representative HPF of CAE-stained liver tissue of 6 and 12 weeks old mice with red stained granulocytes (arrows). (d) Intravital microscopic analysis of sinusoidal leukostasis of healthy livers (control) and livers of STZ/HFD-treated mice at ages of 6, 8, 12, and 20 weeks. Values are given as mean±s.e.m. (control: n=11, 6 weeks: n=10, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=10). (A color version of this figure is available in the online journal.)
Figure 5.
Figure 5.
Analysis of hepatic stellate cell activation. Quantitative analysis of hepatic stellate cells by means of (a) IVM, (b) αSMA Western blot, and (c) collagen 1α staining. (a) In vivo quantification of vitamin A-positive cells of control mice and STZ/HFD treated mice at ages of 6, 8, 12, and 20 weeks. Depicted are representative images (200× magnification) of a healthy mouse and an eight weeks old mouse treated with STZ/HFD showing the decrease in vitamin A autofluorescent spots during NAFLD progression. Values are given as mean±s.e.m. (control: n=10, 6 weeks: n=11, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=12). Significance of differences between the groups was tested by one-way ANOVA on Ranks (Kruskal–Wallis), *P<0.05 vs. control. (b) Quantitative analysis of hepatic protein expression of αSMA relative to GAPDH protein expression of STZ/HFD-treated mice at an age of 6, 8, 12, and 20 weeks. Data are presented as box plots indicating the median, the interquartile range in form of a box, and the minimum and maximum as whiskers (6 weeks: n=7, 8 weeks: n=7, 12 weeks: n=9, 20 weeks: n=7). (c) Quantitative analysis of collagen 1α-staining in livers of 6, 8, 12, and 20 weeks old mice treated with STZ/HFD and representative images of collagen 1α immunohistochemistry (200× magnification). Values are given as mean±s.e.m. (6 weeks: n=7, 8 weeks: n=7, 12 weeks: n=8, 20 weeks: n=7). Significance of differences between the groups was tested by one-way ANOVA (Holm-Sidak), §P<0.05 vs. eight weeks. (A color version of this figure is available in the online journal.)
Figure 6.
Figure 6.
Progression of liver injury during NAFLD. (a) ALT and GLDH activities in plasma of all analyzed STZ/HFD-treated mice at the ages from 4 to 20 weeks given as mean±s.e.m. (n=6–26). (b) NAFLD activity score including subscores (ballooning, inflammation and steatosis) of STZ/HFD treated mice given as mean±s.e.m. (6 weeks: n=7, 8 weeks: n=7, 12 weeks: n=9, 20 weeks: n=7). (c) In vivo analysis of apoptotic hepatocytes in control and STZ/HFD treated mice by means of IVM and representative in vivo images (200× magnification) of a control liver and a liver of a STZ/HFD treated (12 weeks) mouse displaying apoptotic hepatocytes (arrows) appearing as bright spots due to chromatin condensation. Data are presented as box plots indicating the median, the interquartile range in form of a box, and the minimum and maximum as whiskers (control: n=10, 6 weeks: n=11, 8 weeks: n=10, 12 weeks: n=10, 20 weeks: n=11). Significance of differences between the groups was tested by one-way ANOVA on Ranks (Kruskal–Wallis), *P<0.05 vs. control. (d) Quantitative immunohistochemical analysis of TUNEL-positive cells (arrows) and representative images of TUNEL-stained liver sections (200× magnification). Data are presented as box plots indicating the median, the interquartile range in form of a box, and the minimum and maximum as whiskers (n=7 per group). In healthy animals, almost no TUNEL-positive cells are present. (e) Representative images of H&E-stained liver sections of HFD/STZ-treated mice at ages of 6, 8, 12, and 20 weeks (200× magnification). (A color version of this figure is available in the online journal.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dietrich P, Hellerbrand C. Non-alcoholic fatty liver disease, obesity and the metabolic syndrome. Best Pract Res Clin Gastroenterol 2014; 28:637–53 - PubMed
    1. Calzadilla LB, Adams L. The natural course of non-alcoholic fatty liver disease. Int J Mol Sci 2016; 17:774. - PMC - PubMed
    1. World Health Organization. Global health estimates 2015: deaths by cause, age, sex, by country and by region, 2000–2015, Geneva.
    1. White DL, Kanwal F, El-Serag HB. Non-alcoholic fatty liver disease and hepatocellular cancer: a systematic review. Clin Gastroenterol Hepatol 2012; 10:1342–59 - PMC - PubMed
    1. Day CP, James OF. Steatohepatitis: a tale of two ‘hits’? Gastroenterology 1998; 114:842–5 - PubMed

Publication types

  NODES
Association 1
twitter 2