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Comparative Study
. 2014 Dec 23;9(12):e115922.
doi: 10.1371/journal.pone.0115922. eCollection 2014.

Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology

Wen Liang  1 , Aswin L Menke  2 , Ann Driessen  3 , Ger H Koek  4 , Jan H Lindeman  5 , Reinout Stoop  6 , Louis M Havekes  1 , Robert Kleemann  6 , Anita M van den Hoek  6
Affiliations
Free PMC article
Comparative Study

Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology

Wen Liang et al. PLoS One. .
Free PMC article

Abstract

Background and aims: The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.

Methods: The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.

Results: The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001).

Conclusions: We established a simple NAFLD scoring system with high reproducibility that is applicable for different rodent models and for all stages of NAFLD etiology.

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Conflict of interest statement

Competing Interests: Aswin L. Menke is employed by TNO-Triskelion. There are no patents, products in development, or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Histological photomicrographs of human NASH and NASH in mice.
Liver histological cross-sections from a healthy control subject (A) and a NASH patient (B). Liver histological cross-sections from a healthy control (C) and a NASH E3L.CETP mouse (D). All photomicrographs: Hematoxylin and eosin; magnification 200x.
Figure 2
Figure 2. Hepatocellular steatosis, hypertrophy and inflammation.
Liver cross-sections are from a NASH patient (A) or a E3L.CETP mouse fed a high fat diet supplemented with cholesterol to induce NASH (B). Macrovesicular steatosis (dotted line arrow): large lipid droplets are present in hepatocytes; microvesicular steatosis (bold arrow): small lipid droplets are present in hepatocytes. Hypertrophy (open arrow): the representative cell is much larger than the surrounding steatotic hepatocytes but has the same cytoplasmic characteristics. Clusters (aggregates) of inflammatory cells (within circles). All photomicrographs: Hematoxylin and eosin; magnification 200x.
Figure 3
Figure 3. Liver fibrosis.
Photomicrograph of cells with collagen staining (Sirius red). Liver histological cross-sections are from a healthy control subject (A) and a NASH patient (B) or a healthy control (C) and a LDLR−/−.Leiden mouse fed a high fat diet to induce NASH (D). All photomicrographs: Sirius red; magnification 100x.
Figure 4
Figure 4. Bland-Altman plot for correlation analysis between two observers for measurement of macrovesicular steatosis, microvesicular steatosis, hypertrophy and inflammation.
X-axis: average measurements of two observers. Y-axis: Difference between measurements of two observers. UL: Upper 95% limits of agreement. LL: Lower 95% limits of agreement.
Figure 5
Figure 5. Bland-Altman plot for correlation analysis between two observations of one observer for measurement of macrovesicular steatosis, microvesicular steatosis, hypertrophy and inflammation.
X-axis: average measurements of two observations. Y-axis: Difference between measurements of two observations. UL: Upper 95% limits of agreement. LL: Lower 95% limits of agreement.
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Publication types

Grants and funding

This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (www.ctmm.nl), project PREDICCT. It was also financially supported by the TNO research program ‘Personalized Prevention and Therapy – Systems Medicine.’ TNO-Triskelion provided support in the form of a salary for author ALM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.