Liver tissue analysis

Paraffin-embedded cross sections were stained with hematoxylin and eosin, and scored by a pathologist using a grading system adapted for NASH.[51] The level of steatosis was determined over two liver cross sections per mouse, and was expressed as a percentage of the liver area analyzed. Hepatic inflammation was assessed by counting the number of inflammatory foci per field at 100 × magnification in five non-overlapping fields per specimen. Histological sections were also assessed using the NAFLD activity score (NAS),[52] defined as the sum of the scores for steatosis (0–3), lobular inflammation (0–3), and liver injury (ballooning; 0–2). Lipids were extracted from liver tissue using the Bligh and Dyer method [53] and were quantified as previously described.[54] Hydroxyproline was measured in freshly prepared liver homogenates using a total collagen assay (Quickzyme, Leiden, the Netherlands) relative to a collagen standard following the manufacturer’s instructions.[40]