Histological Evaluation of NAFLD in Mice

The liver, thymus, and heart were weighed, fixed in 10% formalin overnight, and then embedded in paraffin and sectioned. Five-mm liver sections were stained either with hematoxylin and eosin (H&E) or with 0.1% Sirius red F3B (Sigma, St. Louis, MO, USA) in saturated picric acid (Sigma) [22 (link)]. H&E-stained sections were scored for steatohepatitis grade using the NASH activity score (NAS), which is defined as the sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2) [23 (link)]. Thus, the results were a continuum ranging from 0 to 8. NAS scores of 0–2 are considered not diagnostic of NASH, while scores of 3–4 are considered possible NASH, and scores of 5–8 are considered diagnostic of NASH. Fibrosis, believed to be a consequence of steatohepatitis, is measured separately from the NAS on a scale ranging from stage 0 to stage 4, with stage 4 indicative of cirrhosis. All liver sections were analyzed and scored in a blinded manner by a trained liver pathologist (Icahn School of Medicine at Mount Sinai Department of Pathology).