Histopathological evaluation of liver lesions

Five µm sections of formalin-fixed, paraffin-embedded liver tissue were stained with hematoxylin and eosin (H&E) for histopathological examination. Two independent investigators blinded to treatment groups examined the sections under light microscopy. Hepatic steatosis was graded according to the magnitude of steatosis (both macro- and micro-vesicular fat accumulation) and the degree of liver inflammation severity as described previously (21). Briefly, the degree of steatosis was graded 0–4 (grading 0=<5%, 1=5–25%, 2=26–50%, 3=51–75%, 4=>75%), based on the average percentage area of the liver section that was occupied by fat vacuoles per field at 100× magnification under H&E staining in 20 random fields. Inflammatory foci were evaluated by the number of inflammatory cell clusters in 20 random fields at 100× magnification, which mainly constitute mononuclear inflammatory cells. Twenty fields of view at 100 magnification represent 0.63 cm², and inflammatory foci counts were represented as the number of foci per cm². A ZEISS microscope with a PixeLINK USB 2.0 (PL-B623CU) Digital Camera and PixeLINK µScope Microscopy Software was used for image capture of all histopathological examinations.