Histological assessment of adipocyte size, non‐alcoholic fatty liver disease and villi structure

Protocol from Liang W, Menke AL, Driessen A, Koek GH, Lindeman JH, Stoop R, Havekes LM, Kleemann R, van den Hoek AM (2014) Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology. PLoS One 9: 1–17

Samples were initially sectioned and stained with Hematoxylin and eosin. 

Macrovesicular steatosis 
Macrovesicular statosis was graded, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). Steatosis was evaluated at a 40 to 100× magnification.

Microvesicular steatosis 
Microvesicular steatosis was graded, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). The difference between macrovesicular and microvesicular steatosis was defined by whether the vacuoles displaced the nucleus to the side (macrovesicular) or not (microvesicular). Steatosis was evaluated at a 40 to 100× magnification.

Hypertrophy
Hepatocellular hypertrophy, was defined as cellular enlargement more than 1.5 times the normal hepatocyte diameter, was scored, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). Hypertrophy was evaluated at a 40 to 100× magnification.

Inflammation
Inflammation was evaluated by counting the number of inflammatory foci per field using a 100 x magnification. A focus was defined as a cluster, not a row, of ≥5 inflammatory cells. Five different fields were counted and the average was subsequently scored into the following categories: normal (<0.5 foci) score 0, slight (0.5–1.0 foci) score 1, moderate (1.0–2.0 foci) score 2, severe (>2.0 foci) score 3.

All were blindly analysed by two individuals.