4.4. Histological Analysis

Formalin-fixed, paraffin-embedded liver, and epididymal fat tissue sections were stained with hematoxylin and eosin. Hepatic fibrosis was assessed using Masson–Goldner staining in accordance with the manufacture’s instructions. The liver sections were scored for the severity of steatosis, inflammation, hepatocyte ballooning, and fibrosis in accordance with the scoring method NAFLD activity score (NAS) and fibrosis staging. Briefly, two independent researchers who have enough experiments evaluated the NAS and fibrosis staging as follows: The degree of steatosis (grade 0 ≤ 5%; 1 = 5–33%; grade 2 = 34%–66%; grade 3 ≥ 66%), lobular inflammation (0: no foci, 1 < 2 foci per 200x field, 2: 2 to 4 foci per 200x field, and 3: > 4 foci per 200x field), hepatocyte ballooning (0: None; 1: Rare or few; 2: Many), and fibrosis (0: No fibrosis, 1: Perisinusoidal or periportal fibrosis, 2: Perisinusoidal and portal/perioral fibrosis, 3: Bridging fibrosis, and 4: Cirrhosis)