Histological determination of the effect of fasting on APAP-induced hepatocyte apoptosis in mice. (A,B) Control mice, PAS reaction. (A) In control mice with free access to food, the vast majority of centrilobular hepatocytes exhibit cytoplasmic glycogen (arrow). (B) Mice fasted for 24 h do not exhibit glycogen within hepatocytes. Bars = 20 µm. (C-F) 3 hours after APAP treatment (530 mg/kg). In animals with free access to food (C,D), centrilobular cell loss was observed (average score 1.1). There were apoptotic cells that could be identified based on their morphology in a hematoxylin and eosin-stained section (arrows, C) and the expression of cleaved caspase-3 (arrows, D). Bars = 10 µm. Animals that had been fasted for 24 h before APAP administration showed more extensive hepatocyte loss (average score 1.6). Necrotic hepatocytes were identified in the hematoxylin and eosin-stained section (arrows, E). There was no evidence of cleaved caspase-3 expression by hepatocytes, and the only positive apoptotic cells were scattered leukocytes or Kupffer cells (arrow, F). Bars = 20 µm. Figures are representative of six animals per group carried out in three independent investigations. CV, central vein.