Effect of argon (Ar) combined with hypothermia treatment on expression of cell death, tissue inflammation, and astrocyte activation in the cortex or hippocampus after hypoxia–ischemia. Seven-day-old rat pups were subjected to unilateral carotid artery ligation and then exposed to 8% O₂ balanced with N₂ for 90 min and then exposed to Ar gas (70% Ar balanced with 30% O₂) or N₂ gas (70% N₂ balanced with 30% O₂) under hypothermia (33°C) for 2 h and then room air for 24 h. (A) Coronal sections of the brain 16 h after hypoxia–ischemia are shown. The caspase-3⁺ areas indicated initiation of caspase-3 activation which is shown with green fluorescence and whose intact region was counterstained with nuclear staining propidium iodide (red). Expression of (B) caspase-3 (green fluorescence) and (D) Nuclear factor (NF)-κB (red fluorescence) was assessed by immunofluorescence at 4 h after gas exposure. Fluorescence intensity (% of naive control [NC]) of (C) caspase-3 and (E) NF-κB. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). (F) Glial fibrillary acidic protein (GFAP; green fluorescence) in the hippocampus (hippo) CA1 and CA3 of rat brain of NC, injury treated only, N₂ or Ar combined with hypothermia treated at 24 h; scale bar: 50 μm. (G) Fluorescence intensity (% of NC) of GFAP. Data are represented as mean ± SD (n = 8); *P < 0.05, **P < 0.01, and ***P < 0.001; scale bar: 50 μm. HI = hypoxic–ischemic insult; Hy = hypothermia.