Figure 4

Osmotin inhibits ethanol-induced apoptosis in vivo. P7 rat pups were injected with saline (−) or ethanol (EtOH; 4 mg/g b.w.) and again with osmotin (Osm) or saline 30 min later. (A) The treatments are indicated above the blots. Level of cleaved caspase-3 and β-actin were analyzed in hippocampal part of brain by western blot at 12 h after ethanol administration. The graph shows the quantification of cleaved caspase-3 levels by densitometry. The values were normalized to β-actin signals and represent mean±S.D. (n=3). Significant difference from the ethanol group at P<0.01 is indicated by an asterisk. (B) Apoptotic DNA fragmentation in brain tissues was evaluated with TUNEL staining 12 h after ethanol administration. The osmotin dose was 15 μg/g b.w. The arrows indicate TUNEL-stained nuclei in neurons. Magnification × 40; scale bar, 50 μm. (C) P7 rat pups were injected with saline (Control) or ethanol (EtOH; 4 mg/g b.w.) and again with osmotin (Osm; 15 μg/g b.w.) or saline 30 min later. Pups were killed at 12 h after ethanol injection for analysis of neurodegeneration by propidium iodide (PI) and Fluoro-Jade B (FJB) staining. (C) Shown is stained sections from the CA1 region of the hippocampus. Magnification × 60; scale bar, 20 μm. (D) Shown are stained sections from the anterior cingulated cortex. (a–f) Magnification × 40; scale bar, 20 μm; (g–i) magnification × 100; scale bar, 10 μm. (E) Quantification of the data represented in (A) and (B). Data represent means±S.D. (n=4, hippocampus; n=3, cortex). Sample pairs that were significantly different at P<0.05 are indicated. (F) Shown are views of the CA1, CA3, and DG regions of hippocampal after PI staining at low ( × 20; scale bar=20μm) and high ( × 100; scale bar=10 μm) magnification. Rectangles indicate the CA1 regions that are shown in the magnified views. Arrows indicate shrunken and damaged neurons