Figure 5

Chronic ethanol enhances NF-κB mRNA and protein expression. C57BL/6 and NF-κB enhanced GFP mice were treated with water or ethanol (5 g/kg, i.g.) daily for 10 days and sacrificed 24 hrs after the last dose of ethanol. (A) C57BL/6 mouse brain mRNA expression of NF-κB-p65 is significantly increased by 10 doses of ethanol exposure 24 hrs following ethanol treatment compared with water controls, as determined by real-time PCR. (B) Representative images are from the dentate gyrus of NF-κB enhanced GFP mice 24 hrs after water or ethanol treatment. Ethanol treated mouse brain had more NF-κB-GFP (green fluorescence) than water control brain. Scale bar = 20 μm. (C) NF-κB reporter mice were injected with dehydroethidium (10 mg/kg, i.p.) 23.5 hrs after ethanol treatment. Brains were harvested 30 min later and frozen sections (15 μm) were processed and immunostained for Iba1, Neu-N or GFAP to analyze cell phenotype for NF-κB activation and ROS production. Confocal results reveal that NF-κB activation and ROS production predominantly occurred in microglia and neurons as shown by NF-κB (green) and ROS (red) are triple labeled with Iba-1+IR cells (blue) or with MAP-2 +IR neurons (blue). Triple-labeling is shown in write with arrows. Double-labeled images are shown in yellow with arrowheads indicating the colabeling of NF-κB (green) and ROS (red). The images presented are from dentate gyrus of ethanol treated mice. Scale bar = 10 μm. * P < 0.05, compared with the water control mice.