To investigate the relationship among proinflammatory gene induction, microglial activation, oxidative stress, and neuronal cell death, we assessed the cell death markers, activated caspase-3 and Fluoro-Jade B. Ethanol, poly I:C and sequential ethanol-poly I:C increased caspase-3 + IR cells in both cortex and hippocampus (Figure 9). Ethanol-poly I:C caused significantly greater increases in caspase-3 + IR cells than either alone. Double immunohistochemistry with confocal microscopy revealed that caspase-3 + IR was colocalized with NeuN, a neuronal marker, in both cortex and hippocampus (Figure 9E), suggesting neuronal cell death. Fluoro-Jade B, another cell death marker, was also increased by ethanol, poly I:C and sequential ethanol-poly I:C treatment in both cortex and hippocampus (Figure 10). Pretreatment with ethanol more than doubled poly I:C increases in Fluoro-Jade B staining, compared to poly I:C alone group (Figure 10A). Confocal microscopy indicated that most Fluoro-Jade B-positive cells were colocalized with NeuN + IR in both cortex and hippocampal dentate gyrus (Figure 10B). These results indicate that markers of neuronal death are increased by ethanol, poly I:C and sequential ethanol-poly I:C in parallel with induction of neuroinflammatory genes and oxidative stress.
