Inflammasome-dependent caspase-1 activity has been shown to cause a rapid inflammatory form of cell death called pyroptosis, in which cytoplasmic content and pro-inflammatory cytokines, including IL-1β and IL-18, are released (Miao et al., 2011). However, apoptosis can also be induced by mitochondria stress and NLRP3 activation (Morizot, 2012), and our previous studies have indicated that ethanol can cause activation of caspase-3-dependent apoptosis and necrosis in the cerebral cortex (Alfonso-Loeches et al., 2010). Therefore, in order to evaluate whether ethanol-induced TLR4-dependent mitochondrial ROS production and NLRP3 activation promote pyroptosis and/or apoptosis, treated and untreated astrocytes were labeled with different staining and then, the proportion of those cells that died by apoptosis, necrosis or pyroptosis were evaluated using an In Cell Analyzer. As shown in Figure 7A, treatment of the astrocytes with ATP or LPS or ethanol 10 mM or ethanol 50 mM promotes 10.2, 15, 15.9, and 9.5% of pyroptosis (PI+ and FLICA 660-YVAD-FMK+ cells), respectively, when compared with untreated control cells.
