FIGURE 6.

Dose and time-dependent increase in ROS levels by PX is not reversed by ZVAD. Monolayers of BLECs were treated with PX for 48 h in a dose response experiment. (A) A representative time course experiment of CellRox green staining, the fluorescence intensity of ROS-positive cells normalized to the whole population is presented as the ratio of green object integrated intensity to phase confluence (a representative figure, out of three time course independent experiments is shown, n = 6 for each time point). ^∗Marks the first time in which the PX treatment is significantly different from control, #marks the first time in which the PX treatment is significantly different from 900 μM PX; after 38 h 900 μM PX+ZVAD is not significantly different from 900 μM PX according to plot color. ^∗p < 0.05 vs. Control, #p < 0.05 vs. PX at 900 μM. (B) Co-cultures of BBB in vitro models were treated at the luminal side with 900 μM PX with or without TEMPOL at 150 μM for 24 h. LDH release at the luminal side of the co-culture BBB system was measured. (C) Pe of NaF from luminal to abluminal side was measured (n = 9–12 Transwell inserts from 3 independent experiments). Data presented as mean ± SEM. ^∗p < 0.05 and ^∗∗∗p < 0.001 vs. control, ^###p < 0.001 vs. PX alone (Ordinary one way ANOVA with Dunnett’s multiple comparisons test). NS, no statistical differences.