21.409; P,0.0001]; indicated that as the training progressed the RA gp91phox_/Y and RA C57BL6/J mice could reach the hidden platform and covered the shortest distance when compared to the distance covered by their littermates exposed to IH in a Morris water maze (Figure 2B). In addition repeated measures MANOVA with swim speed, groups and conditions on the swim speed showed no significant differences between the groups and treatments (Figure 2C). Elevated Plus Maze IH-C57BL6/J mice showed significant differences in the percentage of time spent in the open arm [F = 64.13; p,0.001] and in the number of entries into the closed arm [F = 14.74; p,0.001] (Figure 4). The results of the elevated plus maze showed that IH-C57BL6J spent significantly less time in the open arms (Figure 4; group effect, [F = 18.354; p,0.001] and significantly more time in the center area (Figure 4; group effect, [F = 26.945; p,0.001]. The number of entries into the closed arms was significantly increased (Figure 4; condition effect, [F = 11.533; p,0.001]. Although, the percentage of time spent in the open arm is commonly used as a measure of anxiety, the time spent on the center platform of the maze and the closed arm entries all reflect anxiety-like behaviors in mice. [79,87]. Forced Swim Test. IH-C57BL6/J mice had significantly higher immobility durations during the last 4 min of the FST NADPH Oxidase and Intermittent Hypoxia [F = 25.54; p,0.001] when compared to all other treatment groups, including IH- gp91phox_/Y (Figure 5). NADPH Oxidase Expression and Activity. Frontal cortical tissues from IH- and RA-exposed mice were subjected to quantitative RT-PCR. P47phox expression was increased in IH starting at 3 days and sustained thereafter (Figure 6a). Similarly, NADPH oxidase activity was significantly increased in IH-exposed wild type mice (Figure 6b and 6d). Furthermore, such IH-induced increases of NOX activity were attenuated in gp91-/- mice, although the increase was still significant (p = 0.024; n = 6/group; Figure 6c and 6d). Lipid Peroxidation. After the behavioral experiments, cortical tissues and hippocampus were harvested and processed for assessment of lipid peroxidation as indicated by MDA levels. Figure 7 shows MDA concentrations in homogenates of cerebral cortex from all treatment groups. A significant increase in MDA levels was observed in IH-C57BL6/J mice [F = 10.38; p,0.001] in the cortex and [F = 35.416; p,0.001] in the hippocampus when compared to all other groups. 8-OHDG Levels. The levels of 8-OHDG in homogenates of cerebral cortex and the hippocampus were significantly higher in IH-C57BL6/J mice [F = 32.50; p,0.001] and [F = 22.214; p,0.001] respectively; when compared to all other groups (Figure 8). However there were no significant differences in the levels of 8-OHDG in cortex of IH- gp91phox_/Y when compared to RA controls. Figure 4. Exposure to IH induces anxiety in mice. C57BL6/J mice exposed to IH spend significantly less time in the open arm of the elevated plus maze compared to RA C57BL6/J, or gp91phox_/Y mice exposed to either RA or IH (A). A reduced number of closed-arm entries emerged in wild type mice exposed to IH (B). (C) Time spend in the Center Area was increased in wild type mice exposed to IH (n = 12/experimental group; *P,0.05). doi:10.1371/journal.pone.0019847.g004 PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19847