was used to generate cDNA templates and TaqManH Master Mix Reagent Kit (Applied Biosystems, Foster City, CA) was used to amplify and quantify the p47phox transcript in 25 ml reactions. Triplicate PCR reactions were performed in 96-well in parallel with the 18S rRNA. The steps involved in the reaction program included: the initial step of 2 minutes at 50uC; denaturation at 95uC for 10 min, followed by 45 thermal cycles of denaturation (15 seconds at 95uC) and elongation (1 min at 60uC). Expression values were obtained from the cycle number (Ct value) using the Biosystems analysis software. P47phox and 18S rRNA were performed in triplicates to determine the Ct-diff. These Ct values were averaged and the difference between the 18S Ct (Avg) and the gene of interest Ct (Avg) was calculated (Ct-diff). The relative expression p47phox was analyzed using the 2-DDCT method. Quantitative results were expressed as the mean 6standard deviation (SD). Statistical significance was evaluated by the Student’s t-test. Measurement of NADPH oxidase activity. NADPH oxidase activity was measured using the standard cytochrome c reduction method, as described previously [50]. Isolated frontal cortices from wild type mice exposed to either IH or RA were homogenized in a RIPA buffer. The homogenate was subjected to centrifugation at 250 g for 10 min to remove cellular debris. Supernatant was then centrifuged at 20,000 g for 20 min at 4 uC to eliminate mitochondria, lysosomes, peroxisomes, Golgi membranes, and rough endoplasmic reticulum. The resulting supernatant was centrifuged at 100,000 g for 60 min at 4uC. The pelleted plasma membrane fraction containing Nox was dissolved in a buffer containing 8 mM piperazine-N,N’-bis 2-ethanesulfonic acid (pH 7.2), 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and proteolytic inhibitors. The solubilized membrane fraction samples were placed in a multiwell plate, and flavin adenine dinucleotide (FAD; 0.01 mM), acetylated cytochrome c (0.1 mM), and GTPcS (0.01 mM, as an activating agent for NADPH oxidase) were added to the samples. Superoxide dismutase (SOD; 100 U/ml) was included to block cytochrome c reduction in half of the samples. After the samples were kept at room temperature for 2 min, sodium dodecyl sulfate (0.1 mM) as an additional activating agent was added. After incubation for 3 min, NADPH (0.2 mM) was added to initiate cytochrome c reduction. Absorbance at 550 nm (A550) in samples incubated and not incubated with SOD was measured for 10 min on a FlexStation III microplate reader (Molecular Devices, Sunnyvale, CA, USA). NOX activity was calculated as the SOD-inhibitable reduction of cytochrome c. All chemicals were from Sigma (St. Louis, MO). Lipid Peroxidation Assay. MDA-586 kits (OxisResearch, Portland OR) were used to measure the relative malondialdehyde (MDA) production, a commonly used indicator of lipid peroxidation (50), in frontal brain cortex according to the manufacturer’s instructions. Briefly, after anesthesia with pentobarbital (50 mg/kg intraperitoneally), mice were perfused with 0.9% saline buffer for 5 minutes and the cortex was dissected, snap frozen in liquid nitrogen, and stored at –80uC until assay the following day. Cortical tissues were homogenized in 20 mM phosphate buffer (pH 7.4) containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. After protein concentration measurements, equal amounts of proteins (2.0– 2.5 mg protein from each sample) were used in triplicate to react with chromogenic reagents at 45uC in 500 mL buffer for 1 to 2 hours. The samples were then centrifuged and clear supernatants measured at 586 nm. The level of MDA production was then calculated with the standard curve obtained from the kit according to the manufacturer’s instructions. NADPH Oxidase and Intermittent Hypoxia 8-hydroxydeoxyguanosine (8-OHDG). Levels of 8-OHDG were measured in frontal brain cortex using a commercially available assay (Cell Biolabs, San Diego, CA). Briefly, cortical samples or 8-OHDG standards were first added to an 8-OHDG/ BSA conjugate preabsorbed enzyme immunoassay plate. After a brief incubation, an anti–8-OHDG mAb was added, followed by an horseradish peroxidase-conjugated secondary antibody. The 8- OHDG content in the cortical samples was then determined by comparison with the 8-OHDG standard curve. Data Analysis. To elucidate the nature of interactions between IH and RA conditions, all data were analyzed by one way ANOVA. First, overall statistical significance was determined for the entire training period between the treatment groups. In addition, two-way repeated measures ANOVA were used to analyze each trial blocks, followed by post-hoc Tukey tests. Similar statistical approaches were used to compare probe trial, reference memory, EPM and FST. For all comparisons, a p value ,0.05 was considered to achieve statistical significance. In all the experimental conditions, the data were divided into 6 blocks (containing 3 trials/day). We used a multivariate MAN- OVA model (SPSS software 11; Chicago) that included latency, pathlength and swim speed and Two between factors: (1) Groups (four levels): RA C57BL6J, IH C57BL6J, RA gp91phox_/Y and IH gp91phox_/Y (2) Condition (two levels): RA or IH. All F statistics are reported using Pillai’s Trace. The interaction of three different factors, i.e., time, condition and group were determined using this mixed model repeated measures MANOVA. Results Spatial Learning Performance. On a standard place discrimination task, wild type mice exposed to 14 days of IH (IH-C57BL6/J) exhibited longer latencies and pathlengths to locate the hidden platform when compared to room air controls RA-C57BL6/J, RA- gp91phox_/Y and gp91phox_/Y mice exposed to 14 days IH (IH- gp91phox_/Y) animals (n = 12 per experimental condition; Figures 2A and B). Overall latency analysis for the entire trial blocks revealed significant changes between the different treatment groups, [F = 41.14; p,0.001] and pathlength, [F = 16.44; p,0.001] indicating that IH adversely affected task performance. Significant differences in latencies were observed during blocks 2 [F = 4.91; p,0.006], 3 [F = 8.38; p,0.001], 4 [F = 3.35; p,0.03], 5 [F = 7.06; p,0.001] and 6 [F = 4.457; p,0.01]. There were no significant differences in Block 1. Repeated measures ANOVA revealed significant differences in pathlengths during blocks 3 [F = 5.25; p,0.004], 4 [F = 4.36; p,0.01], 5 [F = 6.73; p,0.001] and 6 [F = 2.99; p,0.04], with no significant differences in Blocks 1 and 2. There were no significant differences in swim speed in these mice. In the probe-trial test, one-way ANOVA revealed a significant effect of treatment [IH vs. RA: F = 12.87; p,0.001]. The magnitude of impairment was greatest in IH-C57BL6/J mice (Figure 2C). In the reference memory tests, IH-C57BL6/J mice exhibited significant deficits in memory retention in both latency [F = 19.61; p,0.001] and pathlength [F = 12.84; p,0.001]. However, the IH- gp91phox_/Y mice performed similar to normoxic controls (Figure 3). Repeated measures MANOVA with latency, groups and conditions [F(5,39) = 72.54; P,0.0001]; revealed that RA gp91phox_/Y and RA C57BL6/J mice required significantly less time than their littermates exposed to IH to find the hidden platform in a Morris water maze (Figure 2A); Repeated measures MANOVA with pathlength, groups and conditions [F(5,39) = PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19847