Influence of O₂ tension, 2-ME and serum on release of β-hexosaminidase.

<p>Differentiated THP-1 cells constitutively release β-hexosaminidase that is measurable in the conditioned medium (supernatant) after 24 h (A) or 48 h (B) of culture. β-Hexosaminidase is also detected in cell lysates (C). β-Hexosaminidase activity per well was normalized to the concentration of protein in the same well as determined using the BCA protein assay. Data are presented as mean ± SEM (n = 3 independent experiments). *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂<i>versus</i> 5% O₂) by Student’s <i>t</i>-test. *, ^#, ^▴<i>p</i><0.05; **, ^##,^▴▴<i>p</i><0.01; ***, ^###,^▴▴▴<i>p</i><0.001.</p

PeakXYAUC

Fig.1-

Results of the visual cue test.

<p>(A) Escape latency on d 7 of training and during the visual cue test expressed as a percentage of baseline escape latency (escape latency on the first training day). Additional parameters that influence performance in the MWM were assessed during the visual cue test including: (B) mean swim velocity and (C) rest time, both of which are presented as a function of training day. No statistically significant differences were identified using paired t-test (A) or repeated measures ANOVA (B,C).</p

MWM training increases transcription of genes associated with synaptic plasticity in multiple brain regions.

<p>Transcript levels of spinophilin (Spn), activity-regulated cytoskeleton-associated protein (ARC), neurogranin (RC3), Homer1a and Homer1b/c were analyzed in total RNA harvested from the cortex, cerebellum, and hippocampus of weanling mice after behavioral studies were completed. Data are presented as fold-change in transcript expression relative to non-maze-trained littermates as calculated by the Pfaffl equation, normalized to the housekeeping gene 18S rRNA (n = 9–12 animals per group). The dashed line represents a fold-change of 1, which indicates no difference in gene expression between MWM-trained animals and untrained littermate controls.</p

Weanling mice exhibit spatial memory after MWM training.

<p>Spatial memory was assessed in a probe trial administered on training day 8 with respect to: (A) percentage of time or (B) percentage of path length spent in the target quadrant relative to non-target quadrants. Data in panels A and B are presented as the mean ± SEM (n = 16 animals). Significantly different from target quadrant at ^a<i>p</i> < 0.05, ^c<i>p</i> < 0.001 as determined by repeated measures ANOVA with the Greenhouse-Geisser correction for LSD <i>post hoc</i> tests. Effect sizes: partial η² for % time = 0.66; partial η² for % path length = 0.68. Observed power: 100% for % time; 100% for % path length.</p

Weanling mice exhibit spatial learning in the Morris water maze (MWM).

<p>Spatial learning was assessed as a function of training day with respect to the following parameters: (A) escape latency, (B) percentage of time spent in the target quadrant, and (C) percentage of total path length spent in the target quadrant. Data are presented as the mean ± SEM (n = 16 animals). Since sex differences were not identified for any of the behavioral parameters shown in this Fig., data from males and females were combined to calculate mean values. Significantly different from training d 1 (A) or d 2 (B and C) at ^a<i>p</i> < 0.05, ^b<i>p</i> < 0.01, ^c<i>p</i> < 0.001 as determined using repeated measures ANOVA with LSD <i>post hoc</i> test. Effect sizes: partial η² for latency = 0.31, partial η² for % time = 0.21; partial η² for % path length = 0.17. Power: 99% for latency, 94% for % time, 84% for % path length. Note that escape latency was the only data collected on the first day of training because of a computer malfunction in collecting data on the first training day.</p

Influence of O₂ tension, 2-ME and serum on the rate of THP-1 differentiation.

<p>Differentiation of THP-1 cells from monocytic to macrophage cells is associated with transition from a non-adherent to an adherent cell type. To determine whether culture conditions affect PMA-stimulated differentiation of THP-1 cells to macrophages, cell adhesion was assessed at 3 and 24 h after addition of PMA (20 ng/ml) to the culture medium. Data are presented as the mean ± SEM of the protein concentration of adherent cells at 3 h as a percentage of the protein concentration of adherent cells at 24 h (n = 4 independent experiments). *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂<i>versus</i> 5% O₂) by Student’s <i>t</i>-test. **, ^##,^▴▴<i>p</i><0.01; ***, ^###,^▴▴▴<i>p</i><0.001.</p

Influence of O₂ tension, 2-ME and serum on proliferation of undifferentiated THP-1 cells.

<p>Monocytic THP-1 cells were synchronized by serum deprivation for 48 h and then cultured in hyperoxic (18% O₂) or normoxic (5% O₂) with or without 2-ME and/or FBS. Cell density was determined using a hemocytometer at 24 h (A) and 48 h (B) after synchronization. Data are presented as the mean ± SEM (n = 5 independent experiments). *Significantly different from cultures with 2-ME and FBS under the same oxygen tension at <i>p</i><0.05 (one-way <i>ANOVA</i> with <i>post hoc</i> Tukey’s test).</p

Oxygen tension influences redox in LPS-induced NF-κB activation in PMA-differentiated THP-1 cells.

<p>Undifferentiated THP-1 XBlue cells, which express an NF-κB reporter gene linked to secreted embryonic alkaline phosphatase (SEAP) were synchronized by serum deprivation for 48 h, and then differentiated with PMA (20 ng/ml) for 48 h. Differentiated THP-1 XBlue cells were then cultured in varying concentrations of DPI (A) or NGA (B) for 4 h followed by LPS (1 µg/ml) stimulation for an additional 24 h in either 18% or 5% O₂. SEAP activity was quantified by QuantiBlue at 630 nm. Data are presented as the mean ± SEM (n = 2 independent experiments). *Significantly different from baseline (SEAP activity in the absence of inhibitor) at the same oxygen tension; **<i>p</i><0.01; ***<i>p</i><0.001 (one-way <i>ANOVA</i> with <i>post hoc</i> Tukey’s test). ^#Significantly different from 5% O₂ at same antioxidant concentration; ^#<i>p</i><0.05; ^##<i>p</i><0.01 (Student’s <i>t</i>-test).</p

Influence of oxygen tension on phagocytosis.

<p>THP-1 cells were cultured with PMA for 25 h to promote macrophage differentiation. In a subset of the cultures, the oxygen tension was switched from normoxic to hyperoxic or from hyperoxic to normoxic for the last hour of the incubation period. Phagocytosis was assessed as the uptake of <i>E.coli</i> BioParticles®. Data are presented as mean ± SEM (n = 3 per treatment group). *Significantly different from 25 h at 18% O₂ at <i>p</i><0.05; and ^ΔΔΔSignificantly different from 25 h at 5% O₂ and from 24 h at 18% O₂ → 1 h @ 5% O₂ at <i>p</i><0.001 (one-way <i>ANOVA</i> with <i>post hoc</i> Tukey’s analysis).</p