Effect of CCI and CCI+mHW on Brain Cytokine Levels.

<p>*p<0.05 & **p<0.01 for CCI vs Sham and for CCI+mHW vs Sham; #p<0.05 & ## p<0.01 for CCI+mHW vs CCI.</p><p>Mean ± SE (pg/mg of brain protein).</p><p>Effect of CCI and CCI+mHW on Brain Cytokine Levels.</p

Changes in Brain Expression with CCI and mHW: Molecular Function.

<p>Categories listed consisted of a minimum of 6 genes, had at least 3 genes that were changed, and a Z-score of ≥2.0.</p><p>Changes in Brain Expression with CCI and mHW: Molecular Function.</p

Effects of CCI and mHW on Cyclophillin A, APP, and Amyloid Beta Peptide Levels.

<p>The upper left panel shows that protein levels of APP were decreased 7 days after CCI and that mHW did not protect from CCI. Amyloid beta peptide_1–40 (upper right panel) was increased on day 7 and amyloid beta peptide_1–42 (lower right panel) was decreased 24 h after CCI, but mHW did not alter these effects of CCI. CypA was decreased 7 days after CCI and this decrease was enhanced by mHW (lower left panel).</p

Reduction of CCI-induced pathological tau by mHW in frontal cortex.

<p>CCI induced increases in AT8 and Alz50 immunoreactivity extend to the frontal cortex and are attenuated by treatment with hydrogen water. Low power representative images demonstrate increased AT8 immunoreactivity in the frontal cortex of CCI (B) compared to sham treated (A) mice. C–F, high power images of the cortical region shown in B (arrow) demonstrate AT8 (C,D) and Alz50 (E,F) immunoreactivity in CCI (C,E) and HW treated (D,F) mice. Scale bars: 500 µm, A,B; 50 µm, D–F.</p

Reduction of CCI-induced pathological tau expression in hippocampus and cortex by mHW.

<p>The CCI-induced increase in AT8 and Alz50 immunoreactivity is attenuated by treatment with hydrogen water. Representative images of immunostaining in brains from sham (A, D, G, H), CCI (B, E, I, J), and CCI+mHW treated (C, F, K, L) animals. Low power representative images depict AT8 (A–C) and Alz50 (D–F) immunoreactivity. G–L, high power images of the cortical region designated by arrows, immunostained for AT8 (G,I,K) and Alz50 (H, J, L). Scale bars: 500 µm, A–F; 50 µm, G–L.</p

Treatment with Molecular Hydrogen Does Not Rescue Respiration After ETC Inhibition.

<p>ImBPCs were subjected to a mitochondrial stress test in the presence of the ATP synthase inhibitor (oligomycin; 3 µM), the uncoupling agent (FCCP; 3 µM), the complex I inhibitor (rotenone; 3 µM), and the complex III inhibitor (antimycin A; 1.5 µM). Once respiration was inhibited, ImBPCs were exposed to hydrogen water (red) or control (dI water; blue), and OCR was measured for 30 min (Panel A) or 6 h (Panel B). ECAR was also examined for 30 min (Panel C). ATP levels were measured after 10 min, 6 h, or 24 h of treatment with molecular hydrogen water. Results presented here are from one experiment completed, which is representative of the 3 experiments that were done. Results are represented as a mean ± SEM. ** is p<0.01 and *** is p<0.001.</p

The CCI induced increase in Iba1 immunoreactivity is attenuated by treatment with hydrogen water.

<p>Representative images are of Iba1 immunoreactivity in the cortex adjacent to the site of injury (A–C) and the hippocampus (D–F) of sham (A,D), CCI (B,E) and CCI/HW treated (C,F) animals. Scale bars: 50 µm, A–C; 100 µm, D–F.</p

Changes in Brain Expression with CCI and mHW: Cellular Component.

<p>Categories listed consisted of a minimum of 6 genes, had at least 3 genes that were changed, and a Z-score of ≥2.0.</p><p>Changes in Brain Expression with CCI and mHW: Cellular Component.</p

Effects of CCI and mHW on Brain Edema.

<p>Main figure shows that the percent of brain weight that was water increased 24 h (day 1) after CCI and that mHW significantly reduced the water content. Inset shows brain edema index calculated by the Keep method at 24 h; CCI increased the edema index, while mHW was protective. **p<0.01, ***p<0.001.</p

CCI-induced Elevations in Six Cytokines in Brain Were Affected by mHW.

<p>IL-6 (not shown; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108034#pone-0108034-t001" target="_blank">Table 1</a> for results) had a pattern similar to G-CSF. Except for the day 7 IL-1α value, the statistically significant effects of mHW were to further increase brain cytokine levels. *p<0.05; **p<0.01; ***p<0.001.</p