Molecular hydrogen in drinking water protects against neurodegenerative changes induced by traumatic brain injury.

Traumatic brain injury (TBI) in its various forms has emerged as a major problem for modern society. Acute TBI can transform into a chronic condition and be a risk factor for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, probably through induction of oxidative stress and neuroinflammation. Here, we examined the ability of the antioxidant molecular hydrogen given in drinking water (molecular hydrogen water; mHW) to alter the acute changes induced by controlled cortical impact (CCI), a commonly used experimental model of TBI. We found that mHW reversed CCI-induced edema by about half, completely blocked pathological tau expression, accentuated an early increase seen in several cytokines but attenuated that increase by day 7, reversed changes seen in the protein levels of aquaporin-4, HIF-1, MMP-2, and MMP-9, but not for amyloid beta peptide 1-40 or 1-42. Treatment with mHW also reversed the increase seen 4 h after CCI in gene expression related to oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, ATP and nucleotide binding. Finally, we found that mHW preserved or increased ATP levels and propose a new mechanism for mHW, that of ATP production through the Jagendorf reaction. These results show that molecular hydrogen given in drinking water reverses many of the sequelae of CCI and suggests that it could be an easily administered, highly effective treatment for TBI

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

GFAP immunoreactivity is decreased in the hippocampus (CA1 stratum radiatum) of CCI mice treated with hydrogen water.

<p>Representative images demonstrate similar levels of hippocampal GFAP immunoreactivity in sham (A) and CCI (B) mice. Immunostaining for GFAP is greatly reduced in the hippocampus of animals treated with hydrogen water (C). Scale bars: 50 µm.</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

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

Pretreatment with Molecular Hydrogen Enhances Mitochondrial Aerobic Metabolism.

<p>ImBPCs were subjected to a mitochondrial stress test after treatment with molecular hydrogen water (red) or control (dI water; blue) for 24 h. Oxygen consumption rates (OCR) (Panel A) and extracellular acidification rates (ECAR) (Panel C) were measured using the Seahorse XF24 analyzer 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). Analysis of OCR data showed molecular hydrogen increased basal respiration, reserve capacity, and non-mitochondrial (non-mt) respiration but had no effects on ATP production rate as assessed indirectly by OCR analysis, proton leak, or maximal respiration (Panel B). Direct measurement of ATP levels (Panel D) in other cells not exposed to the metabolic stressors showed higher levels of ATP after 24 h but not after 10 min or 6 h of exposure to molecular hydrogen. 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

Effects of CCI and mHW on Serum Cytokine Levels.

<p>Only G-CSF was elevated in blood after CCI and only at 24 h. There was no statistically significant effect of mHW on serum G-CSF levels. *p<0.05; **p<0.01.</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