Isolation of bone marrow-derived microglia and resident microglia from hypothalamic tissue and comparison of expression of various molecules in chronic PS-loaded and sham-treated mice.

<p>(A) Representative FACS chart in chronic PS-loaded mice with whole body radiation and the number of isolated GFP^-CD45^low (resident microglia) and GFP⁺CD45^low (bone marrow-derived microglia) from mice with whole body radiation and the radiation with head protection (<i>n</i> = 4−6). Total events in FACS were 20000. Data are expressed as mean ± sem. *<i>P</i> < 0.05 with two-tailed Student’s t-test. (B−E) mRNA expression of chemokine receptors: CCR2 (<i>n</i> = 6−8), CX₃CR1 (<i>n</i> = 3−5), CXCR4 (<i>n</i> = 3−5) (B), exiting amino acid transporters: EAAT1 (<i>n</i> = 4−5), EAAT2 (<i>n</i> = 3−4) (C), purinergic P2X receptors: P2X4 (<i>n</i> = 3) and P2X7 (<i>n</i> = 3) and P2Y receptors: P2Y1 (<i>n</i> = 3), P2Y12 (<i>n</i> = 3−4) (D), IL-1β (<i>n</i> = 4−6) and TNF-α (<i>n</i> = 3−5) (E). (F) The length of axis of GFP⁺Iba-1⁺ microglia (bone marrow-derived microglia, BMDM) and GFP^-Iba-1⁺ microglia (resident microglia. RM) in chronic PS-loaded and sham mice (<i>n</i> = 4). Scale bars: 10 µm. Data are expressed as mean ± sem. *<i>P</i> < 0.05, **<i>P</i> < 0.01 with ANOVA followed by Tukey’s multiple comparison.</p

Three dimensional image of immunofluorescence staining of IL-1β, pNMDA receptor, and IL-1 receptor in PVN from chronic PS-loaded mouse.

<p>(A) GFP-positive cells (green) overlapped with IL-1β (red). (B, C) Positive reactions for pNMDAR (pink) and IL-1R (pink) detected on the membrane of neurons (red) adjacent to bone marrow-derived microglia (green) in PVN. Scale bars: 10 µm. </p

Schematic drawing of brain-bone marrow axis in chronic psychological stress conditions.

<p>When brain is exposed to chronic PS (1), the information is mediated to bone marrow through adrenergic nerves (2). Bone marrow niche cells innervated with sympathetic nerves decrease the expression of SDF-1 through β₃-adrenergic receptor (3). CXCR4^high monocytes egress to peripheral circulation from the bone marrow by reduction of SDF-1 at the bone marrow niche (4). Neurons in the PVN express MCP-1 under chronic PS condition, then accelerate infiltration of CCR2⁺ bone marrow-derived microglia into PVN (5).</p

Chronic psychological stress (PS) increases the infiltration of bone marrow-derived microglia into the PVN.

<p>(A, B) After chronic PS-loading for five days, the numbers of GFP-positive cells (green) in the PVN were significantly increased compared with sham-treated mice. Data are expressed as mean ± sem (PS: <i>n</i> = 6−7, sham: <i>n</i> = 5−11). <b><i>#</i></b><i>P</i> < 0.05 with ANOVA followed by Tukey’s multiple comparison. *<i>P</i> < 0.05 with two-tailed Student’s <i>t</i>-test. (C) GFP-positive cells (green) overlapped with Iba-1 (red) in PVN from chronic PS-loaded mice but did not overlap with GFAP (red) in PVN from chronic PS-loaded mice. (D) Photograph of mice received irradiation with head protection and non-irradiated mice. (E, F) After chronic PS-loading for five days, the number of GFP-positive cells (green) in the PVN of mice received the irradiation with head protection was significantly increased compared with sham-treated mice. Data are expressed as mean ± sem. **<i>P</i> < 0.001 for PS (<i>n</i> = 8) to sham (<i>n</i> = 4) with two-tailed Student’s <i>t</i>-test. (G) GFP-positive cells (green) overlapped with Iba-1 (red) in PVN from chronic PS-loaded mice received the irradiation with head protection. PVN shown by dotted line. Scale bars: 50 µm.</p

MCP-1/CCR2 axis in hypothalamus and peripheral blood, and effects of CCR2 blockade on the infiltration of bone marrow-derived microglia into the PVN and anxiety-like behavior induced by chronic PS.

<p>(<b>A</b>) mRNA expression of chemokines in hypothalamic tissue from chronic PS-loaded and sham-treated mice (<i>n</i> = 4). Data are expressed as mean ± sem. *<i>P</i> < 0.05 with two-tailed Student’s <i>t</i>-test. (B) Immunofluorescence staining with MCP-1 (red) and NeuN (pink) in PVN from chronic PS-loaded and sham-treated mice. Arrows indicate MCP-1⁺NeuN⁺ cells and arrow heads indicate MCP-1^-NeuN⁺ cells. Scale bars: 20 µm. Data are expressed as mean ± sem (n = 7). *<i>P</i> < 0.05 with two-tailed Student’s <i>t</i>-test. (C) Immunofluorescence staining with MCP-1 (red) and GFAP (pink) in PVN from chronic PS-loaded and sham-treated mice. Scale bars: 20µm. Data are expressed as mean ± sem (n = 4−6). *<i>P</i> < 0.05 with two-tailed Student’s t-test. (D) Frequency of GFP⁺CCR2⁺ cells in the peripheral blood obtained by FACS (<i>n</i> = 4−6). Data are expressed as mean ± sem. *<i>P</i> < 0.05 with two-tailed Student’s <i>t</i>-test. (E) The number of CCR2⁺ cells in the hypothalamus obtained by FACS (<i>n</i> = 4). Total events in FACS were 20000. Data are expressed as mean ± sem. *<i>P</i> < 0.05 with ANOVA followed by Tukey’s multiple comparison. (F) Effects of a CCR2 antagonist, RS102895, on infiltration of bone marrow-derived microglia in PVN. PVN is shown by dotted line. Scale bars: 50 µm. Number of GFP-positive cells in a section including PVN is shown. (G) Effect of RS102895 on the anxiety-like behavior induced by chronic PS. Data are expressed as mean ± sem. *<i>P</i> < 0.05 with ANOVA followed by Tukey’s multiple comparison (<i>n</i> = 4). </p

The hypothetical model of H₂ action in glucose excursion.

<p>H₂ promotes glucose uptake into skeletal muscle by stimulating Glut4 translocation by activating phosphatidylinositol-3-OH kinase (PI3K), atypical protein kinase C (aPKC), and AMP-activated protein kinase (AMPK) under conditions of severe insulin deficiency. H₂ has little effect on glucose excursion under conditions of hyperinsulinemia and insulin resistance.</p

Laboratory investigations in the chronic p.o. administration of H₂ experiment with STZ-induced T1DM mice.

<p>Data are expressed as mean ± standard error (SE). Comparisons with control group were performed by Dunnett’s multiple comparison test. As described in Materials and Methods, we lost several mice accidentally and therefore combined two groups [LHW group (n = 2) and HHW group (n = 2)] data together. STZ = streptozotocin; T1DM = type 1 diabetes mellitus; LHW = low content hydrogen water; HHW = high content hydrogen water; NHW = natural hydrogen water; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NEFA = free fatty acids; BUN = blood urea nitrogen; AST = aspartate aminotransferase; ALT = alanine aminotransferase; γ-GTP = γ-glutamyl transpeptidase.</p>*<p>P<0.05,</p>**<p>P<0.01.</p

Laboratory investigations in the chronic p.o. administration of H₂ experiment with high-fat diet-induced T2DM mice.

<p>Data are expressed as mean ± standard error (SE). Comparisons with control group were performed by Dunnett’s multipule comparison test. T2DM = type 2 diabetes mellitus; HHW = high content hydrogen water; NHW = natural hydrogen water; AUC = area under the curve; IPGTT = intraperitoneal glucose tolerance test; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NEFA = free fatty acids.</p>**<p>P<0.01.</p

The effect of H₂ on glucose uptake into C2C12 cells.

<p>(A) 2-DG uptake into C2C12 cells after 30 or 60 min exposure to high content hydrogen water (HHW) was significantly increased over control (n = 6 for each group). (B) Natural hydrogen water (NHW) significantly increased 2-DG uptake into C2C12 cells over control, while degassed NHW did not increase 2-DG uptake (n = 7 for each group). (C, D, E) After incubation with or without each pharmacological inhibitor for 30 min, the cells were exposed to pure water or SHW for another 30 min. The addition of LY-2940002, a phosphatidylinositol-3-OH kinase (PI3K) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 6 for each group). The addition of chelerythrine, a protein kinase C (PKC) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 6 for each group). The addition of Compound C (6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine), an AMP-activated protein kinase (AMPK) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 10 for each group). (F, G) Western blot analysis was performed as described in the Materials and Methods. HHW increased membrane Glut4 (n = 6 for each group) and phosphorylated AMPK (p-AMPK) (n = 13 for each group) in C2C12 cells. (H, I) There was no significant difference in total AMPK in C2C12 cells (n = 8 for each group) or membrane Glut2 in Hep-G2 cells between groups (n = 6 for each group). Comparisons with controls were performed by unpaired Student’s t test between two groups and Dunnett’s multipule comparison test among more than two groups. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Laboratory investigations in the chronic i.p. administration of H₂ experiment with STZ-induced T1DM mice.

<p>Data are expressed as mean ± standard error (SE). Statistical differences between groups were analyzed by Student’s <i>t</i>-test. STZ = streptozotocin; HHS = high content hydrogen saline; HDL = high-density lipoprotein; BUN = blood urea nitrogen; AST = aspartate aminotransferase; ALT = alanine aminotransferase; γ-GTP = γ-glutamyl transpeptidase.</p>*<p>P<0.05,</p>**<p>P<0.01.</p