Figure 1.
Hydrogen treatment suppressed mechanical allodynia in the PSNL model.
Neuropathic mechanical allodynia was analysed by the von Frey test. Hydrogen was supplied for the whole experimental period right after PSNL or sham operation. The control group received distilled water from day 0 to 21. (A) The hydrogen-treated mice exhibited higher threshold on the ipsilateral side to ligation than controls (n = 12 mice for each group). (B) Sham operation did not caused mechanical allodynia regardless of hydrogen treatment (n = 12 mice for each group). We used a two-way RM ANOVA with a Bonferroni post-hoc test; *P<0.05, **P<0.01, ***P<0.001 vs. contralateral side of control mice, #P<0.05 vs. ipsilateral side of control mice.
Figure 2.
Hydrogen treatment suppressed thermal hyperalgesia in the PSNL model.
Thermal hyperalgesia was analysed by the plantar test. The same sets of mice used for the von Frey test were used in the plantar test. (A) The reduction of paw withdrawal latency was attenuated in mice with hydrogen compared with those without hydrogen (n = 12 mice for each group). (B) Sham operation did not cause hyperalgesia regardless of hydrogen treatment (n = 12 mice for each group). We used a two-way RM ANOVA with a Bonferroni post-hoc test; *P<0.05 vs. contralateral side of control mice, #P<0.05 vs. ipsilateral side of control mice.
Figure 3.
Hydrogen treatment was effective against the induction phase but not against the maintenance phase of mechanical allodynia.
(A) Hydrogen water was supplied from day 0 to 4, and after then changed to distilled water. Paw withdrawal threshold was significantly lower in the H2 (+) group when compared with the H2 (–) group (n = 12 mice for each group). Note that the lower threshold in the H2 (+) group lasted beyond the termination of hydrogen treatment. (B) Hydrogen water was supplied from day 4 to 21. There was no significant difference in paw withdrawal threshold between the H2 (+) and the H2 (–) groups (n = 12 mice for each group). These experiments were performed concurrently with those in Figure 1. We used a two-way RM ANOVA with a Bonferroni post-hoc test; *P<0.05, **P<0.01, ***P<0.001 vs. contralateral side of same mice. #P<0.05 vs. ipsilateral side of H2 (–) mice.
Figure 4.
Hydrogen treatment was effective against both induction and maintenance phases of thermal hyperalgesia.
(A) When hydrogen water was supplied from day 0 to 4, paw withdrawal latency was longer in the H2 (+) group when compared with the H2 (–) group (n = 12 mice for each group). Note that the lower threshold in the H2 (+) group lasted beyond the termination of hydrogen treatment. (B) When hydrogen water was supplied from day 4 to 21, there was a significant difference in paw withdrawal latency between the H2 (+) and the H2 (–) groups (n = 12 mice for each group). These experiments were performed concurrently with those in Figure 2. We used a two-way RM ANOVA with a Bonferroni post-hoc test; *P<0.05 vs. contralateral side of same mice. #P<0.05 vs. ipsilateral side of H2 (–) mice.
Figure 5.
Hydrogen suppressed the development of oxidative stress in the spinal cord dorsal horn.
(A–F) Representative images of immunohistochemical staining for the oxidative stress markers 4-HNE (A–C) and 8-OHdG (D-F) in the spinal cord dorsal horn at the level of L5. Staining for 4-HNE was increased in the spinal cord at the end of 4 days period after PSNL (A) compared with sham operation (C). (B) The number of 4-HNE positive cells was decreased in mice with hydrogen when compared with the H2 (–) group (A). Similarly, staining for 8-OHdG was also increased in the spinal cord from mice with PSNL (D) compared with sham operation (F). (E) The number of 8-OHdG positive cells was decreased in mice with hydrogen at the end of 4 days period after PSNL when compared with the H2 (–) group (D). (G, H) The numbers of positive cells for both markers were significantly decreased when compared with the H2 (–) group. *P<0.05 compared with control (t test, n = 6 mice for each). Scale bar: 200 µm.
Figure 6.
Hydrogen suppressed the development of oxidative stress in the DRG.
(A–C) Representative images of immunohistochemical staining for oxidative stress markers 4-HNE in the DRG at the level of L5. Weak staining for 4-HNE (arrows) was observed in the DRG from mice without hydrogen at the end of 4 days period after PSNL (A), whereas staining for 4-HNE was not observed in sham operation mice (C). (B) Staining for 4-HNE was reduced in mice with hydrogen at the end of 4 days period after PSNL. (D) The proportion of 4-HNE positive cells was significantly decreased when compared with the H2 (–) group (A). Results are expressed as percent of total neurons. *P<0.05 compared with control (t test, n = 6 mice for each). Scale bar: 20 µm.
Figure 7.
4-HNE were mainly produced on oligodendrocyte in the spinal cord 4 days after PSNL.
(A–E) Double immunostaining of 4-HNE with cellular markers for neurons (A), astrocyte (B), oligodendrocyte (C, D), and microglia (E). 4-HNE signals were mainly colocalized with the oligodendrocyte marker NG2 and olig2, but not with NeuN, GFAP, and Iba1 in the spinal cord at the end of 4 days period after PSNL. Scale bar: 20 µm.