Effects of MNS-PC6 or i.pag. orexin on the mouse hot-plate test and their interactions with OX1R, CB1R, opioid receptor antagonists, and a DAGL inhibitor. Antinociceptive effects of MNS-PC6 (A, B, E, and F) or i.pag. orexin A (C and D) with i.p. (A and B) or i.pag. (E and F) pretreatment or i.pag. coadministration (C and D) with an OX1R antagonist (SB 334867), CB1R antagonist (AM251), DAGL inhibitor (THL), or vehicle. (A, C, and E) Time courses of antinociceptive effects are expressed as the percentage of MPE (%MPE). Arrows indicate drug administration; horizontal bars indicate MNS-PC6. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control (A), vehicle alone (C), or sham (E) group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the MNS/vehicle (A and E) or orexin A/vehicle (C) group (two-way ANOVA with repeated measures over time/Bonferroni’s post hoc test). (B, D, and F) Antinociceptive effects are expressed as the AUC. Comparison groups are the same as in A, C, and E, except *^,#P < 0.05/n, **^,##P < 0.01/n, ***^,###P < 0.001/n (n was 6, 8 and 10 in B, D, and F, respectively; Kruskal–Wallis test/Mann–Whitney U post hoc test with the Bonferroni correction with n independent hypotheses; see also SI Appendix, Table S2). (Inset in F) Microinjection sites taken from all mice with successful injections in the vlPAG depicted in two PAG sections at bregma −4.72 and −4.84 mm, respectively. Injection sites were confirmed by trypan blue injected through the microinjection cannula after the hot-plate test. The numbers in parentheses over the bars in B, D, and F indicate the number of animals used in each group.

MNS-PC6 increased the number of c-Fos–expressing orexin neurons in the LH and PFA as well as orexin A levels in the vlPAG. (A) Merged confocal micrographs of mouse LH sections double-immunofluorescently labeled with anti-orexin A antibody (green) and anti–c-Fos antibody (red) in the control (Left), non-MNS (Center), and MNS-PC6 (Right) groups. Hypothalamic cells labeled with green in the cytoplasm and red in the nucleus area are indicative of orexin A and c-Fos coexpression (arrows). (Scale bar: 50 μm.) (Lower Right) A lower-magnification confocal micrograph of a mouse hypothalamus section taken from the MNS-PC6 control group. The central hole is the third ventricle. (Scale bar: 100 μm.) (B–D) The total number of orexin A-immunoreactive neurons (OX) (B), the percentage of c-Fos–expressing orexin neurons (Fos-OXA) among total orexin neurons (OX) (C), and the number of c-Fos–expressing neurons (c-Fos) (D) in one side of the LH and PFA in the control, non-MNS, and MNS-PC6 groups. Double immunofluorescent staining was conducted in LH and PFA tissue sections harvested 2 h after MNS-PC6 termination. Fos-OXA, OXA, and c-Fos neurons were counted using the Stereo Investigator (MBF Bioscience). ^##P < 0.01; ***P < 0.001 vs. control; ^###P < 0.001 vs. non-MNS (one-way ANOVA/Tukey’s post hoc test). The numbers in parentheses over the bars in B–D indicate the number of mice used in each group.

MNS-PC6 increased orexin A levels and decreased GABA levels in the vlPAG in a manner prevented by systemic SB 334867 and AM251. (A) Orexin A levels in vlPAG homogenates prepared from mice in the MNS-PC6, non-MNS, and control groups. Immediately after MNS-PC6 termination, the vlPAG in each mouse was micropunched bilaterally and homogenized. Orexin A levels in the vlPAG homogenate were measured with EIA. *P < 0.05 vs. control (one-way ANOVA/Tukey’s post hoc test). (B) GABA levels in vlPAG microdialysates of anesthetized mice in the MNS-PC6, non-MNS, and control groups. GABA levels in the vlPAG microdialysate sampled from 75–55, 55–35, and 20–0 min before, during, and every 20 min after MNS-PC6 or non-MNS treatment for 1 h in each mouse were measured by HPLC and expressed as the percentage of the baseline level in each mouse, which was the mean GABA concentration before treatment. Results from control mice receiving only anesthesia were also compared. *P < 0.05, ***P < 0.001 vs. control and ^#P < 0.05, ^###P < 0.001 vs. non-MNS (two-way ANOVA with repeated measures over time/Bonferroni’s post hoc test), n = 6. (C and D) GABA levels in vlPAG microdialysates of mice before and after MNS-PC6 (C) or non-MNS (D) with or without pretreatment with SB 334867 or AM251. The antagonist was given by i.p. injection (arrows) at 35 min before MNS-PC6 or non-MNS (horizontal bars). In C, ***P < 0.001 vs. MNS-PC6 (two-way ANOVA with repeated measures over time/Bonferroni’s post hoc test), n = 6. The numbers in parentheses over the bars in A indicate the number of mice used in each group.

Effects of MNS-PC6 on the hot-plate test in Cnr1^−/− and WT mice. All protocols, statistical analyses, and data presentation, unless stated otherwise, are the same as in Fig. 2. (A) The antinociceptive effects of MNS-PC6 in WT and Cnr1^−/− mice. Time courses of antinociceptive effects are expressed as the paw-withdrawal latency. ***P < 0.001 vs. the sham group (two-way ANOVA with repeat measures over time/Bonferroni’s post hoc test). (B) MNS-PC6-IA as indicated by AUCs in WT and Cnr1^−/− mice. *P < 0.05/3 vs. the sham group; ^&&P < 0.01/3 vs. the MNS/WT group (Kruskal–Wallis test/Mann–Whitney U post hoc test with the Bonferroni correction). The numbers in parentheses over the bars in B indicate the number of mice used in each group.

MNS-PC6 is equivalent to DMNS. (A) Timeline for procedures (drug administration, anesthesia, and blood and brain tissue sampling) before and after DMNS on the surgically exposed median nerve. After 1 h of acclimation, mice were anesthetized under 2% isoflurane. Their median nerves were surgically exposed, and they were given DMNS for 20 min. DMNS was performed via electrical stimulation (2 Hz, 1 mA, 0.15 ms) by placing the stimulating electrode next to the surgically exposed right median nerve. The sham-DMNS group received the same procedure as the DMNS group, but electrical stimulation was omitted. The control group received anesthesia only. In the DMNS+lidocaine group, a drop of lidocaine solution (2%) was applied directly onto the median nerve before commencing DMNS. vlPAG homogenates were prepared immediately after DMNS termination for orexin A measurement. (B) Orexin A levels in vlPAG homogenates prepared from mice in the DMNS, sham-DMNS, DMNS+lidocaine, and control groups. Immediately after DMNS termination, the vlPAG in each mouse was micropunched bilaterally and homogenized. The orexin A level in the vlPAG homogenate was measured with EIA. **P < 0.01, ***P < 0.001 vs. control; ^&&&P < 0.001 vs. sham-DMNS; ^###P < 0.001 vs. DMNS (one-way ANOVA/Tukey’s post hoc test). (C) Timeline for procedures, the median nerve block, and the hot-plate tests before and after MNS-PC6. Briefly, the procedure was as shown in Fig. 1, except that lidocaine solution (2%, 10 µL) was injected 2.0 mm proximal to the PC6 acupoint before commencing MNS-PC6. (D) The effect of median nerve block by lidocaine injection before MNS-PC6 on MNS-PC6-IA. Time courses of antinociceptive effects are expressed as the percentage of MPE (%MPE). The arrow indicates lidocaine injection; the horizontal bar indicates MNS-PC6. ***P < 0.001 vs. MNS-PC6+lidocaine (two-way ANOVA with repeated measures over time/Bonferroni’s post hoc test) (E) Antinociceptive effects, expressed as the AUC in the MNS-PC6 groups without (MNS-PC6) or with median nerve block by lidocaine (MNS-PC6+lidocaine) and in the group treated with lidocaine only. Comparison groups are as in D; ***P < 0.001 vs. MNS-PC6+lidocaine (Dunnett’s multiple comparisons test; also see SI Appendix, Table S2). The numbers in parentheses over the bars in B and E indicate the number of mice used in each group.

The analgesic effect of MNS-PC6 was not reversed by pretreatment with naloxone or naltrexone. All protocols, statistical analyses, and data presentation, unless stated otherwise, are the same as in Fig. 2. Antinociceptive effects in the MNS-PC6, non-MNS, and vehicle groups of mice without or with i.p. pretreatment with naloxone (1 mg/kg) or naltrexone (1 mg/kg) are shown as percent MPE (A) (two-way ANOVA with repeat measures over time/Bonferroni’s post hoc test) and as AUC (B) (Kruskal–Wallis test/Mann–Whitney U post hoc test with the Bonferroni correction with n independent hypotheses; also see SI Appendix, Table S2). The numbers in parentheses over the bars in B indicate the number of mice used in each group.

MNS-PC6 attenuated CCI-induced mechanical allodynia via OX1Rs and CB1Rs but not via opioid receptors. (A) Mice received CCI surgery on day 0 and developed neuropathy, evidenced by mechanical allodynia of the injured hind paw, by day 7. MNS-PC6, sham-PC6, and non-MNS carried out as in Fig. 1 were applied to CCI mice on day 8. Mechanical sensitivity of the hind paw to a von Frey filament (0.16 g) was measured. (B) Mechanical allodynia, measured by the percentage of positive nociceptive responses (withdrawal, flinching, or licking) during 10 trials of von Frey filament stimulation, developed in CCI mice. Note that mechanical allodynia developed gradually and peaked 7 d after CCI. On day 8, MNS-PC6 significantly reduced mechanical allodynia in CCI mice compared with the control group (CCI mice receiving anesthesia only). (C) Allodynic responses in mice not receiving CCI (non-CCI) or in CCI-mice receiving anesthesia only (control), sham-PC6 (sham), non-MNS, and MNS-PC6 procedures, as well as in the MNS-PC6 group i.p. pretreated with an OX1R antagonist (SB 334867, 15 mg/kg, i.p.), a CB1R antagonist (AM251, 1.1 mg/kg, i.p.), an opioid receptor antagonist (naloxone, 1 mg/kg, i.p.) or vehicle. Note that CCI-induced mechanical allodynia was significantly reduced by MNS-PC6 but not by non-MNS. The MNS-PC6–induced antiallodynic effect was prevented by SB 334867 or AM251 but not by naloxone. **P < 0.01, ***P < 0.001 vs. control; ^##P < 0.01, ^###P < 0.001 vs. MNS-PC6/vehicle (one-way ANOVA with Tukey’s post hoc test).