Effects of suvorexant on the amount of sleep
Mice underwent an electroencephalogram/electromyogram (EEG/EMG) recording head-mount implantation and partial sciatic nerve ligation (PSNL) surgery under general anesthesia with isoflurane. One week after surgery, we repeated oral administration of vehicle (0.5% methyl cellulose) or suvorexant (30 mg/kg) 15 min before Zeitgeber time (ZT) 0 for 7 days (Figure 1a). We calculated the time change every 2 h of the percentage of sleep and the amount of non-REM and REM sleep (Figure 1b), and compared the values that were added up in the light and dark phases (Figure 1c). The PSNL-vehicle group showed a significant decrease in the percentage of sleep (PSNL-vehicle vs. sham-vehicle: 51.8% vs. 72.1%; P = 0.0051) during the light phase, which is the rest period for mice. Both non-REM (PSNL-vehicle vs. sham-vehicle: 5.92 h vs. 7.93 h; P = 0.040) and REM sleep (PSNL-vehicle vs. sham-vehicle: 0.29 h vs. 0.72 h, P = 0.0015) decreased in the PSNL-vehicle group during the light phase. Daily suvorexant administration significantly normalized the percentage of sleep (76.3%, P = 0.0007; vs. PSNL-vehicle), non-REM sleep (8.46 h, P = 0.0070; vs. PSNL-vehicle), and REM sleep (0.69 h, P = 0.0028; vs. PSNL-vehicle) in nerve-ligated mice during the light phase.
Effects of mirtazapine on the amount of sleep
We repeated intraperitoneal injection of the vehicle (5% dimethyl sulfoxide) or mirtazapine (1 mg/kg) 1 week after head-mount implantation and nerve ligation surgery on the same schedule as that of suvorexant (Figure 1a). We calculated the time change every 2 h of the percentage of sleep and the amount of non-REM and REM sleep (Figure 1d), and compared the values that were added up in the light and dark phases (Figure 1e). The PSNL group showed a significant decrease in the percentage of sleep (47.0% vs. 72.7%, P = 0.0079), and the amount of non-REM (5.12 h vs. 8.25 h, P = 0.0017) and REM sleep (0.20 h vs. 0.70 h, P = 0.039) compared to that in the sham group during the light phase, similarly to the previous suvorexant experiment. Daily mirtazapine injection also significantly normalized the percentage of sleep (74.1%, P = 0.0050; vs. PSNL-vehicle), non-REM sleep (7.71 h, P = 0.0094; vs. PSNL-vehicle), and REM sleep (1.17 h, P < 0.0001; vs. PSNL-vehicle) in nerve-ligated mice during the light phase.
Effects of suvorexant and mirtazapine on the duration of arousal and sleep
We analyzed the EEG data to calculate the duration of arousal, non-REM sleep, and REM sleep every 6 h. The sham group showed a long duration of arousal during the dark phase, which is an active period for mice. A significant reduction in the duration of wakefulness was detected in nerve-ligated mice (PSNL-vehicle vs. sham-vehicle: 4.94 m vs. 24.1 m, P < 0.0001 at ZT 12–18; 3.53 m vs. 15.9 m, P = 0.0053 at ZT 18–24; Figure 2a, left; and PSNL-vehicle vs. sham-vehicle: 4.83 m vs. 29.0 m, P = 0.0098 at ZT 12–18; 4.27 m vs. 27.8 m, P = 0.012 at ZT 18–24; Figure 2b, left), which was improved with suvorexant (15.8 m, P = 0.012 at ZT 12–18 and 15.2 m, P = 0.0063 at ZT 18–24; vs. PSNL-vehicle; Figure 2a, left) and mirtazapine (33.4 m, P = 0.0030 at ZT 12–18 and 30.6 m, P = 0.0067 at ZT 18–24; vs. PSNL-vehicle; Figure 2b, left). Analysis of the duration of non-REM sleep revealed fragmented sleep in the PSNL group at almost all periods, which was improved by suvorexant (PNSL-vehicle vs. PSNL-suvorexant: 2.63 m vs. 8.75 m, P = 0.0001 at ZT 0–6; 3.05 m vs. 9.36 m, P < 0.0001 at ZT 6–12; 2.96 m vs. 7.17 m, P = 0.0088 at ZT 12–18; 2.96 m vs. 6.88 m, P = 0.0162 at ZT 18–24; Figure 2a, middle) and mirtazapine (PNSL-vehicle vs. PSNL-mirtazapine: 2.28 m vs. 6.06 m, P < 0.0001 at ZT 0–6; 2.35 m vs. 5.19 m, P = 0.0021 at ZT 6–12; 1.94 m vs. 5.11 m, P = 0.0005 at ZT 12–18; 2.04 m vs. 4.92 m, P = 0.0018 at ZT 18–24; Figure 2b, middle). There was a downward trend or a significant decrease in the duration of REM sleep with nerve ligation only during the light phase. Suvorexant (PNSL-vehicle vs. PSNL-suvorexant: 0.60 m vs. 1.24 m, P = 0.022 at ZT 0–6; 0.65 m vs. 1.23 m, P = 0.042 at ZT 6–12; Figure 2a, right) and mirtazapine (PNSL-vehicle vs. PSNL-mirtazapine: 0.52 m vs. 1.53 m, P < 0.0001 at ZT 0–6; 0.54 m vs. 1.10 m, P = 0.023 at ZT 6–12; Figure 2b, right) significantly increased REM sleep duration in the PSNL group during the light phase.
Effects of suvorexant and mirtazapine on the normalized power density of delta (δ) waves
We analyzed the power density of δ waves, which is one of the factors that define the depth of non-REM sleep. The power density of the δ wave during non-REM sleep was calculated every 3 h, and the ratio with the average value during the entire day was calculated for each individual. In the sham group, the power density of the δ wave in non-REM sleep was particularly strong during the early light phase. The δ power tended to weaken from the late light phase to the early dark phase and became stronger again toward the latter half of the dark phase. However, in the PSNL-vehicle group, diurnal fluctuation of delta power became ambiguous; in particular, the power density of the δ wave in the early light phase was significantly reduced (PSNL-vehicle vs. sham-vehicle: 0.99 vs. 1.13, P = 0.020 at ZT 0; Figure 3a; and PSNL-vehicle vs. sham-vehicle: 0.98 vs. 1.09, P = 0.0042 at ZT 0; Figure 3b). Both suvorexant (1.15, P = 0.0074; vs. PSNL-vehicle; Figure 3a) and mirtazapine (1.12, P = 0.0004; vs. PSNL-vehicle; Figure 3b) improved the reduction in δ power density.
Effects of suvorexant and mirtazapine on the voluntary activity
We placed the mice in cages with a running wheel and recorded the number of wheel rotations. PSNL surgery significantly reduced the wheel rotation compared to sham surgery from day 7 to 14 after surgery (sham vs. PSNL: 18,650/day vs. 11,801/day, P = 0.0040 at day 7; 19,010/day vs. 11,500/day, P = 0.0015 at day 17; Figure 4a). The drug was administered daily for 1 week after PSNL surgery, and the wheel rotation was evaluated at certain test points, as shown in Figure 4b. Daily oral suvorexant administration reduced the number of wheel rotations (8,463/day, P = 0.0073 at test point D; 9,759/day, P = 0.045 at test point E; vs. test point A, Figure 4c), and the reduction was sustained for 1 week after the end of administration compared to that at the baseline (9,799/day, P = 0.048 at test point F; vs. test point A, Figure 4c). However, daily intraperitoneal injection of mirtazapine did not influence the number of wheel rotations (Figure 4d).
Effects of suvorexant and mirtazapine on pain behavior
We administered vehicle or drug daily for 1 week from postoperative day 7 and evaluated mechanical allodynia and thermal hyperalgesia using the von Frey test and Hargreaves test, respectively, at certain test points shown in Figure 5a. Weekly suvorexant administration improved the paw withdrawal latency for mechanical stimuli with von Frey filament (PSNL-vehicle vs. PSNL-suvorexant: 4.98 s vs. 10.1 s, P < 0.0001 at test point C; Figure 5b); however, the effect did not last until the next day. There was no significant difference in the response to thermal stimuli (Figure 5c). However, weekly injection of mirtazapine improved both mechanical allodynia (PSNL-vehicle vs. PSNL-mirtazapine: 4.20 s vs. 9.74 s, P < 0.0001 at test point C; Figure 5d) and thermal hyperalgesia (PSNL-vehicle vs. PSNL-mirtazapine: 4.87 s vs. 9.70 s, P < 0.0001 at test point C; Figure 5e), and these effects persisted until the next day (PSNL-vehicle vs. PSNL-mirtazapine: 5.06 s vs. 7.23 s, P = 0.014 [Figure 5d]; 5.80 s vs. 7.33 s, P = 0.0440 [Figure 5e] at test point D).