Animals
The study is reported in accordance with ARRIVE guidelines. All animal experiments were approved by the Niigata University Ethics Committee for Animal Experiments (SA01010) and conducted in accordance with the guidelines of the Science Council of Japan.
C57BL/6J mature male mice (7–8 weeks old, 24–27 g/body) were obtained from Asazuma Animal Medical Instruments (Niigata City, Japan). The mice were housed in groups of 1–5 mice per cage under an environment with a stable temperature of 25°C, a 12-h light/dark cycle, and free access to water and food.
Drugs and administration
Yokukansan (Yokukansan extract granules; YKS, TJ-54, Lot No. 2110054010, Tsumura & Co. Tokyo, Japan), which is manufactured and provided by Tsumura & Co. and approved for ethical use by the Ministry of Health, Labor, and Welfare of Japan, is a dried extract of the following botanical raw materials: Atractylodes lancea rhizome, Poria sclerotium, Cnidium rhizome, Uncaria hook, Japanese Angelica root, Bupleurum root, Glycyrrhiza (Table 1). The seven medical herbs were extracted with purified water at 95˚C for 1 h, and the extract solution was separated from the insoluble waste and concentrated by removing water under reduced pressure. Spray drying was used to produce a dried extract powder. Seven and a half grams of YKS contains 3.25 g of a dried extract mentioned above and inactive ingredients (lactose hydrate and magnesium stearate). For YKS treatment, a dry powder extract of YKS was suspended in 0.25 ml distilled water. YKS was administered by oral gavage using a sonde (KN-348, Natsume Seisakusho, Tokyo, Japan) at doses of 0.25, 0.5, and 1.0 g/kg, based on a previous study [14]. The same volume of distilled water (0.25 ml) was administered orally as a control.
Para-Chlorophenylalanine (PCPA, R&D Systems, Inc. Minneapolis, USA) depletes serotonin (5-hydroxytryptamine, 5-HT) by inhibiting tryptophan hydroxylase, a serotonin synthesis enzyme [15]. To deplete serotonin, the mice were treated with PCPA (150 mg/kg/day, diluted with distilled water) administered subcutaneously for 7 days under anesthesia (the same anesthetic method used to create the postoperative pain model) via an osmotic pump (Cat# 1007D, Alzet Osmotic Pumps, Cupatino, CA, USA) surgically implanted subcutaneously in the back of the mice. The control group received the same volume of distilled water as the PCPA-administered group. Mice receiving PCPA and distilled water were each marked with an individual-colored marker and kept in mixed conditions.
Surgical protocol
A modified version of Brennan’s procedure, established in a previous study [13], was used to create a mouse model of postoperative pain. All mice were anesthetized with 3% isoflurane administered through a nose cone, and the left plantar region was disinfected with 10% povidone–iodine solution. A 5-mm incision was made in the left hind paw, starting 2 mm from the proximal edge of the heel. The underlying flexor muscle was elevated and transversely incised. The surgical wound was closed by suturing the skin at the incision site at two places using a 7 − 0 nylon thread. Sham mice were only disinfected under the same anesthesia as the model mice, without incision.
Study groups
The following groups were used to demonstrate the effects of YKS on postoperative pain: 1) Group B (Brennan’s plantar incision), which received a plantar incision and were the postoperative pain model mice; 2) Group BY, which received YKS after plantar incision. To confirm that the effects of YKS were mediated by the serotonergic nervous system, we prepared the following groups: 3) the PB group, which received para-chlorophenylalanine (PCPA), to deplete serotonin, and plantar incision; 4) the PY group, which received PCPA and YKS; and 5) the PBY group, which received PCPA, plantar incision, and YKS.
The YKS dose was determined based on a previous study. The dose of YKS was determined according to the estimated ED50, based on the dose–response relationship of the analgesic effect of YKS on thermal stimulation in normal postoperative pain model mice (group B mice).
Behavioral experiments
Behavioral experiments were performed by assessors blinded to the drug administration in the soundproof room. To avoid affecting circadian rhythms, all behavioral experiments were conducted at 9:00 AM, and the animals were allowed to acclimatize for at least 30 min. The von Frey (mechanical stimulation test) [16] and Hargreaves (thermal stimulation test) [17] tests were used to evaluate the paw-withdrawal threshold. For the mechanical stimulation test, the mice were placed in a red transparent plastic chamber on a wire grid and the paw-withdrawal threshold of the mechanical stimuli was measured. The Semmes–Weinstein set of monofilaments (von Frey filaments) was used to stimulate the central part of the left plantar region. The paw-withdrawal threshold was defined as the value of the smallest filament that elicited withdrawal reflexes in two of 10 stimulations.
For the heat stimulation test, the mice were placed in a red transparent plastic chamber on a wire grid and allowed to acclimate for at least 30 min. Paw-withdrawal latency induced by heat stimulation was measured. The left hind paw was irradiated using radiant heat (Hargreaves apparatus model 7370, Ugo Basile, Comerio, Italy). The average of three measurements taken at 3–5-min intervals was used as the latency to escape pain. Both heat and mechanical stimulations were performed immediately before YKS/distilled water administration and 30, 60, 90, 120, 180, 240 and 480 min after administration. The heat stimulation test for PCPA-treated mice was started before PCPA was administered via the osmotic pump (for control), immediately before plantar incision on the day before the heat stimulation test, and continued for an 8-hour period as well.
Immunostaining
The mice were euthanized by isoflurane anesthesia inhalation overdose. Immediately, 25 mL of saline followed by an equal amount of 4% paraformaldehyde (Mildform® 10N, Fujifilm Wako Pure Chemical Company, Osaka, Japan) were injected via transcardial perfusion. Each lumbar enlargement of spinal cord was resected and post-fixed at 4°C overnight in the 4% paraformaldehyde and subsequently equilibrated in 20% sucrose in 0.1 M phosphate-buffered saline (PBS) overnight at 4°C for cryoprotection. Spinal cord tissue was embedded in FSC22 (frozen-section medium; Leica Biosystems, Wetzlar, Germany) and frozen at -70°C until sectioning. Spinal cord sections (10 µm) were prepared on a frozen microtome (CM1520, Leica Biosystems) at -20°C, mounted on glass slides (Matsunami GlassInd. Ltd., Osaka, Japan) and stored at -70°C.
The sections were washed twice with TNT buffer (10% 1 M Tris-HCl, pH 7.5, 5% 3 M NaCl, 0.03% Tween 20) and incubated with Blocking One Histo (Nacalai tesque, Kyoto, Japan) at room temperature (23–25°C) for 1 h. After removing the blocking buffer, the sections were incubated in a humidified chamber at 4°C for 2 days with rabbit anti-5-HT (Serotonin) IgG (1:20,000; Cat. #; 20080, Immunostar, Wisconsin, USA), which were diluted with 0.1% Tween 20 in TNB buffer (0.1 M Tris-HCl buffered saline, pH 7.5, containing 1% blocking reagent. Sections were then rinsed twice with TNT buffer and incubated overnight in a humidified chamber at 4°C with Cy3-conjugated goat anti-rabbit IgG (1:1000; Cat. #; 111-167-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for anti-5-HT IgG. The sections were washed twice with TNT buffer, embedded using VECTASHIELD Antifade Mounting Medium (Vector Laboratories Inc., Newark, CA, USA), and visualized using a fluorescence microscope (BX 53, Olympus, Tokyo, Japan) equipped with a digital camera system (DP73, Olympus, Tokyo, Japan).
Statistical analysis
The sample size was determined based on previous behavioral and immunohistological studies. We did not calculate statistical power a priori. A post-hoc power analysis was conducted using G* Power version 3.1.7 [18], which indicated that a sample of five to six mice/group for behavioral experiments was sufficient to reach statistical significance with power (1 - β) set at 0.8 and α = 0.05. Data are presented as mean ± standard error of the mean.
The dose–response relationship of YKS to mechanical and heat stimuli was fitted using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA) according to the following equation:
where Y is the relative analgesic effect normalized to the maximal analgesic effect, D is the dose of YKS (mg/kg), n is the Hill coefficient, and EC50 is the half-maximal effective concentration for analgesic effect of YKS.
We used repeated-measures (RM) two-way analysis of variance (ANOVA) for behavioral data after tests of homoscedasticity and normality of the datasets using the Shapiro–Wilk test. Repeated-measures analyses were performed without sphericity assumption, and the Greenhouse– Geisser correction was applied. If the RM two-way ANOVA revealed statistical significance, multiple comparisons using Dunnett's test with reference to B or PB groups were performed. All statistical analyses were performed using GraphPad Prism9 1software. Statistical significance was set at P < 0.05.