Device

ait® (AT-04) is a minimally invasive device developed by Peace of Mind Co., Ltd. (Kumamoto, Japan) that consists of a controller and dual-coil emitter assembly (Fig. 1). The dual emitter simultaneously generates alternating magnetic fields at 2 kHz and 83.3 MHz with field strengths of 20–30 µT and 400–700 nT, respectively. A magnetic field has both a magnitude and a direction; an alternating (oscillating) magnetic field exhibits a change in the magnitude and polarity of the field without a change in the direction. The overall energy approximates one-third of terrestrial magnetism. The controller has a timer function designed to discontinue power 30 min after the device is turned on. The sham device has an identical resin case and controller unit but does not generate any alternating magnetic fields.

Fig. 1

ait® (AT-04), a minimally invasive device developed by Peace of Mind Co., Ltd., that consists of a controller and dual-coil emitter assembly, irradiating a mixed alternating magnetic fields

Animals

All experiments involving animals were carried out following the ‘Guidelines for the Proper Conduct of Animal Experiments’ established by the Science Council of Japan, at the animal testing facility of Arcrize Japan Co., Ltd. (21,005, 22,006, 22,007, 22,002, Fukuoka, Japan). Male Sprague–Dawley rats weighing 200–230 g were purchased from Nippon Clare Co. (Tokyo, Japan). Rats were kept at room temperature of 23 ± 2 °C under a 12-h light/dark cycle and had free access to food and water. Animal rearing was carried out in 4 animals/cage (W 263 × D 426 × H 202 mm). One animal/cage (W 263 × D 426 × H 202 mm) was also kept during the study.

Pain thresholds were measured prior surgery, and animals that did not respond to the von Frey test (pain threshold; > 60 g) and those that were hyper-responsive (pain threshold; < 6 g) were excluded.

PSL Model Preparation

The PSL model was established according to previously published methods (Seltzer et al. 1990). Rats were anesthetized with isoflurane by the Univentor 400 anesthesia unit (Univentor, Zejtun, Malta). Intraoperative rat body temperature was measured by a rectal thermometer and maintained at + 37 °C using a BWT-100 Temperature controller (Bio Research Center, Aichi, Japan). The hair of the surgical site in the middle of the rat right thigh was shaved and a 2–3-cm incision was made to expose the sciatic nerve. The sciatic nerve was separated from the surrounding connective tissue and part of the sciatic nerve (approximately 30%) was ligated with 8–0 nylon thread. After sciatic nerve ligation, the incision was sutured. Sham surgery (sham procedure) was performed until the sciatic nerve was exposed and detached from the connective tissue; the sciatic nerve was not ligated and the incision was sutured. After surgery, the rats were individually returned to their cages and left to recover for at least five days until the experiment.

Grouping

Three days after surgery, pain thresholds were measured using the von Frey test, and the animals with pain thresholds of 5 g or below were selected, and they were assigned to groups to ensure equal distribution of mean and standard error values. However, randomization for random assignment was not conducted in this study.

The sample size for this study was determined through power analysis. A priori power analysis was conducted using an effect size estimate derived from our previous studies, the desired level of statistical power (80%), and the significance level (0.05). The power analysis was performed using G*Power 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Germany). However, the results from this preliminary analysis were found to be beyond the physically achievable range. Therefore, in order to establish a realistic sample size, we utilized the maximum feasible upper limit within practical constraints.

Magnetic Fields Irradiation with AT-04

After undergoing PSL model surgery, the rats were followed by a recovery period of 5 days. Prior to the first irradiation, their hair was shaved from the irradiated area near the buttocks and thighs. The rats were then placed in a restraint bag (see Fig. 2) and subjected to magnetic fields irradiation twice a day for 30 min per session, with a 6-h interval between the first and second irradiations. This treatment was repeated for several days.

Fig. 2

Rat restraint method. The rats were restrained in a restraint bag, and the AT-04 or sham pads were placed near the buttocks and thighs for 30 min of magnetic fields irradiation

In the evaluation of the analgesic effect of AT-04, AT-04 irradiation was initiated after the postoperative recovery period following surgery, and irradiation with AT-04 for 30 min twice daily was performed for a week (Table 1).

Table 1 The protocol for the study

In the evaluation of the antagonistic effects of serotonin and noradrenaline receptor antagonists on the analgesic effect of AT-04, AT-04 irradiation was also initiated after the postoperative recovery period following surgery and irradiation with AT-04 for 30 min twice daily was performed for a week (Table 3).

In the evaluation of the antagonistic effect of the opioid receptor antagonist on the analgesic effect of AT-04, AT-04 irradiation was also initiated after the postoperative recovery period following surgery and irradiation with AT-04 for 30 min twice daily was performed for 3 days (Table 8).

In the microdialysis of serotonin and norepinephrine in spinal cords, AT-04 irradiation was also initiated after the postoperative recovery period following surgery. In the single irradiation group, AT-04 was irradiated once during microdialysis (Table 5). On the other hand, in the repeat irradiation group, AT-04 was also irradiated during microdialysis after 5 days of AT-04 irradiation, following the same protocol as in the single irradiation group (Table 6).

Drugs Administered

Serotonin receptor antagonist WAY100635 (sc-391296, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), alpha-2 receptor antagonist Yohimbine (Y3125-1G, Sigma-Aldrich Co. LLC, St. Louis, MO, USA), and μ-opioid receptor antagonist Naloxone (144–09411, FUJIFILM Wako Chemicals, Osaka, Japan) were used in this study, with their doses determined based on references (Di Cesare Mannelli et al. 2017; Sakakiyama et al. 2014; Shiiba et al. 2012; Liu et al. 2014). All drugs were dissolved in 0.9% saline and administered intraperitoneally at a dose of 1 mL/kg, 10 min prior to each irradiation with the AT-04 or Sham machine. The drugs or saline were administered twice a day for each irradiation. A control group received the same volume of saline.

Quantification of Pain Thresholds by Von Frey Test

To quantify pain threshold, we utilized the von Frey test by assessing the escape behavior of the rats in response to stimuli. Prior to the test, the rats were acclimated to the experimental platform with a mesh-like floor surface for approximately 30 min. The von Frey filaments of different diameters (Aesthesio, DanMic Global, LLC, San Jose, CA, USA) were sequentially stimulated to the plantar region of the right hind paw. Each filament was pressed against the paw’s plantar surface for approximately 6 s until it curved. We observed whether escape behavior occurred and recorded the smallest filament stimulus that elicited such a response. This procedure was repeated five times, and the average value was determined as the paw withdrawal threshold (PWT) (Kim et al. 1997; Field et al. 1999; Dowdall et al. 2005).

All behavioral tests were conducted by researchers who maintained the confidentiality of the treatment group. The pain evaluation experiments were carried out at the animal testing facilities of Arcrise-Japan Ltd., in accordance with the Guidelines for the Appropriate Conduct of Animal Experiments set forth by the Science Council of Japan. The pain assessments were performed in a blinded manner, ensuring that the experimenters were unaware of the group assignments of the animals being evaluated.

Spinal Microdialysis

The microdialysis experiment was performed using anesthetized rats (Nakajima et al. 2012; Ito et al. 2018). Isoflurane anesthesia was performed using the anesthesia unit (Univentor 400, Univentor, Zejtun, Malta). The isoflurane concentration at the time of induction was set to 3–4%, and the isoflurane concentration during the maintenance of anesthesia was set to 1–2%. The body temperature of the rats was measured by a rectal thermometer and maintained at + 37 °C using the temperature controller (BWT-100, Bio Research Center, Aichi, Japan). After shaving the surgical site and exposing the L4 to L5 vertebral arch by cutting, the rats were fixed in a positioning device (SR-7R-HT, Narishige, Tokyo, Japan).

After opening the dura mater with a 30-gauge needle, a microdialysis probe (outer diameter 0.315 mm, membrane length 1 mm, cut-off value 15 kDa, MDP, Arcrize Japan, Japan) was inserted into the right-side root of the rat spinal cord at a 25-degree angle with a micromanipulator (SMM-100, Narishige). The inlet and outlet tubes of the probe were connected to a microinjection pump (55–4143, Harvest Apparatus, Holliston, MA, USA) equipped with a 2.5-mL syringe and an fraction collector (ARJ-MPC11, Arcrize Japan), respectively. The probe was perfused with artificial cerebrospinal fluid (148-mM NaCl, 4-mM KCl, 0.8-mM MgCl2, 1.4-mM CaCl2, 1.2-mM Na2HPO4, 0.3-mM NaH2PO4, pH 7.2) at a flow rate of 0.5 µL/min.

The perfusion fluid was infused 4 h prior to the beginning of irradiation. After 3 h of pre-perfusion, samples were taken every 30 min (15 μL each) using an ARJ-MPC11 fraction collector, starting 1 h before the start of irradiation. The first two samples, including the time 0 sample, were used to determine the baseline levels of 5-HT and NA. The test equipment (G1 and G3 used the Sham machine, G2 and G4 used the AT-04) was then irradiated for 30 min and one sample was taken (15 μL each). Six additional samples were collected every 30 min until 3 h after the end of irradiation (15 μL each). To prevent sample degradation, 0.1-M phosphate buffer (pH 3.0) was added to the recovered samples in a 2:1 sample to acid ratio.

HPLC Analysis

The concentration of 5-HT and NA was measured using an HPLC-electrochemical detector system (ECD-700, Eicom, Kyoto, Japan) (Ito et al. 2018; Yoshitake et al. 2014). The HPLC system consisted of a pulsed-free low flow rate pump, a degasser, a column oven, and an anodic detector equipped with a graphite electrode subjected to + 0.45 V relative to a silver/silver chloride reference electrode. The samples were injected using an autosampler (AS-4150, Jasco Corp., Tokyo, Japan), with an injection volume of 15 μL. Chromatograms were recorded and processed using data processing software (Clarity, DataApex, Prague, Czech Republic). The separation of 5-HT and NA was performed using a cation-exchange column (CAX, 200 × 2.0 I.D. mm CAX column, Eicom), and the mobile phase consisted of a mixture of 0.1-M phosphate buffer (pH 6.0) and methanol (7:3, v/v) with the addition of 30-mM potassium chloride and 50-mg/L EDTA-2Na. The detection limit (S/N ratio = 3) of 5-HT and NA was 0.5 fmol per 10 μL of sample.

Statistical Analysis

The raw data for pain threshold was entered into a Microsoft Excel data file. All data were verified prior to analysis, and the mean ± standard deviation (SD) along with the 95% confidence interval (CI) were calculated for each measurement of the pain threshold.

Normality and variance homogeneity was performed ANOVA as well as additional tests, including the Brown–Forsythe test and Bartlett’s test. The comparison of pain thresholds before and after sciatic nerve ligation was performed using t tests. The comparison of pain thresholds between different time points and the assessment of group differences for each day were conducted using two-way ANOVA. In the microdialysis test, the comparison of 5-HT and NA at each time within each group was also analyzed using two-way ANOVA. Statistical significance was considered when P < 0.05.

The individual data point and analyzed data for each experiment have been documented in each Supplemental Information.