Animals
Adult male Sprague–Dawley rats (Hyochang, Seoul, Korea) weighing 250–340 g were used. Animals were housed at constant humidity (40–60%) and temperature (22 ± 2 °C) with 12 h light/dark cycle and allowed free access to food and water. All experiments were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee (IACUC) at the Daegu Haany University.
Chemicals
Evans blue dye (EBD; 50 mg/ml saline; Sigma-Aldrich, MO, USA); rabbit anti-c-Fos primary antibody (sc-52, Santa cruz, CA, USA); donkey anti-rabbit Alexa Fluor 594 (A21207, Life Technologies, CA, USA); naloxone (4 mg/ml saline, Sigma-Aldrich; a non-specific opioid receptor antagonist) were used in this study.
Detection of neurogenic spots in the skin by EBD injection
Cutaneous Neuro-Sps were visualized by injecting Evans blue dye (EBD; 50 mg/kg, 50 mg/ml saline) as described previously [9]. While the rats were immobilized by the cone-shaped bags, the distal portion of the tail was dipped into 40 °C warm water for at least 30 s. EBD was then injected into the tail vein with a catheter (26 gauge), and skin color changes were observed up to 2 h after the injection. The blue-dyed spots on the skin were photographed and compared with an acupoint chart based on the transpositional method, which locates acupoints on the surface of animal skin corresponding to the anatomic site of human acupoints [16].
Development of a novel mechano-electrical acupuncture instrument (MEA)
An MEA device was developed to stimulate acupuncture needles electrically and/or mechanically. This device consisted of a program control unit and two stimulation units (Fig. 1a). In the control unit, 2 pairs of mechanical and electrical drive circuits were mounted on printed circuits boards (PCB; Fig. 1b), packaged with a 3-dimensional (3D) printed plastic cage and controlled by our custom-made program. In the stimulation unit, a vibrator (approximately 80 rotations/sec; MB-0412 V, Motor bank, Korea) was combined with electrodes and a rubber grommet was fixed to the needle at a distance of 3 mm from the tip for controlling the depth of needle insertion (0.10 mm in diameter, 10 mm in length of needle and 10 mm in length of handle; Dongbang Medical Co., Korea) (Fig. 1c).
For acupuncture treatment, the rat was restrained in a cone-shaped plastic bag and acupuncture needles were inserted into Neuro-Sps and stimulated with MA, EA or combined MA + EA. For EA stimulation, electrical stimulation (2 Hz, 0.5 mA, 0.1 ms, triangular pulses) was applied to the needles for 10 min. For MA treatment, the needles were vibrated for 6 s every 30 s for a total of 10 min. For combined MA + EA treatment, intermittent MA (6 s every 30 s) was given during continuous stimulation of EA for 10 min. Control group (Con) were lightly restrained in the same manner as the acupuncture treatment, but without needle insertion. Non-neurogenic spots group received combined MA + EA at the surrounding tissue 3–5 mm distant from the neurogenic spots.
Measurement of intensities of mechanical or electrical stimulation
To measure the intensities of mechanical stimulation (vibration) in MA mode, the tip of acupuncture needle was attached to a force transducer (FT-100, iWorx/CB Sciences Inc., NH, USA) and the signals during vibration were fed into bridged amplifiers (ETH-200, CB Sciences Inc., Dover, NH, USA), filtered between 10 and 200 Hz and quantified using a LabChart & Scope program (AD Instruments). To determine the electrical frequencies and intensities generated in EA mode, the electrodes of MEA stimulation units were connected to bridged amplifiers (ETH-200, CB Sciences Inc., Dover, NH, USA) and recorded using a LabChart & Scope program (AD Instruments).
Recordings of ultrasonic vocalizations (USVs)
Ultrasonic vocalizations (USVs) emitted by rats in response to acupuncture stimulation were recorded using customized sound-attenuating chambers as previously described [17]. The chamber consisted of two boxes to minimize exterior noise (inside box: 60 × 42 × 42 cm, outside box: 68 × 50 × 51 cm). The ultrasonic microphone was positioned at the center of the ceiling of the chambers and recorded with the Avisoft-RECORDER software (Avisoft Bioacoustics). For 22-kHz USVs, the signals were band-filtered between 18 and 32 kHz and analyzed using Avisoft-SASLab Pro (version 4.2, Avisoft Bioacoustics). Animals (n = 6) were habituated for at least 30 min in the chambers prior to experiments. After the USVs were recorded for 2 min as baseline (Base), the acupuncture needles were bilaterally inserted into the wrist area and stimulated for 2 min in EA, MA or combined EA + MA mode (Fig. 1e). All rats received 4 treatments (Con, EA, MA, or MA + EA) over 4 days in random order. Data were calculated by subtracting the basal USVs (Base) from the numbers of USVs emitted during 2-min of acupuncture stimulation (Fig. 1f).
Immobilization-induced hypertension and measurement of blood pressure
Hypertension was induced by immobilization with a cone-shaped polyethylene bag, as described previously [18]. Systolic blood pressure (BP) was measured non-invasively with a tail cuff blood pressure monitor (Model 47, IITC Inc., CA, USA). Briefly, the rat was placed in a chamber kept at 27 °C, and an occluding cuff and a pneumatic pulse transducer were positioned on the base of the tail. A programmed electrosphygmomanometer (Narco Bio-Systems Inc., TX, USA) was inflated and deflated automatically, and the tail cuff signals from the transducer were automatically collected every 10 min using an IITC apparatus (Model 47, IITC Inc.). The mean of two readings was taken at each BP measurement.
Immunohistochemistry of c-Fos in ventrolateral periaqueductal gray (vlPAG) or rostral ventrolateral medulla (rVLM)
After measurement of blood pressure, the brains were taken out, fixed in paraformaldehyde (PFA), cryo-protected, cryo-sectioned 30 μm thick and incubated in blocking solutions containing 0.3% Triton X-100, 5% normal goat serum in 0.1 M PBS at room temperature for 1 h. The sections were incubated with primary antibody for c-Fos (1:200) overnight at 4 °C, followed by an incubation of secondary antibody with donkey anti-rabbit Alexa Fluor 594. All sections were cover-slipped with a mounting medium (Vector laboratories, Burlingame, CA, USA) and imaged in vlPAG or rVLM under a 20 × objective using a confocal microscope (Zeiss Axioskop, Oberkochen, Germany).
Microinjection of naloxone into the rVLM
The head of the rat was fixed on a stereotaxic frame in prone position. For microinjection into rVLM (stereotaxic coordinates: posterior, − 12.72 mm; lateral, + 2 mm; deep, − 10 mm), the nose was deflected ventrally so that the dorsal surface of medulla could be leveled horizontally. A 1.0 mm burr hole was made − 12.72 mm to the bregma and ± 2 mm to the midline, a 26 gauge needle connected to a Hamilton syringe was inserted − 10 mm deep into bilateral rVLM. Naloxone (10 nM) was infused at a constant rate of 0.1 μl/min (CMA 100, Microinjection pump; kdScientific, MA, USA). After termination of experiment, the brain stem was removed, fixed in PFA for 2 h and immersed in 30% sucrose overnight. The brains were cryo-sectioned 30 μm thick and stained with toluidine blue. Injection site was identified under a microscope.
In vivo extracellular single-unit recordings of rVLM neurons
Single-unit discharges of rVLM neurons were recorded in anesthetized rats, as described previously [19] with slight modifications. In brief, 1 h after immobilization, rats (n = 14) were anesthetized with an intraperitoneal (i.p.) injection of urethane (1.5 g/kg). A carbon-filament glass microelectrode (0.4–1.2 MΩ, Carbostar-1, Kation Scientific, USA) was stereotaxically advanced to the rVLM (stereotaxic coordinates: posterior, − 11.96 to − 12.80 mm; lateral, + 1.9– + 2.4 mm; deep, 9.8–10.6 mm). Single-unit activity from the discharges was discriminated, recorded and analyzed via a CED 1401 Micro3 device and Spike2 software (Cambridge Electronic Design, UK). After recording stable baseline for at least 5 min, the rat received either saline (n = 7) or naloxone (n = 7; 1 mg/kg, i.p.) and recorded for 5 min. After 2 min of electrical acupuncture stimulation (EA; 2 Hz, 0.5 mA, 0.1 ms, triangular pulses), single-unit discharges were recorded for 5 min. Only EA treatment was carried out during single-unit recording of rVLM neurons, because electromagnetic noise generated during running of DC motor in MA interfered electrical signals of rVLM neurons.
Data analysis
Statistical analysis was carried out using SigmaPlot 12.5 software. All data are presented as means ± standard error of the mean (S.E.M) and analyzed by one- or two-way repeated measurement analysis of variance (ANOVA) followed by the post-hoc Tukey test. P < 0.05 was considered statistically significant.