Animals
Male Sprague-Dawley rats weighing 210-260 g ( National Institutes for Food and Drug Control, Beijing, China, Certificate number: SYXK 2014-0013) were housed at 20-26 °C in plastic cages (size: 461 x 274 x 229 mm; 3-4 rats per cage) on soft bedding with ad libitum water and food under a 12h/12h light/dark cycle for at least one week before the experiment. Every effort was made to minimize the number of animals used. Numbers of animals were selected according to previous experience, i.e. a trade-off between reaching routine sample sizes for field experiments while minimizing numbers of animals for pain experiments. Experiments were performed on 100 animals (5-10 rats/group). Rats were randomized into treatment groups before their assessment. All experiments, analysis and reporting were ARRIVE-compliant (Animals in research: reporting in vivo experiments). Animal experiments were performed according to the ethical guidelines set by the International Association for the Study of Pain[47]. The protocols applied here for animal care and use were approved by the Animal Experimentation Ethics Committee of Beijing Institute of Traditional Chinese Medicine (Approval number: 2018080102).
Dural cannulation and inflammatory soup injection
The protocols used for the dural cannulation were similar to those described previously[13, 23]. A previous study found that the effects of inflammatory soup (IS) application on the dura were due to the activation of trigeminal afferents and not to a systemic [48]. Rats were briefly anesthetized with sodium pentobarbital (40 mg/kg, ip). The dura of rats was exposed through a 1 mm hole (1 mm left of midline, 1 mm anterior to bregma) and a guide cannula, extending 0.5 mm from the skull surface to avoid irritation of the dural tissue, was inserted into the hole and secured into place with dental acrylic (see Additional file: Figure S1A). One week post-surgery, injections (20 µL, 1µL/minute) of IS or artificial cerebrospinal fluid (aCSF) were performed under brief anesthesia (isoflurane, 3% induction, 1.5% maintenance) through the cannula at 2-days intervals during the 7-days injection protocol[13, 49] (see Additional file: Figure S1B). Twenty microliters of aCSF were injected as in rats which form the control (Con) group. After recovery, rats were gently placed into the glass chamber for behavioral testing. IS was formulated with histamine, serotonin and bradykinin, all at 2 mM, and prostaglandin E2 (PGE2), at 0.2 mM, respectively, in 10 mM HEPES buffer at pH 5.0, and aCSF contained124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.0 mM MgCl2, 2.0 mM CaCl2, 25 mM NaHCO3 and 10 mM glucose.
Direct transcutaneous intrathecal injection
Intrathecal injection was performed as described previously[50]. One week after dural cannulation, rats received direct transcutaneous intrathecal injection before each IS application at 2-days intervals during the 7-days injection protocol. Under isoflurane induced anesthesia (3% induction, 1.5% maintenance), a 25-G needle connected to a 25-μl Hamilton syringe was inserted percutaneously into the vertebral canal between L5 and L6. A tail-flick reaction denoted a successful puncture. A rat was injected intrathecally with 20 μl of 1% Chicago sky blue (C8678, Sigma-Aldrich, USA) to determine whether the drug injected intrathecally was able to reach the L6-S1 spinal cord level. After that, SB269970 (5-HT7R antagonist, 7μL, 5mM dissolved in distilled water; Sigma-Aldrich, USA) or AS-19 (5-HT7R agonist, 15μL, 10μM dissolved in saline; Sigma-Aldrich, USA) was administered intrathecally with an injection rate of 1µL/minute [51, 52]. Rats that exhibit signs of motor impairment are excluded from the experiment.
Electroacupuncture
Rats in GB20+GB34, GB20+SJ17, GB34+ST36, IS+EA, and IS+AS19+EA group received EA stimulation under anesthetization with isoflurane (3% induction, 1.5% maintenance). The Con, IS and IS+SEA group were also anesthetized at the same time. After immobilization, the rats were scrubbed with 75% alcohol disinfectant at the EA sites before EA was being performed by the acupuncturist with acupuncture needles. For the EA stimulation, four needles, each sized at 0.25 × 13 mm (Suzhou Medical Appliance Factory, Suzhou, China) were inserted at a depth of 1.5 mm in the bilateral head and hind leg at acupoints corresponding to Fengchi (GB20), Yifeng (SJ17), Yanglingquan (GB34), Zusanli (ST36) (see Additional file: Figure S2 and Table S1), as described previously[53-56]. The needles were connected to HANS LH202H Han’s acupoint nerve stimulator (Beijing Hua Wei Industrial Development Co, Beijing, China). Electroacupuncture stimulation (2-15 Hz alternating wave, 1.0 mA) of 15 minutes was initiated before IS injection. This process was repeated every 2 days (Fig. 1A). Sham electroacupuncture (SEA) was acupuncture needle insertion into non-acupoints (approximately 10 mm and 15mm above the iliac crest) without electrical stimulation.
Experimental procedures
Experiment I
To find the best treatment protocol, we compared the effects of electroacupuncture at different acupoints, including GB20+GB34, GB20+SJ17, and GB34+ST36, in changing the cutaneous hyperalgesia and 5-HT7R expression of IS rats. We performed the 50% facial mechanical withdrawal threshold (50%FMWT), 50% paw mechanical withdrawal threshold (50%PMWT), tail-flick latency (TFL), hot-plate latency (HPL), and cold-plate behaviors (CPB)[57] to evaluate the mechanical and thermal hyperalgesia. 5-HT7R expression in trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) were detected by real time polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence analyses. The rats were randomly divided into 6 groups: repeated dural injection of aCSF (Con group), repeated dural injection of IS (IS group), IS with sham EA (IS+SEA group), IS with EA at GB20 and GB34 point (GB20+GB34 group), IS with EA at GB20 and SJ17 point (GB20+SJ17 group), and IS with EA at GB34 and ST36 point (GB34+ST36 group) group. (n = 10 per group).
Experiment II
To observe the therapeutic effects of electroacupuncture, we examined whether EA can alleviate cutaneous hyperalgesia and neuronal hyperexcitability in TNC, which induced by repeated dural injection of IS, using behavioral testing and electrophysiological recordings. The rats were assigned at random to Con group, IS group, IS+SEA group, IS+EA (best treatment protocol from experiment I) group (n = 10 per group).
Experiment Ⅲ
To investigate whether electroacupuncture affects 5-HT7R-mediated PKA and ERK1/2 phosphorylation signaling pathway, we examined 5-HT7R, cAMP production, the total amount of PKA, ERK1/2 and CREB, phosphorylation of PKA, ERK1/2, CREB, and c-Fos expression by RT-PCR, Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses in TG and TNC. The animal groups in experiment III were the same as those in experiment II (n = 5 per group).
Experiment Ⅳ
To determine whether 5-HT7R-mediated PKA and ERK1/2 phosphorylation signaling pathways are involved in EA’s therapeutic effects, we tested cutaneous hyperalgesia, neuronal hyperexcitability in TNC, and 5-HT7R related protein using behavioral testing, electrophysiological recordings, Western blot and ELISA analyses. The rats were randomly divided into 4 groups: IS group, IS+SB269970 group (IS with 5-HT7R antagonist SB269970), IS+AS19+EA group (IS+EA with 5-HT7R agonist AS19), IS+EA group (n = 5 per group).
Behavioral testing
Before their behavioral testing, the rats were acclimatized to the experiment’s environment by being exposed to it for 30 minutes a day for two days, and those with an abnormal baseline were excluded. To avoid experimenter bias, the investigator who conducted the behavioral study did not know the type of treatment applied to the rats. On each of the testing days, rats were brought into the testing room for 30 minutes to acclimate them to the environment. As the rats were acclimated to the environment, they did not show signs of anxiety such as defecation or urination), nor did they attempt to escape by climbing onto the plastic cylinder or cage. After the determination of the baseline response (day 0), all behavioral testing was assessed on days 1, 3, 5, 7 after 1 hours of IS injection, and day 8 without IS injection.
Mechanical Withdrawal Threshold (MWT)
Von Frey filaments (Stoelting, Wood Dale, IL) were used to measure the 50% withdrawal threshold using the up-and-down method reported by Dixon[58] and Chaplan[59]. Tests were conducted during the day portion of the circadian cycle only (06:00-18:00 h). For 50% facial mechanical withdrawal threshold (50%FMWT) testing, the rats were placed in a plastic cylinder with two rectangle open windows which allowed full access to the bilateral face. For 50% paw mechanical withdrawal threshold (50%PMWT) testing, the rats were placed in a plastic cylinder with a wire mesh bottom that allowed for full access to the bilateral paws. Behavioral accommodation was allowed for approximately 15 minutes, until cage exploration and major grooming activities ceased. The areas tested were face and mid-plantar hind paw. The face and paw were touched with one of a series of 10 von Frey filaments with logarithmically incremental stiffness (0.4, 0.6, 1, 2, 4, 6, 8, 15, 26 and 60 g). A series of filaments, starting with one that had a buckling weight of 2 g, was applied in a consecutive sequence on the bilateral facial and hind paw with a pressure causing the filament to buckle and held for approximately 6-8 seconds.
The von Frey filaments were presented perpendicular to the facial and plantar surface with sufficient force to cause slight buckling against the bilateral face and paw. Stimuli were presented at intervals of 10 seconds, allowing for apparent resolution of any behavioral responses to previous stimuli. A positive response was noted if the face and paw were sharply withdrawn. Based on previous studies, the cut-off of a 60-gram filament was selected as the upper limit for testing, since stiffer filaments tended to move the entire face or limb rather than to buckle, substantially changing the nature of the stimulus.
According to Dixon[58], optimal threshold calculation by this method requires 6 responses in the immediate vicinity of the 50% threshold. Since the threshold is not known, strings of similar responses may be generated as the threshold is approached from either direction. Accordingly, although all responses were noted, counting of the critical 6 data points did not begin until the response threshold was first crossed, at which time the 2 responses straddling the threshold were retrospectively designated as the first 2 responses of the series of 6. Four additional responses to the continued presence of stimuli that were varied sequentially up or down, based on the rat's response, constituted the remainder of the series [60].
Tail-flick Latency (TFL)
The tail-flick test was used to assess thermal hyperalgesia in the rats. The rats were placed in a plastic cylinder from which its tail protruded. The presence of tail flicking was considered as withdrawal latency. The intensity of the light from the thermal stimulator (IITC Inc., Woodland Hills, CA) was adjusted at the start of the experiment such that average baseline latencies were approximately 3 to 4 seconds, and a cutoff latency of 16 seconds was established. The heat was directed to the distal tail (20 mm from the tip). Three trials were done at intervals of 5 minutes, and the latency (seconds) was the average of 3 trials[61].
Hot-plate Latency (HPL)
The hot-plate test was used to assess thermal hyperalgesia in the rats. The rats were placed on a heated (50 ± 1°C) surface one at a time. Paw licking or jumping was considered as withdrawal latency. A cutoff latency of 50 seconds was established. Three trials were done at intervals of 20 minutes, and the latency (seconds) was the average of 3 trials [62, 63].
Cold Plate Behaviors (CPB)
The cold-plate test was used to assess thermal hyperalgesia in rats. The plate was cooled down to 4 ± 1 ℃. Rats were then placed on the plate for 5 minutes and scored. Rats that were walking freely on the 4 ℃ cold plate for 5 minutes did not exhibit any signs of skin injury, given that this was a mild nociceptive stimulus. The number of paw lifts quantified the response to cold during an observation period of 5 minutes. Three trials were done at intervals of 10 minutes, and the cold plate behaviors (times) was the average of 3 trials[64, 65].
Electrophysiological recordings in the trigeminal nucleus caudalis
Animal preparation
The last IS injection occurred on day 7 and electrophysiological recordings were performed at 2 hours post-injection. As previously described, rats were anesthetized with isoflurane (3% induction, 1.5% maintenance) mixed with oxygen. After the trachea was cannulated and the carotid artery and external jugular vein catheterized, rats were paralyzed by intravenous perfusion of vecuronium bromide (2.4 mg/h) and artificially ventilated with a volume-controlled pump (54-55 strokes/minute). Levels of isoflurane, O2, N2 and end-tidal CO2 (3.5-4.5%) were measured by an anesthetic gas analyzer (Drager Vamos) during the entire experimental period. The mean arterial blood pressure was continuously monitored (90 to 110 mmHg). The colorectal temperature was kept constant at 38 ± 0.5°C. The eyes of the rat were protected with erythromycin ointment.
The rats were placed in a stereotaxic frame with the head fixed in a ventro-flexed position. The TNC was exposed by removing the overlying musculature, atlanto-occipital membrane, dura mater and a cervical laminectomy, and then flooded with aCSF. The tungsten recording electrode with a diameter of 75 μm, tip diameter of 3-4 μm, and an impedance of 10-15 kΩ (TM33CCINS, World Precision Instruments Shanghai Trading Co., Ltd. China) was carefully implanted into a depth of 0.5-1.5 mm using a microelectrode manipulator (SR-1N, NARISHIGE Co., Ltd. Japan) (Fig.2A).
Electrophysiological Recording
Single-unit activities were sampled and analyzed at 100 Hz-50 kHz and were amplified 1k times and displayed on oscilloscopes. The activities went into a window discriminator connected to a CED 1401plus interface (Cambridge Electronic Design) and a computer (Spike 2 version 7.03a software, CED, Cambridge, UK), which allowed sampling and analysis of the spontaneous and evoked neuronal activity. Wide-dynamic range (WDR) neurons were recognized based on their responses to mechanical non-noxious (brushing with a soft brush) and noxious (pinch with forceps) stimulations of their receptive fields as previously described. Specifically, each neuron that responded in a graded manner with increasing firing rates to the stimulus range from non-noxious to noxious intensity was classified as a WDR cell (see Additional file: Figure S3). Once a neuron was identified, electrical square-wave stimuli (1.5ms duration) were applied through a pair of stainless-steel needle electrodes subcutaneously placed into the center of the receptive field; the thresholds for eliciting A- and C-fiber-evoked responses were determined. In poststimulus time histograms (PSTHs), A- and C-fiber-evoked responses were distinguished by their latencies, but only C-fiber-evoked responses were considered in the detailed analysis. Previous research showed that burst discharges at latencies ≥ 30ms are elicited by C-fibers[58, 66]. Therefore, all spikes occurring between 30 and 200ms after a stimulus were considered to be C-fiber-evoked. The C-fiber thresholds (Tc) were defined as the stimulation intensities required to evoke neuronal activity with a conductive velocity of 0.4-2 m.s-1 [67] . Electrostimulation (ES) of 1Tc (2mA) and 2.5Tc (5mA) were applied to the facial region in this part of the experiment[68]. ES comprising square waves consisted of low-intensity (2mA, 0.75Hz) and high-intensity (5mA, 0.75Hz) stimulation, and ES being applied once for 1.5ms in order, to the most sensitive portion of the cutaneous receptive field. The responsiveness of electrical stimulation was assessed by recording the responses to 1.5ms application of ES at 2mA and 5mA. Spontaneous activity (spikes.s-1 for 60 seconds, 30 minutes after finding the activated neurons for stabilization) was also recorded. Only one cell was tested in each animal, and only cells with spike amplitude more than 3mV showing no change in amplitude or waveform throughout the experimental procedure were considered and sorted.
Immunofluorescence
Tissue processing for immunofluorescence
Upon deep anesthesia by intraperitoneal injection of sodium pentobarbital (150 mg/kg), the rat was perfused transcardially with normal saline followed by 4% paraformaldehyde in PBS for 20 minutes. Brain and trigeminal ganglia were isolated and post-fixed 30 minutes in the same fixative at room temperature. All specimens were dehydrated for cryoprotection in 30% sucrose in 0.1 M phosphate buffer (0.08 M K2HPO4, 0.02 M NaH2PO4, pH 7.4). TG and TNC in random orientation were cut with a cryostat into 20μm sections and mounted directly onto gelatin-coated slides. From 5 TG and TNC sections (100mm apart) in each rat, total 25 sections were selected for measurement in each group.
Single-labeling immunofluorescence
The sections obtained were washed in Tris-buffered saline (TBS, 25 mM Tris, pH 7.5) and then TBS containing 0.3% Triton X-100 (TBST), sections were treated with 0.3% hydrogen peroxide in TBS to exhaust endogenous hydrogen peroxidase activity until no bubbles. Nonspecific binding was blocked by 3% normal serum (from an animal species same as the secondary antibody) and 2% bovine serum albumin in TBST for 1.5 hours. Sections were incubated with primary antibodies goat anti-CGRP (1:100, ab36001, Abcam, Cambridge, UK) in TBST at room temperature overnight containing 5% normal serum. After overnight reaction with the primary antibody, the sections were washed with TBST and then incubated for 1.5 hours with secondary antibodies rabbit anti-goat IgG labeled with FITC (1:200, RAG001, MultiSciences Biotech Co., Ltd, Hangzhou, China).
Double-labeling immunofluorescence
The following procedures were used for two primary antibodies derived from different species. Sections were processed similarly to that described under the section ‘Single-antigen immunohistochemistry’. Primary antibodies included rabbit anti-5-HT7R (1:200, ab61562, Abcam, Cambridge, UK), mouse anti-NeuN (1:1000, ab104224, Abcam, Cambridge, UK). Secondary antibodies included goat anti-rabbit IgG labeled with Alexa 488 (1:200, A0432, Beyotime Biotech Inc., Shanghai, China) and goat anti-mouse IgG labeled with Cy3 (1:200, A0432, Beyotime Biotech Inc., Shanghai, China). Sections on slides were dehydrated through an ethanol gradient/xylene and cover-slipped in antifade mounting medium with DAPI (H-1500, Vector Laboratories, Inc., Burlingame, CA). The images were captured using a Leica semi-automatic light microscope (Leica DM5500B), and processed with the Adobe Photoshop CS2 software. The CGRP-, 5-HT7R-, and NeuN- positive fluorescence images were measured in squares at 20 × magnification. ImageJ 1.40g (National Institutes of Health, Bethesda, MD) was used as the software to circle the immunoreactive regions on the soma of TG and TNC neurons.
Western blot
ImageJ 1.40g (National Institutes of Health, Bethesda, MD) was used as the software to circle the immunoreactive regions on the soma of TH and TNC neurons. Punches of TNC regions were selected using a magnifying glass with stainless steel cannulae of 1000 μm inner diameter and subsequently pooled. The tissue was homogenized on the ice with the lysis buffer. The supernatant was collected after centrifugation at 4°C for 20 minutes at 12000 rpm. After being heated at 99°C for 5 minutes, the samples (20 μg/sample) were loaded onto a 5% stacking/10% separating SDS-polyacrylamide gel and then electrophoretically transferred onto a PVDF membrane (Millipore, MA). The membranes were incubated first in the blocking buffer (3% nonfat milk plus 0.1% Tween-20 in the Tris-buffered saline) for 2 hours at room temperature and then in the primary antibodies at 4℃ overnight. The primary antibodies included: rabbit anti-5-HT7R (1:2000, ab128892, Abcam, Cambridge, UK), mouse anti-c-Fos (1:2000, ab190289, Abcam, Cambridge, UK), rabbit anti-PKA (1:2000, 4782, CST, USA), rabbit anti-p-PKA (1:2000 5661, CST, USA), rabbit anti-ERK1/2 (1:2000 , 4695, CST, USA), rabbit anti-p-ERK1/2 (1:2000, 4370, CST, USA), rabbit anti-CREB (1:2000, 4820, CST, USA), rabbit anti-p-CREB (1:2000, 9198, CST, USA), mouse anti-β-actin (1:2000, C1313, Applygen Technologies Inc., Beijing, China). After the membranes were incubated with either the horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:200, 70-GAM007, MultiSciences, Biotech Co., Ltd, Hangzhou, China) at room temperature for 1 hour, the enhanced chemiluminescence reagent (Bio-Rad Laboratories) were used to visualize the proteins. ChemiDoc XRS and System with Image Lab software (Bio-Rad Laboratories) were performed to generate the images. ImageJ 1.40g software (National Institutes of Health, Bethesda, MD) was used to quantify the intensity of the images. The band intensities for 5-HT7R and c-Fos proteins were normalized to β-actin, and those for PKA, ERK1/2, CREB phosphorylation proteins were normalized to these total proteins.
Enzyme-linked immunosorbent assay
The endogenous levels of cAMP were measured in TG and TNC using an enzyme immunoassay kit (EXP110254, Expandbio, Beijing, China) according to the manufacturer's instructions. The concentrations of cAMP were expressed as pmol/mg of protein concentrations calculated in these supernatants.
Real time polymerase chain reaction
Total RNA was extracted from TG and TNC with Trizol reagent (Tiangen Biotech Co., Ltd, Beijing, China). cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (RR047B, TaKaRa, Dalian, China). RT-PCR was performed in a real-time PCR system (ABI7500; Applied Biosystems, Foster City, CA) using the SYBR Premix Ex Taq II (Tli RNaseH Plus) ROX plus (RR42LR, TaKaRa, Dalian, China). The PCR cycle parameters consist of an initial 30s incubation at 95°C, followed by 40 cycles of 95°C for 5 s, 60°C for 40 s; followed by a melt from 60°C to 99°C. Each primer sequence was as follows: 5-ht7r: 5’- GGT GAG GCA AAA TGG GAA ATG -3’ (forward) and 5’- ACC GCA GTG GAG TAG ATC GTG TAG -3’ (reverse); Gapdh: 5’- TGG AGT CTA CTG GCG TCT T -3’ (forward) and 5’- TGT CAT ATT TCT CGT GGT TCA -3’(reverse). The Ct value of each gene was normalized against that of Gapdh. Relative levels of expression were calculated using the comparative (2-△△Ct) method.
Statistical analyses
All statistical analyses were performed using SAS version 9.4 software. Statistical differences were determined using one-way analysis of variance for Immunofluorescence, Western blot, ELISA, RT-PCR and, and two-way repeated-measures analysis of variance was applied for the data from the behavioral testing. When four groups were compared for Electrophysiology, a Kruskal-Wallis test was used, and when only two groups were compared, a Mann-Whitney test was used. Data in the text and figures were shown as mean ± SEM. The level of significance was set at P < 0.05.