Animals and grouping
A total of 50 male Wistar rats (240–300 g) were obtained from the Experimental Animal Center of Peking Union Medical College (Beijing, China), and housed within the animal care facilities in the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences. Animals were randomly assigned (n = 10 in each group) to sham ligature (CON), CCI pain model (CCI) and CCI plus three, five, and twelve sessions of EA treatment (EA3d, EA5d, and EA12d, respectively) groups. In order to observe the involvement of NK cells in EA analgesic effect, EA + Anti-asialo-ganglio-N-tetraosylceramide (anti-asialo-GM1) (EA + anti-asialo-GM1) group and EA + normal rabbit serum (EA + serum) group were added. All experimental procedures were approved by the Institute of Acupuncture and Moxibustion of China Academy of Chinese Medical Sciences, and performed according to the “Guidelines for Laboratory Animal Care and Use” of the Chinese Ministry of Science and Technology (2006).
CCI pain model and pain threshold detection
The CCI model was established by ligating the unilateral sciatic nerve, as previously reported [23]. Briefly, under anesthesia (25% urethane plus 1.5% chloralose, 0.4 mL/100 g body weight) and routine sterilization, the left sciatic nerve was exposed at the mid-thigh level by blunt dissection through the biceps femoris. Four constrictive ligatures (4–0 non-absorbable suture) were tied around the nerve at the distal end close to the bifurcation site (about 1 mm space between every two ligatures). The ligature was all right until a moderate muscular contraction of the leg was seen. The same procedure was performed for rats in the CON group but without nerve ligature.
The paw withdrawal latency (PWL) (i.e. the thermal pain threshold) of the bilateral hind paws was determined using a 37370 Algesia Detector (Ugo, Italy) 5 days after CCI, and every day thereafter prior to EA treatment. The radiant heat source was focused on the plantar surface of the hindpaw, and light intensity was preset to obtain a baseline latency of approximately 15 sec. Each rat underwent two trials with a five-min interval, and the mean value of the two trials was used as the PWL. To minimize differences in individual animals, the difference value of PWL (PWLD) between the healthy and the affected hindpaws was calculated.
EA intervention
According to the theory of traditional Chinese medicine, Zusanli (ST36) and Yanglingquan (GB34) are considered to be the most effective acupoints for treating lower back pain, and are commonly used in modern research to study the effects of acupuncture on various physiological regulatory and control systems. ST36 is located 5 mm beneath the capitulum fibulae and lateral-posterior to the knee-joint. GB34 is approximately 5 mm superior-lateral to ST36. In the present study, 7 days after CCI, animals from the three EA groups were stimulated with EA at bilateral ST36 and GB34. The acupoints were punctured with stainless steel filiform needles (diameter 0.35 mm, length 40 mm, Huatuo; Suzhou Medical Appliance Manufactory, Jiangsu, China) to a depth of approximately 2–3 mm, and stimulated electrically for 30 min using a Han’s EA Stimulator (LH202; Neuroscience Research Center, Peking University, Beijing, China). The intensity and frequency of EA were 1 mA and 15 Hz, respectively. During EA stimulation, the animals were conscious and constrained with a special cloth bag. The treatment was administered once daily continuously for 3, 5 or 12 consecutive days. The other two groups underwent the same procedure but without EA stimulation.
At the end of the EA treatment, all rats from the five groups were deeply anesthetized. Blood samples (3–5 mL) were obtained through carotid intubation and placed separately in three heparinized tubes. Spleen and hypothalamus tissues were rapidly removed on an ice plate and weighed.
Lymphocyte preparation and fluorescence-activated cell sorting (FACS)
Spleen tissue was washed with 5 mL RPMI-1640 medium (Gibco, Grand Island, USA) and then minced with two sterilized glass slides, passed through a 70-μm nylon cell strainer, and washed. The suspension was differentially centrifuged over a Percoll gradient (EMB Biosciences, La Jolla, CA) (40% Percoll, 1200 r/min, 10 min, 25°C). The lymphocytes were washed with phosphate-buffered saline (PBS), and then suspended in RPMI-1640 medium, and 106 single cell suspensions were prepared.
Whole blood (100 μL) and splenic lymphocytes (106) were triple-stained with (1) APC-anti-CD3, FITC-anti-CD45RA and PE-anti-CD161a, or (2) APC-anti-CD3, PE-anti-CD4 and FITC-anti-CD8a (all from BD Biosciences, San Jose, CA, USA) for 30 min at 4°C in the dark, then washed with PBS and resuspended in FACS staining buffer (BD Biosciences). Three-color analysis was performed on a FACSCalibur (BD Biosciences) with a 100,000–200,000 event count. Data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA).
Multiplex enzyme-linked immunosorbent assay (ELISA) analysis of plasma and splenic cytokine concentrations
To prepare splenic aqueous extract for the cytokine concentration assay, the spleen was removed rapidly on an ice plate, and homogenized by a glass tissue homogenizer in an ice cold water bath. The homogenate was then centrifuged (4500 r/min for 30 min, at 4°C). The supernatant was collected and stored at -80°C until future use. Plasma was separated by centrifugation (3000 r/min for 30 min, at 4°C) and stored at -80°C until use. Concentrations of plasma gamma-interferon (IFN-γ), interleukin-1 beta (IL-1β), IL-2, IL-6, IL-4, IL-10, and transforming growth factor beta (TGF-β), and splenic IL-2, IL-12, IL-15 and IFN-γ were simultaneously quantified in samples using an ELISA-based bead multiplex assay (Linco Research, St Charles, MO, USA), according to the manufacturer’s instruction. Samples were analyzed with a Lumine × 100 plate reader (Linco Research, St Charles, Missouri, USA) to determine the cytokine concentrations. Concentration of each cytokine in the multiplex assay was calculated from calibration curves using individual recombinant proteins as standards. All specimens were tested in duplicate wells to assess inter-assay variability.
NK cell and cytotoxic T lymphocyte (CTL) activities assay
Splenic CD8+ T cell subset and NK cells were isolated using MagCellect Rat CD8+ T Cell Isolation Kit (R&D Systems, Minneapolis, MN) and Rat NK Cell Isolation Kit (Yanjin Biotec, Shanghai, China) according to the manufacturer’s instructions. The activities of NK cells and CTLs were respectively detected by methyl thiazolyl tetrazolium colorimetric (MTT) method [24]. Briefly, NK cells of each group were collected and seeded in 96 well plates as effect cells at a concentration of 1 × 105/ml and cultured 5 × 105 YAC-1 used as target cells. Co-cultured NK and YAC-1 cells in 96-well plate at a 50:1 ratio of effector to target (E:T) were simultaneously incubated at 37°C in 5% CO2 for 4 hours in 96 well plates. Then the supernatants were discarded and fresh medium containing 200 μL MTT was added to each well and incubated for 4 hours. After the supernatants were discarded, 20 μL dimethylsulfoxide was added to each well and mixed thoroughly to dissolve the blue crystals at room temperature. Ten minutes later, when all the crystals dissolved, the plates were read on a plate reader (Bio-Rad, Hercules, CA, USA) at 560 nm. For CTL activity, the effector cells were 2.5 × 106/ml splenic CD8+ T lymphocytes and cultured SP2/0 (5 × 105) cells used as target cells. Co-cultured CD8+ T and SP2/0 cells at a 50:1 ratio of effector to target (E:T) were incubated for 24 hours. All MTT assays were performed in triplicate. The activities of CTL and NK cell were calculated using the following formula: NK (or CTL) activity = [1-(OD (E + T)-ODE)/ODT] × 100% (ODE, optical density of effect cells; ODT, optical density of target cells; OD (E + T), optical density of co-cultured cells).
Beta-endorphin (β-EP) assay
The hypothalamus tissue samples were placed in tubes containing 1 mol/L (0.5 mL) glacial acetic acid to be homogenized on ice. The tissue lysate was centrifuged (3500 r/min, at 4°C, for 20 min). The concentration of β-EP was measured by the β-EP 125I radioimmunoassay kit for rat (Sino-UK Institute of Biological Technology, Beijing, China) according to the instruction. All specimens were tested three times to minimize inter-assay variability.
Depletion of NK cells in vivo
For observing the effect of NK cells on EA analgesia in CCI rats, other 20 Wistar rats were equally divided into EA + normal rabbit serum group and EA + Anti-asialo-GM1 rabbit antibody group. The procedures of CCI and EA were the same to those mentioned above. The Anti-asialo-GM1 rabbit antibody (Abcam Cambridge, UK) was intravenously given at a dose of 80 μg on the day before the first EA treatment and once again every 4 days thereafter. Normal rabbit serum was used as control.
Statistical analysis
All data are presented as the mean ± standard deviation (SD), and were analyzed by one-way analysis of variance followed by Fisher-Hayter for multiple comparisons with Stata 12.0 software (Stata Corp, College Station, USA). All P values were 2-sided, and P < 0.05 was considered significant.