Animals
Male Wistar rats (240–300 g) were obtained from the Experimental Animal Center of the 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. Food and water were available ad libitum. The animals were allowed to adapt to the experimental conditions in the laboratory for at least 2 h before pain testing. All experimental procedures were approved by the Institute of Acupuncture and Moxibustion of China Academy of Chinese Medical Sciences and were identical to those recommended in the Guidelines for Laboratory Animal Care and Use from the Chinese Ministry of Science and Technology (2006). Efforts were made to minimize the number and suffering of the animals used.
Experimental design
The study consisted of the following two experiments: Experiment 1 was designed to examine the effects of EA on MEK1 activation. For this experiment, rats with CCI were randomized into eight groups (n = 10 in each group): sham operation control (CON), CCI model, and CCI plus an increasing number of daily EA treatment (EA2d, EA4d, EA6d, EA8d, EA10d, and EA12d); Experiment 2 was designed to validate the effects of MEK1 depletion on the cumulative effects of EA intervention. Rats that received CCI and implantation of guide cannulas in the contralateral hippocampus were divided into four groups and treated with either a microinjection of dimethyl sulfoxide (DMSO for control; two groups, CCI + DMSO, CCI + DMSO + EA) or PD98059 (a MEK1 inhibitor, two groups, CCI + PD98059, CCI + PD98059 + EA). To observe the different effects of PD98059 at different stages of EA intervention, a 3-day cycle hippocampal microinjection was given on the first 3 days of EA or given on day 8, 9, and 10 of EA separately (n = 10 in each group). To examine the effect of a combination of microinjection and EA, rats received a microinjection 4.5 h prior to EA stimulation. To clarify the effect of PD98059, a CON + PD98059 group was added (n = 10). Randomization scheme was created using the standard = RAND() function in Microsoft Excel.
CCI pain model and behavioral tests
The CCI model was established by unilaterally ligating the sciatic nerve, as previously reported [22]. Briefly, under anesthesia (25% urethane plus 1.5% chloralose, 0.4 mL/100 g body weight) and using routine sterile procedures, 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) equally spaced by approximately 1 mm were tied around the nerve at the distal end close to the bifurcation site. The ligatures were tightened until a moderate muscular contraction of the leg was observed. For the CON group, the rats underwent the same procedure but without nerve ligation.
One day before ligation, 10 days after ligation, and everyday within 0.5 h before EA stimulation, the paw withdrawal latencies (PWL, i.e., thermal pain threshold) of both hind paws were determined using a 37370 Algesia Detector (Ugo, Italy). A radiant heat source was focused on the plantar surface of a hind paw, and a light intensity was preset to obtain a baseline latency of approximately 25 s. Each rat underwent three trials with a 5-min interval, and the mean value of three trials was used as the PWL.
Electroacupuncture
According to traditional Chinese medicine theory, Zusanli (ST36) and Yanglingquan (GB34) are considered the most effective acupoints for treating low back pain and are commonly used to study the acupuncture effects on various physiological regulatory and control systems in modern scientific research. ST36 is located 5 mm beneath the capitulum fibulae and lateral posterior to the knee joint, and GB34 is approximately 5 mm superior-lateral to ST36 [18]. In the present study, the animals in the EA group were treated with 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 EA stimulation parameters were 1 mA and 2 and 15 Hz alternating frequencies (automatically shifting between 2 Hz and 15 Hz stimulation for 3 s each), respectively. EA stimulation started from the 10th day after CCI. During EA stimulation, the animals were awake and constrained with a special cloth bag. As neither the ipsilateral nor the contralateral PWLs were influenced by the repeated constraining process in CCI rats, the non-EA groups (CCI and CON groups) underwent the same constraining procedure, but without EA stimulation.
Microinjection procedure
Under anesthesia with urethane and chloralose, after CCI operation, two 6-mm-long stainless steel guide cannulas (Small Parts Inc., Hialeah, Florida, USA) were stereotaxically implanted into the contralateral hippocampus (3.3–3.6 mm posterior to the bregma, 2.4–2.7 mm lateral to the midline, and 3.0–3.5 mm below the cortical surface) based on the coordinates in the atlas completed by Paxinos and Watson [23], with the tip retained about 2 mm above the dura. The guide cannulas were anchored to the cranium with dental cement. The animals were allowed to recover for 10 days. The guide cannulas were plugged with a stainless steel stylet, which was removed before drug administration. PD98059 (Abcam, New Territories, HK) dissolved in 5% (v/v) DMSO (Abcam) was administered to the PD98059 groups. The DMSO groups received the same volume of 5% (v/v) DMSO as control. Drugs were administered into the hippocampus through the guide cannulas using an injection needle (gauge 27) connected by polyethylene tubing to a 10-μL Hamilton microsyringe while the rats were anesthetized under isoflurane anesthesia. PD98059 (10 μg/10 μL) or DMSO (5% (v/v)/10 μL) was injected over 3 min, 4 h prior to behavioral testing.
At the end of each set of experiments, the microinjection sites of one rat in each group were marked with 2 μL of a saturated solution of Pontamine sky blue (Sigma Chemical Co. St. Louis, MO, USA) to determine the distribution of the injection. After fixation with 10% (v/v) formalin, the brain was sectioned and counterstained with cresyl violet. The microinjection sites were histologically verified and plotted based on the Paxinos and Watson stereotaxic atlas coordinates. A representative microinjection site is shown in Fig. 1. For all animals examined, the cannula tips were located just in the target region, and the injection tracks were limited to the CA region or dentate gyrus of the hippocampus.
Tissue harvest and western blot analysis
After termination of the behavioral test, rats in each group were deeply anesthetized and killed by decapitation. The right and left hypothalamus and hippocampi were removed rapidly on an ice plate and then washed with normal saline. Tissue extracts were obtained using mechanical tissue disruption in RIPA lysis buffer with 1× Complete Anti-Protease Cocktail (Roche, Indianapolis, IN, USA), 1 mM PMSF, and incubation for 10 min at 4 °C. Lysates were cleared by centrifugation at 10,000 g for 10 min at 4 °C. Total protein (30 μg) was loaded onto 10% (w/v) gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were then incubated with primary antibodies (anti-MEK1, 1:1000; anti-phospho-MEK1, 1:800; anti-GAPDH, 1:500; Cell Signaling, Beverly, MA, USA) overnight at 4 °C with 2% (w/v) bovine serum albumin in PBST. The membranes were then washed three times with PBST (10 min each time), incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, followed by three washes with PBST. Immunoreactive bands were then revealed by enhanced chemiluminescence (Amersham Biosciences UK Limited, Buckinghamshire, England) using standard x-ray film (Eastern Kodak Co., Rochester, NY, USA). The relative intensities of the detected protein bands were analyzed with a Personal Densitometer SI (Amersham Biosciences) linked to ImageQuant 5.2 software (Amersham Biosciences).
Statistical analysis
SPSS 17.0 software was used to perform the statistical analysis. The results are expressed as the mean ± SD. PWLs were analyzed using two-way repeated measures analysis of variance (ANOVA) with one between-subject factor (EA intervention) and one within-subject factor (time) followed by the least significant difference (LSD) analysis. Western blot data were analyzed using one-way analysis of variance and the LSD test was performed to compare the differences between two groups. All P values were derived from two-sided tests, and P < 0.05 was considered statistically significant.