2.1 Animal preparation
Male Sprague-Dawley rats weighing 280–320 g (Harlan, Indianapolis, IN) were kept under controlled conditions (22°C, relative humidity 40%–60%, 12-hour alternate light-dark cycles, food and water ad libitum). The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland School of Medicine.
2.2 Experimental design
Four experiments were conducted. In Experiment 1 we measured plasma ACTH levels to examine whether EA regulates ACTH secretion. Rats were divided into four groups (n = 6 per group): CFA (0.08 ml) + EA, CFA + sham EA, CFA + no treatment, and naive + EA. EA was administered at 10 Hz, 3 mA, 0.1 ms pulse width for two 20-min periods, once at the beginning and once at the end of a 2-h period starting immediately after CFA injection. Blood samples (0.5 ml) were taken from each rat at baseline (before inflammation and/or EA) and 2 h and 5 h after inflammation. In Experiment 2, an ACTH antagonist, ACTH(11–24) (Bachem), was used to study the effects of ACTH on the therapeutic actions of EA. Rats were divided into four groups (n = 7 per group): 1) CFA (0.06 ml) + Vehicle + Sham EA, 2) CFA + Vehicle + EA, 3) CFA + ACTH(11–24) + Sham EA and 4) CFA + ACTH(11–24) + EA. A 2 mg/ml concentration of ACTH(11–24) was dissolved in saline, and 2 mg/kg of ACTH(11–24) or vehicle was intravenously (i.v.) injected 5 min before each of two 10 Hz EA treatments. The degree of edema, indicative of the intensity of inflammation, was quantified by measuring paw thickness with a Laser Sensor (AR200-50, Acuity, Portland, OR) 2 h and 5 h after a CFA injection into a hind paw. The paw withdrawal latency (PWL) test was conducted at the same time points. The investigator who conducted the measurement was blinded to the treatment assignments. Experiment 3 was to determine whether a CRH receptor antagonist, astressin, influences EA action. CFA-inflamed rats were divided into four groups (n = 7 per group): 1) CFA + Vehicle + Sham EA, 2) CFA + Vehicle + EA, 3) CFA + Astressin (Sigma) + Sham EA and 4) CFA + Astressin + EA. The paw thickness was measured with the same Laser Sensor. A 2 mg/ml concentration of astressin was dissolved in saline, and 0.2 mg/kg of astressin or vehicle was injected (i.v.) 5 min before each of two 10 Hz EA treatments. Experiment 4 was to determine whether EA activates CRH-containing neurons in the PVN. This was done by demonstrating double-labeling of CRH and phosphorylated NR1, an essential subunit of the N-methyl-D-aspartic acid (NMDA) receptor, the phosphorylation of which is known to modulate NMDA receptor activity and can be used as an indicator of NMDA receptor activation. To avoid the effect of CFA-induced inflammation on the PVN neurons, 3 naive rats given EA treatment and 2 untreated naive rats were used in this experiment.
2.3 Intravenous cannulation and blood sample collection
For intravenous cannulation, animals were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally (i.p.) and surgically implanted with a subcutaneous jugular catheter (Braintree Scientific, Inc). The catheter was secured with Mersilene surgical mesh (General Medical, New Haven, CT) at the jugular vein. It exited at the animal's back through a 22-gauge tubing secured with mesh. Antibiotic ointment was applied to the wound. To prevent clogging, catheters were flushed every third day with 0.15 ml of gentamicin (120 μg/ml). At baseline and 2 h and 5 h post-CFA, 0.5 ml of blood was withdrawn, and the lost volume was replaced by an equal volume of saline. Blood was centrifuged (1310 g) for 15 min at 4°C. The plasma was collected and stored at -80°C until assayed. ACTH levels were measured with a commercially available ELISA kit (MD Biosciences, Inc.) using the procedure recommended by the manufacturer. The detection limit of the kit is 0.46 pg/ml. The antibody in the kit specifically reacts with ACTH and has less than 1% cross-reactivity with alpha-MSH (Melanocyte-stimulating hormone) and beta-endorphin. ACTH concentrations (pg/ml) were determined by comparing samples to a standard curve generated with the kit.
2.4 Inflammation and hyperalgesia testing
Inflammation was induced by subcutaneously injecting CFA (0.5 mg/ml heat-killed Mycobacterium tuberculosis suspended in an 1:1 oil/saline emulsion; Sigma, St. Louis, MO) into the plantar surface of one hind paw of each rat using a 25-gauge hypodermal needle . Inflammation appeared within 2 h of the injection and peaked between 6–24 h. PWL was tested with a previously described method [12, 13]. Each rat was placed under an inverted clear plastic chamber on the glass surface of the Paw Thermal Stimulator System (UCSD, San Diego) and allowed to acclimatize for 30 min before the test. A radiant heat stimulus was applied to the plantar surface of each hind paw from underneath the glass floor with a projector lamp bulb (CXL/CXR, 8 V, 50 W). PWL to the nearest 0.1 sec was automatically recorded when the rat withdrew its paw from the stimulus. Stimulus intensity was adjusted to derive a baseline PWL of approximately 10.0 s in naive animals. Paws were alternated randomly to preclude "order" effects. A 20-sec cut-off was used to prevent tissue damage. Four tests were conducted, with a 5-min interval between each test. Mean PWL was established by averaging the tests.
2.5 Acupuncture Treatment Procedures
To maximize the anti-inflammatory effect and to treat animals prophylactically, the EA treatment was given twice, for 20 min each, once immediately after the administration of CFA and again 2 h post-CFA . EA parameters of 10 Hz, 3 mA, 0.1 ms pulse width, which showed significant anti-inflammatory and anti-hyperalgesic effects on the rat inflammation model in our previous studies [2, 3], were used in the present study.
The equivalent of the human acupoint Huantiao (GB30) was chosen for bilateral needling based on traditional Chinese medicine (TCM) meridian theory , on its successful use in our previous studies, and on studies by others [3, 15, 16]. Based on our previous point-specificity study , EA produced better anti-hyperalgesia at GB30 than at acupoint Waiguan (the fifth acupoint on the Triple Energizer Meridian, TE 5) on the forepaw or at two non-specific points, an abdominal point and a point on the quadriceps opposite to GB30. Waiguan is located dorsally between the radius and ulna, two units (based on the standard acupuncture measurement of twelve units between the transverse cubital crease and the transverse wrist crease) above the transverse crease of the wrist. Underneath are the posterior interosseous nerve and the anterior interosseous nerve. In humans, GB30 is located at the junction of the lateral 1/3 and medial 2/3 of the distance between the greater trochanter and the hiatus of the sacrum; underneath are the sciatic nerve, inferior gluteal nerve and gluteal muscles . GB30 was located on the rat's hind limbs using the comparable anatomical landmarks. After cleaning the skin with alcohol swabs, a disposable acupuncture needle (0.25 mm thickness × 0.5 inch length) was inserted obliquely approximately 0.5 inch deep at GB30 on each of the animal's hind limbs, and a pair of electrodes was attached to the handles of the needles. The needles and electrodes were stabilized with adhesive tape.
EA stimulation was delivered by an electrical stimulator (A300 Pulsemaster, World Precision Instruments) via an isolator (A360D Stimulus Isolator, World Precision Instruments) that delivers steady direct current. This bilateral, cross-limb connection has been used previously by our team and others with no adverse effects [3, 18]. Similar connections are frequently used in clinic, and no adverse effects have been reported [19, 20].
While the EA frequency was held constant, intensity was adjusted slowly over the period of approximately 2 min to the designated level of 3 mA, which is the maximum EA current intensity that a conscious animal can tolerate . Mild muscle twitching was observed. During EA treatment, each rat was placed under an inverted clear plastic chamber (approximately 5 × 8 × 11 inches) but was neither restrained nor given anesthetic. The animals remained awake and still during treatment and showed no observable signs of distress. For sham treatment control, acupuncture needles were inserted bilaterally into GB30 without electrical stimulation or manual needle manipulation. This procedure produced no anti-hyperalgesic effect on this animal model in our previous study . Since it is comparable to the treatment procedure but lacks therapeutic effect, we used it as sham control in this study.
Rats in experiment 4 were deeply anesthetized after EA treatment with sodium pentobarbital (60 mg/kg, i.p.) and immediately perfused transcardially with 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (PB) at pH 7.4. The hypothalamus was removed, immersed in the same fixative for 2 h at 4°C, and transferred to 30% sucrose (w/v) in PB saline (PBS) overnight for cryoprotection. Forty micron-thick sections were cut on a cryostat and were double-stained for p-NR1 and CRH using the immunofluorescence method. Sections were blocked in PBS with 10% normal donkey serum for 60 min, incubated overnight at room temperature with a mixture of goat polyclonal antibody against CRH (Santa Cruz Biotechnology, Inc., 1:100) and rabbit polyclonal antibody against P-NR1 (Upstate, Serine 896, 1:100). After three 10-minute washings in PBS, sections were incubated in a mixture of CY2-conjugated donkey anti-rabbit (1:100, Jackson ImmunoResearch Laboratories) and CY3-congugated donkey anti-goat (1:100) for 1 h at room temperature. Control sections were similarly processed, except that the primary antisera were omitted. The stained sections were mounted on gelatin-coated slides, coverslipped with aqueous mounting medium (Biomeda Corp., CA) and examined under a Nikon fluorescence microscope. Control sections without primary antiserum showed no immunoreactive staining.
2.7 Data Analysis
For clarity, plasma ACTH level data are presented as percent changes of the baseline level: [(level2–5 h-levelbaseline)/levelbaseline] × 100%. Paw thickness data were presented as mean ± SEM. PWL data are presented as mean ± SEM or (PWL2–5 h - PWLbaseline)/PWLbaseline × 100%. Data were analyzed using analysis of variance (ANOVA) with repeated measures followed by post-hoc Tukey's multiple comparisons (GraphPad InStat). P < 0.05 was set as the level of statistical significance.