4.1 Experimental Design
Four experiments were conducted. In experiment 1, dorsolateral funiculus-lesioned and sham-operated rats were injected with Freund’s adjuvant and randomly divided into the following groups (n=9 per group): 1) dorsolateral funiculus lesion plus EA; 2) dorsolateral funiculus lesion plus sham EA; 3) sham operation plus EA; 4) sham operation plus sham EA. EA was applied at 10 Hz, 3 mA, 0.1 ms pulse width for two 20-minute periods, once at the beginning and once at the end of a 2 hour period starting immediately after a Freund’s adjuvant injection. Paw withdrawal latency to a noxious thermal stimulus was tested at baseline and 20 minutes after the EA treatment to determine thermal hyperalgesia. In experiment 2, rats were grouped into the same four groups as in experiment 1 (n=6 per group), and their spinal cords were removed after EA treatment to measure Fos protein immunoreactivity, a marker of neuronal activation. Rats used in the immunohistochemical study were not tested for paw withdrawal latency in order to avoid possible radiant stimuli-induced effects on Fos expression. In experiment 3, EA was given to a group of rats (n=3) to determine whether EA activates neurons in the supraspinal brain stem, the nucleus raphe magnus and locus coeruleus, and whether EA-induced Fos is co-localized with serotonin in the nucleus raphe magnus and with tyrosine hydroxylase in the locus coeruleus. In experiment 4, EA was given to a group of rats (n=3) in which the retrograde tracer Fluorogold (4% in H2O) was injected into the lumbar spinal cord. The brain stem was removed after EA treatment to determine whether EA-induced Fos is expressed in Fluorogold-labeled neurons in the nucleus raphe magnus and locus coeruleus.
4.2 Dorsolateral funiculus lesion
Male Sprague–Dawley rats weighing 200-220g (Harlan) were kept under controlled conditions (22°C ± 0.5°C, relative humidity 40-60%, 7 am to 7 pm alternate light-dark cycles, food and water ad libitum). Under anesthesia with pentobarbital sodium (50 mg/kg, i.p.), dorsolateral funiculus lesion was performed on the spine at T10–12. The skin at T10–12 was incised, and a laminectomy was performed under a surgical microscope. The dura mater was cut open with iris scissors, and a 0.1 ml 2% lidocaine solution was applied to the dorsal surface of the cord. Five minutes later, bilateral dorsolateral funiculus lesion was performed by cutting a portion of the dorsolateral quadrant of the spinal cord with a sharp knife. The wound was closed with sutures. For sham rats, the dura mater was opened, but the spinal cord was not lesioned. Post-operatively, rats were carefully monitored and rats with motor impairment were excluded from the study. The extent of the spinal lesions was verified in all animals at the conclusion of the experiments.
4.3 Induction of Hyperalgesia
Seven days after dorsolateral funiculus lesioning, complete Freund’s adjuvant (Sigma, St Louis, MO; suspended in an 1:1 oil/saline emulsion, 0.08 ml, 0.04 mg Mycobacterium tuberculosis
) was injected subcutaneously into the plantar surface of one hind paw of each rat using a 25-gauge hypodermic needle (Iadarola et al., 1988
; Lao et al., 2001
; Lao et al., 2004
). The inflammation, manifesting as redness, edema, and hyper-responsiveness to noxious stimuli, was limited to the injected paw, appeared shortly after the injection, and lasted for about two weeks. Hyperalgesia was determined by a decrease in paw withdrawal latency to a noxious thermal stimulus.
4.4 Acupuncture Treatment
To obtain maximum anti-hyperalgesia, two 20-minute EA treatments were given, a prophylactic treatment immediately after the Freund’s adjuvant injection and a second treatment at the end of a 2-hour period starting immediately after a Freund’s adjuvant injection (Lao et al., 2004
). Previously determined EA parameters of 10 Hz at 3 mA and 0.1 ms pulse width, which showed significant anti-hyperalgesic effects on the rat inflammation model (Lao et al., 2001
; Lao et al., 2004
; Zhang et al., 2004
), were chosen for the present study. EA was applied bilaterally to the equivalent of the human acupoint GB 30 (Cheng, 1999
) on the rat’s hind limbs, the negative pole being ipsilateral to the Freund’s adjuvant injection and the positive pole being contralateral.
GB 30 was chosen based on traditional Chinese medicine (TCM) meridian theory (O’connor and Bensky, 1981
) and its successful use in previous studies (Lao et al., 2001
; Lao et al., 2004
; Xu et al., 1993
). In humans, GB 30 is located at the junction of the lateral one-third and medial two-thirds of the distance between the greater trochanter and the hiatus of the sacrum; underneath are the sciatic nerve, inferior gluteal nerve, and gluteal muscles (Cheng, 1999
). Comparable landmarks were used to locate GB 30 in the rats. Our previous study (Lao et al., 2004
) demonstrated that EA at acupoint GB 30 produced significant anti-hyperalgesia, while EA at the point comparable to the human acupoint TE 5 on the foreleg and at sham points, including one at the opposite aspect of GB 30 and one on the abdomen, did not.
EA treatment was given as reported previously (Lao et al., 2004
). Briefly, an acupuncture needle was inserted into GB 30 on each flank of the animal. The needles were connected to an electrical stimulator (A300 Pulsemaster, World Precision Instruments). While frequency was held constant, intensity was adjusted slowly over a period of two minutes to the designated level of 3 mA, which is the maximum intensity 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”) but was neither restrained nor given any anesthetic. The animals remained awake and still, and gave no observable signs of distress during treatment (Lao et al., 2004
For the sham EA control, acupuncture needles were inserted bilaterally into GB 30 without electrical or manual stimulation. This procedure showed little anti-hyperalgesia in our previous study (Lao et al., 2004
) and seems to be an appropriate control for non-specific needling effects.
4.5 Behavioral Testing
Rats were tested for hind paw thermal hyperalgesia by a method developed previously (Hargreaves et al., 1988
; Lao et al., 2004
). Briefly, rats were placed under a clear plastic chamber on the glass surface of the Paw Thermal Stimulator System (UCSD, San Diego) and allowed to acclimatize for 30 minutes. A radiant heat stimulus, directed onto the plantar surface of each hind paw, was applied from underneath the glass floor with a high intensity projector lamp bulb (CXL/CXR, 8 V, 50 W), and paw withdrawal latency to the nearest 0.1 second was automatically determined. A 20-second cut-off was used to prevent tissue damage.
Mean paw withdrawal latencies were established by averaging the latency of four tests with a five-minute interval between each test. Paw withdrawal latency measurements were made pre- dorsolateral funiculus lesion, post-lesioning (pre-Freund’s adjuvant), and 2.5 hours post-Freund’s adjuvant. The investigator who performed the behavioral tests was blind to the treatment assignments.
4.6 Immunohistochemistry and immunofluorescence
Rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused transcardially with 100 ml of saline followed by 500 ml of 4% paraformaldehyde in 0.1 mol/L phosphate buffer at pH 7.4. The lumbar (L4-5) spinal cord was removed, immersed in the same fixative for 2 hours and transferred to a solution of 30% sucrose in a phosphate buffer for overnight cryoprotection. Thirty micrometer-thick sections were cut with a cryostat at −20°C. Free-floating tissue sections were rinsed in phosphate-buffered saline (PBS) with 0.75% Triton X-100 and 1% H2O2 for one hour and then in PBS with 3% normal goat serum (NGS) for 30 minutes. The sections were incubated overnight in primary antiserum against Fos (Oncogene, 1:20,000) and incubated for 1 hour in biotinylated goat anti-rabbit IgG (Vector, 1:400) and then ABC (Vector, 1:200) solution, respectively. Then, the sections were visualized with the nickel-diaminobenzidine method (DAB substrate kit, Vector labs). Tissue sections were washed twice for 20 minutes in PBS between the antibody incubations. Finally, the sections were mounted on gelatin-coated slides, air-dried, dehydrated in graded alcohol, cleared in xylene, and coverslipped with DPX (Electron Microscopy Sciences). Control sections without primary antiserum showed no immunoreactive staining. The Fos-immunoreactive neurons were counted on ten randomly selected sections of laminae I-II and V-VI of each rat; the totals were averaged separately for each animal and then averaged for the group. Similarly, brain stem sections were immunostained for Fos, and five sections were selected to count the Fos-immunoreactive neurons of the RVM and locus coeruleus.
For double immunofluorescence labeling, spinal cord sections were blocked in PBS with 10% normal donkey serum for 60 minutes, incubated overnight at room temperature with a mixture of rabbit polyclonal antibody against Fos (Oncogene, 1:1000) and mouse monoclonal antibodies against TH (Chemicon, 1:500), or goat polyclonal antibody against 5-HT (ImmunoStar, 1:250). 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-mouse (1:400) or CY3-congugated donkey anti-goat (1:500) for 1 hour 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.
For Fos and Fluorogold double labeling, Fluorogold (4%, 0.2 μl) was injected into the lumbar spinal cord 4 days before EA treatment after a laminectomy under anesthesia with pentobarbital sodium (50 mg/kg, i.p.). After EA treatment, the rats were perfused as above. The spinal cord sections were double stained for Fos and Fluorogold with the avidin-biotin-peroxidase (ABC) method. The sections were treated with 2% hydrogen peroxide for 30 minutes to remove the endogenous peroxidase and then incubated in 5% normal goat serum with 0.3% Triton X-100 in PBS for 1 hour. They were incubated overnight in primary antiserum against Fos (Oncogene, 1:15,000) and incubated for 1 hour in biotinylated goat anti-rabbit IgG (Vector, 1:200) and ABC (Vector, 1:100) solution, respectively. Finally they were developed with the nickel-diaminobenzidine method (DAB substrate kit, Vector labs), which results in a black stain. The primary and secondary antibodies were diluted in 5% normal goat serum with 0.3% Triton X-100 in PBS; the ABC was diluted in PBS alone. Tissue sections were washed 3 times, 10 minutes each, in PBS between antibody incubations. All incubations were carried out at room temperature. Fluorogold immunostaining was identical to the above methods, with the exception that the primary antibody was rabbit anti-Fluorogold (Chemicon, 1:5000) and the peroxidase was reacted in diaminobenzidine solution (DAB substrate kit, Vector labs) to yield a brown stain.
4.7 Data Analysis
Paw withdrawal latency data were presented as mean ± SE and analyzed using analysis of variance (ANOVA) with repeated measures followed by Dunnett’s post-hoc comparisons (GraphPad InStat). The immunohistochemical data were analyzed with one-way between-subject ANOVA followed by the Dunnett’s post hoc test. P<0.05 was considered significant in all cases.