A total of 96 adult male Sprague-Dawley rats (8–10 weeks; weighing 210–250 g) were provided by the animal center of Anhui Medical University (Hefei, China) and housed in the animal facility for 3–4 days prior to experiments. All rats were fed with a 12-h light/dark cycle at a constant room temperature of 22±2°C and relative humidity of 60–80%. The animals had access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Experimental Ethics Committee of Anhui Medical University. All procedures were performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Anhui Medical University.

A total of 96 rats were randomly divided into four groups (n=24 in each): Normal saline (NS), RF, RF + EA (RF/EA) and RF + sham acupuncture (RF/EA-sham). Rats were anesthetized with 30 mg/kg pentobarbital intraperitoneally. The Huantiao and Yanglingquan acupoints or corresponding sham acupoints were stimulated by EA in the RF/EA and RF/EA-sham groups, respectively, during incision and medication procedures. Plantar incisional pain was induced in each group. As appropriate, NS (0.8 ml/h for 60 min) and RF (Yichang Renfu Pharmaceutical, Yichang, China; batch no. 6130502; 0.08 mg/kg at 0.8 ml/h for 60 min) (25,26) were injected intravenously with a pump (Fig. 1).

Stainless steel acupuncture needles (0.18×30 mm) were inserted 5 mm into the right hind leg at the Huantiao (GB30, posterior upper edge of the hip joint) and Yanglingquan (GB34, 5 mm below capitulum fibulae) acupoints, as previously described (27,28) (Fig. 2). Stimulation was performed with a constant current pulse generator model EL-608 (NKL Electronic Products, Brusque, Brazil) for ~90 min (prior to incision until the end of RF administration). The stimuli were set as 0.3 msec wide square waves at a frequency of 2 Hz. Current intensity was increased in a stepwise fashion until a muscle twitch was observed (~1 mA at 2 Hz) as described previously (29,30). Rats in the RF/EA-sham group underwent the same procedure but needles were inserted 0.5 cm right to the correct acupoints (31).

Plantar incision was performed as previously described by Brennan (32). Following sterilization of the right hind paw with 10% iodophor (Aitefu Co., Ltd., Huai'an, China), a 1 cm longitudinal incision was made through the skin and fascia of the plantar aspect, starting 0.5 cm from the proximal edge of the heel and extending toward the toes. The plantaris muscle was elevated and incised longitudinally. The muscle origin and insertion remained intact. Following hemostasis with gentle pressure, the skin was apposed with mattress sutures. The wound was covered with an ointment containing polymyxin B, neomycin and bacitracin (Zhejiang Reachall Pharmaceutical Co., Ltd., Dongyang, China) (32).

Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed 24 h prior to RF infusion and at 4, 12, 24 and 48 h following the completion of RF infusion. Rats were placed in individual wire cages with a mesh bottom and allowed to adapt for 60 min prior to testing.

Mechanical hyperalgesia was assessed using an electronic Von Frey filament (Harvard Apparatus, Holliston, MA, USA) as described by Yuan et al (33). The filament was applied vertically to the area adjacent to the wound on the right hind paw and pressure was increased until a positive response occurred. The effective pressure was then as the PWT. The test was repeated three times at 5-min intervals. A positive response was defined as clear paw withdrawal, licking or squeaking. A cutoff pressure of 60 g was used to prevent tissue damage.

Thermal hyperalgesia was measured using YLS-6B intelligent hotplate equipment (Zhenghua Biologic Apparatus Facilities Co., Ltd., Huaibei, China) as described by Yuan et al (33). Rats were placed on a 50°C hotplate until a positive response was observed. The response time was recorded as the PWL. The test was repeated three times at 10-min intervals. A positive response was defined as a clear paw withdrawal and a cutoff time of 40 sec was used to prevent tissue damage.

Following RF infusion and behavioral tests, a total of 6 rats per group were sacrificed at each time point. TNF-α (cat. no. SC-52746), IL-1β (cat. no. SC-12742) and IL-6 (cat. no. SC-57315) levels were measured in the spinal cord at lumbar segments (L_4–5) using ELISA kits (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) according to the manufacturer's protocol.

At 4, 24 and 48 h following the completion of RF infusion, following behavior testing, 2 rats were anesthetized with an intraperitoneal injection of 350 mg/kg chloral hydrate. The chest was opened and the right atrium was cut, 200 ml saline was perfused rapidly into the left ventricular at 4°C and then the right atrium was perfused with 200 ml paraformaldehyde at 4% for 6 h. The spinal arch plate was cut off, the spinal cord was exposed, and L_4–5 lumbar segments were dissected and removed. Specimens were fixed in 4% paraformaldehyde at room temperature for 24 h and then embedded in paraffin. Each paraffin-cut section was 4 mm in thickness. CD11b expression was measured in the spinal cord at lumbar segments (L_4–5) using immunostaining with the primary antibody OX-42 (cat. no. ab33827; 1:50; Abcam, Cambridge, MA, USA) at 4°C for 12 h. Spinal cord sections were washed and incubated for 30 min at 37°C with horseradish peroxidase-labeled secondary antibodies (cat. no. PV-6000; 1:50; OriGene Technologies, Inc., Beijing, China). A total of 6–10 images were captured for each sample using an inverted microscope with a magnification of ×600. The area of positive staining for CD11b was assessed using computerized morphometry (Image-Pro Plus software version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, USA). Differences were assessed using one-way analysis of variance with a post hoc least-significant difference test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

PWT and PWL values were similar in all groups prior to infusion and decreased gradually following infusion, with the lowest values at 24 h (Fig. 3). PWT and PWL values were significantly decreased in the RF and RF/EA-sham groups at 4, 12, 24 and 48 h following RF infusion compared with the NS group (all P<0.05; Fig. 3), indicating RF-induced hyperalgesia. Higher PWT and PWL values were observed in the RF/EA group compared with the RF/EA-sham group at 4, 12, 24 and 48 h following RF infusion (all P<0.05; Fig. 3). These findings suggest that EA alleviates RF-induced hyperalgesia. No significant differences in PWT and PWL values were observed between the NS and RF/EA groups, or between the RF and RF/EA-sham groups. These results suggest suggested that RF-induced hyperalgesia was almost completely reversed following after EA treatment, while sham EA had no effect.

CD11b levels in the RF and RF/EA-sham groups were significantly increased compared with those of the NS group at 4, 24 and 48 h following RF infusion (all P<0.05; Fig. 4), which indicates that RF treatment increased the amounts of spinal microglia. Compared with the RF group, CD11b levels were significantly decreased at 4, 24 and 48 h following RF infusion in the RF/EA group (all P<0.05; Fig. 4), suggesting that EA decreased the number of spinal microglia. No significant differences in CD11b expression were observed between the NS and RF/EA groups or between the RF and RF/sham groups at any time point. These results demonstrate that EA treatment is able to completely recover the number of spinal microglia following RF infusion, while sham EA has no significant effect.

TNF-α, IL-1β and IL-6 levels in the spinal cord at lumbar segments (L_4–5) were significantly higher in the RF and RF/EA-sham groups compared with the NS control group at 24 and 48 h following RF infusion (P<0.05; Fig. 5), indicating that RF-induced hyperalgesia was associated with spinal inflammation. Furthermore, TNF-α, IL-1β and IL-6 levels in the RF/EA group were significantly reduced compared with those of the RF/EA-sham group at 24 and 48 h following infusion (P<0.05; Fig. 5), which suggests that EA suppresses the inflammatory response in the spine. No significant differences were observed in TNF-α, IL-1β or and IL-6 levels between the NS and RF/EA groups at any time point. These results suggest that EA completely suppresses the inflammatory response in the spine following RF infusion.