Electroacupuncture-induced analgesia in a rat model of ankle sprain pain is mediated by spinal alpha-adrenoceptors

Abstract

In a previous study, we showed that electroacupuncture (EA) applied to the SI-6 point on the contralateral forelimb produces long-lasting and powerful analgesia in pain caused by ankle sprain in a rat model. To investigate the underlying mechanism of EA analgesia, the present study tested the effects of various antagonists on known endogenous analgesic systems in this model. Ankle sprain was induced in anesthetized rats by overextending their right ankle with repeated forceful plantar flexion and inversion of the foot. When rats developed pain behaviors (a reduction in weight-bearing of the affected hind limb), EA was applied to the SI-6 point on the contralateral forelimb for 30 min under halothane anesthesia. EA significantly improved the weight-bearing capacity of the affected hind limb for 2h, suggesting an analgesic effect. The alpha-adrenoceptor antagonist phentolamine (2mg/kg, i.p. or 30 microg, i.t.) completely blocked the EA-induced analgesia, whereas naloxone (1mg/kg, i.p.) failed to block the effect. These results suggest that EA-induced analgesia is mediated by alpha-adrenoceptor mechanisms. Further experiments showed that intrathecal administration of yohimbine, an alpha(2)-adrenergic antagonist, reduced the EA-induced analgesia in a dose-dependent manner, whereas terazosin, an alpha(1)-adrenergic antagonist, did not produce any effect. These data suggest that the analgesic effect of EA in ankle sprain pain is, at least in part, mediated by spinal alpha(2)-adrenoceptor mechanisms.

Fig. 1

Schematic drawings showing the locations of sprained ankle and electroacupuncture (EA). (A) The sprain was produced on the right ankle, and EA was applied to the acupoints on the left forelimb. (B) A detailed view of the left forelimb showing the locations of EA application. EA was applied with a stimulation needle set consisting of two acupuncture needles separated by 1 mm, and electrical current was applied between the two needles. The SI-6 point was used as the main point, and the LI-4 point was used as a control (sham stimulation) point.

Fig. 2

Effects of EA in rats with sprained ankles. Graphs show analog output for the stepping force of the forelimb (F) and hind limb (H) on one side (the right side) of two rats (A, B, C for rat #1 and D, E, F for rat #2) obtained during walking. Weight-bearing force (WBF) is expressed as a percentage of each rat's body weight. In normal conditions (A, D), the hind limb bore about 50−60% of the body weight (height labeled as “a”). One day after ankle sprain (B, E), the WBF of the hind limb with the sprained ankle was reduced to about half (height labeled as “b”). One hour after a 30-minute application of EA to the SI-6 point, WBF significantly recovered in rat #1 (C, height labeled as “c”), whereas the same EA applied to the LI-4 point had no effect in rat #2 (F). For group data treatment, percentage maximum recovery value after EA was calculated as follow using the height values labeled as “a–c” in panels A–C: percentage maximum recovery = ([c – b]/[a – b]) × 100. Therefore, recovery is 100% (full) when the height of c reaches that of a, and is 0% (no recovery) when the height of c remains equal to that of b.

Fig. 3

Group data on EA-induced analgesia on ankle sprain pain expressed as percentage maximum recovery. Graphs are plotted for the average values (± SEM) of percentage of maximum recovery value (100% means full recovery of weight-bearing force [WBF] to pre-ankle sprain levels). (A) Comparison of effects of EA applied to the SI-6 and LI-4 points in two groups of rats (10 rats in each group). EA was applied 1 day after ankle sprain. EA to the SI-6 point (for 30 minutes) produced significant recovery of WBF to about 40% of full recovery lasting about 2 hours. On the other hand, identical EA applied to the LI-4 point produced no recovery of WBF. An asterisk (*) indicates a value significantly (p < 0.05) different from the equivalent value with LI-4 stimulation by the two-way repeated measures ANOVA (time factor repetition) followed by Tukey multiple comparisons. (B) Comparison of effects of EA applied to the SI-6 points repeated in two consecutive days (1st and 2nd post-sprain days) on a group of 7 rats. Two EA applications produced a comparable recovery of WBF (group factor with 2-way repeated-measures ANOVA [two-factor repetition]: F = 3.062, p = 0.131).

Fig. 4

Effects of systemic application of α-adrenergic and opioid receptor antagonists on EA-induced analgesia in rats with sprained ankles. Graphs are plotted for the average values (± SEM) of percentage of maximum recovery value. (A) Effects of an α-adrenergic receptor antagonist on EA-induced analgesia (n = 8 rats). Phentolamine (2 mg/kg in 0.2 ml saline) or the same volume of saline was given intraperitoneally immediately after termination of EA (applied to the SI-6 point on the contralateral forelimb for 30 minutes). The experiment was done with the cross-over design so that all 8 rats received both phentolamine and saline alone within a two-day span (1st and 2nd post-sprain days). There was no significant improvement in weight bearing force (WBF) in the phentolamine-injected group, whereas the saline injection group showed a long-lasting recovery of WBF. An asterisk (*) indicates a value significantly (p < 0.05) different from the equivalent value after phentolamine injection, using cross-over repeated ANOVA with the least square method. (B) Effects of an opioid antagonist on EA-induced analgesia in rats with sprained ankles (n = 7 rats). Either naloxone (1 mg/kg in 0.2 ml of saline) or the same volume of saline was given intraperitoneally immediately after termination of EA (applied to the SI-6 point on the contralateral forelimb for 30 minutes). The experiment was done with the cross-over design so that all 7 rats received both naloxone and saline in the two-day span (1st and 2nd post-sprain days). Both the saline and naloxone group showed a long-lasting recovery of WBF following EA treatment (group factor with cross-over repeated ANOVA: F = 0.62, p = 0.443).

Fig. 5

Effects of intrathecal application of α-adrenergic receptor antagonists on EA-induced analgesia in rats with sprained ankles. Graphs are plotted for the average values (± SEM) of percentage of maximum recovery value. A: Effects of intrathecal application of a mixed α-adrenergic receptor antagonist on EA-induced analgesia (n = 7 rats). Phentolamine (30 μg in 10 μl of saline) or the same volume of saline was given intrathecally (through an implanted catheter) immediately after termination of EA (applied to the SI-6 point on the contralateral forelimb for 30 minutes). The experiment was done with the cross-over design so that all 7 rats received both phentolamine and saline alone in a two-day span (1st and 2nd post-sprain days). There was no significant improvement in weight bearing in the phentolamine-injected group, whereas the saline injection group showed a long-lasting recovery of WBF. An asterisk (*) indicates a value significantly (p < 0.05) different from the equivalent value after phentolamine injection, using cross-over repeated ANOVA with the least square method. (B) Effects of intrathecal application of specific α₁- and α₂-adrenergic receptor antagonists on EA-induced analgesia (n = 8 rats). Yohimbine (an α₂-adrenoceptor blocker, 10 μg/10 μl of saline) or terazosin (an α₁-adrenoceptor blocker, 10 μg/10 μl of saline) was given intrathecally through an implanted catheter immediately after termination of EA (applied to the SI-6 point on the contralateral forelimb for 30 minutes). The experiment was done with the cross-over design so that all 8 rats received both yohimbine and terazosin in a two-day span (1st and 2nd post-sprain days). There was no significant improvement of weight bearing in the yohimbine-injected group, whereas the terazosin injection group showed a long-lasting recovery of WBF. An asterisk (*) indicates a value significantly (p < 0.05) different from the equivalent value after yohimbine injection, using cross-over repeated ANOVA with the least square method.