The average 50% foot withdrawal threshold of the rat hind paw to von Frey stimuli was the von Frey value of 5.27, equivalent to a bending force of 18.7g, before capsaicin injection. One hour after capsaicin injection, mechanical thresholds were markedly decreased to the von Frey values of 4.25 ± 0.01 (1.79 ± 0.03 g, n=36) at the injection site (primary hyperalgesia; P area in ) and 4.30 ± 0.01 (1.96 ± 0.03 g, n=36) at the base or the proximal part of the third and fourth digits (secondary hyperalgesia; S area in ). The mechanical thresholds remained at significantly reduced levels for up to 4-5 hr following injection, as shown in and .
Fig. 2 The effect of EA applied to different sites on capsaicin-induced secondary hyperalgesia (behavioral tests done by using the site “S” in ). (A) The effects of EA at SI3-TE8 on capsaicin-induced hyperalgesia. Immediately after measurement (more ...)
Fig. 3 The effects of ipsilateral and contralateral EA at SI3-TE8 on capsaicin-induced primary and secondary hyperalgesia. A and B are experiments with ipsilateral EA at SI3-TE8 while C and D are with contralateral EA at the same points. A and C show the threshold (more ...)
3.1 Experiment 1 - Are there any acupoints that can produce antihyperalgesic effects on capsaicin-induced secondary hyperalgesia?
As an initial step, the effective analgesic EA points on capsaicin-induced hyperalgesia were identified by applying EA at various points and then measuring the secondary hyperalgesia. A total of 5 pairs of acupoints (GB30-GB34, BL40-BL60, GV2-GV6, LI3-LI6 and SI3-TE8) were tested. Testing of each pair of acupoints was done on 12 rats, randomly divided into 2 groups of control (n=6) and acupuncture (n=6). On the day of the experiment, rats were placed in the behavioral testing chamber and baseline foot withdrawal thresholds to mechanical stimuli were measured. After measuring baseline thresholds, each rat received a capsaicin injection. One hour after capsaicin injection, mechanical thresholds were measured again and then EA was applied (begun 1.5 hr after capsaicin injection) for 30 min at a chosen pair of points ipsilateral to the capsaicin-injected paw. Behavioral tests were performed at 0.5, 1.0, 1.5 and 2 hr after the termination of acupuncture (up to 4 hr after capsaicin). Control rats received anesthesia only without EA stimulation.
EA at BL40-BL60 significantly increased mechanical thresholds only at the time point of 2 hr after termination of EA compared to the control. EA application to GB30-GB34 and GV2-GV6 failed to show any significant changes in mechanical thresholds compared to the non-EA groups (data not shown). On the other hand, EA at SI3-TE8 points greatly reduced the capsaicin-induced secondary hyperalgesia to von Frey value of 4.9 ± 0.10 (8.00 ± 0.18 g, n=6) at 30 min after termination of EA. This von Frey value is significantly different when compared to the pre-EA values of 4.28 ± 0.12 (1.92 ± 0.61 g, P<0.01) or non-treated control of 4.26 ± 0.15 (1.83 ± 0.65 g, P<0.01). These effects lasted longer than 2 hr (). To examine which one of those 2 points is critical for analgesic effects, EA was applied to a single point, either SI3 or TE8, and the results are shown in . The magnitude of antihyperalgesic effect of EA at SI3 (n=6) is similar to that at TE8 (n=6) but little less than EA to both of the points.
We also tested EA at a pair of LI3 and LI6 on the forelimb. As shown in , EA at LI3-LI6 did not produce any analgesia at all. Accordingly, we chose pairs of SI3-TE8 and LI3-LI6 for EA and EA-control groups, respectively, in the following experiment.
3.2 Experiment 2 - Electroacupuncture at ipsilateral SI3-TE8 induces antihyperalgesic effects on capsaicin-induced secondary hyperalgesia
To further evaluate the effects of EA at SI3-TE8 on capsaicin-induced hyperalgesia, we extended the time for behavior testing of secondary hyperalgesia until EA-induced antihyperalgesic effects dissolve. In addition, the primary hyperalgesia was also measured to explore EA effects on peripheral sensitization. shows the effects of EA at ipsilateral SI3-TE8 on capsaicin-induced primary and secondary hyperalgesia. EA at ipsilateral LI3-LI6 was used as a control group. EA at ipsilateral SI3-TE8 markedly increased the withdrawal thresholds on the secondary hyperalgesia site up to 2 hr after EA compared to the control group (), thus confirming the previous results shown in . On the other hand, there was no significant change on primary hyperalgesia (). EA applied to ipsilateral LI3-LI6 did not change either the primary or the secondary hyperalgesia induced by capsaicin ().
We also examined the effect of EA applied to the contralateral SI3-TE8 or LI3-LI6 points on capsaicin-induced hyperalgesia. In contrast to the antihyperalgeic effect of EA on ipsilateral SI3-TE8, EA at contralateral SI3-TE8 (n=6) did not produce any effect on capsaicin-induced hyperalgesia (). EA at contralateral LI3-LI6 (n=6) had no effect on capsaicin-induced hyperalgesia ().
3.3 Experiment 3 - The endogenous opioid system mediates EA-induced antihyperalgesia
To explore the possible involvement of endogenous adrenergic or opioid system in EA-induced antihyperalgesia, we examined the effect of systemically injected phentolamine (PTL, a non-specific adrenergic receptor antagonist; 5 mg/kg in 5 mg/ml saline) or naltrexone hydrochloride (NTX, a non-specific opioid receptor antagonist; 10 mg/kg in 10 mg/ml saline) on EA-induced antihyperalgesia.
When phentolamine (PTL, 5 mg/kg, i.p., n=5) was administered at 10 min after the beginning of EA to the ipsilateral SI3-TE8 in capsaicin treated rats, there was no significant change in mechanical thresholds as compared to the saline injected control group (n=6) in either primary () or secondary () hyperalgesia. Thus, the results showed that systemic phentolamine failed to block the development of EA-induced antihyperalgesic effect on the secondary hyperalgesia. In addition, when phentolamine was injected during the maintenance period of EA-induced antihyperalgesia (n=8), it did not change EA-induced antihyperalgesic effects as compared to the saline (n=5) control group in either primary () or secondary () hyperalgesia.
Fig. 4 Effect of systemic phentolamine (PTL, a non-specific adrenergic receptor antagonist) on capsaicin-induced primary and secondary hyperalgesia. A and B show the effect of pretreatment of systemic PTL (i.p., 5 mg/kg) on EA-induced antihyperalgesic effect (more ...)
On the other hand, when naltrexone (NTX; a non-specific opioid receptor antagonist) was administered systemically (10 mg/kg, i.p., n=6) 10 min after the initiation of EA, EA-induced antihyperalgesic effect on the secondary hyperalgesia was completely blocked (). When naltrexone was administered after EA-induced antihyperalgesia was completely developed (0.5 hr after the termination of EA), it showed a slightly delayed inhibition of the EA-induced antihyperalgesic effect (n=6, ). Naltrexone did not show any change in capsaicin-induced primary hyperalgesia (). These data suggest that the endogenous opioid system is involved in mediating EA-induced antihyperalgesic effects on capsaicin-induced secondary hyperalgesia.
Fig. 5 Effect of systemic naltrexone (NTX; a non-specific opioid receptor antagonist) on capsaicin-induced primary and secondary hyperalgesia. A and B show the effect of pretreatment of systemic NTX (i.p., 10 mg/kg) on EA-induced antihyperalgesic effect on the (more ...)
3.4 Experiment 4 - The spinal opioid system is critical for EA-induced antihyperalgesia
Since it was determined that the endogenous opioid system is involved in the EA-induced antihyperalgesic effect, the possible involvement of the spinal opioid system was tested by injecting naltrexone intrathecally. In capsaicin-treated rats, EA was applied at ipsilateral SI3-TE8 (n=15) and then NTX (10 μg in 30 μl saline, n=8) was injected intrathecally 10 min before the termination of EA. The remaining 7 rats received saline intrathecally instead of NTX. Mechanical thresholds were measured for up to 4 hr after termination of EA. EA at SI3-TE8 did not affect primary hyperalgesia in either the NTX or saline groups (). Intrathecal NTX almost completely inhibited EA-induced antihyperalgesic effects on secondary hyperalgesia (). The data suggest that antihyperalgesic effects of EA on capsaicin-induced secondary hyperalgesia are mediated mainly by the endogenous spinal opioid system.
Fig. 6 Effects of intrathecal naltrexone on capsaicin-induced primary and secondary hyperalgesia. A and B show the effect of intrathecal NTX (10 μg in 30 μl saline) on EA-induced antihyperalgesic effect on the primary (A) and secondary (B) hyperalgesia. (more ...)
3.5 Experiment 5 - μ- and δ-opioid receptors are involved to mediate EA-induced antihyperalgesia
To explore which subtypes of opioid receptors are involved in mediating EA-induced antihyperalgesic effects in the spinal cord, we examined the effects of intrathecally injected specific μ-, δ- and κ-opioid receptor antagonists on EA-induced antihyperalgesia. The drugs used include a μ-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP; 3 μg), a κ-opioid receptor antagonist, nor-binaltorphimine dihydrochloride (nBNI; 7.3 μg) and a δ-opioid receptor antagonist, naltrindole hydrochloride (NTI; 9 μg). The test set for each drug consisted of 12 rats randomly divided into 2 groups of drug treatment (n=6) and saline control (n=6). Each drug was dissolved in saline and administered in a volume of 30 μl and injected intrathecally 10 min before termination of EA at SI3-TE8 in capsaicin-injected rats. The same volume of saline was injected in control groups. The results are shown in . The intrathecal injection of CTAP almost completely blocked the development of antihyperalgesic effect by EA on secondary hyperalgesia (). The κ opioid receptor antagonist, nBNI, did not influence the EA-induced antihyperalgesic effects () but a δ opioid receptor antagonist, NTI, reduced EA-induced antihyperalgesic effects moderately for about one hour as compared to the saline control group (P<0.01, ). EA at SI3-TE8 did not change primary hyperalgesia in either compounds or saline groups (). These data suggest that EA analgesia on capsaicin-induced secondary hyperalgesia is mediated by spinal μ- and δ-opioid receptors, but not by κ-opioid receptors.
Fig. 7 Effects of intrathecal injection of subtypes of opioid antagonists on capsaicin-induced primary and secondary hyperalgesia. A and B show the effect of intrathecal D-Phe-Cys-Tyr-DTrp-Arg-Pen-Thr-NH2 (CTAP; μ-opioid receptor antagonist, 3 μg), (more ...)