In the present study, we demonstrate that one single injection of RTX substantially reduced thermal sensitivity but produced profound and persistent tactile allodynia in adult rats. These symptoms are similar to those seen in patients with PHN [1, 4]. EA at 2 and 15 Hz, but not 100 Hz, significantly recovered the thermal sensitivity of RTX-treated rats 4 weeks after EA treatment. EA at 2 and 15 Hz also significantly decreased the tactile allodynia of RTX-treated rats after 2 weeks of EA treatment. Consistently, EA with 2 and 15 Hz attenuated the loss of TRPV1-positive DRG neurons and the central terminals of afferent fibers in the superficial dorsal horn of RTX-treated rats. EA at 2 and 15 Hz also significantly attenuated the loss of unmyelinated fibers and the damage of the myelinated nerve fibers of RTX-treated rats. Furthermore, EA with 2 and 15 Hz, but not 100 Hz, inhibited the sprouting of myelinated afferent terminals into lamina II of the spinal dorsal horn of RTX-treated rats. Thus, our study suggests that EA improves thermal and mechanical sensitivities in a rat model of PHN by attenuating RTX-induced damage to sensory nerves and associated anatomical plasticity in the peripheral nerve and spinal dorsal horn.
TRPV1 plays an important role in detecting thermal nociception . Resiniferotoxin (RTX), originally isolated from the cactus-like plant Euphorbia resinifera, is an ultrapotent TRPV1 agonist . Systemic injection of RTX ablates TRPV1-expressing sensory neurons and induces a long-lasting impairment of thermal nociception in adult rats [5, 12]. In this study, EA treatment had no significant effect on thermal impairment in RTX-treated rats until 4 weeks after EA treatment. The thermal withdrawal threshold of the hindpaw tested was significantly increased within 2 days after RTX administration, whereas EA was applied only 1 week after RTX treatment when most of TRPV1-positive afferent neurons had been depleted by RTX . EA may not rescue TRPV1-positive DRG neurons already damaged by RTX, which may explain no obvious effect of EA until 4 weeks after treatment. However, at the end of 4 weeks of EA treatment, 2 and 15 Hz EA alleviated the reduction of TRPV1-positive primary sensory neurons and their central terminals in the superficial dorsal horn. Moreover, 2 and 15 Hz EA recovered the loss of unmyelinated fibers caused by RTX. Therefore, EA may improve thermal sensitivity by promoting the regeneration and recovery of TRPV1-positive sensory neurons and their central terminals.
EA at 2 and 15 Hz had a more pronounced effect than 100 Hz EA on recovering TRPV1-expressing unmyelinated afferent terminals damaged by RTX. EA can modulate TRPV1 receptors and thermal sensitivity [13–15]. Previous studies have shown that 2 Hz EA can inhibit the streptozotocin- or NGF-induced thermal hyperalgesia and increase TRPV1 expressions in the spinal cord or hindpaw skin [13, 14]. Also, EA can promote nerve regeneration after the transection of the sciatic nerve and function of denervated muscle tissues after sciatic nerve lesion . Thus, low frequency EA may be more effective than high frequency EA on regeneration of physically or chemically injured sensory neurons and nerves. EA also could promote restoration of sensory function following partial DRG ganglionectomies by enhancing nerve regeneration from the spared DRG [16, 17]. Nevertheless, the biochemical mechanism underlying the beneficial effect of EA in promoting the recovery of sensory neurons and axonal regeneration remains to be explored .
We found that EA at 2 and 15 Hz were more effective than 100 Hz on alleviating RTX-induced tactile allodynia. This is consistent with the report by Hwang et al.  showing that 2 Hz EA relieves the sign of mechanical allodynia in a rat model of neuropathic pain, while 100 Hz EA has no effect . To determine the potential sites of the action of EA, we examined the effect of EA on the peripheral nerve and the topographical projection of myelinated afferent terminals in the spinal dorsal horn. Our analyses showed that EA attenuated ultrastructural damage to the myelinated fibers in the sciatic nerve in RTX-treated rats. This finding suggests that the therapeutic effect of EA may not be confined to the unmyelinated C-fibers. Since TRPV1 receptors are also located in 30% of myelinated A-fiber afferent neurons , damage to this population of myelinated afferent nerves may play an important role in allodynia development induced by RTX. Previous studies have shown that injured myelinated afferent nerves develop ectopic activities that could alter and amplify the sensory input so that an innocuous stimulus could be interpreted as being painful [22, 23]. Thus, these ectopic discharges from damaged myelinated afferents could induce and maintain a state of hypersensitivity of spinal dorsal horn neurons, thereby resulting in allodynia. In our study, 2 and 15 Hz EA significantly reduced the tactile allodynia in RTX-treated rats, which could be explained, at least in part, by attenuating the damage to myelinated afferent fibers by RTX.
Another salient finding of our present study is that 2 and 15 Hz, but not 100 Hz, EA inhibited sprouting of CTB-labeled terminals of myelinated afferent terminals in the spinal lamina II in RTX-treated rats, which is parallel to the inhibitory effect of EA on tactile allodynia. To our knowledge, this is the first study showing that the association of the EA effects on tactile allodynia and sprouting of myelinated afferent fibers in a PHN model. The relationship between sprouting of myelinated fibers and allodynia has been well demonstrated previously. Pain hypersensitivity after chronic constriction nerve injury is associated with the sprouting of myelinated fibers into the lamina II . Moreover, it has been demonstrated that excessive sprouting of myelinated fibers within the dorsal horn after spinal cord injury was associated with neuropathic pain . In our study, using CTB as a tracer for myelinated afferent fibers, we confirmed that myelinated fibers sprouted into lamina II after RTX treatment. Lamina II normally receives mostly nociceptive C-fiber afferent inputs, and the expansion of myelinated afferent fibers into lamina II could alter the peripheral input to produce a painful response to touch [26, 27]. Because 2 and 15 Hz EA inhibited the sprouting of myelinated nerve terminals 5 weeks after EA treatment, EA may reduce tactile allodynia through inhibition of primary afferent nerve sprouting in the spinal lamina II . However, the mechanism through which EA regulates myelinated fiber sprouting remains to be defined. In RTX-treated rats, TRPV1-expressing unmyelinated afferent nerve terminals in the lamina II are largely removed, and the presence of vacant synaptic sites within the superficial dorsal horn may promote sprouting from neighboring intact myelinated terminals [26, 27]. Because EA treatment recovered TRPV1-expressing unmyelinated afferent terminals in RTX-treated rats, EA could decrease vacant synaptic sites and thus minimize axonal sprouting of myelinated afferent fibers. Therefore, EA-produced reorganization of the spinal dorsal horn circuitry may constitute a neuroanatomic basis for the potent therapeutic effect of EA on nociceptive processing in PHN.