Pulsed Radiofrequency Inhibits Expression of P2X3 Receptors and Alleviates Neuropathic Pain Induced by Chronic Constriction Injury in Rats
Neuropathic pain (NP) is a complex and debilitating condition that arises as a direct consequence of a lesion or disease affecting the somatosensory system. It is characterized by spontaneous pain, hyperalgesia, and allodynia, presenting a significant public health challenge. Despite extensive research into the neuroanatomical and neurophysiological mechanisms underlying NP, specific details remain elusive, and no targeted treatments or breakthrough therapies have been developed to date. This has necessitated the exploration of novel treatment strategies, one of which is pulsed radiofrequency (PRF).
PRF is a minimally invasive interventional technique that provides a novel and effective treatment strategy for NP. Unlike continuous radiofrequency (CRF), PRF does not damage nerves and avoids sensory loss after treatment. PRF operates at an emission frequency of 2 Hz, an output of 45 V, and a 500-kHz high-frequency alternating current during each session. The RF current lasts 20 ms with an intervening 480 ms interval, allowing the dispersion of heat generated by the RF current through the nerve tissue. This prevents the temperature at the electrode tip from exceeding 42°C, thereby avoiding the associated degeneration of local tissues. PRF has been widely applied to treat various NP conditions, including trigeminal neuralgia, occipital neuralgia, chronic inguinal neuralgia, and post-herpetic neuralgia. However, the mechanism through which PRF exerts its analgesic effects remains unclear.
An increasing body of evidence suggests that the expression of the P2X ligand-gated ion channel 3 (P2X3) receptor is closely related to NP. The P2X3 receptor, a member of the P2X family, is highly selectively expressed in the peripheral and central axons of the primary afferent sensory neurons associated with nociceptive transmission. During peripheral nerve injury, the nerve releases ATP, which activates the P2X3 receptor on the postsynaptic membrane, leading to membrane depolarization and subsequent rapid synaptic transmission. The released ATP also activates the P2X3 receptor on the presynaptic membrane, resulting in a pain signal that passes through dorsal root ganglion (DRG) neurons and enters the spinal cord via C and Ad nerve fibers, ultimately arriving at the pain center in the brain. Peripheral nerve injury facilitates the release of endogenous ATP to reinforce the activation of P2X3 receptors, amplifying nociceptive signaling. Therefore, activation of the neuronal P2X3 receptor is engaged in the initiation and development of chronic neuropathic pain. Inhibition of P2X3 in the brainstem or the spinal dorsal horn reduces hypersensitivity following nerve injury, suggesting that P2X3 plays a role in pathological pain.
This study aimed to investigate whether the expression of the P2X3 receptor is involved in NP relief due to PRF. A total of 36 healthy adult male Sprague-Dawley (SD) rats were randomly divided into three groups: Sham group, chronic constriction injury (CCI) group, and PRF group. The right sciatic nerve was ligated in the CCI and PRF groups to establish a CCI model, while the right sciatic nerve was separated but not ligated in the Sham group. On day 14 after the operation, PRF was administered to the ligated sciatic nerve in the PRF group (42°C, 45 V, 2 min). A non-live electrode was placed at the exposed sciatic nerve for the rats in the Sham and CCI groups. The hindpaw withdrawal threshold (HWT) and thermal withdrawal latency (TWL) were measured at the right hindpaw at different time points before and after PRF or sham therapy. On day 28 after treatment, the DRG and spinal dorsal horn of the right L4–6 were harvested from each group to determine the mRNA and protein levels of the P2X3 receptor.
Behavioral testing revealed that no significant between-group difference was observed in the HWT before sciatic nerve ligation. On day 7 after ligation, the HWT showed a significant decrease in the CCI and PRF groups, decreasing to 2.71 ± 0.53 g and 2.69 ± 0.60 g, respectively, on day 14 after ligation. No significant difference was found between the CCI and PRF groups, but the HWT in these two groups was significantly lower than that in the Sham group (9.55 ± 0.64 g). Similarly, no significant between-group difference was observed in the TWL before sciatic nerve ligation. On day 7 after ligation, the TWL showed a significant decrease in the CCI and PRF groups, decreasing to 11.42 ± 1.45 s and 11.04 ± 1.54 s, respectively, on day 14 after ligation. No significant difference was found between the CCI and PRF groups, but the TWL in these two groups was significantly lower than that in the Sham group (27.80 ± 1.51 s).
On day 7 after PRF treatment, the HWT was significantly higher in the PRF group than in the CCI group. From day 14 to day 28, the HWT continued to increase in the PRF group and was significantly higher than that in the CCI group. During the first 14 days after PRF treatment, the HWT gradually increased in the PRF group but remained significantly lower than that in the Sham group. From day 21 to day 28 after PRF treatment, no significant difference in the HWT was observed between the PRF and Sham groups. In the PRF group, the TWL gradually increased after PRF treatment and was significantly higher than that in the CCI group on day 7 after treatment. This difference was even greater from day 14 to day 28 after treatment. The TWL increased in the PRF group by day 7 after treatment but remained significantly lower than that in the Sham group. By day 14 after treatment, the TWL increased further in the PRF group but remained lower than that in the Sham group. From day 21 after PRF treatment through the end of the experiment, however, no significant difference was observed in the TWL between the PRF and Sham groups.
RT-qPCR detection of the mRNA expression of the P2X3 receptor revealed that on day 28 after treatment, the relative mRNA expression of the P2X3 receptor in the DRG was 1.12 times higher in the CCI group than in the Sham group. The relative mRNA expression of the P2X3 receptor in the DRG in the PRF group was 23.7% lower than that in the CCI group and 60.3% higher than that in the Sham group. The relative mRNA expression of the P2X3 receptor in the spinal dorsal horns was 1.21 times higher in the CCI group than in the Sham group, and the relative mRNA expression of the P2X3 receptor in the spinal dorsal horns in the PRF group was 22.7% lower than that in the CCI group and 71.3% higher than that in the Sham group.
Western blotting analysis of the protein expression of the P2X3 receptor showed that on day 28 after sham treatment, the relative protein expression of the P2X3 receptor in the DRG was 1.39 times higher in the CCI group than in the Sham group. On day 28 after PRF treatment, the relative protein expression of the P2X3 receptor in the DRG in the PRF group was 27.8% lower than that in the CCI group and 89.6% higher than that in the Sham group. On day 28 after sham treatment, the relative protein expression of the P2X3 receptor in the spinal dorsal horns was 1.75 times higher in the CCI group than in the Sham group. On day 28 after PRF treatment, the relative protein expression of the P2X3 receptor in the spinal dorsal horns in the PRF group was 35.6% lower than that in the CCI group and 76.9% higher than that in the Sham group.
The results of this study suggest that PRF effectively alleviates NP after its administration to the compressed peripheral nerve, with a long-lasting effect. The mechanism might involve down-regulating the expression of the P2X3 receptor in the DRG and spinal dorsal horns, subsequently reducing the transmission of pain signals to the central nervous system. PRF improved hyperalgesia after application to the ligated sciatic nerve in CCI rats, as evidenced by the significant increase in HWT and TWL in the PRF group compared to the CCI group. The mRNA and protein expression levels of the P2X3 receptor in the DRG and spinal dorsal horns of the ligated side of CCI rats were significantly lower in the PRF group than in the untreated CCI group on day 28 after PRF treatment. This suggests that PRF alleviates NP in CCI rats by down-regulating the expression of the P2X3 receptor on the pain pathway, reducing or inhibiting the transmission of pain signals to the central nervous system, and modulating pain transmission.
Despite the significant pain relief observed in the PRF group, the expression of the P2X3 receptor in the DRG and spinal dorsal horns remained significantly higher and pain thresholds were lower in the PRF group than in the Sham group without sciatic nerve ligation. This indicates that one session of PRF treatment with the standard setting has limited effects. Additional research is needed to investigate the effect and mechanism of multiple PRF sessions or PRF treatments with different parameters on NP. Combining PRF treatment with other methods to reduce the expression of the P2X3 receptor on the pain pathway may further improve the efficacy of PRF in alleviating NP.
In conclusion, PRF is a safe and effective treatment for NP, providing significant and lasting pain relief by down-regulating the expression of the P2X3 receptor in the DRG and spinal dorsal horns. This study provides valuable insights into the mechanism of PRF in alleviating NP and highlights the potential of PRF as a novel treatment strategy for NP. However, further research is needed to optimize PRF treatment parameters and explore its potential in combination with other therapeutic approaches to enhance its efficacy in managing NP.
doi.org/10.1097/CM9.0000000000000302
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