Pulsed radiofrequency (PRF) is a popular pain treatment modality. It is a technique in which electromagnetic waves (20-millisecond pulses of 500 kHz) are applied close to the dorsal root ganglion (DRG) or the sensory nerve to increase the mean temperature to a maximum of 42°C. This technique was introduced by Sluijter1 with the aim of dissociating the effect of electromagnetic waves from the thermal destruction that is caused by continuous radiofrequency. Clinical reports have described successful treatment episodes that have produced pain relief for weeks or months after the application of PRF current.2,3 Van Zundert et al.4 have suggested that PRF procedures of the cervical DRG may provide pain relief for patients with chronic cervical radicular pain. However, Erdine et al.5 have demonstrated that PRF current is not an effective method of pain treatment for idiopathic trigeminal neuralgia. Thus, the effect of PRF current on neuropathic pain is controvertible.
Postherpetic neuralgia (PHN) is neuropathic pain with skin changes in a dermatomal distribution subsequent to acute herpes zoster, which can produce degenerative change in primary neurons in the DRG.6 PHN is characterized by deep aching or burning pain and profound mechanical allodynia. Allodynia, produced by innocuous cutaneous stimuli, such as touch, is present in the majority of patients with PHN; it may persist indefinitely and can be the most unbearable part of PHN for many patients. It is always resistant to known treatments, such as nonsteroidal antiinflammatory drugs.6 Considerable efforts have been made to improve the medical and surgical approaches to treat PHN. Nerve blocks are effective for the relief of acute herpes zoster pain and for a reduction in the incidence of PHN.7 However, once PHN is established, invasive local anesthetic blocks usually provide no more than temporary relief. At present, treatment strategies for PHN include the use of tricyclic antidepressants, anticonvulsants, opioids, and the topical application of lidocaine.6 Additionally, spinal cord stimulation seems to offer a therapeutic option for pharmacological nonresponders.8
Rodent models of neuropathic pain display increased sensitivity to both thermal and mechanical stimuli after peripheral nerve injury. However, paradoxical changes in thermal and mechanical sensitivity are present in patients with small fiber neuropathies, in particular, PHN.9,10 Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been used to study the action of nociceptive C fiber afferents.11 Pan et al.12 have found that depletion of TRPV1-sensitive unmyelinated afferents with RTX produces long-lasting paradoxical changes that diminish the thermal sensitivity but increase the sensitivity to tactile stimulation in adult rats, which mimic the unique clinical feature of PHN.
The objective of this study was to investigate the effect of the PRF current on mechanical allodynia induced with RTX in rats, especially regarding the influence of the duration of allodynia before PRF procedures and that of exposure time to PRF.
After receiving approval from the Animal Care Committee of the University of Miyazaki, rats were individually housed in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle and were permitted free access to food and water. Adult male Sprague-Dawley rats weighing 250 to 400 g were divided into the groups described below.
In the first series of experiments, we investigated the influence of PRF on mechanical allodynia according to the duration of allodynia before PRF procedures. Rats in group S2 (n = 5) were assigned to receive PRF current to the right sciatic nerve for 2 minutes 1 week after RTX treatment (short duration of allodynia before PRF treatment); rats in group M2 (n = 6), PRF current for 2 minutes 3 weeks after RTX treatment (medium duration of allodynia before PRF treatment); and rats in group L2 (n = 7), PRF current for 2 minutes 5 weeks after RTX treatment (long duration of allodynia before PRF treatment).
In a second series of experiments, we investigated the influence of PRF on mechanical allodynia according to the PRF exposure time. In addition to group S2, rats in group S4 (n = 5) were assigned to receive PRF current to the right sciatic nerve for 4 minutes 1 week after RTX treatment; rats in group S6 (n = 5), PRF current to the right sciatic nerve for 6 minutes 1 week after RTX treatment; and rats in group S0 (n = 3), no PRF current was delivered. Instead, the needle and electrode were inserted at proper points for 6 minutes 1 week after RTX treatment.
RTX (LC Laboratories, Woburn, MA) was dissolved by a mixture of 10% Tween-80 and 10% ethanol in normal saline. Rats received a single intraperitoneal injection of RTX in a dose of 200 μg/kg under 2% to 3% sevoflurane anesthesia. Rats were transferred to their cages after the recovery period.
All rats were evaluated for sensitivity to mechanical stimulation with von Frey (VF) filaments (Stoelting, Wood Dale, IL) and for sensitivity to thermal stimulation with a thermal testing apparatus (UGO Basile, Comerio, Italy) by an experienced observer who was unaware of the treatment. This was done before and 1 week after each RTX treatment and 1, 2, 3, 4, and 5 weeks after the PRF procedures.
Mechanical sensitivity was examined by testing the paw withdrawal threshold with VF filaments. The paw withdrawal threshold with VF filaments was determined by placing each rat in suspended chambers on a mesh floor. After an acclimation period of 30 minutes, a series of calibrated VF filaments was applied perpendicularly to the plantar surface of the right and left hindpaws with sufficient force to bend the filament for 5 seconds. Brisk withdrawal or paw flinching was considered a positive response. In the absence of a response, the filament of next greater force was applied. After a response, the filament of next lower force was applied. The tactile stimulus producing a 50% likelihood of withdrawal response was calculated using the up-down method.13 The threshold evaluated with VF filaments <4.00 g was assumed to indicate allodynia.13
Thermal sensitivity was examined by testing paw withdrawal latency in response to noxious heat stimulation. Paw withdrawal latency was determined by placing each rat in an individual Plexiglas enclosure on a transparent glass. After a 30-minute acclimation period, the heat-emitting projector lamp of a thermal testing apparatus was activated after focusing the beam directly onto the plantar surface of the hindpaw. A built-in digital timer was used to record the paw withdrawal latency. The mean value of the withdrawal latency on 3 consecutive trials was calculated. A cutoff of 30 seconds was used to avoid potential tissue damage.
In addition, all rats were evaluated for motor function using placing and grasping reflexes before and 1 week after each RTX treatment and 1, 2, 3, 4, and 5 weeks after the PRF procedures.14 To elicit the placing reflex, the dorsal surface of the hindpaws was brought in contact with the edge of a tabletop and the reflex response of placing the hindpaw on the tabletop was recorded. To elicit the grasping reflex, hindpaws were placed on a wire grid and the grasping response was recorded. Five trials were given for both tests.
Rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, Abbott Park, IL) dissolved at 50 mg/mL; the anesthetic was administered to the rats in a dose of 50 mg/kg. They were then positioned prone on the operation table, and the lumbar skin was prepared with an antiseptic solution. The indifferent electrode connected to a radiofrequency generator was attached to the abdominal skin. All rats were unilaterally treated at 1 sciatic nerve on the right side. A 54-mm, 22-gauge guiding needle with a 4-mm activate tip (Ac-4; Hakko, Tokyo, Japan) was introduced percutaneously at an anatomically defined region known as the sciatic notch. This location (between the greater trochanter and ischial tuberosity) has been used for neurobehavioral experiments.15 After the puncture, the stylet of the needle was replaced with a radiofrequency probe, tissue impedance was measured, and the presence of muscle contractions was checked using 3-Hz electrical stimulation to a maximum of 1.0 V. If muscle contractions were observed with a lower output than 0.5 V, the electrode was pulled back 1 mm. If muscle contractions were observed with a higher output than 1.0 V or no contraction was observed, the electrode was advanced 1 mm. The procedure was repeated until muscle contractions were observed with proper output between 0.5 and 1.0 V. This criterion indicates that the electrode was near the sciatic nerve but did not penetrate it.16 After proper electrode placement, the PRF procedures were performed. A radiofrequency generator with standard clinical specifications (model JK3; RDG Medical, Surrey, UK) was used. PRF current was applied in 20-millisecond pulses every 500 milliseconds (20 milliseconds of 500-kHz RF pulses, delivered at a rate of 2 Hz). The maximum temperature was automatically controlled to 42°C.
The last behavioral tests were performed 5 weeks after PRF procedures, and rats were killed with an intraperitoneal injection of a lethal dose (150 mg/kg) of sodium pentobarbital.
Within each group, the results of repeated measurements were analyzed by analysis of variance for repeated measures, followed, where appropriate, by Scheffé test. PRF-treated hindpaw and PRF-untreated hindpaw data were compared using a 2-tailed paired t test. We compared the ipsilateral–contralateral paw withdrawal threshold among the 3 groups in each series of experiments. Comparisons among the 3 groups and the 6 groups according to baseline data were made by using 1-way analysis of variance, followed by Scheffé test. All tests were conducted using StatView software (SAS Institute, Inc., Cary, NC). Data are expressed as means ± SEM. P < 0.05 was considered statistically significant.
The mean impedance values were 443 ± 22 Ω in group S2, 535 ± 79 Ω in group M2, 433 ± 18 Ω in group L2, 518 ± 72 Ω in group S4, 489 ± 16 Ω in group S6, and 533 ± 81 Ω in group S0, and there was no significant difference among the 6 groups.
Rats injected with RTX exhibited immediate behavioral reactions, including hyperexcitability and restlessness. However, these reactions were transient and gradually subsided within 1 to 3 hours.
The paw withdrawal thresholds of both hindpaws 1 week after RTX treatment were significantly lower than the pre-RTX baseline in all groups (Figs. 1 and 2). The paw withdrawal threshold of the right hindpaw before PRF procedures (time 0) was 4.2 ± 1.1 g in group S2, 2.7 ± 0.6 g in group M2, 2.1 ± 0.3 g in group L2, 4.2 ± 0.4 g in group S4, 4.0 ± 0.8 g in group S6, and 4.0 ± 0.6 g in group S0, and there was no significant difference among the 6 groups.
The placebo PRF in group S0 had no effect on both ipsilateral and contralateral paw withdrawal threshold with VF filaments. In groups S2 and M2, after PRF procedures, ipsilateral paw withdrawal thresholds significantly increased (Fig. 1). A statistically significant difference was detected between the PRF-treated and PRF-untreated hindpaws from 1 week to 5 weeks after PRF procedures. Compared with the pre-RTX baseline, no significant difference was found 4 and 5 weeks after PRF procedures on the PRF-treated right hindpaw in group S2. However, in group L2, no increase of the ipsilateral paw withdrawal thresholds after PRF procedures was observed, and no significant difference was observed between the PRF-treated and -untreated hindpaws.
The ipsilateral–contralateral paw withdrawal thresholds in group S2 were significantly higher than those in group L2 after PRF procedures (Fig. 3). These thresholds in group S2 were also significantly higher than those in group M2 1, 3, 4, and 5 weeks after PRF procedures. Between groups M2 and L2, a statistically significant difference was found 1, 2, 4, and 5 weeks after PRF procedures.
In groups S4 and S6, after PRF procedures, ipsilateral paw withdrawal thresholds significantly increased (Fig. 2). A statistically significant difference was detected between the PRF-treated and PRF-untreated hindpaws from 1 week to 5 weeks after PRF procedures. Compared with the pre-RTX baseline, no statistically significant difference was found from 2 weeks to 5 weeks after PRF procedures in group S4 and from 1 week to 5 weeks after PRF procedures in group S6.
The ipsilateral–contralateral paw withdrawal thresholds in group S6 were significantly higher than those in groups S2 and S4 5 weeks after PRF procedures (Fig. 4). No significant difference was found between groups S2 and S4 at any time.
In response to heat stimulation, the withdrawal latency of both tested hindpaws was increased within 1 week after RTX treatment. After PRF procedures, no statistically significant difference in the withdrawal latency after heat stimulation was detected between the PRF-treated right and PRF-untreated left hindpaws through the entire period of the study in all groups (Fig. 5).
In all groups, placing reflex scores and grasping reflex scores of the PRF-treated right and PRF-untreated left hindpaws were 5.0 ± 0.0 and 5.0 ± 0.0, and 5.0 ± 0.0 and 5.0 ± 0.0, respectively, through the entire period of the study.
The goal of this study was to investigate the effect of PRF current on mechanical allodynia induced with RTX in rats, especially to examine the influence of the duration of allodynia before PRF procedures and the influence of the PRF exposure time. PRF current applied for 2 minutes 1 week after RTX treatment showed a predominant increase in the ipsilateral–contralateral paw withdrawal thresholds, suggesting that PRF current has an antiallodynic effect. However, this antiallodynic effect of PRF current weakened gradually with the delay of PRF procedures. Increasing the exposure time to PRF significantly increased the ipsilateral–contralateral paw withdrawal thresholds, and PRF current did not result in major motor impairment of the PRF-treated right hindlimb. Therefore, PRF current applied for 6 minutes was the most effective way to reverse RTX-induced allodynia induced without motor impairment in this study.
In this study, we used RTX-treated rats as a model of PHN. A single intraperitoneal injection of RTX substantially reduced thermal sensitivity but increased mechanical sensitivity in adult rats12; similar sensitivity is observed in patients with PHN. The reduced thermal sensitivity by RTX is attributed to depletion of TRPV1-sensitive afferent neurons and fibers, and the sustained mechanical allodynia by RTX is likely caused by damage to myelinated afferent fibers and their abnormal sprouting into the spinal lamina II.12 Chen and Pan17 have demonstrated that gabapentin administered systemically and spinally can effectively relieve mechanical allodynia in this PHN model.
To re-create true clinical conditions, we applied PRF through the needle introduced percutaneously. Impedance could exceed 1000 Ω if PRF is applied to the exposed nerve or exposed DRG.4 A high impedance would result in a diminished biological effect of the pulsed electric field.1 Although percutaneous PRF application has the possibility of causing nerve injury, in the present study, impedance had a value of approximately 500 Ω, close to that in a clinical procedure, and motor paralysis caused by nerve injury was not detected.
Controlled or uncontrolled clinical reports of the treatment of neuropathic pain have described the use of PRF current for 2 minutes.5,18 However, a few studies with longer exposure time have also been reported.19,20 Kim et al.19 and Liliang et al.20 arbitrarily chose a PRF exposure time of 6 minutes. The optimal exposure time of PRF is unknown, and the exposure time is arbitrarily chosen in clinical procedures. Therefore, we compared 2, 4, and 6 minutes of PRF current and found that PRF exposure for 6 minutes was the most effective for mechanical allodynia. Özsoylar et al.21 compared 2 and 6 minutes of percutaneous PRF and reported that the application for 2 minutes effectively reduced neuropathic pain symptoms. However, they found no significant antiallodynic effect from the 6-minute application. The discrepancy between their study and the present study could be attributed to the animal models used and parts exposed to PRF current. Özsoylar et al.21 applied PRF current percutaneously to the allodynia-detected hindpaw, which is far from the DRG. We applied PRF adjacent to the sciatic nerve at an anatomical sciatic notch. To our knowledge, the present study is the first to compare the effectiveness of different exposure times to PRF current adjacent to the sciatic nerve.
The effects of PRF current on PHN have been investigated in only 1 human study. Kim et al.19 reported a decrease in pain intensity of 55% 4 weeks after PRF procedures adjacent to the DRG. They arbitrarily chose a PRF exposure time of 6 minutes. They showed that the duration of pain before PRF procedures did not influence the outcome. However, we demonstrated that the duration of allodynia before PRF procedures influenced the effect of PRF current; thus, the antiallodynic effect of PRF current for 2 minutes weakened gradually by the delay of PRF procedures. We examined the differences in outcome between the delay interval in application of PRF for a 2-minute application only. The antiallodynic effect of PRF current for 6 minutes may be maintained to some degree even if the duration of allodynia before PRF procedures is delayed.
The mechanisms of PRF current to neuropathic pain are thought to be a neuromodulatory effect caused by a pulsed electric field.1 Evidence suggests that the electric fields reversibly disrupt the transmission of nerve impulses across unmyelinated C fibers.22 In the PRF procedures of medial branches for the treatment of chronic facet joint pain, the inhibition of excitatory C fiber responses is an important mechanism. However, our study using a rat model produced with RTX suggests that TRPV1-sensitive C fiber afferents are not required in the antiallodynic effect of PRF current, because TRPV1-sensitive afferents are depleted by RTX. The antiallodynic effect of PRF current may result from the stimulation of Aδ- or Aβ-fiber afferents by a pulsed electric field. Van Zundert et al.23 demonstrated c-Fos expression in the dorsal horn 7 days after PRF to the DRG. Hamann et al.24 demonstrated the upregulation of activating transcription factor 3 expression as an indicator of cellular stress and the down-regulation of calcitonin gene-related peptide expression in DRG neurons 14 days after PRF applied close to the DRG. PRF current does not produce any obvious cellular changes in the DRG neurons when applied far from the DRG.24 In the present study, the direct effect of the pulsed electric field might have induced changes in the DRG neurons and the dorsal horn in the spinal cord because PRF current was applied adjacent to the sciatic nerve at the sciatic notch, which is nearer to the DRG than the midthigh24 and the hindpaw.21 However, which mechanism has a prominent role in the antiallodynic effect of PRF current has not been identified. Further studies are needed to determine the mechanisms underlying the antiallodynic effect of PRF current.
A limitation of this study is that we used RTX-treated rats as a model of PHN. This PHN model has no connection with a varicella zoster virus infection. Therefore, our finding might not completely correspond to clinical practice. Another limitation of this study is that we evaluated the motor effects of PRF current using placing and grasping reflexes and did not measure the force of the hindlimb directly, such as extensor postural thrust25; extrapolation of our findings to clinical practice must proceed cautiously. However, consistent with our findings, the study by Kim et al.19 showed that no neurological or other complications were observed after PRF procedures for 6 minutes.
The results of this study revealed the effect of PRF current on mechanical allodynia induced with RTX in rats. PRF treatment was more effective when applied in the early stages of mechanical allodynia (1 week) in rats. Increased exposure time to PRF current from 2 to 6 minutes showed a significant antiallodynic effect. We propose the application of PRF current for 6 minutes adjacent to the PHN nerve as soon as possible after allodynia occurs.
NT helped with study design, data analysis, and conduct of study; MY helped with conduct of study; ST and TU helped with study design; IT helped with data analysis; and MT helped with data analysis and manuscript preparation.
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