Cell Mol Neurobiol (2014) 34:195–203 DOI 10.1007/s10571-013-0003-z
ORIGINAL RESEARCH
Pulsed Radiofrequency Reduced Complete Freund’s Adjuvant-induced Mechanical Hyperalgesia via the Spinal c-Jun N-terminal Kinase Pathway Kuan-Hung Chen • Chien-Hui Yang • Sin-Ei Juang • Hui-Wen Huang • Jen-Kun Cheng • Shyr-Ming Sheen-Chen Jiin-Tsuey Cheng • Chung-Ren Lin
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Received: 12 September 2013 / Accepted: 28 October 2013 / Published online: 17 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund’s adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly Jiin-Tsuey Cheng and Chung-Ren Lin have contributed equally to this project and should be considered co-corresponding authors.
Electronic supplementary material The online version of this article (doi:10.1007/s10571-013-0003-z) contains supplementary material, which is available to authorized users. K.-H. Chen C.-H. Yang S.-E. Juang H.-W. Huang C.-R. Lin (&) Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No.123, Dapi Rd., Niaosong Dist., Kaohsiung 833, Taiwan e-mail: [email protected] K.-H. Chen H.-W. Huang J.-T. Cheng (&) Department of Biological Sciences, National Sun Yat-Sen University, No.70 Lienhai Rd., Gushan Dist., Kaohsiung 80424, Taiwan e-mail: [email protected]
attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P \ .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P \ .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn. Keywords Inflammation MAPK pathway Mechanical allodynia Dorsal root ganglion Spinal dorsal horn
Introduction Growing evidence suggests that pulsed radiofrequency (PRF) is effective in the treatment of various painful conditions such as radicular pain, facet joint pain, sacroiliac joint pain, and other chronic pain syndromes (Chua et al. 2011; Malik and Benzon 2008). PRFs popularity has increased significantly in recent years because of non-neurodestructive effects and clinical efficacy as compared to continuous radiofrequency J.-K. Cheng Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan S.-M. Sheen-Chen Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan C.-R. Lin Department of Anesthesiology, National Taiwan University College of Medicine, Taipei, Taiwan
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(CRF) therapy (Van Zundert et al. 2007; Shabat et al. 2006; Eyigor et al. 2010). Continuous radiofrequency provides a constant highfrequency electric field output to produce temperatures between 60 and 80 °C, which results in neuroablative thermocoagulation. By contrast, we used PRF to deliver a radiofrequency electric field (500 kHz) in 20-ms pulses, at a cycle frequency of 2 Hz. The relatively long silent phase between pulses allowed time for heat dissipation, to maintain the target tissue temperature below 42 °C and so minimize nerve damage (Byrd and Mackey 2008; Erdine et al. 2005; Podhajsky et al. 2005; Protasoni et al. 2009; Bogduk 2006). Most preclinical studies that investigate antinociceptive effects report on PRFs possible mode of action in different animal neuropathic pain models (Hamann et al. 2006; Tanaka et al. 2010; Ozsoylar et al. 2008; Aksu et al. 2010; Perret et al. 2011). However, the PRF mechanism of action has not yet been fully elucidated. The rapidly changing electric fields produced by PRF and not temperature, may modulate the transmission of pain signals via a pathway involving the immediate-early gene c-Fos, possibly some pain-inhibiting mechanisms such as long-term depression and inhibition of excitatory C fibers (Van Zundert et al. 2005; Higuchi et al. 2002; Sandkuhler et al. 1997). However, little is understood of the multifactorial etiology and complexity of the pathophysiological interrelationship characterized by neuropathic pain. Increasing evidence suggests that the three major members of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinases (JNK), play important roles in regulating neural plasticity and inflammatory responses after tissue and nerve injury by altering gene expression, leading to neuromodulation and pain hypersensitivity (Ji et al. 2009; Obata and Noguchi 2004; Gao and Ji 2008). In our preliminary study, we examined the effect of PRF on ERK and p38 activities in the spinal dorsal horn of CFA-induced inflammatory pain; however, we did not find any significant results (Supplemental file). In this study, we examined the direct effect of PRF applied proximal to the dorsal root ganglion (DRG), associated with peripheral inflammatory injury, on alleviation of inflammation-induced mechanical hyperalgesia and on the involvement of the JNK signal transduction pathway. In doing so, we aimed to uncover the possible molecular mechanism of action of PRF in a complete Freund’s adjuvant (CFA)-induced inflammatory pain model.
Materials and Methods Animals and the Inflammatory Pain Model All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee
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(IACUC) of the Chang Gung Memorial Hospital, Taiwan. Adult male Sprague-Dawley (SD) rats (350–375 g; National Science Council, Taiwan) were housed with a 12-hour light–dark cycle and with free access to food and water ad libitum. Rats were allowed seven days acclimation in a similar environment before data collection began. Rats were anesthetized in a Plexiglas box containing an atmosphere of 3 % isoflurane in a 1:1 oxygen/room air mixture. Inflammatory pain was induced in the unilateral hind paw by intradermal injection of 0.2 ml of CFA (Sigma; MO, USA) into the plantar surface of the left hind paw (n = 75). Following experimentation, five animals were each sacrificed on days 1, 3, 7, and 14 post-CFA injection (n = 5 for each group). The bilateral dorsal horns of the lumbar region of the rat spinal cords (L4–L5) were taken as specimens for western blots analysis. Assessment of Thermal Hyperalgesia Animals (n = 20) were placed on a glass plate, and a radiant heat (Plantar Test Apparatus; Ugo Basile, Milan, Italy) was applied to the plantar surface of the left hind paw. The withdrawal latency and duration were recorded; the minimum stimulus duration was set to 0.1 s and the maximum value (cut-off latency) to 30 s to avoid paw injury. Each rat was tested three times at 5-min intervals, and the mean values were used for analysis. Assessment of Mechanical Hyperalgesia Animals (n = 20) were placed in a chamber, and a servocontrolled mechanical stimulus (Dynamic Plantar Aesthesiometer; Ugo Basile, Milan, Italy) was applied to the plantar surface of the hind paw at 5-min intervals with increasing punctate pressure until the rat withdrew its paw. Both hind paws were assessed in turn. The maximum cutoff force was set to 50 g to prevent tissue damage. The threshold was assessed three times for each time point, and the mean values were used for analysis. Application of Pulsed Radiofrequency After induction of CFA-induced peripheral inflammatory pain, rats were randomly separated into three treatment groups and sacrificed on days 1, 3, 7, and 14 post-treatment. Group 1: proximal PRF treatment (PRF applied adjacent to DRG just distal to the intervertebral foramen) (n = 5 for each time group). Group 2: distal PRF treatment (PRF applied to the sciatic nerve, distal to DRG) (n = 5 for each time group). Group 3: no-PRF group (n = 5 for each time group). On the basis of a method reported by Podhajsky et al. (2005), PRF was delivered adjacent to the L5 DRG or the sciatic nerve using a 10-cm long electrode (22 gauge, 5 mm
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active tip; SMK-10, Radionics). The electrode was powered by a PRF generator to deliver a 500 kHz electric field in 20-ms pulses at a rate of two pulses per second (2 Hz). Generator output was automatically increased until the electrode tip reached a temperature of 42 °C and was then maintained for a further 120 s. In no-PRF rats, identical electrode placement was performed, but no-PRF electric current was applied.
the injected paw at one day post-injection (P \ .05), which lasted for the entire 14-day testing period (Fig. 1b) (P \ .05). However, we did not observe any significant change in TPWL for the contralateral hind paw at any time.
Western Blotting
In CFA rats, no-PRF applied proximal to DRG (n = 5) had no effect on either ipsilateral or contralateral MPWT (Fig. 2a, b). By contrast, CFA rats receiving PRF close to DRG (n = 5) showed significant increases in the ipsilateral MPWT compared to no-PRF rats. MPWT continued to increase throughout the 14-day testing period (Fig. 2a). No significant differences were seen in contralateral MPWT between PRF-treated and no-PRF rats throughout the study period (Fig. 2b). A statistically significant difference in ipsilateral TPWL was observed between the PRF-treated group and no-PRF group hind paws at one day post-PRF treatment (Fig. 2c). No significant differences were seen in
For the western blot study, the bilateral dorsal horns of L4– L5 spinal cord segments were homogenized in a lysis buffer. Protease inhibitor (Sigma, MO, USA) was added to prevent protein degradation. The protein content of the supernatant was determined by BCA protein assay (Pierce, Rockford, IL USA). Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose blots. The blots were then blocked with 5 % milk and incubated overnight at 4 °C with rabbit polyclonal antibody against phosphorylated JNK (pJNK, 1:1,000; Cell signaling, MA, USA), and mouse monoclonal antibody against GAPDH (1:7,500; Santa Cruz Biotechnology, CA, USA). Blots were further incubated with horseradish peroxidase-conjugated secondary antibody for one hour at room temperature. Color molecular weight standard markers were run on each gel. Western blot results were quantified by densitometry analysis.
Attenuation of CFA-Induced Mechanical Hyperalgesia by PRF Applied Close to DRG
Statistical Analysis To quantify western blot results, the densities of specific bands for pJNK1 and JNK1 (46 kDa), pJNK2 and JNK2 (54 kDa), p-c-Jun, b-tubulin, and GAPDH were measured with imaging analysis software (Image J, NIH). The levels of pJNK1/2 and JNK1/2 were normalized to loading controls (GAPDH). Western result data and animal behavior data were analyzed using one-way ANOVA, followed by the Newman–Keuls test for post-hoc analysis. All data are presented as mean (SD), and P \ .05 were considered statistically significant in all cases.
Results CFA Injection Produced Marked Inflammatory Pain In CFA plantar-injected animals (n = 20), the baseline mechanical paw withdrawal threshold (MPWT) was 17.4 ± 1.26 g, and this decreased by 88, 76, 83, and 77 % on days 1, 3, 7, and 14 post-CFA injection, respectively (Fig. 1a) (P \ .05). Mechanical hyperalgesia was also observed in the contralateral paw, albeit at reduced magnitude than that seen in the ipsilateral paw. There was a decrease in the thermal paw withdrawal latency (TPWL) in
Fig. 1 Time course of a CFA-induced mechanical hyperalgesia and b thermal hyperalgesia in rats. CFA produces rapid and persistent mechanical hyperalgesia in both the ipsilateral and contralateral paws. CFA induces ipsilateral heat hyperalgesia, which largely recovers after two weeks. Each value is presented as mean (SD); *P \ .05, compared to the pre-CFA baseline (day 0), n = 5 for each group
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Fig. 2 Time course of CFA-induced mechanical and thermal hyperalgesia determined for both the ipsilateral (a, c) and contralateral hind paws (b, d) in CFA rats receiving PRF treatment proximal to DRG. Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared
to no-PRF group rats; n = 5 for each group. PRF applied proximal to DRG immediately after the appearance of inflammation (a) inhibits CFA-induced mechanical hyperalgesia in the ipsilateral hind paw (day 1 to day 14), and (c) inhibits thermal hyperalgesia (day 1)
contralateral TPWL between PRF-treated and no-PRF rats throughout the study period (Fig. 2d).
days 1, 3, 7, and 14 post-CFA injection (n = 5). CFA treatment induced a rapid increase in pJNK1 and pJNK2 levels in the spinal cord both the ipsilateral and contralateral to the injection site (Fig. 4, 5) Increases in pJNK1 and pJNK2 were maintained throughout the 14 days of the study. Levels of pJNK1 levels were much greater than those of pJNK2 levels for all time points, suggesting that pJNK1 is the major form of pJNK present in the spinal cord. PRF applied distal to DRG did not induce any change in pJNK expression compared with no-PRF rats (Fig. 4). By contrast, PRF applied proximal to DRG caused a significant down-regulation of pJNK1 and pJNK2 in the spinal dorsal horn. The expression of pJNK1 significantly reduced to 72, 66, 69, and 69 % (P \ .05) that of the no-PRF group on days 1, 3, 7, and 14, respectively (Fig. 5c). The expression of pJNK2 was reduced to 88 % (P [ .05), 77, 80, and 68 % (P \ .05) that of no-PRF rats on days 1, 3, 7, and 14, respectively (Fig. 5e). Therefore, our studies revealed that PRF administered proximal to DRG attenuates CFA-induced inflammatory pain and might also have an effect on activated JNK expression.
No Significant Effect on CFA-Induced Inflammatory Pain by PRF Applied Distal to DRG No-PRF applied distal to DRG (n = 5) had no effect on either ipsilateral or contralateral MPWT (Fig. 3a, b). There were no significant differences in ipsilateral MPWT and TPWL between rats receiving PRF distal to DRG (n = 5) and no-PRF rats (Fig. 3a, c). Contralateral MPWT values in rats receiving PRF were not statistically different from those in control rats that received the no-PRF procedure (Fig. 3b). No significant difference was seen in contralateral TPWL between PRF-treated and no-PRF rats (Fig. 3d). PRF Attenuates Activation of JNK in the Spinal Cord After CFA-Induced Inflammation We examined JNK activation in the bilateral dorsal horn of the lumbar spinal cord (L4–L5) by western blot analysis at
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Fig. 3 Time course of CFA-induced mechanical and thermal hyperalgesia in both the ipsilateral (a, c) and contralateral hind paws (b, d) of CFA rats that received PRF treatment distal to DRG. Analyzed
by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as means (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
Discussion
growth factors, Toll-like receptors, increases in intracellular Ca2?, and several neurodegenerative conditions (Bonny et al. 2005) are involved in the activation of downstream kinases such as cdc42, Rac1, and PAK1. These kinases in turn activate their downstream MAPK kinases (MAP3Ks) which activate their own downstream MAPK kinases (MAP2Ks). Finally, the MAP2Ks phosphorylate and activate their downstream MAPK, the JNK proteins. The activated JNKs translocate to the nucleus where they phosphorylate a range of substrates such as the transcription factors c-Jun, Elk-1, p53, activating transcription factor 2 (ATF2), c-Myc, and the nuclear factor family of activated T cells (NFAT), leading to gene transcription. The protein products of these genes include tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and cyclooxygenase 1 and 2 (COX-1/2), and are responsible for enhancement of sensitivity to pain (Gao and Ji 2008). Although PRF applied adjacent to DRG attenuates CFAinduced mechanical hyperalgesia as evidenced by our work, and in agreement with previous studies (Van Zundert et al. 2005; Aksu et al. 2010; Perret et al. 2011; Hamann et al. 2006), the PRF mechanism of action remains poorly
We made some interesting findings in this study; firstly, the application of PRF adjacent to DRG just distal to the intervertebral foramen, but not to the mid-thigh sciatic nerve, results in a significant reduction in CFA-induced mechanical hyperalgesia. Secondly, the CFA-induced activation of JNK in the spinal cord dorsal horn is significantly suppressed by the application of PRF proximal to DRG. According to a previous study (Gao et al. 2010), JNK1 is the predominant JNK isoform present in spinal cord astrocytes, suggesting that JNK1 activation in spinal astrocyte networks plays an important role in mediating bilateral mechanical allodynia by producing proinflammatory and pronociceptive mediators (Gao and Ji 2008; Scholz and Woolf 2007), or by modulating glutamate uptake (Chiang et al. 2007). JNK has three isoforms: JNK1, JNK2, and JNK3. Of these, JNK1 and JNK2 are constitutively expressed in the spinal cord (Ji et al. 2006), and phosphorylated JNK1 is the predominant active form in the spinal cord following nerve injury (Zhuang et al. 2006). Initially, cellular stress, proinflammatory cytokines,
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Fig. 4 Western blot analysis showing the time course of CFAinduced pJNK1 and pJNK2 expression in a ipsilateral and b contralateral hind paws, and the effect of PRF treatment distal to DRG on expression of JNK1 and JNK2 in the c, e ipsilateral and d, f
contralateral dorsal horn of the spinal cord. Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
understood. Several theories have been postulated to explain PRFs temperature-independent pain modulating effect; the characteristic low intensity electric field generated by PRF only causes alteration in cellular transmembrane potentials and temporary electroporation, rather than direct disruption of cell membranes (Cosman and Cosman 2005; Bogduk 2006; Chua et al. 2011). In theory, this could result in conditioning stimulation and long-term depression (LTD) of pain-related synaptic transmission, such the involvement of excitatory C fibers (Cosman and Cosman 2005; Sandkuhler et al. 1997). However, this neurophysiological theory does not account for the analgesic effects of PRF applied to regions where there is no nervous tissue, such as the application of intra-articular PRF (Sluijter et al. 2008; Karaman et al. 2011; Al-Badawi et al. 2004). Additionally, alterations in dorsal horn neuronal activity in response to PRF treatment have been demonstrated (Hamann et al. 2006; Higuchi et al. 2002; Van Zundert et al. 2005). PRF applied to DRG induced increases in c-Fos immunoreactive neurons in the superficial laminas of the
spinal dorsal horn, suggesting temperature-independent activation of spinal dorsal horn neurons (Higuchi et al. 2002). Another study revealed up-regulated c-Fos expression in cells of the spinal dorsal horn seven days after application of either CRF or PRF to DRG, demonstrating late, temperature-independent cellular activity and neuronal activation (Van Zundert et al. 2005). Furthermore, Hamann et al. (2006) demonstrated a heat-independent increase in the expression of activated transcription factor 3 (ATF3) in a specific population of DRG neurons in response to PRF applied proximal to DRG. Collectively these studies’ findings do not provide a reasonable explanation for PRFs observed analgesic effects. Expression of c-Fos and ATF3 are no more than markers of increased cellular metabolic activity (Bogduk 2006) and nerve injury (Tsujino et al. 2000), respectively. They are not even specific to the nociceptive pathway, they are not involved in pain modulation and do not distinguish between inhibitory and excitatory activity (Bogduk 2006; Richebe et al. 2005).
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Fig. 5 Western blot analysis showing the time course of CFAinduced pJNK1 and pJNK2 expression in a ipsilateral and b contralateral hind paws, and the effect of PRF treatment proximal to DRG on expression of JNK1 and JNK2 in the c, e ipsilateral and d, f contralateral dorsal horn of the spinal cord. The pJNK1 and pJNK2 proteins were rapidly activated in the dorsal horn of the lumbar spinal cord, and their expression was maintained for the 14 day duration of the study in both the ipsilateral and contralateral lumbar spinal cord.
PRF applied proximal to DRG reduced the induction of pJNK1 (day 1 to day 14 on ipsilateral and day 7 to day 14 on contralateral spinal dorsal horn) and pJNK2 (day 3 to day 14 on both ipsilateral and contralateral spinal dorsal horn). Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
We provide herein, a more logical and convincing theory to explain the possible mechanism underlying PRFs observed antinociceptive and neuromodulation effects. In our study, the application of 500 kHz RF in 20-ms pulses at a rate of 2 Hz adjacent to DRG created an electric field at the tip of the electrode. This caused local heating, which we limited to a maximum temperature of 42 °C to prevent tissue damage from occurring. The delivery of brief electrical pulses or electromagnetic waves adjacent to DRG was sufficient to inhibit JNK activation in spinal dorsal horn cells, probably mainly in astrocytes (Gao et al. 2010). The inhibition of JNK activation further suppresses JNK downstream gene expression, such as proinflammatory and pronociceptive mediators (Scholz and Woolf 2007; Gao and Ji 2008) that in conjunction enhance pain sensitivity in the dorsal horn of the spinal cord, termed ‘‘central sensitization’’ (Ji et al. 2003; Latremoliere and Woolf 2009). This
central suppression effect induced by PRF treatment eventually leads to the attenuation of pain hypersensitivity, as evidenced by our behavioral findings. Furthermore, previous study demonstrated that exposure of neurons to PRF resulted in an acute neuromodulatory action such as transient inhibition of evoked excitatory transmission that might be able to block or attenuate the peripheral excitatory transmission (Cahana et al. 2003). Besides, PRF applied adjacent to DRG might work in a similar manner to electroacupuncture, activating both the spinal and supraspinal mechanisms which might further reduce pain perception (Carlsson 2002). In addition, a recent study revealed that PRF applied adjacent to DRG resulted in a significant suppression of spinal excitatory amino acids release and attenuation of mechanical allodynia (Yang et al. 2013). In our work, we evaluated the analgesic effects of PRF applied to two different locations, proximal and distal to
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DRG. PRF applied adjacent to DRG produced significant reductions only in mechanical allodynia for the entire 14 day experimental period. However, the attenuation of thermal hyperalgesia was only significant in the first day following application of PRF proximal to DRG. This observation is similar, albeit in greater detail, to a previous study by Hagiwara et al. (2009), which demonstrated that application of PRF (2 bursts per second of 500-kHz RF with a duration of 20 ms per burst and temperature of 42 °C for 180 s) to the sciatic nerve effectively reduces CFA-induced thermal hyperalgesia during the first three consecutive days following treatment. This reduction in hyperalgesia results from enhancement of the descending inhibitory pathways as the PRF mechanism of action. Three possible explanations for the discrepancies between our results and Hagiwaras’ are: (1) different age/weight SD rats (250–300 vs. 350–375 g); (2) different amount of CFA injected (0.15 vs. 0.2 ml); (3) different energy duration of PRF (180 vs. 120 s) applied to the sciatic nerve. The differences in these research regimens might influence intracellular pathophysiological events (Bogduk 2006; Cosman and Cosman 2005) and further affect the analgesic effect of PRF in inflammatory pain model. Furthermore, we did not evaluate the effect of PRF on CFA-induced immediateearly behavioral responses (e.g., 2 or 6 h) because the most significant reductions in pain usually occur in the days following PRF treatment (Malik and Benzon 2008; Hagiwara et al. 2009; Werner et al. 2012). Our PRF animal study was designed to be clinically oriented, and so we considered it reasonable that behavioral observation should commence at 1 day post-CFA injection. There are some inconsistencies in our findings, for example, PRF treatment did not reduce CFA-induced contralateral mechanical hyperalgesia or thermal hyperalgesia, but did reduce CFA-induced increases in p-JNK expression in the contralateral dorsal horn. We can account for these apparent inconsistencies as follows: according to previous reports, unilateral CFA injection in a hind paw induces not only bilateral mechanical hyperalgesia, but also ipsilateral heat hyperalgesia (Schepers et al. 2008; Gao et al. 2010). This is in accord with our results, which showed no significant alterations in the contralateral hind paw in response to thermal stimulation compared to basal level TPWL, or between the no-PRF and PRF groups. Therefore, although the application of PRF significantly suppressed the expression of contralateral JNK, we did not see a significant difference in thermal behavioral test responses between groups. And this is supported by previous study which mentioned that spinal JNK induction is accompanied with mechanical response and spread of pain and which degree of JNK induction might induce the mechanical hyperalgesia in ipsilateral and contralateral is not known at this point (Gao et al. 2010).
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Conclusion In conclusion, PRF applied proximal to DRG provides an effective treatment for inflammatory pain by attenuating mechanical hyperalgesia by modulation of the spinal dorsal horn to suppress JNK activation, and potentially down-regulating the expression of downstream pain-enhancing genes. Acknowledgments This work was supported in part by Grant Nos. 880891, 880892, 880893, 891251, and 8A1011 from Chang Gung Memorial Hospital Research, Kaohsiung, Taiwan, and by Grant Nos. 96-2628-B-182A-005-MY3, 98-2314-B-182A-035-MY2, 100-2314B-182A-037, 101-2314-B-182A-014-, and 101-2314-B-182A-068MY3 from the Taiwan National Science Council, Taipei, Taiwan. Conflict of interest
The authors declare no conflict of interest.
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203 root: a morphological light and transmission electron microscopy study at acute stage. Eur Spine J 18(4):473–478. doi:10.1007/ s00586-008-0870-z Richebe P, Rathmell JP, Brennan TJ (2005) Immediate early genes after pulsed radiofrequency treatment: neurobiology in need of clinical trials. Anesthesiology 102(1):1–3 Sandkuhler J, Chen JG, Cheng G, Randic M (1997) Low-frequency stimulation of afferent Adelta-fibers induces long-term depression at primary afferent synapses with substantia gelatinosa neurons in the rat. J Neurosci 17(16):6483–6491 Schepers RJ, Mahoney JL, Gehrke BJ, Shippenberg TS (2008) Endogenous kappa-opioid receptor systems inhibit hyperalgesia associated with localized peripheral inflammation. Pain 138(2):423–439. doi:10.1016/j.pain.2008.01.023 Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10(11):1361–1368. doi:10. 1038/nn1992 Shabat S, Pevsner Y, Folman Y, Gepstein R (2006) Pulsed radiofrequency in the treatment of patients with chronic neuropathic spinal pain. Minim Invasive Neurosurg 49(3):147–149. doi:10.1055/s-2006-944238 Sluijter ME, Teixeira A, Serra V, Balogh S, Schianchi P (2008) Intraarticular application of pulsed radiofrequency for arthrogenic pain—report of six cases. Pain Pract 8(1):57–61. doi:10.1111/j. 1533-2500.2007.00172.x Tanaka N, Yamaga M, Tateyama S, Uno T, Tsuneyoshi I, Takasaki M (2010) The effect of pulsed radiofrequency current on mechanical allodynia induced with resiniferatoxin in rats. Anesth Analg 111(3):784–790. doi:10.1213/ANE.0b013e3181e9f62f Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, Yonenobu K, Ochi T, Noguchi K (2000) Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol Cell Neurosci 15(2):170–182. doi:10.1006/mcne 1999.0814 Van Zundert J, de Louw AJ, Joosten EA, Kessels AG, Honig W, Dederen PJ, Veening JG, Vles JS, van Kleef M (2005) Pulsed and continuous radiofrequency current adjacent to the cervical dorsal root ganglion of the rat induces late cellular activity in the dorsal horn. Anesthesiology 102(1):125–131 Van Zundert J, Patijn J, Kessels A, Lame I, van Suijlekom H, van Kleef M (2007) Pulsed radiofrequency adjacent to the cervical dorsal root ganglion in chronic cervical radicular pain: a double blind sham controlled randomized clinical trial. Pain 127(1–2):173–182. doi:10.1016/j.pain.2006.09.002 Werner MU, Bischoff JM, Rathmell JP, Kehlet H (2012) Pulsed radiofrequency in the treatment of persistent pain after inguinal herniotomy: a systematic review. Reg Anesth Pain Med 37(3):340–343. doi:10.1097/AAP.0b013e31824bea4e Yang CH, Chen KH, Huang HW, Sheen-Chen SM, Lin CR (2013) Pulsed radiofrequency treatment attenuates increases in spinal excitatory amino acid release in rats with adjuvant-induced mechanical allodynia. NeuroReport 24(8):431–436. doi:10.1097/ WNR.0b013e32836164f5 Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, Decosterd I, Ji RR (2006) A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J Neurosci 26(13):3551–3560. doi:10.1523/ JNEUROSCI.5290-05.2006
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ORIGINAL RESEARCH
Pulsed Radiofrequency Reduced Complete Freund’s Adjuvant-induced Mechanical Hyperalgesia via the Spinal c-Jun N-terminal Kinase Pathway Kuan-Hung Chen • Chien-Hui Yang • Sin-Ei Juang • Hui-Wen Huang • Jen-Kun Cheng • Shyr-Ming Sheen-Chen Jiin-Tsuey Cheng • Chung-Ren Lin
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Received: 12 September 2013 / Accepted: 28 October 2013 / Published online: 17 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund’s adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly Jiin-Tsuey Cheng and Chung-Ren Lin have contributed equally to this project and should be considered co-corresponding authors.
Electronic supplementary material The online version of this article (doi:10.1007/s10571-013-0003-z) contains supplementary material, which is available to authorized users. K.-H. Chen C.-H. Yang S.-E. Juang H.-W. Huang C.-R. Lin (&) Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No.123, Dapi Rd., Niaosong Dist., Kaohsiung 833, Taiwan e-mail: [email protected] K.-H. Chen H.-W. Huang J.-T. Cheng (&) Department of Biological Sciences, National Sun Yat-Sen University, No.70 Lienhai Rd., Gushan Dist., Kaohsiung 80424, Taiwan e-mail: [email protected]
attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P \ .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P \ .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn. Keywords Inflammation MAPK pathway Mechanical allodynia Dorsal root ganglion Spinal dorsal horn
Introduction Growing evidence suggests that pulsed radiofrequency (PRF) is effective in the treatment of various painful conditions such as radicular pain, facet joint pain, sacroiliac joint pain, and other chronic pain syndromes (Chua et al. 2011; Malik and Benzon 2008). PRFs popularity has increased significantly in recent years because of non-neurodestructive effects and clinical efficacy as compared to continuous radiofrequency J.-K. Cheng Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan S.-M. Sheen-Chen Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan C.-R. Lin Department of Anesthesiology, National Taiwan University College of Medicine, Taipei, Taiwan
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(CRF) therapy (Van Zundert et al. 2007; Shabat et al. 2006; Eyigor et al. 2010). Continuous radiofrequency provides a constant highfrequency electric field output to produce temperatures between 60 and 80 °C, which results in neuroablative thermocoagulation. By contrast, we used PRF to deliver a radiofrequency electric field (500 kHz) in 20-ms pulses, at a cycle frequency of 2 Hz. The relatively long silent phase between pulses allowed time for heat dissipation, to maintain the target tissue temperature below 42 °C and so minimize nerve damage (Byrd and Mackey 2008; Erdine et al. 2005; Podhajsky et al. 2005; Protasoni et al. 2009; Bogduk 2006). Most preclinical studies that investigate antinociceptive effects report on PRFs possible mode of action in different animal neuropathic pain models (Hamann et al. 2006; Tanaka et al. 2010; Ozsoylar et al. 2008; Aksu et al. 2010; Perret et al. 2011). However, the PRF mechanism of action has not yet been fully elucidated. The rapidly changing electric fields produced by PRF and not temperature, may modulate the transmission of pain signals via a pathway involving the immediate-early gene c-Fos, possibly some pain-inhibiting mechanisms such as long-term depression and inhibition of excitatory C fibers (Van Zundert et al. 2005; Higuchi et al. 2002; Sandkuhler et al. 1997). However, little is understood of the multifactorial etiology and complexity of the pathophysiological interrelationship characterized by neuropathic pain. Increasing evidence suggests that the three major members of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinases (JNK), play important roles in regulating neural plasticity and inflammatory responses after tissue and nerve injury by altering gene expression, leading to neuromodulation and pain hypersensitivity (Ji et al. 2009; Obata and Noguchi 2004; Gao and Ji 2008). In our preliminary study, we examined the effect of PRF on ERK and p38 activities in the spinal dorsal horn of CFA-induced inflammatory pain; however, we did not find any significant results (Supplemental file). In this study, we examined the direct effect of PRF applied proximal to the dorsal root ganglion (DRG), associated with peripheral inflammatory injury, on alleviation of inflammation-induced mechanical hyperalgesia and on the involvement of the JNK signal transduction pathway. In doing so, we aimed to uncover the possible molecular mechanism of action of PRF in a complete Freund’s adjuvant (CFA)-induced inflammatory pain model.
Materials and Methods Animals and the Inflammatory Pain Model All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee
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(IACUC) of the Chang Gung Memorial Hospital, Taiwan. Adult male Sprague-Dawley (SD) rats (350–375 g; National Science Council, Taiwan) were housed with a 12-hour light–dark cycle and with free access to food and water ad libitum. Rats were allowed seven days acclimation in a similar environment before data collection began. Rats were anesthetized in a Plexiglas box containing an atmosphere of 3 % isoflurane in a 1:1 oxygen/room air mixture. Inflammatory pain was induced in the unilateral hind paw by intradermal injection of 0.2 ml of CFA (Sigma; MO, USA) into the plantar surface of the left hind paw (n = 75). Following experimentation, five animals were each sacrificed on days 1, 3, 7, and 14 post-CFA injection (n = 5 for each group). The bilateral dorsal horns of the lumbar region of the rat spinal cords (L4–L5) were taken as specimens for western blots analysis. Assessment of Thermal Hyperalgesia Animals (n = 20) were placed on a glass plate, and a radiant heat (Plantar Test Apparatus; Ugo Basile, Milan, Italy) was applied to the plantar surface of the left hind paw. The withdrawal latency and duration were recorded; the minimum stimulus duration was set to 0.1 s and the maximum value (cut-off latency) to 30 s to avoid paw injury. Each rat was tested three times at 5-min intervals, and the mean values were used for analysis. Assessment of Mechanical Hyperalgesia Animals (n = 20) were placed in a chamber, and a servocontrolled mechanical stimulus (Dynamic Plantar Aesthesiometer; Ugo Basile, Milan, Italy) was applied to the plantar surface of the hind paw at 5-min intervals with increasing punctate pressure until the rat withdrew its paw. Both hind paws were assessed in turn. The maximum cutoff force was set to 50 g to prevent tissue damage. The threshold was assessed three times for each time point, and the mean values were used for analysis. Application of Pulsed Radiofrequency After induction of CFA-induced peripheral inflammatory pain, rats were randomly separated into three treatment groups and sacrificed on days 1, 3, 7, and 14 post-treatment. Group 1: proximal PRF treatment (PRF applied adjacent to DRG just distal to the intervertebral foramen) (n = 5 for each time group). Group 2: distal PRF treatment (PRF applied to the sciatic nerve, distal to DRG) (n = 5 for each time group). Group 3: no-PRF group (n = 5 for each time group). On the basis of a method reported by Podhajsky et al. (2005), PRF was delivered adjacent to the L5 DRG or the sciatic nerve using a 10-cm long electrode (22 gauge, 5 mm
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active tip; SMK-10, Radionics). The electrode was powered by a PRF generator to deliver a 500 kHz electric field in 20-ms pulses at a rate of two pulses per second (2 Hz). Generator output was automatically increased until the electrode tip reached a temperature of 42 °C and was then maintained for a further 120 s. In no-PRF rats, identical electrode placement was performed, but no-PRF electric current was applied.
the injected paw at one day post-injection (P \ .05), which lasted for the entire 14-day testing period (Fig. 1b) (P \ .05). However, we did not observe any significant change in TPWL for the contralateral hind paw at any time.
Western Blotting
In CFA rats, no-PRF applied proximal to DRG (n = 5) had no effect on either ipsilateral or contralateral MPWT (Fig. 2a, b). By contrast, CFA rats receiving PRF close to DRG (n = 5) showed significant increases in the ipsilateral MPWT compared to no-PRF rats. MPWT continued to increase throughout the 14-day testing period (Fig. 2a). No significant differences were seen in contralateral MPWT between PRF-treated and no-PRF rats throughout the study period (Fig. 2b). A statistically significant difference in ipsilateral TPWL was observed between the PRF-treated group and no-PRF group hind paws at one day post-PRF treatment (Fig. 2c). No significant differences were seen in
For the western blot study, the bilateral dorsal horns of L4– L5 spinal cord segments were homogenized in a lysis buffer. Protease inhibitor (Sigma, MO, USA) was added to prevent protein degradation. The protein content of the supernatant was determined by BCA protein assay (Pierce, Rockford, IL USA). Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose blots. The blots were then blocked with 5 % milk and incubated overnight at 4 °C with rabbit polyclonal antibody against phosphorylated JNK (pJNK, 1:1,000; Cell signaling, MA, USA), and mouse monoclonal antibody against GAPDH (1:7,500; Santa Cruz Biotechnology, CA, USA). Blots were further incubated with horseradish peroxidase-conjugated secondary antibody for one hour at room temperature. Color molecular weight standard markers were run on each gel. Western blot results were quantified by densitometry analysis.
Attenuation of CFA-Induced Mechanical Hyperalgesia by PRF Applied Close to DRG
Statistical Analysis To quantify western blot results, the densities of specific bands for pJNK1 and JNK1 (46 kDa), pJNK2 and JNK2 (54 kDa), p-c-Jun, b-tubulin, and GAPDH were measured with imaging analysis software (Image J, NIH). The levels of pJNK1/2 and JNK1/2 were normalized to loading controls (GAPDH). Western result data and animal behavior data were analyzed using one-way ANOVA, followed by the Newman–Keuls test for post-hoc analysis. All data are presented as mean (SD), and P \ .05 were considered statistically significant in all cases.
Results CFA Injection Produced Marked Inflammatory Pain In CFA plantar-injected animals (n = 20), the baseline mechanical paw withdrawal threshold (MPWT) was 17.4 ± 1.26 g, and this decreased by 88, 76, 83, and 77 % on days 1, 3, 7, and 14 post-CFA injection, respectively (Fig. 1a) (P \ .05). Mechanical hyperalgesia was also observed in the contralateral paw, albeit at reduced magnitude than that seen in the ipsilateral paw. There was a decrease in the thermal paw withdrawal latency (TPWL) in
Fig. 1 Time course of a CFA-induced mechanical hyperalgesia and b thermal hyperalgesia in rats. CFA produces rapid and persistent mechanical hyperalgesia in both the ipsilateral and contralateral paws. CFA induces ipsilateral heat hyperalgesia, which largely recovers after two weeks. Each value is presented as mean (SD); *P \ .05, compared to the pre-CFA baseline (day 0), n = 5 for each group
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Fig. 2 Time course of CFA-induced mechanical and thermal hyperalgesia determined for both the ipsilateral (a, c) and contralateral hind paws (b, d) in CFA rats receiving PRF treatment proximal to DRG. Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared
to no-PRF group rats; n = 5 for each group. PRF applied proximal to DRG immediately after the appearance of inflammation (a) inhibits CFA-induced mechanical hyperalgesia in the ipsilateral hind paw (day 1 to day 14), and (c) inhibits thermal hyperalgesia (day 1)
contralateral TPWL between PRF-treated and no-PRF rats throughout the study period (Fig. 2d).
days 1, 3, 7, and 14 post-CFA injection (n = 5). CFA treatment induced a rapid increase in pJNK1 and pJNK2 levels in the spinal cord both the ipsilateral and contralateral to the injection site (Fig. 4, 5) Increases in pJNK1 and pJNK2 were maintained throughout the 14 days of the study. Levels of pJNK1 levels were much greater than those of pJNK2 levels for all time points, suggesting that pJNK1 is the major form of pJNK present in the spinal cord. PRF applied distal to DRG did not induce any change in pJNK expression compared with no-PRF rats (Fig. 4). By contrast, PRF applied proximal to DRG caused a significant down-regulation of pJNK1 and pJNK2 in the spinal dorsal horn. The expression of pJNK1 significantly reduced to 72, 66, 69, and 69 % (P \ .05) that of the no-PRF group on days 1, 3, 7, and 14, respectively (Fig. 5c). The expression of pJNK2 was reduced to 88 % (P [ .05), 77, 80, and 68 % (P \ .05) that of no-PRF rats on days 1, 3, 7, and 14, respectively (Fig. 5e). Therefore, our studies revealed that PRF administered proximal to DRG attenuates CFA-induced inflammatory pain and might also have an effect on activated JNK expression.
No Significant Effect on CFA-Induced Inflammatory Pain by PRF Applied Distal to DRG No-PRF applied distal to DRG (n = 5) had no effect on either ipsilateral or contralateral MPWT (Fig. 3a, b). There were no significant differences in ipsilateral MPWT and TPWL between rats receiving PRF distal to DRG (n = 5) and no-PRF rats (Fig. 3a, c). Contralateral MPWT values in rats receiving PRF were not statistically different from those in control rats that received the no-PRF procedure (Fig. 3b). No significant difference was seen in contralateral TPWL between PRF-treated and no-PRF rats (Fig. 3d). PRF Attenuates Activation of JNK in the Spinal Cord After CFA-Induced Inflammation We examined JNK activation in the bilateral dorsal horn of the lumbar spinal cord (L4–L5) by western blot analysis at
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Fig. 3 Time course of CFA-induced mechanical and thermal hyperalgesia in both the ipsilateral (a, c) and contralateral hind paws (b, d) of CFA rats that received PRF treatment distal to DRG. Analyzed
by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as means (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
Discussion
growth factors, Toll-like receptors, increases in intracellular Ca2?, and several neurodegenerative conditions (Bonny et al. 2005) are involved in the activation of downstream kinases such as cdc42, Rac1, and PAK1. These kinases in turn activate their downstream MAPK kinases (MAP3Ks) which activate their own downstream MAPK kinases (MAP2Ks). Finally, the MAP2Ks phosphorylate and activate their downstream MAPK, the JNK proteins. The activated JNKs translocate to the nucleus where they phosphorylate a range of substrates such as the transcription factors c-Jun, Elk-1, p53, activating transcription factor 2 (ATF2), c-Myc, and the nuclear factor family of activated T cells (NFAT), leading to gene transcription. The protein products of these genes include tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and cyclooxygenase 1 and 2 (COX-1/2), and are responsible for enhancement of sensitivity to pain (Gao and Ji 2008). Although PRF applied adjacent to DRG attenuates CFAinduced mechanical hyperalgesia as evidenced by our work, and in agreement with previous studies (Van Zundert et al. 2005; Aksu et al. 2010; Perret et al. 2011; Hamann et al. 2006), the PRF mechanism of action remains poorly
We made some interesting findings in this study; firstly, the application of PRF adjacent to DRG just distal to the intervertebral foramen, but not to the mid-thigh sciatic nerve, results in a significant reduction in CFA-induced mechanical hyperalgesia. Secondly, the CFA-induced activation of JNK in the spinal cord dorsal horn is significantly suppressed by the application of PRF proximal to DRG. According to a previous study (Gao et al. 2010), JNK1 is the predominant JNK isoform present in spinal cord astrocytes, suggesting that JNK1 activation in spinal astrocyte networks plays an important role in mediating bilateral mechanical allodynia by producing proinflammatory and pronociceptive mediators (Gao and Ji 2008; Scholz and Woolf 2007), or by modulating glutamate uptake (Chiang et al. 2007). JNK has three isoforms: JNK1, JNK2, and JNK3. Of these, JNK1 and JNK2 are constitutively expressed in the spinal cord (Ji et al. 2006), and phosphorylated JNK1 is the predominant active form in the spinal cord following nerve injury (Zhuang et al. 2006). Initially, cellular stress, proinflammatory cytokines,
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Fig. 4 Western blot analysis showing the time course of CFAinduced pJNK1 and pJNK2 expression in a ipsilateral and b contralateral hind paws, and the effect of PRF treatment distal to DRG on expression of JNK1 and JNK2 in the c, e ipsilateral and d, f
contralateral dorsal horn of the spinal cord. Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
understood. Several theories have been postulated to explain PRFs temperature-independent pain modulating effect; the characteristic low intensity electric field generated by PRF only causes alteration in cellular transmembrane potentials and temporary electroporation, rather than direct disruption of cell membranes (Cosman and Cosman 2005; Bogduk 2006; Chua et al. 2011). In theory, this could result in conditioning stimulation and long-term depression (LTD) of pain-related synaptic transmission, such the involvement of excitatory C fibers (Cosman and Cosman 2005; Sandkuhler et al. 1997). However, this neurophysiological theory does not account for the analgesic effects of PRF applied to regions where there is no nervous tissue, such as the application of intra-articular PRF (Sluijter et al. 2008; Karaman et al. 2011; Al-Badawi et al. 2004). Additionally, alterations in dorsal horn neuronal activity in response to PRF treatment have been demonstrated (Hamann et al. 2006; Higuchi et al. 2002; Van Zundert et al. 2005). PRF applied to DRG induced increases in c-Fos immunoreactive neurons in the superficial laminas of the
spinal dorsal horn, suggesting temperature-independent activation of spinal dorsal horn neurons (Higuchi et al. 2002). Another study revealed up-regulated c-Fos expression in cells of the spinal dorsal horn seven days after application of either CRF or PRF to DRG, demonstrating late, temperature-independent cellular activity and neuronal activation (Van Zundert et al. 2005). Furthermore, Hamann et al. (2006) demonstrated a heat-independent increase in the expression of activated transcription factor 3 (ATF3) in a specific population of DRG neurons in response to PRF applied proximal to DRG. Collectively these studies’ findings do not provide a reasonable explanation for PRFs observed analgesic effects. Expression of c-Fos and ATF3 are no more than markers of increased cellular metabolic activity (Bogduk 2006) and nerve injury (Tsujino et al. 2000), respectively. They are not even specific to the nociceptive pathway, they are not involved in pain modulation and do not distinguish between inhibitory and excitatory activity (Bogduk 2006; Richebe et al. 2005).
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Fig. 5 Western blot analysis showing the time course of CFAinduced pJNK1 and pJNK2 expression in a ipsilateral and b contralateral hind paws, and the effect of PRF treatment proximal to DRG on expression of JNK1 and JNK2 in the c, e ipsilateral and d, f contralateral dorsal horn of the spinal cord. The pJNK1 and pJNK2 proteins were rapidly activated in the dorsal horn of the lumbar spinal cord, and their expression was maintained for the 14 day duration of the study in both the ipsilateral and contralateral lumbar spinal cord.
PRF applied proximal to DRG reduced the induction of pJNK1 (day 1 to day 14 on ipsilateral and day 7 to day 14 on contralateral spinal dorsal horn) and pJNK2 (day 3 to day 14 on both ipsilateral and contralateral spinal dorsal horn). Analyzed by one-way ANOVA with Newman–Keuls post-hoc analysis. Each value is presented as mean (SD); *P \ .05, compared to no-PRF group rats; n = 5 for each group
We provide herein, a more logical and convincing theory to explain the possible mechanism underlying PRFs observed antinociceptive and neuromodulation effects. In our study, the application of 500 kHz RF in 20-ms pulses at a rate of 2 Hz adjacent to DRG created an electric field at the tip of the electrode. This caused local heating, which we limited to a maximum temperature of 42 °C to prevent tissue damage from occurring. The delivery of brief electrical pulses or electromagnetic waves adjacent to DRG was sufficient to inhibit JNK activation in spinal dorsal horn cells, probably mainly in astrocytes (Gao et al. 2010). The inhibition of JNK activation further suppresses JNK downstream gene expression, such as proinflammatory and pronociceptive mediators (Scholz and Woolf 2007; Gao and Ji 2008) that in conjunction enhance pain sensitivity in the dorsal horn of the spinal cord, termed ‘‘central sensitization’’ (Ji et al. 2003; Latremoliere and Woolf 2009). This
central suppression effect induced by PRF treatment eventually leads to the attenuation of pain hypersensitivity, as evidenced by our behavioral findings. Furthermore, previous study demonstrated that exposure of neurons to PRF resulted in an acute neuromodulatory action such as transient inhibition of evoked excitatory transmission that might be able to block or attenuate the peripheral excitatory transmission (Cahana et al. 2003). Besides, PRF applied adjacent to DRG might work in a similar manner to electroacupuncture, activating both the spinal and supraspinal mechanisms which might further reduce pain perception (Carlsson 2002). In addition, a recent study revealed that PRF applied adjacent to DRG resulted in a significant suppression of spinal excitatory amino acids release and attenuation of mechanical allodynia (Yang et al. 2013). In our work, we evaluated the analgesic effects of PRF applied to two different locations, proximal and distal to
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DRG. PRF applied adjacent to DRG produced significant reductions only in mechanical allodynia for the entire 14 day experimental period. However, the attenuation of thermal hyperalgesia was only significant in the first day following application of PRF proximal to DRG. This observation is similar, albeit in greater detail, to a previous study by Hagiwara et al. (2009), which demonstrated that application of PRF (2 bursts per second of 500-kHz RF with a duration of 20 ms per burst and temperature of 42 °C for 180 s) to the sciatic nerve effectively reduces CFA-induced thermal hyperalgesia during the first three consecutive days following treatment. This reduction in hyperalgesia results from enhancement of the descending inhibitory pathways as the PRF mechanism of action. Three possible explanations for the discrepancies between our results and Hagiwaras’ are: (1) different age/weight SD rats (250–300 vs. 350–375 g); (2) different amount of CFA injected (0.15 vs. 0.2 ml); (3) different energy duration of PRF (180 vs. 120 s) applied to the sciatic nerve. The differences in these research regimens might influence intracellular pathophysiological events (Bogduk 2006; Cosman and Cosman 2005) and further affect the analgesic effect of PRF in inflammatory pain model. Furthermore, we did not evaluate the effect of PRF on CFA-induced immediateearly behavioral responses (e.g., 2 or 6 h) because the most significant reductions in pain usually occur in the days following PRF treatment (Malik and Benzon 2008; Hagiwara et al. 2009; Werner et al. 2012). Our PRF animal study was designed to be clinically oriented, and so we considered it reasonable that behavioral observation should commence at 1 day post-CFA injection. There are some inconsistencies in our findings, for example, PRF treatment did not reduce CFA-induced contralateral mechanical hyperalgesia or thermal hyperalgesia, but did reduce CFA-induced increases in p-JNK expression in the contralateral dorsal horn. We can account for these apparent inconsistencies as follows: according to previous reports, unilateral CFA injection in a hind paw induces not only bilateral mechanical hyperalgesia, but also ipsilateral heat hyperalgesia (Schepers et al. 2008; Gao et al. 2010). This is in accord with our results, which showed no significant alterations in the contralateral hind paw in response to thermal stimulation compared to basal level TPWL, or between the no-PRF and PRF groups. Therefore, although the application of PRF significantly suppressed the expression of contralateral JNK, we did not see a significant difference in thermal behavioral test responses between groups. And this is supported by previous study which mentioned that spinal JNK induction is accompanied with mechanical response and spread of pain and which degree of JNK induction might induce the mechanical hyperalgesia in ipsilateral and contralateral is not known at this point (Gao et al. 2010).
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Conclusion In conclusion, PRF applied proximal to DRG provides an effective treatment for inflammatory pain by attenuating mechanical hyperalgesia by modulation of the spinal dorsal horn to suppress JNK activation, and potentially down-regulating the expression of downstream pain-enhancing genes. Acknowledgments This work was supported in part by Grant Nos. 880891, 880892, 880893, 891251, and 8A1011 from Chang Gung Memorial Hospital Research, Kaohsiung, Taiwan, and by Grant Nos. 96-2628-B-182A-005-MY3, 98-2314-B-182A-035-MY2, 100-2314B-182A-037, 101-2314-B-182A-014-, and 101-2314-B-182A-068MY3 from the Taiwan National Science Council, Taipei, Taiwan. Conflict of interest
The authors declare no conflict of interest.
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