Abstract
Background Transcutaneous electrical nerve stimulation (TENS) is often used for management of chronic pain.
Objective The purpose of this study was to investigate whether TENS altered postincisional allodynia, substance P, and proinflammatory cytokines in a rat model of skin-muscle incision and retraction (SMIR).
Design This was an experimental study.
Methods High-frequency (100-Hz) TENS therapy began on postoperative day 3 and was administered for 20 minutes daily to SMIR-operated rats by self-adhesive electrodes delivered to skin innervated via the ipsilateral dorsal rami of lumbar spinal nerves L1–L6 for the next 27 days. The expressions of substance P, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) in the spinal cord and mechanical sensitivity to von Frey stimuli (4g and 10g) were evaluated.
Results The SMIR-operated rats displayed a marked hypersensitivity to von Frey stimuli on postoperative day 3. In contrast to the SMIR-operated rats, SMIR-operated rats after TENS administration showed a quick recovery of mechanical hypersensitivity. On postoperative days 3, 16, and 30, SMIR-operated rats exhibited an upregulation of substance P and cytokines (TNF-α, IL-6, and IL-1β) in the spinal cord, whereas SMIR-operated rats after TENS therapy inhibited that upregulation. By contrast, the placebo TENS following SMIR surgery did not alter mechanical hypersensitivity and the levels of spinal substance P, TNF-α, IL-6, and IL-1β.
Limitations The experimental data are limited to animal models and cannot be generalized to postoperative pain in humans.
Conclusions The results revealed that TENS attenuates prolonged postoperative allodynia following SMIR surgery. Increased levels of spinal substance P and proinflammatory cytokines, activated after SMIR surgery, are important in the processing of persistent postsurgical allodynia. The protective effect of TENS may be related to the suppression of spinal substance P and proinflammatory cytokines in SMIR-operated rats.
Prolonged postincisional pain is a major factor in reducing quality of life1 and increasing the utilization of health services.2,3 Although postsurgical pain can be managed by administration of adequate parenteral opioids, high-dose opioid therapy is limited due to sedation and respiratory depression. Additionally, postsurgical pain can be increased or initiated by movement, which is less responsive to opioids compared with pain at rest.4,5 There is a growing body of evidence that transcutaneous electrical nerve stimulation (TENS) can alleviate pain.6–10 For instance, the application of high-frequency TENS is able to attenuate activity-related pain in patients undergoing surgery,9 but the underlying mechanism of TENS treatment is unclear.
The pain impulse is transmitted along the C and A(delta) fibers from the peripheral nervous system to the dorsal horn of the spinal cord. Considerable evidence has established that intense, recurrent, or sustained noxious stimulation of C fibers leads to an increase in synaptic efficacy and a wide dynamic range of neuron excitability in the spinal dorsal horn.11–15 Substance P appears to be involved in the mechanisms of hyperexcitability of dorsal horn neurons through potentiation of the excitatory effects of glutamate or through the direct action on the postsynaptic cells in the spinal cord.16,17 Additionally, substance P has been implicated in regulating relatively high-intensity nociceptive transmission occurring with the administration of strong chemical, mechanical, and thermal stimuli. In inflammatory processes, substance P release was upregulated in the spinal cord and peripheral tissues.18,19
Resulting data from animal experiments give definite evidence for the crucial role of cytokines in the onset and maintenance of pain.20–22 In our previous study, we found that rats after skin-muscle incision-retraction (SMIR) surgery elicited mechanical hypersensitivity and the upregulation of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the spinal cord.23 Moreover, interleukin-1beta (IL-1β) content was markedly increased in damaged nerve.24,25 Additionally, it has been shown that in patients who underwent standard posterolateral thoracotomy, the TENS group was associated with a lesser release of IL-6 and TNF-α in comparison with the placebo group.26 Furthermore, TENS application around the edges of the wound improves skin healing by inhibiting proinflammatory cytokines (IL-1β, IL-6, and TNF-α).27 Recently, TENS was shown to inhibit an upregulation of IL-1β in the dorsal root ganglion of rats undergoing SMIR surgery,28 whereas tissue injury induces mediator releases in the spinal cord resulting in pain hypersensitivity. To date, little is known of the impact of high-frequency TENS in the alteration of substance P and proinflammatory cytokine levels in the spinal cord of SMIR-operated rats. It is well-established that the SMIR model does accurately reflect the clinical scenario of postincisional pain (ie, prolonged tissue retraction resulting in persistent pain).29 The purpose of this study was to examine the effect of TENS on mechanical sensitivity as well as the time course of substance P, TNF-α, IL-6, and IL-1β levels in the spinal cord in rats after SMIR surgery.
Materials and Method
Animals
The experimental procedures were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (Tainan, Taiwan) and conducted according to International Association for the Study of Pain (IASP) ethical guidelines.30 Male Sprague-Dawley rats, each weighing 205 to 253 g, were obtained from the Laboratory Animal Center of National Cheng Kung University and kept in the animal housing facilities at National Cheng Kung University, with controlled humidity (approximately 50% relative humidity), room temperature (22°C), and a 12-hour (6:00 am–6:00 pm) light/dark cycle.
SMIR
The SMIR procedure was performed on rats as previously described.29 In brief, animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal), and a 15- to 20-mm incision was made in the skin of the medial thigh approximately 4 mm medial to the saphenous vein to expose the muscle of the thigh. Next, a 7- to 10-mm incision was made in the gracilis muscle layer of the thigh, approximately 4 mm medial to the saphenous nerve. Subsequently, the prongs of a retractor (Cat. No. 13-1090, Biomedical Research Instruments Inc, Silver Spring, Maryland) were inserted into the gracilis muscle to position all prongs underneath the superficial layer of thigh muscle. The skin and superficial muscle of the thigh were then retracted by 2 cm, which exposed the fascia of the underlying adductor muscles. The retraction time was maintained for 1 hour, with gauze pads covering the incision site. Following the SMIR procedure, these tissues in the surgical site were closed with 4.0 Vicryl sutures (Ethicon Inc, a Johnson & Johnson company, Somerville, New Jersey).
TENS Preparation
High-frequency (100-Hz) TENS was applied to rats while they were lightly anesthetized with isoflurane (1.5%–2%). The rats were treated using the TENS machine (Trio 300, Ito Co, Tokyo, Japan) through the self-adhesive surface electrodes, and the stimulator was run continuously without any preprogrammed options. The intensity of TENS stimulation was set at 80% of that needed to elicit visible muscle contractions (30–40 μA delivered through 45 mm × 5 mm electrodes). The pulse duration was kept at 100 microseconds, and the treatments lasted for 20 minutes.31 Surface electrode placement was on the chemically denuded and presumably uninvolved skin overlying the dorsal thigh musculature, and the stimulated skin was innervated by the dorsal rami of lumbar spinal cord segments 1 through 6.32
Mechanical Sensitivity
Mechanical allodynia was measured using von Frey filaments.29 All behavioral measurements were tested between 9:00 am and 11:00 am, and rats were evaluated for mechanical allodynia after a period of at least 3 days of habituation to the testing environment and experimenters. In brief, rats were placed individually in a clear plexiglass chamber (23 cm [length] × 17 cm [width] × 14 cm [height]) and supported by a wire mesh floor (40 cm [width] × 50 cm [length]). Mechanical sensitivity was evaluated using 2 von Frey filaments with bending forces of 4g and 10g (Linton Instrumentation, Norfolk, United Kingdom). In ascending order of force, each filament was applied 10 times vertically to the mid-plantar area of the hind paw. This procedure was done carefully to avoid stimulating the same spot repeatedly within this region and to avoid stimulating the tori and footpads themselves. Withdrawal responses caused by mechanical stimulation were determined, including foot lifting, shaking, licking, and squeaking. Paw movements associating with weight shifting or locomotion were not counted. For consistency, an experienced investigator, who was blinded to the groups, was responsible for handling all of the animals and for behavioral assessment.
Cytokine (TNF-α, IL-1β, and IL-6) Analysis
Rats were anesthetized with urethane (1.67 g/kg, intraperitoneal) and sacrificed to obtain the L3–L5 segments of rat spinal cord on postoperative days 3, 16, and 30. The nerve specimen was immediately stored at −80°C for the protein assay. Ice cold (4°C) homogenization buffer was freshly prepared by adding protease inhibitor (P8340 cocktail, Sigma-Aldrich, St Louis, Missouri) to T-PER Tissue Protein Extraction Reagent (Pierce Chemical Co, Rockford, Illinois) prior to tissue lysis. After adding the buffer (300 μL to each spinal nerve), a homogenization probe (Tissue Tearor, Polytron, Biospec Products Inc, Bartlesville, Oklahoma) was applied for 20 seconds on ice at 21,000 rpm. The homogenized samples were then centrifuged for 40 minutes at a speed of 13,000 rpm at 4°C, stored at −80°C, and used subsequently for protein quantification. The protein concentration in the supernatant was quantified using the Lowry protein assay. Samples were pipetted as duplicates (1 μL/50 μL/well) in a 96-well microtiter plate (Costar, Sigma-Aldrich). Each plate was inserted into a plate reader (Molecular Device Spec. 383, Sunnyvale, California) to read the optical density of each well at an absorbance of 750 nm. Data were analyzed using Ascent Software (London, United Kingdom) for iEMS Reader.
The concentrations of TNF-α, IL-1β, and IL-6 in the supernatants were determined by the DuoSet ELISA Development Kit (R&D Systems, Minneapolis, Minnesota).25,33 All experimental procedures were practiced in accordance with the manufacturer's recommended protocols. Plates were individually inserted into the plate reader for reading optical density by a 450-nm filter. Data were analyzed using Ascent Software for iEMS Reader and a 4-parameter logistics curve-fit and were expressed in pg/mg protein of duplicate samples.
Substance P Assay
Tissues (the L3–L5 spinal segments) were homogenized in 200-μL RIPA buffer and 10 μL protease inhibitor (P8340, Sigma-Aldrich) using a glass homogenizer. After incubating on ice for 1 hour, the lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C with a High-Speed Micro Refrigerated Centrifuge (model 3740, Kubota Corp, Tokyo, Japan). The supernatant was collected and determined the protein concentration using a protein assay. Then, we added 25 μL with Laemmli Sample Buffer (Bio-Rad, Hercules, California) into lysates and heated it at 100°C for 5 minutes. An ELISA reader was used to assay protein with bovine serum albumin as standard at 620 nm.
Protein samples (30 μg/lane) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis at a constant voltage of 75 V. These electrophoresed proteins were transferred to a polyvinylidene difluoride membrane with a 0.45-μm pore size (Millipore, Bedford, Massachusetts) by a transfer apparatus (Bio-Rad, Hercules). The polyvinylidene diflouride membrane was then blocked in Tris-buffered saline (TBS) (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% fat-free milk (Difco, Detroit, Michigan) for 1 hour. The primary antibody of substance P (Millipore, Billerica, Massachusetts) and the primary antibody of actin were diluted to 1:2,000 in antibody binding buffer overnight at 4°C. The membrane was washed 3 times with TBS (10 minutes per wash) and incubated for 1 hour with goat-anti-mouse IgG-HRP (Santa Cruz Biotechnology Inc, Santa Cruz, California) and diluted 5,000-fold in TBS buffer at 4°C. The membrane was washed in TBS buffer for 10 minutes, 3 times. Immunodetection for substance P was performed by the enhanced chemiluminescence ECL Western blotting luminal reagent (Santa Cruz Biotechnology Inc, Dallas, Texas), and the membrane was quantified using a Gel-Pro Analyzer (version 4.0, Media Cybernetics Inc, Rockville, Maryland). Actin was used as a loading control, so it adjusted the substance P values as a ratio to actin to compensate for unequal protein that was loaded in wells.
Groups and Design
Animals were randomly divided into 4 groups. The first group of rats (n=8) received only SMIR. The second group of rats underwent the same procedure with the exception of the skin-muscle retraction (sham group, n=8). The third group of rats (SMIR-TENS group, n=8) received SMIR surgery to the right (ventral) thigh and high-frequency TENS through stimulating electrodes positioned on skin overlying the dorsal region of right thigh musculature (ipsilateral to injury in the SMIR-operated rats). The fourth group of rats was treated exactly like the SMIR-operated rats that received TENS, including isoflurane administration, without administering TENS (SMIR-Placebo-TENS group, n=8).
On day 3 after surgery, the rats received the treatment (TENS) that they did not receive the previous day. The TENS was delivered to the SMIR-operated rats for 20 minutes once a day commencing immediately on day 3 after the SMIR surgery and then delivered daily for 20 minutes for the next 27 days. Significant mechanical allodynia in animals began 3 days after rats had received SMIR surgery and lasted for up to 1 month.23
All rats were evaluated twice for mechanical sensitivity on the day before surgery, and the 2 measurements were averaged to obtain a single baseline mechanical sensitivity value for each evaluation. These rats were subsequently evaluated on days 3, 9, 16, 23, and 30 after SMIR surgery or on the analogous day if SMIR surgery did not occur. The SMIR surgery, which induced significant mechanical hypersensitivity in the ipsilateral hind paw, was seen by postoperative day 3 as previously described.29 The 5-day postsurgery evaluation was performed at 23 hours after the final TENS therapy or isoflurane anesthesia. The last TENS therapy occurred 29 days after SMIR surgery.
On postoperative days 3 (24 rats; n=6 rats in each group for tissue analysis), 16 (24 rats; n=6 rats in each group for tissue analysis), and 30 (32 rats; n=8 rats in each group for the behavior testing and some of them for tissue analysis), a total of 78 rats were used for this study. The behavior testing used 8 rats and tissue analysis used 6 rats per time point (6 rats per group for cytokines analysis and some of them [4 rats per group] for substance P analysis).
Data Analysis
Experimental data are presented as the mean (± standard error of the mean) number of observations unless noted otherwise. The difference in number withdrawn from stimulus (Fig. 1) was analyzed by 2-way analysis of variance (ANOVA) for repeated measures, followed by Bonferroni post hoc comparison. The TENS application was the between-subjects factor, and time was the repeated measure. The differences in substance P and cytokines (Fig. 2) were determined using 1-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. SPSS for Windows (version 17.0, SPSS Inc, Chicago, Illinois) was used for all statistical analyses. In each case, statistical significance was set at P<.05.
The behavioral time courses of number of rats withdrawn from mechanical stimuli. The graphs exhibit mean (± standard error of the mean) of the number of rat hind paw withdrawals out of 10 stimuli with (A) von Frey 4g stimuli and (B) von Frey 10g stimuli. For all time points, n=8 in each of the 4 experimental groups: sham-operated rats (sham group), skin-muscle incision-retraction (SMIR)-operated rats (SMIR group), rats that received transcutaneous electrical nerve stimulation (TENS) after SMIR surgery (SMIR-TENS group), and rats that received placebo TENS after SMIR surgery (SMIR-Placebo-TENS group). The asterisk (*) indicates P<.05 when the SMIR or SMIR-Placebo-TENS group was compared with the SMIR-TENS group; the plus symbol (+) indicates P<.05 when the groups were compared with the sham group (2-way analysis of variance for repeated measures followed by Bonferroni post hoc test).
The level of substance P (A–C), tumor necrosis factor alpha (TNF-α) (D), interleukin-1beta (IL-1β) (E), and Interleukin-6 (IL-6) (F) on postoperative days 3 (A), 16 (B), and 30 (C) in the sham-operated rats (sham group), skin-muscle incision-retraction (SMIR)-operated rats (SMIR group), rats that received transcutaneous electrical nerve stimulation (TENS) after SMIR surgery (SMIR-TENS group), and rats that received placebo TENS after SMIR surgery (SMIR-Placebo-TENS group). The values are presented as mean (± standard error of the mean) for 4 to 6 rats per group. Compared with the sham group or SMIR-TENS group, the SMIR group showed a significant increase in substance P level in the spinal cord (P<.05) on postoperative days 16 and 30. The asterisk (*) indicates P<.05 when the SMIR or SMIR-Placebo-TENS group was compared with the SMIR-TENS group; the plus symbol (+) indicates P<.05 when the groups were compared with the sham group (1-way analysis of variance followed by Bonferroni post hoc test).
Results
High-Frequency TENS Suppresses SMIR-Induced Mechanical Allodynia
Rats in the SMIR and SMIR-Placebo-TENS groups demonstrated mechanical hypersensitivity on postoperative days 3, 9, 16, 23, and 30 (P>.05, 2-way repeated-measures ANOVA; Fig. 1). Rats following SMIR surgery and SMIR-Placebo-TENS rats demonstrated a prolonged mechanical hypersensitivity, which significantly was found in the SMIR ipsilateral paw response to the von Frey hair test (4g and 10g) in comparison with the sham-operated rats on postoperative days 3, 9, 16, 23, and 30 (P<.05, 2-way repeated-measures ANOVA, Bonferroni post hoc comparison; Fig. 1). Figure 1A shows the effects of TENS application on SMIR-evoked ipsilateral mechanical hypersensitivity in which the response to von Frey 4g stimuli was markedly inhibited compared with SMIR rats without TENS treatment or SMIR-Placebo-TENS rats on postoperative days 9, 16, 23, and 30 (P<.05, 2-way repeated-measures ANOVA). The second, third, and fourth weeks of TENS therapy also significantly inhibited SMIR-evoked mechanical hypersensitivity compared with SMIR rats without TENS treatment or SMIR-Placebo-TENS rats for the response to von Frey 10g stimuli (P<.05, 2-way repeated-measures ANOVA; Fig. 1B). There was no significant difference between SMIR-TENS and sham-operated rats on postoperative day 30, whereas SMIR-TENS rats on postoperative days 9, 16, and 23 showed mechanical hypersensitivity compared with sham-operated rats (P<.05, 2-way repeated-measures ANOVA; Fig. 1).
High-Frequency TENS Prevents Substance P Upregulation in the Spinal Cord
Figure 2 (graphs A–C) reveals the expression of substance P in the spinal cord on postoperative days 3, 16, and 30 in the 4 groups. Substance P levels in the spinal cord were significantly increased in the SMIR group (P<.01) and SMIR-Placebo-TENS group (P<.01) than in the sham-operated group on postoperative days 3 (Fig. 2A), 16 (Fig. 2B), and 30 (Fig. 2C). The SMIR-operated rats underwent a 2-week (P<.01; Fig. 2B) or 4-week (P<.05; Fig. 2C) TENS program that showed the substance P content was lower than the content in SMIR-operated rats without TENS therapy. There was no significant difference in substance P levels of the spinal cord between SMIR and SMIR-Placebo-TENS animals on postoperative days 3, 15, and 30 (Fig. 2).
High-Frequency TENS Decreases Excess Proinflammatory Cytokine Levels in the Spinal Cord
Figure 2 (graphs D–F) depicts the levels of IL-1β, IL-6, and TNF-α in the spinal cord of sham-operated, SMIR, SMIR-TENS, and SMIR-Placebo-TENS rats on postoperative days 3, 16, and 30. The expression of TNF-α (Fig. 2D), IL-1β (Fig. 2E), and IL-6 (Fig. 2F) in the spinal cord on postoperative days 3, 16, and 30 was significantly increased in the SMIR group (P<.05) and the SMIR-Placebo-TENS group (P<.05) compared with the sham-operated group. The SMIR-Placebo-TENS and SMIR rats showed similar cytokine (TNF-α, IL-6, and IL-1β) levels in the spinal cord. By comparison, the SMIR-operated rats undergoing TENS therapy demonstrated markedly lower TNF-α (P<.05; Fig. 2D), IL-1β (P<.05; Fig. 2E), and IL-6 (P<.05; Fig. 2F) levels than those in SMIR-operated rats without TENS treatment on postoperative days 16 and 30.
Discussion
In the present study, we demonstrated for the first time that high-frequency TENS diminishes SMIR-evoked allodynia and upregulation of proinflammatory cytokines in the spinal cord of the SMIR-operated rat. These results support the findings of our previous study in which exercise decreased postsurgical pain and cytokine expression in rats after SMIR surgery.23 Another interesting finding is that high-frequency TENS therapy in SMIR-operated rats lowered the increased spinal substance P content induced by SMIR surgery. Overall, these data presume that TENS inhibits the progress of prolonged postincisional allodynia, which possibly relates to attenuate spinal substance P and proinflammatory cytokine releases.
High-Frequency TENS Retards the Progress of SMIR-Evoked Allodynia
Transcutaneous electrical nerve stimulation has been used to control postoperative pain following various surgical procedures,9,10,26 but the ambiguous conclusions of several studies suggested that the location of the electrodes, the mode of TENS application, and the disease stage in patients may influence the therapeutic results of these procedures.34–37 In this case, it is unclear whether TENS can treat SMIR-evoked allodynia and its amelioration. There is credible evidence that TENS attenuates postoperative pain by requiring less analgesic during the first 3 days after the major surgery.38 That way, the efficacy of TENS is dose-dependent and requires a very strong sensation of currents. In this study, we set the intensity of high-frequency TENS to evoke 80% of visible muscle contractions. We also observed that the SMIR-operated rats that received TENS therapy showed a reduction in mechanical hypersensitivity. However, this case did not display normal sensitivity to the mechanical stimulation. In contrast to the sham-operated rats, the degree of reduction (<50%) in postoperative mechanical/tactile hypersensitivity by TENS was small. Therefore, this finding indicates the relevance of the findings in relation to the presence of postincisional allodynia. Interestingly, the protective effect of TENS was not found to be too intense while SMIR-operated rats were recovering.
Assuredly, it is interesting to note that there was a temporal window, namely from postoperative day 16 to postoperative day 30, in which TENS therapy markedly alleviated mechanical hypersensitivity. Meanwhile, high-frequency TENS also significantly suppressed tactile allodynia from postoperative day 9 to postoperative day 30. These results suggested that the 1-week outcomes remained unsatisfactory and further intervention was necessary, whereas TENS therapy could maintain the 2- to 4-week clinical outcomes. It appears that approximately 2 weeks of TENS intervention is required before the differences in pain behavior can occur. The pain behaviors, which are altered by TENS, may have been dependent on the von Frey stimuli (4g versus 10g). This assumption possibly could relate to the healing postoperative phase (acute inflammation versus proliferation) in which TENS may be more beneficial toward recovery later rather than earlier.
High-Frequency TENS Inhibits the Upregulation of Spinal Substance P Release Caused by SMIR Surgery
Central sensitization occurs when there is an increase in the excitability of neurons within the central nervous system (ie, spinal cord), so that a normal input from the peripheral nervous system begins to evoke abnormal responses.39 Substance P as a neuromodulator or neurotransmitter is involved in the transmission of nociceptive sensitization in the spinal cord.40,41 It has been shown that substance P levels in the spinal cord and peripheral tissues were upregulated in the inflammatory processes.18,19 In the present study, we noticed SMIR-operated rats not only exhibited a significant increase in spinal substance P levels but also developed aggrandized plantar responsiveness from the mechanical stimuli. We presumed that neuropeptide (substance P), as a neurotransmitter, was released into the dorsal horn of the spinal cord to evoke postoperative tactile allodynia. It is possible that a lack of this substance attenuates the intensity of the inflammatory reaction, thereby providing a second mechanism for the reduced thermal and mechanical sensitization.42
To investigate the mechanisms of the therapeutic effects of TENS application on postoperative allodynia, we explored a possible role played by substance P in the spinal cord of the SMIR-operated rats. We demonstrated that increased production of substance P after SMIR surgery could be reversed by high-frequency TENS therapy. Thus, the disruption of substance P signaling lowered the responses to the main nociceptive mediators and reduced the production of few of the similar mediators. More evidence suggested that TENS could produce a significant suppression of chronic hyperalgesia43 and formalin-induced pain,44 which is accompanied by a reduction of the substance P level in the spinal cord. Although the effect of TENS was frequency dependent, acupuncture or TENS altered the noxious nerve stimulation-induced release45,46 and production44 of substance P in the spinal cord.47 These results suggested that TENS suppressed central sensitization that was induced by nerve (tissue) damage and inflammation by diminishing the upregulation of substance P.
High-Frequency TENS Attenuates SMIR-Induced Increased Proinflammatory Cytokines in the Spinal Cord
Several cytokines are released from a variety of immune cells and later evoke powerful pain. Although there is no direct evidence that cytokines affect the excitability of sensory fibers, it is well-established that the effects of messages can be delivered to the brain by the activation of vagal afferents and cutaneous nerves can be activated through cytokines.48 In the current study, we investigated spinal cytokine levels in SMIR-operated rats, which has not been reported previously. We wanted to give an objective measurement of the value of TENS therapy, considering that cytokines play an important role in the immunologic and inflammatory responses to the surgical injury. Our study has demonstrated an essential role of spinal proinflammatory cytokines in rats following SMIR surgery. Proinflammatory cytokines are well-established to reflect the degree of surgical injuries because they are the markers of inflammatory response.26
Although we cannot be sure whether the mechanisms of proinflammatory cytokines are involved, it is generally believed that proinflammatory cytokine induces the pain behavior after spinal injection.49 Moreover, spinal IL-1β was shown to enhance C-fiber-evoked responses and windup in wide-dynamic-range dorsal horn neurons50 while the intrathecal injection of IL-6 neutralizing antibody and the inhibition of TNF-α synthesis by intraperitoneal thalidomide markedly delayed the onset of pain.51 On the contrary, there is controversy regarding the role of IL-6 in pain regulation.52 Our data supported that increased IL-6 levels are aligned with SMIR-induced allodynia.
In the spinal cord, each of the proinflammatory cytokines has been shown to be involved in pain facilitation.53–55 Moreover, there is a growing body of evidence that TENS suppresses proinflammatory cytokines.26,27,56–58 For instance, TENS application has been shown to enhance the increase in blood supply,58 and the epidermal growth factor (EGF) and the decrease in TNF-α explains the lower rate of mucositis.57 Furthermore, transcutaneous electrical acupoint stimulation, transcutaneous auricular vagus nerve stimulation, and vagus nerve stimulation have been shown to activate the cholinergic anti-inflammatory pathway by inhibiting the proinflammatory cytokine levels in an endotoxemic rat model.56 Additionally, TENS therapy attenuated the upregulation of proinflammatory cytokines in rats following skin incision.27 Our data also confirmed the findings of a previous study on the efficacy of TENS in controlling postthoracotomy pain with a decrease in cytokine production.26 Therefore, our finding of reduced proinflammatory cytokines with TENS intervention is important because healthy individuals receiving surgery demonstrate increased inflammation,26 suggesting that the increased inflammation is responsible for the delay of the healing process caused by surgery.
Essentially, proinflammatory cytokines activate the glia and neurons through binding to their respective receptors.53 Spinal glial cells have been involved in producing hypersensitivity after surgical incisions.59–62 In this situation, previous studies have shown that vagus nerve stimulation was implicated in increased activation of the enteric glia cells, resulting in the reduction of burn-induced intestinal barrier damage.63 Although we did not indicate how TENS (peripheral) intervention leads to alterations in the glial activation, a recent study showed that the spinal cord stimulation (central) can regulate nociceptive input at the spinal cord through multiple inhibitory neurotransmitters that subsequently inhibit glial cell activation.64
A previous experiment exhibited low-frequency (10 Hz) and high-frequency (130 Hz) TENS that were highly effective as analgesic treatments.65 Both abolished inflammatory pain (hyperalgesia), and the participation of endogenous opioids on TENS-induced analgesia was confirmed in low-frequency instead of high-frequency TENS-treated rats.65 It had been shown that low-frequency TENS and acupuncture are ineffective in the animals' tolerance toward morphine.66,67 Nevertheless, high-frequency TENS is still effective in morphine-tolerated rats.66 On the other hand, high-frequency electroacupuncture is effective in the rats' tolerance to δ-opioid agonists, although this treatment was ineffective in animals that tolerated a κ-opioid receptor agonist called dynorphin.68 Evidence has suggested that repeated administration of high- or low- frequency TENS for 6 days elicited a μ- and δ-opioid– mediated analgesic tolerance in a rat model of carrageenan-induced hind paw inflammation, respectively.69 Although we did not find that repeated administration of high-frequency TENS can cause an analgesic tolerance in this SMIR-evoked allodynic rat model, it needs to be tested with regard to future findings that may show the different methods of repeated application of TENS leading to the development of analgesic tolerance in SMIR-operated rats.
We conclude that high-frequency TENS alleviates the development of persistent postoperative or postincisional allodynia evoked by SMIR surgery. High-frequency TENS therapy inhibits the upregulation of spinal substance P and proinflammatory cytokines, and it may show the TENS-therapeutic mechanisms to manage prolonged postsurgical allodynia. The treatment strategy of using TENS to prevent the process of postoperative allodynia warrants further research.
Footnotes
Dr Chen, Dr Hung, and Dr Wang provided concept/idea/research design. Dr Chen, Dr Tzeng, and Dr Hung provided writing. Dr Tzeng and Ms Lin provided data collection. Dr Chen, Dr Tzeng, Ms Lin, and Dr Hung provided data analysis and consultation (including review of manuscript before submission). Dr Chen and Dr Hung provided project management and fund procurement. Dr Hung provided facilities/equipment, institutional liaisons, and administrative support.
The authors acknowledge the financial support provided by grants NSC 100-2314-B-039-017-MY3 and NSC 101-2314-B-006-037-MY3 from the National Science Council, Taiwan.
- Received July 17, 2013.
- Accepted August 28, 2014.
- © 2015 American Physical Therapy Association
References
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