Transcutaneous Electrical Nerve Stimulation Attenuates
Post-surgical Allodynia
and Suppresses Spinal Substance P and
Pro-inflammatory Cytokines Release in Rats
Yu-Wen Chen1,2, Ph.D., Jann-Inn Tzeng3,4, M.S., M.D., Min-Fei Lin5, M.S., Ching-Hsia Hung5,*, Ph.D., Jhi-Joung Wang2, M.D., Ph.D.
1 Department of Physical Therapy, China Medical University, Taichung, Taiwan 2 Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan 3 Department of Food Sciences and Technology, Chia Nan University of Pharmacy
and Sciences, Jen-Te, Tainan City, Taiwan
4 Department of Anesthesiology, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan
5 Institute & Department of Physical Therapy, National Cheng Kung University, Tainan, Taiwan
Institution: This work was done in National Cheng Kung University, Tainan,
Taiwan.
Running Header (< 45 characters): TENS decreasespostoperative allodynia
Funding: The financial support provided for this study by grants NSC
100-2314-B-039-017-MY3 and NSC 101-2314-B-006-037-MY3 from the National Science Council, Taiwan.
*Corresponding author:
Ching-Hsia Hung, Ph.D.
National Cheng Kung University,
Institute & Department of Physical Therapy, No.1 Ta-Hsueh Road, Tainan, Taiwan Phone: 886-6-2353535 ext 5939
The Bottom Line
What do we already know about this topic?
The available evidence suggests that TENS therapy is effective in the treatment of mechanical hypersensitivity. Although the early phase of central sensitization
reflects changes in the spinal cord, the mechanism of action of TENS therapy on post-surgical allodynia is unclear.
What new information does this studyoffer?
This study gives the first information demonstrating the important role of substance P in the antinociceptive effect of SMIR operation. In addition, the antinociceptive effect of SMIR operation also appears to involve in the regulation of spinal TNF-α, IL-6, and IL-1β release.
If you’re a patient, what might these findings mean for you?
The experiment demonstrates that TENS, one of the world’s most commonly used modalities, can inhibit SMIR-evoked mechanical hypersensitivity. Thus, physical therapists may advise the patients to use the TENS before physical therapy sessions in order to decrease the painful motion and ambulation.
Background. Transcutaneous electrical nerve stimulation (TENS) was used for management of chronic pain.
Objective. We investigated whether TENS altered postincisional allodynia, substance P, and proinflammatory cytokines in a rat model of skin–muscle incision retraction (SMIR).
Design. This was an experimental study.
Methods. High-frequency (100Hz) TENS therapy began on postoperative day 3 (POD3) and then daily for 20 min to SMIR rats by self-adhesive electrodes delivered to skin innervated via the ipsilateral dorsal rami of lumbar spinal nerves L1-L6 and for the next 27 days. The expressions of substance P, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in the spinal cord and mechanical sensitivity to von Frey stimuli (10g and 4g) were evaluated.
Results. SMIR rats displayed a marked hypersensitivity to von Frey stimuli on POD3. In contrast with SMIR rats, SMIR-operated rats after TENS administration showed a quick recovery of mechanical hypersensitivity. OnPODs 3, 16 and 30, SMIR-operated rats exhibited an up-regulation 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 up-regulation. 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. Our results reveal thatTENS attenuates prolonged postoperative allodyniafollowing SMIR operation. Increased levels of spinal substance P and proinflammatory cytokines, activated after SMIR surgery, are important in the processing of persistent post-surgical allodynia. The protective effect by TENS may be relating to the suppression of spinal substance P and proinflammatory cytokines in SMIR rats.
Key words: Transcutaneous electrical nerve stimulator, Postincisional allodynia,
Substance P, Tumor necrosis factor-α, Interleukin-6, Interleukin-1β
Introduction
treated by administration of adequate parenteral opioids, high dose opioid therapy was limited due to sedation and respiratory depression. Additionally, postsurgical pain can be increased or initiated by movement which is less responsive to opioids when 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 surgery patients,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 established that intense, recurrent and/or sustained noxious stimulation of C fibers leads to an increase in synaptic efficacy and wide dynamic range neuron excitability in the spinal dorsal horn.11-15 Substance P appears to be involved in the mechanisms of hyper-excitability 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, the 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 up-regulated in the spinal cord and peripheral tissues.18, 19
role of cytokines in the onset and maintenance of pain.20-22 In our previous study, we revealed that rats after SMIR surgery elicited mechanical hypersensitivity and the up-regulation of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the spinal cord.23 Moreover, IL-1β content was markedly increased in damaged nerve.24, 25 Additionally, it has been shown that in patients underwent standard posterolateral thoracotomy, the TENS group is 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 has been known to inhibit an
up-regulation of IL-1β in the dorsal root ganglion of skin/muscle incision and retraction (SMIR) rats,28 while 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 the SMIR rats. It is well-established that the SMIRmodel does accurately reflect the clinical scenarios of postincisional pain, i.e. 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 thetime-courseof substance P, TNF-α, IL-6, and IL-1βlevelsin the spinal cord in rats after SMIR operation.
Materials and Methods
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 IASP ethical guidelines.30 Male Sprague-Dawley rats, each
Cheng Kung University and kept in the animal housing facilities at National Cheng Kung University, with controlled humidity (approximately 50% relative humidity), room temperature (22C), and a 12-hour (6:00 AM to 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 (50mg/kg, i.p.), and a 15 – 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. Then, 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 retractor (Cat. No. 13-1090, Biomedical Research Instruments Inc, USA) were inserted into the gracilis musclein orderto 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 exposesthe fascia of the underlying adductor muscles. Then, the retraction time was maintained for 1 hour with covering the incision site with the gauze pads to cover the incision site. Following the SMIR procedure, these tissues in the surgical site were closed with 4.0 Vicryl® sutures.
TENS preparation
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 used to run continuously through without any preprogrammed options. The intensity of TENS stimulation was set at 80% of that needed to elicit visible muscle contractions (30-40 microamperes delivered through 45 mm 5 mm electrodes). The pulse duration was kept at 100 μs and the treatments were lasted for 20 min.31 Surface electrode placement was on the chemically-denuded and presumably-uninvolved skin overlying the dorsal thigh musculature while the stimulated skin was innervated by the dorsal rami of lumbar spinal cord segments one-six.32
Mechanical sensitivity
Mechanical allodynia was measured using the von Frey filaments.29 All
behavioral measurements were tested between 9:00 a.m. and 11:00 a.m., and rats were evaluated for mechanical allodynia after a period of at least three days of habituation to the testing environment and experimenters. In brief, rats were placed individually in a clear plexiglass chamber (23 cm [length] x 17 cm [width] x 14 cm [height]) and supported by a wire mesh floor (40 cm [width] x 50 cm [length]). Mechanical sensitivity was evaluated by two von Frey filaments with bending forces of 4g and 10g (Linton Instruments, UK). In ascending order of force, each filament was applied
10 times vertically, to the mid-plantar area of the hind paw. This was takencarefully to avoid stimulating the same spot repeatedly within this region and alsoto avoid stimulating the tori/footpads themselves. Withdrawal responses caused by mechanical stimulation were determined including foot lifting, shaking, licking and squeaking. Paw movements associatingwith weight shifting or locomotion were not counted. For consistency, an experienced investigator, who was blinded to the groups, was
responsible for handling all the animals and behavioral assessment. Cytokines (TNF-α, IL-1β, and IL-6) analysis
Rats were anesthetized with urethane (1.67 g/kg, i.p.) and sacrificed to obtain the L3-L5 segments of rat spinal cordon PODs 3, 16 and 30.The nerve specimen was immediately stored at −80℃ for the protein assay. Ice cold (4ºC) homogenization buffer was freshly prepared by adding protease inhibitor (P 8340 cocktail, Sigma-Aldrich, St. Louis, MO) to T-PER™ Tissue Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL) prior to tissue lysis. After adding the buffer (300 μl/each spinal nerve), a homogenization probe (Tissue Tearor, Polytron; Biospec Products, Inc., Bartlesville, OK, USA) was applied for 20 seconds on ice at 21,000 rpm. Then the homogenized samples were 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). Each plate was inserted into a plate reader (Molecular Device Spec 383, Sunnyvale, CA, USA) to read the optical density of each well at an absorbance of 750 nm. Data were analyzed using Ascent Software (London, UK) 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, MN).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. Then, data were then analyzed using Ascent Software for iEMS Reader and a four-parameter logistics curve-fitand were expressed in pg/mg protein of duplicate samples.
Substance P assay
Tissues (the L3-L5 spinal segments) were homogenized in 200μl RIPA buffer and 10 μl protease inhibitor (P3840, Sigma, St. Louis, MO, USA) using a glass homogenizer. After incubating on ice for 1 hour, the lysates were centrifuged at 12000 rpm for 10 min at 4℃ with High Speed Micro Refrigerated Centrifuge (Model 3740, KUBOTA Corp., Tokyo, Japan). The supernatant was collected and determined theprotein concentration using a protein assay. Then, we added 25μl with Laemmli Sample Buffer (Bio-Rad, Hercules, CA) into lysates, and heated at 100℃ for 5
minutes. An ELISA reader was used to assay protein with bovine serum albumin as standard at 620nm.
Protein samples (30 μg/lane) were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 75 V. These electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane with a 0.45 μm pore size (Millipore, Bedford, MA) by a transfer apparatus (Bio-Rad, Hercules, CA, USA). Then the PVDF membrane was blocked in TBS (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% fat-free milk (Difco, Detroit, MI) for 1 hour. The primary antibody of substance P (Millipore, Billerica, MA, USA) and the primary antibody of actin were diluted to 1:2,000 in antibody binding buffer overnight at 4ºC. The membrane was then washed 3 times with TBS (10 minutes per wash) and incubated for 1 hour with goat-anti-mouse IgG-HRP (Santa-Cruz, Santa Cruz, CA) 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) and then the membrane was quantified by a Gel-Pro Analyzer (version 4.0; Media Cybernetics, USA). 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 four groups. The first group of SMIR rats (n = 8)received skin/muscle incision and retraction. The second group of sham rats underwent the same procedure with the exception of the skin/muscle retraction(n = 8). The third group of SMIR-TENS rats(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 rightthigh musculature (ipsilateral to injuryin SMIR rats), and the fourth group of SMIR-Placebo-TENS rats was treated exactly like SMIR rats receiving TENS, including isoflurane administration, without administeringTENS (n = 8).
On day 3 after surgery, the rats received the treatment (TENS) in which they did not receive the previous day. TENS was delivered to the SMIR rats for 20 minutes once a day commencing immediately on day 3 after the SMIRsurgery and then daily for 20 minutes for the next 27 days. In our hands, significant mechanical allodynia in animals began 3 days after rats had been 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
day 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 hindpaw, was seen by postoperative day 3 (POD3) as previously described.29 The 5-day postsurgery evaluation was performed at 23 hours after the final TENS therapy and/or isoflurane anesthesia. The last TENS therapy occurred 29 days after SMIR surgery.
On PODs 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].
Statistical analysis
Experimental data are presented as the mean ± S.E.M. of N observations unless noted otherwise. The difference in No. withdraw from stimulus (Fig. 1) was analyzed by 2-way analysis of variance (ANOVA) of repeated measures, followed by
Bonferroni post hoc comparison. TENS application was the between-subjects factor, and time was the repeated measure. The differences in substance P (Fig. 2) and cytokines (Fig. 3) were determined using 1-way ANOVA followed by post hoc
Bonferroni test for multiple comparisons. SPSS for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. In each case, statistical significance was set at P < 0.05.
Results
High-frequency TENS suppresses SMIR-induced mechanical allodynia
SMIR and SMIR-Placebo-TENS rats demonstrated similarly to mechanical hypersensitivity on PODs 3, 9, 16, 23 and 30 (Fig. 1, P > 0.05; two-way repeated measures ANOVA). Rats following SMIR operation or MIR-Placebo-TENS rats caused a prolonged mechanical hypersensitivity, which was significantly found in the SMIR ipsilateral paw response to the von Frey hair test (10 and 4g), in comparison
with the sham-operated rats on PODs 3, 9, 16, 23 and 30 (Fig. 1, P < 0.05, two-way repeated measures ANOVA, Bonferroni’s post-hoc). Fig. 1A showed the effects of TENS application on SMIR-evoked ipsilateral mechanical hypersensitivity, which the response to von Frey 4 g was markedly inhibited compared with SMIR rats without TENS treatment or MIR-Placebo-TENS rats on PODs 9, 16, 23 and 30 (P < 0.05; two-way repeated measures ANOVA). The second, third and fourth weeks of TENS therapy also significantly inhibited SMIR-evoked mechanical hypersensitivity when compared with SMIR rats without TENS treatment or MIR-Placebo-TENS rats for the response to von Frey 10 g (Fig. 1B, P < 0.05; two-way repeated measures ANOVA). There was no significant difference between SMIR-TENS and sham-operated rats on POD 30, whereas SMIR-TENS rats on PODs 9, 16 and 23 showed mechanical hypersensitivity compared with sham-operated rats (Fig. 1, P < 0.05; two-way repeated measures ANOVA).
High-frequency TENS prevents substance P upregulation in the spinal cord
Figure 2 A-C reveals the expression of substance P in the spinal cord on PODs 3, 16 and 30 in four different groups. Substance P levels in the spinal cord were
significantly increased in the SMIR group (P < 0.01) and SMIR-Placebo-TENS group (P < 0.01) than in the sham group onPODs 3 (Fig. 2A), 16 (Fig. 2B) and 30 (Fig. 2C). The SMIR-operated rats underwent a 2-week (P < 0.01, Fig. 2B) or 4-week(P <
0.05, Fig. 2C) TENS program that showed the substance P contentwaslower 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 PODs 3, 15 and 30 (Fig. 2).
High-frequency TENS decreases excess proinflammatory cytokine levels in the spinal cord
Figure 2 (D-F) depicts the levels of IL-1β, IL-6 and TNF-α in the spinal cord of sham, SMIR, SMIR-TENS, and SMIR-Placebo-TENS rats on PODs 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 PODs 3, 16 and 30 was significantly increased in the SMIR (P < 0.05) and Placebo-TENS (P < 0.05) group as compared to the sham 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 < 0.05, Fig. 2D), IL-1β (P < 0.05, Fig. 2E), and IL-6 (P < 0.05, Fig. 2F) levels than those in SMIR-operated rats without TENS treatment on PODs 16 and 30.
Discussion
In this present study, we revealed for the first time that high-frequency TENS diminished SMIR-evoked allodynia and the upregulation of proinflammatory cytokines in the spinal cord of the SMIR rat. In the same way, this resembles to our previous experiment that exercise decreased post-surgical pain and cytokine
expression in rats after SMIR operation.23 In fact, another interesting finding is that high-frequency TENS therapy in SMIR rats lower the increased spinal substance P content induced by SMIR surgery. Overall, these resulting data presume that TENS
attenuate spinal substance P and proinflammatory cytokine releases. High-frequency TENS retards the progress of SMIR-evoked allodynia
TENS 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 pain-like procedures.34-37 In this case, it is unclear whether or not TENS can treat SMIR-evoked allodynia and its amelioration. There is credible evidence that TENS attenuates postoperative pain by less analgesic requirement during the first three days after the major surgery.38 That way, the efficacy of TENS is dose-dependent and requires a much strong sensation of currents. In this study, we set the intensity of high-frequency TENS to evoke an 80% of visible muscle contractions. We also observed that SMIR-operated rats, which received TENS therapy, showed a reduction in mechanical hypersensitivity. However, this case did not display normal sensitivity to the mechanical stimulation. In contrast with the sham-operated rats, the degree of reduction (< 50%) in postoperative mechanical/tactile hypersensitivity by TENS was small. Therefore, this indicates the relevance of the findings in relation to the current presence of postincisional
allodynia. Interestingly, the protective effect of TENS was found to be not too intense while SMIR rats were recovering.
Assuredly, it is interesting to notice that there is a temporal window, namely from POD16 to POD30, which TENS therapy markedly alleviated mechanical hypersensitivity. Meanwhile, high-frequency TENS also significantly suppressed tactile allodynia from POD9 to POD30. These results suggested that the one-week period outcomes remained unsatisfactory and further intervention was necessary; whereas, TENS therapy could maintain the 2- to 4-week period clinical outcomes. It appears that approximately two weeks of TENS intervention are required before the differences in pain behavior can occur. The pain behaviors, which are altered by TENS, may have been dependent based on these stimuli (4g vs 10 g). This assumption possibly could relate to the healing postoperative phase (acute inflammation vs. proliferation) in which TENS may be more beneficial towards recovery later rather than earlier.
High-frequency TENS inhibits the upregulation of spinal substance P release caused by SMIR operation
Central sensitization occurs when there is an increase in the excitability of neurons within the central nervous system (i.e. 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
levels in the spinal cord and peripheral tissues were upregulated in the inflammatory processes.18, 19 In this present study, we noticed SMIR 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, released into the dorsal horn of the spinal cord to evoke postoperative tactile allodynia. It is possible that alack of this substance attenuates the intensity of the inflammatory reaction thereby providing a second mechanism for the reduced thermal and mechanical sensitization.42
In order 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 hyperalgesia 43 and formalin-induced pain,44 which is
accompanied with a reduction of the substance P level in the spinal cord. While the effect of TENS was frequency dependent, acupuncture or TENS altered the noxious nerve stimulation-induced release 45, 46 and production 44 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 via diminishing the up-regulation 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 fibres, 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 investigate spinal cytokine levels in SMIR rats through which has not been stated before. Hopefully, we want to give an objective measurement of the values 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 for 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-induced 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 neurons,50 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 suppressed proinflammatory cytokines.26, 27, 56-58 For instance, TENS application enhanced the increase in blood supply,58 and the epidermal growth factor (EGF) and the decrease in TNF-α explained the lower rate of mucositis.57 Furthermore,
transcutaneous electrical acupoint stimulation (TEAS), transcutaneous auricular vagus nerve stimulation (ta-VNS), or vagus nerve stimulation (VNS) activated 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 with the previous study regarding the efficacy of TENS in controlling post-thoracotomy pain with a decrease in cytokine production.26 Therefore, our finding of reduced pro-inflammatory cytokines by TENS intervention is important
because normal subjects receiving surgeries demonstrate increased inflammation,26 suggesting that the increased inflammation is responsible for the delay of the healing process caused by surgeries.
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 VNS 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 (SCS; central) can regulate nociceptive input at the spinal cord through multiple inhibitory
neurotransmitters that subsequently inhibited glial cell activation.64
The previous experiment exhibited low- (10 Hz) and high- (130 Hz) frequency 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- instead of high-frequency (TENS) treated rats.65 It had been shown that low frequency TENS or acupuncture is ineffective in the animals’ tolerance towards morphine.66, 67 Nevertheless, high
frequency electroacupuncture is effective in the rats’ tolerance to δ-opioid agonists. However, this was ineffective in which animals were tolerated towards a κ-opioid receptor agonist called dynorphin.68 Evidence suggested that repeated administration of high- or low- frequency TENS for six days elicited a μ- and δ-opioid mediated analgesic tolerance in a rat model of carrageenan-induced hindpaw inflammation, respectively.69 Although we did not find repeated administration of high-frequency TENS that can cause an analgesic tolerance in this SMIR-evoked allodynic rat model, it needs to be tested with regards to the future findings that can show
the different methods of repeated application of TENS leading to the development of analgesic tolerance in SMIR rats.
Conclusion
We conclude that high-frequency TENS alleviates the development of persistent postoperative/postincisional allodynia evoked by SMIR operation.High-frequency TENS therapy inhibits the up-regulation of spinal substance P and proinflammatory cytokines in which may show the TENS-therapeutic mechanisms to manage
prolonged post-surgical allodynia. The treatment strategy by using TENS to prevent the process of postoperative allodynia possibly may occur sometime in the future.
Acknowledgments
We gratefully 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.
References
1. Gianesello L, Pavoni V, Barboni E, Galeotti I, Nella A. Perioperative
pregabalin for postoperative pain control and quality of life after major spinal surgery. Journal of neurosurgical anesthesiology. 2012;24:121-126.
2. Gallagher RM. Primary care and pain medicine. A community solution to the public health problem of chronic pain. Med Clin North Am. 1999;83:555-583.
3. Becker A, Held H, Redaelli M, et al. Low back pain in primary care: costs of care and prediction of future health care utilization. Spine. 2010;35:1714-1720.
4. Tverskoy M, Oren M, Dashkovsky I, Kissin I. Alfentanil dose-response relationships for relief of postoperative pain. Anesth Analg. 1996;83:387-393.
after abdominal surgery. Anesthesiology. 1999;91:639-647.
6. Somers DL, Clemente FR. Contralateral high or a combination of high- and low-frequency transcutaneous electrical nerve stimulation reduces mechanical allodynia and alters dorsal horn neurotransmitter content in neuropathic rats. J Pain. 2009;10:221-229.
7. Somers DL, Clemente FR. High-frequency transcutaneous electrical nerve stimulation alters thermal but not mechanical allodynia following chronic constriction injury of the rat sciatic nerve. Arch Phys Med Rehabil. 1998;79:1370-1376.
8. Somers DL, Clemente FR. Transcutaneous electrical nerve stimulation for the management of neuropathic pain: the effects of frequency and electrode position on prevention of allodynia in a rat model of complex regional pain syndrome type II. Phys Ther. 2006;86:698-709.
9. Kara B, Baskurt F, Acar S, et al. The effect of TENS on pain, function, depression, and analgesic consumption in the early postoperative period with spinal surgery patients. Turkish neurosurgery. 2011;21:618-624.
10. Desantana JM, Sluka KA, Lauretti GR. High and low frequency TENS reduce postoperative pain intensity after laparoscopic tubal ligation: a randomized controlled trial. Clin J Pain. 2009;25:12-19.
118.
12. Sandkuhler J, Liu X. Induction of long-term potentiation at spinal synapses by noxious stimulation or nerve injury. Eur J Neurosci. 1998;10:2476-2480.
13. Svendsen F, Tjolsen A, Hole K. AMPA and NMDA receptor-dependent spinal LTP after nociceptive tetanic stimulation. Neuroreport. 1998;9:1185-1190.
14. Willis WD, Jr. Is central sensitization of nociceptive transmission in the spinal cord a variety of long-term potentiation? Neuroreport. 1997;8:iii.
15. Willis WD. Long-term potentiation in spinothalamic neurons. Brain Res Brain Res Rev. 2002;40:202-214.
16. Kangrga I, Randic M. Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice. J Neurosci. 1990;10:2026-2038.
17. De Koninck Y, Henry JL. Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation. Proc Natl Acad Sci U S A. 1991;88:11344-11348.
18. Calvino B, Couraud JY, Besson JM. Prevaccination with diluted Freund adjuvant prevents the development of chronic pain and transient release of cerebrospinal fluid substance P in adjuvant-induced arthritis in rats. Pain. 1994;58:211-217.
experimental arthritis model in rats. Pain. 1993;55:367-377.
20. Khodorova A, Montmayeur JP, Strichartz G. Endothelin receptors and pain. J Pain. 2009;10:4-28.
21. Thacker MA, Clark AK, Marchand F, McMahon SB. Pathophysiology of peripheral neuropathic pain: immune cells and molecules. Anesth Analg. 2007;105:838-847.
22. Barr TP, Kam S, Khodorova A, Montmayeur JP, Strichartz GR. New
perspectives on the endothelin axis in pain. Pharmacol Res. 2011;63:532-540.
23. Chen YW, Lin MF, Chen YC, Hung CH, Tzeng JI, Wang JJ. Exercise Training Attenuates Postoperative Pain and Expression of Cytokines and N-methyl-D-aspartate Receptor Subunit 1 in Rats. Reg Anesth Pain Med. 2013;38:282-288.
24. Lavand'homme PM, Eisenach JC. Perioperative administration of the alpha2-adrenoceptor agonist clonidine at the site of nerve injury reduces the
development of mechanical hypersensitivity and modulates local cytokine expression. Pain. 2003;105:247-254.
25. Chen YW, Li YT, Chen YC, Li ZY, Hung CH. Exercise training attenuates neuropathic pain and cytokine expression after chronic constriction injury of rat sciatic nerve. Anesth Analg. 2012;114:1330-1337.
transcutaneous electrical nerve stimulation: effect on serum cytokine levels, visual analogue scale, pulmonary function and medication. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 2012;41:861-868.
27. Gurgen SG, Sayin O, Cetin F, Tuc Yucel A. Transcutaneous Electrical Nerve Stimulation (TENS) Accelerates Cutaneous Wound Healing and Inhibits Pro-inflammatory Cytokines. Inflammation. 2014;37:775-784.
28. Chen YW, Tzeng JI, Lin MF, Hung CH, Hsieh PL, Wang JJ. High-Frequency Transcutaneous Electrical Nerve Stimulation Attenuates Postsurgical Pain and Inhibits Excess Substance P in Rat Dorsal Root Ganglion. Reg Anesth Pain Med. 2014.
29. Flatters SJ. Characterization of a model of persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR). Pain. 2008;135:119-130.
30. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109-110.
31. Sluka KA, Bailey K, Bogush J, Olson R, Ricketts A. Treatment with either high or low frequency TENS reduces. Pain. 1998;77:97-102.
1994;168:85-88.
33. Chen YW, Hsieh PL, Chen YC, Hung CH, Cheng JT. Physical exercise induces excess hsp72 expression and delays the development of hyperalgesia and allodynia in painful diabetic neuropathy rats. Anesth Analg.
2013;116:482-490.
34. Gossrau G, Wahner M, Kuschke M, et al. Microcurrent transcutaneous electric nerve stimulation in painful diabetic neuropathy: a randomized placebo-controlled study. Pain Med. 2011;12:953-960.
35. Pertovaara A. Experimental pain and transcutaneous electrical nerve stimulation at high frequency. Applied neurophysiology. 1980;43:290-297.
36. Eriksson MB, Rosen I, Sjolund B. Thermal sensitivity in healthy subjects is decreased by a central mechanism after TNS. Pain. 1985;22:235-242.
37. Ekblom A, Hansson P. Thermal sensitivity is not changed by acute pain or afferent stimulation. Journal of neurology, neurosurgery, and psychiatry. 1987;50:1216-1220.
38. Bjordal JM, Johnson MI, Ljunggreen AE. Transcutaneous electrical nerve stimulation (TENS) can reduce postoperative analgesic consumption. A meta-analysis with assessment of optimal treatment parameters for postoperative pain. Eur J Pain. 2003;7:181-188.
hypersensitivity. Nature. 1983;306:686-688.
40. Zubrzycka M, Janecka A. Substance P: transmitter of nociception (Minireview). Endocrine regulations. 2000;34:195-201.
41. Mantyh PW. Neurobiology of substance P and the NK1 receptor. J Clin Psychiatry. 2002;63:6-10.
42. Sahbaie P, Shi X, Guo TZ, et al. Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision. Pain. 2009;145:341-349.
43. Liu HX, Tian JB, Luo F, et al. Repeated 100 Hz TENS for the Treatment of Chronic Inflammatory Hyperalgesia and Suppression of Spinal Release of Substance P in Monoarthritic Rats. Evid Based Complement Alternat Med. 2007;4:65-75.
44. Rokugo T, Takeuchi T, Ito H. A histochemical study of substance P in the rat spinal cord: effect of transcutaneous electrical nerve stimulation. J Nippon Med Sch. 2002;69:428-433.
45. Du J, He L. Alterations of spinal dorsal horn substance P following electroacupuncture analgesia--a study of the formalin test with
immunohistochemistry and densitometry. Acupuncture & electro-therapeutics research. 1992;17:1-6.
Ann N Y Acad Sci. 1991;632:488-489.
47. Shen S, Bian JT, Tian JB, Han JS. [Frequency dependence of substance P release by electroacupuncture in rat spinal cord]. Sheng li xue bao : [Acta physiologica Sinica]. 1996;48:89-93.
48. Nagahiro I, Andou A, Aoe M, Sano Y, Date H, Shimizu N. Pulmonary function, postoperative pain, and serum cytokine level after lobectomy: a comparison of VATS and conventional procedure. Ann Thorac Surg. 2001;72:362-365.
49. Kawasaki Y, Zhang L, Cheng JK, Ji RR. Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord. J Neurosci. 2008;28:5189-5194.
50. Reeve AJ, Patel S, Fox A, Walker K, Urban L. Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat. Eur J Pain.
2000;4:247-257.
51. Wei XH, Na XD, Liao GJ, et al. The up-regulation of IL-6 in DRG and spinal dorsal horn contributes to neuropathic pain following L5 ventral root
nociceptive transmission following neuropathy. Brain Res. 2003;984:54-62.
53. Wieseler-Frank J, Maier SF, Watkins LR. Central proinflammatory cytokines and pain enhancement. Neuro-Signals. 2005;14:166-174.
54. Milligan ED, Twining C, Chacur M, et al. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci. 2003;23:1026-1040.
55. Watkins LR, Milligan ED, Maier SF. Glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain. Adv Exp Med Biol. 2003;521:1-21.
56. Zhao YX, He W, Jing XH, et al. Transcutaneous auricular vagus nerve stimulation protects endotoxemic rat from lipopolysaccharide-induced inflammation. Evid Based Complement Alternat Med. 2012;2012:627023.
57. Pimenta Amaral TM, Campos CC, Moreira dos Santos TP, et al. Effect of salivary stimulation therapies on salivary flow and chemotherapy-induced mucositis: a preliminary study. Oral surgery, oral medicine, oral pathology and oral radiology. 2012;113:628-637.
58. Hargitai IA, Sherman RG, Strother JM. The effects of electrostimulation on parotid saliva flow: a pilot study. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. 2005;99:316-320.
kinase in spinal microglia contributes to incision-induced mechanical allodynia. Anesthesiology. 2009;110:155-165.
60. Romero-Sandoval A, Eisenach JC. Spinal cannabinoid receptor type 2 activation reduces hypersensitivity and spinal cord glial activation after paw incision. Anesthesiology. 2007;106:787-794.
61. Obata H, Eisenach JC, Hussain H, Bynum T, Vincler M. Spinal glial
activation contributes to postoperative mechanical hypersensitivity in the rat. J Pain. 2006;7:816-822.
62. Fu D, Guo Q, Ai Y, Cai H, Yan J, Dai R. Glial activation and segmental upregulation of interleukin-1beta (IL-1beta) in the rat spinal cord after surgical incision. Neurochem Res. 2006;31:333-340.
63. Costantini TW, Bansal V, Krzyzaniak M, et al. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. American journal of physiology. Gastrointestinal and liver physiology. 2010;299:G1308-1318.
64. Sato KL, Johanek LM, Sanada LS, Sluka KA. Spinal cord stimulation reduces mechanical hyperalgesia and glial cell activation in animals with neuropathic pain. Anesth Analg. 2014;118:464-472.
inflammatory edema and pain. Eur J Pharmacol. 2004;504:217-222.
66. Sluka KA, Judge MA, McColley MM, Reveiz PM, Taylor BM. Low frequency TENS is less effective than high frequency TENS at reducing inflammation-induced hyperalgesia in morphine-tolerant rats. Eur J Pain. 2000;4:185-193.
67. Han JS, Li SJ, Tang J. Tolerance to electroacupuncture and its cross tolerance to morphine. Neuropharmacology. 1981;20:593-596.
68. Chen XH, Han JS. Analgesia induced by electroacupuncture of different frequencies is mediated by different types of opioid receptors: another cross-tolerance study. Behavioural brain research. 1992;47:143-149.
69. Chandran P, Sluka KA. Development of opioid tolerance with repeated transcutaneous electrical nerve stimulation administration. Pain. 2003;102:195-201.
A.
0 3
9
16
23
30
N
o
.
w
ith
d
ra
w
fr
o
m
s
tim
u
lu
s
0
2
4
6
8
10
Sham SMIR SMIR-TENS SMIR-Placebo-TENSDay after operation
4g
*
*
+
*
+
*
+
+
*
*
*
*
+
+
+
+
+
+
+
+
+
+
B.
Day after operation
0 3
9
16
23
30
N
o
. w
ith
d
ra
w
fr
o
m
s
tim
u
lu
s
0
2
4
6
8
10
Sham SMIR SMIR-TENS SMIR-Placebo-TENS10g
+
*
+
*
+
*
+
*
**
+
+
+
+
+
+
+
+
+
+
+
Fig. 1.
Figure Legends
Fig. 1. The behavioral time courses of no. withdraw from mechanical stimuli. The
graphs exhibit mean ± S.E.M. of the number of rat hindpaw withdrawals out of 10 stimuli with (A) von Frey 4g and (B) von Frey 10g. For all time points n = 8 rats per group, including sham-operated (sham), skin–muscle incision retraction (SMIR)-operated (SMIR), transcutaneous electrical nerve stimulation (TENS) after SMIR surgery (SMIR-TENS), and placebo-TENS after SMIR surgery (TENS) groups. The asterisk (*) indicates P < 0.05 when the SMIR or SMIR-Placebo-TENS group was compared with the SMIR-SMIR-Placebo-TENS group; the plus symbol (+)
indicates P < 0.05 when the groups were compared with the sham group (2-way ANOVA of repeated measures followed by post hoc Bonferroni’s test).
Fig. 2. The level of substance P (A-C), TNF-α (D), IL-1β (E), and IL-6 (F) on
postoperative days 3 (A), 16 (B) and 30 (C) after skin–muscle incision retraction (SMIR) in the spinal cord in sham, SMIR, SMIR-TENS, and SMIR-TENS-Control rats, where sham = sham operated; SMIR = skin–muscle incision retraction (SMIR)-operated; SMIR-TENS = transcutaneous electrical nerve stimulation (TENS) after SMIR surgery; SMIR-Placebo-TENS = placebo-TENS after SMIR surgery. The values are presented as mean ± S.E.M. for 4-6 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<0.05) on postoperative days 16 and 30. TNF-α means tumor necrosis factor-α; IL-6 means interleukin-6; IL-1β means interleukin-1β. The asterisk (*) indicates P < 0.05 when the SMIR or SMIR-Placebo-TENS group
when the groups were compared with the sham group (1-way ANOVA followed by post hoc Bonferroni’s test).
