Effect of transcutaneous electrical stimulation on ...

International Journal of Neuroscience, 2013; 123(7): 507–515 Copyright © 2013 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.768244

Effect of transcutaneous electrical stimulation on nociception and edema induced by peripheral serotonin Cristiane M. F. Santos,1 Janetti N. Francischi,2 Patr´ıcia Lima-Paiva,2 Kathleen A Sluka,3 and Marcos A. Resende1 1

Post Graduate Program in Sciences of Rehabilitation, Department of Physical Therapy Belo Horizonte, MG, Brazil; Pharmacology Department of the Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 3 Pain Research Program, Graduate Program in Physical Therapy and Rehabilitation Science, University of Iowa, Iowa City, IA, USA

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Transcutaneous electrical nerve stimulation (TENS) is defined as the application of an electrical current to the skin through surface electrodes for pain relief. Various theories have been proposed in order to explain the analgesic mechanism of TENS. Recent studies have demonstrated that part of this analgesia is mediated through neurotransmitters acting at peripheral sites. The aim of this study was to investigate the effects of low frequency (LF: 10 HZ) TENS and high frequency (HF: 130 HZ) TENS on hyperalgesia and edema when applied before the serotonin (5-HT) administered into the rat paw. LF and HF TENS were applied to the right paw for 20 min, and 5-HT was administered immediately after TENS. The Hargreaves method was used to measure nociception, R ) was used to measure edema. Neither HF nor LF TENS inhibited while the hydroplethysmometer (Ugo Basile 5-HT-induced edema. However, LF TENS, but not HF TENS, completely reduced 5-HT-induced hyperalgesia. Pre-treatment of the paw with naltrexone, prior to application of TENS, (Nx: 50μg; I.pl.) showed a complete blockade of the analgesic effect induced by low frequency TENS. Thus, our results confirmed the lack of an anti-inflammatory effect through the use of TENS as well as the participation of peripheral endogenous opioid receptors in LF TENS analgesia in addition to its central action. KEYWORDS: TENS, serotonin, endogenous opioids, analgesia, electrotherapy

Introduction Transcutaneous electrical nerve stimulation (TENS) is defined by the American Physical Therapy Association (APTA) as the application of an electrical current to the skin through surface electrodes for pain relief [1,2]. Treatment of inflammatory and non-inflammatory conditions, as arthritis and neuralgia in humans is usually done with the use of drugs as well as with nonpharmacological agents. One of these agents is TENS, frequently used in physical therapy clinics [3]. Different mechanisms have been proposed to explain the analgesic effect of TENS [4]. Neurophamacological studies suggest the participation of spinal and Received 2 October 2012; revised 7 January 2013; accepted 16 January 2013. Correspondence: Marcos A. Resende: Post Graduate Program in Sciences of Rehabilitation, Department of Physical Therapy, Federal University of Minas ˆ Gerais. Av. Antonio Carlos, 6627. CEP: 31.270–901. Belo Horizonte, MG, Brazil. Tel.: + 55 31 34097483. Fax: + 55 31 34094781. E-mail: mresende@ eeffto.ufmg.br

supraspinal opioid mechanisms underlie the analgesia produced by TENS [2,4,5]. More recently the participation of peripheral opioidergic pathways have been observed [6]. In addition to opioids, serotonin plays a role in the analgesia produced by LF TENS. Specifically, there is an increased release of serotonin in the spinal cord that produces analgesia through activation of spinal 5HT2A and 5-HT3 receptors [4]. The effects of serotonin are complicated but generally it is believed that when released from brainstem structures produces analgesia [7,8]. On the other hand, serotonin injected peripherally produces edema and pain in humans, and hyperalgesia in rodents [9,10]. Peripheral 5-HT is released from plasma, and activates 5-HT1 and 5-HT2 receptors on peripheral blood vessels to enhance vascular permeability [9,11]. This would result in edema and enhance release of pronociceptive and proinflammatory substances such as bradykinin and eicosanoids. Peripherally released serotonin can directly produce pain by activating 5-HT3 receptors found on Aδ/C afferent fibers 507


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[8–10,12]. Thus, the effects of serotonin peripherally are to produce edema and pain. The peripheral effects of TENS can therefore be studied for its ability to modulate edema and pain induced by peripherally applied 5-HT. The current study hypothesized that LF and HF TENS will reduce the edema and hyperalgesia produced by peripheral serotonin through activation of opioid receptors. In addition, we sought in the present study to verify whether the application of low (10 Hz) and high (130 Hz) frequency TENS before the administration of the serotonin into the rat paw would also affect the hyperalgesia and edema.

administered after measuring baseline paw withdrawal threshold and paw volume. To induce nociception and edema, 10 μg/0.1 ml serotonin was intradermally administered (i.d.) into the one paw. The same dose of 5HT (10 μg) was chosen based on dose-response analysis of 5-HT on nociception and edema. The contralateral paw received the same volume of saline. Subcutaneous injection (s.c.) of serotonin antagonists (pizotifen 2 mg/Kg, methysergide 2 mg/Kg and ondansetron 2 mg/Kg) or saline (0.1 ml) were administered into the dorsal neck of the animals based on prior studies [15,16]. Intraplantar naltrexone hydrochloride (50 μg/0.1 ml) or saline (0.1 ml) were injected into ipsilateral paw based on prior work [17].

Materials and methods

Measurement of nociception

Animals Male Hotzman rats weighing 280–310 g were used for the experiments in the Hargreaves algesimetric test [13] in 1998, and 160–200 g for experiments in the hydroplethysmometer. Animals were supplied by the Bioterism Center of the School of Biological Sciences at the Federal University of Minas Gerais (CEBIO), Brazil. The experiments were performed in groups of 4–10 animals, totaling 161 animals throughout the study. The experimental design was previously approved by the Ethics Committee on Animal Experimentation of the same university (UFMG – CETEA; Certificate Nr. 237/08).

Acclimatization of animals Animals were housed in plastic boxes (six per cage) with free access to food and water. They were left to adapt to the testing room, under temperature control (22–24◦ C) and a light-dark cycle of 12 h, in a controlled temperature of 22–24◦ C. Before the procedure the animals also were left to adapt to the algesimetric device for 30 min, as well as to the manipulation imposed by researcher [6,14].

Drugs The substances used in this study were: serotonin (SigmaR , St. Louis, MO, USA), methysergide maleate (SigmaR , St. Louis, MO, USA), pizotifen (SandozR , Taboão da Serra, SP, Brazil), ondansetron (GlaxoR , Rio de Janeiro, RJ, Bazil), naltrexone (SigmaR , St. Louis, MO, USA), and sterile saline solution (NaCl 0.9%).

Administration of drugs All the drugs used were prepared in sterile saline solution immediately before the experiment. They were

To measure paw withdrawal latency to radiant heat, the device Plantar test from Ugo BasileR (model 7370, Comerio, VA, Italy) was used. For the testing, each rat was housed in an individual acrylic cubicle (18 cm × 29 cm × 12.5 cm) placed under a special glass surface, which allowed passage of an infrared light placed under each hind paw, as described in Hargreaves [13]. Animals were acclimated for 5 min to the acrylic cubicles after which an infrared light was shone on each hind paw. The latency to withdrawal from the heat source was recorded. A 30 s maximum was used as cut off time in order to prevent tissue damage. An average of two trials at each time period was assessed for each paw. All measurements were taken by the same researcher, and at the same period of the day. The latency to withdrawal was assessed before and 5, 15, 30, 60 and 120 min after the plantar intradermal injection of serotonin. The values were expressed as the mean ± SEM in seconds.

Measurement of edema In order to measure the increase of hind paw volume caused by 5-HT, a hydroplethysmometer from Ugo BasileR (model 1750, Comerio, VA, Italy) was used. This device measures displacement of water in millimeters. Measurements were taken bilaterally before and 5, 15, 30, 60 and 120 min after 5-HT. Measurements were assessed by the same researcher, who had been previously trained. The values were expressed as the mean ± SEM for the difference of volume (ml) between the hind paws.

Transcutaneous electrical nerve stimulation device The TENS device used to induce antinociception was Neurodyn III/IbramedR (Amparo, SP, Brazil). For calibration, the following parameters were used: low frequency (LF: 10 Hz), and high frequency (HF: 130 Hz). International Journal of Neuroscience

TENS effects on nociception and edema induced by 5-HT.

Pulse duration was set at 130 μs, and the TENS treatments were of 20 min. The intensity of sensory threshold was considered immediately below motor threshold. The intensity was increased to achieve a motor contraction and reduced to just below this level as described by Sluka et al. [18] in 1999 and used previously by us Resende et al. [14] in 2006; Sabino et al. [6] in 2008. Electrodes of 1 cm2 were specially built for these experiments, and attached to the plantar and dorsal surface of the ipsilateral hind paw with adhesive tape. A lace was made of cardboard and fastened around the animals’ neck with an adhesive tape in order to prevent them from damaging the electrodes.

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ure 1A). Withdrawal latency remained significantly decreased through 30 min after injection.

Serotonin-induced edema The intradermal injection of 5-HT (0.1, 1.0, 10 μg) resulted in a significant increase in volume of paw when compared to control (saline). Only the dose of 10 μg significantly increased the volume in 15, 30,60 and 120 min when compared to control (Figure 1B). Therefore the dose of choice to induce nociception and edema was 10 μg.


5-HT antagonists in nociception and edema Experimental protocol To choose the dose of serotonin, different doses of the 5-HT (0.1, 1.0 and 10 μg) [19] were administered into one hind paw in different groups of animals. In a separate experiments, the non-selective 5-HT1 and 5-TH2 antagonist, methysergide [15,16], the 5-HT3 antagonist, ondansetron, or the 5-HT2 antagonist pizotifeno [15] were subcutaneously administered into the dorsal neck of the animals 30 min before serotonin injection (10 μg/0.1 ml). To examine the effects of LF and HF TENS on hyperalgesia and edema caused by serotonin, LF or HF TENS was applied to the right paw 30 min before the animals received intradermal serotonin (10 μg/100 μl). To test the role of opioids in the analgesia produced by TENS, naltrexone (50 μg/100 μl) [17] an opioid antagonist, was administered intraplantar 30 min before LF or HF TENS. A placebo control consisted of switchedoff TENS.

Statistical analysis The software program GraphPad Prism 4.00 under WindowsR was used to prepare database as well as the statistical analysis. Results are represented as the mean ± standard error of the mean. A one-way ANOVA with post-hoc correction by the Bonferroni test was used for the statistical comparison of the means at the different time points. A p value, lower than 0.05 was considered to be statistically significant.

Results Serotonin-induced nociception Intradermal injection of 5-HT (0.1, 1.0, 10 μg) resulted in a significant and dose-dependent decrease in withdrawal latency. The largest dose of 5-HT reduced withdrawal latency by −13.0s 5 min after injection (Fig C

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In order to identify the main receptors involved in the hyperalgesia and edema produced by 5-HT, specific 5HT receptor antagonists were administered systemically 30 min prior to injection. The 5-HT1 and 5-HT2 receptor antagonist methysergide, completely prevented the 5-HT-induced hyperalgesia and edema (Figure 2A and 2B). There was a complete blockade of the 5HT (10 μg)-induced hyperalgesia in animals pre-treated with the 5-HT2 receptor antagonist pizotifen. However, pizotifen had no effect on 5-HT-induced edema. Treatment with the 5-HT3 antagonist ondansentron had no effect on the 5-HT-induced hyperalgesia or edema.

Effect of TENS in nociception and edema Low Frequency TENS pre-treatment of animals completely prevented the hyperalgesia produced by local injection of serotonin (10 μg) when compared to placebo controls (Figure 3A). On the other hand, HF TENS had no effect on the 5-HT-induced hyperalgesia (Figure 3B). Neither LF TENS nor HF TENS had any effect on the 5-HT-induced edema when compared to the group receiving placebo TENS (Figure 3C).

Effect of naltrexone on LF TENS-induced antinociception Considering that TENS treatment was effective in reducing hyperalgesia only when LF TENS was used, and our prior studies show that LF TENS produces analgesia by acting on peripherally located opioid receptors [20], we tested if opioids were responsible for the analgesia produced by LF TENS. Local pre-treatment with naltrexone significantly reversed LF TENS analgesia in serotonin hyperalgesia (Figure 4).

Discussion Our data confirm previous studies on serotonin showing a dose-dependent development of heat hyperalgesia by intradermal injection [9,21]. Previous work also shows



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Figure 1. Dose-response curves of nociception (A) and edema (B) following intradermal injection of 5-HT in rat paws. ♦Saline (control), • serotonin (5-HT 0,1 μg/paw), € serotonin (5-HT 1,0 μg/paw), serotonin (5-HT 10,0 μg). Results were presented as the difference between test and control value±S.E.M. in groups of five animals. The greater the negative y-axis values, the most intense the nociceptive response is (A) and the greater y-axis values, the most volume (B). ∗ indicates p < 0.05 given by one-way ANOVA.

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Effect of pizotifeno, methisergide, ondansetrona on nociception (A) and edema (B) induced by serotonin in rat paw. ♦ Saline + 5-HT, Pizotifeno + 5-HT, Methisergide + 5-HT, • Ondansetron + 5-HT, o saline + saline. Results were presented as the difference between test and control value±S.E.M. in groups of four to ten animals/group. The greater the negative y-axis values, the most intense the nociceptive response is (A). Edema (B) was obtained by volume displacement of hind paws (ml) using a plethysmometer apparatus. ∗ indicates p < 0.05 given by one-way ANOVA.

Figure 2.

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Figure 3. Effect of low (A)- and high (B)-frequency TENS on nociception and edema (C) induced by 5-HT in rat paws. TENS and 5-HT indicates treatments with electrical stimulation and serotonin, respectively. -- Switched-off TENS (placebo group, panels A and B and C). Low frequency TENS + 5-HT, low frequency TENS + saline (panel A). • High frequency TENS + 5-HT, o high frequency TENS + saline (panel B). Low frequency TENS + 5-HT, high frequency TENS + 5-HT (panel C). Results were presented as the difference between test and control value±S.E.M. in groups of six to twelve animals/group. The greater the negative y-axis values, the most intense the nociceptive response is (panels A and B). Edema (panel C) was obtained by volume displacement of hind paws (ml) using a plethysmometer apparatus. ∗ indicates p < 0.05 given by one-way ANOVA.

that serotonin produces mechanical hyperalgesia [19]. In addition in animals with nerve injury or inflammation, peripheral blockade of serotonin receptors 5-HT1 , 5-HT2 or 5-HT3 reverses heat hyperalgesia [12]. Thus, serotonin in peripheral tissues plays a critical role in producing hyperalgesia. Interestingly, we show that methysergide, a 5-HT2 /5HT1 receptor antagonist, and pizotifen, a 5-HT2 receptor antagonist [18,22–24] prevented the hyperalgesia and edema produced by serotonin. In contrast, ondansentron, a 5-HT3 antagonist [9,23,25], had no effect

on 5–HT-induced hyperalgesia or edema. Prior studies have shown the participation of 5-HT1 , 5HT2 and 5HT3 receptors in peripheral serotonin hyperalgesic effect [9,21,26,27]. The current study, on the other hand suggests that 5-HT2 receptors mediate the heat hyperalgesia produced by peripheral injection of serotonin. This agrees with that of Tokunaga et al. [12] in 1998, in which administration of the 5-HT2 antagonist but not 5-HT3 antagonized 5-HT-induced heat hyperalgesia. Our data, however, are in contrast with the findings from Zeitz et al. [27] in 2002, who showed a role for 5-HT3 in International Journal of Neuroscience



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Effect of naltrexone on low frequency TENS following nociception induced by serotonin in rat paws. Nx and TENS and 5-HT indicates treatments with naltrexone and electrical stimulation and serotonin, respectively. Saline was administered 60 min before 5-HT and 30 min before low frequency TENS. Naltrexone was administered 60 min before 5-HT and 30 min before low frequency TENS. Results were presented as the difference between test and control value±S.E.M. in groups of six animals/group. The greater the negative y-axis values, the most intense the nociceptive response is. ∗ indicates p < 0.05 given by one-way ANOVA. Figure 4.

nociception. However, this study showed a reduction in spontaneous behaviors induced by intraperitoneal injection of serotonin, and by intraplantar injection of formalin after blockade of 5-HT3 receptors with odansentron. In 5-HT3 mice, however, there was a reduction in 5HT-induced edema and nociception, and in the second phase of the formalin test. The differences between the current study and prior studies may relate to differences in assessment methods (spontaneous pain, site of serotonin injection), animal species (mice vs. rats), and the drug doses used. Our current study shows that blockade of 5-HT2, but not 5-HT3, receptors reduces 5-HT-induced edema. This is in agreement with prior studies showing that 5HT3 receptors do not play a role in the edema obtained by 5-HT [15,27–29]. On the other hand, 5-HT2 seems to be the major receptor responsible for such an effect agreeing with prior studies [28,29]. Perhaps more importantly, other subtype of receptor as 5-HT2A can be involved [29]. C


The peripheral analgesic mechanism of TENS is not totally clear. The results from this study show a complete inhibition of the hyperalgesia produced by peripheral serotonin by LF (10 Hz) TENS, but not through HF (130 Hz) TENS. This effect was not a result of a reduction in edema since there was no change in edema with either LF or HF TENS. Prior studies similarly show no effect of TENS on edema produced by carrageenan [14,30,31]. Our study shows that local blockade of opioid receptors prevents the analgesia produced by LF TENS. Similarly, our prior study shows that carrageenan-induced LF TENS anti-hyperalgesia is also reversed by local blockade of opioid receptors [6]. A differential mechanism between LF and HF TENS analgesia has also been observed in both the central and peripheral nervous systems [2,6,14]. Centrally, both LF and HF TENS analgesia involves an endogenous release of opioids [32,33]. LF, but not HF, TENS analgesia is reversed through a low dose of naxolone [20], at doses that selectively block mu-opioid receptors [34].


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We believe our dose of naltrexone is local since a prior study showed that 50 μg naltrexone, the same dose we used, administered into rat paw antagonized morphineinduced antinociception [17] when given ipsilaterally but not contralaterally. We show a similar effect where contralaterally administered naltrexone has no effect on LF TENS analgesia when administered ipsilaterally [6]. It has been proposed that μ-opioid receptor antagonists act to inhibit activation of adenylate cyclase on peripheral afferent neurons by inflammatory mediators, such as serotonin and prostaglandin E2, while δ- and κopioid receptor agonists inhibit secretion of proinflammatory substances by sympathetic neurons [35]. Some studies have shown that opioids can produce a potent analgesia through activation of receptors in peripheral sensory neurons [36–38]. Such opioid receptors are found both peripherally and centrally in sensory neurons [38] in animals [38,39] as well as in humans [40]. Activation of those receptors through opioids can inhibit excitatory ion channels such as TRPV1, Ca2+ channels, and the release of proinflammatory neuropeptides such as substance P and calcitonin gene-related peptide (CGRP), resulting in an antinociceptive effect [38]. μ-opioid agonists are considered more potent than δ and κ agonists in inducing peripheral antinociceptive effect [37]. A recent study has described the presence of an immune-reactive β-endorphin on rat skin [41]. Release of endorphin from keratinocytes reduced antinociception by activating μ-opioid receptors on primary afferent neurons [41]. Thus, changes in keratinocyte membranes after LF TENS application could induce release of β-endorphin and contribute to an antihyperalgesic effect [6]. Further immunohistochemical studies are necessary to investigate on such a contribution. The time of analgesia obtained from the application of LF TENS prior to serotonin administration was of 30 min. In the study of Resende et al. [20] in 2004, TENS was applied after carrageenan, and it obtained a 1 h 30 min analgesia. The difference in the analgesia duration is probably due to the difference at the time of the TENS application, which indicates that the analgesia obtained through TENS application after the beginning of the inflammatory process becomes more lasting. It may be that after induction of peripheral inflammation, axonal transport of opioid receptors to peripheral nerve terminations is intensified [42]. Immune cells in the inflamed tissue or that migrate to lesion site, such as granulocytes, monocytes/macrophages and lymphocytes of rodents and humans are also a source of opioid peptides, could induce to analgesia in the injured tissue [43]. These factors may have contributed to a more lasting analgesia when TENS was applied after an inflammation induced by carrageenan than when induced by serotonin in rat paw [20].

Since LF TENS inhibited serotonin-induced nociception through a local activation of opioid receptors, the clinical relevance of the LF TENS is that can be useful to stimulate local analgesic mechanisms in addition to its central action in humans with acute pain. It is possible that in the future, with more basic information, the TENS can be used to modulate predictable acute pain.

Conclusion Our results clearly show the absence of an antiinflammatory effect associated with both LF or HF TENS. We suggest that LF TENS may be a useful analgesic modality that could locally activate peripheral opioid receptors in addition to its central action to enhance analgesia in patients with acute pain.

Declaration of interest The authors report no conflict of interest

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