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The Journal of Pain, Vol 9, No 2 (February), 2008: pp 157-163 Available online at www.sciencedirect.com

elease of Endogenous Opioids Following Transcutaneous lectric Nerve Stimulation in an Experimental Model of Acute nflammatory Pain

eorge S. Sabino,* Cristiane M. F. Santos,† Janetti N. Francischi,‡ and arcos Antônio de Resende*

Rehabilitation Sciences Program, Federal University of Minas Gerais, Minas Gerais, Brazil. Physiotherapist, Belo Horizonte, Minas Gerais, Brazil. Pharmacology Department of the Institute of Biological Sciences at the Federal University of Minas Gerais, Minas erais, Brazil.

Abstract: Transcutaneous electric nerve stimulation (TENS) is a noninvasive treatment used in physiotherapy practice to promote analgesia in acute and chronic inflammatory conditions. The aim of the present study was to investigate the action mechanism of TENS at high (HF: 130 Hz) and low (LF: 10 Hz) frequencies in an inflammation model produced by the injection of carrageenan in rat paws (Cg; 250 �g). After carrageenan administration (0 time), either HF or LF TENS was applied to the inflamed paw of rats for 20 minutes, and hyperalgesia was assessed hourly using the modified Randall-Selitto method (1957). HF and LF TENS inhibited the carrageenan-induced hyperalgesia by 100%. Pretreatment of animals with intraplantar naltrexone (Nx; 50 �g) reversed the analgesic effect of the LF TENS but did not alter the effect of HF TENS. The application of HF and LF TENS to the contralateral paw reversed the hyperalgesia of the inflamed paw similar to that observed when TENS was applied to the inflamed paw. However, LF TENS presented a longer-lasting analgesic effect than HF TENS. Our data demonstrate that HF and LF TENS induced antihyperalgesia. We also report that the antihyperalgesia provoked by LF TENS is partially due to the local release of endogenous opioids. Perspective: This study offers important information about physiotherapy practices aimed at pain relieving. TENS is a noninvasive treatment that promotes analgesia in acute and chronic inflammatory conditions. Scientists, patients, and the general population may benefit from this knowledge.

© 2008 by the American Pain Society

Key words: Antihyperalgesia, endogenous opioids, TENS.

a l

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ranscutaneous electric nerve stimulation (TENS) is a noninvasive form of treatment used to alleviate pain in several forms of acute and chronic inflam-

ation.17 The use of TENS combined with other physio- herapeutic methods favors an increase in the patient’s evel of activity, reduced hospital stay, and improvement n function of the affected region. However, despite be- ng used for over 30 years, mechanisms involved in the

eceived December 6, 2007; Revised August 16, 2007; Accepted Septem- er 27, 2007. ddress reprint requests to Dr. Marcos Antônio de Resende, Physio-

herapy Department of the Physical Education, Physiotherapy, and ccupational Therapy School of the Federal University of Minas Gerais. v. Antônio Carlos, 6627, CEP: 31270-901, Belo Horizonte, M. G., Brazil. -mail: mresende@eeffto.ufmg.br 526-5900/$34.00 2008 by the American Pain Society

toi:10.1016/j.jpain.2007.09.003

nalgesia induced by TENS is not well-defined, and the iterature on the subject remains controversial.37

The first study demonstrating the effectiveness of ENS in controlling pain was carried out by Wall and weet42 in 1967, based on the gate theory of pain previ- usly described by Melzack and Wall22 in 1965. This the- ry postulates that the activation of thick (pressure) erve fibers inhibits the activation of the thin fibers that onduct nociceptive impulses leading from the periphery o the central nervous system (CNS), thereby reducing ain. Moreover, a descending system from higher cen- ers of the CNS to the dorsal horn of the spinal cord could lso contribute to the modulation of pain.23 The activity f dorsal horn cell and spinothalamic tract were reduced uring stimulation by TENS.12,20 However, the neuro- ransmitters involved were not suggested at the time, as

hey were not known.36 Currently, there are more details

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n TENS mechanisms of analgesia, including knowledge n the participation of cholinergic and serotonergic re- eptors to reduce hyperalgesia.25,26

Later studies verified that low (LF: �10 Hz) and high HF: �100 Hz) frequency TENS reduce the nociceptive esponse without altering the edema in the rat paw and hat depending on the frequency used, this analgesic ffect can be modified by the systemic administration of altrexone,29 a specific antagonist of opioid receptors. F and HF TENS induce antihyperalgesia in the CNS, hich is mediated by the opioid receptors � and �, re-

pectively, suggesting that the different TENS frequen- ies provoke antihyperalgesia through activation of spe- ific cell receptors.19,21,34 A number of studies have hown that LF TENS is less effective than HF TENS in re- ucing hyperalgesia in rats that are tolerant to mor- hine.30,35,41

Exogenous as well as endogenous opioids can act on eripheral nociceptors to reduce a local inflammation-

nduced hyperalgesia.3,8 Although the presence of opi- id receptors on the peripheral terminals of primary af- erents has been shown, the mechanisms by which pioid agonists induce peripheral antihyperalgesia re- ain unclear.7,9,10,17,22,42 However, antihyperalgesia in- uced by local administration of opioids may minimize he side effects of a systemic use21 and also prevent tol- rance to a specific opioid substance.41

Despite being directly related to the TENS response, he positioning of the electrodes varies considerably mong the studies.37 In general, TENS electrodes are laced around the site of the pain or over the somatic eceptive field related to the evoked pain.2,43 A number f authors suggest that the electrodes should be placed n the contralateral side of the lesion in situations where he site of the pain is sensitive to touch,18 as in situations f neuropathic pain.37

In the present study, we propose to investigate the echanisms involved in the antinociceptive effect of

ENS through the application of low and high frequency ENS either in the ipsilateral or contralateral side of in- amed rat paws. Participation of opioid receptors in the ffects of TENS was also investigated by treatment of rat aws with the antagonist opioid naltrexone.

aterials and Methods

nimals Female Holtzman rats, weighing between 160 and

80 g, from Bioterism Center (CEBIO, Institute of Biolog- cal Sciences of UFMG, Minas Gerais, Brazil) were used in he present study. The experiments were carried out us- ng 3 to 6 animals per group, totaling 87 animals hroughout the study. Experimental design was previ- usly approved by the local Animal Ethics Committee CETEA/UFMG No. 19/2003) and care and use of the ani- als conformed to national/international guidelines.

cclimatization of the Animals The animals were kept in the experimentation room in

lastic boxes containing a maximum of 6 animals, lined I

ith wood shavings, for 2 days before data collection at controlled temperature (23°–25°C) and 12-hour light-

ark cycle, with free access to water and food. During his period, adaptation to the algesimetric device was arried out for the animals to become adapted to the andling imposed by experimental procedures.

rugs The following substances were used in the present

tudy: Carrageenan (Carrageenan; Sigma-Aldrich, Mu- ich, Germany), naltrexone 50 �g (Naltrexone hydro- hloride; Sigma-Aldrich) and sterile saline solution (NaCl .9%). The carrageenan and naltrexone were diluted in he saline solution so that the volume injected was 0.1 L/paw. Naltrexone dose (50 �g/site) was based on the ork of Rodrigues et al.32

arrageenan Inflammation Model in the aw of the Rat Carrageenan, a sulfated polysaccharide extracted from

lgae, is a phlogogenic agent used in a number of studies o induce hyperalgesia and edema in rat paws.6,10,28,29 In he present study, carrageenan was administered in the ight hind paw of conscious rats through a subcutaneous ose of 250 �g diluted in 0.1 mL of saline solution (pH .2–7.4). Contralateral paws were injected with the same olume of saline as described in Francischi et al10 and esende et al.29 In control (naive) animals, saline solution as injected in the contralateral paw under the same

onditions.

easurement of Hyperalgesia Assessment of hyperalgesia consisted of measurement f the threshold stimulus for reaction (escape or paw ithdrawal) using a weight (maximum limit of 500g) ap- lied to the pads of hind paws by an experimenter using n Ugo Basile apparatus, as described in Randall and elitto27 in 1957. The threshold for pain sensation was easured before (time 0) and 1, 2, 3, 4, and 6 hours after

he intraplantar injection of carrageenan. The response o painful mechanical stimulation has been used in ex- erimental models of acute pain for decades to assess he effectiveness of pharmacological agents.36 The re- ults for each animal were presented by the difference in ressure (in grams) between the measurements per- ormed on the right hind paw (injected with carra- eenan) and those performed on the left hind paw (in-

ected with saline). The reduction in thresholds was nterpreted as hyperalgesia. Greater hyperalgesia stem-

ing from the inflammatory process induced by the car- ageenan in the right paw requires less pressure needed o provoke the removal of the paw from the algesimeter, hereby leading to a more negative value when compar- ng the inflamed and noninflamed paws. All measure-

ents were taken by the same experimenter.

ranscutaneous Electric Nerve timulation Device A TENS device, a commercially available Neurodin III/

bramed, was used to induce antihyperalgesia. For cali-

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159ORIGINAL REPORT/Sabino et al

ration, the following parameters were used: Low (LF: 10 z) or high frequency (HF: 130 Hz), with pulse duration

et at 130 �s. The intensity of electric stimulation was efined by the sensory threshold, that immediately be-

ow the motor level (intensity was increased to achieve he threshold for motor level), as described by Sluka et l33 and used in previous studies of this laboratory.29,30

lectric stimulation was applied before the peak of the nflammatory response induced by carrageenan, which as maintained for 20 minutes. The parameters found in

he present study regarding frequency, pulse duration, pplication time, and intensity are in accordance to hose used in routine physiotherapy practice.17,37 Elec- rodes of 1 cm2 were especially constructed for the ex- eriments and attached to the plantar and dorsal surface f the right hind paw (or left, depending on the experi- entation protocol), using an adhesive tape. To mini- ize danger to the electrodes by experimental animals, paper lace was placed around the neck of control

sham) and test animals during TENS application, which, owever, allowed the animals to move freely in the ages. It had been observed previously that the neck lace id not affect control nociceptive responses to carra- eenan injected in rat paws (unpublished observations).

rotocol The proinflammatory effect of the carrageenan was etermined in previous studies in our laboratory, where he peak of the inflammatory response was observed to ccur between the third and fourth hours after carra- eenan administration.10 Thus, TENS was applied at 2.5 ours of carrageenin administration. Responses (noci- eptive threshold) to mechanical compression of the aws subjected to either LF or HF TENS and injected with arrageenan were compared with those from carra- eenan injected animals (N � 5 animals/group, unless therwise stated). TENS was also administered under the ame above experimental conditions on the contralat-

igure 1. Antihyperalgesic effect of low and high frequency tr ion in inflamed paws. � indicates IPSI HF TENS (130 Hz); ‘ indic arrageenan (250 �g/0.1 mL) in the right paw at 0 time, without er group *Statistically significant difference in relation to con

nalysis).

ral to the inflamed paws and the nociceptive threshold as assessed as previously described. In a second experiment, the intraplantar opioid antag- nist naltrexone (Nx; 50 �g in 0.1 mL) was administered 2 ours after carrageenan to animals which received ei- her LF or HF TENS, ipsilaterally (in the inflamed) or con- ralaterally (in noninflamed paws). In the sole case of ontralateral naltrexone injection, 3 animals per group ere used.

tatistical Analysis The main tendency of the data was carried out using

he average values observed in each group. Analysis of ariance (ANOVA) with post hoc correction by the Bon- erroni test was used for the statistical comparison of the eans at the different time points and were considered

tatistically significant when P � .05. To this end, SPSS ersion 13.0 (SPSS Inc., Chicago, IL) installed in the Win- ows (Microsoft Corp., Redmond, WA) environment, was sed.

esults

ffect of Low (LF) and High (HF) requency TENS Application in the Rat psilateral Paw LF and HF TENS completely reversed the decreased ithdrawal threshold, here described as hyperalgesia, roduced by intraplantar carrageenan administration

mmediately after TENS treatment (at 3 hours), as shown n Fig 1. This reversion indicated that both LF and HF ENS induced a definite antihyperalgesic effect. More- ver, this antihyperalgesic effect remained longer (for 4 ours) in LF TENS-treated animals when compared with hose with HF TENS (P � .05, 1-way ANOVA; Fig 1). Sham nimals, in contrast, showed the characteristic hyperal- esic response to carrageenan, attaining a maximum

taneous electric nerve stimulation (TENS) occurs after applica- IPSI LF TENS (10 Hz); Œ C, control group, inflamed, injected with ric stimulation. Each point represents mean � SEM of 6 animals (P � .05; 1-way ANOVA, followed by Bonferroni test post hoc

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160 Endogenous Opioids in TENS Application

alue at 3 hours, which was gradually reversed to basal evels until 6-hour observation (Fig 1).

ffect of LF and HF TENS Application in he Rat Contralateral Paw The prolonged antihyperalgesic effect verified with LF

ENS application was also observed when LF TENS was pplied in the contralateral, noninflamed paws (Fig 2). imilarly, antihyperalgesia was also obtained with HF ENS applied on noninflamed paws.

ffect of Naltrexone on the Antihyperalgesia nduced by LF and HF TENS

To assess a possible involvement of endogenous opioids n the antihyperalgesic effect of TENS, local (50 �g, in-

igure 2. Antihyperalgesic effect of low and high frequency tran oninflamed paws. Œ C indicates control group, inflamed, injected lectric stimulation. □ CTL LF TENS: (10 Hz) LF TENS applied on the oninflamed paw. Each point represents mean � SEM of 6 animals .05; 1-way ANOVA, followed by Bonferroni test post hoc analys

igure 3. Effect of naltrexone on antihyperalgesic induced by low timulation (TENS). � SAL � IPSI LF TENS, control group, saline (0.1 arrageenan administration, respectively. Œ Nx � LF TENS: Naltrexo fter carrageenan administration, respectively; ‘ SAL � IPSI HF TE 30 (Hz) 2 and 2.5 hours after carrageenan administration, respect lus HF TENS (130 Hz) 2 and 2.5 hours after carrageenan administr

roup. *Statistically significant difference in relation to controls (P � .0

raplantar) naltrexone was administered in the inflamed aws before either LF or HF TENS treatment. As shown in ig 3, naltrexone reversed to control levels the antihyperal- esia induced by LF TENS but not that induced by HF TENS. urprisingly, however, contralateral naltrexone adminis- ration did not affect the otherwise expected antihyperal- esia induced by LF TENS given in the inflamed paw. Surpris-

ngly, however, contralateral naltrexone administration did ot affect the otherwise expected antyhyperalgesia induced y LF TENS give in the inflamed paw (Fig 4).

iscussion With regard to clinical intervention, much of the suc-

ess of physiotherapy relies on the suitability of the

neous electric nerve stimulation (TENS) occurs after application in carrageenan (250 �g/0.1 mL) in the right paw at 0 time, without

inflamed paw; ‘ CTL HF TENS: (130 Hz) HF TENS applied on the roup. *Statistically significant difference in relation to controls (P

uency (LF) and high frequency (HF) transcutaneous electric nerve paw) was administered plus LF TENS (10 Hz) 2 and 2.5 hours after 0 �g/0.1 mL) was administered plus LF TENS (10 Hz) 2 and 2.5 hours ontrol group, saline (0.1 mL/paw) was administered plus HF TENS � Nx � IPSI HF TENS: Naltrexone (50 �g/0.1 mL) was administered , respectively. Each point represents mean � SEM of 6 animals per

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161ORIGINAL REPORT/Sabino et al

ethods used to treat patients.24 Whatsoever, the liter- ture on TENS is as yet rather controversial regarding its fficacy to relieve situations of acute pain.4 It has been uggested that TENS uses distinct analgesic mechanisms, epending on the frequency used.29,30 Most of the stud-

es tend to vary TENS parameters,36 hampering the iden- ification of the exact conditions to influence the ob- erved response.13 Therefore in this study, we chose to ary the frequency of activation current, maintaining ther TENS parameters constant. Our results demonstrated a complete reversal of the ociceptive effect in rat paws by both low (10 Hz) and igh (130 Hz) TENS frequencies. However, LF TENS pro- oted a longer lasting antihyperalgesic effect than that bserved with HF TENS. Indeed, similar results were pre- iously obtained in our 29 as well as in other laboratories, s reviewed by Sluka and Walsh.36

In the next experiment, we sought to verify the impor- ance of the localization of the TENS electrodes. Under onditions of acute pain, TENS electrodes are normally laced around the points of pain. However, in some clin-

cal conditions, electrode placement in the injured place s not feasible. In such cases, the placement of the TENS lectrodes is recommended on the same pathway that nnervates the injured area or over the emergence of the erve from the spinal cord responsible for the affected ermatome.13,38 However, we observed in the current tudy that LF and HF TENS were effective in inducing ntihyperalgesia when the electrodes were placed on he opposite side to the inflammation. These data are in greement with various studies,1,37 who showed LF or HF ENS applied to the gastrocnemius muscle contralateral o the site of inflammation also significantly reduced ntihyperalgesia. Therefore, we may assume that appli- ation of TENS on the contralateral side modified the

igure 4. Ipsilateral and contralateral effect of naltrexone a timulation (TENS)-treated rats. Œ C: Control group, inflamed, in ithout electric stimulation. ‘CTL Nx � IPSI LF TENS: Naltrexo

nflamed and LF (10Hz) TENS-treated paw; � IPSI Nx � IPSI LF TEN ne-half hour before LF (10 Hz) TENS administration. Each po ignificant difference (P � .05, 1-way ANOVA) compared with C

ransmission of pain occurring in the inflamed side. As n

rojections of the inhibitory descending system are bilat- ral, diffuse and connect both sides of the spinal cord,31

hese results may suggest that this pathway was acti- ated by TENS on the contralateral side. Antihyperalge- ia may have actually been evolved from spinal cord, as ther studies have demonstrated that deep spinal affer- nts present bilateral projections in the dorsal horn of he cord.11,13

To verify further the participation of endogenous opi- ids release in the antihyperalgesic effect shown by both requencies of TENS treatment, naltrexone, a specific pioid antagonist, was administered into rat paws be- ore the treatments. In fact, only the antinociceptive ef- ect induced by LF-TENS was modified by treatment of at paws with naltrexone, clearly suggesting that LF and F-TENS bring about an antihyperalgesic effect through- ut different mechanisms of action: One involving en- ogenous opioids release (LF TENS) and the other (HF ENS) involving a nonopioid mediated mechanism. As pposed to our results, however, some studies in the

iterature support the notion that both HF TENS and LF ENS result in the release of endogenous opioids.5,14

F TENS would produce antihyperalgesia through activa- ion of �-opioid receptors and HF TENS through activa- ion of �-opioid receptors in the spinal cord.34 However, F TENS was less effective than HF TENS at reducing hy- eralgesia in morphine-tolerant rats,30 suggesting that pioid receptors of the �-type are involved in the anal- esic mechanisms of LF TENS. Accordingly, �-opioid re- eptors are more potent than �- or �-agonists in inducing eripheral antinociceptive effects.40 Taken together, hese data suggest that the analgesic effect shown by LF ENS-treated animals might be mediated predominantly y �-opioid receptors. Another contribution of the resent study was demonstration that naltrexone did

istration in low frequency (LF) transcutaneous electric nerve d with carrageenan (250 �g/0.1 mL) in the right paw at 0 time, 0 �g/0.1 mL) was administered in the contralateral paw to the altrexone (50 �g/0.1 mL) was administered in the inflamed paw presents mean � SEM of 3 animals per group. *,#Statistically IPSI Nx.

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162 Endogenous Opioids in TENS Application

ot HF TENS when administered in the inflamed paw ipsilateral paw). On the other hand, naltrexone admin- stered in the contralateral paw to the TENS did not pre- ent the antihyperalgesic effect of LF-TENS in the in- amed paw. The latter might suggest that either (a) the

ocal vasodilatation promoted a more efficacious presen- ation of naltrexone into the central nervous system CNS) to antagonize the opioid receptors activated by LF ENS, and/or (b) a partial contribution of local endoge- ous opioids release to the overall antihyperalgesic ef- ect shown by (LF) TENS exist. Experimental and clinical tudies have demonstrated that locally administered opi- ids produce analgesic effects by interacting with pe- ipheral opioid receptors8,39 and that morphine exerts its eripheral analgesic effect by activating ATP-sensitive � channels.32 Indeed, presence of immunoreactive -endorphin in rat skin keratinocytes was recently de-

cribed in the literature,16 which is the most potent en- m

f inflammation. Br J Pharmacol 137:837-844, 2002

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ogenous opioids at �-opioid receptors,15 thus consis- ent with our previous considerations. Alteration of eratinocyte membranes by LF TENS could bring about -endorphin release, further contributing to the overall ntihyperalgesic effect. This contribution, however, de- erves future investigation. Taken together, these data indicate that under some

pecial clinical conditions of acute pain such as it occurs in pen injuries, extensive burns or neuropathic pain, ap- lication of TENS on the opposite side of the injury may educe pain, thus offering a therapeutic alternative for ain-relief, with satisfactory results,38 a goal aimed by oth patients and clinicians.

cknowledgments The authors thank Webster Glayser Pimenta dos Reis

or excellent technical assistance during the experi-

ents.

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