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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 289:840 –846, 1999
Vol. 289, No. 2
Printed in U.S.A.
Spinal Blockade of Opioid Receptors Prevents the Analgesia
Produced by TENS in Arthritic Rats1
KATHLEEN A. SLUKA, MEREK DEACON, ANDREA STIBAL, SHANNON STRISSEL, and AMY TERPSTRA
Physical Therapy Graduate Program (M.D., A.S., S.S., A.T.) and Neuroscience Graduate Program (K.A.S.), The University of Iowa,
Iowa City, Iowa
Accepted for publication December 2, 1998
This paper is available online at http://www.jpet.org
One noninvasive treatment commonly used to manage arthritic pain is transcutaneous electrical nerve stimulation
(TENS). Studies have shown that TENS reduces pain in
people with rheumatoid and osteoarthritis (Manheimer et
al., 1978; Manheimer and Carlsson, 1979; Kumar and Redford, 1982). Although studies have demonstrated the effectiveness of TENS for reducing pain in people with arthritis,
the physiological mechanism by which TENS produces analgesia is unknown. Two different theories have been proposed.
The most popular theory for the mechanism of action of
TENS is the gate control theory of pain (Melzack and Wall,
1965; Kumar and Redford, 1982; Garrison and Foreman,
1994; Hollman and Morgan, 1997). This theory proposes that
stimulation of large-diameter afferent fibers inhibits secondorder neurons in the dorsal horn and prevents pain impulses
carried by small-diameter fibers from reaching higher brain
centers.
Received for publication October 20, 1998.
1
This study was supported by grants from the Central Investment Fund for
Research Enhancement from the University of Iowa and the Arthritis Foundation.
as a control) administration, and after drug (or artificial cerebral
spinal fluid) administration 1 TENS. Either high- (100 Hz) or
low- frequency (4 Hz) TENS produced approximately 100%
inhibition of hyperalgesia. Low doses of naloxone, selective for
m opioid receptors, blocked the antihyperalgesia produced by
low-frequency TENS. High doses of naloxone, which also block
d and k opioid receptors, prevented the antihyperalgesia produced by high-frequency TENS. Spinal blockade of d opioid
receptors dose-dependently prevented the antihyperalgesia
produced by high-frequency TENS. In contrast, blockade of k
opioid receptors had no effect on the antihyperalgesia produced by either low- or high-frequency TENS. Thus, low-frequency TENS produces antihyperalgesia through m opioid receptors and high-frequency TENS produces antihyperalgesia
through d opioid receptors in the spinal cord.
The second explanation for the mechanism of action of
TENS is that it stimulates the release of endogenous opioids.
Naloxone, an opioid receptor antagonist, blocks the analgesia
produced by low-frequency electroacupuncture (,10 Hz),
suggesting it works through the release of endorphins
(Mayer et al., 1977; Woolf et al., 1977; Cheng and Pomeranz,
1979; Ha et al., 1981). Fox and Melzack (1976) compared the
use of TENS and acupuncture in the treatment of lower back
pain and concluded they have the same underlying mechanism of action. Others have demonstrated an increased content of opioid peptides in the cerebrospinal fluid in humans
after administration of TENS (Salar et al., 1981; Hughes et
al., 1984; Almay et al., 1985; Han et al., 1991).
Several studies indicate that high- (.10 Hz) and low- (,10
Hz) frequency TENS work through different mechanisms.
Abram et al. (1981) investigated the role of opioids in analgesia produced by high-frequency TENS. Specifically, no reversal of analgesia was seen after administration of naloxone, suggesting to the authors that high-frequency TENS
does not work through the release of opioids. High-frequency
TENS is, therefore, believed to work through mechanisms
ABBREVIATIONS: TENS, transcutaneous electrical nerve stimulation; PWL, paw withdrawal latency; ACSF, artificial cerebral spinal fluid; MEAP,
Met-enkephalin-Arg-Phe; NSAID: non-steroidal anti-inflammatory.
840
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ABSTRACT
Transcutaneous electrical nerve stimulation (TENS) is commonly used for relief of pain. The literature on the clinical
application of TENS is extensive. However, surprisingly few
reports have addressed the neurophysiological basis for the
actions of TENS. The gate control theory of pain is typically
used to explain the actions of high-frequency TENS, whereas,
low-frequency TENS is typically explained by release of endogenous opioids. The current study investigated the role of m, d,
and k opioid receptors in antihyperalgesia produced by lowand high-frequency TENS by using an animal model of inflammation. Antagonists to m (naloxone), d (naltrinodole), or k (norbinaltorphimine) opioid receptors were delivered to the spinal
cord by microdialysis. Joint inflammation was induced by injection of kaolin and carrageenan into the knee-joint cavity.
Withdrawal latency to heat was assessed before inflammation,
during inflammation, after drug (or artificial cerebral spinal fluid
1999
TENS and Opioids
Materials and Methods
Placement of the Microdialysis Fiber
All experiments were approved by the animal care and use committee at our institution and are in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats (250 –350 g;
n 5 122) were implanted with a microdialysis fiber in the dorsal horn
(L4–L6 spinal level) for delivery of drugs to the spinal cord (Sluka and
Westlund, 1992). A microdialysis fiber (Hospal AN69 with a cutoff of
45 kDa) was prepared by marking a 2-mm gap and then applying an
epoxy coating to the remaining length of the fiber. This allowed
diffusion of the drug to occur only in the 2-mm gap to be positioned
in the dorsal horn of the spinal cord. Rats were initially anesthetized
with sodium pentobarbital (50 mg/kg i.p.) for placement of the microdialysis fiber. A hole was drilled just under the lip of the pedicle
on both sides of the T13 spinal segment with a manual drill. The
prepared microdialysis fiber was then threaded through the holes.
Polyethylene (PE 20) tubing was secured to both ends of the fiber
with super glue gel and epoxy. The fiber was positioned so that the
2-mm section was in the dorsal horn of the spinal cord and then
secured in place with dental cement. The PE 20 tubing was then
sutured to the fascia to prevent any unnecessary movement. Staples
were used to close the incision and the rat was then placed in its cage
for recovery overnight. The next day, rats were divided into the
following treatment groups: 1) Artificial cerebrospinal fluid (ACSF)
and no TENS treatment (n 5 6) control; 2) ACSF 1 low- (n 5 8) or
high-frequency (n 5 6) TENS; 3) Naloxone hydrochloride (Sigma
Chemical Co., St. Louis, MO; 1.0 –10.0 mM) 1 low- (1 mM, n 5 3; 5
mM, n 5 5; 10 mM, n 5 7) or high-frequency (1 mM, n 5 3; 5 mM, n 5
6; 10 mM, n 5 6) TENS; 4) Naltrinodole hydrochloride (Sigma Chemical Co.; 0.01–1.0 mM) 1 low- (1 mM; n 5 6) or high-frequency (0.01
mM, n 5 5; 0.1mM, n 5 3; 1 mM, n 5 6) TENS; or 5) nor-binaltorphimine (nor-BNI; Research Biochemicals International, Natick,
MA; 0.01 mM) 1 low- (n 5 6) or high-frequency (n 5 7) TENS.
All drugs were dissolved in ACSF and pH-corrected (7.2–7.4).
Behavioral Testing and Treatment Protocol
The day after implantation of the microdialysis fiber, withdrawal
latencies of both hindpaws were determined according to the protocol
described by Hargreaves et al. (1988). Rats were placed in clear
plastic cages on an elevated glass plate and allowed to acclimate for
10 to 20 min. A radiant heat source was applied to the posterior
plantar surface of the hindpaw and the time for the rat to withdraw
its paw was measured. The light box had an on/off switch connected
to a timer, which measured the duration of the paw withdrawal
latency (PWL). If the PWL exceeded 20 s, the heat source was turned
off to avoid tissue damage. The average of five trials for each paw
was determined. The examiner was kept blinded to the treatment
groups, both drug treatment and TENS treatment. The knee-joint
circumferences were measured bilaterally with a flexible tape measure around the center of the fully extended knee.
Injection of the Knee Joint. After baseline behavioral measurements, rats were anesthetized with 2 to 4% halothane via a face
mask for approximately 5 min and a solution of 3% kaolin and 3%
carrageenan (0.1 ml; pH 7.4) in sterile saline was injected into the
left knee joint to induce inflammation (Sluka and Westlund, 1993).
Four hours after the injection, the paw withdrawal responses to
heat were tested as before. Spontaneous pain-related behaviors were
rated on a scale from zero to five (0 5 normal, 1 5 curled toes, 2 5
everted foot, 3 5 partial weight bearing, 4 5 nonweight bearing, and
5 5 complete avoidance of limb by lying on side) (Sluka and
Westlund, 1993). The rats were then given either a drug or ACSF for
1 h through the microdialysis fiber. After the 1-h infusion, PWL and
spontaneous pain-related behaviors were assessed.
TENS Treatment. Rats were then lightly anesthetized with halothane (1–2%, 20 min), their knee-joint circumferences were measured, and TENS was applied to the knee joint. Rats received either
1) low-frequency TENS at sensory intensity to the inflamed knee
joint (4 Hz; 20 min.; EMPI Eclipse 1; EMPI, Inc., Minneapolis, MN),
2) high-frequency TENS at sensory intensity to the inflamed knee
joint (100 Hz; 20 min.; EMPI Eclipse 1), or 3) halothane without
TENS. One-inch round pregelled electrodes were placed on the medial and lateral aspects of the shaved knee joint. Sensory-intensity
TENS was determined by increasing the intensity until a palpable
muscle contraction was elicited and then reducing the intensity to
just below that point. The study minimized variability of stimuli by
maintaining a pulse duration constant at 100 ms and an intensity
constant at sensory-level intensity (see Sluka et al., 1998). Thus, only
the frequency of stimulation was varied. These parameters are based
on those used clinically (see Robinson and Snyder-Mackler, 1995)
and those previously published (Sluka et al., 1998).
Immediately after TENS treatment, PWLs were determined and
spontaneous pain-related behaviors were recorded. Finally, rats
were anesthetized again to measure knee-joint circumferences. After
the final measurements were taken, the rats were euthanized with
an overdose of sodium pentobarbitol and the spinal cords were removed and dissected to verify the correct placement of the microdialysis fiber at the L4–L6 level of the spinal cord.
Selectivity of Drugs. To test selectivity of naloxone to opioid
receptors, 13 rats were implanted with microdialysis fibers. The m
opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Glyy-ol5]-enkephalin) (Research Biochemicals International; 1 mM; n 5 4), the d
opioid receptor agonist SNC8O ((1)-4-[(aR)-a-((2S,5R)-4-allyl-2,5dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide)
(Tocris Cookson; 1 mM; n 5 4), or the k opioid receptor agonist
U50,488 (trans-(1)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexy]benzenecetamide) (Tocris Cookson; 0.1 mM, n 5 5) was
infused into the dorsal horn and PWL to radiant heat was tested.
Naloxone (1, 5, or 10 mM) was tested for its ability to antagonize the
analgesia produced by the opioid receptor agonists. Similarly, the
selectivity of the d opioid receptor antagonist naltrinodole (n 5 14)
and the k opioid receptor antagonist nor-BNI (n 5 13) was tested
against DAMGO (1 mM), SNC8O (1 mM), or U50,488 (0.1 mM). The
effects of 0.01, 0.1, and 1 mM naltrinodole and 0.1, 1, and 10 mM
nor-BNI were tested against the opioid agonists.
Statistical Analysis
To minimize variability between groups, data were assessed for
the percentage of inhibition by TENS for PWL with the following
formula: (TENS or drug 2 arthritis)/(base 2 arthritis) 3 100. Thus,
100% inhibition is a full reversal of hyperalgesia and 0% inhibition is
no change from the hyperalgesia measured 4 h after induction of
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proposed by the gate control theory, producing only shortterm analgesia (Garrison and Foreman, 1994; Hollman and
Morgan, 1997). Conversely, low-frequency TENS is proposed
to work through release of endogenous opioids, which causes
a more systemic and long-term response (Sjound and Eriksson, 1979). Some studies, however, have demonstrated that
high-frequency TENS has a longer lasting effect than lowfrequency TENS (Manheimer and Carlsson, 1979; Walsh et
al., 1995; Gopalkrishnan and Sluka, 1998; Sluka et al., 1998).
Furthermore, Woolf et al. (1977) demonstrated that high
doses of naloxone block the analgesia produced by highfrequency TENS in rats. The different mechanisms by which
high- and low-frequency TENS works still remain unclear.
In response to the conflicting results of previous studies
and the lack of research on the mechanisms through which
TENS works, this study investigated the spinal mechanisms
through which low- and high-frequency TENS exert their
antihyperalgesic effects. We hypothesized that low-frequency
TENS activates endogenous opioid receptors in the spinal
cord.
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Sluka et al.
Vol. 298
inflammation. The group effect of TENS and the group effect of drug
on the percentage of inhibition of hyperalgesia were assessed by an
ANOVA (p , .05). Post hoc tests were done with independent t tests
for assessing differences between groups.
TABLE 2
Spontaneous pain-related behavior ratings for control arthritic rats and
those receiving TENS treatment. Ratings were based on a scale from
zero to five, with zero being normal and five representing total
avoidance of the inflammed limb. Values are the median and range.
Results
TABLE 1
Joint circumference measurements (cm) for control arthritic animals
and those receiving either high- or low-frequency sensory TENS
treatment. Values represent mean 6 S.E.M.
Inflamed knee
ACSF, no TENS
ACSF 1 low frequency TENS
ACSF 1 high frequency TENS
10 mM naloxone 1 low TENS
5 mM naloxone 1 low TENS
1 mM naloxone 1 low TENS
10 mM naloxone 1 high TENS
5 mM naloxone 1 high TENS
1 mM naloxone 1 high TENS
1 mM naltrinodole 1 low TENS
1 mM naltrinodole 1 high TENS
0.1 mM naltrinodole 1 high
TENS
0.01 mM naltrinodole 1 high
TENS
10 mM nor BNI 1 low TENS
10 mM nor BNI 1 high TENS
Contralateral knee
ACSF, no TENS
ACSF 1 low frequency TENS
ACSF 1 high frequency TENS
10 mM naloxone 1 low TENS
5 mM naloxone 1 low TENS
1 mM naloxone 1 low TENS
10 mM naloxone 1 high TENS
5 mM naloxone 1 high TENS
1 mM naloxone 1 high TENS
1 mM naltrinodole 1 low TENS
1 mM naltrinodole 1 high TENS
0.1 mM naltrinodole 1 high
TENS
0.01 mM naltrinodole 1 high
TENS
10 mM nor BNI 1 low TENS
10 mM nor BNI 1 high
Baseline
4 h after
Arthritis
After
TENS
cm
cm
cm
5.5 6 0.9
5.6 6 .07
5.5 6 .08
5.8 6 .18
5.7 6 .29
5.9 6 .22
6.0 6 .26
5.8 6 .08
5.6 6 0.1
5.9 6 .03
5.9 6 .07
5.7 6 .03
6.9 6 .21
6.7 6 .12
6.6 6 .16
7.0 6 .26
6.7 6 .14
7.2 6 .15
7.3 6 .21
7.2 6 .15
7.3 6 .33
7.2 6 .11
7.4 6 .23
6.8 6 .15
6.9 6 .23
6.7 6 .16
6.7 6 .16
7.0 6 .22
7.1 6 .22
7.6 6 .13
7.4 6 .25
7.1 6 .16
7.2 6 .20
7.5 6 .08
7.4 6 .19
7.0 6 .21
5.6 6 .07
6.9 6 .16
6.9 6 .15
6.0 6 .06
5.7 6 .13
7.5 6 .08
7.1 6 .10
7.5 6 .09
7.1 6 .07
5.6 6 .09
5.6 6 .10
6.0 6 .22
5.6 6 .08
5.7 6 .19
5.7 6 .29
5.9 6 .25
5.8 6 .09
5.6 6 .07
5.9 6 .08
6.0 6 .08
5.7 6 .07
5.6 6 .10
5.6 6 .09
6.0 6 .23
5.6 6 .08
5.7 6 .19
5.8 6 .33
6.1 6 .23
5.8 6 .13
5.6 6 .09
5.8 6 .03
5.9 6 .06
5.6 6 .03
5.6 6 .11
5.6 6 .07
6.0 6 .22
5.5 6 .07
5.8 6 .19
5.8 6 .32
6.0 6 .28
5.8 6 .12
5.5 6 .06
5.8 6 .04
5.9 6 .06
5.7 6 .03
5.6 6 .08
5.6 6 .09
5.6 6 .06
6.1 6 .04
5.6 6 .06
6.0 6 .02
5.6 6 .08
6.1 6 .03
5.6 6 .04
ACSF, no TENS
ACSF 1 low frequency TENS
ACSF 1 high frequency TENS
10 mM naloxone 1 low TENS
5 mM naloxone 1 low TENS
1 mM naloxone 1 low TENS
10 mM naloxone 1 high TENS
5 mM naloxone 1 high TENS
1 mM naloxone 1 high TENS
1 mM naltrinodole 1 low TENS
1 mM naltrinodole 1 high TENS
0.1 mM naltrinodole 1 high TENS
0.01 mM naltrinodole 1 high TENS
10 mM nor BNI 1 low TENS
10 mM nor BNI 1 high TENS
1 h after
Drug
After
TENS
4 (3–5)
4 (3–4)
4.5 (4–5)
4 (3–5)
4 (3–5)
3 (3–4)
4 (3–4)
4 (3–5)
4 (3–4)
4 (3–5)
4 (3–5)
4 (4)
4 (3–4)
4 (4)
4 (2–4)
4.5 (3–5)
4 (2–5)
4.5 (4–5)
4 (3–4)
5 (3–5)
4 (3–4)
4 (4–5)
4 (4–5)
4 (3–4)
4 (3–5)
4 (3–5)
4 (4)
4 (2–4)
4 (4–5)
4 (3–4)
4 (3–4)
3 (2–4)
4 (3–5)
4 (3–4)
4 (3–5)
4 (3–4)
4 (3–4)
4 (3–4)
4 (3–5)
4 (3–4)
4 (2–5)
4 (3–4)
4 (3–4)
4 (3–5)
4 (3–4)
after induction of inflammation, to 8.3 6 0.26 s after treatment with TENS (baseline 5 9.0 6 0.75 s). Similarly, in the
group of animals treated with high-frequency TENS, the
PWL increased from 7.2 6 0.32 s, 4 h after induction of
inflammation, to 10.1 6 0.42 s after treatment with TENS
(baseline 5 9.8 6 0.31 s). There was an overall significant
effect across time (F1,66 5 131.95; p 5 .001) for changes in
PWL in all of the groups of animals. A significant effect for
group by time occurred for the percentage of inhibition of
hyperalgesia after treatment with TENS (F14,66 5 4.05; P 5
.001) but not after infusion of drug (or ACSF) alone (F14.66 5
1.75; p 5 .08).
The PWL of the contralateral hindpaw remained unchanged after induction of inflammation in animals with
ACSF or those treated with TENS or drug. For example,
baseline PWL for the contralateral paw in control arthritic
animals treated only with ACSF was 9.3 6 0.655 s, and 4 h
after inflammation the PWL remained at 8.7 6 0.655 s. After
treatment with either high- or low-frequency TENS, the contralateral PWL was 10.5 6 0.53 s and 9.3 6 0.55 s, respectively, compared with baseline values of 9.8 6 0.53 s and
9.6 6 0.56 s.
Effects of Naloxone on TENS Analgesia. Spinal infusion of 1 mM naloxone had no effect on the inhibition of
hyperalgesia produced by either high- or low-frequency
TENS; the percentage of inhibition of hyperalgesia remained
at approximately 100%. However, 5 and 10 mM naloxone
prevented the inhibition of hyperalgesia by low- frequency
TENS (Fig. 1). Thus, there was still a decrease in the PWL to
radiant heat after TENS treatment similar to that observed
4 h after induction of inflammation. Only 10 mM naloxone
blocked the inhibition of hyperalgesia produced by high-frequency TENS (Fig. 2).
To test selectivity of naloxone for different opioid receptors,
naloxone was tested against agonists selective for m
(DAMGO), d (SNC80), or k (U50,488) opioid receptors. As Fig.
3A shows, all three agonists produced analgesia as indicated
by the significant increase in PWL (p , .05). After administration of 1 mM naloxone, the PWL remained significantly
increased. After administration of 5 mM naloxone, only the
group receiving DAMGO (m agonist) returned to the baseline.
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Control Arthritic Animals and Effect of TENS. Four
hours after induction of arthritis, there was a significant
decrease in PWL to radiant heat that was maintained
throughout the testing period. There was also an increase in
joint circumference and an increase in spontaneous painrelated behavior ratings 4 h after inflammation. Changes in
joint circumference and spontaneous pain behavior ratings
are given in Tables 1 and 2, respectively. There were no
significant differences between groups for joint circumference or spontaneous pain-related behaviors at any time period (baseline, 4 h after inflammation, after administration of
a drug or ACSF, or after TENS). ACSF had no effect on the
decreased withdrawal latency normally observed after joint
inflammation (Fig. 1). In the group of animals treated only
with ACSF, the withdrawal latency decreased from 9.0 6
0.22 s to 7.2 6 0.24 s, 4 h after induction of arthritis. In
contrast, treatment with either high- or low-frequency TENS
produced approximately 100% reversal of the hyperalgesia
(Figs. 1 and 2). In the group of animals treated with lowfrequency TENS, the PWL increased from 6.7 6 0.32 s, 4 h
4h
Arthritis
1999
TENS and Opioids
843
Fig. 2. The percentage of inhibition of hyperalgesia is represented After
administration of drug or ACSF (E) or treatment with TENS 1 drug or
ACSF (F) in the group of animals treated with high-frequency TENS. A,
inhibition of hyperalgesia after treatment with ACSF (n 5 6), highfrequency TENS (TENS, n 5 6), or naloxone at 1 (n 5 3), 5 (n 5 6), or 10
mM (n 5 6). The percentage of analgesia was significantly increased after
treatment with TENS (p 5 .001) when compared with animals treated
with ACSF and no TENS. Treatment with 10mM (p 5 .0001) naloxone
significantly prevented the analgesia produced by high-frequency TENS.
B, inhibition of hyperalgesia after treatment with 10 mM nor-BNI (n 5 7)
or 1mM naltrinodole (n 5 6). A significant blockade of the inhibition of
hyperalgesia was observed After treatment with 1 mM naltrinodole (p 5
.002) when compared with treatment with high-frequency TENS 1ACSF.
Inset, dose response effect after administration of .01 (n 5 5), .1 (n 5 3),
or 1 naltrinodole (n 5 6). A significant inhibition of hyperalgesia occurred
in the group treated with .1 (p 5 .03) and 1 mM (p 5 .002). *, significantly
different from TENS group. Values are mean 6 S.E.M.
2). The percentage of inhibition of hyperalgesia was similar
to that observed in animals treated with ACSF and TENS
and was not significantly different from that group.
The selectivity of nor-BNI was tested against agonists to m
(DAMGO), d (SNC80), and k (U50,488) opioid receptors. As
Fig. 3C shows, spinal infusion of 10 mM nor-BNI selectively
blocks k opioid receptors. The analgesia produced by spinal
infusion of DAMGO or SNC80 was unaffected by nor-BNI.
Fig. 1. The percentage of inhibition of hyperalgesia is represented after
administration of drug or ACSF (E) or treatment with TENS 1 drug or
ACSF (F) in the group of animals treated with low-frequency TENS. A,
inhibition of hyperalgesia after treatment with ACSF (n 5 6), low-frequency TENS (TENS, n 5 8), or naloxone at 1 (n 5 3), 5 (n 5 5), or 10 mM
(n 5 7). The percentage of analgesia was significantly increased after
treatment with TENS (p 5 .009) when compared with animals treated
with ACSF and no TENS. Treatment with 5 (p 5 .01) or 10mM (p 5 .02)
naloxone significantly prevented the analgesia produced by low-frequency TENS. B, inhibition of hyperalgesia after treatment with 10 mM
nor-BNI (n 5 6) or 1mM naltrinodole (n 5 6). No significant difference
was observed between the group treated with ACSF 1 low-frequency
TENS and those treated with nor-BNI or naltrinodole. *, significantly
different from TENS group. Values are mean 6 S.E.M.
Discussion
The current study supports the theory that TENS works
through release of endogenous opioids at the spinal cord
level. Spinal administration of 5 mM naloxone, which selectively blocks m opioid receptors, significantly reduced the
antihyperalgesic effects of low-frequency TENS. A greater,
nonselective dose of naloxone (10 mM) reduced the antihyperalgesia produced by high-frequency TENS, suggesting the
involvement of endogenous opioids acting at d or k opioid
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Therefore, at a dose of 5 mM, naloxone selectively blocks m
receptors but not k or d opioid receptors. After increasing the
concentration of naloxone to 10 mM, all of the groups’ PWL
returned to the baseline, indicating all opioid receptors (m, d,
and k) were blocked at this dose.
Effects of Naltrinodole on TENS Analgesia. Blockade
of d opioid receptors with 1 mM naltrinodole prevented the
inhibition of hyperalgesia produced by high-frequency but
not low-frequency TENS (Figs. 1 and 2). The effects of naltrinodole on preventing the inhibition of hyperalgesia by
high-frequency TENS were dose-dependent (Fig. 2, inset).
The selectivity of naltrinodole was tested against agonists
to m (DAMGO), d (SNC80), and k (U50,488) opioid receptors.
Figure 3B demonstrates that 1 mM naltrinodole selectively
blocks d opioid receptors. The analgesia produced by spinal
infusion of DAMGO or U50,488 was unaffected by naltrinodole.
Effects of nor-BNI on TENS Analgesia. Blockade of k
opioid receptors with nor-BNI had no effect on the analgesia
produced by either high- or low-frequency TENS (Figs. 1 and
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Sluka et al.
receptors, or both. The antihyperalgesic effect of high-frequency TENS was reduced by blockade of d opioid receptors
but not k opioid receptors. Thus, the current study demonstrated that the analgesia produced by high-frequency sensory TENS is mediated by d opioid receptors spinally, and
that produced by low-frequency sensory TENS is mediated by
m opioid receptors spinally.
Previous studies support the conclusion that both highand low-frequency TENS result in the release of endogenous
opioids. Increased b-endorphin concentrations in the cerebral
spinal fluid were observed after administration of either
high- or low-frequency TENS (Salar et al., 1981; Hughes et
al., 1984; Almay et al., 1985). Han et al. (1991) analyzed the
opioid peptides Met-enkephalin-Arg-Phe (MEAP) and dynorphin A in the cerebral spinal fluid of human subjects after
application of either high- or low-frequency TENS. They
found that high-frequency stimulation produced an increase
in dynorphin A but not in MEAP, whereas low-frequency
TENS increased MEAP but not dynorphin A. Although similar frequencies of stimulation were used by Han et al. (1991;
2 and 100 Hz), the intensity of stimulation was greater,
eliciting a motor contraction in the subjects. Previously, we
demonstrated that increasing intensity of stimulation resulted in an increase in inhibition of hyperalgesia in carrageenan-inflamed rats (Gopalkrishnan and Sluka, 1998). Similarly, Garrison and Foreman (1996) showed an increased
inhibition of responses to noxious stimuli with increased
intensity of stimulation when recording from unsensitized
dorsal horn neurons. Differences between the studies, thus,
could be explained by differences in intensity of stimulation.
Several studies have demonstrated that acupuncture-induced analgesia is reduced by naloxone in normal subjects
and a variety of patient populations (Mayer et al., 1977; Ha et
al., 1981; Homma et al., 1985; Eriksson et al., 1991). Similarly, low-frequency, high-intensity electroacupuncture suppresses responses of dorsal horn neurons to noxious stimuli
and this suppression is reversed by naloxone (Pomeranz and
Cheng, 1979). Sjolund and Eriksson (1979) demonstrated
that analgesia produced by low-frequency, high-intensity
TENS but not high-frequency, low-intensity TENS is reversible by administration of naloxone systemically. The dose
used by Sjolund and Eriksson (1979) was at a concentration
expected to block m opioid receptors. In contrast, high-frequency TENS was unaffected by systemic naloxone in patient
populations (Abram et al., 1981; Freeman et al., 1983). However, analgesia induced in rats by high-frequency TENS was
reversed by high doses of systemic naloxone expected to block
m, d, and k opioid receptors (Woolf et al., 1977; Han et al.,
1984).
The release of endogenous opioids in the spinal cord in
response to TENS stimulation could result from activation of
local circuits within the spinal cord or from activation of
descending inhibitory pathways. Opioid peptides, enkephalin, and dynorphin are contained in spinal dorsal horn neurons (Hokfelt et al., 1977; Glazer and Basbaum, 1981). Likewise, m and d opioid receptors have been localized to the
dorsal horn, both presynaptically on primary afferent fibers
and postsynaptically on dorsal horn neurons (LaMotte et al.,
1976; Atweh and Kuhar, 1983; Cheng et al., 1997). By using
immunohistochemistry, Zhang et al. (1998) demonstrated
that small dorsal root ganglia neurons labeled for the d opioid
receptor also contain Substance P and calcitonin gene-related peptide. Further spinal localization of the d receptor
was reduced by dorsal rhizotomy, suggesting presynaptic
localization on primary afferents. Release of the primary
afferent peptides, Substance P and calcitonin gene-related
peptide, is blocked by opioid agonists (Yaksh et al., 1980;
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 3. Bar graphs representing the selectivity of opioid receptor antagonists to the agonists. The paw withdrawal latency was measured before
(base) and after spinal infusion of opioid agonists (drug), and then agonist
plus increasing doses of the antagonists. A, effects of naloxone at reducing
the increase in PWL induced by DAMGO (n 5 4) to activate m receptors;
SNC80 (n 5 4) to activate d receptors, and U50,488 (n 5 5) to activate k
receptors. A significant reversal of the analgesia produced by DAMGO
occurred after spinal infusion of 5 and 10 mM naloxone. 10mM naloxone
also reversed the analgesia produced by SNC80 and U50,488. B, effects of
increasing doses of naltrinodole on the increased paw withdrawal latency
produced by DAMGO (n 5 4), SNC80 (n 5 5), and U50,488 (n 5 5). A
significant reversal of the analgesia by SNC80 was produced with spinal
infusion of 1mM naltrinodole. C, effects of increasing doses of nor-BNI on
the increased paw withdrawal latency produced by DAMGO (n 5 4),
SNC80 (n 5 4), and U50,488 (n 5 4). A significant reversal of the
analgesia produced by U50,488 occurred with spinal infusion of 10 mM
nor-BNI. *p , .05, significantly decreased from infusion of agonist. Values are mean 6 S.E.M.
Vol. 298
1999
845
ation of mood, mental clouding, and increased tolerance to
the drug with continued use. Thus, the use of TENS in
conjunction with opioids could lower the intake of drugs and
limit side effects.
Herrero and Headley (1996) found that naloxone blocks the
antinociceptive effects of the nonsteroidal anti-inflammatory
drug (NSAID) flunixin in rats with an inflamed paw. They
concluded that naloxone acts as a noncompetitive antagonist
to flunixin and that spinal antinociception caused by the
NSAID was mediated via release of endogenous opioid peptides. This implies that TENS could be an effective alternative to NSAIDs as an analgesic when used in conditions of
acute inflammation. Furthermore, if a patient is taking
NSAIDs, TENS may be less effective.
Conclusions. Both high- and low-frequency TENS at sensory intensity reverse the hyperalgesia produced by kneejoint inflammation. The antihyperalgesic effects of low-frequency TENS are reversed by spinal administration of low
doses of naloxone that are selective for m opioid receptors.
However, the antihyperalgesia produced by high-frequency
TENS is prevented by blockade of d opioid receptors in the
spinal cord. Thus, low-frequency TENS works through activation of m opioid receptors and high-frequency TENS works
through activation of d opioid receptors.
Acknowledgments
We thank Dr. G. F. Gebhart for critically reading the manuscript
and EMPI for providing the TENS units.
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