The Journal of Pain, Vol 12, No 10 (October), 2011: pp 1069-1079

Available online at www.sciencedirect.com

Endogenous Opioids Released During Non-Nociceptive

Environmental Stress Induce Latent Pain Sensitization Via
a NMDA-Dependent Process

Le Roy, Emilie Laboureyras, St

Chloe
ephanie Gavello-Baudy, J

er

emy Chateauraynaud,
Jean-Paul Laulin, and Guy Simonnet

Bordeaux, INCIA, CNRS UMR 5287, F-33076 Bordeaux, France.
Universite

Abstract: Although stress induces analgesia, there is evidence that stressful events may exacerbate
pain syndromes. Here, we studied the effects of 1 to 3 prestressful events (days 0, 2, and 7), such as
non-nociceptive environmental stress, on inflammatory hyperalgesia induced by a carrageenan injection (day 14) in 1 rat hind paw. Changes in nociceptive threshold were evaluated by the paw pressure
vocalization test. The higher the number of stress sessions presented to the rats, the greater was the
inflammatory hyperalgesia. Blockade of opioid receptors by naltrexone before each stress inhibited
stress-induced analgesia and suppressed the exaggerated inflammatory hyperalgesia. Stressed versus nonstressed animals could be discriminated by their response to a fentanyl ultra-low dose
(fULD), that produced hyperalgesia or analgesia, respectively. This pharmacological test permitted
the prediction of the pain vulnerability level of prestressed rats because fULD analgesic or hyperalgesic indices were positively correlated with inflammatory hyperalgesic indices (r2 = .84). In prestressed rats, fULD-induced hyperalgesia and the exaggerated inflammatory hyperalgesia were
prevented NMDA receptor antagonists. This study provides some preclinical evidence that pain intensity is not only the result of nociceptive input level but is also dependent on the individual history,
especially prior life stress events associated with endogenous opioid release.
Perspective: Based on these preclinical data, it would be of clinical interest to evaluate whether
prior stressful events may also affect further pain sensation in humans. Moreover, this preclinical
model could be a good tool for evaluating new therapeutic strategies for relieving pain hypersensitivity.
2011 by the American Pain Society
Key words: Stress, pain sensitization, hyperalgesia, endogenous opioids, NMDA receptors.

pioid-dependent stress decreases acute pain sensitivity, a phenomenon referred to as stressinduced analgesia.1 However, consistent with
the biopsychosocial conceptual framework,15 there is
a compelling body of evidence in humans that unmanaged negative emotion associated with stressful events
is a psychological predictor of exaggerated acute pain;
for example, as postoperative pain.22 In preclinical
Received October 7, 2010; Revised April 21, 2011; Accepted April 30, 2011.

Victor Se

galen Bordeaux 2, Universite

Bordeaux
Supported by Universite
re de lEducation Nationale, de lenseignement supe

rieur et
1, the Ministe
de la Recherche and the Centre National de la Recherche Scientifique
re de la Recherche
(CNRS). C. Le Roy has a fellowship from the Ministe

rieur.
et de lEnseignement Supe
Address reprint requests to Pr Guy Simonnet, Laboratory CNRS UMR 5287,

ostasie-Allostasie-Pathologie-Re

habilitation, Universite


Team Home


tage, 146 rue Le

o Saignat, 33076 BorBordeaux 2 Zone Nord Bat 4A 3eme e
deaux Cedex, France. E-mail: gsimonnet@yahoo.com
1526-5900/$36.00
2011 by the American Pain Society
doi:10.1016/j.jpain.2011.04.011

models, there is a growing body of evidence that repeated exposure of animals to non-nociceptive stressful
situations can elicit a persistent increase in nociception.21,35,37,43 Pain hypersensitivity induced by stress is
a lesser-known phenomenon than stress-induced analgesia, despite the fact it is much more relevant from
the clinical and therapeutic viewpoints since pain hypersensitivity leads to exaggerated pain and enhancement
of medical care. Neural mechanisms by which prior stress
may induce pain aggravation following acute tissue
injury have to be better understood for identifying patients at risk for developing exaggerated pain following
a tissue injury.26 Indeed, clinical studies to date have
failed to distinguish the significant role of stressful
events that occur before or after to acute tissue injuries
in the development of exaggerated pain. Preclinical animal studies have the advantage of allowing one to selectively evaluate the role of prestressful events in pain
hypersensitivity.
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Although numerous endogenous molecules released

by stress, such as glucocorticoids or biogenic amines,
may possibly participate in neuroplastic changes leading
to latent pain hypersensitivity, the possible role of endogenous opioids released by stress is poorly understood. Indeed, previous reports show that a single
exposure to various exogenous opioids, like heroin,30
fentanyl,9 and remifentanil,5 paradoxically induces hyperalgesia for several days following analgesia in nave
rats and strongly enhances postoperative pain in both
animals and humans.2,3,6,17,23
The main purpose of this study is to gain a better understanding of the mechanisms and pathophysiological
relevance of changes in pain sensitivity induced by prior
non-nociceptive environmental stress (NNES). We chose
to examine inflammatory pain because it is a critical
component in various pain syndromes and the evolution
of carrageenan-induced inflammatory pain may be compared with the time-course of postoperative pain.16 A
hind paw inflammation was performed several days
after 1 to 3 exposures to events of NNES. The role of endogenous opioids released during NNES in the development of changes in pain sensitivity was examined by
blocking opioid receptors with naltrexone before each
NNES.
In addition, we asked whether it was possible to detect pain vulnerability in prestressed rats, because it
would be of clinical interest to develop tests for identifying patients at risk for developing exaggerated pain following a tissue injury.26 Interestingly, the administration
of an ultra-low dose of fentanyl (fULD, 50 ng/kg) in rats
preexposed to opioids for relieving prior pain induces
paradoxical hyperalgesia, but not the analgesia observed in rats without prior pain or opioid exposures.38
This finding suggests that behavioral responses to
a fULD administration may be used as a pharmacological
test to detect pain vulnerability (latent pain hypersensitivity) and then to predict exaggerated pain response to
further noxious stimuli. Here, we evaluated whether
a single fULD administration induced analgesia or hyperalgesia in rats preexposed to 1 to 3 NNES. Relationships
between the amplitude of the fULD response (ie, analgesia or hyperalgesia) and the amplitude of the inflammatory hyperalgesia were analyzed in prestressed and
nonstressed rats.
We and others9,32 have previously reported that NMDA
receptor systems play a critical role in the development of
pain hypersensitivity. Therefore, we examined the ability
of 2 NMDA receptor (NMDAR) antagonists, ketamine and
BN2572, to affect changes in pain sensitivity induced by
prior NNES in rats.

AM) and at a constant room temperature of 23 6

2 C. Food and water were available ad libitum. All experiments were performed during the light period.
Pharmacological tests and animal care were conducted
in accordance with the guidelines of the Committee
for Research and Ethical Issues of IASP49 and the local
Ethics Committee For Animal Experimentation at Aquitaine and Poitou-Charentes (France). These experiments were conducted in an authorized laboratory
and under the supervision of a certified researcher,
Emilie Laboureyras.

Drugs
Fentanyl citrate, ketamine hydrochloride, BN2572, naltrexone, and carrageenan l (Sigma-Aldrich, SaintQuentin Fallavier, France) were dissolved in physiological
saline (.9%). BN2572, the gacyclidine enantiomer (-)-(1S2R)-1-[1-(2-thienyl)-2-methylcyclohexyl] piperidine, is an
NMDA receptor antagonist19 with a longer half-life
and inducing more moderated side effects than ketamine.20,38 This drug was obtained from BEAUFOURIPSEN (Les Ulis, France).
Fentanyl (50 ng/kg), ketamine (10 mg/kg), BN2572
(.3 mg/kg), and naltrexone (1 mg/kg) were administered
subcutaneously (s.c.; 1 mL/kg body weight). Control animals received an equal volume of saline injections.

Measurement of the Nociceptive

Threshold
Nociceptive thresholds in handled rats were determined using a modified Randall-Selitto method as previously described:9 a paw pressure vocalization test in
which a constantly increasing pressure is applied to the
hind paw until the rat squeaks. The Basile analgesimeter
(Bioseb, Chaville, France; stylus tip diameter, 1 mm) was
used with a 600-g cut-off value to prevent tissue damage.
In all experiments, nociceptive thresholds were evaluated in both hind paws.

Non-Nociceptive Environmental Stress

(NNES)
Two types of NNES were used.

The Novel Environment Stress (NES)

Methods

The NES consisted of exposing animals to a novel environment for 1 hour, as previously described.38 Rats were
placed in a new experimental room, in new cages with
fresh litter, and were exposed to a light (350 lux) placed
2 meters away from the rat cages. At the end of the stress
session, rats were returned to their home cage in the
usual experimental room. Nonstressed animals were
kept in their home cages.

Animals

The Forced Swim Stress (FSS)

Experiments were performed using adult male

Sprague-Dawley rats (Charles River Laboratories, LArbresle, France) weighing 200 to 225 g before experiments. Rats were housed in groups of 4 animals per
cage with a 12-hour light/dark cycle (lights on at 7:00

The FSS consisted of placing animals in plastic cylinders

(diameter, 30 cm; height, 50 cm) containing water (24
26 C) with a depth of 20 cm for 20 minutes as previously
described.43 The water was changed and the container
thoroughly cleaned between each swimming session.

Le Roy et al
At the end of the stress session, rats were returned to
their home cages. Nonstressed animals were maintained
in their home cages.
Two types of environmental stress were used in the
first part of this study (Experiment 1) to be sure that
changes in pain sensitivity was not specifically associated
with 1 type of stress, especially the NES mainly used in
this study.

Inflammatory Pain Model

On day 14 (D14), rats were placed in a plastic cage and
then anaesthetized with 3% halothane for 3 minutes.
Carrageenan (.2 mL of a 1% carrageenan solution in saline) was then subcutaneously injected into the left
hind paw with a 25-gauge needle.

Experimental Procedure
Following arrival at the laboratory, the animals were
randomly assigned to the different experimental groups
and acclimatized to the animal care unit for 4 days. To
avoid experimental perturbations that may have affected the measurement of nociceptive threshold, the
experiments were performed by the same experimenter
under quiet conditions in a testing room close to the animal care unit. For 8 days prior to the experiments, the
animals were placed in the test room for 2 hours daily
for acclimatization. In this test room, the animals were
weighed daily and gently handled for 5 minutes. All experiments were performed on groups of 8 animals each
during the light cycle. Experimental design is shown in
Fig 1. Nociceptive threshold assessments were performed
for 2 days (ie, on D 2 and D 1) preceding the experimental day (D0) and were repeated on the experimental day
immediately before the first stress event (D0). Experiments were only initiated when there were no statistical
changes in the basal nociceptive threshold for 3 successive days (D 2, D 1 and D0, 1-way ANOVA, P > .05). The
reference value of the nociceptive threshold was selected as the basal value on D0. The experimenter was unaware of the treatment used. On D0, D2 and D7, NNES
were performed. For the novel environment stress
(NES), nociceptive threshold was measured 30 minutes
after the beginning of each stress period and every
hour for 4 hours during the poststress period. For the

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forced swim stress test (FSS), nociceptive threshold measurements were performed at the end of the stress period (20 minutes after the beginning of the stress
event) and every 30 minutes for 2 hours during the poststress period.
For the fULD test, nociceptive threshold measurements
were performed every 30 minutes for 4 hours following
a subcutaneous injection of 50 ng/kg of fentanyl on D8
and D13.
On D14, nociceptive thresholds were measured 2, 4,
and 6 hours after a carrageenan injection and once daily
for 12 to 18 days. The diameter of the inflamed hind paw
was evaluated daily with a caliper rule.

Experiment 1: Effect of Prior Exposure to

Stress on Changes in Nociceptive Threshold
Induced by Inflammation
Stressed rats (n = 8) were exposed to NES or FSS on D0,
D2, and D7. Nonstressed rats (n = 8) were not exposed to
stress sessions. All rats were subjected to inflammatory
pain on D14.

Experiment 2: Effect of Naltrexone Administration Immediately Before Each NES Session

on Changes in Nociceptive Threshold Induced
by Inflammation
Two groups of rats (n = 8 for each group) were subjected to NES on D0, D2, and D7 and were preadministered naltrexone (1mg/kg, s.c.) or saline 30 minutes
before each stress. The nonstressed group (n = 8) received similar saline injections on D0, D2, and D7 but no
exposure to stress. On D14, all groups were subjected to
inflammatory pain.

Experiment 3: Comparative Effect of 1 to 3 NES

Exposures on Changes in Nociceptive Threshold Induced by fULD and Subsequent Inflammatory Pain
Four groups of rats were used in this experiment. In the
first group (n = 8), the rats were not subjected to NES. The
second group (n = 8) was subjected to 1 stress session on
D7. The third group (n = 8) was subjected to 2 stress sessions on D2 and D7, and the fourth group (n = 8) was subjected to 3 stress sessions on D0, D2, and D7. All rats
received a subcutaneous injection of 50 ng/kg fentanyl
on D8 and D13 and were subjected to inflammatory
pain on D14.

Experiment 4: Effect of NMDA Receptor Antagonist Administration on Changes in Nociceptive Threshold Induced by Inflammation
in Prestressed Rats
Figure 1. Experimental design. In rats, nociceptive thresholds
were evaluated by the paw pressure vocalization test. Drugs or
saline (.9% NaCl) were administered subcutaneously. The gray
square indicates experimental day with stress session, drug administration or lesion (see Methods). Fentanyl ULD, Fentanyl
ultra-low dose.

Three groups of rats were exposed to NES on D0, D2, and

D7. On D14, all rats were subjected to inflammatory pain.
Two different NMDA receptor antagonists were studied
to evaluate the role of NMDA receptors. The first is ketamine, a well-known drug widely used in humans for preventing exaggerated postoperative pain18 but that may

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12

induce side effects. The second is BN2572, a drug which

has a longer half-life and induces more moderated side
effects than ketamine.19,20,38 A previous study38 has demonstrated that a single BN2572 injection (.3 mg/kg, s.c.)
has the same effectiveness for preventing opioidinduced hyperalgesia than 3 injections of ketamine
(10mg/kg, s.c.). In the ketamine group (n = 8), the rats
received 3 subcutaneous ketamine (10 mg/kg) injections,
30 minutes before and 4.5 and 9.5 hours after the carrageenan injection. In the BN2572 group (n = 8), the rats received only 1 BN2572 injection (.3 mg/kg, s.c.) 30 minutes
before the carrageenan injection, and 2 saline injections
were administered 4.5 and 9.5 hours later. In the saline
group (n = 8), the rats were subjected to 3 saline
injections.

Experiment 5: Effect of NMDA Receptor

Antagonist Administration on Changes in Nociceptive Threshold Induced by fULD Administration in Prestressed Rats
Three groups of rats were used. All rats were subjected to NES on D0, D2, and D7 and were tested on D8.
The first group of rats (n = 8) received an injection of
the NMDA receptor antagonist, BN2572 (.3 mg/kg), 30
minutes before a saline injection. The second group
(n = 8) received a saline injection 30 minutes before
a subcutaneous injection of 50 ng/kg fentanyl. The rats
in the third group (n = 8) received an injection of
BN2572 (.3 mg/kg) 30 minutes before an injection of
50 ng/kg fentanyl.

Statistical Analysis
The data represent the mean 6 SEM. Repeated measures 2-way analyses of variance (ANOVA), with factors
time (within) and group (between) were performed
with Statistica 5.1 (Statsoft, Maisons-Alfort, France).
When ANOVAs showed a significant time effect (P <
.05), Newman Keuls post hoc test was used to assess
the differences of each time point versus basal value
on D0. When ANOVAs showed a significant group effect
and/or a time-group interaction (P < .05), Newman
Keuls post hoc test was used to assess the differences
between groups. Separated 2-way ANOVA were performed on different experimental periods (see experimental design on Fig 1): from D 2 to D0 for checking
the stability of basal nociceptive threshold before the
beginning of experiment; on D0 (1st stress session),
from D1 to D2, on D2 (2nd stress session), from D3 to
D7, on D7 (3rd stress session), on D8 (1st fULD test),
from D9 to D13, on D13 (2nd fULD test), on D14 (inflammation) and from D15 to D34. For comparing amplitude
of stress-induced analgesia on D0, D2, and D7, a repeated
measures 2-way ANOVA was performed followed by
Newman Keuls post hoc test when ANOVA showed a significant time effect.
Analgesic or hyperalgesic indices, represented by the
area under or above the curve, respectively, were calculated for each rat using the trapezoidal method. As previously reported,9 surface areas were calculated by
summing the nociceptive threshold values measured

every day after the planned experimental day according the formula Surface = S(nociceptive threshold at
Dn11)
basal value  (n 1 1). The n value indicates
the number of days with a nociceptive threshold statistically different (1-way ANOVA and Newman Keuls test,
P < .05) from the nociceptive threshold basal value. Hyperalgesic indexes were expressed as the mean percentage (6 SEM) of the reference index (100% denotes
a value that matches that of the control, ie, nonstressed
rats in Experiments 1 and 2 and saline-treated rats in Experiment 4). By contrast, the absolute values were used
in Experiment 3 for linear correlation analysis. Linear
correlation analyses were conducted between the hyperalgesic indices obtained after fULD administration
and after carrageenan administration. One-way ANOVA was used to compare analgesic or hyperalgesic indices following fULD administration, and hyperalgesic
indices following carrageenan injection. If ANOVA revealed a significant group effect, Newman Keuls post
hoc test was used to determine the differences between groups. Differences were considered significant
at P < .05.

Results
Effect of Prior Exposure to Stress on
Changes in Nociceptive Threshold
Induced by Inflammation
The NES induced analgesia that was limited to the time
of stress exposure (Figs 2A and 2C; Newman Keuls test, P
< .05). The amplitude of analgesia decreased with repetition of the stressor (Newman Keuls test, P < .05). No
change in nociceptive threshold was observed in nonstressed rats (1-way ANOVA, P > .05). In nonstressed
rats, an intraplantar carrageenan injection on D14 induced a decrease in nociceptive threshold in inflamed
and noninflamed hind paws for 4 days (Fig 2A; Newman
Keuls test, P < .05) and 2 days (Fig 2C; Newman Keuls test,
P < .05), respectively. In stressed rats, an intraplantar carrageenan injection on D14 (7 days after the last stress) induced a decrease in nociceptive threshold in inflamed
and noninflamed hind paws for 12 days (Fig 2A; Newman
Keuls test, P < .05) and 9 days (Fig 2C; Newman Keuls test,
P < .05), respectively. The hyperalgesic indices were increased 3.7- to 4.5-fold in inflamed and noninflamed
hind paws, respectively, when compared with the nonstressed group (Figs 2A and 2C, Insets; Newman Keuls
test, P < .05).
The FSS induced analgesia that lasted for 1 hour after
stress exposure (Figs 2B and 2D; Newman Keuls test, P <
.05). The amplitude of the analgesia decreased with repetition of the stressor (Newman Keuls test, P < .05). No
change in nociceptive threshold was observed in nonstressed rats (1-way ANOVA, P > .05). When the nonstressed rats received an intraplantar carrageenan
injection on D14, a decrease in the nociceptive thresholds of inflamed and noninflamed hind paws was observed for 4 days (Fig 2B; Newman Keuls test, P < .05)
and 1 day (Fig 2D; Newman Keuls test, P < .05), respectively. In stressed rats, the intraplantar carrageenan

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1073

Figure 2. Effects of prior exposure to stress on changes in the nociceptive threshold induced by inflammation in rats. Nociceptive
threshold changes were evaluated by the paw pressure vocalization test on inflamed (A, B) and noninflamed (C, D) hind paws. Novel
environment stress (A, C) or forced swim stress (B, D) were performed on D0, D2, and D7 in stressed groups. Stress was not performed in
nonstressed groups. On D14, the nonstressed group (B) and the stressed group () received an intraplantar carrageenan injection.
Mean pressure values to trigger vocalization were expressed in grams 6 SEM (n = 8 for each group). *Newman Keuls test, P < .05
for comparison with nonstressed group. Insets indicate postinflammation hyperalgesic indexes calculated (see Methods) using the
variations of the nociceptive threshold of the inflamed or noninflamed in nonstressed groups (white square) and in stressed groups
(black square). $Newman Keuls test, P < .05 for comparison with nonstressed group.

injection on D14 led to decreases in the nociceptive

thresholds of the inflamed and noninflamed hind
paws for 10 days (Fig 2B; Newman Keuls test, P < .05)
and 4 days (Fig 2D; Newman Keuls test, P < .05), respectively. In the stressed group, the hyperalgesic index was
increased 2.9- and 2.5-fold in both inflamed and noninflamed hind paws, respectively, when compared with
the nonstressed group (Figs 2B and 2D, Insets; Newman
Keuls test, P < .05).
In addition, there was no significant difference between the stressed and nonstressed groups with respect
to body weight curve (data not shown) and the diameter of the inflamed hind paw (Table 1; 1-way ANOVA,
P > .05).

Effect of Naltrexone Administration

Immediately Before Each NES Session on
Changes in Nociceptive Threshold
Induced by Inflammation
When injected into nonstressed rats, naltrexone failed
to induce changes in the basal nociceptive threshold and
in the amplitude and duration of postinflammatory hyperalgesia (data not shown). Naltrexone preadministration completely prevented stress-induced analgesia
(Figs 3A and 3B; Newman-Keuls test, P < .05) and abolished the enhancement of postinflammatory hyperalgesia observed in prestressed rats (Fig 3A; Newman-Keuls
test, P < .05). Indeed, the hyperalgesic index associated

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Evaluation of Diameter of Inflamed Hind Paw in the Nonstressed, the 1 Stress, the 2 Stress,
and the 3 Stress Groups in Experiment 3. Diameter was Evaluated 2 Hours, 4 Hours, and 6 Hours
After the Injection of Carrageenan and on the Subsequent Days. All Data are Expressed as Mean 6
SEM.

Table 1.

Non-stressed
1 Stress
2 Stress
3 Stress

D14 BASAL

D14 2H

D14 4H

D14 6H

D16

D18

D20

D22

3.72 6 0.05
3.66 6 0.04
3.78 6 0.05
3.79 6 0.07

9.58 6 0.36
9.58 6 0.33
9.23 6 0.49
9.20 6 0.42

10.33 6 0.14
10.35 6 0.23
10.08 6 0.25
10.46 6 0.36

9.92 6 0.25
9.78 6 0.36
9.78 6 0.24
9.58 6 0.27

6.35 6 0.20
6.38 6 0.28
5.92 6 0.19
5.73 6 0.13

4.95 6 0.11
4.95 6 0.21
4.70 6 0.12
4.66 6 0.12

4.67 6 0.05
4.75 6 0.17
4.45 6 0.10
4.35 6 0.09

4.23 6 0.09
4.26 6 0.08
4.12 6 0.05
3.97 6 0.08

with postinflammatory hyperalgesia was 344 6 44% in

prestressed rats that received saline versus 131 6 18%
in prestressed rats that received naltrexone before each
stress session (Fig 3A, Insets). Similar results were observed regarding the secondary hyperalgesia observed
in the noninflamed hind paw (Fig 3B, Insets).

Comparative Effect of 1 to 3 NES

Exposures on Changes in Nociceptive
Threshold Induced by fULD and
Subsequent Inflammation
Administration of fULD (50 ng/kg; s.c.), was performed
on D8 and D13 in non-stressed rats. In these rats, fULD induced an increase in nociceptive threshold for 30 minutes (Fig 4; Newman Keuls test, P < .05). In contrast,
fULD led to decreases in nociceptive threshold that lasted
for 2.5 hours (Newman Keuls test, P < .05), 3 hours
(Newman Keuls test, P < .05) and 4 hours (Newman Keuls
test, P < .05) in rats previously subjected to 1, 2, or 3 stress
sessions, respectively (Fig 4). No differences in the analgesic and hyperalgesic indices between D8 and D13
were observed (data not shown; 1-way ANOVA, P > .05).
The higher the number of stress sessions presented to
the rats, the greater the amplitude of the NT decrease induced by a fULD (Newman-Keuls test, P < .05).
In nonstressed rats, an intraplantar carrageenan injection on D14 induced a decrease in nociceptive threshold
in inflamed and noninflamed hind paws that lasted for
4 days (Fig 4A; Newman Keuls test, P < .05) and 1 day
(Fig 4B; Newman Keuls test, P < .05), respectively. In prestressed rats, an intraplantar carrageenan injection on
D14 induced a decrease in nociceptive threshold in the inflamed hind paw for 6 days (Newman Keuls test, P < .05),
10 days (Newman Keuls test, P < .05), and 12 days (Newman Keuls test, P < .05), in rats previously subjected to 1,
2, or 3 stress sessions, respectively (Fig 4A). The noninflamed hind paw displayed decreases in nociceptive
threshold that lasted for 3 days (Newman Keuls test,
P < .05), 6 days (Newman Keuls test, P < .05), and 7 days
(Newman Keuls test, P < .05) in rats previously subjected
to 1, 2, or 3 stress sessions, respectively (Fig 4B). In both
inflamed and noninflamed hind paws, the hyperalgesic
index increased with repetition of the stressor (Newman-Keuls test, P < .05).
A correlation analysis showed that the analgesic or hyperalgesic indices post-fULD (day 13) were closely and
positively correlated with hyperalgesic indices

Figure 3. Effect of naltrexone administration immediately before each NES session on changes in the nociceptive threshold
induced by inflammation. Nociceptive threshold changes
were evaluated by the paw pressure vocalization test on inflamed (A) and noninflamed (B) hind paws. Two groups of
rats were subjected to novel environment stress on D0, D2,
and D7 and received a preadministration of naltrexone (1 mg/
kg, s.c.) (A) or saline (>) 30 minutes before each stress. A nonstressed group received similar saline (B) injections on D0, D2,
and D7 but was not exposed to stress session. On D14, all groups
were subjected to inflammatory pain. Mean pressure values to
trigger vocalization were expressed in grams 6 SEM (n = 8 for
each group). *Newman Keuls test, P < .05 for comparison between Naltrexone-stressed group and stressed group. Insets indicate postinflammation hyperalgesic indexes calculated (see
Methods) using the variations of the nociceptive threshold of
the inflamed or noninflamed in the nonstressed group (black
diamond), the stressed group (dashed square), and the
Naltrexone-stressed group (black square). $Newman Keuls
test, P < .05 for comparison with nonstressed group; 1Newman
Keuls test, P < .05 for comparison with stressed group.

Le Roy et al

Figure 4. Comparative effect of 1 to 3 NES exposures on

changes in the nociceptive threshold induced by fentanyl
ultra-low dose and subsequent inflammatory pain in rats. Nociceptive threshold changes were evaluated by the paw pressure
vocalization test on inflamed (A) and noninflamed hind paws.
Novel environment stress (NES) was performed on D7 in the 1
stress group (-). NES was performed on D2 and D7 in the 2 stress
group (:). NES was performed on D0, D2, and D7 in the 3 stress
group (). NES was not performed in the nonstressed group (B).
In all groups, fULD administration (50 ng/kg, s.c) was performed
on D8 and D13. On D14, animals received an intraplantar carrageenan injection. Mean pressure values to trigger vocalization
were expressed in grams 6 SEM (n = 8 for each group). Insets indicate correlation analysis between analgesic or hyperalgesic indexes post-fentanyl 50 ng/kg (day 13) and hyperalgesic indexes
post-carrageenan in both inflamed (A) and noninflamed (B)
hind paws.
determined during the postcarrageenan period (after
D14) in both inflamed (r2 = .84, Fig 4A, Inset) and noninflamed (r2= .78, Fig 4B, Inset) hind paws.

Effect of NMDA Receptor Antagonist

Administration on Changes in
Nociceptive Threshold Induced by
Inflammation in Prestressed Rats
In rats preexposed to stress, an intraplantar carrageenan injection on D14 induced a decrease in nociceptive threshold for 13 days (Fig 5A; Newman Keuls test,
P < .05) and 10 days (Fig 5B; Newman Keuls test, P <
.05) in inflamed and noninflamed hind paws, respectively. When injected 3 times at 5-hour intervals on D14,
ketamine (10 mg/kg) strongly reduced the decrease in
nociceptive threshold associated with carrageenan injection in pre-stressed rats.

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Figure 5. Effect of NMDA receptor antagonist administration

on changes in the nociceptive threshold induced by inflammation in prestressed rats. Nociceptive threshold changes were
evaluated by the paw pressure vocalization test on inflamed
(A) and noninflamed (B) hind paws. In all groups, novel environment stress (NES) was performed on D0, D2, and D7. On
D14, 3 boluses of ketamine (10 mg/kg, s.c., Ketamine group )
or 1 bolus of BN2572 (.3 mg/kg, s.c., BN2572 group ) or saline
(Control group B) were injected in prestressed rats, then an intraplantar carrageenan injection was performed. Mean pressure values to trigger vocalization were expressed in grams 6
SEM (n = 8 for each group). *Newman Keuls test, P < .05 for
comparison with Control group. Ket, ketamine; BN, BN2572. Insets indicate post-inflammation hyperalgesic indexes calculated
(see Methods) using the variations of the nociceptive threshold
of the inflamed or noninflamed in BN2572 group (black
square), in Ketamine group (gray square) and in Control group
(white square). $Newman Keuls test, P < .05 for comparison
with Control group. 1Newman Keuls test, P < .05 for comparison with Ketamine group. Ket, ketamine; BN, BN2572.

In ketamine-treated rats, the decrease in nociceptive

threshold was reduced to 10 days (Fig 5A; Newman Keuls
test, P < .05) and 7 days (Fig 5B; Newman Keuls test, P < .05)
in inflamed and noninflamed hind paws, respectively.
Moreover, in ketamine-treated rats, the hyperalgesic index was reduced to 71 6 12% and 56.5 6 11% in inflamed
and noninflamed hind paws, respectively, compared with
100 6 10% in saline-treated rats (Fig 5A, Inset).
A single BN2572 injection (.3 mg/kg) performed 30
minutes before the carrageenan injection on D14 reduced the NT decrease induced by carrageenan injection
in prestressed rats (Fig 5). In BN2572-treated rats, longlasting hyperalgesia was reduced to 6 days (Fig 5A; Newman Keuls test, P < .05) and 3 days (Fig 5B; Newman Keuls
test, P < .05) in inflamed and noninflamed hind paws,

1076

The Journal of Pain

Figure 6. Effect of NMDA receptor antagonist administration

on changes in the nociceptive threshold induced by fULD administration in prestressed rats. Nociceptive threshold changes were
evaluated by the paw pressure vocalization test. Mean pressure
values to trigger vocalization were expressed in grams 6 SEM.
Novel environment stress (NES) was performed on D0, D2, and
D7 in all groups. In the Stressed/BN2572/fULD group (A),
BN2572 (.3 mg/kg, s.c.) was injected 30 minutes before fULD
(50 ng/kg, s.c.) administration on D8. In the Stressed/Saline/
fULD group (>), saline administration was performed 30 minutes before the fULD administration (50 ng/kg, s.c). In stressed/
BN2572/Saline group (), rats were injected with BN2572
(.3 mg/kg, s.c.) 30 minutes before saline administration. Mean
pressure values to trigger vocalization were expressed in grams
6 SEM (n = 8 for each group). fULD, fentanyl ultra-low dose
(50 ng/kg) *Newman Keuls test, P < .05 for comparison with nonstressed group.
respectively. Moreover, in BN2572-treated rats, the hyperalgesic index was reduced to 34 6 5% (Fig 5A, Inset)
and 18.5 6 6% (Fig 5B, Inset) in inflamed and noninflamed hind paws, respectively, compared with 100 6
10% in saline-treated rats.

Effect of NMDA Receptor Antagonist

Administration on Changes in
Nociceptive Threshold Induced by fULD
Administration in Prestressed Rats
In prestressed rats, the administration of BN2572 alone
on D8 did not evoke any change in nociceptive threshold
(Fig 6, 1-way ANOVA, P > .05). When injected 30 minutes
before fULD administration, BN2572 completely prevented the decrease in nociceptive threshold induced
by fULD (Fig 6; Newman Keuls test, P < .05).

Discussion
The main finding of this study is that endogenous opioids, which are released during non-nociceptive environmental stress (NNES) and induce acute analgesia,
secondarily induce a long-lasting and latent pain hypersensitivity that leads to an exaggerated pain in response
to further noxious stimulation. Interestingly, the higher
the number of stress sessions presented to the rats, the

Opioid-Dependent Stress and Pain Vulnerability

greater were the amplitudes of the inflammatory hyperalgesia.
It could be paradoxical that stress, which is associated
with an endogenous opioid release, leads to pain hypersensitivity. Of note is the observation that 2 different types
of stress, such as novel environment or forced swim stress,
provoked similar exaggerated inflammatory hyperalgesia. Although numerous endogenous molecules released
by stress, such as glucocorticoids or biogenic amines,
may participate in neuroplastic changes leading to latent
pain hypersensitivity, our study shows that the blockade
of opioid receptors by naltrexone is sufficient to completely prevent exaggerated inflammatory hyperalgesia.
However, though this study demonstrates the critical
role of opioid receptors in the development of pain vulnerability, it has been reported in mice that exogenous
opioid administration induces thermal hyperalgesia independently of prior or concurrent opioid receptor activation or analgesia.24,25,46 Such discrepancies could be
explained by putative differences in endogenous and
exogenous opioid effects on the CNS. Endogenous
opioids would expected to act only at opioid receptors
activated at specific synapses recruited by natural
effectors as environmental stress and not at all opioid
receptors in the CNS, as the case with exogenous opioids.
In the absence of changes in basal nociceptive threshold values in prestressed rats, our results suggest that
the release of endogenous opioids by NNES triggered
a series of neuroplastic changes that led to pain hypersensitivity manifested only in response to a serious tissue
injury. This phenomenon is referred to as latent pain sensitization.4,38 In previous animal experiments, opioiddependent stress has been reported to cause a progressive
and sustained decrease in baseline nociceptive threshold
that persists for several days after stress sessions.21,35,43
The pain hypersensitivity observed in the present study
differs from these data because pain hypersensitivity
manifested only as an enhancement of hyperalgesia
evoked by acute inflammation. Similar data have been
reported in a recent study showing that rats exposed
to unpredictable sound stress exhibit no changes in
mechanical nociceptive threshold after stress but show
a marked increase in hyperalgesia evoked by local
injections of prostaglandins E2 or epinephrine performed several days later.27 Our study indicates that endogenous opioids have effects that are not limited to
the benefits of analgesia but also induce adverse outcomes such as latent pain hypersensitivity. Moreover, indication of individual hyperalgesia levels for each rat
shows that the pain level induced by a tissue injury is
not only the result of nociceptive input level but is also
dependent on individual history, in which the level of
prior life stress events plays a critical role. This could explain some individual differences in pain sensitivity to
acute tissue injury.
The results of the present study show that endogenous opioids released during stress induced biphasic effects: an immediate analgesia, SIA as previously
described, followed by a latent hypersensitivity to noxious stimuli. These results have to be compared to effects
of exogenous opioids. Although exogenous opioids are

Le Roy et al
the analgesic of choice for the treatment of moderateto-severe pain, we and others8,9,28,32 have previously
reported that in animals a long-lasting hyperalgesia
and a latent pain hypersensitivity are observed after analgesia. Clinical studies have also reported that the exogenous opioids used for surgery can unexpectedly
facilitate postoperative hyperalgesia and allodynia after
analgesia.2,17 Consistent with the effects of exogenous
opioids,2,9,32 our results show that the acute and
delayed effects of endogenous opioids are also
opposite in nature.
Interestingly, pain hypersensitivity was not limited to
the injured hind paw, because we also observed an enhancement of secondary hyperalgesia in the contralateral noninflamed hind paw in prestressed rats. Altered
sensitivity in contralateral structures has been observed
in many animal models of pain, especially inflammatory
pain.41 In the absence of differences in hind paw inflammation in prestressed versus nonstressed rats, these results suggest that the latent pain hypersensitivity
induced by NNES is primarily derived from a central origin. Experimental investigations in animals have demonstrated that opioid-induced hyperalgesia may result
from tonic activation of a descending pain facilitatory
pathway in which the rostroventromedial medulla plays
a critical role.45 Such descending facilitation may influence spinal cord networks involved in the expression of
paradoxical pain.
From a mechanistic viewpoint, experimental studies indicate that administration of NMDA receptor antagonists
totally prevented the enhancement of both inflammatory
or surgical hyperalgesia induced by fentanyl in rats.36,39
This suggests that prior exposure to exogenous opioids
has facilitated the development of NMDA receptormediated central changes in synaptic excitability leading
to exaggerated hyperalgesia following tissue injuries.7,8,39
In the present study, the exaggerated inflammatory
hyperalgesia observed in prestressed rats was suppressed
when NMDA receptor antagonists were administered
immediately prior to inflammation. Indeed, a single
BN2572 injection is more effective than 3 ketamine
injections, suggesting that this compound is a better
drug for blocking the activation of pronociceptive
systems. Since we previously reported that NMDA
receptor antagonists did not affect inflammatory
hyperalgesia in nonstressed rats,39 these results suggest
a critical role for NMDA receptor systems in exaggerated
inflammatory hyperalgesia induced by prior stress. It is
well known that m-opioid receptor stimulation by exogenous opioids triggers the activation of NMDA receptors
by reducing Mg21 blockade via the activation of intracellular protein kinase C10,11 leading to hyperalgesia.14,31 This
mechanism may also explain the development of pain
hypersensitivity induced by endogenous opioids.
However, the administration of an NMDA receptor
antagonist in prestressed rats that were returned to
basal nociceptive thresholds after stress-induced analgesia
had no effect per se on basal nociceptive threshold. This
result indicates that pain hypersensitivity, as revealed by
exaggerated inflammatory hyperalgesia, is not associated
with excessive basal activity at the NMDA synaptic cleft. In-

The Journal of Pain

1077

deed, the NMDA receptor system appears to function only

as a trigger able to reactivate memory systems, which by
increasing gain, elicit abnormal pain hypersensitivity.29,47
Activation of NMDA-dependent pronociceptive systems
may be induced by both endogenous and exogenous opioids as previously proposed.10,11 Pharmacological and
transcriptomic approaches are currently in progress to
identify neural, neural-glial, neuroendocrine, and immune systems that could be involved in a cascade of cellular events leading to pain hypersensitivity.
From a clinical viewpoint, an important clinical challenge is to identify patients at risk for developing
pain hypersensitivity and to propose appropriate treatments.26,33,34 Our results indicated that nonstressed
and prestressed rats showed opposite fULD responses,
analgesia versus hyperalgesia, respectively. It is noteworthy that the higher the number of stress sessions
presented to the rats, the greater was the increase in
the severity of the fULD-induced hyperalgesia. Therefore, under our experimental conditions, it is possible
to discriminate between prestressed and nonstressed
animals according to their hyperalgesic or analgesic responses levels to the fULD test. Moreover, the amplitude
of the pharmacological response to fULD administration
was closely and positively correlated with the magnitude
of long-lasting inflammatory hyperalgesia. This result indicates that it is possible, in laboratory rats, to predict the
level of latent pain hypersensitivity, ie, pain vulnerability,
according to individual animal histories, particularly
stressful environmental events. It is noteworthy that
both the fULD-induced hyperalgesia and the exaggerated inflammatory hyperalgesia observed in prestressed
rats were completely prevented by the preadministration of an NMDA receptor antagonist, suggesting common mechanisms.
From a pathophysiological viewpoint, this study provides some preclinical evidence that pain level is not
only the result of nociceptive input level but is also dependent on the individual history, especially prior life
stress events associated with endogenous opioid release.
Given that postoperative pain is one of the most frequently reported pains,13 our preclinical results suggest
that some hyperalgesia syndromes, as exaggerated postoperative pain, may be partly due to latent pain sensitization induced by prior stress events.
The preclinical model of latent pain sensitization induced by NNES developed in this study could be
a good tool for evaluating therapeutic strategies for reducing pain hypersensitivity induced by earlier life
events in individuals. Since it has been recently reported
that anxiolytic drugs as benzodiazepines did not reverse
pain hypersensitivity in animals subjected to chronic
social defeat,37 new therapeutic strategies have to be
developed for specifically blocking pronociceptive systems. Based on our results showing that endogenous
opioid released by NNES play a critical role in the development of latent pain hypersensitivity, we hypothetize
that NMDA pronociceptive systems activated by opioid
receptor stimulation8,11,32 could be good targets for
reducing latent pain sensitization. However, though
our study indicates that NMDA receptor antagonists

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The Journal of Pain

Opioid-Dependent Stress and Pain Vulnerability

could be candidates, their chronic use induce

undesirable side effects. Anti-opioid systems, such as dynorphin, chloecystokinin, and neuropeptide FF might
be good targets because they are involved in opioidinduced hyperalgesia.42,44,48 Recently, it has been
reported that cholecystokinin receptor antagonists
abolish anxiety-induced hyperalgesia in rats.37 Moreover,
polyamine deficient diets, which prevent hyperalgesia induced by endogenous opioid release in pain- and opioid-

experienced rats,40 may provide an useful strategy. Studies are in progress to evaluate these putative therapeutic
approaches.

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