Endogenous Opioids Released Du

Environmental Stress Induce Laten

a NMDA-Dependent Process

G

de

re

1 to

Oinduced analgesia.1 However, consistent with

for example, as postoperative pain.22 In preclinical

at re-ressful

situations can elicit a persistent increase in nocicep-

sensitivity leads to exaggerated pain and enhancement

events that occur before or after to acute tissue injuriesTeam Homeostasie-Allostasie-Pathologie-Rehabilitation, Universite

The Journal of Pain, Vol 12, No 10 (October), 2011: pp 1069-1079Available online at www.sciencedirect.comemeof medical care. Neural mechanisms by which prior stressmay induce pain aggravation following acute tissueinjury have to be better understood for identifying pa-tients at risk for developing exaggerated pain followinga tissue injury.26 Indeed, clinical studies to date havefailed to distinguish the significant role of stressful

ReceivedOctober 7, 2010; Revised April 21, 2011; AcceptedApril 30, 2011.Supported by Universite Victor Segalen Bordeaux 2, Universite Bordeaux1, theMinistere de lEducationNationale, de lenseignement superieur etde la Recherche and the Centre National de la Recherche Scientifique(CNRS). C. Le Roy has a fellowship from the Ministere de la Rechercheet de lEnseignement Superieur.Address reprint requests to Pr Guy Simonnet, Laboratory CNRSUMR5287,the biopsychosocial conceptual framework,15 there isa compelling body of evidence in humans that unman-aged negative emotion associated with stressful eventsis a psychological predictor of exaggerated acute pain;

tion.21,35,37,43 Pain hypersensitivity induced by stress isa lesser-known phenomenon than stress-induced anal-gesia, despite the fact it is much more relevant fromthe clinical and therapeutic viewpoints since pain hyper-Bordeaudeaux Ce

1526-590

2011 bdoi:10.10tion (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 ver-

sus 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 hyperal-

gesic indices were positively correlated with inflammatory hyperalgesic indices (r2 = .84). In pre-

stressed rats, fULD-induced hyperalgesia and the exaggerated inflammatory hyperalgesia were

prevented NMDA receptor antagonists. This study provides some preclinical evidence that pain inten-

sity 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 whetherprior 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 hypersensi-

tivity.

2011 by the American Pain SocietyKey words: Stress, pain sensitization, hyperalgesia, endogenous opioids, NMDA receptors.

pioid-dependent stress decreases acute pain sen-sitivity, a phenomenon referred to as stress-

models, there is a growing body of evidence thpeated exposure of animals to non-nociceptive stnon-nociceptive environmental stress, on inflammatory hyperalgesia induced by a carrageenan injec-Chloe Le Roy, Emilie Laboureyras, StephanieJean-Paul Laulin, and Guy SimonnetUniversite Bordeaux, INCIA, CNRS UMR 5287, F-33076 Bor

Abstract: Although stress induces analgesia, thepain syndromes. Here, we studied the effects ofx 2 Zone Nord Bat 4A 3 etage, 146 rue Leo Saignat, 33076 Bor-dex, France. E-mail: gsimonnet@yahoo.com

0/$36.00

y the American Pain Society

16/j.jpain.2011.04.011ring Non-Nociceptive

t Pain Sensitization Via

avello-Baudy, Jeremy Chateauraynaud,

aux, France.

is evidence that stressful events may exacerbate

3 prestressful events (days 0, 2, and 7), such asin the development of exaggerated pain. Preclinical ani-mal studies have the advantage of allowing one to selec-tively evaluate the role of prestressful events in painhypersensitivity.

1069

exposure to various exogenous opioids, like heroin,309 5

NMDA receptor antagonist19 with a longer half-life

mined using a modified Randall-Selitto method as previ-

The NES consisted of exposing animals to a novel envi-38fentanyl, and remifentanil, paradoxically induces hy-peralgesia for several days following analgesia in naverats and strongly enhances postoperative pain in bothanimals and humans.2,3,6,17,23

The main purpose of this study is to gain a better un-derstanding of the mechanisms and pathophysiologicalrelevance of changes in pain sensitivity induced by priornon-nociceptive environmental stress (NNES). We choseto examine inflammatory pain because it is a criticalcomponent in various pain syndromes and the evolutionof carrageenan-induced inflammatory pain may be com-pared with the time-course of postoperative pain.16 Ahind paw inflammation was performed several daysafter 1 to 3 exposures to events of NNES. The role of en-dogenous opioids released during NNES in the develop-ment of changes in pain sensitivity was examined byblocking opioid receptors with naltrexone before eachNNES.In addition, we asked whether it was possible to de-

tect pain vulnerability in prestressed rats, because itwould be of clinical interest to develop tests for identify-ing patients at risk for developing exaggerated pain fol-lowing a tissue injury.26 Interestingly, the administrationof an ultra-low dose of fentanyl (fULD, 50 ng/kg) in ratspreexposed to opioids for relieving prior pain inducesparadoxical hyperalgesia, but not the analgesia ob-served in rats without prior pain or opioid exposures.38

This finding suggests that behavioral responses toa fULD administration may be used as a pharmacologicaltest to detect pain vulnerability (latent pain hypersensi-tivity) and then to predict exaggerated pain response tofurther noxious stimuli. Here, we evaluated whethera single fULD administration induced analgesia or hyper-algesia in rats preexposed to 1 to 3 NNES. Relationshipsbetween the amplitude of the fULD response (ie, analge-sia or hyperalgesia) and the amplitude of the inflamma-tory hyperalgesia were analyzed in prestressed andnonstressed rats.Weandothers9,32 havepreviously reported thatNMDA

receptor systems play a critical role in the development ofpain hypersensitivity. Therefore, we examined the abilityof 2NMDA receptor (NMDAR) antagonists, ketamine andBN2572, to affect changes in pain sensitivity induced byprior NNES in rats.

Methods

AnimalsExperiments were performed using adult male

Sprague-Dawley rats (Charles River Laboratories, LAr-bresle, France) weighing 200 to 225 g before experi-Although numerous endogenous molecules releasedby stress, such as glucocorticoids or biogenic amines,may possibly participate in neuroplastic changes leadingto latent pain hypersensitivity, the possible role of en-dogenous opioids released by stress is poorly under-stood. Indeed, previous reports show that a single

1070 The Journal of Painments. Rats were housed in groups of 4 animals percage with a 12-hour light/dark cycle (lights on at 7:00ronment for 1 hour, as previously described. Rats wereplaced in a new experimental room, in new cages withfresh litter, and were exposed to a light (350 lux) placed2meters away from the rat cages. At the end of the stresssession, rats were returned to their home cage in theusual experimental room. Nonstressed animals werekept in their home cages.

The Forced Swim Stress (FSS)

The FSS consisted of placing animals in plastic cylinders(diameter, 30 cm; height, 50 cm) containing water (2426C) with a depth of 20 cm for 20 minutes as previouslyously described:9 a paw pressure vocalization test inwhich a constantly increasing pressure is applied to thehind paw until the rat squeaks. The Basile analgesimeter(Bioseb, Chaville, France; stylus tip diameter, 1 mm) wasusedwith a 600-g cut-off value to prevent tissue damage.In all experiments, nociceptive thresholds were evalu-ated in both hind paws.

Non-Nociceptive Environmental Stress(NNES)Two types of NNES were used.

The Novel Environment Stress (NES)and inducing more moderated side effects than ket-amine.20,38 This drug was obtained from BEAUFOUR-IPSEN (Les Ulis, France).Fentanyl (50 ng/kg), ketamine (10 mg/kg), BN2572

(.3 mg/kg), and naltrexone (1 mg/kg) were administeredsubcutaneously (s.c.; 1 mL/kg body weight). Control ani-mals received an equal volume of saline injections.

Measurement of the NociceptiveThresholdNociceptive thresholds in handled rats were deter-AM) and at a constant room temperature of 23 62C. Food and water were available ad libitum. All ex-periments were performed during the light period.Pharmacological tests and animal care were conductedin accordance with the guidelines of the Committeefor Research and Ethical Issues of IASP49 and the localEthics Committee For Animal Experimentation at Aqui-taine and Poitou-Charentes (France). These experi-ments were conducted in an authorized laboratoryand under the supervision of a certified researcher,Emilie Laboureyras.

DrugsFentanyl citrate, ketamine hydrochloride, BN2572, nal-

trexone, and carrageenan l (Sigma-Aldrich, Saint-Quentin Fallavier, France) were dissolved in physiologicalsaline (.9%). BN2572, the gacyclidine enantiomer (-)-(1S-2R)-1-[1-(2-thienyl)-2-methylcyclohexyl] piperidine, is an

Opioid-Dependent Stress and Pain Vulnerabilitydescribed.43 The water was changed and the containerthoroughly cleaned between each swimming session.

At the end of the stress session, rats were returned totheir home cages. Nonstressed animals were maintainedin their home cages.Two types of environmental stress were used in the

first part of this study (Experiment 1) to be sure thatchanges in pain sensitivity was not specifically associatedwith 1 type of stress, especially the NES mainly used inthis study.

Inflammatory Pain ModelOn day 14 (D14), rats were placed in a plastic cage and

then anaesthetized with 3% halothane for 3 minutes.

were performed. For the novel environment stress

Le Roy et alFigure 1. Experimental design. In rats, nociceptive thresholdswere evaluated by the paw pressure vocalization test. Drugs orsaline (.9% NaCl) were administered subcutaneously. The gray(NES), nociceptive threshold was measured 30 minutesafter the beginning of each stress period and everyhour for 4 hours during the poststress period. For theCarrageenan (.2 mL of a 1% carrageenan solution in sa-line) was then subcutaneously injected into the lefthind paw with a 25-gauge needle.

Experimental ProcedureFollowing arrival at the laboratory, the animals were

randomly assigned to the different experimental groupsand acclimatized to the animal care unit for 4 days. Toavoid experimental perturbations that may have af-fected the measurement of nociceptive threshold, theexperiments were performed by the same experimenterunder quiet conditions in a testing room close to the an-imal care unit. For 8 days prior to the experiments, theanimals were placed in the test room for 2 hours dailyfor acclimatization. In this test room, the animals wereweighed daily and gently handled for 5 minutes. All ex-periments were performed on groups of 8 animals eachduring the light cycle. Experimental design is shown inFig 1. Nociceptive threshold assessments were performedfor 2 days (ie, on D2 and D1) preceding the experimen-tal day (D0) and were repeated on the experimental dayimmediately before the first stress event (D0). Experi-ments were only initiated when there were no statisticalchanges in the basal nociceptive threshold for 3 succes-sive days (D2, D1 and D0, 1-way ANOVA, P > .05). Thereference value of the nociceptive threshold was se-lected as the basal value onD0. The experimenterwas un-aware of the treatment used. On D0, D2 and D7, NNESsquare indicates experimental day with stress session, drug ad-ministration or lesion (see Methods). Fentanyl ULD, Fentanylultra-low dose.forced swim stress test (FSS), nociceptive threshold mea-surements were performed at the end of the stress pe-riod (20 minutes after the beginning of the stressevent) and every 30 minutes for 2 hours during the post-stress period.For the fULD test, nociceptive thresholdmeasurements

were performed every 30 minutes for 4 hours followinga subcutaneous injection of 50 ng/kg of fentanyl on D8and D13.On D14, nociceptive thresholds were measured 2, 4,

and 6 hours after a carrageenan injection and once dailyfor 12 to 18 days. The diameter of the inflamed hind pawwas evaluated daily with a caliper rule.

Experiment 1: Effect of Prior Exposure toStress on Changes in Nociceptive ThresholdInduced by Inflammation

Stressed rats (n = 8) were exposed to NES or FSS on D0,D2, and D7. Nonstressed rats (n = 8) were not exposed tostress sessions. All rats were subjected to inflammatorypain on D14.

Experiment 2: Effect of Naltrexone Adminis-tration Immediately Before Each NES Sessionon Changes in Nociceptive Threshold Inducedby Inflammation

Two groups of rats (n = 8 for each group) were sub-jected to NES on D0, D2, and D7 and were preadminis-tered naltrexone (1mg/kg, s.c.) or saline 30 minutesbefore each stress. The nonstressed group (n = 8) re-ceived similar saline injections on D0, D2, and D7 but noexposure to stress. On D14, all groups were subjected toinflammatory pain.

Experiment 3: Comparative Effect of 1 to 3NESExposures on Changes in Nociceptive Thresh-old Induced by fULD and Subsequent Inflam-matory Pain

Four groups of ratswere used in this experiment. In thefirst group (n = 8), the rats were not subjected toNES. Thesecond group (n = 8) was subjected to 1 stress session onD7. The third group (n = 8) was subjected to 2 stress ses-sions on D2 and D7, and the fourth group (n = 8) was sub-jected to 3 stress sessions on D0, D2, and D7. All ratsreceived a subcutaneous injection of 50 ng/kg fentanylon D8 and D13 and were subjected to inflammatorypain on D14.

Experiment 4: Effect of NMDA Receptor An-tagonist Administration on Changes in Noci-ceptive Threshold Induced by Inflammationin Prestressed Rats

Threegroupsof ratswere exposed toNESonD0,D2, andD7. On D14, all rats were subjected to inflammatory pain.Two different NMDA receptor antagonists were studiedto evaluate the role of NMDA receptors. The first is ket-

The Journal of Pain 1071amine, awell-knowndrugwidely used in humans for pre-venting exaggerated postoperative pain18 but that may

a subcutaneous injection of 50 ng/kg fentanyl. The rats

rats, an intraplantar carrageenan injection on D14 in-in the third group (n = 8) received an injection ofBN2572 (.3 mg/kg) 30 minutes before an injection of50 ng/kg fentanyl.

Statistical AnalysisThe data represent the mean 6 SEM. Repeated mea-

sures 2-way analyses of variance (ANOVA), with factorstime (within) and group (between) were performedwith Statistica 5.1 (Statsoft, Maisons-Alfort, France).When ANOVAs showed a significant time effect (P .05). In nonstressed

Opioid-Dependent Stress and Pain Vulnerabilityand 1 day (Fig 2D; Newman Keuls test, P < .05), respec-tively. In stressed rats, the intraplantar carrageenan

Le Roy et alinjection on D14 led to decreases in the nociceptivethresholds of the inflamed and noninflamed hindpaws for 10 days (Fig 2B; Newman Keuls test, P < .05)and 4 days (Fig 2D; Newman Keuls test, P < .05), respec-tively. In the stressed group, the hyperalgesic index wasincreased 2.9- and 2.5-fold in both inflamed and nonin-flamed hind paws, respectively, when compared withthe nonstressed group (Figs 2B and 2D, Insets; NewmanKeuls test, P < .05).In addition, there was no significant difference be-

tween the stressed and nonstressed groups with respectto body weight curve (data not shown) and the diame-ter of the inflamed hind paw (Table 1; 1-way ANOVA,P > .05).

Figure 2. Effects of prior exposure to stress on changes in the nocthreshold changes were evaluated by the paw pressure vocalizationenvironment stress (A, C) or forced swim stress (B, D)were performednonstressed groups. On D14, the nonstressed group (B) and the strMean pressure values to trigger vocalization were expressed in grafor comparison with nonstressed group. Insets indicate postinflammvariations of the nociceptive threshold of the inflamed or noninflam(black square). $Newman Keuls test, P < .05 for comparison with noThe Journal of Pain 1073Effect of Naltrexone AdministrationImmediately Before Each NES Session onChanges in Nociceptive ThresholdInduced by InflammationWhen injected into nonstressed rats, naltrexone failed

to induce changes in the basal nociceptive threshold andin the amplitude and duration of postinflammatory hy-peralgesia (data not shown). Naltrexone preadministra-tion completely prevented stress-induced analgesia(Figs 3A and 3B; Newman-Keuls test, P < .05) and abol-ished the enhancement of postinflammatory hyperalge-sia observed in prestressed rats (Fig 3A; Newman-Keulstest, P < .05). Indeed, the hyperalgesic index associated

iceptive threshold induced by inflammation in rats. Nociceptivetest on inflamed (A, B) and noninflamed (C, D) hind paws. Novelon D0, D2, and D7 in stressed groups. Stress was not performed inessed group () received an intraplantar carrageenan injection.ms 6 SEM (n = 8 for each group). *Newman Keuls test, P < .05ation hyperalgesic indexes calculated (see Methods) using theed in nonstressed groups (white square) and in stressed groupsnstressed group.

duced by a fULD (Newman-Keuls test, P < .05).

Paw in the Nonstressed, the 1 Stress, the 2 Stress,ter was Evaluated 2 Hours, 4 Hours, and 6 Hoursbsequent Days. All Data are Expressed as Mean6

14 6H D16 D18 D20 D22

6 0.25 6.35 6 0.20 4.95 6 0.11 4.67 6 0.05 4.23 6 0.09

6 0.36 6.38 6 0.28 4.95 6 0.21 4.75 6 0.17 4.26 6 0.08

6 0.24 5.92 6 0.19 4.70 6 0.12 4.45 6 0.10 4.12 6 0.05

6 0.27 5.73 6 0.13 4.66 6 0.12 4.35 6 0.09 3.97 6 0.08

Figure 3. Effect of naltrexone administration immediately be-fore each NES session on changes in the nociceptive threshold

Opioid-Dependent Stress and Pain VulnerabilityIn nonstressed rats, an intraplantar carrageenan injec-tion on D14 induced a decrease in nociceptive thresholdin inflamed and noninflamed hind paws that lasted for4 days (Fig 4A; Newman Keuls test, P < .05) and 1 day(Fig 4B; Newman Keuls test, P < .05), respectively. In pre-stressed rats, an intraplantar carrageenan injection onD14 induced a decrease in nociceptive threshold in the in-with postinflammatory hyperalgesia was 344 6 44% inprestressed rats that received saline versus 131 6 18%in prestressed rats that received naltrexone before eachstress session (Fig 3A, Insets). Similar results were ob-served regarding the secondary hyperalgesia observedin the noninflamed hind paw (Fig 3B, Insets).

Comparative Effect of 1 to 3 NESExposures on Changes in NociceptiveThreshold Induced by fULD andSubsequent InflammationAdministration of fULD (50 ng/kg; s.c.), was performed

on D8 and D13 in non-stressed rats. In these rats, fULD in-duced an increase in nociceptive threshold for 30 min-utes (Fig 4; Newman Keuls test, P < .05). In contrast,fULD led to decreases in nociceptive threshold that lastedfor 2.5 hours (Newman Keuls test, P < .05), 3 hours(Newman Keuls test, P < .05) and 4 hours (Newman Keulstest, P < .05) in rats previously subjected to 1, 2, or 3 stresssessions, respectively (Fig 4). No differences in the anal-gesic and hyperalgesic indices between D8 and D13were observed (data not shown; 1-way ANOVA, P > .05).The higher the number of stress sessions presented tothe rats, the greater the amplitude of the NT decrease in-

Table1. Evaluation of Diameter of Inflamed Hindand the 3 Stress Groups in Experiment 3. DiameAfter the Injection of Carrageenan and on the SuSEM.

D14 BASAL D14 2H D14 4H D

Non-stressed 3.72 6 0.05 9.58 6 0.36 10.33 6 0.14 9.92

1 Stress 3.66 6 0.04 9.58 6 0.33 10.35 6 0.23 9.78

2 Stress 3.78 6 0.05 9.23 6 0.49 10.08 6 0.25 9.78

3 Stress 3.79 6 0.07 9.20 6 0.42 10.46 6 0.36 9.58

1074 The Journal of Painflamed hind paw for 6 days (Newman Keuls test, P < .05),10 days (Newman Keuls test, P < .05), and 12 days (New-man Keuls test, P < .05), in rats previously subjected to 1,2, or 3 stress sessions, respectively (Fig 4A). The nonin-flamed hind paw displayed decreases in nociceptivethreshold 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 subjectedto 1, 2, or 3 stress sessions, respectively (Fig 4B). In bothinflamed and noninflamed hind paws, the hyperalgesicindex increased with repetition of the stressor (New-man-Keuls test, P < .05).A correlation analysis showed that the analgesic or hy-

peralgesic indices post-fULD (day 13) were closely andpositively correlated with hyperalgesic indices

induced by inflammation. Nociceptive threshold changeswere evaluated by the paw pressure vocalization test on in-flamed (A) and noninflamed (B) hind paws. Two groups ofrats 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 non-stressed group received similar saline (B) injections on D0, D2,and D7 but was not exposed to stress session. On D14, all groupswere subjected to inflammatory pain. Mean pressure values totrigger vocalization were expressed in grams 6 SEM (n = 8 foreach group). *Newman Keuls test, P < .05 for comparison be-tween Naltrexone-stressed group and stressed group. Insets in-dicate postinflammation hyperalgesic indexes calculated (seeMethods) using the variations of the nociceptive threshold ofthe inflamed or noninflamed in the nonstressed group (blackdiamond), the stressed group (dashed square), and theNaltrexone-stressed group (black square). $Newman Keulstest, P < .05 for comparison with nonstressed group; 1NewmanKeuls test, P < .05 for comparison with stressed group.

Le Roy et aldetermined during the postcarrageenan period (afterD14) in both inflamed (r

2 = .84, Fig 4A, Inset) and nonin-flamed (r2= .78, Fig 4B, Inset) hind paws.

Effect of NMDA Receptor AntagonistAdministration on Changes inNociceptive Threshold Induced byInflammation in Prestressed RatsIn rats preexposed to stress, an intraplantar carra-

geenan injection on D14 induced a decrease in nocicep-tive threshold for 13 days (Fig 5A; Newman Keuls test,P < .05) and 10 days (Fig 5B; Newman Keuls test, P .05). When injected 30 minutesbefore fULD administration, BN2572 completely pre-vented the decrease in nociceptive threshold inducedby fULD (Fig 6; Newman Keuls test, P < .05).

DiscussionThe main finding of this study is that endogenous opi-

oids, which are released during non-nociceptive environ-mental stress (NNES) and induce acute analgesia,secondarily induce a long-lasting and latent pain hyper-sensitivity that leads to an exaggerated pain in responseto further noxious stimulation. Interestingly, the higherthe number of stress sessions presented to the rats, the

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 min-utes 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. Meanpressure values to trigger vocalization were expressed in grams6 SEM (n = 8 for each group). fULD, fentanyl ultra-low dose(50 ng/kg) *NewmanKeuls test, P< .05 for comparisonwith non-stressed group.greater were the amplitudes of the inflammatory hyper-algesia.It could be paradoxical that stress, which is associated

with an endogenous opioid release, leads to pain hyper-sensitivity.Ofnote is theobservation that2different typesof stress, such as novel environment or forced swim stress,provoked similar exaggerated inflammatory hyperalge-sia. Although numerous endogenous molecules releasedby stress, such as glucocorticoids or biogenic amines,may participate in neuroplastic changes leading to latentpain hypersensitivity, our study shows that the blockadeof opioid receptors by naltrexone is sufficient to com-pletely prevent exaggerated inflammatory hyperalgesia.However, though this study demonstrates the criticalrole of opioid receptors in the development of pain vul-nerability, it has been reported in mice that exogenousopioid administration induces thermal hyperalgesia inde-pendently of prior or concurrent opioid receptor activa-tion or analgesia.24,25,46 Such discrepancies could beexplained by putative differences in endogenous andexogenous opioid effects on the CNS. Endogenousopioids would expected to act only at opioid receptorsactivated at specific synapses recruited by naturaleffectors as environmental stress and not at all opioidreceptors in the CNS, as the case with exogenous opioids.In the absence of changes in basal nociceptive thresh-

old values in prestressed rats, our results suggest thatthe release of endogenous opioids by NNES triggereda series ofneuroplastic changes that led topainhypersen-sitivity manifested only in response to a serious tissueinjury. This phenomenon is referred to as latent pain sen-sitization.4,38 In previous animal experiments, opioid-dependent stresshasbeenreported tocauseaprogressiveand sustained decrease in baseline nociceptive thresholdthat persists for several days after stress sessions.21,35,43

The pain hypersensitivity observed in the present studydiffers from these data because pain hypersensitivitymanifested only as an enhancement of hyperalgesiaevoked by acute inflammation. Similar data have beenreported in a recent study showing that rats exposedto unpredictable sound stress exhibit no changes inmechanical nociceptive threshold after stress but showa marked increase in hyperalgesia evoked by localinjections of prostaglandins E2 or epinephrine per-formed several days later.27 Our study indicates that en-dogenous opioids have effects that are not limited tothe benefits of analgesia but also induce adverse out-comes such as latent pain hypersensitivity. Moreover, in-dication of individual hyperalgesia levels for each ratshows that the pain level induced by a tissue injury isnot only the result of nociceptive input level but is alsodependent on individual history, in which the level ofprior life stress events plays a critical role. This could ex-plain some individual differences in pain sensitivity toacute tissue injury.The results of the present study show that endoge-

nous opioids released during stress induced biphasic ef-fects: an immediate analgesia, SIA as previouslydescribed, followed by a latent hypersensitivity to nox-

Opioid-Dependent Stress and Pain Vulnerabilityious stimuli. These results have to be compared to effectsof exogenous opioids. Although exogenous opioids are

the analgesic of choice for the treatment of moderate-to-severe pain, we and others8,9,28,32 have previouslyreported that in animals a long-lasting hyperalgesiaand a latent pain hypersensitivity are observed after an-algesia. Clinical studies have also reported that the exog-enous opioids used for surgery can unexpectedlyfacilitate postoperative hyperalgesia and allodynia afteranalgesia.2,17 Consistent with the effects of exogenousopioids,2,9,32 our results show that the acute anddelayed effects of endogenous opioids are alsoopposite in nature.Interestingly, pain hypersensitivity was not limited to

the injured hind paw, because we also observed an en-hancement of secondary hyperalgesia in the contralat-eral noninflamed hind paw in prestressed rats. Alteredsensitivity in contralateral structures has been observedin many animal models of pain, especially inflammatorypain.41 In the absence of differences in hind paw inflam-mation in prestressed versus nonstressed rats, these re-sults suggest that the latent pain hypersensitivityinduced by NNES is primarily derived from a central ori-gin. Experimental investigations in animals have demon-strated that opioid-induced hyperalgesia may resultfrom tonic activation of a descending pain facilitatorypathway in which the rostroventromedial medulla playsa critical role.45 Such descending facilitation may influ-ence spinal cord networks involved in the expression ofparadoxical pain.From a mechanistic viewpoint, experimental studies in-

dicate that administration of NMDA receptor antagoniststotally prevented the enhancement of both inflammatoryor surgical hyperalgesia induced by fentanyl in rats.36,39

This suggests that prior exposure to exogenous opioidshas facilitated the development of NMDA receptor-mediated central changes in synaptic excitability leadingtoexaggeratedhyperalgesia following tissue injuries.7,8,39

In the present study, the exaggerated inflammatoryhyperalgesia observed in prestressed rats was suppressedwhen NMDA receptor antagonists were administeredimmediately prior to inflammation. Indeed, a singleBN2572 injection is more effective than 3 ketamineinjections, suggesting that this compound is a betterdrug for blocking the activation of pronociceptivesystems. Since we previously reported that NMDAreceptor antagonists did not affect inflammatoryhyperalgesia in nonstressed rats,39 these results suggesta critical role for NMDA receptor systems in exaggeratedinflammatory hyperalgesia induced by prior stress. It iswell known that m-opioid receptor stimulation by exoge-nous opioids triggers the activation of NMDA receptorsby reducing Mg21 blockade via the activation of intracel-lularproteinkinaseC10,11 leading tohyperalgesia.14,31 Thismechanism may also explain the development of painhypersensitivity induced by endogenous opioids.However, the administration of an NMDA receptorantagonist in prestressed rats that were returned tobasalnociceptive thresholdsafter stress-inducedanalgesiahad no effect per se on basal nociceptive threshold. Thisresult indicates that pain hypersensitivity, as revealed by

Le Roy et alexaggerated inflammatory hyperalgesia, is not associatedwithexcessivebasal activity at theNMDAsynaptic cleft. In-deed, theNMDA receptor systemappears to function onlyas a trigger able to reactivate memory systems, which byincreasing gain, elicit abnormal pain hypersensitivity.29,47

Activation of NMDA-dependent pronociceptive systemsmay be induced by both endogenous and exogenous opi-oids as previously proposed.10,11 Pharmacological andtranscriptomic approaches are currently in progress toidentify neural, neural-glial, neuroendocrine, and im-mune systems that could be involved in a cascade of cellu-lar events leading to pain hypersensitivity.From a clinical viewpoint, an important clinical chal-

lenge is to identify patients at risk for developingpain hypersensitivity and to propose appropriate treat-ments.26,33,34 Our results indicated that nonstressedand prestressed rats showed opposite fULD responses,analgesia versus hyperalgesia, respectively. It is note-worthy that the higher the number of stress sessionspresented to the rats, the greater was the increase inthe severity of the fULD-induced hyperalgesia. There-fore, under our experimental conditions, it is possibleto discriminate between prestressed and nonstressedanimals according to their hyperalgesic or analgesic re-sponses levels to the fULD test. Moreover, the amplitudeof the pharmacological response to fULD administrationwas closely and positively correlated with themagnitudeof long-lasting inflammatory hyperalgesia. This result in-dicates that it is possible, in laboratory rats, to predict thelevel of latent pain hypersensitivity, ie, pain vulnerability,according to individual animal histories, particularlystressful environmental events. It is noteworthy thatboth the fULD-induced hyperalgesia and the exagger-ated inflammatory hyperalgesia observed in prestressedrats were completely prevented by the preadministra-tion of an NMDA receptor antagonist, suggesting com-mon mechanisms.From a pathophysiological viewpoint, this study pro-

vides some preclinical evidence that pain level is notonly the result of nociceptive input level but is also de-pendent on the individual history, especially prior lifestress events associated with endogenous opioid release.Given that postoperative pain is one of the most fre-quently reported pains,13 our preclinical results suggestthat some hyperalgesia syndromes, as exaggerated post-operative pain, may be partly due to latent pain sensiti-zation induced by prior stress events.The preclinical model of latent pain sensitization in-

duced by NNES developed in this study could bea good tool for evaluating therapeutic strategies for re-ducing pain hypersensitivity induced by earlier lifeevents in individuals. Since it has been recently reportedthat anxiolytic drugs as benzodiazepines did not reversepain hypersensitivity in animals subjected to chronicsocial defeat,37 new therapeutic strategies have to bedeveloped for specifically blocking pronociceptive sys-tems. Based on our results showing that endogenousopioid released by NNES play a critical role in the devel-opment of latent pain hypersensitivity, we hypothetizethat NMDA pronociceptive systems activated by opioidreceptor stimulation8,11,32 could be good targets for

The Journal of Pain 1077reducing latent pain sensitization. However, thoughour study indicates that NMDA receptor antagonists

26. Kehlet H, Jensen TS, Woolf CJ: Persistent postsurgicalpain: Risk factors and prevention. Lancet 367:1618-1625,11. Chen L, Huang LY: Sustained potentiation of NMDAreceptor-mediated glutamate responses through activationof protein kinase C by a mu opioid. Neuron 7:319-326, 1991

12. Chizh BA, Headley PM, Tzschentke TM: NMDA receptorantagonists as analgesics: Focus on the NR2B subtype.Trends Pharmacol Sci 22:636-642, 2001

13. ChungF,UnV, Su J: Postoperative symptoms24hours afterambulatory anaesthesia. Can J Anaesth 43:1121-1127, 1996

14. Coderre TJ, Katz J: Peripheral and central hyperexcitabil-

ity: Differential signs and symptoms in persistent pain.Behav Brain Sci 20:404-419; discussion 435-513, 19972006

27. Khasar SG, Burkham J, Dina OA, Brown AS, Bogen O,Alessandri-Haber N, Green PG, Reichling DB, Levine JD:1070:35-44, 2006

25. Juni A, Klein G, Pintar JE, Kest B: Nociception increasesduring opioid infusion in opioid receptor triple knock-outmice. Neuroscience 147:439-444, 2007of NMDA-receptor channels as a mechanism of modulation.Nature 356:521-523, 1992

unrelated to opioid activity, analgesia, or tolerance: Evi-dence for multiple diverse hyperalgesic systems. Brain Rescould be candidates, their chronic use induceundesirable side effects. Anti-opioid systems, such as dy-norphin, chloecystokinin, and neuropeptide FF mightbe good targets because they are involved in opioid-induced hyperalgesia.42,44,48 Recently, it has beenreported that cholecystokinin receptor antagonistsabolish anxiety-inducedhyperalgesia in rats.37Moreover,polyaminedeficient diets,whichprevent hyperalgesia in-duced by endogenous opioid release in pain- and opioid-

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Endogenous Opioids Released During Non-Nociceptive Environmental Stress Induce Latent Pain Sensitization Via a NMDA-Depende ... Methods Animals Drugs Measurement of the Nociceptive Threshold Non-Nociceptive Environmental Stress (NNES) The Novel Environment Stress (NES) The Forced Swim Stress (FSS)

Inflammatory Pain Model Experimental Procedure Experiment 1: Effect of Prior Exposure to Stress on Changes in Nociceptive Threshold Induced by Inflammation Experiment 2: Effect of Naltrexone Administration Immediately Before Each NES Session on Changes in Nociceptive Threshold I ... Experiment 3: Comparative Effect of 1 to 3 NES Exposures on Changes in Nociceptive Threshold Induced by fULD and Subsequent ... Experiment 4: Effect of NMDA Receptor Antagonist Administration on Changes in Nociceptive Threshold Induced by Inflammation ... Experiment 5: Effect of NMDA Receptor Antagonist Administration on Changes in Nociceptive Threshold Induced by fULD Adminis ...

Statistical Analysis

Results Effect of Prior Exposure to Stress on Changes in Nociceptive Threshold Induced by Inflammation Effect of Naltrexone Administration Immediately Before Each NES Session on Changes in Nociceptive Threshold Induced by Infl ... Comparative Effect of 1 to 3 NES Exposures on Changes in Nociceptive Threshold Induced by fULD and Subsequent Inflammation Effect of NMDA Receptor Antagonist Administration on Changes in Nociceptive Threshold Induced by Inflammation in Prestresse ... Effect of NMDA Receptor Antagonist Administration on Changes in Nociceptive Threshold Induced by fULD Administration in Pre ...

Discussion Acknowledgments References