1521-0103/351/1/224–232$25.00 http://dx.doi.org/10.1124/jpet.114.215681THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 351:224–232, October 2014Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

Treatment with a Heme Oxygenase 1 Inducer Enhances theAntinociceptive Effects of m-Opioid, d-Opioid, and Cannabinoid 2Receptors during Inflammatory Pain

Mireia Carcolé, Sílvia Castany, Sergi Leánez, and Olga PolGrup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau and Institut de Neurociències, UniversitatAutònoma de Barcelona, Barcelona, Spain

Received April 21, 2014; accepted June 19, 2014

ABSTRACTThe administration of m-opioid receptor (MOR), d-opioid recep-tor (DOR), and cannabinoid 2 receptor (CB2R) agonists attenu-ates inflammatory pain. We investigated whether treatment withthe heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX(CoPP), could modulate the local effects and expression ofMOR, DOR, or CB2R during chronic inflammatory pain. In micewith inflammatory pain induced by the subplantar administrationof complete Freund’s adjuvant, we evaluated the effects ofthe intraperitoneal administration of 10 mg/kg CoPP on theantiallodynic and antihyperalgesic actions of locally administeredMOR (morphine), DOR (DPDPE {[D-Pen(2),D-Pen(5)]-enkephalin}),or CB2R [JWH-015 {(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone}] agonists and its reversion with theHO-1 inhibitor, tin protoporphyrin IX (SnPP). The effect of CoPPtreatment on the dorsal root ganglia expression of HO-1, MOR,

DOR, and CB2R was also assessed. The results show thattreatment with CoPP increased the local antinociceptive effectsproduced by morphine, DPDPE, or JWH-015 during chronic in-flammatory pain, and these effects were blocked by the subplantaradministration of SnPP, indicating the participation of HO-1 inthe antinociceptive actions. CoPP treatment, apart from induc-ing the expression of HO-1, also enhanced the expression ofMOR, did not alter CB2R, and avoided the decreased expres-sion of DOR induced by inflammatory pain. This study showsthat the HO-1 inducer (CoPP) increased the local antinociceptiveeffects of MOR, DOR, and CB2R agonists during inflammatorypain by altering the peripheral expression of MOR and DOR. There-fore, the coadministration of CoPP with local morphine, DPDPE,or JWH-015may be a good strategy for themanagement of chronicinflammatory pain.

IntroductionIt is well known that the local administration of m-opioid

receptor (MOR), d-opioid receptor (DOR), or cannabinoid 2receptor (CB2R) agonists elicits antiallodynic and antihyper-algesic effects during peripheral inflammation (Obara et al.,2009; Negrete et al., 2011; Hervera et al., 2013b). More in-teresting is the fact that under inflammatory pain conditionsthe local antinociceptive effects induced by MOR, DOR, andCB2R agonists are produced by the activation of the periph-eral nitric oxide–cGMP–protein kinase G (PKG)–ATP-sensitivepotassium (KATP) channels signaling pathway. Accordingly,the local antinociceptive effects of a MOR (morphine), DOR(DPDPE {[D-Pen(2),D-Pen(5)]-enkephalin}) or CB2R [JWH-015{(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone}]agonist were significantly reduced by their coadministrationwith selective neuronal or inducible nitric oxide synthases,

L-guanylate cyclase, or PKG inhibitors, while the activation ofthis signaling pathway potentiated the peripheral antinocicep-tive effects produced by these agonists during inflammatorypain (Pacheco and Duarte, 2005; Hervera et al., 2009; Leánezet al., 2009; Cunha et al., 2010; Negrete et al., 2011). In addi-tion, the expression of MOR, DOR, or CB2R after inflammatorydiseases was also regulated by nitric oxide (Pol et al., 2005;Jiménez et al., 2006; Negrete et al., 2011).It is also well known that, similar to nitric oxide, another

gaseous neurotransmitter carbon monoxide synthesized bythe heme oxygenase 1 (HO-1) enzyme also activates thecGMP-PKG pathway to modulate nociception (Steiner et al.,2001; Nascimento and Branco, 2007). Indeed, the adminis-tration of carbon monoxide–releasing molecules—a new classof chemical agents able to reproduce several biologic effects ofHO-1–derived carbon monoxide, or the HO-1 inducer com-pound, cobalt protoporphyrin IX (CoPP)—inhibits inflamma-tion, acute nociception, and neuropathic pain (Ferrándiz et al.,2008; Rosa et al., 2008; Egea et al., 2009; Maicas et al., 2010;Hervera et al., 2013a; Negrete et al., 2014). We have also

This work was supported by the Fondo de Investigación Sanitaria [GrantPS0900968], Madrid, Spain.

dx.doi.org/10.1124/jpet.114.215681.

ABBREVIATIONS: AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide; AM630, [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone; ANOVA, analysis of variance; CB1R, cannabinoid 1 receptor; CB2R,cannabinoid 2 receptor; CFA, complete Freund’s adjuvant; CoPP, cobalt protoporphyrin IX; CTAP, H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2;DPDPE, [D-Pen(2),D-Pen(5)]-enkephalin; DOR, d-opioid receptor; HO-1, heme oxygenase 1; JWH-015, (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone; KATP, ATP-sensitive potassium; MOR, m-opioid receptor; NX-ME, naloxone methiodide; PKG, protein kinase G; SnPP, tinprotoporphyrin IX.

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recently demonstrated that carbon monoxide synthesized byHO-1 modulates the effects of morphine but not of DPDPEand JWH-015 under neuropathic pain conditions (Herveraet al., 2013b), although the role played by CoPP treatment onthe effects and expression of MOR, DOR, and CB2R duringchronic inflammatory pain has not been evaluated.Therefore, in a mouse model of chronic inflammatory pain

induced by the subplantar administration of complete Freund’sadjuvant (CFA), we evaluated 1) the antiallodynic and anti-hyperalgesic effects produced by the subplantar administrationof specificMOR (morphine), DOR (DPDPE), or CB2R (JWH-015)agonists alone or combinedwithCoPP intraperitoneally admin-istered; 2) the antinociceptive effects of morphine, DPDPE, orJWH-015 subplantarly administered, alone or combined, withthe HO-1 inhibitor tin protoporphyrin IX (SnPP); 3) the re-versibility of morphine, DPDPE, and JWH-015 antinociceptiveeffects by their coadministration with specific antagonists; and4) the effect of CoPP treatment on the expression of HO-1,MOR, DOR, and CB2R in the dorsal root ganglia from CFA-injected mice.

Materials and MethodsExperimental Animals. The experiments were performed in

male C57BL/6 mice acquired from Harlan Laboratories (Barcelona,Spain). All mice weighing 21–25 g were housed under 12-hour light/dark conditions in a room with controlled temperature (22°C) andhumidity (66%). Animals had free access to food and water and wereused after a minimum of 7 days acclimatization to the housingconditions. All experiments were performed according with the Guidefor the Care and Use of Laboratory Animals as adopted and pro-mulgated by the US National Institutes of Health and approved bythe local Committee of Animal Use and Care of the AutonomousUniversity of Barcelona. All efforts were made to minimize animalsuffering and to reduce the number of animals used.

Induction of Chronic Inflammatory Pain. Chronic inflamma-tory pain was induced by the subplantar injection of 30 ml CFA(Sigma-Aldrich, St. Louis, MO) into the right hind paw under briefanesthetic conditions with isoflurane, as described in our previousworks (Hervera et al., 2009; Leánez et al., 2009). All experiments wereperformed 10 days after CFA injection. At this time point, all theanimals developed a local inflammatory reaction, allodynia to mechan-ical stimuli, and hyperalgesia to noxious thermal stimuli, as previouslyreported by our group (Negrete et al., 2011).

The development of mechanical allodynia and thermal hyper-algesia was evaluated by using von Frey filaments and plantar tests,respectively. All animals were tested in each paradigm before and at10 days after CFA injection.

Nociceptive Behavioral Tests. Mechanical allodynia was quan-tified by measuring the hind-paw withdrawal response to von Freyfilament stimulation. In brief, animals were placed in methacrylatecylinders (20 cm high, 9 cm diameter; Servei Estació, Barcelona,Spain) with a wire-grid bottom through which the von Frey filaments(North Coast Medical, Inc., San Jose, CA) with a bending force in therange of 0.008–3.5 g were applied by using a modified version of theup–down paradigm, as previously reported by Chaplan et al. (1994).The 0.4-g filament was used first, and the 3.0-g filament was used asa cutoff. Then, the strength of the next filament was decreased orincreased according to the response. The threshold of response wascalculated from the sequence of filament strength used during theup–down procedure by using an Excel program (Microsoft Iberia SRL,Barcelona, Spain) that includes curve-fitting of the data. Clear pawwithdrawal or shaking or licking of the paw was considered to bea nociceptive-like response. Both ipsilateral and contralateral hind

paws were tested. Animals were allowed to habituate for 1 hour beforetesting to allow an appropriate behavioral immobility.

Thermal hyperalgesia was assessed as previously reported byHargreaves et al. (1988). Paw withdrawal latency in response toradiant heat was measured using a plantar test apparatus (UgoBasile, Varese, Italy). Briefly, mice were placed in methacrylatecylinders (20 cm high � 9 cm diameter) positioned on a glass surface.The heat source was positioned under the plantar surface of the hindpaw and activated with a light beam intensity chosen in preliminarystudies to give baseline latencies from 8–10 seconds in control mice. Acutoff time of 12 seconds was used to prevent tissue damage in theabsence of a response. The mean paw withdrawal latencies fromthe ipsilateral and contralateral hind paws were determined from theaverage of three separate trials, taken at 5-minute intervals to preventthermal sensitization and behavioral disturbances. Animals were ha-bituated to the environment for 1 hour before the experiment to allowthem to become quiet and to permit testing.

Western Blot Analysis. Animals were sacrificed at 0 days (naïve)and after CFA injection by cervical dislocation. Tissues from theipsilateral section of the dorsal root ganglia (L3 to L5) were removedimmediately after killing, frozen in liquid nitrogen, and stored at280°C until assay. Samples from five animals were pooled into oneexperimental sample to obtain enough protein levels for performingWestern blot analysis. The HO-1, MOR, DOR, and CB2R proteinlevels were analyzed by Western blot. Tissues were homogenized inice-cold lysis buffer (50 mM Tris·Base, 150 nMNaCl, 1% NP-40, 2 mMEDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 Triton X-100, 0.1%SDS, 1 mM Na3VO4, 25 mM NaF, 0.5% protease inhibitor cocktail,and 1% phosphatase inhibitor cocktail). All reagents were purchased atSigma-Aldrichwith the exception of NP-40 fromCalbiochem (Darmstadt,Germany). The crude homogenate was solubilized for 1 hour at 4°C,sonicated for 10 seconds and centrifuged at 4°C for 15 minutes at 700g.The supernatant (60 mg of total protein) was mixed with 4� Laemmliloading buffer and then loaded onto 4% stacking/10% separating SDSpolyacrylamide gels.

The proteins were electrophoretically transferred onto polyvinyli-dene fluoride membranes for 120 minutes, blocked with phosphate-buffered saline 1 5% nonfat dry milk, and subsequently incubatedovernight at 4°C with polyclonal rabbit anti–HO-1 (1:300; Stressgen,Ann Arbor, MI), anti-MOR (1:1000; Chemicon-Millipore, Billerica,MA), anti-DOR (1:2500; Chemicon-Millipore), or anti-CB2R (1:500;Abcam, Cambridge, UK). The proteins were detected by a horseradishperoxidase–conjugated anti-rabbit secondary antibody (GE Health-care, Little Chalfont, Buckinghamshire, UK) and visualized withchemiluminescence reagents (ECL kit; GE Healthcare) and by expo-sure onto hyperfilm (GE Healthcare). The intensity of the blots wasquantified by densitometry. The membranes were stripped andreproved, with a monoclonal rabbit anti–b-actin antibody (1:10,000;Sigma-Aldrich) used as a loading control.

Experimental Procedure. In a first set of experiments, we as-sessed the expression of inflammatory pain induced by the subplantaradministration of CFA, as previously used in our studies (Leánez et al.,2009). After the habituation period, baseline responses were establishedin the following sequence: von Frey filaments and plantar test. Afterbaseline measurements, inflammatory pain was induced, and animalswere again tested in each paradigm at day 10 after CFA injection byusing the same sequence as for the baseline responses. The contralateralpaws were used as controls (n 5 6 animals per group).

In a second set of experiments, we evaluated the mechanical anti-allodynic and thermal antihyperalgesic effects of the subplantaradministration of different doses of a specific MOR (morphine), DOR(DPDPE), or CB2R (JWH-015) agonist and their respective vehicles inthe contralateral and ipsilateral paw of CFA-injected animals (n 5 6animals per group).

In a third set of experiments, we investigated the mechanicalantiallodynic and thermal antihyperalgesic effects produced by theintraperitoneal administration of 10 mg/kg CoPP alone or combinedwith the subplantar administration of a low dose of morphine (50 mg),

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DPDPE (50 mg), or JWH-015 (30 mg) in the contralateral and ipsilat-eral paw of CFA-injected animals (n 5 6 animals per group).

In another set of experiments, we evaluated the mechanicalantiallodynic and thermal antihyperalgesic effects produced by thesubplantar administration of 290 mg SnPP alone or combined withthe subplantar administration of a high dose of morphine (100 mg),DPDPE (150 mg), or JWH-015 (300 mg) in the contralateral and ip-silateral paw of CFA-injected animals (n 5 6 animals per group).

The doses of CoPP and SnPP combined with morphine, DPDPE,or JWH-015 were selected in accordance to our previous studies(Hervera et al., 2013a,b). The doses of all tested opioid and canna-binoid receptor agonists subplantarly administered were chosen fromthe dose-response curves performed in this study, as the ones thatproduced aminimal or amaximal antinociceptive effect in CFA-injectedmice.

The reversibility of the antinociceptive effects produced by thesubplantar administration of morphine (100 mg), DPDPE (150 mg), orJWH-015 (300 mg), as doses that produce the maximal antiallodynicand antihyperalgesic effects after peripheral inflammation by theirsubplantar coadministration with specific (120 mg CTAP [H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2]; 50 mg naltrindole; and 60 mgAM630 ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone)) and an unspecific peripheral opioidantagonist (50 mg naloxone methiodide [NX-ME]) or a cannabinoid 1receptor (CB1R) antagonist (150 mg AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide])(Hervera et al., 2010, 2011), was also evaluated (n5 6 animals per group).The doses of all tested opioid and cannabinoid receptor antagonists wereselected according to our previous data obtained in animals withchronic pain (Hervera et al., 2009, 2013b; Negrete et al., 2011).

Finally, in another set of experiments we evaluated the effects ofCoPP on the expression of HO-1, MOR, DOR, and CB2R in the ipsi-lateral site of the dorsal root ganglia from CFA-injected mice by use ofWestern blot assay. In these experiments, mice treated with vehiclewere used as controls (n 5 5 samples per group). The total number ofanimals used in this study was 291.

Drugs. CoPP and SnPP were purchased from Frontier Scientific(LivchemGmbH&Co, Frankfurt, Germany). Morphine hydrochloridewas obtained from Alcaiber S.A. (Madrid, Spain), and DPDPE, CTAP,naltrindole, and NX-ME were acquired from Sigma-Aldrich. JWH-015, AM630, and AM251 were purchased from Tocris (Ellisville, MI).

CoPP and SnPP were dissolved in dimethylsulfoxide (1% solutionin saline). JWH-015, AM630, AM251 were dissolved in dimethylsulf-oxide (50% solution in saline). Morphine hydrochloride, DPDPE,

TABLE 1Mechanical response (von Frey filaments strength, grams) and thermalresponse (withdrawal latency, seconds) in the contralateral andipsilateral paw of mice after the subplantar administration of CFAResults are shown as mean 6 S.E.M.; n = 6 animals per experimental group.

Paw Mechanical Response(von Frey Filaments Strength)

Thermal Response(Withdrawal Latency)

g s

Contralateral 2.4 6 0.1 9.5 6 0.3Ipsilateral 1.4 6 0.1* 3.5 6 0.2*

*P , 0.01 denotes statistically significant difference between ipsilateral andcontralateral paw (paired Student’s t test) for each test.

Fig. 1. Effects of the subplantar administration of morphine, DPDPE, or JWH-015 on the mechanical allodynia and thermal hyperalgesia induced byperipheral inflammation. Mechanical antiallodynic and thermal antihyperalgesic effects produced by the subplantar administration of different doses ofmorphine (A and B), DPDPE (C and D), or JWH-015 (E and F) and their respective vehicles in the ipsilateral paw of CFA-injected mice. Data areexpressed as mean value of maximal possible effect (%) 6 S.E.M. (six animals for dose). *Statistically significant difference versus their respectivevehicle treated animals (P , 0.05, one-way ANOVA followed by the Student-Newman-Keuls test) for each test, drug, and dose.

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CTAP, NX-ME, and naltrindole were dissolved in saline solution (0.9%NaCl). All drugs were freshly prepared before use. CoPP was intra-peritoneally administered 3–4 hours before testing in a final volume of10ml/kg. SnPP,morphine,DPDPE, JWH-015, CTAP,NX-ME, naltrindole,AM630, andAM251were administered into the plantar side of the rightpaw 30minutes before behavioral testing, in a final volume of 30 ml. Foreach group treated with a drug, the respective control group receivedthe same volume of vehicle.

Statistical Analysis. Data are expressed as mean 6 S.E.M. Thestatistical analysis was performed by use of SPSS (version 17 forWindows; IBM España, Madrid, Spain). All comparisons were run astwo-tailed testing.

We used a paired Student’s t test to compare the mechanical andthermal responses induced by peripheral inflammation in the ipsilateralpaw of CFA-injected mice with the effects produced in the contralateralpaws of mice. For each test and drug evaluated, the comparison of theeffects produced by the subplantar administration of different doses ofmorphine, DPDPE, JWH-015, or their corresponding vehicle was eval-uated by using one-way analysis of variance (ANOVA) followed by theStudent-Newman-Keuls test.

For each behavioral test, the comparison of the effects produced bythe administration of CoPP or SnPP on the local antinociceptiveeffects produced by morphine, DPDPE, or JWH-015 was evaluated byusing one-way ANOVA followed by the Student-Newman-Keuls test.

In these experiments, antinociception in von Frey filaments andplantar tests is expressed as the percentage ofmaximal possible effect,where the test latencies before (baseline) and after drug administra-tion are compared and calculated according to the following equation:

Maximal possible effectð%Þ5 ½ðdrug2baselineÞ=ðcutoff 2baselineÞ�� 100

For each test, the reversal of the local antinociceptive effects producedby morphine, DPDPE, or JWH-015 with their respective antagonistsand the effects produced by these antagonists administered alonewere also analyzed by use of one-way ANOVA followed by theStudent-Newman-Keuls test.

Changes on the expression of HO-1, MOR, DOR, and CB2R in thedorsal root ganglia from naïve and CFA-injected mice treated withvehicle or CoPP were also analyzed by using one-way ANOVA followedby the Student-Newman-Keuls test. P , 0.05 was considered statisti-cally significant.

ResultsInduction of Inflammatory Pain. In accordance with our

previous findings, the subplantar administration of CFAproduced unilateral mechanical allodynia and thermal hyper-algesia (Table 1). Indeed, peripheral inflammation led to asignificant decrease of the threshold for evoking pawwithdrawalto a mechanical stimulus and a decrease of paw withdrawallatency to thermal stimulus in the ipsilateral paw as comparedwith the contralateral paw (P , 0.01, paired Student’s t test).Effects of the Subplantar Administration of Morphine,

DPDPE, and JWH-015 on the Mechanical Allodynia andThermal Hyperalgesia Induced by Peripheral Inflam-mation in Mice. The subplantar administration of morphine

Fig. 2. Effects of CoPP on the antiallodynic and antihyperalgesic responsesto morphine. Mechanical antiallodynic (A) and thermal antihyperalgesic (B)effects produced by the subplantar administration of 50 mg of morphine orvehicle in the ipsilateral paw of CFA-injectedmice pretreated with 10mg/kgCoPP. The effect of the intraperitoneal administration of CoPP alone is alsoshown.Data are expressed asmean values of themaximal possible effect (%)6S.E.M. (6 animals per group). *Statistically significant difference versuscontrol group treated with vehicle (P, 0.05, one-way ANOVA followed byStudent-Newman-Keuls test) for each behavioral test. +Statistically sig-nificant difference versus control group treated with morphine (P , 0.05,one-way ANOVA followed by the Student-Newman-Keuls test) for eachbehavioral test. #Statistically significant difference versus group treatedwith CoPP plus vehicle (P, 0.05; one-way ANOVA followed by the Student-Newman-Keuls test) for each behavioral test.

Fig. 3. Effects of CoPP on the antiallodynic and antihyperalgesic responsesto DPDPE. Mechanical antiallodynic (A) and thermal antihyperalgesic (B)effects produced by the subplantar administration of 50 mg DPDPE orvehicle in the ipsilateral paw of CFA-injectedmice pretreated with 10mg/kgCoPP. The effect of the intraperitoneal administration of CoPP alone is alsoshown.Data are expressed asmean values of themaximal possible effect (%)6S.E.M. (6 animals per group). *Statistically significant difference versuscontrol group treated with vehicle (P , 0.05, one-way ANOVA followed byStudent-Newman-Keuls test) for each behavioral test. +Statistically signif-icant difference versus control group treated with DPDPE (P, 0.05, one-wayANOVA followed by the Student-Newman-Keuls test) for each behavioraltest. #Statistically significant difference versus group treated with CoPP plusvehicle (P , 0.05; one-way ANOVA followed by the Student-Newman-Keulstest) for each behavioral test.

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(50–100 mg) dose-dependently inhibited the mechanical allo-dynia (Fig. 1A) and thermal hyperalgesia (Fig. 1B) induced byperipheral inflammation. Indeed, the mechanical antiallodynicand thermal antihyperalgesic effects produced by high doses ofmorphine (75 or 100 mg) in the ipsilateral paw of CFA-injectedmice were statistically significantly higher than those pro-duced by a low dose of the same drug or their correspondingvehicle-treated animals (P , 0.001, one-way ANOVA followedby the Student-Newman-Keuls test).The subplantar administration of DPDPE (50–150 mg) dose-

dependently inhibited the mechanical allodynia (Fig. 1C) andthermal hyperalgesia (Fig. 1D) induced by peripheral inflam-mation. Indeed, the mechanical antiallodynic and thermalantihyperalgesic effects produced by high doses of DPDPE(75, 100, or 150 mg) in the ipsilateral paw of CFA-injectedmicewere statistically significantly higher than those produced bya low dose of the same drug or their corresponding vehicle-treated animals (P , 0.001, one-way ANOVA followed by theStudent-Newman-Keuls test).In a similar way, the subplantar administration of JWH-

015 (30–300 mg) also dose-dependently inhibited the mechan-ical allodynia (Fig. 1E) and thermal hyperalgesia (Fig. 1F)induced by peripheral inflammation. That is, the mechanicalantiallodynic and thermal antihyperalgesic effects producedby high doses of JWH-015 (75, 150, or 300mg) in the ipsilateral

paw of CFA-injectedmicewere statistically significantly higherthan those produced by 30 mg of the same drug or their corre-sponding vehicle-treated animals (P, 0.001, one-way ANOVAfollowed by the Student-Newman-Keuls test).The subplantar administration of morphine, DPDPE, JWH-

015, or vehicle did not elicit any antinociceptive effect in thecontralateral paw of CFA-injected mice (data not shown).Effects of CoPP on the Antiallodynic and Antihyper-

algesic Responses to Morphine, DPDPE, and JWH-015during Peripheral Inflammation. The effects of the in-traperitoneal administration of 10 mg/kg CoPP on the me-chanical antiallodynic and thermal antihyperalgesic effectsproduced by the subplantar administration of a subanalgesicdose of morphine (50 mg), DPDPE (50mg), JWH-015 (30 mg), orvehicle in CFA-injected mice were investigated. For mor-phine, our results showed that the intraperitoneal adminis-tration of CoPP alone statistically significantly attenuatedthe mechanical allodynia (Fig. 2A) and thermal hyperalgesia(Fig. 2B) induced by peripheral inflammation (P, 0.001, one-way ANOVA versus control vehicle–treatedmice). Our resultsalso demonstrate that treatment with CoPP significantly in-creased the local antiallodynic (Fig. 2A) and antihyperalgesic(Fig. 2B) effects produced by the subplantar administration ofmorphine in the ipsilateral paw of CFA injected mice (P ,0.001, one-way ANOVA versus control group treated withvehicle or morphine or CoPP plus vehicle).

Fig. 4. Effects of CoPP on the antiallodynic and antihyperalgesic re-sponses to JWH-015. Mechanical antiallodynic (A) and thermal anti-hyperalgesic (B) effects produced by the subplantar administration of30 mg JWH-015 or vehicle in the ipsilateral paw of CFA-injected micepretreated with 10 mg/kg CoPP. The effect of the intraperitoneal ad-ministration of CoPP alone is also shown. Data are expressed as meanvalues of the maximal possible effect (%) 6 S.E.M. (6 animals per group).*Statistically significant difference versus control group treated withvehicle (P , 0.05, one-way ANOVA followed by Student-Newman-Keulstest) for each behavioral test. +Statistically significant difference versuscontrol group treated with JWH-015 (P , 0.05, one-way ANOVA followedby the Student-Newman-Keuls test) for each behavioral test. #Statisticallysignificant difference versus group treated with CoPP plus vehicle (P ,0.05; one-way ANOVA followed by the Student-Newman-Keuls test) foreach behavioral test.

Fig. 5. Effects of SnPP treatment on the antiallodynic and antihyper-algesic responses to morphine, DPDPE, or JWH-015. Mechanical anti-allodynic (A) and thermal antihyperalgesic (B) effects of the subplantaradministration ofmorphine (100 mg), DPDPE (150mg), or JWH-015 (300 mg)combinedwith SnPP (290mg) in the ipsilateral paw of CFA-injectedmice areshown. The effects of the subplantar administration of morphine, DPDPE,JWH-015, SnPP, or vehicle alone are also represented. Data are expressedas mean values of the maximal possible effect (%) 6 S.E.M. (6 animals pergroup). *Statistically significant difference versus control group treated withvehicle (P , 0.05, one-way ANOVA followed by Student-Newman-Keulstest) for each behavioral test. +Statistically significant difference versuscontrol group treated with SnPP (P, 0.05, one-way ANOVA followed by theStudent-Newman-Keuls test) for each behavioral test. #Statistically signif-icant difference versus group treated with morphine, DPDPE, or JWH-015plus vehicle (P , 0.05, one-way ANOVA followed by the Student-Newman-Keuls test) for each behavioral test.

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For DPDPE, treatment with CoPP also statistically sig-nificantly attenuated the mechanical allodynia (Fig. 3A) andthermal hyperalgesia (Fig. 3B) induced by peripheral in-flammation in the ipsilateral paw of CFA-injected mice (P ,0.001, one-way ANOVA versus control vehicle–treated mice).Moreover, although treatment with CoPP did not alter themechanical antiallodynic (Fig. 3A) effects of DPDPE, it sta-tistically significantly increased the thermal antihyperalgesic(Fig. 3B) effects produced by the subplantar administration ofDPDPE in the ipsilateral paw of CFA-injected mice (P, 0.001,one-way ANOVA versus control group treated with vehicle orDPDPE or CoPP plus vehicle).Regarding JWH-015, similar to what occurred with mor-

phine, treatment with CoPP statistically significantly en-hanced the antiallodynic (Fig. 4A) and antihyperalgesic (Fig.4B) effects produced by the subplantar administration ofJWH-015 in the ipsilateral paw of CFA-injected mice (P ,0.001, one-way ANOVA versus control group treated withvehicle or JWH-015 or CoPP plus vehicle).The subplantar administration of morphine, DPDPE, or

JWH-015 alone or combined with CoPP had no statisticallysignificant effect on the contralateral paw of CFA-injectedanimals (data not shown).Effects of the HO-1 Inhibitor, Tin Protoporphyrin IX,

on the Antinociceptive Responses to Morphine, DPDPE,and JWH-015 in CFA-Injected Mice. The effects of thesubplantar administration of SnPP (290 mg) on the mechan-ical antiallodynic and thermal antihyperalgesic effects pro-duced by the subplantar administration of morphine (100 mg),DPDPE (150 mg), or JWH-015 (300 mg) in CFA-injected micewere assessed.Formorphine,DPDPE, and JWH-015, and each test evaluated,

our results show that although the subplantar administration

of SnPP alone did not alter the mechanical allodynia (Fig. 5A)or thermal hyperalgesia (Fig. 5B) induced by peripheral in-flammation, their local coadministration with a high dose ofmorphine, DPDPE, or JWH-015 statistically significantly de-creased the local antiallodynic (Fig. 5A) and antihyperalgesic(Fig. 5B) effects produced by these drugs on the ipsilateral paw ofCFA-injected mice (P , 0.001, one-way ANOVA versus grouptreated with morphine, DPDPE, or JWH-015 plus vehicle).The subplantar administration of morphine, DPDPE, or

JWH-015 alone or combined with SnPP had no statisticallysignificant effect on the contralateral paw of CFA-injectedanimals (data not shown).Reversal of the Antinociceptive Effects of Morphine,

DPDPE,andJWH-015bySpecificAntagonistsafterPeriph-eral Inflammation. The antiallodynic and antihyperalgesiceffects produced by the subplantar administration of 100 mgof morphine in the ipsilateral paw of CFA-injected mice werecompletely reversed by its subplantar coadministration withselective MOR (CTAP, 120 mg) or peripheral opioid receptor(NX-ME, 50 mg) antagonists (P , 0.001; one-way ANOVA,followed by Student-Newman-Keuls test) (Table 2). In asimilar way, the antiallodynic and antihyperalgesic effectsproduced by 150 mg of DPDPE in the ipsilateral paw of CFA-injected mice were completely reversed by its subplantarcoadministration with a selective DOR (naltrindole, 50 mg) ora peripheral opioid receptor (NX-ME, 50 mg) antagonist (P ,0.001, one-way ANOVA, followed by Student-Newman-Keulstest). In addition, the antinociceptive effects produced by300 mg of JWH-015 in the ipsilateral paw of CFA-injected micewere also completely reversed by its subplantar coadministra-tion with a selective CB2R antagonist (AM630, 60 mg; P ,0.001, one-way ANOVA, followed by Student-Newman-Keulstest). The subplantar administration of AM251 (a selective

TABLE 2Effects of the subplantar administration of morphine (100 mg), DPDPE (150 mg), or JWH-015(300 mg) alone or combined with CTAP (120 mg) or NX-ME (50 mg), naltrindole (50 mg) or NX-ME (50 mg), or AM630 (60 mg) or AM251 (150 mg), respectively, on the mechanical allodyniaand thermal hyperalgesia induced by the subplantar administration of CFA in the ipsilateralpaw of miceResults are shown as mean 6 S.E.M.; n = 6 animals per experimental group.

Treatments Mechanical Allodynia(von Frey Filaments Strength)

Thermal Hyperalgesia(Withdrawal Latency)

g s

Vehicle Vehicle 1.4 6 0.1 3.5 6 0.4Vehicle 2.4 6 0.1* 9.4 6 0.6*

Morphine CTAP 1.3 6 0.2 3.5 6 0.4NX-ME 1.4 6 0.1 3.6 6 0.2

Vehicle CTAP 1.3 6 0.1 3.4 6 0.5NX-ME 1.2 6 0.1 3.6 6 0.2

Vehicle Vehicle 1.3 6 0.1 3.4 6 0.5Vehicle 2.4 6 0.1* 9.2 6 0.7*

DPDPE Naltrindole 1.2 6 0.2 3.7 6 0.4NX-ME 1.1 6 0.1 3.5 6 0.2

Vehicle Naltrindole 1.3 6 0.1 3.4 6 0.6NX-ME 1.2 6 0.1 3.6 6 0.5

Vehicle Vehicle 1.4 6 0.1 3.8 6 0.3Vehicle 2.5 6 0.2* 9.2 6 0.2*

JWH-015 AM630 1.2 6 0.1 4.1 6 0.4AM251 2.4 6 0.1* 9.0 6 0.3*AM630 1.3 6 0.1 3.7 6 0.2

Vehicle AM251 1.4 6 0.1 3.6 6 0.4

*P, 0.05 denotes statistically significant differences versus their respective vehicle plus vehicle treatedgroup (one-way ANOVA, followed by the Student-Newman-Keuls test) for each test and drug tested.

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CB1R antagonist, 150 mg) was unable to revert the local anti-allodynic and antihyperalgesic effects produced by JWH-015.The subplantar administration of the different antagonists

alone in the ipsilateral paw of CFA-injected mice (Table 2) aswell as in the contralateral paw of these animals (data notshown) had no statistically significant effect on the differentnociceptive responses evaluated in this study. In addition, thesubplantar administration of all tested agonists alone or com-bined with their respective antagonists produced no statisti-cally significant effect in the contralateral paw of CFA-injectedmice (data not shown).Effect of CoPP Treatment on HO-1, MOR, DOR, and

CB2R Protein Expression in the Dorsal Root Gangliafrom CFA-Injected Mice. The protein levels of HO-1, MOR,DOR, and CB2R in the dorsal root ganglia from CFA-injectedmice treated with vehicle or CoPP as well as from control micetreated with vehicle are shown in Figs. 6 and 7. Our resultsshow that the dorsal root ganglia expression of HO-1 (Fig. 6A)was statistically significantly increased by CoPP treatment(P , 0.001, one-way ANOVA versus control vehicle and CFA-injected mice treated with vehicle). Similarly, the expressionof MOR (Fig. 6B) was also statistically significantly increasedby CoPP treatment (P , 0.049, one-way ANOVA versuscontrol mice). Moreover, the decreased protein levels of DORin the dorsal root ganglia from CFA-injected mice did notoccur with CoPP treatment (P , 0.001, one-way ANOVAversus control mice and CFA-injected mice treated withCoPP) (Fig. 7A), and the unchanged peripheral expression ofCB2R in the dorsal root ganglia from CFA-injected miceremained unaltered with CoPP treatment (Fig. 7B).

DiscussionIn this study, we demonstrated that treatment with an HO-1

inducer compound (CoPP) increased the local antinociceptiveeffects produced by a MOR, DOR, or CB2R agonist by en-hancing the peripheral expression of MOR, leaving CB2R un-altered, and avoiding the decreased protein levels of DORinduced by peripheral inflammation.The antinociceptive and anti-inflammatory effects produced

by substances that can liberate carbon monoxide during in-flammatory diseases have previously been shown (Guillénet al., 2008; Negrete et al., 2014). In accordance, our resultsfurther demonstrated that the administration of CoPP (anHO-1 inducer) also inhibited the mechanical and thermal hy-persensitivity induced by chronic peripheral inflammation inmice. As expected, treatment with CoPP increased the expres-sion of HO-1, indicating that carbon monoxide synthesized byHO-1 is principally responsible for the antinociceptive actionsproduced by CoPP during chronic inflammatory pain (Rosaet al., 2008).Our study also revealed, for first time, that treatment with

CoPP significantly enhanced the local antinociceptive effectsproduced by morphine, DPDPE, and JWH-015 after chronicperipheral inflammation. Moreover, the peripheral antiallodynicand antihyperalgesic effects produced by these drugs wereFig. 6. Effect of CoPP on HO-1 and MOR protein expression from CFA-

injected mice. The protein expression of HO-1 (A) and MOR (B) in theipsilateral site of the dorsal root ganglia from CFA-injected mice treatedwith vehicle (CFA-vehicle) or CoPP (CFA-CoPP) are represented. Theexpression of these receptors in the dorsal root ganglia from naïve micetreated with vehicle (naïve-vehicle) has been also represented as controls.*Statistically significant difference when compared versus naïve vehicletreatedmice (P, 0.05, one-way ANOVA followed by Student-Newman-Keuls

test) for each protein. Representative examples of Western blots for HO-1andMOR proteins, in which b-actin was used as a loading control, are alsoshown. Data are expressed as mean 6 S.E.M.; n = 5 samples per group.

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significantly decreased by the subplantar administration ofthe HO-1 inhibitor SnPP, indicating that HO-1 participates inthe local antinociceptive effects produced by a MOR, DOR, orCB2R agonist during chronic inflammatory pain. Theseresults expand our and others’ previous data revealing thatthe peripheral antinociceptive effects of morphine, DPDPE, orJWH-015 during peripheral inflammation are produced bythe activation of the cGMP-PKG-KATP channels signalingpathway triggered by nitric oxide (Pacheco and Duarte, 2005;Leánez et al., 2009; Hervera et al., 2009; Cunha et al., 2010;Negrete et al., 2011), further demonstrating that the activationof this pathway triggered by carbon monoxide synthesizedby HO-1 also participates in this action.Curiously, under neuropathic pain conditions, CoPP treat-

ment likewise improved the local antinociceptive effects ofmorphine but decreased those produced by DPDPE and JWH-015 (Hervera et al., 2013a). These results suggest thatalthough MOR agonists use the same mechanism of actionto produce peripheral antinociception during inflammatoryand neuropathic pain—that is, the activation of the periph-eral carbon monoxide/nitric oxide-cGMP-PKG-KATP chan-nels signaling pathway—DOR and CB2R agonists did notactivate the same way to produce peripheral antinociceptionin both types of pain. Therefore, because the activation of thecarbonmonoxide/nitric oxide-cGMP-PKG-KATP channels sig-naling pathway enhanced the local antinociceptive effectsof DOR and CB2R agonists during inflammation, the acti-vation of this pathway limits their effects under sciatic nerveinjury conditions. These data revealed the different mecha-nisms implicated in the inhibitory effects produced by DORand CB2R agonists under inflammatory or neuropathic painconditions.The specificity of the antiallodynic and antihyperalgesic

effects produced by the local administration of morphine andDPDPEafter chronic peripheral inflammationwas demonstratedby the complete reversion of their effects with their coadmin-istration with selective antagonists (CTAP and naltrindole).These effects are produced by interaction with peripheral opi-oid receptors as demonstrated by the reversal of their effectswith the coadministration with a nonselective peripherally-acting opioid receptor antagonist (NX-ME). Finally, the speci-ficity of the local antinociceptive effects of JWH-015 after chronicperipheral inflammation was also proven by the complete rever-sion of its actions with their coadministration with a selectiveCB2R (AM630), but not with a CB1R (AM251), antagonist.In accordance with other studies, our results demonstrated

that the dorsal root ganglia expression of MOR increased, theexpression of DOR decreased, and those of CB2R did notchange after chronic peripheral inflammation (Puehler et al.,2004; Obara et al., 2009; Gavériaux-Ruff and Kieffer, 2011;Negrete et al., 2011). Moreover, the present study also re-vealed that although treatment with CoPP enhanced theperipheral expression of MOR, it avoided the decreased ex-pression of DOR and did not modify the protein levels of CB2Rin the dorsal root ganglia from animals with peripheral in-flammation. These data indicate that although the enhancedperipheral expression of MOR induced by CoPP treatment

Fig. 7. Effect of CoPP on DOR and CB2R protein expression from CFA-injected mice. The protein expression of DOR (A) and CB2R (B) in theipsilateral site of the dorsal root ganglia from CFA-injected mice treatedwith vehicle (CFA-vehicle) or CoPP (CFA-CoPP) are represented. Theexpression of these receptors in the dorsal root ganglia from naïve micetreated with vehicle (naïve-vehicle) is also represented as controls.*Statistically significant difference when compared versus naïve vehicletreated mice and CFA-injected mice treated with CoPP (P, 0.05, one-wayANOVA followed by Student-Newman-Keuls test) for each protein.Representative examples of Western blots for DOR and CB2R proteins,

in which b-actin was used as a loading control, are also shown. Data areexpressed as mean 6 S.E.M.; n = 5 samples per group.

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might be responsible for the increased local antiallodynic andantihyperalgesic effects produced by morphine combined withCoPP during chronic inflammation, the undecreased expres-sion of DOR observed in CoPP-treated mice could also ex-plained the enhanced antihyperalgesic effects produced byDPDPE in these animals.Regarding CB2R, the fact that CoPP treatment enhanced

their antinociceptive actions without altering their expressioncould be related to the fact that during chronic inflammatorypain the antiallodynic and antihyperalgesic effects producedby JWH-015 are completed by the activation of the peripheralnitric oxide-cGMP-PKG-KATP channels signaling pathwaymediated by local endogenous opioids (Negrete et al., 2011).As a consequence, the increased protein levels of MOR and/orthe undecreased expression of DOR induced by CoPP treat-ment could be involved in the enhanced effects produced bya CB2R agonist during inflammatory pain although non-changes in the expression of their receptors could be demon-strated in CFA-injected mice treated with CoPP. Nonetheless,a reduction of the overall inflammation produced by CoPP(Megias et al., 2009; Fan et al., 2011) could be also implicatedin the enhanced local antinociceptive effects produced by mor-phine, DPDPE, and JWH-015 in CoPP-treated mice.In summary, this study suggests, for first time, that treat-

ment with an HO-1 inducer enhances the local antinociceptiveeffects of morphine, DPDPE, and JWH-015 through regulat-ing the peripheral opioid receptor expression. We propose thecoadministration of CoPP with MOR, DOR, or CB2R agonistsas a promising strategy to improve their antinociceptive ef-fects during chronic inflammatory pain.

Authorship Contributions

Participated in research design: Pol.Conducted experiments: Carcolé, Castany, Leánez.Performed data analysis: Carcolé, Pol.Wrote or contributed to the writing of the manuscript: Pol.

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Address correspondence to: Dr. Olga Pol, Grup de NeurofarmacologiaMolecular, Institut de Neurociències, Facultat de Medicina, Edifici M2-115,Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.E-mail: opol@santpau.es

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