Analgesic properties of oleoylethanolamide (OEA) in visceral and inflammatory pain

Pain 133 (2007) 99–110 www.elsevier.com/locate/pain

Analgesic properties of oleoylethanolamide (OEA) in visceral and inflammatory pain Margarita Suardı´az

b

a,*

, Guillermo Estivill-Torru´s a, Carlos Goicoechea b, Ainhoa Bilbao a, Fernando Rodrı´guez de Fonseca a,*

a Fundacio´n IMABIS, Unidad de Investigacio´n, Hospital Universitario Carlos Haya, Ma´laga 29010, Spain Unidad Farmacologı´a, Departamento de Ciencias de la Salud III, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Avda. Atenas s/n, Alcorco´n, Madrid 28922, Spain

Received 22 July 2006; received in revised form 15 January 2007; accepted 7 March 2007

Abstract Oleoylethanolamide (OEA) is a natural fatty acid amide that mainly modulates feeding and energy homeostasis by binding to peroxisome proliferator-activated receptor-alpha (PPAR-a) [Rodrı´guez de Fonseca F, Navarro M, Go´mez R, Escuredo L, Navas F, Fu J, et al. An anorexic lipid mediator regulated by feeding. Nature 2001;414:209–12; Fu J, Gaetani S, Oveisi F, Lo Verme J, Serrano A, Rodrı´guez de Fonseca F, et al. Oleoylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-a. Nature 2003;425:90–3]. Additionally, it has been proposed that OEA could act via other receptors, including the vanilloid receptor (TRPV1) [Wang X, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1. J Physiol 2005;564:541–7.] or the GPR119 receptor [Overton HA, Babbs AJ, Doel SM, Fyfe MC, Gardner LS, Griffin G, et al. Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents. Cell Metab 2006;3:167–175], suggesting that OEA might subserve other physiological roles, including pain perception. We have evaluated the effect of OEA in two types of nociceptive responses evoked by visceral and inflammatory pain in rodents. Our results suggest that OEA has analgesic properties reducing the nociceptive responses produced by administration of acetic acid and formalin in two experimental animal models. Additional research was performed to investigate the mechanisms underlying this analgesic effect. To this end, we evaluated the actions of OEA in mice null for the PPAR-a receptor gene and compared its actions with those of PPAR-a receptor wild-type animal. We also compared the effect of MK-801 in order to evaluate the role of NMDA receptor in this analgesia. Our data showed that OEA reduced visceral and inflammatory responses through a PPAR-a-activation independent mechanism. Co-administration of subanalgesic doses of MK-801 and OEA produced an analgesic effect, suggesting the participation of glutamatergic transmission in the antinociceptive effect of OEA. This study represents a novel approach to the examination of the effectiveness of OEA in nociceptive responses and provides a framework for understanding its biological functions and endogenous targets in visceral and inflammatory pain.  2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Mice; Rat; Oleoylethanolamide; PPAR-alpha receptor; Cannabinoid; Inflammation; NMDA receptor

1. Introduction The fatty-acid ethanolamides (FAEs) are a family of naturally occurring lipids that are present in both plant *

Corresponding authors. Tel.: +34 669426548; fax: +34 951030447. E-mail address: [email protected] (F. Rodrı´guez de Fonseca).

and animal tissues. Animal cells synthesize and release FAEs in a stimulus-dependent manner, in response to a variety of physiological and pathological stimuli, suggesting that these compounds may participate in cell-tocell communication (Rodrı´guez de Fonseca et al., 2001; Piomelli, 2003; Lo Verme et al., 2005b). Many FAEs appear to play major roles in the modulation of pain sensitivity and inflammatory processes

0304-3959/$32.00  2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2007.03.008

100

M. Suardı´az et al. / Pain 133 (2007) 99–110

(Walker et al., 2002). Their participation in the regulation of pain responses was supported by in vitro and in vivo demonstrations of their interactions with signalling systems known to regulate pain and inflammatory mechanisms (Malan and Porreca, 2005): while several FAEs inhibit pain responses, others appear to enhance pain sensitivity (Walker et al., 2002). This is perhaps not surprising, since many of the recently discovered FAEs are derived from polyunsaturated fatty acids, including arachidonic acid, key metabolic precursor for numerous pro-inflammatory/pronociceptive compounds including the prostanoids and leukotrienes (Walker et al., 2005). Oleoylethanolamide (OEA) is a recently described FAE that regulates feeding and lipid metabolism (Rodrı´guez de Fonseca et al., 2001). Although structurally and functionally related to endocannabinoids, OEA does not bind to cloned cannabinoid CB1 and/or CB2 receptors. OEA was recently characterized to be the natural ligand of the peroxisome proliferator-activated receptor-alpha (PPAR-a) (Fu et al., 2003). Although its action on appetite seems to depend on the activation of vagal sensory afferent neurones, the mechanisms involved in exciting these sensory cells remain unclear (Ahern, 2003). Wang and coworkers (2005) have postulated that OEA directly excites vagal sensory neurons via activation of the capsaicin receptor TRPV1. The identification of other receptors that respond to OEA suggests that the family of FAE receptors is in fact significantly more extensive than the long-accepted cannabinoid CB1/CB2 receptor pairing and the PPAR nuclear receptor (Overton et al., 2006). Because FAEs such as anandamide and palmitoylethanolamide cause profound antinociception and antiinflammatory effects (Calignano et al., 2001; Lo Verme et al., 2005a; Malan and Porreca, 2005), we investigated whether OEA also has these antinociceptive properties in two animal models of visceral and inflammatory pain (writhing and formalin tests). 2. Materials and methods 2.1. Animals Adult (25–30 g) male, CD1 (Charles River, Spain), PPAR-a / (129S4/SvJae-Pparatm1Gonz) (KO) (See Genotyping Protocol for PPAR-a) and wild-type 129S1/SvImJ mice (WT) (Jackson Laboratories, USA) were used in writhing experiments. For the formalin test, we used adult (200–250 g) male Wistar rats (Charles River, Spain), and WT and PPAR-a KO mice. The animals were housed in clear plastic cages under standard laboratory conditions: controlled temperature of 23 C, 12/12-h light/dark cycle and free access to food and water. Spontaneous behaviour was observed in the cages before starting the experimental procedures; rodents showing aggressiveness or alterations in motility were discarded (2%).

This investigation was conducted following a protocol that was approved by the University of Ma´laga Ethics Committee and in accordance with the guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Experiments were conducted under strict adherence to the European Community Council Directive 86/609/EEC regulating animal research. 2.2. Drugs We synthesized OEA as previously described (Rodrı´guez de Fonseca et al., 2001). Acetic acid and formalin were purchased from Panreac Quı´mica SA, Spain. PPAR-a agonist Wy14643 (WY), opioid agonist Morphine (M), the non-competitive NMDA receptor antagonist MK-801 (MK) and cannabinoid agonist WIN55,212-2 (WIN) were obtained from Sigma– Aldrich, Spain. OEA, WY and WIN were firstly dissolved in ethanol 1 mg/ 1 ml and subsequently in a solution of 2 mg of Tween 80 and 1 ml of ethanol, after which the ethanol was evaporated and further dissolved in saline (Pertwee et al., 1992). Solutions of acetic acid (0.6%) and formalin (2.5% for rats and 3% for mice) were prepared in physiological saline. M and MK were dissolved in physiological saline as well. 2.3. Genotyping protocol for PPAR-a Mice homozygous for the Pparatm1Gonz targeted mutation (129S4/SvJae-Pparatm1Gonz) were obtained from Jackson Laboratories (Bar Harbor, ME). Adult mice from wild-type and homozygous females were genotyped for PPAR-a deletion using DNA isolated from a small part of the tail and following the protocol from the supplier (http:// www.jax.org). Schemes of PCR assay for detection of wildtype allele (143 bp) and targeted PPAR-a deletion containing fragment from bacterial neomycin resistance gene (280 bp) are shown in Fig. 1. Expected product sizes were obtained after use of genomic DNA (50 ng/ll/reaction), added as a template in a 25 ll PCR (heating up to 94 C for 3 min followed by 12 cycles of 94 C decreased in 0.5 C per cycle for 20 s, 64 C for 30 s and 72 C for 35 s, and 25 cycles of 94 C for 30 s, 58 C for 30 s, and 72 C for 35 s, with a final incubation of 72 C for 2 min before cooling to 10 C) containing BioThermMix reagent (Genecraft GmbH) and with the following primers: IMR0013 (5 0 -CTTGGGTGGAGAGGCTATTC-3; Tm = 59 C), IMR0014 (5 0 -AGGTGAGATGACAGGAGATC-3 0 ; Tm = 54 C), IMR11999 (5 0 -CCATCCAGATGACACCTTCC-3 0 ; Tm = 60 C), IMR1200 (5 0 -TCTCTTGCAACAGTGGGTGC -3 0 ; Tm = 62 C).

2.4. Western blot analysis Nuclear extracts of mouse brain and liver were prepared as follows. One gram of tissue was frozen in liquid nitrogen, crushed and further homogenized in 0.5 M sucrose, 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 25 mM KCl solution. After lysis in 0.5% Triton X-100 for 30 min, the homogenates were layered on a 0.9 M sucrose, 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 25 mM KCl solution, and centrifuged at 1800g for 20 min. Nuclei were finally resuspended from pellet

M. Suardı´az et al. / Pain 133 (2007) 99–110

Fig. 1. (a) PCR assay for genotyping of mice. Photograph depicts expected product sizes for wild-type allele (143 bp; lanes 1 and 2) and targeted PPAR-a deletion containing fragment from bacterial neomycin resistance gene (280 bp; lanes 3 and 4). Neomycin gene insertion replaced the coding region of exon 8 of the gene avoiding the amplification of wild-type allele. Expected product sizes were obtained after use of genomic DNA (50 ng/ll/reaction) added as a template in a 25 ll PCR and containing BioThermMix reagent (Genecraft GmbH) and the primers. (b) Western blot analysis of PPAR-a expression in liver (lanes 1 and 3) and brain (lanes 2 and 4) nuclear extracts from wild-type (1 and 2) and knock-out (3 and 4) mice. Thirty micrograms per lane was loaded in SDS gel and detected after blotting using a rabbit antiPPAR-a antibody. A major band with an expected size of 52 kDa was observed in wild-type extracts by contrast of KO nuclei. Densitometry analysis, showed as optical density (OD) at bottom, confirmed the absence of specific signal in KO extracts, wherein signal was similar to background or negative controls (not shown). Molecular weight markers for DNA (a) or protein (b) parallel run onto lane 5.

in 40% glycerol, 50 mM Tris–HCl (pH 8), 5 mM MgCl2, and 0.1 mM EDTA and protein concentration determined by Bradford reactive. Thirty micrograms of proteins from each sample was loaded in Laemmli buffer, separated by SDS– PAGE (10%) and electrotransferred to a PVDF membrane. Non-specific binding was blocked by 1 h incubation in phosphate buffered saline containing 5% non-fat dry milk and 0.1% Tween 20. Membranes were then exhaustively washed in PBS and incubated overnight at 4 C in rabbit polyclonal anti-PPAR-Alpha (RDI Divison of Fitzgerald Industries Intl) diluted in blocking buffer. Immunoreaction was specifically visualized by use of Opti-4CN amplification system (BioRad). Optical density measurement for each band was estimated using OptiQuant v.4.00 software (Packard Instruments Co.) after membrane scanning. Omission of primary antibody resulted in no specific signal. 2.5. Locomotor activity 2.5.1. Open field test Although the effect of OEA on locomotor activity has already been described in rats (Rodrı´guez de Fonseca et al.,

101

2001), we decided to also evaluate exploratory and locomotor activity in mice treated with OEA in order to discard potential motor effect that could mask analgesic responses. Experiments were conducted between 9:00 and 12:00 h. Mice were moved into the behavioural testing room at least 1 h prior to testing. The open field consisted of a 40 · 40 cm arena divided in 25 squares by lines drawn on the floor of the apparatus. The 9 squares not bounded by the walls of the test were referred to as centre squares. Mice were habituated for 30 min to the open field 24 h. before behavioural observation. Immobility time was recorded during the 30 min session. The apparatus was cleaned between mice with a weak acetic acid solution. Illumination of the test room was the same as the mouse colony room (100 lux). They were monitored by a video-tracking system equipped with a camera (Smart, Panlab, Barcelona) that records the animal’s horizontal activity (Bilbao et al., 2006). OEA (vehicle, 1, 5, 10 and 20 mg/kg) was injected i.p. 30 min before placing the animal in the open field. After OEA injection, each mouse was placed into the central square of the arena and allowed to freely explore the field for 30 min. 2.6. Analgesic activity The analgesic activity of OEA was measured against chemical stimulus. 2.6.1. Acetic acid-induced abdominal writhes or writhing test The test was performed as described by Collier et al. (1968). Nociception was induced by an intraperitoneal (i.p.) injection of 10 ml/kg of 0.6% acetic acid solution in mice. The number of writhes was cumulatively counted over a 10-min period, starting 5 min after the administration of the acetic acid solution. A writhe was defined as a contraction of the abdominal muscles accompanied by an elongation of the body and extension of the hind limbs. Animals were randomly allocated to receive one of the following treatments: • CD1 mice were i.p. treated with OEA (0.5, 1, 5, 10 and 20 mg/kg), M (1, 2, 5 and 10 mg/kg), WIN (0.5, 1 and 2 mg/kg) or corresponding vehicle solutions, 15 min before the acetic acid administration, at a total volume of 10 ml/kg. Control animals received a similar volume of saline solution. • Wild-type and PPAR-a KO mice received i.p. OEA (5 and 10 mg/ kg), its vehicle solution or saline (control animals), 15 min before the acetic acid administration at the same volume (10 ml/kg). • Wild-type animals were i.p. treated with MK (0.1, 0.05 and 0.025 mg/kg) or saline (control animals), 15 min before the acetic acid administration at the same volume (10 ml/kg). We also evaluated the effects of OEA (1 and 5 mg/kg, i.p.) administered 15 min after MK-801 (0.025 mg/kg, i.p.) in this test.

Each animal was used once and received only one dose of the drugs tested. All observations during the assay were performed in a randomized manner by a blind observer and 10–12 animals were used in each treatment group. Data are expressed as means ± standard error of the mean and percent inhibition (%) compared to the mean number of writhes observed in control animals.

M. Suardı´az et al. / Pain 133 (2007) 99–110

102

2.6.2. Formalin test

3. Results

The procedure was similar to that described previously (Hunskaar and Hole, 1987), and consisted of the injection of a solution of formalin in saline into the ventral surface of the hind paw of the rodents. The initial nociceptive behaviour normally peaked 3 min after formalin injection (early phase) and 15– 30 min after formalin injection (late phase), representing both the neurogenic and inflammatory pain responses, respectively. Before drug or vehicle administration, the animals were placed individually in a Plexiglas cage suspended above mirrors to allow clear observation of the paws of the animals. After a 15-min period of acclimatization to individual observation cages, 20 ll of 2.5% (rats) or 10 ll of 3% (mice) formalin solution was injected subcutaneously (s.c.) into the ventral surface of the hind paw and the rodents were then returned to the clear observation cages. Fifteen minutes before the formalin injection, animals were randomly allocated to receive one of the following i.p. treatments:

3.1. Effect of OEA in locomotor activity

(1) Rats: Physiological saline (control animals), OEA (0.5, 5, 10 or 20 mg/kg), WY (5 or 20 mg/kg) or corresponding vehicle solution, at a total volume of 2 ml/kg. (2) Wild-type and PPAR-a KO mice: Physiological saline (control animals), OEA (5 or 10 mg/kg) or corresponding vehicle solution, at a total volume of 10 ml/kg. (3) Wild-type mice: MK (0.025 mg/kg) or saline (control animals) at the same volume (10 ml/kg). We also evaluated the effects of OEA (0.5 and 5 mg/kg) administered 15 min after MK (0.025 mg/kg) in this test.

The number of shakings and lickings of the injected paw, considered as indicative of pain, was cumulatively counted over a continuous period of 60 min (rats) or 36 min (mice) after the formalin injection. Data are expressed as means ± standard error of the mean of the total number of lickings and shakings in both phases. Each animal was used only once and received one dose of the drugs tested. The number of animals per individual experimental group was at least 10–12. An observer who was blind to drug treatment conducted all the behavioural assays. 2.7. Statistical analysis Data are expressed as means ± standard error of the mean (SEM). Statistical analyses for differences between groups in the acetic acid test were performed using the Student’s t-test. Statistical analyses for differences between multiple groups in the formalin test were performed by analysis of variance (ANOVA) followed, when appropriate, by the Newman–Keuls test or Bonferroni test. A value of p < 0.05 was considered as statistically significant. Comparisons were established using as control values those obtained in the physiological saline treated animals.

Considering that OEA has been described to decrease food intake and locomotor activity at very high doses (Rodrı´guez de Fonseca et al., 2001) in rats, we firstly tested the locomotor activity in mice after an acute dose of OEA in order to identify the range of doses for analgesia that might avoid motor depression. Table 1 shows the results obtained in the open field test in mice. Only the highest dose used (20 mg/kg) was able to modify the locomotor activity in mice. 3.2. Effect of OEA in writhing test Intraperitoneal injection of acetic acid (0.6%) evoked a stereotypical writhing response in CD1 mice (control animals: writhes: 32.2 ± 1.8, n = 13). No differences were found when control animals were compared with mice treated with physiological saline (31.3 ± 1.6, n = 13). These writhing episodes were significantly reduced by M treatment in a dose-dependent manner (Fig. 2a). The highest dose of cannabinoid agonist WIN evaluated (2 mg/kg, i.p.) also inhibited the acetic-acid induced writhing responses in mice, in a statistically significant manner. In control experiments, the vehicle of WIN had no effect modifying the pain behaviour evoked by the acid (Fig. 2b). Treatment with OEA produced antinociception decreasing the writhing response induced by acetic acid. The nociceptive response was significantly inhibited by OEA at doses of 1, 5 and 10 mg/kg in a dose-dependent manner (Fig. 2c). A plateau was reached for the highest dose used (20 mg/kg). In order to test the involvement of the PPAR-a receptor in this antinociceptive effect of OEA, we performed the acetic acid test in WT and PPAR-a KO mice. No significant differences were observed in the nociceptive responses induced by the irritant between WT and KO mice (Figs. 3a and b). The administration of the vehicle did not induce any modification in none of the three types of mice (CD-1, WT and KO) compared with control. The i.p. injection of OEA reduced the writhing response induced by acetic acid in WT animals at all doses evaluated (5 and 10 mg/kg) (Fig. 3a). Interestingly, systemic administration of those doses of OEA was also able to significantly inhibit the nociceptive response in KO mice dose-dependently (Fig. 3b), sug-

Table 1 Effect of oleoylethanolamide (OEA) (1, 5, 10 and 20 mg/kg. i.p. n = 11–16/group) and vehicle of OEA (v) on open field test in mice Treatment

OEA vehicle

OEA 1 mg/kg

OEA 5 mg/kg

OEA 10 mg/kg

OEA 20 mg/kg

Inmobility (sc)

450.2 ± 40.8

515.8 ± 44.4

558.6 ± 36.6

583.6 ± 45.3

1125.1 ± 67.5***

***

p < 0.0001 vs OEA vehicle (v) by t-test.

M. Suardı´az et al. / Pain 133 (2007) 99–110

a

a

40

30

Number of Writhings

Number of Writhings

35

*

25 20

** *

15 10

** *

5

25

100%

Saline

20

- 51.84%

15

***

*** 5

***

M1

M5

M2

Saline

M10

b

30

Number of Writhings

25

100%

Number of Writhings

35 30 25 20 15

***

10 5 0 WIN Vehicle

WIN 0.5

WIN 1

WIN 2

OEA 10

4.45%

20

- 56.83% 15

*** - 85.91%

10

*** 5

Saline

Treatment (mg/kg) 40

OEA 5

OEA 10

Fig. 3. Effect of oleoylethanolamide (OEA) (5 and 10 mg/kg. i.p. n = 10–12/group) on abdominal constrictions (writhings) caused by i.p. injection of 0.6% acetic acid in (a) wild-type and (b) PPAR-a knock-out mice. Drugs were administered 15 min prior to acetic acid injection. Bars represent mean values ± SEM and percent inhibition (%) compared to the control acetic-acid treated animals. *Statistically significant difference compared to control acetic acid treated animals (n = 12). (***p < 0.001 vs saline by t-test.)

* ***

20

OEA Vehicle

Treatment (mg/kg)

35

Number of Writhings

OEA 5

0 Saline

25

OEA Vehicle

Treatment (mg/kg)

40

30

- 78.54%

10

Treatment (mg/kg)

c

5.23%

0

0

b

103

15

*** ***

10 5 0

Saline OEA Vehicle OEA 0.50 OEA 1

OEA 5

OEA 10

OEA 20

Treatment (mg(kg)

Fig. 2. Effect of (a) Morphine (M) (1, 2, 5 and 10 mg/kg. i.p. n = 12– 13/group), (b) the cannabinoid agonist WIN 55,212-2 (WIN) (0.5, 1 and 2 mg/kg. i.p. n = 12/group) and vehicle of WIN and (c) oleoylethanolamide (OEA) (0.5, 1, 5, 10 and 20 mg/kg. i.p. n = 10–13/group) and vehicle of OEA on abdominal constrictions (writhing) caused by i.p. injection of 0.6% acetic acid in CD1 mice. Drugs were administered 15 min prior to acetic acid injection. Bars show mean values ± SEM. *Statistically significant difference compared to control acetic acid treated animals (n = 12). (***p < 0.001, *p < 0.05 vs saline by t-test.)

gesting that this effect is not mediated by selective activation of the PPAR-a receptor. To evaluate the possible implication of the NMDA receptor, we also examined the effect of the MK-801 (MK), a non-competitive NMDA receptor antagonist

(0.1, 0.05 and 0.025 mg/kg, i.p.), in this nociceptive test (Fig. 4a). The analgesic effect of OEA (5 mg/kg, i.p.) was potentiated using a subanalgesic dose of MK (0.025 mg/ kg, i.p.), and moreover, when this subanalgesic dose of MK was co-administered with a subanalgesic dose of OEA (1 mg/kg, i.p.) the two drugs act synergistically, and mice showed a statistically significant analgesia (Fig. 4b). 3.3. Effect of OEA in formalin test In the formalin test in rats, 20 ll of 2.5% formalin solution was s.c. injected into the ventral hind paw to induce a characteristic biphasic nociceptive response (Hunskaar and Hole, 1987). Data were recorded as the number of cumulatively counted injected-paw formalin-induced shaking and licking episodes. The nociceptive episodes were significantly reduced by all doses of OEA evaluated (0.5–20 mg/kg) (Fig. 5a). Figs. 5b and c show the effect of OEA on the behaviour in rats

M. Suardı´az et al. / Pain 133 (2007) 99–110

104

a

30

10%

100% Number of Writhings

25 20 15

- 83.91%

10

***

5

- 87.82%

***

0 Saline

MK 0.1

MK 0.05

MK 0.025

Treatment (mg/kg)

b

30

10% 100%

- 25.21%

Number of Writhings

25

-6 .52%

*

#

20

- 60%

15

***

10

- 93.47%

+++

5

***

0 Saline

MK 0.025

OEA 5

MK 0.025+OEA 5

OEA 1 MK0.025+OEA 1

Treatment (mg/kg)

Fig. 4. Effect of (a) NMD antagonist MK 801 (MK) (0.1, 0.05 and 0.025 mg/kg. i.p. n = 8–10/group) and (b) MK (0.025 mg/kg) and oleoylethanolamide (OEA) (1 and 5 mg/kg. i.p. n = 8–10/group) on abdominal constrictions (writhings) caused by i.p. injection of 0.6% acetic acid in wild-type mice. Drugs were administered 15 min prior to acetic acid injection. Bars represent mean values ± SEM and percent inhibition (%) compared to the control acetic-acid treated animals. *Statistically significant difference compared to control acetic acid treated animals (n = 12). (***p < 0.001, *p < 0.05 vs saline, +++ p < 0.001 vs OEA 5, #p < 0.05 vs OEA 1, by t-test.)

where the data were averaged over the early phase (0– 3 min) and the late phase (15–27 min). OEA reduces the nociceptive response induced by the irritant in a dose-dependent manner in both phases. There were no significant differences in data recorded in control rats (i.p., physiological saline) and in OEA vehicle treated rats (Figs. 3b,c). Moreover, we tested the analgesic properties of the PPAR-a agonist WY in the formalin test in rats. WY (5 and 20 mg/kg) did not affect the early phase of the formalin test (Figs. 6a and b) whereas it slightly decreased the late phase, in which a 20% reduction in the number of shaking and licking episodes was observed (Figs. 6a and c), indicating a partial analgesic profile. In order to evaluate the underlying mechanisms in the antinociceptive effect of OEA in inflammatory pain, we investigated its effects on the formalin-evoked nociceptive behaviour in WT and PPAR-a KO mice. The formalin solution (10 ll; 3%) was s.c. injected into the ventral hind paw to induce the characteristic nociceptive

response in mice. Formalin injection induced similar biphasic responses in WT and KO animal (Figs. 7a and 8a, respectively). In control experiments, i.p. administration of vehicle solution of OEA produced no changes in this nociceptive response compared to baseline (physiological saline) either in WT or in KO mice (Figs. 7a, 8a). All doses of OEA evaluated were able to reduce both the early and late phases of formalin-evoked biphasic nociceptive response in these animals. Figs. 7b and c show the effects of formalin on the behaviour of WT mice, where the data were averaged over the early phase (0–3 min) and the late phase (15–27 min). Intraperitoneal administration of OEA (5 and 10 mg/kg) induced significantly dose-dependent antinociception. OEA significantly inhibited the number of licking and shaking episodes in KO animals. The inhibition was similar at all doses tested (5 and 10 mg/ kg, i.p.) in the early and in the late phases (Figs. 8b and c). To test the implication of the NMDA receptor in inflammatory pain, we examined the effect of the subanalgesic dose of MK (0.025 mg/kg, i.p.) in formalin test in mice (Figs. 9a, b and c). We also investigated the effect of the systemic co-administration of the subanalgesic dose of MK (0.025 mg/kg) and two doses of OEA (0.5 and 5 mg/kg). At the dose used in this study, MK did not modify the effect of either the subanalgesic (0.5 mg/kg) or analgesic (5 mg/kg) dose of OEA in the early phase of formalin-evoked nociceptive response in mice (Fig. 9b). However, the two drugs acted synergistically in the late phase and animals showed a statistically significant analgesia either with subanalgesic or analgesic doses of OEA (Fig. 9c). 4. Discussion Many fatty acid amides appear to play a role in pain and inflammation. The field of lipid signalling has added important new dimensions to how we think about neuronal and immune signalling in neuroinflammation. Oleoylethanolamide, the naturally occurring amide of ethanolamine and oleic acid and structurally related to the endogenous cannabinoid anandamide, is a new member of this family with interesting pharmacological properties. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling. OEA does not bind to cannabinoid receptors and its molecular targets seem to be more complex than initially thought (Fu et al., 2003; Wang et al., 2005; Overton et al., 2006). Oleoylethanolamide is an endogenous lipid that modulates feeding, body weight and lipid metabolism by binding with high affinity to the nuclear receptor PPAR-a (Rodrı´guez de Fonseca et al., 2001; Fu et al.,

M. Suardı´az et al. / Pain 133 (2007) 99–110

a

105

40

Saline OEA 0.5 mg/kg OEA 5 mg/kg

Shaking + Licking

30

OEA 10 mg/kg OEA 20 mg/kg

** ***

*

***

*** 0-3

3 -6

6- 9

9 -12

**

**

*** *

0

*

** *

10

*

*

20

***

***

**

**

*** ***

12-15 15 -18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 Time (min)

c

40

225 200

Shaking+Licking

Shaking + Licking

b

30

** 20

*** ***

10

***

175

**

150 125

**

**

100

**

75 50 25

0

0

Saline

OEA Vehicle OEA 0.5

OEA 5

OEA 10

OEA 20

Treatment (mg/kg)

Saline

OEA Vehicle

OEA 0.5

OEA 5

OEA 10

OEA 20

Treatment (mg/kg)

Fig. 5. (a) Effect of oleoylethanolamide (OEA, 0.5, 5, 10 and 20 mg/kg) and physiological saline (n = 8–10/group) on the formalin-induced nociceptive responses caused by intraplantar injection of 2.5% formalin in Wistar rats. Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Shaking and licking behaviours after formalin injection were summarized as (b) early phase 1 (0–3 min) and (c) late phase 2 (15–27 min). Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Lines/columns show mean values ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001 vs saline by (a) one way ANOVA and (b, c) t-test.)

2003). In addition, OEA excites peripheral vagal sensory nerves, but the mechanisms by which this occurs and the molecular targets of OEA are unclear (Ahern, 2003). The aim of this work was to test the effect of OEA in two different nociceptive animal models, inflammatory and visceral pain. We demonstrate for the first time that OEA has antinociceptive effects in these models. In visceral pain, our results support the previous demonstration that analgesic compounds like the opioid agonist M and the cannabinoid agonist WIN produce antinociception, inhibiting the writhes induced by an irritant (Honore et al., 2002; Ulugol et al., 2006). Data also showed that the i.p. administration of OEA produces antinociceptive effect inhibiting the writhes induced by acetic acid in CD1 mice. This antinociceptive effect was induced in a dose-dependent manner and reached maximum effect at 10 mg/kg, suggesting this dose as the ceiling effect for OEA analgesia.

We can discard that the antinociception induced by the administration of OEA could be related with modifications in the locomotor activity or spontaneous activity since no modifications were found, at the analgesic doses, when the open field test was carried out in mice and rats (Rodrı´guez de Fonseca et al., 2001). In order to investigate the underlying mechanisms in these antinociceptive effects, we used WT and KO mice for the putative receptor of OEA, PPAR-a. In this group of experiments, data showed that all doses of the amide tested were also able to significantly inhibit, in a dose-dependent manner, the nociceptive response induced by the acetic acid. Interestingly, the doses evaluated (5 and 10 mg/kg) were as effective reducing the writhes in PPAR-a KO as in WT mice. These findings suggest that OEA has a role in visceral pain, modulating nociceptive pathways, and that this effect does not require the activation of the PPAR-a receptor.

M. Suardı´az et al. / Pain 133 (2007) 99–110

106

a

40

Saline WY 5mg/kg

Shaking + Licking

30

WY 20mg/kg

20

* ** **

10

0 0- 3

3-6

6-9

9 -12

12 -15

15 -18

18-21

21-24

24-27

27-30

30-33

33-36

36-39

39-42

42-45

Time (min)

b

c

30

180

140

Shaking + Licking

Shaking + Licking

160

20

10

**

120 100 80 60 40 20

0

0

Saline

WY 5

WY 20

Treatment (mg/kg)

Saline

WY 5

WY 20

Treatment (mg/kg)

Fig. 6. (a) Effect of the PPAR-a agonist WY14643 (5 and 20 mg/kg) and physiological saline (n = 7–5/ group) on the formalin-induced nociceptive responses caused by intraplantar injection of 2.5% formalin in Wistar rats. Shaking and licking behaviours after formalin injection were summarized as (b) early phase 1 (0–3 min) and (c) late phase 2 (15–27 min). Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Lines/columns show mean values ± SEM. (*p < 0.05, **p < 0.01 vs saline by (a) one way ANOVA and (b, c) t-test.)

Taking into account that some types of non-opioid analgesia (as stress-induced analgesia) can be reversed by administration of NMDA antagonist MK-801 (Mogil et al., 1993), and that neuroprotective effect of cannabinoids can rely on inhibition of glutamatergic transmission (Morisset and Urban, 2001), we tested the possible participation of glutamate receptors in the OEA induced antinociception in visceral pain. Interestingly, when subanalgesic doses of OEA and MK-801 were co-administered in mice, the mixture induced an analgesic effect, showing a synergistic effect that suggests a participation of the glutamate pathways in the analgesic effect of OEA. Although this relationship merits a more detailed research, it presents a novel and interest-

ing proposal for the mechanisms involved in the analgesic effect of OEA. Previous studies have proposed that OEA could directly excite vagal sensory neurones and induce, at elevated doses (25 mg/kg), visceral pain via activation of TRPV1 (Wang et al., 2005), a vanilloid receptor with a well-described role in pain signalling and essential for the development of inflammatory thermal hyperalgesia in mice (Caterina et al., 2000; Davis et al., 2000; Caterina and Julius, 2001). In our hands, OEA not only did not induce any pain-associated behaviour, but, on the contrary, showed a clear dose-dependent antinociceptive effect. This difference could be related with differences of dosage (10 mg/kg vs 25 mg/kg), or with the difference in

M. Suardı´az et al. / Pain 133 (2007) 99–110

a

107

70

Saline

60

OEA Vehicle

Shaking + Licking

50

40

30

OEA 5mg/kg

***

OEA 10mg/kg

***

20

**

10

*

0

0-3

3-6 m

6-9 m

*

9-12 m 12-15 m 15-18 m 18-21 m 21-24 m 24-27 m 27-30 m 30-33 m 33-36 m

Time (min)

b

80

c

50

70 40

50

***

40

***

30 20

Shaking + Licking

Shaking + Licking

60

30

***

20

*** 10

10 0

0 Saline

EA Vehicle

OEA 5

OEA 10

Treatment (mg/kg)

Saline

EA Vehicle

OEA 5

OEA 10

Treatment (mg/kg)

Fig. 7. (a) Effect of oleoylethanolamide (OEA) (5 and 10 mg/kg. i.p. n = 8–10/group), vehicle of OEA and physiological saline on the formalininduced nociceptive responses caused by intraplantar injection of 3% formalin in wild-type mice. Shaking and licking behaviours after formalin injection were summarized as (b) early phase 1 (0–3 min) and (c) late phase 2 (15–27 min). Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Lines/columns show mean values ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001 vs saline by (a) one way ANOVA and (b, c) t-test.)

the evaluation of the effect of OEA, since in the work of Wang their conclusion is only based in the behaviour of the mice after the administration of OEA (a ‘‘hunched, recumbent posture’’), and here we show the effect after two well-known and well-accepted nociceptive tests. In the inflammatory pain model, our results substantiate the typical biphasic behavioural response seen after s.c. formalin injection in rodents (Hunskaar and Hole, 1987). High nociceptive scores were recorded during the first 3 min after s.c. formalin administration and were followed by a reduction in scores for several minutes. A later increase was maintained until the end of the test period (Jaggar et al., 1998). The early phase seems to be caused predominantly by C-fibre activation due to the peripheral stimulus, while the late phase appears to be dependent on the combination of an

inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord. These functional changes seem to be initiated by the C-fibre barrage during the early phase (Tjolsen et al., 1992). Our results demonstrate that systemic administration of OEA dose-dependently reduces the early and the late phases of the behavioural response to s.c. formalin injection in rats. Moreover, intraperitoneal injection of OEA was able to induce significantly antinociception in a dose-dependent manner not only in the early phase but also in the early part of late phase in mice. Taken together, these results suggest that OEA may participate in the peripheral nociceptive pathway, maybe modulating or modifying both the altered C-fibre activation and/or the inflammatory process in the peripheral

M. Suardı´az et al. / Pain 133 (2007) 99–110

108

a

70

60

Saline OEA Vehicle

Shaking + Licking

50

40

OEA 5 mg/kg OEA 10 mg/kg

***

30

*** 20

10

0 0-3

3-6

6-9

9-12

12-15

15-18

18-21

**

*

**

24-27

21-24

*

27-30

30-33

33-36

Time (min)

b 80

c

40

60 50

***

40

***

30 20

Shaking + Licking

Shaking + Licking

70

50

30

20

***

10

***

10 0

0 Saline

OEA Vehicle

OEA 5

OEA 10

Treatment (mg/kg)

Saline

OEA Vehicle

OEA 5

OEA 10

Treatment (mg/kg)

Fig. 8. (a) Effect of oleoylethanolamide (OEA) (5 and 10 mg/kg. i.p.), vehicle of OEA and physiological saline (n = 8–9/group) on the formalininduced nociceptive responses caused by intraplantar injection of 3% formalin in PPAR-a knock-out mice. Shaking and licking behaviours after formalin injection were summarized as (b) early phase 1 (0–3 min) and (c) late phase 2 (15–27 min). Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Lines/columns show mean values ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001 vs saline by (a) one way ANOVA and (b, c) t-test.)

tissues implicated in this test. Moreover, the antinociceptive effect of OEA in the late phase suggests that it could intervene inhibiting the functional changes in the dorsal horn of the spinal cord induced by the peripheral injection of formalin. To test whether PPAR-a activation contributes to the analgesic property of OEA, we used mice deficient in PPAR-a. PPAR-a KO animals treated with formalin presented similar nociceptive responses to WT mice. Moreover, experiments developed in these mice demonstrated that OEA also reduced formalin-induced nociception in KO mice. We also evaluated the possible role of PPAR-a in inflammatory pain, testing the analgesic properties of the PPAR-a agonist WY in the formalin test in rats. WY slightly decreased the late phase, indicating a partial analgesic profile. These results suggest that OEA may reduce inflamma-

tory pain in this test through a PPAR-a-activation-independent mechanism. Because the antinociceptive effect of NMDA antagonists in the formalin test is well-recognised (Berrino et al., 2003; Ha Yoon et al., 2005) we evaluated the possible role of glutamate receptors in the OEA induced antinociception in inflammatory pain. When doses of OEA and MK-801 were co-administered, the mixture did not modify the effect of OEA in the formalin induced early phase. However, our data demonstrate that systemic co-administration of subanalgesic doses of MK and OEA produces a significant antinociceptive effect during the late phase after formalin injection. Furthermore, when analgesic and subanalgesic doses of OEA and MK, respectively, were co-administered, the antinociceptive effect of OEA was potentiated by the

M. Suardı´az et al. / Pain 133 (2007) 99–110

109

80 70

Saline

Shaking + Licking

60

MK 0.025 OEA 0.5

50

MK 0.025 + OEA 0.5

*

OEA 5

40

MK 0.025 + OEA 5

** 30 20

10

***

0 0-3

3-6

6-9

9-12

12-15

***

15-18

***

***

***

21-24

18-21

***

***

24-27

**

27-30

30-33

Time

19.31%

Shaking + Licking

70

12.58%

60

- 2.93%

100%

- 3.75%

0.79% - 29.34%

50

100%

60

-36.03%

**

50

-23.27%

*

40 30

Shaking + Licking

80

###

***

40 30

- 68.91% - 83.20%

***

20

20

***

10

10 0

+++

0 Saline

MK 0.025

OEA 0.5

MK 0.025 + OEA 0.5

OEA 5

MK 0.025 + OEA 5

Saline

MK 0.025

OEA 0.5

MK 0.025 + OEA 0.5

OEA 5

MK 0.025 + OEA 5

Treatment (mg/kg)

Treatment (mg/kg)

Fig. 9. (a) Effect of NMDA antagonist MK 801 (MK) (0.025 mg/kg. i.p. n = 6–8/group) and oleoylethanolamide (OEA) (0.5 and 5 mg/kg. i.p.) and physiological saline (n = 8–9/group) on the formalin-induced nociceptive responses caused by intraplantar injection of 3% formalin in mice. Shaking and licking behaviours after formalin injection were summarized as (b) early phase 1 (0–3 min) and (c) late phase 2 (15–27 min). Drugs were intraperitoneal (i.p.) administered 15 min prior to formalin injection. Lines/columns show mean values ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001 vs saline, ###p < 0.001 vs OEA 0.5, +++p < 0.001 vs OEA 5, by (a) one way ANOVA and (b, c) t-test.)

NMDA antagonist, showing a synergistic effect. These results suggest a participation of the glutamate pathways in the antinociceptive effect of OEA, maybe inhibiting the functional changes in the dorsal horn of the spinal cord induced by the peripheral injection of formalin. In summary, this study represents a first approach to the examination of the effectiveness of the fatty acid amide, OEA, at reducing the nociceptive responses produced by administration of acetic acid and formalin in two experimental animal models. The results demonstrate that the administration of OEA antagonizes the writhes and licking and shaking induced by the irritant agents in rodents. These effects were similar in mice deficient in PPAR-a, suggesting that the nuclear receptors

might not play an important role in these properties of OEA. These findings provide a framework for understanding the biological functions of the naturally occurring fatty acid ethanolamide and may help researchers to identify novel pharmacological targets for the treatment of pain.

Acknowledgements This work was supported by MEC SAF 2004/07762, Plan Nacional Sobre Drogas, FIS 05/0997, REDES RTARD 06/001 Fundacio´n Eugenio Rodriguez Pascual and proyectos de excelencia Consejerı´a de Innovacio´n Ciencia y Empresa, Junta de Andalucı´a.

110

M. Suardı´az et al. / Pain 133 (2007) 99–110

References Ahern GP. Activation of TRPV1 by the satiety factor oleoylethanolamide. J Biol Chem 2003;278:30429–34. Berrino L, Oliva P, Massimo F, Aurili C, Maione S, Grella A, et al. Antinociceptive effect in mice of intraperitoneal N-methyl-Daspartate receptor antagonists in the formalin test. Eur J Pain 2003;7:131–7. Bilbao A, Cippitelli A, Martin AB, Granado N, Ortiz O, Bezard E, et al. Absence of quasi-morphine withdrawal syndrome in adenosine A2A receptor knockout mice. Psychopharmacology (Berl) 2006;185:160–8. Calignano A, La Rana G, Piomelli D. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur J Pharmacol 2001;419:191–8. Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. [Review]. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. Collier HDJ, Dinnin LC, Johnson CA, Schneider C. The abdominal response and its suppression by analgesic drugs in the mouse. Br J Pharmacol Chemother 1968;32:295–310. Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000;405:183–7. Fu J, Gaetani S, Oveisi F, Lo Verme J, Serrano A, Rodrı´guez de Fonseca F, et al. Oleoylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-a. Nature 2003;425:90–3. Ha Yoon M, Beom Bae H, Choi J, Wook Jeong S, Chung S, Yeon Yoo K, et al. Evaluation of interaction between intrathecal adenosine and MK801 or NBQX in a rat formalin pain model. Pharmacology 2005;75:157–64. Honore P, Mikusa J, Bianchi B, McDonald H, Cartmell J, Faltynek C, et al. TNP-ATP, a potent P2X3 receptor antagonist, blocks acetic acid induced abdominal constriction in mice: comparison with reference analgesics. Pain 2002;96:99–105. Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987;30:103–4. Jaggar SI, Hasnie FS, Sellaturay S, Rice AS. The anti-hyperalgesic actions of the cannabinoid anandamide and the putative CB2 receptor agonist palmitoylethanolamide in visceral and somatic inflammatory pain. Pain 1998;76:189–99. Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A, et al. The nuclear receptor peroxisome proliferator-activated

receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol Pharmacol 2005a;67:15–9. Lo Verme J, Gaetani S, Fu J, Oveisi F, Burton K, Piomelli D. Regulation of food intake by oleoylethanolamide. Cell Mol Life Sci 2005b;62:708–16. Malan TP, Porreca F. Lipid mediators regulating pain sensitivity. Prostaglandins Other Lipid Mediat 2005;77:123–30. Mogil JS, Marek P, Yirmiya R, Balian H, Sadowski B, Taylor AN, et al. Antagonism of the non-opioid component of ethanolinduced analgesia by the NMDA receptor antagonist MK-801. Brain Res 1993;602:126–30. Morisset V, Urban L. Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord. J Neurophysiol 2001;86:40–8. Overton HA, Babbs AJ, Doel SM, Fyfe MC, Gardner LS, Griffin G, et al. Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents. Cell Metab 2006;3:167–75. Pertwee RG, Stevenson LA, Elrick DB, Mechoulam R, Corbett AD. Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea-pig small intestine. Br J Pharmacol 1992;105:980–4. Piomelli D. The molecular logic of endocannabinoid signalling. Nat Rev Neurosci 2003;4:873–84. [Review]. Rodrı´guez de Fonseca F, Navarro M, Go´mez R, Escuredo L, Navas F, Fu J, et al. An anorexic lipid mediator regulated by feeding. Nature 2001;414:209–12. Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain 1992;51:5–17. [Review]. Ulugol A, Ozyigit F, Yesilyurt O, Dogrul A. The additive antinociceptive interaction between WIN 55,212-2, a cannabinoid agonist, and ketorolac. Anesth Analg 2006;102:443–7. Walker JM, Krey JF, Chu CJ, Huang SM. Endocannabinoids and related fatty acid derivatives in pain modulation. Chem Phys Lipids 2002;121:159–72. Walker JM, Krey JF, Chen JS, Vefring E, Jahnsen JA, Bradshaw H, Huang SM. Targeted lipidomics: fatty acid amides and pain modulation. Prostaglandins & other Lipid Mediators 2005;77: 35–45. Wang X, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1. J Physiol 2005;564:541–7. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.