Implication of allopregnanolone in the antinociceptive effect of N-palmitoylethanolamide in acute or persistent pain

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PAIN 153 (2012) 33–41

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Implication of allopregnanolone in the antinociceptive effect of N-palmitoylethanolamide in acute or persistent pain Oscar Sasso a, Roberto Russo a, Sergio Vitiello b,c, Giuseppina Mattace Raso a, Giuseppe D’Agostino a, Anna Iacono a, Giovanna La Rana a, Monique Vallée b,c, Salvatore Cuzzocrea d,e, Pier Vincenzo Piazza b,c, Rosaria Meli a,⇑, Antonio Calignano a a

Department of Experimental Pharmacology, University of Naples ‘‘Federico II’’, via D. Montesano 49, 80131 Naples, Italy INSERM U862, Institut F. Magendie, Bordeaux, France Université de Bordeaux, Bordeaux, France d Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy e IRCCS Centro Neurolesi ‘‘Bonino-Pulejo,’’ Messina, Italy b c

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

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Article history: Received 29 July 2010 Received in revised form 2 August 2011 Accepted 8 August 2011

Keywords: Allopregnanolone Cytochrome p450 side-chain cleavage Neurosteroid N-Palmitoylethanolamide Peroxisome proliferator-activated receptor alpha Steroidogenic acute regulatory protein

a b s t r a c t We investigated the involvement of de novo neurosteroid synthesis in the mechanisms underlying the analgesic and antihyperalgesic effects of N-palmitoylethanolamine (PEA) in two models of acute and persistent pain, the formalin test and carrageenan-induced paw edema. The pivotal role of peroxisome proliferator-activated receptor (PPAR)-a in the antinocifensive effect of PEA was confirmed by the lack of this effect in PPAR-a-null mice. PEA antinociceptive activity was partially reduced when the animals were treated with aminoglutethimide or finasteride, implying that de novo neurosteroid synthesis is involved in the effect of PEA. Accordingly, in the spinal cord, the allopregnanolone (ALLO) levels were increased by PEA treatment both in formalin- and carrageenan-exposed mice, as revealed by gas chromatography– mass spectrometry. In agreement with those data, in both pain models, PEA administration in challenged mice specifically restored the expression of two proteins involved in neurosteroidogenensis, the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) in the ipsilateral horns of spinal cord, without affecting their expression in the contralateral side. These results provide new information about the involvement of de novo neurosteroid synthesis in the modulation of pain behavior by PEA. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction N-Palmitoylethanolamine (PEA), the endogenous amide of palmitic acid and ethanolamine, belongs to the family of N-acylethanolamines, a class of lipid mediators. PEA exerts antinociceptive effects in several animal models [8,9,38,51] and inhibits peripheral inflammation and mast cell degranulation in rodents [3,34]. In the carrageenan-induced rat paw edema model, the anti-inflammatory effects of PEA have been associated with strong reduction of the levels of the inflammatory markers cyclooxygenase-2, inducible nitric oxide synthase, and malondialdehyde [11]. Among the molecular mechanisms proposed to explicate the effects of PEA, we have previously demonstrated that its anti-inflammatory and analgesic effects are dependent on peroxisome

⇑ Corresponding author. Tel.: +39 081 678413; fax: +39 081 678403. E-mail address: [email protected] (R. Meli).

proliferator-activated receptor (PPAR)-a. In fact, PEA failed to exert these properties in PPAR-a knockout mice [12,13,19,29–31]. We demonstrated that PEA interacts with this receptor with potency comparable with that of the synthetic PPAR-a agonist Wy14643, without activating other PPAR isoforms [29,30]. PPAR-a, as well as PPAR-c, is able to regulate inflammatory responses both in the periphery and in the central nervous system (CNS) [14,15,26]. Neurosteroids play a key role in cognitive and emotive functions and show analgesic and anti-inflammatory properties in several pathophysiologic conditions [7,17,22]. The first and rate-limiting step in the biosynthesis of all steroid hormones is the conversion of insoluble cholesterol to soluble pregnanolone, which is accomplished by cleavage of the cholesterol side chain, catalyzed by the mitochondrial cytochrome P450 enzyme (P450scc). This enzyme functions within the mitochondria [4] after delivery of cholesterol to the inner mitochondrial membrane due to steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine-type receptor [21,47,48].

0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.08.010

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Recently, we have demonstrated that PEA modulates the hypnotic effect induced by pentobarbital through a mechanism that involves neurosteroids, in particular the synthesis of 3a-hydroxy5a-pregnan-20-one (allopregnanolone, ALLO) [43]. The 5a-reduced neurosteroids act on GABAA (gamma-aminobutyric acid), as well as on peripheral benzodiazepine-type receptor, modulating GABA-induced Cl currents that result in neuronal hyperpolarization and exert several rapid effects, including the modulation of hypnosis [23,35]. Among neuroactive steroids, ALLO displays anxiolytic, sedative, analgesic, and anesthetic properties [42]. We hypothesized that neurosteroid formation could contribute to the antinociceptive effects of PEA. To test this possibility, we analyzed the effects of two inhibitors of neurosteroid biosynthesis, aminoglutethimide and finasteride, on PEA-induced analgesia in acute and persistent inflammatory pain models in mice. We also evaluated the capability of peripheral injection of PEA to modulate pain perception through de novo synthesis of neurosteroids in the spinal cord.

2. Methods 2.1. Animals Male Swiss mice (20–25 g; Harlan, Udine, Italy) were housed in stainless steel cages in a room kept at 22 ± 1°C on a 12/12-h light/ dark cycle. The animals were acclimated to their environment for 1 week with ad libitum access to water and food. Mice (4–5 weeks old; 20–22 g) with a targeted disruption of the PPAR-a gene (PPAR-a knockout) and their wild-type littermate controls (PPAR-a wild type) were purchased from Jackson Laboratories (Harlan Nossan, Udine, Italy). Mice homozygous for the PparatniJGonz-targeted mutation are viable and fertile, and they seem normal in appearance and behavior [18]. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (DM 116192), as well as with European Economic Community regulations (OJ of E.C. L 135 358/1 12/18/1986). 2.2. Chemicals

administered by i.p. injection in 200 lL of the same vehicle 30 min before formalin. After injections, animals were immediately transferred to a transparent observation chamber where pain behavior (time spent licking and biting the injected paw) was continuously monitored for 45 min. In the mouse, intraplantar injection of formalin produces a biphasic behavioral reaction (phase I, 0–15 min; phase II, 15– 45 min). This nocifensive behavior consists of an initial phase, with a peak occurring about 5 min after the injection, and then a second phase that peaks between 30 and 40 min. The intensities and durations of these behaviors are dependent on the concentration of formalin that is administered [2,10,31,41]. 2.3.2. Carrageenan-induced hyperalgesia Carrageenan-induced paw edema was induced by injecting kcarrageenan (1% w/v in sterile water, 20 lL) subcutaneously into the plantar surface of the left hind paw of mice. Animals were used 24 h after the paw injection, a period during which animals exhibited a clear mechanical and thermal heat hyperalgesia; they were treated with PEA (50 lg/10 lL; intraplantarly) dissolved in 80% of 0.9% sterile saline/10% polyethylene glycol 400/10% Tween 80. Neurosteroid enzymes inhibitors were administered by i.p. injection in 200 lL of vehicle the 30 min before PEA administration. Mechanical and thermal hyperalgesia were measured 30 min after vehicle or PEA administration. Mechanical hyperalgesia was determined by measuring the latency in seconds to withdraw the paw away from a constant mechanical pressure exerted onto the dorsal surface. We used an apparatus that we made ourselves following the description reported by Lo Verme et al. [31]. Briefly, a 15-g calibrated glass cylindrical rod (diameter 10 mm) chambered to a conical point (diameter 3 mm) was used to exert the mechanical force. The weight was suspended vertically between two rings attached to a stand and was free to move vertically. A cutoff time of 180 s was used. Withdrawal thresholds were measured 30 min after i.pl. PEA administration. Thermal hyperalgesia was assessed by the method of Hargreaves et al. [20] by measuring the latency to withdraw the hind paw from a focused beam of radiant heat (thermal intensity: infrared 3.0) applied to the plantar surface with a plantar test apparatus (Ugo Basile, Comerio, Italy). The cutoff time was set at 35 s.

N-Palmitoylethanolamine (PEA) was purchased from Tocris Cookson (UK), and aminoglutethimide (AMG), 3a-hydroxy-5apregnan-20-one (allopregnanolone, ALLO), and finasteride (FIN) from Steraloids (Newport, RI). For quantitative analysis of ALLO, deuterium-labeled standards (P98% chemical purity) 3a-hydroxy5a-pregnan-20-one-d4 (allopregnanolone-d4) was a gift from Dr Purdy (Scripps Research Institute, La Jolla, CA). Derivatizing agents including pyridine, O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (Florox), N,O-bis(trimethylsilyl) trifluoroacetamide with 1%; trimethylchlorosilane (BSTFA + TMCS), k-carrageenan, and formaldehyde were purchased from Sigma–Aldrich (St Louis, MO, USA). Fresh drug solutions were prepared immediately before use in a vehicle 80% of 0.9% sterile saline/10% polyethylene glycol 400/10% Tween 80.

2.3.3. Rotarod To evaluate motor neurologic deficits of mice, we used a rotarod test, which measured balance, coordination, and motor control. The rotarod apparatus (Ugo Basile, Comerio, Italy) consisted of a suspended rod able to turn at a constant or accelerating speed. All mice were exposed to a 5-min training period at a constant speed (4.5 rpm) immediately before the test to familiarize them with the apparatus. During the test, the mice had to remain on the rod for as long as they could. The time spent by the animals on the rotarod was recorded (a 60-s maximal trial was used for the test) 30 and 60 min after i.p. injection of both neurosteroid enzyme inhibitors.

2.3. Behavioral testing

In another set of experiments, the spinal cord (9 animals/group) from animals subjected to formalin test (15 and 45 min after formalin administration) or to carrageenan edema at 24 h (30 min after PEA or vehicle treatment) was exposed by laminectomy, and the lumbar cord was excised, bisected longitudinally into ipsilateral and contralateral halves, and homogenized on ice in lysis buffer (20 mM Tris–HCl pH 7.5, 10 mM NaF, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, leupeptin and trypsin inhibitor 10 lg/mL; 0.25 mL per 50 mg tissue).

2.3.1. Formalin test We injected a 5% formalin solution (10 lL) subcutaneously into the plantar surface of the left hind paw of mice with a 27-gauge needle fitted to a microsyringe. This solution contained PEA (0.01–50 lg) or its vehicle (80% of 0.9% sterile saline/10% polyethylene glycol 400/10% Tween 80). Stock solution of formalin was obtained in sterile saline. Neurosteroid enzymes inhibitors were

2.4. Western blot analysis of total tissue protein extracts from spinal cord

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Fig. 1. (A) Time course of PEA effect on formalin test and (B) evaluation of PEA antinocifensive effect on early (phase I, 0–15 min) or late (phase II, 15–45 min) phase. PEA (0.01–50 lg/10 lL) was dissolved in the formalin solution and i.pl. injected. Thereafter, animals (n = 6) were immediately transferred to a transparent observation chamber, where nocifensive behavior (time spent licking and biting the injected paw) was continuously monitored for 45 min. Nocifensive response in phase I (0–15 min) and phase II (15–45 min) were expressed as mean ± SEM. (C) Effect of PEA (50 lg/10lL) on formalin test in wild-type (+/+) and (D) PPAR-a knockout ( / ) mice. Control animals received i.pl. vehicle. ⁄⁄P < .01 and ⁄⁄⁄P < .001 vs vehicle.

After 1 h, lysates prepared from ipsilateral or contralateral halves were centrifuged at 100,000g for 15 min at 4°C, and supernatant was stored at 80° until use. All procedures were performed on ice with ice-cold reagents. Protein concentration of the extracts were estimated by a protein assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as standard. Total lysate (for StAR and P450scc) containing equal amount of protein was separated on sodium dodecyl sulfate–

polyacrylamide minigels and transferred onto nitrocellulose membranes (Protran Nitrocellulose Transfer Membrane Schleicher & Schuell Bioscience, Dassel, Germany), blocked with phosphate-buffered saline (PBS) containing 5% nonfat dried milk for 45 min at room temperature, and incubated at 4°C overnight in the presence of commercial antibodies for StAR (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:500); or P450scc (Chemicon International Inc., Temecula, CA, USA; dilution 1:500) in PBS containing 5% nonfat dried milk, 0.1% Tween 20. The secondary antibody (anti-mouse IgG, or anti-rabbit IgG, peroxidase conjugate; Jackson Laboratories, Bar Harbor, ME, USA) was incubated for 1 h at room temperature. Blots were washed with PBS, developed using enhanced chemiluminescence detection reagents (Amersham Pharmacia, Piscataway, NJ, USA) following the manufacturer’s instructions, and exposed to X-Omat film (Eastman Kodak, Rochester, NY, USA). Protein bands for StAR (30 kDa) and P450scc (50 kDa), were quantified with a model GS-700 imaging densitometer (Bio-Rad, Hercules, CA, USA). To ascertain that blots were loaded with equal amounts of protein lysates were analyzed for the expression of a-tubulin protein (dilution 1:1000; Sigma–Aldrich, Milan, Italy). 2.5. Quantitative analysis of neurosteroids

Fig. 2. Effect of steroidogenic protein inhibitors on PEA effect in formalin test: aminoglutethimide (AMG, 20 mg/kg), a P450scc inhibitor, or finasteride (FIN, 20 mg/kg), a 5a-reductase inhibitor, was i.p. injected 30 min before formalin challenge. After injections, nocifensive behavior was measured in mice (n = 6 in each group) and expressed as mean ± SEM. Control animals received i.pl. vehicle. ⁄ P < .05, ⁄⁄P < .01, and ⁄⁄⁄P < .001 vs vehicle, and °P < .05 vs PEA.

ALLO in spinal cord samples from animals subjected to formalin test (15 min and 45 min after PEA and formalin administration) or to carrageenan paw edema (30 min after PEA treatment), was isolated by solid-phase extraction on C18 columns (Varian Inc., Palo Alto, CA, USA) preconditioned with 2 mL methanol and 2 mL of 5%

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methanol in water. The loaded columns were eluted with 2 mL methanol. The eluate was dried under a nitrogen stream and derivatized. For the evaluation of recovery and reproducibility, and for the generation of calibration curves, 200 lL of a spinal cord (20 mg of tissue) was used for each analysis. The response was linear in the range of 0.5–32 pg; a standard curve generated with 20 pg of internal standard was interpolated for quantification of neurosteroid in samples. Derivatized samples and standards were analyzed by gas chromatography–mass spectrometry according to the method of Valleé et al. [50] with slight modifications. Briefly, neurosteroids were Q3 reacted with 40 lL pyridine and 20 lL Florox at 5 mg/mL to the evaporated spinal cord samples at 70°C for 45 min. The reaction mixture was evaporated off under a stream of nitrogen, and trimethylsilyl derivatives were formed by adding 15 lL of ethyl acetate and 15 lL of BSTFA and reacted for 20 min at 70°C. The derivatized samples were injected directly into a gas chromatograph–mass spectrometer (GC–MS; QP2010; Shimadzu, Tokyo, Japan) via an autosampler. The instrument was used in negative ion chemical ionization (NICI) mode with methane as reactant gas (Air Products, Walton on Thames, UK). A 15-m Rtx-5Sil MS (Restek, Bellefonte, PA, USA) with a 0.25-mm inside diameter and 0.1-lm film thickness was used for analytic resolution. Helium (ultrahigh purity, Linde Gas) at a linear velocity set at 60 cm/s at 160°C was used as the carrier gas. Splitless injections of 1-lL volume were made at injector temperature of 260°C. Splitless injection conditions were maintained for 0.5 min after the sample injection. The capillary column temperature was maintained at 160°C for 1 min, and the injector contents were split at a ratio of 25:1. The column oven was heated to 230°C at a rate of 60°C/min. The column oven temperature was subsequently raised to 260°C at a rate of 4°C/min and finally to 290°C at 60°C/min. The transfer line temperature was maintained at 290°C. Derivatized neurosteroids were first analyzed qualitatively by full scan in the mass range of 150–600. For quantification, the mass spectrometer was operated in the selected ion monitoring mode. The limit of quantification of GC–MS(LOQ) is defined as the signal equivalent to 10 times the noise (signal-to-noise ratio of 10:1).

respond to PEA compared to wild-type (+/+) animals. No significant differences in licking time were shown between wild-type and knockout mice in both phases. 3.2. Role of neurosteroids on PEA-induced antinociceptive effect The P450scc inhibitor, AMG, or the 5a-reductase inhibitor, FIN, was used to determine whether neurosteroids were involved in PEA antinociceptive effect. PEA effect was partially reversed in phases I and II by both inhibitors (P < .05, Fig. 2), when mice were pretreated with AMG or FIN (20 mg/kg) 30 min before challenge. The doses of AMG and FIN we used are able to block P450scc and 5a-reductase in CNS [1,16,25]. In preliminary experiments, we created dose–response curves for both inhibitors and to find the dose (20 mg/kg) that was inactive on the pain tests (formalin and carrageenan) when administered alone (Supplementary Data, Figs. 1 and 2). Moreover, we evaluated the effect of the chosen dose on the rotarod test to exclude a possible muscle relaxant or sedative effect for both inhibitors. Both drugs at the doses used did not modify locomotor activity, suggesting the absence of a sedative effect (Supplementary Table 1). 3.3. Antihyperalgesic action of PEA and neurosteroid contribution in carrageenan-challenged mice To evaluate the antihyperalgesic role of PEA in the carrageenan model of inflammatory pain, we measured nociceptive thresholds in untreated and carrageenan-treated mice after vehicle or PEA

2.6. Statistical analysis The significance of differences among groups was determined by 2-way analysis of variance followed by Bonferroni post-hoc test for multiple comparisons.

3. Results 3.1. Involvement of PPAR-a in the antinociceptive effect PEA on formalin test We first tested PEA’s ability to reduce the nocifensive behavior in a formalin test (Fig. 1A and B). As reported in Fig. 1A, obtained analyzing PEA effect at increasing doses every 5 min, the 2 phases of test are clearly marked. An intense early phase subsides approximately 5–15 min after formalin injection with a peak at about 5 min. The behaviors reappear and last another 35 min or longer, with a second peak at about 35 min. Here, we showed that PEA induced a dose-dependent reduction of nocifensive behavior (0.01– 50 lg/paw), which was significant at the highest doses (5 and 50 lg/paw) at different early and delayed interval times. Fig. 1B illustrates the dose-dependent reduction in both early and late phases of formalin-induced nociception by PEA. PEA effect was significant at 5 and 50 lg/paw (P < .01 and P < .001, respectively). As evidenced in Fig. 1C and D, the obligatory role of PPAR-a in the antinociceptive effect of PEA (50 lg/paw) was demonstrated by the finding that PPAR-a knockout animals ( / ) failed to

Fig. 3. Effect of neurosteroidogenic protein inhibitors on PEA antinociceptive effect in carrageenan-challenged mice: aminoglutethimide (AMG, 20 mg/kg), a P450scc inhibitor, or finasteride (FIN, 20 mg/kg), a 5a-reductase inhibitor, was i.p. injected 30 min before PEA administration. After neurosteroidogenic protein inhibitors and/ or PEA administration, withdrawal latency for mechanical (A) and thermal heat hyperalgesia (B) was measured in mice (n = 6 in each group) and expressed as mean ± SEM. Control animals received i.pl. vehicle. ⁄⁄P < .01 and ⁄⁄⁄P < .001 vs vehicle, °P < .05 vs PEA, and ###P < .001 vs baseline.

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(50 lg/paw) administration. As expected (Fig. 3A and B), 24 h after the injection of the phlogogen agent in the hind paw, we observed a significant reduction of mechanical and thermal threshold values (P < .001). Both hyperalgesic parameters were strongly reduced by PEA (Fig. 3A and B), as evidenced by the significant increase in withdrawal latency of hind paws (P < .001). To confirm neurosteroid involvement in the PEA antihyperalgesic effect, animals were treated with either AMG or FIN (20 mg/kg) 30 min before administration of PEA or vehicle. AMG and FIN did not themselves modify the mechanical and thermal thresholds; however, they were able to partially (33%) reduce the antihyperalgesic effect of PEA in both measurements (Fig. 3A and B). 3.4. Effect of PEA on StAR and P450scc expression in the spinal cord To highlight the involvement of de novo neurosteroid synthesis in PEA-induced analgesia, we evaluated the expression of StAR and P450scc in ipsilateral and contralateral halves of spinal cord, the first station of pain signal traveling to brain. Both these proteins are involved in cholesterol metabolism and have been implicated

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in the early steps of neurosteroidogenesis. Although StAR is a key protein, indicating an indirect rate of cholesterol transfer into the mitochondria, the expression of P450scc, which is the first and rate-limiting enzyme in steroidogenesis, represents the net steroidogenic capacity. Western blot analysis of StAR and P450scc was performed 15 and 45 min after formalin injection downstream the peaks of phases I and II to evaluate the biochemical modifications that occurred throughout both phases (Fig. 4A and B). In formalin-challenged mice, we observed a significant reduction of both proteins only in the ipsilateral halves of the spinal cord, which was prevented by PEA. A similar pattern was observed in the carrageenan test, where 30 min after PEA treatment the reconstitution of StAR and P450scc in ipsilateral halves was noted (Fig. 5A and B). No modification was observed in the contralateral side in either test. 3.5. PEA induces ALLO synthesis in spinal cord of challenged mice We have previously reported that PEA, in another experimental model, modulates the pattern of neurosteroids biosynthesis, in

Fig. 4. Expression of StAR (A) and P450scc (B) in spinal cord bisected longitudinally into ipsilateral and contralateral halves of formalin-insulted mice killed 15 and 45 min after PEA administration. Representative immunoblots of StAR and P450scc are shown. The densitometric quantification of all determinations (n = 9 mice in each group) of StAR and P450scc expression are also reported. All data are expressed as mean ± SEM. Basal expression of both proteins was also reported. Equal loading was confirmed by atubulin staining. ⁄⁄P < .01 vs vehicle.

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Fig. 5. Expression of StAR (A) and P450scc (B) in spinal cord bisected longitudinally into ipsilateral and contralateral halves of carrageenan-insulted mice killed 30 min after PEA administration. Representative immunoblots of StAR and P450scc are shown. The densitometric quantification of all determinations (n = 9 mice in each group) of StAR and P450scc expression are also reported. All data are expressed as mean ± SEM. Basal expression of both proteins was also reported. Equal loading was confirmed by atubulin staining. ⁄⁄P < .01 vs vehicle.

particular ALLO [43]. Here, we measured the changes in ALLO levels in the whole spinal cord after PEA treatment in formalin- or carrageenan-challenged animals (Fig. 6A and B, respectively). Although pregnanolone is the first metabolite of cholesterol, ALLO is synthesized from progesterone via two enzymatic reactions: 5a reduction of progesterone, yielding dihydroprogesterone, followed by 3a reduction of the C3 ketone, mediated by 3a-hydroxysteroid dehydrogenase. As depicted in Fig. 6A, when coadministered with formalin, PEA induced a significant increase of ALLO in spinal cord both in phase I and II of the formalin test, as measured at 15 and 45 min. PEA did not affect ALLO levels after 15 and 45 min without formalin challenge. At 24 h after carrageenan treatment (Fig. 6B), ALLO levels were significantly reduced in the spinal cord (P < .01); in this persistent and inflammatory pain, PEA prevented the decrease of ALLO. 4. Discussion In the present study, we have demonstrated that the antinociceptive effects of PEA in two models of acute and persistent pain are partially due to the activation of a neurosteroid pathway. PEA

exerts a dose-dependent analgesic effect, which is significantly reduced by the 5a-reductase inhibitor FIN, suggesting the involvement of a 5a-reduced metabolite in this effect. Moreover, our results confirm the previously defined role for PPAR-a in pain modulation by PEA [29] because the antinociceptive effect of this endogenous ethanolamide is absent in PPAR-a knockout mice. Although the key role of PPAR-a in several effects of PEA is well established [12,13,29–31], the molecular mechanisms underlying PEA activity remain to be fully clarified. It has been suggested that ligand-activated PPAR-a may suppress pain responses by altering the gating properties of IKCa and BKCa channels in dorsal root ganglia neurons, in agreement either with the presence of PPAR-a in this first relay station of pain or with the suppression of nocifensive behavior in the formalin test, where PEA was administered into the paw [31]. However, we have investigated other mechanisms that could be involved after PEA activation of PPAR-a. The present study provides once again concrete evidence that PPAR-a and its putative endogenous ligand, PEA, could regulate neurosteroidogenesis in the spinal cord, which has the enzymatic machinery for neurosteroid biosynthesis. These findings also confirm our recent data on the relationship between PEA and de novo

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Fig. 6. Spinal cord ALLO levels, expressed as ng/g of tissue, were obtained from naive, untreated (vehicle), or PEA-treated (50 lg/10 lL) animals (n = 6 mice in each group) 15 or 45 min after formalin solution injection (A) or 30 min after PEA administration in carrageenan-challenged mice (B). All data are expressed as mean ± SEM. Basal level of neurosteroid was also reported. ⁄⁄⁄P < .001 vs formalin or carrageenan; ##P < .01 vs naive; °P < .05, °°°P < .001 vs PEA.

neurosteroidogenesis in CNS [43]. We showed that PEA modulates pentobarbital-evoked hypnosis by activating PPAR-a and subsequently inducing de novo neurosteroid synthesis. In particular, the increased ALLO levels in the brain stem in PEA-treated mice could lead to a positive modulation of GABAA receptor, reinforcing the hypnotic effect of pentobarbital [43]. Among neuroactive steroids, ALLO seems to share many of the effects showed by PEA, including analgesic, anticonvulsant, and antiallodynic activities [25,29–31,53]. This hormone has been recognized to be a positive activator of the GABAA receptor, increasing the inward Cl currents that lead to neuronal hyperpolarization, also achieved by PEA through the opening of the IKCa and BKCa channels. We hypothesized that two separate but converging mechanisms could contribute to the antinociceptive effect of PEA, an early molecular control through IKCa and BKCa channels opening [31], and thereafter a reinforcing effect mediated by gene transcription and hence neurosteroid synthesis. Therefore, hypothesizing a role for the de novo synthesis of ALLO in PEA effect, we used AMG, an inhibitor of P450scc, a key enzyme in the biosynthesis of all neurosteroids and FIN as inhibitor of 5a-reductase, to reduce the availability of 5a-reduced neurosteroids, including ALLO. Indeed, AMG, beyond its inhibitory activity on P450scc, is also considered a weak inhibitor of aromatase. It is well known that the inhibitors of aromatase have a hyperalgesic activity as a result of their capability to inhibit estradiol synthesis, and subsequently they reduce its protective activity on dorsal root ganglia [45]. AMG did not show any hyperalgesic effect in either test (Supplementary Data). AMG and FIN were used at doses able to block the P450scc and the 5a-reductase in CNS [1,16,25]. We created dose–response curves for both inhibitors (Supplementary Data) and used the doses that were inactive on the both pain tests when administered alone. The significant antinociceptive effect of both inhibitors at the highest doses (30–50 mg/kg) highlighted a role for both enzymes and hence for neurosteroids in pain perception.

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Moreover, we tested their effect on via the rotarod test to exclude possible muscle relaxant or sedation activities. Both drugs at the doses we used did not modify locomotor activity, suggesting the absence of a sedative effect (Supplementary Data). As expected, the inhibitors partially reduced the PEA effect, suggesting a contribution of ALLO in the antinociceptive effects of PEA on acute or persistent inflammatory pain. This finding was supported by the observation that the levels of ALLO in the spinal cord were increased by PEA. The biosynthesis of steroids in steroidogenic tissues starts with the enzymatic conversion of cholesterol to pregnenolone. This reaction is catalyzed by P450scc, which is located on the matrix side of the inner mitochondrial membrane. Here, we report data indicating that PEA-induced increase in ALLO biosynthesis is related to a selective increase in P450scc expression of the ipsilateral rather than the contralateral dorsal horns. The rate-limiting step of steroidogenesis, which was previously thought to be the production of pregnenolone, is now attributed to the prior step of cholesterol transfer from the outer to the inner mitochondrial membrane, where the P450scc is located [27,37,39]. It has been suggested that StAR, localized in mitochondria, is a critical protein in the regulation of cholesterol availability in peripheral and central steroidogenic cells [24,36,46]. StAR, synthesized as a 37-kDa preprotein, is rapidly imported into the mitochondria and processed to the inactive 30-kDa form, found in the mitochondrial matrix. The Cterminal region contains a hydrophobic pocket that might be involved in desorption of cholesterol from the sterol-rich outer membrane to the sterol-poor inner membrane. The N-terminal domain targets StAR in the mitochondria, and its cleavage effectively terminates the delivery of cholesterol to P450scc and therefore the synthesis of pregnenolone [6,33,48,49]. In our experimental conditions, carrageenan and formalin reduced the expression of StAR and P450scc in the spinal horn involved in pain process, resulting in the decrease of ALLO level, whereas PEA administration restores and/or increases protein expression and ALLO content. Interestingly, the modulation of both proteins involved in cholesterol metabolism are specifically modified in the ipsilateral side and not in the contralateral one, confirming that only the fired neurons are involved in pain transmission at spinal level. Therefore, only in the ipsilateral horns does PEA restore the expression of StAR and P450scc, without affecting the contralateral side. These data strongly suggest that even in pain models characterized by the activation of different pathways in the CNS and timing in nocifensive behavior, the analgesic effect of PEA is mediated by ALLO. In fact, the formalin test induces tonic, long-lasting pain and different behavioral responses mediated at different levels of the CNS (ie, flinching, a phasic short contraction of the leg, present also in spinalized animals; flexing, a tonic contraction of the injected paw; licking, mediated at higher supraspinal levels), whereas in carrageenan-induced edema, gene transcription plays a crucial role in pain control [28,44]. Our data are consistent with our recent findings about the pivotal role that PPAR-a plays in the transcriptional control of proteins involved in cholesterol metabolism in C6 cells or astrocytes stimulated by PEA [40]; their expression was induced by PEA and blunted by PPAR-a antagonism or PPAR-a silencing by RNA interference. Accordingly, ALLO levels were increased in supernatant of PEA-treated astrocytes, and this effect was inhibited by PPAR-a antagonist, GW6471. On the basis of this evidence, we demonstrated that PEA could act in a coordinated manner to induce steroidogenesis and increase the ALLO level, with which it shares a similar pharmacologic profile. We cannot exclude the involvement of other 5a-reduced neurosteroids (ie, dihydroprogesterone, 3a,5a-tetrahydrodeoxycorticosterone) or even sex hormones (testosterone or estradiol) known to play a part

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in pain perception [32,52], such as testosterone and its antihyperalgesic effects [5]. In conclusion, this study further clarifies the role of PEA in pathologic status and illustrates a novel aspect of the mechanism of action of PEA after PPAR-a activation. PEA could be a candidate for a drug that would control pain perception as a result of the activation of both the nongenomic and genomic pathways that cooperate in nociception maintenance. Conflict of interest statement This study was performed without any financial or other contractual agreements that may cause conflict of interest. Acknowledgments We thank Giovanni Esposito and Angelo Russo for animal care and assistance. We are grateful to Dr Claudio Fiorelli for nuclear magnetic resonance analysis. This study was supported by a grant from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica PRIN 2007, Italy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pain.2011.08.010. References [1] Ahmad B, Nicholls PJ. Development of tolerance to the CNS effects of aminoglutethimide in mice. Eur J Pharmacol 1990;182:237–44. [2] Aloisi AM, Albonetti ME, Carli G. Behavioural effects of different intensities of formalin pain in rats. Physiol Behav 1995;58:603–10. [3] Berdyshev E, Boichot E, Corbel M, Germain N, Lagente V. Effects of cannabinoid receptor ligands on LPS-induced pulmonary inflammation in mice. Life Sci 1998;63:PL125–9. [4] Black SM, Harikrishna JA, Szklarz GD, Miller WL. The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc. Proc Natl Acad Sci USA 1994;91:7247–51. [5] Borzan J, Fuchs PN. Organizational and activational effects of testosterone on carrageenan-induced inflammatory pain and morphine analgesia. Neuroscience 2006;143:885–93. [6] Bose HS, Lingappa VR, Miller WL. The steroidogenic acute regulatory protein, StAR, works only at the outer mitochondrial membrane. Endocr Res 2002;28:295–308. Neuroreport 1996;8:139–41. [7] Burade VS, Jain MR, Khan FA, Saha SG, Subhedar N. Involvement of corticosteroid-like neurosteroids in pentobarbital-induced sleep. Neuroreport 1996;8:139–41. [8] Calignano A, La Rana G, Giuffrida A, Piomelli D. Control of pain initiation by endogenous cannabinoids. Nature 1998;394:277–81. [9] Calignano A, La Rana G, Piomelli D. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur J Pharmacol 2001;419:191–8. [10] Clavelou P, Dallel R, Orliaguet T, Woda A, Raboisson P. The orofacial formalin test in rats: effects of different formalin concentrations. Pain 1995;62:295–301. [11] Costa B;Conti S;Giagnoni G;Colleoni M. Therapeutic effect of the endogenous fatty acid amide, palmitoylethanolamide, in rat acute inflammation: inhibition of nitric oxide and cyclo-oxygenase systems. Br J Pharmacol 2002;137:413–20. [12] D’Agostino G, La Rana G, Russo R, Sasso O, Iacono A, Esposito E, Raso GM, Cuzzocrea S, Lo Verme J, Piomelli D, Meli R, Calignano A. Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist, modulates carrageenan-induced paw edema in mice. J Pharmacol Exp Ther 2007;322:1137–43. [13] D’Agostino G, La Rana G, Russo R, Sasso O, Iacono A, Esposito E, Mattace Raso G, Cuzzocrea S, Lo Verme J, Piomelli D, Meli R, Calignano A. Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-kappaB nuclear signalling in dorsal root ganglia. Eur J Pharmacol 2009;613:54–9. [14] Delerive P, Fruchart JC, Staels B. Peroxisome proliferator-activated receptors in inflammation control. J Endocrinol 2001;169:453–9. [15] Devchand PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature 1996;384:39–43. [16] Gabriel KI, Cunningham CL, Finn DA. Allopregnanolone does not influence ethanol-induced conditioned place preference in DBA/2J mice. Psychopharmacology (Berl) 2004;176:50–6.

[17] Gazouli M, Yao ZX, Boujrad N, Corton JC, Culty M, Papadopoulos V. Effect of peroxisome proliferators on Leydig cell peripheral-type benzodiazepine receptor gene expression, hormone-stimulated cholesterol transport, and steroidogenesis: role of the peroxisome proliferator-activator receptor alpha. Endocrinology 2002;143:2571–83. [18] Genovese T, Mazzon E, Di Paola R, Cannavo G, Muia C, Bramanti P, Cuzzocrea S. Role of endogenous ligands for the peroxisome proliferators activated receptors alpha in the secondary damage in experimental spinal cord trauma. Exp Neurol 2005;194:267–78. [19] Haller VL, Cichewicz DL, Welch SP. Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice. Eur J Pharmacol 2006;546:60–8. [20] Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32:77–88. [21] Hauet T, Liu J, Li H, Gazouli M, Culty M, Papadopoulos V. PBR, StAR, and PKA: partners in cholesterol transport in steroidogenic cells. Endocr Res 2002;28:395–401. [22] Jung-Testas I, Baulieu EE. Steroid hormone receptors and steroid action in rat glial cells of the central and peripheral nervous system. J Steroid Biochem Mol Biol 1998;65:243–51. [23] Keller AF, Breton JD, Schlichter R, Poisbeau P. Production of 5alpha-reduced neurosteroids is developmentally regulated and shapes GABA(A) miniature IPSCs in lamina II of the spinal cord. J Neurosci 2004;24:907–15. [24] King SR, Manna PR, Ishii T, Syapin PJ, Ginsberg SD, Wilson K, Walsh LP, Parker KL, Stocco DM, Smith RG, Lamb DJ. An essential component in steroid synthesis, the steroidogenic acute regulatory protein, is expressed in discrete regions of the brain. J Neurosci 2002;22:10613–20. [25] Kokate TG, Banks MK, Magee T, Yamaguchi S, Rogawski MA. Finasteride, a 5alpha-reductase inhibitor, blocks the anticonvulsant activity of progesterone in mice. J Pharmacol Exp Ther 1999;288:679–84. [26] Kostadinova R, Wahli W, Michalik L. PPARs in diseases: control mechanisms of inflammation. Curr Med Chem 2005;12:2995–3009. [27] Lambeth JD, Xu XX, Glover M. Cholesterol sulfate inhibits adrenal mitochondrial cholesterol side chain cleavage at a site distinct from cytochrome P-450scc. Evidence for an intramitochondrial cholesterol translocator. J Biol Chem 1987;262:9181–8. [28] Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev 2001;53:597–652. [29] Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A, Piomelli D. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol Pharmacol 2005;67:15–9. [30] Lo Verme J, La Rana G, Russo R, Calignano A, Piomelli D. The search for the palmitoylethanolamide receptor. Life Sci 2005;77:1685–98. [31] Lo Verme J, Russo R, La Rana G, Fu J, Farthing J, Mattace-Raso G, Meli R, Hohmann A, Calignano A, Piomelli D. Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-alpha. J Pharmacol Exp Ther 2006;319:1051–61. [32] Manson JE. Pain: sex differences and implications for treatment. Metabolism 2010;59:S16–20. [33] Mathieu AP, Fleury A, Ducharme L, Lavigne P, LeHoux JG. Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR. J Mol Endocrinol 2002;29:327–45. [34] Mazzari S, Canella R, Petrelli L, Marcolongo G, Leon A. N-(2hydroxyethyl)hexadecanamide is orally active in reducing edema formation and inflammatory hyperalgesia by down-modulating mast cell activation. Eur J Pharmacol 1996;300:227–36. [35] Mendelson WB, Martin JV, Perlis M, Wagner R. Sleep and benzodiazepine receptor sub-types. J Neural Transm 1987;70:329–36. [36] Mensah-Nyagan AG, Kibaly C, Schaeffer V, Venard C, Meyer L, Patte-Mensah C. Endogenous steroid production in the spinal cord and potential involvement in neuropathic pain modulation. J Steroid Biochem Mol Biol 2008;109:286–93. [37] Miller WL. Steroidogenic enzymes. Endocr Dev 2008;13:1–18. [38] Petrosino S, Palazzo E, de Novellis V, Bisogno T, Rossi F, Maione S, Di Marzo V. Changes in spinal and supraspinal endocannabinoid levels in neuropathic rats. Neuropharmacology 2007;52:415–22. [39] Privalle CT, McNamara BC, Dhariwal MS, Jefcoate CR. ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P450scc. Regulation of intramitochondrial cholesterol transfer. Mol Cell Endocrinol 1987;53:87–101. [40] Raso GM, Esposito E, Vitiello S, Iacono A, Santoro A, D’Agostino G, Sasso O, Russo R, Piazza PV, Calignano A, Meli R. Palmitoylethanolamide stimulation induces allopregnanolone synthesis in c6 cells and primary astrocytes: involvement of peroxisome-proliferator activated receptor-a. J Neuroendocrinol 2011;23:591–600. [41] Rosland JH, Tjølsen A, Maehle B, Hole K. The formalin test in mice. effect of formalin concentration. Pain 1990;42:235–42. [42] Rupprecht R, di Michele F, Hermann B, Ströhle A, Lancel M, Romeo E, Holsboer F. Neuroactive steroids: molecular mechanisms of action and implications for neuropsychopharmacology. Brain Res Brain Res Rev 2001;37:59–67. [43] Sasso O, La Rana G, Vitiello S, Russo R, D’Agostino G, Iacono A, Russo E, Citraro R, Cuzzocrea S, Piazza PV, De Sarro G, Meli R, Calignano A. Palmitoylethanolamide modulates pentobarbital-evoked hypnotic effect in

Ò

O. Sasso et al. / PAIN 153 (2012) 33–41

[44]

[45]

[46] [47] [48]

mice. involvement of allopregnanolone biosynthesis. Eur Neuropsychopharmacol 2010;20:195–206. Sawynok J, Reid A. Antinociception by tricyclic antidepressants in the rat formalin test: differential effects on different behaviours following systemic and spinal administration. Pain 2001;93:51–9. Schaeffer V, Meyer L, Patte-Mensah C, Eckert A, Mensah-Nyagan AG. Sciatic nerve injury induces apoptosis of dorsal root ganglion satellite glial cells and selectively modifies neurosteroidogenesis in sensory neurons. Glia 2010;58:169–80. Sierra A. Neurosteroids: the StAR protein in the brain. J Neuroendocrinol 2004;16:787–93. Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 2001;63:193–213. Stocco DM. Tracking the role of a star in the sky of the new millennium. Mol Endocrinol 2001;15:1245–54.

41

[49] Thomson M. Does cholesterol use the mitochondrial contact site as aconduit to the steroidogenic pathway? Bioessays 2003;25:252–8. [50] Valleé M, Rivera JD, Koob G, Purdy RH, Fitzgerald RL. Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry. Anal Biochem 2000;287:153–66. [51] Wallace VC, Segerdahl AR, Lambert DM, Vandevoorde S, Blackbeard J, Pheby T, Hasnie F, Rice AS. The effect of the palmitoylethanolamide analogue, palmitoylallylamide (L-29) on pain behaviour in rodent models of neuropathy. Br J Pharmacol 2007;151:1117–28. [52] Wiesenfeld-Hallin Z. Sex differences in pain perception. Gend Med 2005;2:137–45. [53] Zheng P. Neuroactive steroid regulation of neurotransmitter release in the CNS: action, mechanism and possible significance. Prog Neurobiol 2009;89:134–52.