Gen. Pharmac. Vol. 31, No. 5, pp. 753–758, 1998 ISSN 0306-3623/98 $–see front matterCopyright 1998 Elsevier Science Inc. PII S0306-3623(98)00108-6Printed in the USA. All rights reserved

Effect of Acetylsalicylic Acidon Formalin Test and on Serotonin System in the Rat Brain

Giovanni Vitale,1 Luigi-Alberto Pini,1*Alessandra Ottani2 and Maurizio Sandrini2

1Department of Internal Medicine and2Department of Biomedical Sciences, University of Modena, Modena, Italy

ABSTRACT. 1. Acetylsalicylic acid (ASA; 400 mg/kg, IP) increased serotonin (5-HT) content inrat brain but did not modify the number or the affinity of 5-HT1A receptors in the pons and the cerebralcortex, whereas the number of cortical 5-HT2 receptors decreased significantly.

2. Pretreatment with parachlorophenylaline (100 mg/kg/day for 4 days) depleted 5-HT brain con-tent but modified neither the serum levels of salicylates nor the 5-HT2 cortical receptor characteristics,and it abolished the antinociceptive effect of ASA, 400 mg/kg, in the first phase of the formalin test.

3. These data support the involvement of the central serotonergic system in the antinociceptive ac-tivity of ASA. gen pharmac 31;5:753–758, 1998. 1998 Elsevier Science Inc.

KEY WORDS. Nociception, ASA, formalin test, serotonin

INTRODUCTION 5-HT modulates the antinociceptive activity of drugs by apparentlyacting on different targets (Taber and Lantrany, 1981).The antinociceptive effects of nonsteroidal anti-inflammatory drugs

These data suggest that the antinociceptive activity of ASA could(NSAIDs) are widely held to be manifested in the periphery and todepend, at least in this experimental model, on the brain serotoner-be basically a consequence of the inhibition of prostaglandin syn-gic system.thesis (Vane, 1971; Vane, 1995). Dissociation between the antino-

The aim of this study was to investigate the influence of pretreat-ciceptive and the anti-inflammatory activity of several NSAIDs hasment with PCPA on the changes, induced by ASA, in antinocicep-been reported (McCormack and Brune, 1991). Interference withtion, in brain 5-HT content and in the number of 5-HT1A and 5-HT2G-protein-mediated signal transduction may form the basis of anreceptor subtypes in the cortical and pontine areas of the rat brain.analgesic mechanism unrelated to inhibition of prostaglandin syn-

thesis (Cashman, 1996), and a central component of the analgesicaction has been suggested by many authors (Abramson and Weiss- MATERIALS AND METHODSman, 1989; Bjorkman, 1995; Jurna and Brune, 1990; Malmberg and Animals and drug treatmentYaksh, 1992).

Adult male Wistar rats (Harlan-Nossan, SPF, Corezzano, Italy)The involvement of central monoaminergic pathways in the anti-weighing 180–190 g, were housed five per cage with free access tonociceptive mechanisms has been emphasized (Shyu and Lin,food and water, under controlled environmental conditions (tem-1985), and the role of serotonin (5-HT) in the modulation of theperature 228C, humidity 60%), and a 12-hr light–dark cycle (lightnociceptive stimuli has also been demonstrated (Pelissier et al.,on at 07.00). All the procedures were carried out according to the1995; Pini et al., 1995; Warner et al., 1990).EEC ethical regulations for animal research (EEC Council 86/609;The central serotonergic pathways have been claimed to exertD.L. 27/1/1992, No. 116).antinociceptive effects in defined brain areas through their receptor

Acetylsalicylate of lysine was injected intraperitoneally (IP) dis-subtypes, notably 5-HT1A (Eide and Hole, 1991) and 5-HT2 (Al-solved in sterile saline at a dose equivalent to 200, 300, and 400 mg/heider, 1991; Meller et al., 1991), as well as through interactionskg of ASA in a volume of 2 ml/kg body weight. The rats, dividedwith opioid (Nichols et al., 1989; Wigdor and Wilcox, 1987) andinto groups of eight, were subjected to the formalin test 30 ming-aminobutyric acid pathways (Tunnicliff, 1991).thereafter.We previously demonstrated that the antinociceptive effect of

PCPA (100 mg/kg, IP, in 2 ml/kg of sterile saline) or saline wasphenazone (Sandrini et al., 1993) and acetylsalicylic acid (ASA) ininjected daily for four consecutive days; ASA (400 mg/kg) or salinethe hot-plate test is prevented by treatment with parachloropheny-was administered 2 hr after the last dose, and the rats were thenlalanine (PCPA) (Pini et al., 1993, 1995); at the same time, the totaltested in the formalin test.number of 5-HT receptors in brain membranes changes, decreasing

At the end of the test, the rats were killed under ethyl ether anes-during the antinociceptive effect. Moreover, the injection of ASA in-thesia; their blood was collected and the sera were stored at 2808Ccreases serotonin and 5-hydroxyindolacetic acid concentrations inuntil required for assay, whereas the brain regions (pons and cerebralbrain (Groppetti et al., 1988), whereas pretreatment with PCPA re-cortex) were immediately dissected on an ice-cold surface, weighedduces the relative antinociceptive activity of morphine, indicating thatand stored at 2808C prior to membrane preparation or serotonindetermination.

*To whom correspondence should be addressed at: Clinical Pharmacology Two more groups of eight rats, treated with ASA, 400 mg/kg, orUnit, via del Pozzo, 71, I-41100 Modena Italy [Tel: 39 59 424 065; Fax: 39saline, were killed after the first phase of the formalin test (1 min59 424 069; E-mail: pinila@unimo.it].

Received 4 August 1997; accepted 28 April 1998. after drug administration).

754 G. Vitale et al.

Four additional groups of animals were tested with regard to theirbehavioral profile by using either ASA (200, 300 and 400 mg/kg)or saline.

One more group was treated with morphine, 8 mg/kg, SC, andsubjected to the formalin test 20 min thereafter to compare the anti-nociceptive potency of ASA with that of a reference compound.

Behavioral test

Behavioral tests were performed between 9:00 and 12:00 a.m. in asound-proof room by experienced observers unaware of the treat-ments.

Motor activity was measured in an activity cage by means of anultrasound apparatus (Cibertec, S.A., Barcelona, Spain) placed onthe lid of the cage. The number of movements was recorded contin-

FIGURE 1. Dose–response effect of acute treatment of ASA 200,uously for 1 hr after an adaptation period of 30 min.300 and 400 mg/kg, IP, or morphine 8 mg/kg, SC (MORP) ad-ministered 30 and 20 min, respectively, before the formalin test.

Formalin test Each histogram represents the total number of flinches (mean6SEM) of eight rats in phases 1, 2A and 2B. SAL, saline. *P,0.05Two hours before testing, the animals were placed individually in versus SAL (ANOVA followed by Student-Newman-Keuls test).

standard cages and, after an adaptation period, 50 ml of 5% formalinsolution was injected subcutaneously into the dorsal surface of theright hind paw by using a microsyringe with a 26-gauge needle. Pain cific activity 142.9 Ci/mmol, 0.18–6.0 nM; [3H]ketanserin, specific ac-behavior was monitored over a period of 60 min, the number of tivity 87.5 Ci/mmol, 0.09–3.0 nM.flinches or shakes of the injected paw being summed at 5-min inter-vals starting at time 0. Two phases of spontaneous flinching behav- Serotonin determinationior were observed: phase 1 from immediately after formalin injection

The brain areas were assayed for 5-HT determination by reverse-to 10 min thereafter; phase 2 began at time 10 min and a maximumphase high performance liquid chromatography, according to Grossiresponse was observed about 25 to 35 min after formalin injection.et al. (1990) with modifications.For the purpose of data analysis, the second phase was further di-

The thawed areas were homogenized, with an ultrasonic dismem-vided into 2A (10–39 min) and 2B (40–60 min) (Malmberg andbranator, in 0.1 M HClO4 containing 4 mM NaHSO4, 10 ml/mg ofYaksh, 1992). To prevent possible interference of room temperaturewet weight) and centrifuged at 2,000g for 15 min at 48C. After cen-on skin temperature, all experiments were performed at a room tem-trifugation, the acid supernatant was filtered on 0.22-mm filters be-perature of 22618C (Hole and Tjølsen, 1993).fore analysis.

5-HT assays were performed with a Beckman System Gold high-Membrane preparation and binding assay performance liquid chromatograph (Beckman Instruments, Inc.,

San Ramon, CA, USA) equipped with a ESA Coulochem II Multi-The binding of 5-HT1A receptors was determined according to theElectrode high-sensitivity electrochemical detector (ESA Inc., Bed-method of Gulati and Bhargava (1990). The binding of 5-HT2 re-

ceptors was evaluated according to the method of Leysen et al.(1981) with minor modifications. Brain regions were homogenizedin 5 ml of ice-cold 0.25 M sucrose (12 strokes of a Teflon pestle at120 rpm) and centrifuged at 1,300g for 10 min at 48C.

This procedure was repeated; then the combined sucrose surna-tants were diluted with 10 ml 50 mM Tris HCl, pH 7.7, and the sus-pension was centrifuged at 3,500g for 10 min. The pellet was resus-pended in 20 ml of Tris HCl buffer and centrifuged once at 50,000gfor 10 min. The resultant pellet was then resuspended in the samebuffer and frozen at 2208C. On the day of assay, the frozen mem-branes were allowed to thaw at room temperature and were then ho-mogenized and diluted in Tris HCl (about 300 mg protein/ml). Ali-quots of membranes (800 ml) were placed in plastic test tubescontaining [3H]ketanserin (100 ml; six concentrations in 10% etha-nol), methysergide (100 ml; 1 mM, dissolved in 10% ethanol to de-fine nonspecific binding) or ethanol at 378C for 15 min. The mix-ture was filtered under reduced pressure through GF/B filters(previously soaked in 0.5% polyethyleneimine for 10 min) by using

FIGURE 2. Effect of ASA 200, 300 and 400 mg/kg, IP, on sero-a Millipore vacuum pump and rapidly rinsed twice with 5 ml ice- tonin content in cortical and pontine brain areas. Rats were killedcold Tris buffer. The filters were transferred to plastic vials con- immediately after the formalin test; brain areas were weighed andtaining 6 ml of Opti-fluor (Packard) and shaken. The vials were stored frozen at 2808C until assayed. Values are expressed as means6for 20 hr at 48C in the dark. The following concentrations were used: SEM of eight rats for each group. SAL, saline. *P,0.05 versus

SAL (ANOVA followed by Student-Newman-Keuls test).[3H]8-hydroxy-2-(d-n-propyl-amino)-tetralin ([3H]8-OH-DPAT), spe-

ASA and 5-HT-Mediated Antinociception 755

TABLE 1. Influence of the treatment of ASA on the number and affinity of 5-HT1A receptorsin the cortical and pontine membranes of rat brain

Cortex Pons

Treatment Bmax KD Bmax KD

(mg/kg IP) (fmol/mg/prot) (nM) (fmol/mg/prot) (nM)

Saline 285.3 6 18.0 2.2 6 1.7 199.7 6 25.3 3.8 6 0.9ASA 200 252.3 6 12.5 2.0 6 0.3 N.P. N.P.ASA 300 246.1 6 17.9 2.5 6 0.2 207.8 6 21.7 3.5 6 1.3ASA 400 231.9 6 18.9 2.4 6 0.6 205.3 6 20.8 3.2 6 0.5

The brain membranes were collected 90 min after ASA IP administration. Values are expressed as mean 6SEM of seven brains.

N.P., not performed; P . 0.05 (ANOVA).

ford, MA, USA) with conditioning cell set at 20.75 V, detector 1 set rized as arithmetic means and were correlated by using ANOVA fol-lowed by the Student-Newman-Keuls test. A two-way ANOVA wasat 10.05 V and detector 2 set at 10.25 V, response 2, gain 1035.

A reverse-phase C-18 10 cm34.6 mm Hypersil column (Labser- used to analyze the effects of PCPA pretreatment, ASA treatmentand their interaction, followed by a 232 factorial analysis based onvice Analytical, Bologna, Italy) packed with 3-mm ODS was used.

The mobile phase, composed of methanol 15%, acetonitrile 8% orthogonal comparisons.The data were evaluated with the Student-Newman-Keuls testand 50 mM NaH2PO4, pH 2.8, with 0.2 mM ethylendiaminete-

traacetic acid disodium salt and 200 mg/l sodium octyl sulfate, was when the evaluation concerned PCPA or ASA single effects.pumped at a rate of 1 ml/min.

3,4-Dihydroxybenzylamine (DHBA; the internal standard) and Chemicals5-HT were used as standards. Standards and samples were quanti-

Acetylsalicylate of lysine was provided by Sanofi-Wintrop (Milan,tated according to analite/DHBA ratio in a calibration curve.Italy). Morphine sulfate, PCPA, DHBA hydrobromide salt and 5-HThydrochloride were purchased from Sigma Chemical Co. (St. Louis,

Drug assay MO, USA). [3H]8-OH-DPAT and [3H]ketanserin were from DuPont NEN, Co. Ltd (Milan, Italy). Formalin was obtained throughSerum salicylates were determined by fluorescence polarization im-

munoassay; the analyses were performed with a TDx analyzer (Ab- Bracco Chemical Co. (Milan, Italy).bot Laboratories, Chicago IL, USA).

RESULTSData analysis In the formalin test, ASA showed a dose–response effect, depending

the phase studied: an antinociceptive effect in phase 2A at the doseThe data were expressed as means6SEM. The results obtained inthe behavioral test were analyzed by using one-way analysis of vari- of 200 mg/kg was observed, whereas, in phase 1 and in phase 2B,

ASA was active only at 300 and 400 mg/kg (Fig. 1).ance (ANOVA) for motor activity.The data obtained from formalin experiments as well as the val- These results were obtained without modifying motor activity:

the number of movements was 1,423678, 1,429676, 1,436680 andues of 5-HT cerebral levels were evaluated by one-way ANOVA fol-lowed by the Student-Newman-Keuls test, or Student’s t-test, when 1,435667 for 200, 300, and 400 mg/kg of ASA- or saline-treated

rats, respectively (ANOVA; P.0.05).appropriate.The results of binding experiments were analyzed according to Figure 2 shows that ASA increases the serotonin content in corti-

cal areas at the dose of 300 mg/kg and in cortical and pontine areasthe method of Rosenthal (1967). The equilibrium dissociation con-stant (KD) and the maximum number of binding sites (Bmax) were at the dose of 400 mg/kg. The number of 5-HT1A receptors, as well

as the affinity for [3H]8-OH-DPAT, was not affected at the maxi-evaluated individually for each sample by using six concentrationsof labeled drug. mum dose used (Table 1). On the other hand, at the dose of 300

mg/kg, ASA decreased the number of 5-HT2 receptors with the useAll assays were performed in duplicate; KD and Bmax were summa-

TABLE 2. Influence of the treatment of ASA on the number and affinity of 5-HT2 receptorsin the cortical and pontine membranes of rat brain

Cortex Pons

Treatment Bmax KD Bmax KD

(mg/kg IP) (fmol/mg/prot) (nM) (fmol/mg/prot) (nM)

Saline 230.4 6 13.4 1.7 6 0.2 44.5 6 5.9 1.6 6 0.4ASA 200 205.5 6 14.3 1.1 6 0.1 N.P. N.P.ASA 300 168.7 6 16.7* 1.3 6 0.2 37.2 6 1.5 1.1 6 0.3ASA 400 152.5 6 11.8* 1.5 6 0.1 38.5 6 3.3 1.4 6 0.3

The brain membranes were collected 90 min after ASA IP administration. Values are expressed as mean 6SEM of seven brains.

N.P., not performed; * P . 0.05 versus saline (ANOVA followed by Student-Newman-Keuls test).

756 G. Vitale et al.

TABLE 3. The effect of the treatment with 400 mg/kg ASA on the number and affinity ofcortical 5-HT2 receptors and on 5-HT levels in the rat brain after the first phase of the formalintest

Cortical 5-HT2 receptors 5-HT levels

Treatment Bmax KD Cortex Pons(mg/kg IP) (fmol/mg/prot) (nM) (ng/g) (ng/g)

Saline 221.9 6 17.2 1.4 6 0.1 123.5 6 9.8 285.7 6 12.3ASA 400 137.4 6 16.6* 1.3 6 0.1 181.1 6 11.5* 344.8 6 11.2*

ASA (400 mg/kg) or saline were administered intraperitoneally, and the rats were killed 1 min thereafter.Values are expressed as mean 6 SEM of five brains.* P , 0.05 versus saline (Student’s t-test).

DISCUSSIONof [3H]ketanserin as ligand in the cerebral cortex, whereas the num-ber of receptors in pontine areas remained unchanged with respect The formalin test explores the response to prolonged nociceptiveto the control rats. The affinity constant (KD) was not affected by stimulus. The division of the observation period into three phases,any of the treatments (Tables 1 and 2). as suggested by Malmberg and Yaksh (1992), enables the first non-

Moreover, the changes in the number of 5-HT2 receptors and in inflammatory phase and the second inflammatory and pain-sus-5-HT levels induced by ASA at the dose of 400 mg/kg also occur tained phase to be evaluated; in particular, various NSAIDS are15 min after the formalin injection, which coincides with the end most active in reducing the behavioral response to formalin duringof the first phase of the formalin test (Table 3). the first period of the second phase of the test (phase 2A).

Pretreatment with PCPA interacted with the effect of ASA (400 Previously, we demonstrated that ASA, 400 mg/kg, exerts a maxi-mg/kg) and significantly antagonized the drug’s activity on the num- mal antinociceptive activity in the hot-plate test (Pini et al., 1995);ber of flinches in the first phase of the formalin test [F(1.28)59.36: the present data show that, in the formalin test, the antinociceptiveP,0.01], whereas it did not significantly affect the action of ASA activity is comparable with that of morphine, 8 mg/kg, SC, and thatin phases 2A and 2B (Fig. 3).

phase 2A, when the inflammatory reaction is at its peak, is the mostPCPA prevented the decrease in the number of 5-HT2 receptors

sensitive to the action of ASA. These data agree with those of Malm-provoked by ASA in the cerebral cortex [F(1.24)54.53; P,0.05],berg and Yaksh (1992), who showed that several NSAIDS suppress thewhereas it did not change per se the number of 5-HT2 receptors.prolonged pain behavior of the second phase of the formalin test.Moreover, pretreatment with PCPA decreased the serotonin levels

When brain serotonin is decreased by PCPA treatment, the anti-in both areas studied; the depletion of 5-HT induced by PCPAnociceptive effect of ASA disappears both in the hot-plate test (Piniblocked the effect of ASA on 5-HT content both in the pons andet al., 1995) and (present data) in the first phase of the formalin testin the cerebral cortex (Table 4).but not in the inflammatory phase (2A and 2B). The prevention ofThe serum levels of salicylates 90 min after IP injection rangedthe analgesic effect of ASA by PCPA cannot be attributed to its hyper-between 200 and 400 mg/ml, corresponding to the values reportedalgesic action, because this drug does not alter the basal reaction values.in the literature (Hunskaar et al., 1986), and were not affected by

The 5-HT2 receptors are widely distributed in many brain areaspretreatment with PCPA (432.6641.0 and 415.6632.0 mg/ml forincluding cortical areas (Leysen and Pawels, 1990), the cerebral cor-ASA, 400 mg/kg, and PCPA1ASA, respectively; P.0.05, Stu-tex being an important end point for axonal projections of seroton-dent’s t-test).ergic neurons originating from the brainstem; 5-HT1A receptors arefound particularly in hippocampus, septum, raphe nuclei and thespinal cord (Millan and Colpaert, 1991). We therefore decided tostudy brain areas such as the cerebral cortex and the pons becausethey are important targets of the serotonergic system net.

ASA significantly increased the serotonin levels in cortical areasat the doses of 300 and 400 mg/kg, whereas, in the pontine areas,serotonin levels changed significantly only at the dose of 400 mg/kg.The number of 5-HT2, but not of 5-HT1A, receptors showed a reduc-tion in the cortical membranes induced by 300 and 400 mg/kg ASA,whereas, in pontine areas, neither of these receptors were affectedby ASA treatment (Tables 1 and 2).

Pretreatment with PCPA prevented the increase in serotoninlevels in the cerebral areas examined and the decrease in the num-ber of cortical 5-HT2 receptors. These data indicate that the antino-ciceptive activity of ASA was mediated by the 5-HT system mainlyin noninflammatory pain, thus confirming the results obtained in

FIGURE 3. Influence of PCPA treatment on the action of ASA, the hot-plate test. On the other hand, the antinociceptive activity400 mg/kg, IP, in the formalin test. ASA was administered 2 hr exerted by ASA, when the pain is sustained by inflammation, was notafter the last injection of PCPA (100 mg/day, IP, for 4 days), and

impaired by the depletion of brain serotonin content. There is evi-the rats were tested 30 min thereafter. Each histogram representsdence to suggest that different serotonin receptor subtypes may af-the total number of flinches (mean6SEM) of eight rats in phases 1,fect nociception (Eide and Hole, 1991; McKenna and Peroutka,2A and 2B. SAL, saline. *P,0.05 versus SAL; jP,0.05 versus

SAL1ASA (ANOVA followed by Student-Newman-Keuls test). 1989), and the present data suggest that ASA exerts its antinocicep-

ASA and 5-HT-Mediated Antinociception 757

TABLE 4. Influence of pretreatment with PCPA on the effect of 400 mg/kg ASA on thenumber and affinity of cortical 5-HT2 receptors and on 5-HT levels in the rat brain

Cortical 5-HT2 receptors Serotonin levels

Treatment Bmax KD Cortex Pons(mg/kg IP) (fmol/mg/prot) (nM) (ng/g) (ng/g)

SAL 1 SAL 228.8 6 10.7 1.1 6 0.1 118.1 6 10.2 265.8 6 13.3PCPA 1 SAL 238.3 6 11.3 1.4 6 0.1 27.4 6 4.6* 37.6 6 8.3*SAL 1 ASA 164.7 6 9.8* 1.2 6 0.2 184.7 6 9.4* 389.4 6 29.5*PCPA 1 ASA 219.3 6 10.5 1.2 6 0.1 57.3 6 12.5* 145.3 6 25.2*

PCPA (100 mg/kg/day) or saline were injected IP for 4 days; ASA (400 mg/kg) or saline were administeredintraperitoneally 120 min after the last treatment with PCPA, and the rats were tested 30 min thereafter.

Values are expressed as mean 6 SEM of seven brains.* P , 0.05 versus SAL 1 SAL (ANOVA followed by Student-Newman-Keuls test).

tive activity through the serotonergic system and probably through creased 5-HT content in the cerebral membranes, it did not modifythe 5-HT2 receptors, as previously reported (Alheider, 1991). On the the number or the affinity of 5-HT1A receptors in the pons and theother hand, Backus et al. (1990) reported a functional interaction be- cerebral cortex. On the other hand, the number of 5-HT2 receptorstween central 5-HT2 and 5-HT1A receptors by using a behavioral test. decreased significantly in the cortex by using [3H]ketanserin as li-

It is well known that a persistent exposure to agonists or endoge- gand, whereas it did not change in the pons.nous neurotransmitters results in receptor down-regulation. Indeed, Pretreatment with PCPA (100 mg/kg/day for 4 days) depleted theafter agonist exposure, the 5-HT2 receptor system is able to induce 5-HT brain content, whereas it did not modify the serum levels ofa down-regulation in a relatively short period of time, as shown by salycilates or induce modifications of cortical 5-HT2 receptor char-Darmani et al. (1992). Thus, ASA, might induce a down-regulation acteristics. PCPA treatment abolished the antinociceptive effect ofof 5-HT2 receptors through an indirect increase in the concentra- ASA, 400 mg/kg, in the first phase of the formalin test, preventingtion of 5-HT. the increase in 5-HT content and the reduction in 5-HT2 receptors

This observation is supported by our results showing that the induced by ASA in the cerebral cortex.down-regulation of 5-HT2 receptors and the increase in 5-HT levels These data suggest that central 5-HT and, specifically, 5-HT2 re-are obtained also 1 min after ASA injection, which is the time point ceptors play a pivotal role in the antinociceptive activity of ASA.that coincides with the noninflammatory antinociceptive phase of

This work was supported in part by a grant from the Ministero dell’ Universita’the formalin test.della Ricerca Scientifica e Tecnologica (MURST) and from CNR, Roma, Italy.Recently, we demonstrated that the depletion of brain serotonin

by PCPA is effective in preventing the antinociceptive activity ofparacetamol in phase 1 but not in phase 2 of the formalin test (Pini

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