Research in Veterinary Science 80 (2006) 194200 www.elsevier.

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Involvement of opioidergic and a2-adrenergic mechanisms in the central analgesic eects of non-steroidal anti-inammatory drugs in sheep
Ignacio Lizarraga *, J.P. Chambers
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand Accepted 14 June 2005

Abstract The level within the central nervous system where non-steroidal anti-inammatory drugs (NSAIDs) produce analgesia and the mechanisms by which they mediate this eect are still uncertain. This study assessed the central analgesic eects of ketoprofen, phenylbutazone, salicylic acid and tolfenamic acid in sheep implanted with indwelling intrathecal (i.t.) catheters and submitted to mechanical noxious stimulation. The sheep received i.t. cumulative concentrations (0.375200 lM; 100 lL) as well as a single intravenous (i.v.) dose (3, 8, 10 and 2 mg/kg, respectively) of each NSAID. The sheep were also given i.t. naloxone (5.49 mM; 100 lL) and atipamezole (4.03 mM; 100 lL) prior to i.v. ketoprofen. None of the i.t. NSAIDs increased mechanical thresholds. Intravenously, only ketoprofen and tolfenamic acid raised the pain thresholds. The hypoalgesic eect of i.v. ketoprofen was prevented by i.t. naloxone or atipamezole. Although NSAIDs had no direct eect on the spinal cord, their analgesic action appeared to be spinally mediated. 2005 Elsevier Ltd. All rights reserved.
Keywords: Analgesics; Non-steroidal anti-inammatory drugs; Spinal cord; Intrathecal administration

1. Introduction The analgesic eects of non-steroidal anti-inammatory drugs (NSAIDs) in the periphery are well known, but there is also evidence suggesting that they exert central analgesic eects although the level within the central nervous system at which these eects occur is unkown. Neurophysiological (Bustamante et al., 1997) and behavioural studies (Malmberg and Yaksh, 1992; Pelisaz-Reval sier et al., 1996; Dolan and Nolan, 1999; D et al., 2004) in laboratory animals as well as reports on human beings (Lauretti et al., 1998) suggest that the site of analgesic action is the spinal cord. However,
Corresponding author. Tel.: +64 6 356 9099x7444; fax: +64 6 350 5699. E-mail address: I.Lizarraga@massey.ac.nz (I. Lizarraga). 0034-5288/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.06.001
*

electrophysiological (Vanegas et al., 1997) and behavaz-Reval et al., 2004) ioural (Tortorici et al., 1996; D studies in laboratory animals have also demonstrated a supraspinal site of action. The central analgesic mechanisms of action of NSAIDs are also poorly dened. Analgesia with intrathecal (i.t.) NSAIDs given at concentrations (nM) sucient to inhibit cyclo-oxygenase (COX) activity (Malmberg and Yaksh, 1992) and at the same time that spinal prostaglandin (PG) production inhibition takes place (MuthSelbach et al., 1999) has been reported. However, similar central analgesic eects for both S(+)- and R()-urbiprofen enantiomers [the former inhibits PG synthesis but the later does not and does not undergo signicant chiral inversion to S(+)-urbiprofen] were observed in various algesiometric tests in rats (Neugebauer et al., 1995), suggesting an additional mode of action other

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than prostanoid synthesis inhibition for the antinociceptive eects of some NSAIDs. Descending inhibitory inuences on the spinal transmission of nociceptive inputs have also been proposed (Chambers et al., 1995; az-Reval Pelissier et al., 1996; Tortorici et al., 1996; D et al., 2004). There are at least two COX isozymes: COX-1 and COX-2. The potency for each individual NSAID to inhibit these isozymes varies according to the species, the tissue, the test used to characterise COX inhibition and the drug itself. However, COX-1 and COX-2 inhibition is achieved with lM drug concentrations (Ricketts et al., 1998; Warner et al., 1999). The current study sought to determine the direct spinal analgesic eects of the NSAIDs ketoprofen, phenylbutazone, salicylic acid and tolfenamic acid given at concentrations sucient to inhibit COX activity in vitro (Ricketts et al., 1998); assess the analgesic eects of these drugs when injected systemically; and characterise the spinal contribution of opioidergic and a2-adrenergic mechanisms. Sheep implanted with indwelling i.t. cervical catheters, an established way of determining the spinal analgesic action of drugs (Waterman et al., 1988), were subjected to mechanical noxious stimulation of a forelimb, which has been shown to be sensitive to the analgesia produced by NSAIDs in this species (Chambers et al., 1995).

Chambers et al. (1995), which pressed a 2-mm diameter blunted-ended pin against the sheeps leg with increasing force. The response measured was the minimum force needed to elicit a clear lifting of the left forelimb, at this point the force was recorded and immediately removed. If the sheep did not lift its leg at 20 Newtons (N), the stimulus was terminated to prevent tissue damage and this value recorded. In all the experiments, at least four consistent threshold values were measured at 23 min intervals before the administration of drugs. Treatments were given in blocks as specied in Table 1, and at least 1 week was allowed between trials on each sheep. The operator was blind to the drug and combinations the sheep were given, although he knew the route by which the drugs were given. 2.2. Experiment 1: Eect of intrathecally administered NSAIDs Ketoprofen, phenylbutazone, salicylic acid and tolfenamic acid (all n = 5) were administered i.t. in cumulative doses (0.375200 lM) and repetitive doses of preservative-free normal saline (n = 5; 10 doses) were injected i.t. as control. These NSAIDs inhibited COX activity in vitro within this rage concentration in dogs (Ricketts et al., 1998). The cumulative dosing of a NSAID in a sheep took place on the same day and started immediately after recording control thresholds, starting with the lowest concentration and increasing up to 200 lM. Thresholds were measured at 5 min intervals for 30 min after injection of each dose. The same procedure was carried out for the repetitive administration of 0.9% saline. Each dose was given in a 100 lL volume. 2.3. Experiment 2: Eect of intravenously administered NSAIDs Ketoprofen (3 mg/kg; n = 6), phenylbutazone (8 mg/kg; n = 5), salicylic acid (10 mg/kg; n = 5) and tolfenamic acid (2 mg/kg; n = 6) were injected into a jugular vein. Doses for the rst three NSAIDs are sucient to inhibit PG production in sheep (Nolan et al., 1990; Cheng et al., 1998; Landoni et al., 1999). Since no studies on tolfenamic acid in sheep were found, the manufacturers recommended dose for producing analgesia in cattle was used. All NSAIDs were made up to a nal volume of 3 mL by adding 0.9% sterile saline solution as necessary. Normal saline solution i.v. (3 mL; n = 5) was used as control. After administration of the drug being tested, thresholds were recorded at 5 min intervals for the rst hour, every 10 min for the second hour and every 30 min for up to 6 h, or until values returned to baseline levels.

2. Materials and methods After approval by the Massey University Animal Ethics Committee, 15 Romney-cross, non-lame, healthy ewes (24 years old, 4070 kg) were housed indoors and food and water were provided ad libitum. The sheep had chronic indwelling cervical i.t. catheters, terminating at the level of the fth cervical vertebra, implanted under general anaesthesia at least 2 weeks prior to the experiments (Kyles et al., 1992). Before any experiment was carried out, xylazine (10 or 50 lg/100 lL) was injected through the catheters to check that the tips were in the expected place; xylazine has been shown to produce good spinal analgesia in sheep (Waterman et al., 1988). Sheep in which the xylazine did not produce analgesia were not used (four sheep). 2.1. Mechanical nociceptive testing The sheep were placed individually in steel, mobile crates (86 cm high 112 cm long 53 cm wide) and were always kept in pairs. The skin on the cranial aspect of the left foreleg was clipped and the mechanical nociceptive device attached to the lower end of the radius. The sheep were given a 1520 min period to acclimatise. Threshold responses to a mechanical stimulus were measured using a device similar to that described by

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Table 1 Distribution of sheep used in the experiments Treatmenta Sheep number 1 Experiment 1 Saline i.t. Salicylic acid i.t. Phenylbutazone i.t. Ketoprofen i.t. Tolfenamic acid i.t. Experiment 2 Saline i.v. Salicylic acid i.v. Phenylbutazone i.v. Ketoprofen i.v. Tolfenamic acid i.v. Experiment 3 Saline i.t. + ketoprofen i.v. Naloxone i.t. + ketoprofen i.v. Atipamezole i.t. + ketoprofen i.v. Naloxone i.t. + saline i.v. Atipamezole i.t. + saline i.v. Saline i.t. + saline i.v.
a

n 3 p p p p p p p p p p p p p p p p 4 p p p p p p p p p p p p p p p p 5 6 9 93 p p p p p p p p p p p p p p p 404 p p p p p p p p p 1019 1572 5 5 5 5 5 5 5 5 6 6 6 6 6 4 4 5

2 p p p

p p p p p p p p p p p

p p p p p p

p p

p p p

Treatments were given in blocks with at least a 1-week wash out period in between.

2.4. Experiment 3: Antagonism studies Sheep were given the following treatments: (a) i.v. ketoprofen (3 mg/kg) injected 10 min after i.t. naloxone (100 lL, 5.49 mM) (n = 6); (b) i.v. ketoprofen (3 mg/kg) injected 10 min after i.t. atipamezole (100 lL, 4.03 mM) (n = 6); (c) i.v. ketoprofen (3 mg/kg) injected 10 min after i.t. saline solution (100 lL, 0.9%) (n = 6); (d) i.v. saline solution (3 mL, 0.9%) injected 10 min after i.t. naloxone (100 lL, 5.49 mM) (n = 4); (e) i.v. saline solution (3 mL, 0.9%) injected 10 min after i.t. atipamezole (100 lL, 4.03 mM) (n = 4); (f) i.v. saline solution (3 mL, 0.9%) injected 10 min after i.t. saline solution (100 lL, 0.9%) (n = 5). The concentrations for i.t. naloxone and atipamezole were selected because they inhibited the analgesic response to i.v. fentanyl (10 lg/kg) and i.v. xylazine (20 lg/kg) in sheep, without aecting mechanical thresholds when administered alone (Lizarraga, 2000). The nociceptive testing was carried out at 5 min intervals for 60 min, and administration of the i.v. treatment was taken as time zero. 2.5. Behavioural eects of NSAIDs The behavioural eects produced were subjectively assessed during the mechanical nociceptive testing. Animals were observed for signs of increased or decreased

movement, head droop, ataxia or recumbency, agitation, vocalisation, chewing movements, changes in respiratory patterns and salivation. 2.6. Drugs Intrathecal administration: ketoprofen, naloxone HCl, phenylbutazone, salicylic acid, tolfenamic acid and xylazine HCl were purchased from Sigma (St. Louis, MO, USA), atipamezole HCl (Antisedane) was obtained from Ciba-Geigy (New Zealand) Ltd. (Auckland, New Zealand). All NSAIDs were dissolved in preservative-free saline and NaOH. Atipamezole, naloxone and xylazine were dissolved in preservative-free saline only. Once dissolved, drugs were ltered (0.2 lm syringe lter; Sartorius, Minisart CE, Goettingen, Germany) and then injected i.t. in a 100 lL volume followed by a wash-in injection of 300 lL preservative-free saline (the volume of the i.t. catheter system). This volume has been shown to limit drug distribution to about 3 or 4 vertebral segments either side of the catheter tip (Waterman et al., 1988). Intravenous administration: ketoprofen (Ketofen 10%, rieux, Lyon, France), phenylbutazone (Bute ne Me Rho IV, Virbac Laboratories (NZ) Ltd., Auckland, New toquinol Zealand) and tolfenamic acid (Tolfedine CS, Ve Veterinary Pharmaceuticals, Lure, France) were administered. Sodium salicylate (Sigma, St. Louis, MO, USA) was dissolved in 3 mL 0.9% saline solution and then passed through a sterile 0.2 lm lter.

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2.7. Statistical analysis Data were expressed as the mean s.e.m. The eects of treatment on mechanical thresholds were compared with their respective mean pre-drug administration values and also were compared over time with other treatments. Repeated measures ANOVA with Dunnetts or Tukeys post hoc tests were used as appropriate. The areas under the threshold vs. time curve values for 30 and 60 min (AUC30 and AUC60) after i.t and i.v. drug administration, respectively, for individual sheep (with the mean baseline subtracted) were calculated using the trapezoidal method. This gives an estimate of the total eect of the drug in a single gure, although a graph of threshold plotted against time (such as Fig. 2) gives useful information on peak eects and duration of action. One-way ANOVA followed by Tukeys test were used to compare AUC values. GraphPad Prism version 4.0a for Macintosh, GraphPad Software, USA, was used for these purposes. Dierences with P < 0.05 were considered signicant.

not signicantly dierent when i.t. saline was administered repetitively up to 10 times (data not shown) or when cumulative concentrations of any NSAID were given i.t. (P = 0.75) (Fig. 1). 3.2. Experiment 2: Eect of intravenously administered NSAIDs Intravenous saline, salicylic acid or phenylbutazone did not signicantly increase mechanical thresholds (P P 0.083). Thresholds were signicantly raised after ketoprofen from 3.76 0.04 N to a maximum of 6.59 0.12 N at 25 min (P < 0.01) (Fig. 2(a)), returning to baseline by 180 min. A second, smaller peak (5.55 0.16 N), not signicantly dierent from pre-drug administration levels (P > 0.05), was observed at 240 min. Tolfenamic acid behaved similarly, although only one peak was observed. Thresholds were signicantly raised from 3.78 0.04 N up to 6.83 0.66 N at 30 min (P < 0.01) (Fig. 2(a)), and returned to baseline by 210 min. When the eects on mechanical thresholds were compared over time, a signicant dierence was found between ketoprofen and control, and tolfenamic acid and control from 10 to 120 min after drug injection (P < 0.05). No signicant dierences were observed between ketoprofen and tolfenamic acid treatments at any time (P > 0.05) (Fig. 2(a)). No signicant dierence over time was found when phenylbutazone and salicylic acid were compared to saline (P > 0.05). Comparison of the AUC60s revealed no signicant dierence between saline (12.38 2.77 N min), salicylic acid (21.87 9.35 N min), and phenylbutazone

3. Results 3.1. Experiment 1: Eect of intrathecally administered NSAIDs Mechanical thresholds were not signicantly raised after repetitive i.t. administration of saline or after cumulative i.t. injection of NSAIDs at dierent concentrations (0.375200 lM) (P P 0.26). The AUC30s were

Fig. 1. Area under the threshold vs. time curves for the rst 30 min of healthy sheep given cumulative intrathecal concentrations of ketoprofen ((a) 0.75200 lM), phenylbutazone ((b) 0.375200 lM), salicylic acid ((c) 0.375200 lM) and tolfenamic acid ((d) 0.375200 lM) (n = 5, mean s.e.m.). No signicant dierences were found.

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Fig. 2. Eects of intravenously administered ketoprofen (3 mg/kg), tolfenamic acid (2 mg/kg), phenylbutazone (8 mg/kg), salicylic acid (10 mg/kg) and saline (3 mL) on mechanical nociceptive thresholds of healthy sheep (n = 56, mean s.e.m.). (a) Mechanical nociceptive thresholds over the rst 2 h after treatment (time 0). *,,Signicant dierence between ketoprofen and tolfenamic acid in comparison to saline at each given time (P < 0.05, P < 0.001, and P < 0.01, respectively). (b) Area under the threshold change vs. time curves for the rst 60 min. Signicant dierence between treatment and saline (P < 0.001).

Fig. 3. Area under the threshold change vs. time curves for the rst 60 min (AUC60) of healthy sheep given intravenous (i.v.) ketoprofen (3 mg/kg) alone or 10 min after either intrathecal (i.t.) saline (100 lL), naloxone (5.49 mM, 100 lL) or atipamezole (4.03 mM, 100 lL) (n = 6, mean s.e.m.). AUC60s for the combinations i.t. naloxone and atipamezole plus i.v. saline were not dierent from that of i.t. saline plus i.v. saline (n = 45, mean s.e.m.). *Signicant dierence between treatment and ketoprofen i.v. alone (P < 0.001).

(30.65 16.89 N min) (P > 0.05). However when ketoprofen and tolfenamic acids AUC60s (111.31 5.42 and 120.38 14.58 N min, respectively) were compared to those of saline or the other NSAIDs, a signicant difference was found (P < 0.001). No signicant dierence was observed between ketoprofen and tolfenamic acids AUC60s (P > 0.05) (Fig. 2(b)). 3.3. Experiment 3: Antagonism studies The AUC60 for i.t. saline plus i.v. ketoprofen was similar to i.v. ketoprofen alone (P > 0.05), and both were signicantly dierent to i.v. saline alone (108.57 9.82 and 111.31 5.42 vs. 12.38 2.77 N min, respectively, P < 0.001). Intrathecal naloxone or atipamezole 10 min before i.v. ketoprofen signicantly reduced the AUC60 by 85.28% and 91.57%, respectively, when compared to that of i.t. saline plus i.v. ketoprofen (15.98 13.66 and 9.15 8.08 vs. 108.57 9.82 N min; P < 0.001) (Fig. 3). The AUC60s for i.v. saline alone (12.38 2.77 N min), i.t. saline plus i.v. saline (6.53 2.35 N min), naloxone i.t. plus i.v. ketoprofen (15.98 13.66 N min), i.t. atipamezole plus i.v. ketoprofen (9.15 8.08 N min), saline i.v. plus naloxone i.t. (1.44 4.50 N min), and saline i.v. plus atipamezole i.t. (10.19 9.31 N min) were not signicantly dierent to each other (P > 0.05). (Fig. 3).

3.4. Behavioural eects of NSAIDs No signs of distress, excessive salivation, altered chewing movements or changes on movement patterns, head positioning and respiratory patterns were observed after any drug given by either route.

4. Discussion Intrathecal injection of ketoprofen, tolfenamic acid, phenylbutazone and salicylic acid produced no change in mechanical nociceptive thresholds of non-lame healthy sheep whereas a rise in thresholds was detected after i.v. ketoprofen and tolfenamic acid, but not phenylbutazone and salicylic acid. Intrathecal opioid or a2-adrenergic receptor antagonists reversed the hypoalgesic eect of an i.v. NSAID. These results suggest that the central analgesic eects of NSAIDs in sheep are mediated by endogenous inhibitory mechanisms. PGs and all the necessary enzymes for their synthesis are present in the spinal cord and they can be released by activation of high-threshold nociceptive aerent inputs, direct administration of neurotransmitters or peripheral inammatory stimuli (for review see Svensson and Yaksh, 2002). Intrathecal administration of PGs can enhance nociception by a wide variety of mechanisms.

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Analgesic eects have been seen in laboratory animals (Malmberg and Yaksh, 1992) and people (Lauretti et al., 1998) after spinal administration of NSAIDs; these eects have been attributed to spinal PG production inhibition (Malmberg and Yaksh, 1992; Muth-Selbach et al., 1999). Analgesia was not seen in our sheep, which were given drug concentrations high enough to inhibit COX-1 and COX-2 activity as reported in in vitro studies (e.g., reported drug concentrations (in lM) needed to inhibit 50% of dogs COX-1 and COX-2 activity were 0.029 and 0.123 for ketoprofen, 0.206 and 0.014 for tolfenamic acid, >100 and 3.79 for phenylbutazone and 34.3 and >100 for acetyl salicylic acid) (Ricketts et al., 1998). This discrepancy is probably due to dierences in analgesia tests. In most experiments on central analgesic eects of NSAIDs, nociception was assessed using models of inammatory pain, but no analgesia was observed when models of acute pain were utilised in laboratory animals (Malmberg and Yaksh, 1992; Dolan and Nolan, 1999) and people (Eisenach et al., 2002). Therefore, NSAIDs seem capable of producing a direct spinal analgesic eect only when inammation is present. During inammation a large variety of neurotransmitters and neuromodulators are released, which act at a large number of receptor types including the NMDA receptor (Malmberg and Yaksh, 1992). Nonetheless, Pelissier et al. (1996) reported that i.t. paracetamol produced antinociception in rats with no inammation subjected to a paw pressure test. However, this eect was only seen using mM concentrations: NSAIDs at nM or lM concentrations have failed to increase mechanichal nociceptive thresholds (Dolan and Nolan, 1999). Since COX inhibition by NSAIDs only requires nM to lM concentrations (Ricketts et al., 1998), it seems reasonable to suggest that mechanisms other than, or complementary to, prostanoid synthesis inhibition might mediate central antinociception. There are dierences between the analgesic ecacy of NSAIDs and their COX inhibition potency (McCormack, 1994). COX inhibition seems to be specic for dierent species and tissues. Since data describing NSAID-mediated COX inhibition in sheep spinal neurones have not been published, the possibility exists that drug concentrations utilised in experiment 1 may not have been high enough to inhibit PG production in the sheep spinal cord. It could also be argued that the 30 min for which each i.t. concentration was assessed was not long enough to inhibit COX enzymes in sheep spinal neurones, since COX time-dependent inhibition has been reported for some NSAIDs (Ricketts et al., 1998). However, no modication of pain thresholds was observed even up to 5 h after i.t. administration of some NSAIDs (cumulative administration). Moreover, analgesia was observed almost immediately after i.v. administration of NSAIDs in sheep (Chambers et al., 1995; this study), indicating that despite being weak acids and therefore polar mole-

cules, the drugs are able to reach their site of action quickly. This suggests that 30 min should be a reasonable time to observe any analgesic eect after i.t. injection of these drugs. The characterisation of PG synthesis inhibition in sheep spinal cord neurones by NSAIDs may clarify these issues. Results from experiment 1 conrmed that i.t. administration of NSAIDs at nM to lM concentrations does not produce analgesia in animals with no inammation. However, these results do not rule out a possible central analgesic eect for NSAIDs mediated by COX inhibition either. In contrast to i.t. injected NSAIDs, i.v. administration of ketoprofen and tolfenamic acid, but not of phenylbutazone and salicylic acid, raised mechanical thresholds in healthy sheep. This is in accordance with Chambers et al. (1995), who found that i.v. administration of the NSAIDs unixin (2.2 mg/kg) and dipyrone (25 mg/kg) produced mechanical analgesia in sheep. These results conrm that NSAIDs can produce hypoalgesia in the absence of inammation (Chambers et al., 1995; Pelissier et al., 1996; Bustamante et al., 1997). Failure of phenylbutazone to signicantly increase mechanical thresholds in this study contrasts with its analgesic action in sheep subjected to stie surgery (Dowd et al., 1998), although they administered phenylbutazone chronically rather than using a single dose. The reduction in inammation may have contributed to the analgesic eect. Aspirin has been shown to inhibit COX in sheep, as assessed by decreased serum thromboxane B2 (TxB2) values (Nolan et al., 1990); however this may be a direct eect on platelets. In this study, i.v. salicylic acid (10 mg/ kg) had no analgesic action although this may have been related to dose. Nociceptive testing along with quantication of serum TxB2 before and after salicylic acid injection at larger doses may clarify this point. Since no peripheral inammation was present in our sheep, the eects of i.v. ketoprofen and tolfenamic acid cannot be explained by inhibition of COX enzymes in the periphery. After systemic administration, some NSAIDs cross the bloodbrain barrier and can be found in the cerebrospinal uid at the time that they inhibit prostanoid synthesis in the dorsal horn of the spinal cord (Muth-Selbach et al., 1999), which supports the possibility of a central analgesic mechanism of action for NSAIDs by COX inhibition. Opioidergic and adrenergic mechanisms have been linked to the analgesic eects of some NSAIDs (Chambers et al., 1995; Tortorici et al., 1996). In this study, both naloxone and atipamezole prevented the analgesic eects of i.v. ketoprofen, demonstrating that both opioidergic and adrenergic mechanisms were involved. Although receptor-binding studies were not carried out, it seems unlikely that ketoprofen interacted directly with opioid and a2-adrenergic receptors since other

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NSAIDs have shown no anity to these and other receptors involved in nociception at clinical concentrations (Pelissier et al., 1996). This study suggests that NSAIDs activate supraspinal mechanisms, which indirectly cause descending inhibitory inuences on the spinal transmission of nociceptive inputs. Intracerebroventricular (i.c.v.) injection of ketoprofen may help to elucidate this in sheep, although some experiments in rats failed to produce analgesia after i.c.v. injection (Bustamante et al., 1997). In conclusion, these results showed that although NSAIDs did not have a direct eect on the spinal cord, their analgesic action appeared to be spinally mediated, probably by activating inhibitory descending opioidergic and adrenergic mechanisms. This raises the possibility of exploring drug combinations of NSAIDs and other agents that act on these pathways with the hope of nding better ways to control pain. Acknowledgements We thank Dr Sean Johnson for skilled assistance. I.L. was the recipient of a NZODA Study Award. References
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