Comparative actions of the opioid analgesics morphine, methadone and codeine in rat models of peripheral and central neuropathic pain

Pain 116 (2005) 347–358 www.elsevier.com/locate/pain

Comparative actions of the opioid analgesics morphine, methadone and codeine in rat models of peripheral and central neuropathic pain Helle Kirsten Erichsena, Jing-Xia Haob, Xiao-Jun Xub, Gordon Blackburn-Munroa,* a Department of Pharmacology, NeuroSearch A/S, 93 Pederstrupvej, DK-2750 Ballerup, Denmark Section of Clinical Neurophysiology, Karolinska University Hospital-Huddinge, Karolinska Institutet, S-14186 Stockholm, Sweden

b

Received 15 September 2004; received in revised form 11 April 2005; accepted 3 May 2005

Abstract Controversy persists in relation to the analgesic efficacy of opioids in neuropathic pain. In the present study the effects of acute, subcutaneous administration of the m-opioid receptor agonists morphine, methadone and codeine were examined in rat models of peripheral and central neuropathic pain. In the spared nerve injury (SNI) and chronic constriction injury (CCI) models of peripheral neuropathic pain, both morphine (6 mg/kg) and methadone (3 mg/kg) attenuated mechanical allodynia, mechanical hyperalgesia and cold allodynia for up to 1.5 h post-injection (P!0.05); codeine (30 mg/kg) minimally alleviated mechanical hypersensitivity in SNI, but not CCI rats. When administered to rats with photochemically-induced spinal cord injury (SCI), morphine (2 and 6 mg/kg) and methadone (0.5–3 mg/kg) robustly attenuated mechanical and cold allodynia for at least 2 h post-injection (P!0.05). Codeine (10 and 30 mg/kg) also attenuated mechanical and cold allodynia in this model for at least 3 h after injection. The magnitude of opioid-mediated antinociception was similar between SNI, SCI and non-injured rats as measured in the tail flick test. At antinociceptive doses, no motor impairment as determined by the rotarod test was observed. The therapeutic window (based on antiallodynia versus ataxia) obtained for codeine, was vastly superior to that obtained with morphine or methadone in SNI and SCI rats. Furthermore, the therapeutic window for codeine in SCI rats was 4-fold greater than in SNI rats. Our results further support the efficacy of m-opioid receptor agonists in alleviating signs of neuropathic pain in animal models of peripheral and especially central nerve injury. q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Allodynia; Chronic constriction injury; Hyperalgesia; Opioid; Spared nerve injury; Spinal cord injury

1. Introduction Neuropathic pain can arise as a result of damage to the peripheral or central nervous system and includes a variety of conditions that differ in aetiology as well as location. Sensory abnormalities which often manifest as allodynia (pain evoked by normally non-noxious stimuli), and hyperalgesia (an increased response to a noxious stimuli) are routinely observed in human neuropathic pain conditions as well as in relevant animal models (BlackburnMunro, 2004; Dworkin et al., 2003). The management of neuropathic pain, especially that of central origin as exemplified by spinal cord injury, remains a major clinical * Corresponding author. Tel.:C45 44 60 83 33; fax: C45 44 60 80 80. E-mail address: [email protected] (G. Blackburn-Munro).

challenge. This is in part a consequence of an inadequate understanding of the mechanisms involved in disease aetiology, but also a result of the relative absence of clinically effective treatments (Dworkin et al., 2003; Finnerup and Jensen, 2004). Opioids are among the most powerful analgesics in clinical use for the treatment of nociceptive pain. Nevertheless, opioid treatment of neuropathic pain is often discouraged due to concerns relating to the development of analgesic tolerance, the risk of addiction, and other debilitating adverse effects (Carver and Foley, 2001; Rowbotham, 2001). However, evidence is now accumulating to suggest that opioids are effective in relieving some symptoms of pain in patients with neuropathic pain of primarily peripheral origin (Gimbel et al., 2003; Raja et al., 2002; Watson et al., 2003). Significantly less evidence

0304-3959/$20.00 q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2005.05.004

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exists to support the use of opioids in the treatment of central neuropathic pain (Attal et al., 2002; Rowbotham et al., 2003; Siddal et al., 2000). Indeed, opioid administration in patients with spinal cord injury has been anecdotally reported to precipitate neuropathic pain (Parishod et al., 2003). Numerous studies in various animal models of peripheral or central nerve injury suggest that opioids are effective in alleviating neuropathic pain behaviours. However, inconsistencies are apparent. Whilst systemic administration of morphine attenuates allodynia and hyperalgesia in the chronic constriction injury and spinal nerve ligation (SNL) models (Backonja et al., 1995; Kayser et al., 1995; Lee et al., 1995), intrathecal morphine is apparently ineffective (Bian et al., 1995; Mao et al., 1995; Ossipov et al., 1995). In contrast, electrophysiological recordings made from convergent dorsal horn neurones suggest an enhanced antinociceptive potency of intrathecally applied morphine in SNL rats compared with controls (Suzuki et al., 1999). In rats with spinal cord injury both systemic and intrathecal morphine attenuate mechanical allodynia (Kim et al., 2003), although systemically-mediated antinociception may be compromised by sedation (Hao et al., 1998). Thus, the neuropathic model and nociceptive behaviours studied, together with the route of opioid administration utilised appear to have an important bearing on therapeutic outcome. To address some of the inconsistencies reported in the literature pertaining to the effectiveness of opioids in animal neuropathic pain models of diverse aetiology, we have compared the effects of the m-opioid receptor agonists morphine, methadone and codeine after acute, systemic administration on nociceptive behaviours in rat models of peripherally and centrally-induced nerve injury.

2. Materials and methods 2.1. Animals Except where mentioned, adult male Sprague–Dawley rats (Harlan Scandinavia, Denmark) were used. Animals were housed together in groups with unrestricted access to food and water, and kept on a 12:12 h light–dark cycle (lights on at 0600 h) with all experiments performed during the light period. Animals were housed in the same room in which the testing procedure was performed to try and minimise any stress response associated with novel environmental cues. All procedures were performed according to the Ethical Guidelines of the International Association for the Study of Pain (Zimmermann, 1983), and were approved by either the Danish or Swedish Committee for Experiments on Animals. All experiments were performed blind by the observer. In experiments using neuropathic rats, a crossover paradigm was employed such that each rat typically received an injection of both vehicle and the drug being tested. Accordingly, drug wash-out periods of at least 2–3 days were used between experiments.

2.2. Peripheral nerve injury A spared nerve injury (SNI) was performed in rats as described previously (Decosterd and Woolf, 2000). The rats (body weight 180–220 g at the time of surgery) were anaesthetized with a gaseous mixture of isoflurane (Baxter A/S, Denmark), oxygen and nitric oxide. The skin of the lateral left thigh was incised and the cranial and caudal parts of the biceps femoris muscle were separated and held apart by a retractor to expose the sciatic nerve and its three terminal branches: the sural, common peroneal and tibial nerves. The tibial and common peroneal nerves, were tightly ligated with 4/0 silk and 2–3 mm of the nerve distal to the ligation was removed. Any stretching or contact with the intact sural nerve was avoided. A chronic constriction injury (CCI) was produced in rats as described previously (Bennett and Xie, 1988). Rats (body weight 150–180 g at the time of surgery) were anaesthetized with chloral hydrate (400 mg/kg, i.p., Merck, Denmark) supplemented as necessary with a gaseous mixture of halothane and O2, and the sciatic nerve exposed at the mid-thigh level proximal to the sciatic trifurcation. Four chromic gut ligatures (4/0) were tied loosely around the nerve, 1–2 mm apart such that the vascular supply was not compromised. For both nerve injury procedures, the overlying muscle was closed in layers with 4/0 synthetic absorbable surgical suture. The skin was closed and sutured together with 4/0 silk thread using hidden stitches to avoid any opening of the wound by biting. Only animals which recovered completely with no overt behavioural deficits unrelated to the surgical procedure were included in these studies. 2.3. Spinal cord injury A spinal cord injury (SCI) was performed in adult female Sprague–Dawley rats (Møllegaard, Denmark) (body weight 150– 200 g at time of surgery), as described previously (Hao et al., 1991). The rats were anaesthetized with chloral hydrate (300 mg/kg, i.p., Sigma, USA) and a catheter was inserted into the jugular vein. A midline skin incision was then made along the back to expose the T11-L2 vertebrae. The animals were positioned beneath a tunable argon ion laser (Innova model 70, Coherent Laser Products Division, CA, USA) operating at a wavelength of 514 nm with an average power of 0.17 W. The laser light was focused into a thin beam covering the single T13 vertebra, which was irradiated for 10 min. Immediately before the irradiation erythrosin B (Aldrich, 32.5 mg/kg dissolved in 0.9% saline) was injected intravenously via the jugular catheter. Due to rapid metabolism of erythrosin B the injection was repeated after 5 min in order to maintain adequate blood concentrations. During irradiation body core temperature was maintained at 37–38 8C by a heating pad. After irradiation the wound was closed in layers and the skin sutured together. 2.4. Behavioural testing of animals with peripheral nerve injury SNI and CCI rats were routinely tested for the presence of painlike behaviours from 2–3 days after surgery. During testing the animals were placed on an elevated metal grid allowing stimulation of the plantar surface of the paw (lateral aspect in SNI animals since this is the area of the paw innervated by the intact sural nerve). Stimulation of the injured hindpaw was then initiated in the following order (von Frey, pin prick and

H.K. Erichsen et al. / Pain 116 (2005) 347–358

cold stimulation). Animals were allowed to recover for 1–2 min between the testing procedures. To test for the presence of mechanical allodynia a set of von Frey monofilaments (0.073–21.8 g, modal value was 21.8 g prior to surgery; Stoelting, IL, USA) was used. The monofilaments (hairs) were applied to the plantar surface of the hindpaw with increasing force until the rat withdrew the paw. Lifting of the paw due to normal locomotor behaviour was ignored. Each von Frey hair was applied five times. The threshold force (g) was taken as the lowest force that caused at least three withdrawals out of the five consecutive stimuli (Hao et al., 1999). The presence of mechanical hyperalgesia was determined by pressing the mid-plantar glabrous surface of the hindpaw with the point of a safety pin, 3 cm in length (Tal and Bennett, 1994). The intensity applied was sufficient to produce a reflex withdrawal response in normal unoperated animals, but insufficient to penetrate the skin. The duration of the normal pin prick-evoked withdrawal response was too short to time with a stopwatch and was arbitrarily assigned a duration of !0.5 s. A cut-off time of 15 s was applied to long withdrawals often seen for the paw ipsilateral to the nerve injury (Decosterd et al., 1998; Tal and Bennett, 1994). The stimulus was repeated twice to obtain an averaged paw withdrawal duration value (s). To test for the presence of cold allodynia, ethyl chloride (Perstrops, Sweden) was sprayed onto the lateral plantar surface of the hindpaw. The response was observed and classified according to the following scale: 0, no visible response; 1, startle response without paw withdrawal; 2, clear withdrawal of the paw (!5 s); 3, prolonged withdrawal (5–30 s) often combined with flinching and licking of the paw; 4, prolonged repetitive withdrawal (O30 s) and/or vocalization (Hao et al., 1999). 2.5. Behavioural testing of animals with spinal cord injury SCI rats were routinely tested for the presence of pain-like behaviours from 3–4 weeks after surgery. The fur of the animals was shaved at least a day prior to examination of the cutaneous pain threshold to avoid sensitization of the skin receptors. During testing the rats were gently held in a standing position by the experimenter and the flank area and hindlimbs were examined for hypersensitivity to sensory stimulation. On the day of drug testing, SCI rats were administered drug according to the experimental schedule and the time course of pain-like behaviours measured. Ethical issues prevented the inclusion of vehicle-treated SCI rats in the current experiments. The presence of mechanical allodynia was determined by a series of calibrated von Frey hairs (0.61–170 g, modal value was 95 g prior to surgery; Stoelting, IL, USA) that were applied to the skin of the animal until it became bent corresponding to a certain pressure force applied (g). Care was taken not to sensitize skin receptors to von Frey hair application by successively testing different body regions. Although the animals showed reactions as twitching of the skin in response to the von Frey hair stimulation, only inducement of consistent vocalization over a relatively large skin area was considered as representing pain-like behaviours (Hao et al., 1991). The responses of rats to tactile stimulation was tested with the blunt point of a pencil by gently brushing the skin in a rostral-caudal direction. The frequency of the stimulation was approximately 1 Hz and responses were graded according to the following scale: 0, no observable response; 1,

349

transient vocalization and moderate effort to avoid pencil tip; 2, consistent vocalization and aversive reactions; 3, sustained and prolonged vocalizations, aggressive behaviours. To test for the presence of cold allodynia, ethyl chloride was sprayed onto the skin of the animals which had been determined to be sensitive to mechanical stimulation. The subsequent response to cold stimulation was observed and classified according to the following scale: 0, no visible response; 1, localized response (skin twitch) without vocalization; 2, transient vocalization; 3, sustained vocalization. 2.6. Tail flick test To test for a preferential antinociceptive action of either morphine, methadone or codeine in normal nociceptive processing as compared with nerve injury-induced nociceptive processing, drug effects were evaluated in normal uninjured rats, and in SNI and SCI rats by means of the tail flick test (Blackburn-Munro et al., 2004; Hao et al., 1998). A radiant heat source (Denmark-Ugo Basile, Comerio, Italy; Sweden-IITC, Woodland Hills, CA, USA) was focused on the tail 2–3 cm from its distal end in normal, SNI and SCI rats (body weights 220–330 g). The individual sets of apparatus were calibrated to give a tail flick latency of approximately 4–5 s (15 s cut-off) and 2–3 s (10 s cut-off) prior to drug injection, respectively, enabling increases or decreases in tail flick latency to be measured to the nearest 0.1 s. Baseline measurements (two measurements separated by 5 min) were made 2 days prior to testing, to familiarize the animals with the testing procedure. Two further baseline tail flick latency measurements were obtained on the day of drug testing to ensure consistent reflex responses were present. Animals were then administered drug or vehicle according to the experimental paradigm, and the tail flick latency response was determined at 30, 60, 90, 120 and 180 min post-injection. 2.7. Rotarod test In normal, uninjured rats (body weight 220–330 g) changes in motor performance after drug administration were measured using an accelerating rotarod (Ugo Basile, Comerio, Italy) as described previously (Blackburn-Munro et al., 2004). The rotarod speed was increased from 3–30 rpm over a 180 s period, with the maximum time spent on the rod set at 180 s. Rats received two training trials (separated by 3–4 h) on two separate days prior to drug testing for acclimatization purposes. On the day of testing, a baseline response was obtained, and rats subsequently administered drug or vehicle and the time course of motor performance was tested 30 and 60 min after injection. The minimum time possible to spend on the rod was 0 s. 2.8. Drugs Morphine hydrochloride was obtained from either Mecobenzon (Denmark) or Pharmacia and UpJohn (Sweden). Methadone was purchased from Kabi Pharmacia (Sweden) or Nycomed (Denmark). Codeine phosphate was obtained from Apoteksbolaget (Sweden) or Merck (Germany). Morphine hydrochloride (Pharmacia and UpJohn) and methadone were obtained as pre-made solutions ready for injection, while morphine hydrochloride (Mecobenzon) and codeine phosphate were dissolved in

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2.9. Calculation of ED50 values and statistical analysis To enable a comparison of drug effects on acute nociceptive responses in normal, SNI and SCI rats, allodynic responses in SNI and SCI rats, and on motor performance in normal rats, raw data was converted to percent maximum possible effect (%MPE) according to the equation: %MPEZ([Post-treatment value]K[Pre-treatment value])!100/ceiling value of assayK[Pre-treatment value]. %MPE values are presented as meanGSEM. Sigmoidal non-linear regression curve fitting was used to construct dose response curves from %MPE data and ED50 values obtained (GraphPad Prism v3.0 for Windows, GraphPad software, CA, USA). Statistical analysis of data was performed using Sigmastat 2.03 for Windows (SPSS Inc., Germany). Data obtained after pin prick stimulation and in the tail flick and rotarod tests are presented as meanGSEM. Data for von Frey hair stimulation, cold observation and tactile response scores are expressed as medianGmedian derived absolute devision (MAD). Repeated measures (RM) analysis of variance (ANOVA) was used to analyse the overall effects of parametric data obtained after pin prick stimulation, and in the rotarod and tail flick tests. When the F value was significant this was followed by Bonferroni’s t-test. To analyse the overall effects of non-parametric data obtained after von Frey, cold and tactile stimulation, Kruskal Wallis ANOVA on ranks was used. When the H value was significant this was followed by Wilcoxon signed rank test. %MPE values were compared using one way ANOVA followed by Bonferonni’s t-test. P!0.05 was considered to be statistically significant.

(3G0 compared with 0–1 prior to surgery) of the injured trunk area in response to ethyl chloride spray stimulation was also observed in SCI rats. 3.2. Effects of morphine, methadone and codeine on mechanical allodynia in SNI, CCI and SCI rats In SNI rats, the withdrawal threshold in response to von Frey hair stimulation of the injured hindpaw was significantly increased (H[3]Z14.528, PZ0.002) by morphine (1–6 mg/kg, s.c.), (Fig. 1(A)). For the highest dose tested A 20

Withdrawal threshold (g)

physiological saline. All three drugs were administered s.c. in a dosing volume of 1 ml/kg and doses expressed for the salts as milligram per kilogram body weight.

Vehicle Morphine 1 mg/kg Morphine 2 mg/kg Morphine 6 mg/kg

*

10

* *

0 Base

30

60

90

120

180

Time (min) B 20

Withdrawal threshold (g)

350

Vehicle Methadone 0.5 mg/kg Methadone 1 mg/kg Methadone 3 mg/kg

* 10

* *

** 0

3. Results

Base

30

60

90

120

180

Time (min)

In SNI and CCI rats (nZ28 and nZ33, respectively) pronounced mechanical allodynia (0.6G0.4 and 1.0G0.4 g) in response to von Frey hair stimulation of the injured hindpaw was observed, compared with pre-surgery levels which typically ranged from 8.4–21.8 g. Prior to surgery the paw withdrawal duration in response to pin prick stimulation was always !0.5 in both SNI and CCI rats. After nerve injury paw withdrawal duration values in SNI and CCI rats were 11.8G0.5 and 14.2G0.3 s, respectively, indicative of marked mechanical hyperalgesia. Cold allodynia (3G0 and 3G0 in SNI and CCI rats, respectively, compared with 0–1 prior to surgery) of the injured hindpaw in response to ethyl chloride spray stimulation was also observed. In SCI rats (nZ36) mechanical allodynia was observed as a reduction in the vocalization threshold to von Frey hair stimulation of the trunk (3.5G1.1 g compared with 28.8–95 g prior to surgery). Light tactile stimulation initiated painful reactions in some, but not all rats (range 1.5–2 compared with 0 before injury). Cold allodynia

C 20 Withdrawal threshold (g)

3.1. Development of neuropathic pain in SNI, CCI and SCI rats

Vehicle Codeine 5 mg/kg Codeine 10 mg/kg Codeine 30 mg/kg

10

*

*

0 Base

30

60

90

120

180

Time (min) Fig. 1. The effects of morphine, methadone and codeine on mechanical allodynia in SNI rats. The withdrawal threshold (g) in response to von Frey hair stimulation of the injured hindpaw was determined as a measure of mechanical allodynia. Immediately after the baseline response had been obtained either drug or vehicle was injected s.c. and the time course of drug actions followed. (A) Morphine (1, 2 and 6 mg/kg), (B) methadone (0.5, 1 and 3 mg/kg), and (C) codeine (5, 10 and 30 mg/kg). Typical pre-surgery values for von Frey stimulation were in the range 8.4–21.8 g (mode; 21.8 g). Data are presented as medianGMAD. Six to eight animals were included in each group. *P!0.05 vs. baseline, **P!0.01 vs. baseline (Kruskal–Wallis one way ANOVA on Ranks followed by Wilcoxon signed rank test).

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351

Table 1 Effects of morphine, methadone and codeine on nociceptive behaviours in normal, SNI and SCI rats Dose (mg/kg)

Normal

Tail flick

Von Frey

SNI

SCI

SNI

SCI

27.4G12.8 37.6G10.7* 96G2.5*** 2.2 6.4

17.3G6.1 54.4G12.2*** 100G0*** 1.8 7.8

5.3G2.7 22.9G6.4** 93.7G4.2*** 2.8 5.0

0.1G0.8 10.4G5.6 45.4G11.7*** 6.6 2.1

2.3G1.3 20.5G6.6* 93.5G5.9*** 2.9 4.9

Methadone 0.5 1 3 ED50 TI

16.1G5.1 27.3G5** 94G6*** 1.3 2.6

8.4G4 52.4G10.5*** 100G0*** 1 3.4

10.9G2.7 16.5G4.9 65.8G15.6*** 2.2 1.5

0.9 G 0.7 13.2 G 8.4 56.4G15.9*** 2.6 1.3

3G1.3 20.1G7.6* 93.5G6*** 2.9 1.1

Codeine 5 10 30 ED50 TI

10.6G4.6 29.2G9.1 54.1G11.2*** 25.1 38.2

11G6.3 39G16 66.1G13.8** 16.8 57.1

12.5G3.1 19.6G3.5 41.7G12.7*** 43.4 22.1

0.8G0.4 7.3G5.8 24.8G10* 63.9 15

12.5G3.9 24.5G10.5 88.6G10.2*** 14.6 65.8

Morphine 1 2 6 ED50 TI

% MPE values (see Section 2 for details of calculation) were measured from 30–90 min after drug injection. Therapeutic index (TI) values were calculated as ED50 in rotarod (14.1, 3.4 and O960 mg/kg for morphine, methadone and codeine, respectively) divided by ED50 in nociceptive test. All groups nZ6–9 rats. Data are presented as meanGSEM. *P!0.05, **P!0.01, ***P!0.001 vs. corresponding baseline value prior to drug injection; one way ANOVA followed by Bonferroni’s t-test.

(6 mg/kg), the maximal increase achieved (Table 1) was observed 30 min after injection and this increase remained significantly different from baseline for a further 60 min. Administration of methadone (0.5–3 mg/kg, s.c.) also induced a significant increase (H[3]Z18.812, P!0.001) in the withdrawal threshold of the injured hindpaw (Fig. 1(B)). Similar to morphine, the greatest increase observed for methadone was from 30 min after injection (Table 1), and this increase remained significantly different from baseline until 60 min post-injection. A significant antiallodynic effect of codeine (5–30 mg/kg, s.c.) on the withdrawal threshold of the injured hindpaw (H[3]Z 21.633, P!0.001) was also observed (Fig. 1(C)). As with morphine and methadone, the maximal increase in withdrawal threshold after codeine administration was obtained with the highest dose tested (60 mg/kg) 30 min postinjection. The rank order of antiallodynic potency was 2.6!6.6!63.9 mg/kg for methadone, morphine and codeine, respectively (Table 1). In CCI rats, ANOVA on Ranks revealed significant dosedependent antiallodynic effects for morphine (2 and 6 mg/kg, s.c.; H[2]Z39.442, P!0.001) and methadone (1 and 3 mg/kg, s.c.; H[2]Z28.676, P!0.001) in response to von Frey hair stimulation of the injured hindpaw. In contrast, codeine (10 and 30 mg/kg, s.c.) had no effect on mechanical allodynia (H[2]Z1.528, PZ0.466). For the highest dose of morphine (6 mg/kg) and methadone tested (3 mg/kg) the %MPE of antiallodynic effects observed in CCI rats were very similar (69.8G0.3% and 70.8G0.4%, respectively, both P!0.001 vs. vehicle). Notably, these were approximately 5-fold higher than the 13.9G6.7%

non-significant antiallodynic increase obtained after injection of codeine (30 mg/kg). In SCI rats, morphine (1–6 mg/kg, s.c.) injection produced a dose-dependent increase (H[2]Z36.148, P!0.001) in the vocalization threshold induced by von Frey hair stimulation (Fig. 2(A)). The highest dose of morphine (6 mg/kg) increased the vocalisation threshold 30 min after administration, and this increase remained significantly different from baseline until 120 after injection (all time points P!0.05). Administration of methadone (0.5– 3 mg/kg, s.c.) also produced a dose-dependent increase (H[2]Z23.224, P!0.001) in the vocalization threshold from 30 min after injection (Fig. 2(B)). The magnitude of increase was greatest at 90 min post-injection for the highest dose of methadone tested (3 mg/kg), and remained different from baseline until 120 min after administration (P!0.05). A significant effect of codeine (5–30 mg/kg, s.c.) on the vocalization threshold was observed from 30 min after injection (H[2]Z25.215, P!0.001; Fig. 2(C)). The magnitude of increase in the vocalization threshold (147.5G2.5 g) observed for the highest dose of codeine (30 mg/kg) tested was essentially identical to that observed for both morphine and methadone (Fig. 2(C) and Table 1). Furthermore, the vocalization threshold in response to von Frey hair stimulation remained significantly different from baseline throughout the 180 min test period (all time points P!0.05). Tactile sensitivity in response to brush stimulation was also reduced by administration of morphine (H[2]Z39.914, P!0.001) in SCI rats. At the highest dose tested (6 mg/kg) the inhibition was evident from 30–120 min post-injection (all time points P!0.05 vs. baseline; Fig. 3(A)).

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H.K. Erichsen et al. / Pain 116 (2005) 347–358 Morphine 1 mg/kg

200

*

*

*

*

100

Morphine 2 mg/kg Morphine 6 mg/kg

2

1

0

* Base

30

*

* 60

90

Base

30

60

90

120

180

B

180

Methadone 0.5 mg/kg

3

B

Methadone 1 mg/kg

Methadone 0.5 mg/kg

200

*

*

Methadone 3 mg/kg

Response score

Methadone 1 mg/kg Methadone 3 mg/kg

*

2

1

*

100

0 Base

** *

0 Base

30

30

60

90

** C 60

90

120

180

Codeine 5 mg/kg

3

Codeine 10 mg/kg Codeine 30 mg/kg

200

*

*

Codeine 10 mg/kg

1

* Base

30

60

90

120

180

Time (min)

**

**

*

*

0 30

2

0

*

*

Response score

Codeine 5 mg/kg

Base

180

Codeine 30 mg/kg

C

100

120

Time (min)

Time (min)

Vocalization threshold (g)

* 120

Time (min) Time (min)

Vocalization threshold (g)

Morphine 1 mg/kg

3

*

* 0

A

Morphine 2 mg/kg Morphine 6 mg/kg

Response score

Vocalization threshold (g)

A

60

90

120

180

Time (min) Fig. 2. The effects of morphine, methadone and codeine on mechanical allodynia in SCI rats. The vocalization threshold (g) in response to von Frey hair stimulation of the trunk was determined as a measure of mechanical allodynia. Immediately after the baseline response had been obtained either morphine, methadone or codeine was injected s.c. and the time course of drug actions followed. (A) Morphine (1, 2 and 6 mg/kg), (B) methadone (0.5, 1 and 3 mg/kg), and (C) codeine (5, 10 and 30 mg/kg). Typical presurgery values for von Frey stimulation were in the range 28.8–95 g (mode; 95 g). Data are presented as medianGMAD. Six to eight animals were included in each group. *P!0.05 vs. baseline, **P!0.01 vs. baseline (Kruskal–Wallis one way ANOVA on Ranks followed by Wilcoxon signed rank test).

The sensitivity to tactile stimulation was also significantly attenuated by both methadone (H[2]Z27.115, P!0.001) and codeine (H[2]Z9.915, PZ0.01) in SCI rats. However, post-hoc analysis failed to reveal which time points were significantly different from baseline after methadone

Fig. 3. The effects of morphine, methadone and codeine on tactile allodynia in SCI rats. The response score in response to tactile stimulation with the blunt end of a pencil brushed against the trunk was determined as a measure of tactile allodynia. Immediately after the baseline response had been obtained either morphine, methadone or codeine was injected s.c. and the time course of drug actions followed. (A) Morphine (1, 2 and 6 mg/kg), (B) methadone (0.5, 1 and 3 mg/kg), and (C) codeine (5, 10 and 30 mg/kg). Typical pre-surgery values for tactile allodynia were 0. Data are presented as medianGMAD. Six to eight animals were included in each group. *P! 0.05 vs. baseline (Kruskal–Wallis one way ANOVA on Ranks followed by Wilcoxon signed rank test).

administration (Fig. 3(B)). A shorter duration of antinociceptive action was observed for codeine (30 mg/kg; 30 min time point P!0.05 vs. baseline) after tactile stimulation, compared with that observed for morphine (Fig. 3(C). 3.3. Effects of morphine, methadone and codeine on mechanical hyperalgesia in SNI and CCI rats In SNI rats, withdrawal duration of the injured hindpaw in response to pin prick stimulation was significantly

H.K. Erichsen et al. / Pain 116 (2005) 347–358 Vehicle Morphine 6 mg/kg Methadone 3 mg/kg Codeine 30 mg/kg

Withdrawal duration (s)

20

10

** ** **

** ** * *

0 Base

30

60

90

120

180

Time (min) Fig. 4. The effects of morphine, methadone and codeine on mechanical hyperalgesia in SNI rats. The withdrawal duration (s) in response to pin prick stimulation of the injured hindpaw was determined as a measure of mechanical hyperalgesia. Immediately after the baseline response had been obtained either morphine (1, 2 and 6 mg/kg), methadone (0.5, 1 and 3 mg/kg), codeine (5, 10 and 30 mg/kg) or vehicle was injected s.c. and the time course of drug actions followed. However, for ease of representation only the highest dose of each drug is shown, since lower doses had no effect on mechanical hyperalgesia. Pre-surgery values for pin prick stimulation were always !0.5 s. Data are presented as meanGSEM. Six to eight animals were included in each group. ***P!0.001 vs. baseline (two way RM ANOVA followed by Bonferroni’s t-test).

attenuated by both morphine and methadone administration (F[5,191]Z5.036, P!0.001 and F[5,191]Z2.965, P!0.05, respectively). For both morphine and methadone, significant antihyperalgesic effects were only observed for the highest dose of each drug tested (6 and 3 mg/kg, respectively) from 30–60 min post-injection as compared with corresponding baseline levels (13.5G0.8 s and 14.7G0.3 s, respectively; Fig. 4). In contrast, codeine (5–30 mg/kg) failed to affect mechanical hyperalgesia in SNI rats at any of the time points measured (Fig. 4). In CCI rats, two way RM ANOVA also revealed significant antihyperalgesic effects for morphine (2 and 6 mg/kg; F[5,107]Z15.502, P!0.001) and methadone (1 and 3 mg/kg; F[5,107]Z22.141, P!0.001) in response to pin prick stimulation of the injured hindpaw. No time effect of codeine was observed in CCI rats although a significant effect of treatment (F[2,179]Z6.219, P!0.01) on mechanical hyperalgesia was observed. This antihyperalgesic effect was observed for the highest dose tested (30 mg/kg) from 60–180 min after administration compared with vehicle (all time points P!0.05). 3.4. Effects of morphine, methadone and codeine on cold allodynia in SNI and SCI rats Administration of morphine (1–6 mg/kg), methadone (0.5–3 mg/kg) and codeine (5–30mg/kg) to SNI rats revealed weak but significant attenuating effects of treatment on the cold allodynia observed in response to ethyl chloride stimulation of the hindpaw (H[3]Z14.528, P!0.01; H[3]Z9.940, P!0.05 and H[3]Z8.867,

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P!0.05, respectively. However, further analysis failed to reveal any additional significant effects of any of the three drugs tested at specific time points up to 180 min after injection (Fig. 5(A)). In SCI rats, administration of morphine (1–6 mg/kg) attenuated the cold allodynia observed over the trunk region after stimulation with ethyl chloride cold spray (H[2]Z35.992, P!0.001). In particular, the highest dose of morphine (6 mg/kg) significantly attenuated cold observation scores from 30–120 min post-injection (all P!0.05, Fig. 5(B)). After injection of methadone (0.5–3 mg/kg) a dose-dependent decrease in cold hypersensitivity was apparent in rats with spinal injury (H[2]Z21.990, P!0.001). More specifically, injection of methadone (1 mg/kg) significantly attenuated the cold observation score at 30 min after injection as compared with baseline (P!0.05), while the highest dose tested (3 mg/kg) significantly reduced cold observation scores from 30 min and up to 120 min post-injection (all P!0.05; Fig. 5(C)). Administration of codeine (5–30 mg/kg) also attenuated cold observation scores in SCI rats (H[2]Z8.09, P!0.001). Thus, at 30 min post-injection cold observation scores were reduced by 10 mg/kg codeine as compared with baseline, and this effect remained for the highest dose tested until 90 min post-injection (all time points P!0.05; Fig. 5(D)). 3.5. Effects of morphine, methadone and codeine on tail flick responses in uninjured, SNI and SCI rats Subcutaneous injection of morphine (1–6 mg/kg) had a marked antinociceptive effect on the reflex response to a noxious stimulus applied to the tail in normal (F[5,191]Z 28.568, P!0.001), SNI (F[5,185]Z37.653, P!0.001) and SCI (F[5,113]Z26.148, P!0.001) rats. When expressed as a %MPE value, the antinociceptive effect for morphine was dose-dependent and appeared equipotent across all three treatment groups (Table 1). Two way RM ANOVA also revealed a robust dose-dependent antinociceptive effect for methadone (0.5–3 mg/kg) in normal (F[5,191]Z40.814, P!0.001) and SNI (F[5,185]Z39.825, P!0.001) rats. Although an antinociceptive effect of methadone was also observed in SCI rats (F[5,113]Z19.418, P!0.001), it was not dose-dependent. Correspondingly, the %MPE antinociceptive increase obtained was less marked for the highest dose of methadone (3 mg/kg) tested in SCI rats, and this was reflected in a 2-fold lower antinociceptive potency for methadone as compared with SNI rats (Table 1). Codeine (5–30 mg/kg) administration was also associated with an antinociceptive effect on the tail flick latency response in normal (F[5,191]Z9.348, P!0.001), SNI (F[5,185]Z 16.693, P!0.001) and SCI (F[5,113]Z12.436, P!0.001) rats. Once again, the %MPE of antinociception achieved was most marked in SNI rats; codeine was 2.5-fold less potent in SCI rats (Table 1).

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H.K. Erichsen et al. / Pain 116 (2005) 347–358 Vehicle Morphine 6 mg/kg Methadone 3 mg/kg Codeine 30 mg/kg

A

Cold score

4

2

0 Base

30

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90

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Morphine 1 mg/kg Morphine 2 mg/kg Morphine 6 mg/kg

Cold score

4

2

*

* *

0 Base

30

60

* 90

180

Time (min) Methadone 0.5 mg/kg Methadone 1 mg/kg

4

Cold score

Methadone 3 mg/kg

* 2

* *

*

*

Our data show that acute, systemic administration of the m-opioid receptor preferring agonists morphine, methadone and codeine alleviate a wide range of pain-like behaviours in animal models of both peripheral and central neuropathic pain. In particular, administration of all three opioids, especially the weak opioid receptor agonist codeine produced unexpectedly pronounced antiallodynic effects in animals with spinal injury as compared to those with peripheral nerve injury. 4.1. Peripheral nerve injury

0 Base

30

60

90

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Time (min) D

Codeine 5 mg/kg Codeine 10 mg/kg Codeine 30 mg/kg

4

Cold score

To evaluate whether the antinociceptive doses of morphine, methadone and codeine were associated with any toxic side effects, motor function before and after drug administration was assessed in normal, uninjured rats in the rotarod test. Subcutaneous administration of morphine (6, 12 and 24 mg/kg) produced a dose-dependent ataxia (F[2,95]Z26.043, P!0.001). This was most evident for the highest dose of morphine tested 60 min after administration (P!0.001 vs. baseline), (Fig. 6). Methadone (1, 3 and 6 mg/kg, s.c.) administration also impaired motor function (F[2,95]Z22.695, P!0.001), (Fig. 6). The highest dose of methadone tested produced marked motor impairment at 30 and 60 min post-injection (both time points P!0.001 vs. baseline). Administration of codeine (120, 480 and 960 mg/kg, s.c.) had no effect on motor function (Fig. 6), at either 30 or 60 min after administration as compared with corresponding baseline values.

4. Discussion 120

C

3.6. Effects of morphine, methadone and codeine on motor function in uninjured rats

*

2

*

*

Both SNI and CCI rats developed marked hypersensitivity of the injured hindpaw in response to mechanical and cold stimulation (Bennett and Xie, 1988; Decosterd and Woolf, 2000). The attenuation by morphine and methadone of evoked pain-like behaviours in SNI and CCI rats for up to 1–1.5 h after injection corresponds favourably with previous observations obtained in these and other animal models of peripheral nerve injury (Backonja et al., 1995; Erichsen and Blackburn-Munro, 2002; Pelissier et al., 2003; Zhao et al., 2004). In contrast, only a moderate attenuation 3

0 Base

30

60

90

120

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Time (min) Fig. 5. The effects of morphine, methadone and codeine on cold allodynia in SNI and SCI rats. The cold observation score in response to ethyl chloride stimulation of the injured hindpaw or trunk was determined as a measure of cold allodynia in SNI rats and SCI rats, respectively. (A) In SNI rats, immediately after the baseline response had been obtained either morphine (1, 2 and 6 mg/kg), methadone (0.5, 1 and 3 mg/kg), codeine (5, 10 and

30 mg/kg) or vehicle was injected s.c. and the time course of drug actions followed. However, for ease of representation only the highest dose of each drug is shown. In SCI rats, immediately after the baseline response had been obtained either (B) morphine (1, 2 and 6 mg/kg), (C) methadone (0.5, 1 and 3 mg/kg), or (D) codeine (5, 10 and 30 mg/kg) was injected and the time course of drug actions followed. Typical pre-surgery values for cold allodynia were in the range of 0–1 (mode; 0). Data are presented as medianGMAD. Six to eight animals were included in each group. *P!0.05 vs. baseline, **P!0.01 (Kruskal-Wallis one way ANOVA on Ranks followed by Wilcoxon signed rank test).

H.K. Erichsen et al. / Pain 116 (2005) 347–358 100

Vehicle Morphine Methadone Codeine

*** ***

80

%MPE

60 40

*

20 0 –20 0.1

1

10

100

1000

Dose (mg/kg) Fig. 6. The effects of morphine, methadone and codeine on motor performance in the rotarod test. Immediately after a baseline response had been obtained either vehicle, morphine (6, 12 and 24 mg/kg), methadone (1, 3 and 6 mg/kg) or codeine (120, 480 and 960 mg/kg) were administered s.c. to normal, uninjured rats and effects on motor performance (represented as %MPE) determined 30 and 60 min later. Morphine and methadone significantly impaired motor function (ED50Z 14.1 and 3.4 mg/kg, respectively). In contrast, codeine had no effect on motor function at any of the doses tested (ED50O960 mg/kg). Data are presented as meanGSEM. Six to eight animals were included in each group. *P!0.05, ***P!0.001 vs. corresponding baseline injection.

of pain-like behaviours was observed after administration of codeine in SNI and CCI rats. These findings appear to correlate with the level of antinociception (measured by the tail flick latency) conferred by these drugs in SNI rats; methadoneRmorphineOOcodeine. Methadone has been reported to act as a non-competitive antagonist at N-methylD aspartate receptors (Ebert et al., 1995), and this mechanism of action has been suggested to contribute to its antiallodynic action in a rat model of peripheral nerve injury (Bulka et al., 2002a). Ultimately however, the rank order of antinociceptive efficacy observed for morphine, methadone and codeine after acute administration agrees reasonably well with their respective binding affinities for the cloned human m-opioid receptor (2.0!5.6!65 nM, respectively), (Gourlay, 1999). Various studies have reported reduced antinociceptive efficacy of morphine in animal models of peripheral nerve injury. In particular, intrathecally administered morphine has been reported to be less effective in suppressing the tail flick reflex in SNL rats, and is ineffective against tactile allodynia at fully antinociceptive doses (Bian et al., 1995; Ossipov et al., 1995). Recently, these findings have been challenged by Zhao and colleagues who have reported antiallodynic effects of both systemic and intrathecally administered morphine in both the SNI and SNL models (Zhao et al., 2004). We have shown here that the magnitude of antinociception observed for each of the three opioids tested was similar between SNI rats and uninjured control rats (Table 1). However, within SNI rats the antiallodynic potency of each opioid was approximately 2–3-fold less than the corresponding antinociceptive potency, supporting

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a reduced potency of opioids against nerve injury-induced pain-like behaviours in this model. Several mechanisms have been proposed to explain the reduced analgesic efficacy and/or potency of opioids in animal models of neuropathic pain (Dickenson and Suzuki, 2004). These include loss of functional spinal m-and d-opioid binding sites, NMDA receptor-induced excitation of spinal neurones, antagonism of inhibitory opioid actions by cholecystokinin and activation of descending facilitatory controls (Besse et al., 1992; Bian et al., 1999; Porreca et al., 2002; Wiesenfeld-Hallin et al., 2002). Based on tail flick data obtained in SNI and uninjured rats, our results do not readily support the concept that m-opioid receptor-mediated signalling within the spinal dorsal horn is reduced after nerve injury (Besse et al., 1992; Zhang et al., 1998). However, it is possible that any injury-induced changes in m-opioid receptor expression may be specifically localised to the segmental level of hindlimb afferents rather than tail afferents (Porreca et al., 1998). Crucially, this might explain the reduced antiallodynic potency of the opioids tested in SNI rats. 4.2. Spinal cord injury Fewer studies have addressed the analgesic role of opioids after spinal cord injury. Nevertheless, spinal administration of morphine effectively eliminates chronic mechanical allodynia after ischaemic spinal cord injury or T13 spinal hemisection in rats (Kim et al., 2003; Yu et al., 1997). Similarly, systemic administration of morphine in both models attenuates mechanical allodynia, albeit at effective doses of 5 and 10 mg/kg in the SCI model which were also shown to induce sedation in the open field test (Xu et al., 1992; Yu et al., 1997). However, in the T13 spinal hemisection model Kim and colleagues reported no effect of morphine (5 mg/kg) on motor function using a modified behavioural score (Kim et al., 2003). Notably, when we administered morphine, methadone or codeine to normal, uninjured rats at the highest doses tested in the nerve injury models we saw no impairment of motor function up to 60 min post-injection. Whilst this does not exclude the possibility that rats were sedated in the current study, we believe that in the SCI rats any sedative component of the administered opioids would have dissipated 2–3 h after injection. The attenuating actions of all three opioids on both mechanical and cold allodynia in SCI rats were clearly superior to those observed in either SNI or CCI rats. An intriguing facet of the broad spectrum antiallodynic actions observed for morphine, was that they remained completely undiminished throughout the 3 h duration of the experiment; a time far in excess of its plasma half-life (approximately 115 min) in rats (Iwamoto et al., 1977). More remarkable was the improved antiallodynic potency of codeine in relation to morphine and methadone in SCI rats, compared with either its antiallodynic potency in SNI rats or

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antinociceptive potency in normal animals as measured in the tail flick test. This translated into a therapeutic index (ratio of motor impairment/antiallodynia) for codeine of 65.8 in SCI rats, which far exceeds those measured for morphine or methadone (4.9 and 1.1, respectively). Although morphine has been proposed to be the active metabolite of codeine (Chen et al., 1988), the pronounced antiallodynic effect of codeine (in spite of a weaker binding affinity to the m-opioid receptor than morphine) suggests that codeine may act via other mechanisms to mediate its antiallodynic actions in spinally-injured animals (Vree et al., 2000). Methodological limitations may provide a more parsimonious explanation. The differences in codeine sensitivity between rats with peripheral or central neuropathic pain could be attributed to sex differences in opioidmediated antinociception (Terner et al., 2003). However, this is unlikely here, since both low and high efficacy opioids have been reported to induce a more potent antinociception in male rather than female rats of various strains (Terner et al., 2003). Differential opioid-mediated antinociception between substrains of Sprague–Dawley rats supplied by different vendors have also been reported (Bulka et al., 2002b; Bulka et al., 2004). In the current study, Sprague–Dawley rats used in peripheral and central neuropathic pain experiments were obtained from two different sources (Harlan and Møllegaard, respectively). Whilst we cannot easily discount this factor, we noted that ED50 values obtained for all three opioids in the tail flick test in non-injured rats from Harlan compared favourably with those previously reported for rats obtained from Møllegaard (Bulka et al., 2004). 4.3. Clinical implications A number of recently completed clinical trials support the use of opioids in the treatment of peripheral neuropathic pain (Gimbel et al., 2003; Raja et al., 2002; Watson and Babul, 1998; Watson et al., 2003). Unfortunately, the evidence to support the use of opioids in central pain associated with multiple sclerosis, stroke or spinal cord injury is much less convincing (Attal et al., 2002; Kalman et al., 2002; Rowbotham et al., 2003). However, intravenous administration of morphine reduces brush-induced mechanical allodynia in central pain patients (Attal et al., 2002), a finding which was essentially replicated in the present study in SCI rats. Thus, opioids may be of some use for attenuating specific symptoms of neuropathic pain in subgroups of patients with central neuropathic pain. To our knowledge the effects of codeine on neuropathic pain of central origin have never been reported. Nevertheless, some insight may be gained from human models of experimental pain where codeine has been reported to inhibit temporal pain summation after repetitive electrical stimulation of the sural nerve and to relieve cold pressor pain (Enggaard et al., 2001). Temporal summation may be a key feature of neuropathic pain, suggesting that codeine may have some

utility in the treatment of such pain, if dosing and study design problems can be circumvented (Max et al., 1988).

5. Conclusions The present results confirm previous findings describing the analgesic efficacy of systemically administered morphine in animal models of neuropathic pain. We have extended these observations to show that systemic administration of methadone and codeine also profoundly attenuate pain-like behaviours in the SNI, CCI and SCI animal models of neuropathic pain. However, our results partially contrast with reported opioid efficacy in corresponding clinical neuropathic pain conditions of peripheral and especially central aetiology. This raises the issue as to whether a single animal model of nerve injury can sufficiently encapsulate the plethora of pathophysiological mechanisms which contribute to the signs and symptoms of neuropathic pain in corresponding human conditions. Finally, bearing in mind some of the adverse effects often associated with prolonged opioid administration, the current results suggest that weaker affinity opioids such as codeine, may have some utility in the clinical treatment of central neuropathic pain conditions.

Acknowledgements We would like to thank Nete Ibsen and Paula Lindberg for expert technical assistance. Support was provided by the Danish University of Pharmaceutical Sciences.

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