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Increased nociceptive response in mice lacking the adenosine A1 receptor
Pain 113 (2005) 395–404 www.elsevier.com/locate/pain
Increased nociceptive response in mice lacking the adenosine A1 receptor Wei-Ping Wua, Jing-Xia Haoa, Linda Halldnerb, Cecilia Lo¨vdahlb, Gary E. DeLanderb,c, Zsuzsanna Wiesenfeld-Hallina, Bertil B. Fredholmb, Xiao-Jun Xua,* a
Department of Neurotec, Division of Clinical Neurophysiology, Karolinska Institutet, Karolinska University Hospital-Huddinge, S-141 86 Stockholm, Sweden b Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, S-141 86 Stockholm, Sweden c Department of Pharmacology, College of Pharmacy, Oregon State University, Corvallis, OR 97311-3507, USA Received 27 August 2004; received in revised form 5 November 2004; accepted 22 November 2004
Abstract The role of the adenosine A1 receptor in nociception was assessed using mice lacking the A1 receptor (A1RK/K) and in rats. Under normal conditions, the A1RK/K mice exhibited moderate heat hyperalgesia in comparison to the wild-type mice (A1RC/C). The mechanical and cold sensitivity were unchanged. The antinociceptive effect of morphine given intrathecally (i.t.), but not systemically, was reduced in A1RK/K mice and this reduction in the spinal effect of morphine was not associated with a decrease in binding of the m-opioid ligand DAMGO in the spinal cord. A1RK/K mice also exhibited hypersensitivity to heat, but not mechanical stimuli, after localized inflammation induced by carrageenan. In mice with photochemically induced partial sciatic nerve injury, the neuropathic pain-like behavioral response to heat or cold stimulation were significantly increased in the A1RK/Kmice. Peripheral nerve injury did not change the level of adenosine A1 receptor in the dorsal spinal cord in rats and i.t. administration of R-PIA effectively alleviated pain-like behaviors after partial nerve injury in rats and in C57/BL/6 mice. Taken together, these data suggest that the adenosine A1 receptor plays a physiological role in inhibiting nociceptive input at the spinal level in mice. The C-fiber input mediating noxious heat is inhibited more than other inputs. A1 receptors also contribute to the antinociceptive effect of spinal morphine. Selective A1 receptor agonists may be tested clinically as analgesics, particularly under conditions of neuropathic pain. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Hyperalgesia; Inflammation; Mice; Morphine; Neuropathic pain; Nociception
1. Introduction Adenosine is a ubiquitous endogenous neurotransmitter/modulator (Dunwiddie and Masino, 2001). It acts on specific membrane-bound G-protein coupled receptors (Klinger et al., 2002; Ribeiro et al., 2003) and four subtypes of adenosine receptors (AR), A1R, A2AR, A2BR, and A3R, have been cloned and pharmacologically characterized (Fredholm et al., 2001). A1R and A2AR binding sites have been found in the dorsal horn of the spinal cord, particularly in the substantia gelatinosa (Choca et al., 1987, 1988). The A1R is mostly located on intrinsic dorsal horn neurons, but there are also * Corresponding author. Tel.: C46 8 58582213; fax: C46 8 7748856. E-mail address: [email protected] (X.-J. Xu).
some A1R on dorsal root ganglion (DRG) cells (Schulte et al., 2003). Although some A2AR can be detected in spinal cord, the mRNA for A2AR is not found there, but in the DRG (Kaelin-Lang et al., 1998, 1999). Activation of A1R primarily produces inhibition of neuronal activity in the spinal cord and DRG (Deuchars et al., 2001; Dolphin et al., 1986; Li and Perl, 1994; Patel et al., 2001; Reeve and Dickenson, 1995; Salter et al., 1993). Systemic or spinal administration of adenosine analogs produces antinociception in a wide range of tests (Karlsten et al., 1990; Keil and DeLander, 1992; Post, 1984; see Sawynok and Liu, 2003; Sawynok et al., 1986 for review). Adenosine analogs also produce motor effect when given spinally, effects that are nonetheless distinguishable from antinociception (Karlsten et al., 1990; Sawynok and Poon, 1999). It is believed that the A1R is responsible for
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.11.020
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the antinociceptive effect of adenosine analogues (Lee and Yaksh, 1996; Nakamura et al., 1997; Poon and Sawynok, 1998; but see DeLander and Keil, 1994). Adenosine analogues are effective in treating neuropathic pain in animal models (Cui et al., 1997; Gomes et al., 1999; Lavand’homme and Eisenach, 1999; Lee and Yaksh, 1996; Von Heijne et al., 2000) and in clinical studies (Belfrage et al., 1995, 1999; Karlsten and Gordh, 1995). Spinal release of adenosine has also been implicated in spinal and supraspinal morphine antinociception (Delander and Hopkins, 1986; Sweeney et al., 1987, 1989; see Sawynok and Liu, 2003 for review). Although extensive data have been gathered concerning the effect of exogenous adenosine on nociception, relatively little is known about the physiological significance of adenosine and its respective receptors. Mice with a targeted deletion of receptor genes provided a new avenue for analyzing the physiology of adenosine receptors. Mice lacking A2AR were shown to be hypoalgesic, probably reflecting a peripheral pro-nociceptive function (Ledent et al., 1997). We showed that mice lacking A1Rs exhibited moderate hyperalgesia to heat stimulation (Johansson et al., 2001). The present study aimed to extend our previous studies by examining the responses of A1R knock-out (A1RK/K) mice to inflammation or partial sciatic nerve injury. We also assessed the contribution of A1R in spinal vs. systemic morphine antinociception. The effect of intrathecal (i.t.) R-phenyl-isopropyl adenosine (R-PIA), a somewhat A1R selective agonist, on neuropathic pain-like behaviors in mice and rats with partial nerve injury was also examined as was the density of A1R binding after nerve injury in rats.
2. Materials and methods 2.1. Animals All experiments were approved by the regional research ethics committees. The animals used were A1RK/K, C/K and C/C (129/OlaHsd!C57BL) mice of both sexes, male C57BL/6 mice (B&K Universal, Sollentuna, Sweden) and male Sprague–Dawley rats (Mo¨llega˚rd, Denmark). 2.2. Generation of mice lacking the A1 receptors A1R knock-out animals were generated as previously described (Johansson et al., 2001). Each experiment was carried out using siblings with different genotypes. Genotyping of the offspring was performed using PCR essentially as described (Turner et al., 2003). The lack of A1R was confirmed using in situ hybridization and by the absence of high affinity binding sites for the selective adenosine A1R antagonist DPCPX in several areas of the brain and spinal cord using quantitative autoradiography (not shown).
was injected subcutaneously into the plantar area of the left or right hindpaws, respectively. The effect of carrageenan or saline on the response of the mice to mechanical and heat stimuli was determined 24 h after the injection. Paw thickness was measured with a caliper at metatarsal level before and 24 h after the injection. 2.4. Photochemically-induced sciatic nerve injury in mice and rats The methods used to produce photochemical sciatic nerve injury in mice or rats have been described in detail previously (Hao et al., 2000; Kupers et al., 1998). In brief, the animals were anesthetized by chloral hydrate (300 mg/kg, i.p.) and the left sciatic nerve was exposed and irradiated by an argon ion laser in the presence of a photosensitizing dye, erythrosine B, injected i.v. The irradiation time was 45 s for mice and 2 min for rats. The wounds were then closed in layers. The animals were returned to the cages and behavioral tests determining mechanical, cold and heat sensitivity of the hind paws were conducted up to 90 days post-injury. 2.5. Behavioral tests 2.5.1. Heat tests 1. Tail flick. The mice were gently held in the experimenter’s hand and a radiant heat source was focused 1–2 cm from the tip of the tail and the latency to tail flick was recorded automatically. 2. Hargreaves test. The heat response of hind paws after nerve injury or inflammation was tested with a radiant heat source (IITC, Woodland Hills, CA, USA) aimed at the plantar surface of the hind paw through the metal mesh floor and the latency to withdrawal of the stimulated paw was measured. 2.5.2. Mechanical test To test of sensitivity to mechanical stimulation, the mice or rats were placed in plastic cages with a metal mesh floor. The plantar surface of the hind paws was stimulated with a set of calibrated nylon monofilaments (von Frey hairs, Stoelting, IL, USA) with increasing force until the animal withdrew the limb. Each monofilament was applied 5 times. The withdrawal threshold was taken as the force at which the animal withdrew the paw from at least 3 out of 5 consecutive stimuli. 2.5.3. Cold test The response of mice to cold stimulation was tested by gently touching the plantar skin of the hind paws with an acetone bubble formed at the tip of a 1 ml syringe. Cold response in rats was tested by spraying ethyl chloride spray onto the plantar skin. The responses were classified according to the following scale: 0, no response; 1, startle response without paw withdrawal; 2, withdrawal of the paw; 3, withdrawal of the paw often combined with flinching and licking the paw and/or vocalization; 4, above as 3 with responses persist more than 10 s. 2.6. Implantation of i.t. catheter in mice and rats
2.3. Carrageenan-induced inflammation The mice were anesthetized with chloral hydrate (300 mg/kg, i.p.). l-carrageenan (2% in 20 ml saline) or 20 ml normal saline
For i.t. administration of R-PIA and morphine a catheter was implanted chronically into mice and rats under chloral hydrate anesthesia. The skin overlying the vertebra L5-6 was cut and for
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and DPCPX, and 20 mM 2-chloroadenosine for CGS 21680 binding. Incubations were terminated by washing twice in 0 8C Tris–HCl buffer, followed by one wash in 0 8C water. Slides were dried overnight and apposed to Hyperfilm-3H film (Amersham) for 4–8 weeks.
rats, a PE 10 catheter (0.61 mm outer diameter) was inserted into the subarachnoid space through a guide cannula connected to a 20 gauge needle that punctured the muscle and dura at the level of the cauda equina. For mice, a hole was made by a needle in the overlaying muscle and a stretched PE10 catheter with a guide wire inside punctured the dura, upon penetration of the dura, which is accompanied by typical tail twitch, the wire was withdrawn from the catheter. The catheter was then carefully implanted rostrally, aiming its tip at the lumbar cord enlargement. The method for chronic lumbar catheter implantation in mice was presented in detail recently (Wu et al., 2004). The proper location of the catheter was examined by assessing sensory and motor blockade after the i.t. injection of lidocaine (Xylocain 20 or 50 mg/ml, Astra, So¨derta¨lje, Sweden). Only animals exhibiting bilateral motor and sensory blockade were used.
Morphine hydrochloride was from Apoteksbolaget (Stockholm, Sweden). R-PIA and 2 chloro adenosine was from Sigma (St Louis, MO, USA). Data are presented as medianGmedian absolute deviation (MAD) or meanGS.E.M. where appropriate. In morphine experiments, % maximal possible effect (% MPE) in the tail flick test was calculated based on the formula
2.7. DAMGO binding in A1R knock-out mice
%MPE Z
Spinal cord was dissected out from 1 wild type and 1 knockout mice and rapidly frozen on dry ice. Sections were cut using a cryostat and slide mounted. Autoradiography was carried out essentially as described by Unterwald et al. (1998), using concentrations between 0.5 and 10 nM [3H]-DAMGO. Nonspecific binding was defined using 10 mM naloxone. Non-specific binding did not exceed 15% of total binding even at the highest concentration examined. Measurements were made in the dorsal horn. Each data-point is based on the average of five to six sections.
The data were analyzed with the Kruskal-Wallis ANOVA, Wilcoxon Signed Ranks test, Mann–Whitney U-test, ANOVA with repeated measures followed by Fisher’s PLSD test as well as paired- or unpaired t-test. P!0.05 was considered to be statistically significant.
2.8. A1R level in nerve injured rats
As described previously (Johansson et al., 2001), the A1RK/K mice are viable, fertile and do not exhibit gross behavioral abnormality. The A1RK/K mice also did not differ from A1RC/C mice in terms of mechanical withdrawal threshold and cold response scores (Fig. 1A and B). However, the A 1RK/K mice did exhibit significantly shortened tail flick latency in comparison to A1RC/C mice (Fig. 1C).
Partial sciatic nerve ligation was induced unilaterally on the right side, while the rat was under halothane-induced anesthesia, as described (DeLander et al., 1997). Animals with sciatic nerve lesions developed mechanical allodynia, as determined with von Frey hairs, within 24 h after surgery. Using this model, we have observed allodynia lasting longer than 28 days. Animals used in the present experiments were sacrificed after anesthesia 30 min to 28 days following surgery. The lumbar section of the spinal cord was rapidly dissected, frozen on dry ice and stored at K70 8C until sectioned. Tissue slices (10 mm) from the lumbar cord were obtained from L4 or high L5 and thaw mounted on gelatin coated slides for autoradiography and stored at K20 8C. [3H]-Cyclohexyl adenosine and [3H]-CGS 21680 were used in saturation isotherms and competition assays as standard selective agonists for A1R and A2AR, respectively. A saturation isotherm was also determined for [3H]-DPCPX, an A1R antagonist. In addition, autoradiography using the selective A2AR antagonist [3H]-SCH 58261 was carried out at 0.3 and 1 nM ligand concentration. Receptor autoradiography was carried out essentially as described previously (Fredholm et al., 1998; Johansson et al., 1993; Parkinson and Fredholm, 1990). Briefly, tissue sections were pre-incubated for 30 min at 37 8C in 170 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA and 2 U adenosine deaminase/ml. Following two 10 min washes in Tris–HCl, tissues were incubated for 2 h at room temperature. The incubation mixture was composed of 170 mM Tris–HCl buffer, tritiated ligand and 2 U adenosine deaminase/ml. Incubation mixture for experiments with CGS 21680 and DPCPX also included 10 mM or 1 mM MgCl2, respectively. Non-specific binding was determined in the presence of 100 mM R-PIA for CHA
2.9. Drugs and statistics
post drug latency baseline !100 cut off baseline
3. Results 3.1. Basal nociceptive sensitivity
3.2. Effect of i.t. and systemic morphine and DAMGO binding in A1RK/K mice As previous reported in part (Johansson et al., 2001), systemic (i.p.) administration of morphine similarly and significantly increased tail flick latency in A1RC/C and A1RK/K mice (Fig. 2A). In contrast, the effect of i.t. morphine was significantly reduced in the A1RK/K mice compared to A1RC/C mice (Fig. 2B). No significant difference was observed in DAMGO binding between A1RC/C mice and A1RK/K mice (Fig. 2C). 3.3. Carrageenan-induced inflammation The inflamed paw exhibited edema 24 h after carrageenan administration and no difference was observed between A1RC/C, C/K and K/K mice (Fig. 3A). Carrageenan also induced bilateral mechanical hypersensitivity which was not significantly different among the three groups of
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Fig. 1. Paw withdrawal threshold to von Frey hair stimulation (A), cold response score (B) and tail flick latency to heat stimulation (C) in A1RC/C, C/K and K/K mice. The data are expressed as meanGSEM. The genotypes and number of mice studied are indicated under the columns. Kruskal–Wallis test showed no significant overall difference was seen among the three groups for mechanical and cold stimulation whereas ANOVA indicated overall significant differences for tail flick latency (F(2,72)Z4.7, P!0.05). *P!0.01 compared to A1RC/C mice with Fisher PLSD test.
mice (Fig. 3B). However, significant hypersensitivity to radiant stimulation was observed for the ipsilateral paw in A1RK/K mice, but not in the other two groups (Fig. 3C).
Fig. 2. Effects of i.p. (A) and i.t. (B) morphine on the tail flick latency in A1RC/C and A1RK/K mice. The effect is expressed as % possible maximal effect and shown as meanGS.E.M. Seven to twelve mice were included in each group. ANOVA with repeated measures indicated that morphine in all four groups induced an overall increase in tail flick latency and for i.p. injection, no difference is found between the A1RK/K and A1RC/C mice in the effect of morphine. In contrast, ANOVA indicated significant difference between the two groups for i.t. morphine (F(1,78)Z9.9, **P!0.01). (C) Binding of 3H-DAMGO to spinal cord. Quantitative autoradiography was carried out as described. Kd values were 2.6G1.6 in A1RC/C and 3.7G1.6 in A1RK/K mouse spinal cord. The corresponding Bmax values were 20.9G4.8 and 25.2G4.6 fmol/mg, respectively (nZ5K6).
3.4. Partial sciatic nerve injury in mice As described previously, mechanical, cold and heat hypersensitivity develops in mice after photochemically induced sciatic nerve ischemic injury (Hao et al., 2000). The changes in the ipsilateral side are more profound and consistent, although a contralateral effect was also observed.
For mechanical hypersensitivity, no significant difference was found between A1RC/C and K/K mice at most time points after injury (Fig. 4) whereas heat hypersensitivity was significantly greater in the A1RK/K than in A1RC/C (Fig. 5). The most striking difference was seen for cold
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Fig. 3. Effect of unilateral subcutaneous injection of carrageenan on paw thickness (A), paw withdrawal threshold to mechanical stimulation (B) and paw withdrawal latency to heat stimulation (C) ipsilterally (L) and contralaterally (R) 24 h after injection. The control value (C) is the average of both sides before carrageenan. The data are expressed as meanGSEM for A and C and as medianGMAD for B. The genotypes and number of mice are indicated under the columns, *P!0.05, **P!0.01 and ***P! 0.001 compared to control value with paired t-test in A and C and with Wilcoxon signed-ranks test in B.
hypersensitivity where A1RC/C mice only exhibited a weak and transient effect. Both A1RC/K mice and K/K mice had significantly increased cold hypersensitivity over A1RC/C mice (Fig. 6).
Fig. 4. Mechanical hypersensitivity in A1RC/C (nZ9), C/K (nZ11) and K/K mice (nZ11) after partial sciatic nerve ischemic injury. The data are expressed as medianGMAD. *P!0.05 and **P!0.01 compared to normal value (time 0) with Wilcoxon signed-rank test. †P!0.05 compared to A1RC/C mice with Mann–Whitney U-test.
3.5. A1R level in the spinal cord in rats after nerve section Binding of various adenosine receptor ligands was studied in rat spinal cord to determine the type of receptor and if there were changes after partial ligation of the sciatic nerve. The selective A2AR antagonist [3H]SCH 58261 did not show any specific binding (not shown). By contrast, the agonist CGS 21680 did show binding to the dorsal horn, which coincided with that observed with the A1R agonist [3H]CHA and the A1R antagonist [3H]-DPCPX (not shown). However, the binding of CGS 21680 showed an unusual pharmacology, being displaced by low nM concentrations of CHA, DPCPX and XAC, but only weakly displaced by A2AR
antagonists including chlorostyrylcaffeine and KF 17387. Thus, the binding of CGS 21680 appeared to be to the atypical site previously described (Cunha et al., 1996) and not to A2AR. The binding of the A1R antagonist DPCPX had a Kd value of 0.3 nM in the presence or absence of GTP, but the Bmax was markedly increased in the presence of the guanine nucleotide, in agreement with observations made in brain (Fastbom and Fredholm, 1990; Parkinson and Fredholm, 1992). The binding in the absence of GTP is decreased because endogenous adenosine binds quasi-irreversibly to the receptor. Therefore, in the experiments to determine if there are changes with sciatic nerve ligation, banding was performed both
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Fig. 5. Heat hypersensitivity in A1RC/C (black circles, nZ9). C/K (triangels, nZ11) and K/K (open circles, nZ11) mice after partial nerve injury. ANOVA indicated significant overall difference among the three groups (F(2,512)Z7, P!0.01). Individual comparisons are made with paired or unpaired unpaired t-test, *P!0.05 and **P!0.01 compared to time 0 and †P!0.05 compare to A1RC/C group.
in the presence and in the absence of GTP. The results, presented in Fig. 7, show that there are no clear changes in A1 receptors upon sciatic lesion. 3.6. Effects of i.t. R-PIA on neuropathic pain-like behavior in mice and rats after partial sciatic nerve injury The experiment was conducted 7–14 days after photochemically-induced partial sciatic nerve injury when animals were exhibiting stable pain-related behaviors. i.t. R-PIA effectively alleviated mechanical and cold hypersensitivity in nerve injured C57/BL/6 mice already at a dose of 0.1 nmol i.t. and the effect was further increased at higher doses (Fig. 8A and B). A similar effect was observed in rats, although higher doses were required (1–10 nmol i.t.) (Fig. 8C and D). No motor effect was observed after i.t. R-PIA administration at any of the doses employed.
4. Discussion 4.1. Hyperalgesa in A1RK/K mice In the tail flick test we consistently observed a shortened latency in A1RK/K mice, indicating hyperalgesia to heat stimulation. These mice, however, did not exhibit increased response to innocuous or noxious mechanical and cold stimulation under basal condition. Thus, it is likely that adenosine, via A1R, exerts a tonic inhibitory effect on noxious heat input, but not on input mediating mechanical or cold stimulation, in normal mice. Release of adenosine in response to noxious stimulation occurred in the spinal cord (Cahill et al., 1993a; Sweeney et al., 1989) and adenosine deaminase
Fig. 6. Cold hypersensitivity in A1RC/C (nZ9), C/K (nZ11) and K/K mice (nZ11) after partial sciatic nerve ischemic injury. The data are expressed as medianGMAD. *P!0.05 and **P!0.01 compared to normal value (time 0) with Wilcoxon signed-rank test. †P!0.05 compared to A1RC/C mice with Mann–Whitney U-test.
and particularly adenosine kinase inhibitors induced antinociception, suggesting that released adenosine serves to inhibit noxious input in rodents (Keil and DeLander, 1992, 1996; Kowaluk, 1998; Poon and Sawynok, 1998). Further evidence for an ongoing release of adenosine can be observed after i.t. administration of adenosine receptor antagonists. Thus, several antagonists, with limited receptor selectivity, induce hyperalgesia or a pain-like behavioral response (Jurna, 1984; Keil and DeLander, 1996; Nagaoka et al., 1993; Sawynok et al., 1986). Our results imply that the adenosine A1R in the spinal cord is an important link in such an action. Detailed morphological studies have localized A1Rs in a subpopulation of superficial dorsal horn interneurons.
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et al., 2001). Interestingly, two previous studies have shown that hyperalgesia after systemic or i.t. administration of theophylline is blocked by NMDA receptor antagonists (Nagaoka et al., 1993; Paalzow, 1994). 4.2. A1R is important for the spinal, but not the supraspinal, antinociceptive effect of morphine
Fig. 7. Binding of [3H]-DPCPX (1 nM) to dorsal spinal cord at different times after unilateral ligation of the sciatic nerve. Binding experiments were performed in the presence and absence of 1 mM GTP. Mean of 4–8 determinations. The standard deviations were close to 10%.
They do not express numerous markers, including glutamic acid decarboxylase (GAD), glycine and nitric oxide synthase (Schulte et al., 2003), suggesting that these neurons are not inhibitory interneurons. Adenosine caused postsynaptic inhibition in neurons in substantia gelatinosa through membrane hypopolarization and depression of excitatory postsynaptic currents (De Koninck and Henry, 1992; Li and Perl, 1994; Salter et al., 1993), suggesting that these A1R positive neurons are likely to be excitatory interneurons containing glutamate. Thus, it is probable that the hyperalgesia observed in A1RK/K mice may be due to a reduced inhibition of glutamatergic neuronal activity, especially as inhibition of glutamatergic excitatory neurotransmission is a well known feature of A1R stimulation (see Dunwiddie and Masino, 2001; Johansson
It has been reported that morphine releases adenosine from brain slices and that some effects of morphine in brain are mediated by adenosine (Fredholm and Vernet, 1978; Phillis et al., 1980). Extensive studies, many done by Sawynok and coworkers, have shown that this is also the case for spinal morphine (see Sawynok and Liu, 2003). Thus, morphine induced a calcium-dependent release of adenosine from dorsal spinal cord in vivo and in vitro (Cahill et al., 1993b; Sweeney et al., 1987, 1989) and antinociceptive effect of i.t. morphine is blocked by adenosine receptor antagonists (Delander and Hopkins, 1986; Sawynok et al., 1989). In the present study we found that the effect of a moderate dose of i.t. morphine was significantly reduced in A1RK/K mice whereas that of a high dose of morphine was unchanged. Since A1R deletion did not alter DAMGO binding in the spinal cord, it is unlikely that this reduction in the effect of i.t. morphine is due to changes in the level of m-opioid receptors. This suggests that the spinal antinociceptive effect of morphine is linked to the activation of A1Rs in mice, supporting earlier findings of morphineinduced spinal release of adenosine. It also suggests that i.t. morphine also possesses adenosine-independent effects that
Fig. 8. Effect of i.t. R-PIA on mechanical (A, C) and cold (B, D) hypersensitivity in mice (A, B) and rats (C, D) after partial sciatic nerve injury. The data are expressed as medianGMAD and 7–9 animals were included in each group. *P!0.05 and **P!0.01 compared to control value at time 0 with Wilcoxon signed-ranks test.
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are able to fully compensate the loss of A1R at high doses. The effect of systemic morphine was unchanged in the A1RK/K mice. Since systemic morphine acts through spinal and supraspinal sites, our results suggest that A1R plays little or no role in the supraspinal antinociceptive effect of morphine. 4.3. Carrageenan-induced inflammation No difference was seen among A1RC/C, C/K and K/K mice in paw edema after carrageenan-induced localized inflammation, indicating that A1R may not be involved in localized non-neurogenic inflammatory response in the periphery. This is in contrast to the findings that deletion of A3Rs reduces carrageenan-induced inflammatory edema and plasma extravasation (Wu et al., 2002). Adenosine possesses anti-inflammatory properties mainly mediated by the A2ARs rather than the A1Rs (Cronstein, 1994; Montesinos et al., 2002). Carrageenan inflammation in our present paradigm produced bilateral mechanical hypersensitivity, which has been observed in several strains of mice (Wu et al., 2002; Xu et al., 1997). This is likely an indication of central integration of inflammatory sensitization (Koltzenburg et al., 1999). A1R depletion does not affect mechanical hypersensitivity, but A1RK/K mice exhibited significant heat hyperalgesia whereas the wild-type and heterozygotes did not. This would suggest a selective inhibitory effect by A1R on input from noxious heat stimulation after inflammation similar to that observed previously in normal A1RK/K mice. Such inhibition may be exerted at the spinal level or in the periphery since activation of A1R in the periphery has been shown to produce antinociception in inflamed tissue (Sawynok and Poon, 1999; Sawynok and Liu, 2003). 4.4. Physiological role of the A1 receptor in nerve injured mice A1RK/K mice differ little from wild-type mice in mechanical allodynia following partial nerve injury. Thus, A1Rs are physiologically involved in modulation of mechanical input after nerve injury, which involves activation of large diameter Ab-fibers (Field et al., 1999; Ossipov et al. 1999). Nevertheless R-PIA is active in alleviating mechanical allodynia in nerve injured mice, suggesting that A1Rs are present. A1RK/K mice exhibited more marked heat hyperalgesia than A1RC/C mice after nerve injury. This agrees with what is seen in normal and inflamed rats, supporting a role for A1R in capsaicin-sensitive C-fiber input (Field et al., 1999; Ossipov et al. 1999). Interestingly, there was a marked increase in cold allodynia in A1RK/K as well as A1RC/K mice, suggesting that cold input after nerve injury is particularly sensitive to adenosinergic control. The characteristics of cold afferent input in mice under normal conditions and after nerve injury are unknown and studies in
rats have suggested that cold input and cold allodynia may be mediated by C-fibers (Hao et al., 1996; Leem et al., 1993). Other studies have suggested that cold is transmitted via a special population of superficial dorsal horn neurons in a manner that is different from other sensory modalities (Craig and Hunsley, 1991; Han et al., 1998). It would therefore be of interest to determine the relationship between the A1R containing neurons and those neurons involved in cold transmission. Our results support an ongoing release of adenosine and tonic activation of A1R after nerve injury as implied by studies in which adenosine kinase inhibitors were shown to reduce pain-like behaviors in rat models of neuropathic pain (Lavand’homme and Eisenach, 1999; Suzuki et al., 2001; Zhu et al., 2001). Furthermore, spontaneous GTP-g-S binding is elevated in dorsal spinal cord in rats with spinal nerve ligation, suggesting an altered interaction between A1R and G proteins in neuropathic states (Bantel et al., 2002). 4.5. Activation of A1 receptors alleviates neuropathic pain-like behavioral responses in rats and mice Peripheral nerve injury induces complex plastic changes in the levels of neurotransmitters, as well as their receptors, in DRG cells and in the spinal cord (Ho¨kfelt et al., 1994). Some of these plastic changes have been shown to be related to the potency and efficacy of analgesics (Ho¨kfelt et al., 1994). Little is known about the changes in A1Rs after peripheral nerve injury, although earlier work has shown that dorsal rhizotomy does not markedly alter A1R binding (Choca et al., 1988). Bantel et al. recently reported that in rats after spinal nerve ligation there is no change in R-PIA stimulated GTP-g-S binding in the spinal cord, indicating that nerve injury does not reduce signal transduction through the A1R. Our present results showing that axotomy did not reduce the level of DPCPX binding in rats support this conclusion. In accordance with the unaltered level of A1R in the spinal cord after peripheral nerve injury, spinal administration of adenosine analogues probably acting on the A1R (Johansson et al., 2001) alleviated neuropathic pain-like behaviors in rats after partial peripheral nerve injury or spinal cord injury (Cui et al., 1997; Lee and Yaksh, 1996; Von Heijne et al., 2000). In both rat and mouse, the antinociceptive effect of R-PIA in nerve injured animal occurs at lower doses than motor effects. These observations indicate that drugs directly, or indirectly, stimulating A1Rs may be beneficial in neuropathic pain.
Acknowledgements The present study was supported by the Swedish Science Council (02553, 07913, 12168), the Clinical Research Center at the Karolinska University Hospital-Huddinge and the Research Funds of the Karolinska Institutet.
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Ribeiro JA, Sebastiao AM, de Mendonca A. Adenosine receptors in the nervous system: pathophysiological implications. Prog Neurobiol 2003; 68:377–92. Salter MW, de Koninck Y, Henry JL. Physiological roel for adenosine and ATP in synaptit transmission in the spinal dorsal horn. Prog Neurobiol 1993;41:125–56. Sawynok J, Liu XJ. Adenosine in the spinal cord and periphery, release and regulation of pain. Prog Neurobiol 2003;69:313–40. Sawynok J, Poon A. Purines: potential for development as analgesic agents. In: Sawyonk J, Cowan A, editors. Novel aspects of pain management. New York: Wiley-Liss; 1999. p. 229–48. Sawynok J, Sweeney MI, White TD. Classification of adenosine receptors mediating antinociception in the rat spinal cord. Br J Pharmacol 1986; 88:923–30. Sawynok J, Sweeney MI, White TD. Adenosine release may mediate spinal analgesia by morphine. Trends Pharmacol Sci 1989;10:186–9. Schulte G, Robertson B, Fredholm BB, DeLander GE, Shortland P, Grant G, Molander C. Distribution of nociceptive adenosine A1 receptors in the rat spinal cord dorsal horn, and relationship to primnary afferents and neuronal subpopulations. Neuroscience 2003;121:907–16. Suzuki R, Stanfa LC, Kowaluk EA, Williams M, Jarvis MF, Dickenson A. The effect of ABT-702, a novel adenosine kinase inhibitor, on the responses of spinal neurones follwing carrageenan inflammation and peripheral nerve injury. Br J Pharmacol 2001;132:1615–23. Sweeney MI, White TD, Jhamandas KH, Sawynok J. Morphine releases endogenous adenosine from the spinal cord in vivo. Eur J Pharmacol 1987;141:169–70. Sweeney MI, White TD, Sawynok J. Morphine, capsaicin and KC release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord. J Pharmacol Exp Ther 1989;2248:447–54. Turner CP, Seli M, Ment L, Stewart W, Yan H, Johansson B, Fredholm BB, Blackburn M, Rivkees SA. A1 adenosine receptors mediate hypoxiainduced ventriculomegaly. Proc Natl Acad Sci USA 2003;100: 11718–22. Unterwald EM, Anton B, To T, Lam H, Evans CJ. Quantitative immunolocalization of mu opioid receptors: regulation by naltrexone. Neuroscience 1998;85:897–905. Von Heijne M, Hao J-X, Sollevi A, Xu X-J, Wiesenfeld-Hallin Z. Marked enhancement of anti-allodynic effect by combined intrathecal administration of the adenosine A1-receptor agonist R-phenylisopropyladenosine and morphine in a rat model of central pain. Acta Anaesthesiol Scand 2000;44:665–71. Wu W-P, Hao J-X, Halldner-Henriksson L, Xu X-J, Jacobson MA, Wiesenfeld-Hallin Z, Fredholm BB. Decreased inflammatory pain due to reduced carrageenan-induced inflammation in mice lacking adenosine A3 receptors. Neuroscience 2002;114:523–7. Wu W-P, Xu X-J, Hao J-X. Chronic lumbar catheterization of the spinal subarachnoid space in mice. J Neurosci Methods 2004;133:65–9. Xu XJ, Hao JX, Andell-Jonsson S, Poli V, Bartfai T, Wiesenfeld-Hallin Z. Nociceptive responses in interleukin-6-deficient mice to peripheral inflammation and peripheral nerve section. Cytokine 1997;9:1028–33. Zhu CZ, Mikusa J, Chu KL, Cowart M, Kowaluk EA, Jarvis MF, McGaraughty SA. A-134974: a novel adenosine kinase inhibitor, relieves tactile allodynia via spinal sites of action in peripheral nerve injured rats. Brain Res 2001;905:104–10.
Increased nociceptive response in mice lacking the adenosine A1 receptor Wei-Ping Wua, Jing-Xia Haoa, Linda Halldnerb, Cecilia Lo¨vdahlb, Gary E. DeLanderb,c, Zsuzsanna Wiesenfeld-Hallina, Bertil B. Fredholmb, Xiao-Jun Xua,* a
Department of Neurotec, Division of Clinical Neurophysiology, Karolinska Institutet, Karolinska University Hospital-Huddinge, S-141 86 Stockholm, Sweden b Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, S-141 86 Stockholm, Sweden c Department of Pharmacology, College of Pharmacy, Oregon State University, Corvallis, OR 97311-3507, USA Received 27 August 2004; received in revised form 5 November 2004; accepted 22 November 2004
Abstract The role of the adenosine A1 receptor in nociception was assessed using mice lacking the A1 receptor (A1RK/K) and in rats. Under normal conditions, the A1RK/K mice exhibited moderate heat hyperalgesia in comparison to the wild-type mice (A1RC/C). The mechanical and cold sensitivity were unchanged. The antinociceptive effect of morphine given intrathecally (i.t.), but not systemically, was reduced in A1RK/K mice and this reduction in the spinal effect of morphine was not associated with a decrease in binding of the m-opioid ligand DAMGO in the spinal cord. A1RK/K mice also exhibited hypersensitivity to heat, but not mechanical stimuli, after localized inflammation induced by carrageenan. In mice with photochemically induced partial sciatic nerve injury, the neuropathic pain-like behavioral response to heat or cold stimulation were significantly increased in the A1RK/Kmice. Peripheral nerve injury did not change the level of adenosine A1 receptor in the dorsal spinal cord in rats and i.t. administration of R-PIA effectively alleviated pain-like behaviors after partial nerve injury in rats and in C57/BL/6 mice. Taken together, these data suggest that the adenosine A1 receptor plays a physiological role in inhibiting nociceptive input at the spinal level in mice. The C-fiber input mediating noxious heat is inhibited more than other inputs. A1 receptors also contribute to the antinociceptive effect of spinal morphine. Selective A1 receptor agonists may be tested clinically as analgesics, particularly under conditions of neuropathic pain. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Hyperalgesia; Inflammation; Mice; Morphine; Neuropathic pain; Nociception
1. Introduction Adenosine is a ubiquitous endogenous neurotransmitter/modulator (Dunwiddie and Masino, 2001). It acts on specific membrane-bound G-protein coupled receptors (Klinger et al., 2002; Ribeiro et al., 2003) and four subtypes of adenosine receptors (AR), A1R, A2AR, A2BR, and A3R, have been cloned and pharmacologically characterized (Fredholm et al., 2001). A1R and A2AR binding sites have been found in the dorsal horn of the spinal cord, particularly in the substantia gelatinosa (Choca et al., 1987, 1988). The A1R is mostly located on intrinsic dorsal horn neurons, but there are also * Corresponding author. Tel.: C46 8 58582213; fax: C46 8 7748856. E-mail address: [email protected] (X.-J. Xu).
some A1R on dorsal root ganglion (DRG) cells (Schulte et al., 2003). Although some A2AR can be detected in spinal cord, the mRNA for A2AR is not found there, but in the DRG (Kaelin-Lang et al., 1998, 1999). Activation of A1R primarily produces inhibition of neuronal activity in the spinal cord and DRG (Deuchars et al., 2001; Dolphin et al., 1986; Li and Perl, 1994; Patel et al., 2001; Reeve and Dickenson, 1995; Salter et al., 1993). Systemic or spinal administration of adenosine analogs produces antinociception in a wide range of tests (Karlsten et al., 1990; Keil and DeLander, 1992; Post, 1984; see Sawynok and Liu, 2003; Sawynok et al., 1986 for review). Adenosine analogs also produce motor effect when given spinally, effects that are nonetheless distinguishable from antinociception (Karlsten et al., 1990; Sawynok and Poon, 1999). It is believed that the A1R is responsible for
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.11.020
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the antinociceptive effect of adenosine analogues (Lee and Yaksh, 1996; Nakamura et al., 1997; Poon and Sawynok, 1998; but see DeLander and Keil, 1994). Adenosine analogues are effective in treating neuropathic pain in animal models (Cui et al., 1997; Gomes et al., 1999; Lavand’homme and Eisenach, 1999; Lee and Yaksh, 1996; Von Heijne et al., 2000) and in clinical studies (Belfrage et al., 1995, 1999; Karlsten and Gordh, 1995). Spinal release of adenosine has also been implicated in spinal and supraspinal morphine antinociception (Delander and Hopkins, 1986; Sweeney et al., 1987, 1989; see Sawynok and Liu, 2003 for review). Although extensive data have been gathered concerning the effect of exogenous adenosine on nociception, relatively little is known about the physiological significance of adenosine and its respective receptors. Mice with a targeted deletion of receptor genes provided a new avenue for analyzing the physiology of adenosine receptors. Mice lacking A2AR were shown to be hypoalgesic, probably reflecting a peripheral pro-nociceptive function (Ledent et al., 1997). We showed that mice lacking A1Rs exhibited moderate hyperalgesia to heat stimulation (Johansson et al., 2001). The present study aimed to extend our previous studies by examining the responses of A1R knock-out (A1RK/K) mice to inflammation or partial sciatic nerve injury. We also assessed the contribution of A1R in spinal vs. systemic morphine antinociception. The effect of intrathecal (i.t.) R-phenyl-isopropyl adenosine (R-PIA), a somewhat A1R selective agonist, on neuropathic pain-like behaviors in mice and rats with partial nerve injury was also examined as was the density of A1R binding after nerve injury in rats.
2. Materials and methods 2.1. Animals All experiments were approved by the regional research ethics committees. The animals used were A1RK/K, C/K and C/C (129/OlaHsd!C57BL) mice of both sexes, male C57BL/6 mice (B&K Universal, Sollentuna, Sweden) and male Sprague–Dawley rats (Mo¨llega˚rd, Denmark). 2.2. Generation of mice lacking the A1 receptors A1R knock-out animals were generated as previously described (Johansson et al., 2001). Each experiment was carried out using siblings with different genotypes. Genotyping of the offspring was performed using PCR essentially as described (Turner et al., 2003). The lack of A1R was confirmed using in situ hybridization and by the absence of high affinity binding sites for the selective adenosine A1R antagonist DPCPX in several areas of the brain and spinal cord using quantitative autoradiography (not shown).
was injected subcutaneously into the plantar area of the left or right hindpaws, respectively. The effect of carrageenan or saline on the response of the mice to mechanical and heat stimuli was determined 24 h after the injection. Paw thickness was measured with a caliper at metatarsal level before and 24 h after the injection. 2.4. Photochemically-induced sciatic nerve injury in mice and rats The methods used to produce photochemical sciatic nerve injury in mice or rats have been described in detail previously (Hao et al., 2000; Kupers et al., 1998). In brief, the animals were anesthetized by chloral hydrate (300 mg/kg, i.p.) and the left sciatic nerve was exposed and irradiated by an argon ion laser in the presence of a photosensitizing dye, erythrosine B, injected i.v. The irradiation time was 45 s for mice and 2 min for rats. The wounds were then closed in layers. The animals were returned to the cages and behavioral tests determining mechanical, cold and heat sensitivity of the hind paws were conducted up to 90 days post-injury. 2.5. Behavioral tests 2.5.1. Heat tests 1. Tail flick. The mice were gently held in the experimenter’s hand and a radiant heat source was focused 1–2 cm from the tip of the tail and the latency to tail flick was recorded automatically. 2. Hargreaves test. The heat response of hind paws after nerve injury or inflammation was tested with a radiant heat source (IITC, Woodland Hills, CA, USA) aimed at the plantar surface of the hind paw through the metal mesh floor and the latency to withdrawal of the stimulated paw was measured. 2.5.2. Mechanical test To test of sensitivity to mechanical stimulation, the mice or rats were placed in plastic cages with a metal mesh floor. The plantar surface of the hind paws was stimulated with a set of calibrated nylon monofilaments (von Frey hairs, Stoelting, IL, USA) with increasing force until the animal withdrew the limb. Each monofilament was applied 5 times. The withdrawal threshold was taken as the force at which the animal withdrew the paw from at least 3 out of 5 consecutive stimuli. 2.5.3. Cold test The response of mice to cold stimulation was tested by gently touching the plantar skin of the hind paws with an acetone bubble formed at the tip of a 1 ml syringe. Cold response in rats was tested by spraying ethyl chloride spray onto the plantar skin. The responses were classified according to the following scale: 0, no response; 1, startle response without paw withdrawal; 2, withdrawal of the paw; 3, withdrawal of the paw often combined with flinching and licking the paw and/or vocalization; 4, above as 3 with responses persist more than 10 s. 2.6. Implantation of i.t. catheter in mice and rats
2.3. Carrageenan-induced inflammation The mice were anesthetized with chloral hydrate (300 mg/kg, i.p.). l-carrageenan (2% in 20 ml saline) or 20 ml normal saline
For i.t. administration of R-PIA and morphine a catheter was implanted chronically into mice and rats under chloral hydrate anesthesia. The skin overlying the vertebra L5-6 was cut and for
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and DPCPX, and 20 mM 2-chloroadenosine for CGS 21680 binding. Incubations were terminated by washing twice in 0 8C Tris–HCl buffer, followed by one wash in 0 8C water. Slides were dried overnight and apposed to Hyperfilm-3H film (Amersham) for 4–8 weeks.
rats, a PE 10 catheter (0.61 mm outer diameter) was inserted into the subarachnoid space through a guide cannula connected to a 20 gauge needle that punctured the muscle and dura at the level of the cauda equina. For mice, a hole was made by a needle in the overlaying muscle and a stretched PE10 catheter with a guide wire inside punctured the dura, upon penetration of the dura, which is accompanied by typical tail twitch, the wire was withdrawn from the catheter. The catheter was then carefully implanted rostrally, aiming its tip at the lumbar cord enlargement. The method for chronic lumbar catheter implantation in mice was presented in detail recently (Wu et al., 2004). The proper location of the catheter was examined by assessing sensory and motor blockade after the i.t. injection of lidocaine (Xylocain 20 or 50 mg/ml, Astra, So¨derta¨lje, Sweden). Only animals exhibiting bilateral motor and sensory blockade were used.
Morphine hydrochloride was from Apoteksbolaget (Stockholm, Sweden). R-PIA and 2 chloro adenosine was from Sigma (St Louis, MO, USA). Data are presented as medianGmedian absolute deviation (MAD) or meanGS.E.M. where appropriate. In morphine experiments, % maximal possible effect (% MPE) in the tail flick test was calculated based on the formula
2.7. DAMGO binding in A1R knock-out mice
%MPE Z
Spinal cord was dissected out from 1 wild type and 1 knockout mice and rapidly frozen on dry ice. Sections were cut using a cryostat and slide mounted. Autoradiography was carried out essentially as described by Unterwald et al. (1998), using concentrations between 0.5 and 10 nM [3H]-DAMGO. Nonspecific binding was defined using 10 mM naloxone. Non-specific binding did not exceed 15% of total binding even at the highest concentration examined. Measurements were made in the dorsal horn. Each data-point is based on the average of five to six sections.
The data were analyzed with the Kruskal-Wallis ANOVA, Wilcoxon Signed Ranks test, Mann–Whitney U-test, ANOVA with repeated measures followed by Fisher’s PLSD test as well as paired- or unpaired t-test. P!0.05 was considered to be statistically significant.
2.8. A1R level in nerve injured rats
As described previously (Johansson et al., 2001), the A1RK/K mice are viable, fertile and do not exhibit gross behavioral abnormality. The A1RK/K mice also did not differ from A1RC/C mice in terms of mechanical withdrawal threshold and cold response scores (Fig. 1A and B). However, the A 1RK/K mice did exhibit significantly shortened tail flick latency in comparison to A1RC/C mice (Fig. 1C).
Partial sciatic nerve ligation was induced unilaterally on the right side, while the rat was under halothane-induced anesthesia, as described (DeLander et al., 1997). Animals with sciatic nerve lesions developed mechanical allodynia, as determined with von Frey hairs, within 24 h after surgery. Using this model, we have observed allodynia lasting longer than 28 days. Animals used in the present experiments were sacrificed after anesthesia 30 min to 28 days following surgery. The lumbar section of the spinal cord was rapidly dissected, frozen on dry ice and stored at K70 8C until sectioned. Tissue slices (10 mm) from the lumbar cord were obtained from L4 or high L5 and thaw mounted on gelatin coated slides for autoradiography and stored at K20 8C. [3H]-Cyclohexyl adenosine and [3H]-CGS 21680 were used in saturation isotherms and competition assays as standard selective agonists for A1R and A2AR, respectively. A saturation isotherm was also determined for [3H]-DPCPX, an A1R antagonist. In addition, autoradiography using the selective A2AR antagonist [3H]-SCH 58261 was carried out at 0.3 and 1 nM ligand concentration. Receptor autoradiography was carried out essentially as described previously (Fredholm et al., 1998; Johansson et al., 1993; Parkinson and Fredholm, 1990). Briefly, tissue sections were pre-incubated for 30 min at 37 8C in 170 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA and 2 U adenosine deaminase/ml. Following two 10 min washes in Tris–HCl, tissues were incubated for 2 h at room temperature. The incubation mixture was composed of 170 mM Tris–HCl buffer, tritiated ligand and 2 U adenosine deaminase/ml. Incubation mixture for experiments with CGS 21680 and DPCPX also included 10 mM or 1 mM MgCl2, respectively. Non-specific binding was determined in the presence of 100 mM R-PIA for CHA
2.9. Drugs and statistics
post drug latency baseline !100 cut off baseline
3. Results 3.1. Basal nociceptive sensitivity
3.2. Effect of i.t. and systemic morphine and DAMGO binding in A1RK/K mice As previous reported in part (Johansson et al., 2001), systemic (i.p.) administration of morphine similarly and significantly increased tail flick latency in A1RC/C and A1RK/K mice (Fig. 2A). In contrast, the effect of i.t. morphine was significantly reduced in the A1RK/K mice compared to A1RC/C mice (Fig. 2B). No significant difference was observed in DAMGO binding between A1RC/C mice and A1RK/K mice (Fig. 2C). 3.3. Carrageenan-induced inflammation The inflamed paw exhibited edema 24 h after carrageenan administration and no difference was observed between A1RC/C, C/K and K/K mice (Fig. 3A). Carrageenan also induced bilateral mechanical hypersensitivity which was not significantly different among the three groups of
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Fig. 1. Paw withdrawal threshold to von Frey hair stimulation (A), cold response score (B) and tail flick latency to heat stimulation (C) in A1RC/C, C/K and K/K mice. The data are expressed as meanGSEM. The genotypes and number of mice studied are indicated under the columns. Kruskal–Wallis test showed no significant overall difference was seen among the three groups for mechanical and cold stimulation whereas ANOVA indicated overall significant differences for tail flick latency (F(2,72)Z4.7, P!0.05). *P!0.01 compared to A1RC/C mice with Fisher PLSD test.
mice (Fig. 3B). However, significant hypersensitivity to radiant stimulation was observed for the ipsilateral paw in A1RK/K mice, but not in the other two groups (Fig. 3C).
Fig. 2. Effects of i.p. (A) and i.t. (B) morphine on the tail flick latency in A1RC/C and A1RK/K mice. The effect is expressed as % possible maximal effect and shown as meanGS.E.M. Seven to twelve mice were included in each group. ANOVA with repeated measures indicated that morphine in all four groups induced an overall increase in tail flick latency and for i.p. injection, no difference is found between the A1RK/K and A1RC/C mice in the effect of morphine. In contrast, ANOVA indicated significant difference between the two groups for i.t. morphine (F(1,78)Z9.9, **P!0.01). (C) Binding of 3H-DAMGO to spinal cord. Quantitative autoradiography was carried out as described. Kd values were 2.6G1.6 in A1RC/C and 3.7G1.6 in A1RK/K mouse spinal cord. The corresponding Bmax values were 20.9G4.8 and 25.2G4.6 fmol/mg, respectively (nZ5K6).
3.4. Partial sciatic nerve injury in mice As described previously, mechanical, cold and heat hypersensitivity develops in mice after photochemically induced sciatic nerve ischemic injury (Hao et al., 2000). The changes in the ipsilateral side are more profound and consistent, although a contralateral effect was also observed.
For mechanical hypersensitivity, no significant difference was found between A1RC/C and K/K mice at most time points after injury (Fig. 4) whereas heat hypersensitivity was significantly greater in the A1RK/K than in A1RC/C (Fig. 5). The most striking difference was seen for cold
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Fig. 3. Effect of unilateral subcutaneous injection of carrageenan on paw thickness (A), paw withdrawal threshold to mechanical stimulation (B) and paw withdrawal latency to heat stimulation (C) ipsilterally (L) and contralaterally (R) 24 h after injection. The control value (C) is the average of both sides before carrageenan. The data are expressed as meanGSEM for A and C and as medianGMAD for B. The genotypes and number of mice are indicated under the columns, *P!0.05, **P!0.01 and ***P! 0.001 compared to control value with paired t-test in A and C and with Wilcoxon signed-ranks test in B.
hypersensitivity where A1RC/C mice only exhibited a weak and transient effect. Both A1RC/K mice and K/K mice had significantly increased cold hypersensitivity over A1RC/C mice (Fig. 6).
Fig. 4. Mechanical hypersensitivity in A1RC/C (nZ9), C/K (nZ11) and K/K mice (nZ11) after partial sciatic nerve ischemic injury. The data are expressed as medianGMAD. *P!0.05 and **P!0.01 compared to normal value (time 0) with Wilcoxon signed-rank test. †P!0.05 compared to A1RC/C mice with Mann–Whitney U-test.
3.5. A1R level in the spinal cord in rats after nerve section Binding of various adenosine receptor ligands was studied in rat spinal cord to determine the type of receptor and if there were changes after partial ligation of the sciatic nerve. The selective A2AR antagonist [3H]SCH 58261 did not show any specific binding (not shown). By contrast, the agonist CGS 21680 did show binding to the dorsal horn, which coincided with that observed with the A1R agonist [3H]CHA and the A1R antagonist [3H]-DPCPX (not shown). However, the binding of CGS 21680 showed an unusual pharmacology, being displaced by low nM concentrations of CHA, DPCPX and XAC, but only weakly displaced by A2AR
antagonists including chlorostyrylcaffeine and KF 17387. Thus, the binding of CGS 21680 appeared to be to the atypical site previously described (Cunha et al., 1996) and not to A2AR. The binding of the A1R antagonist DPCPX had a Kd value of 0.3 nM in the presence or absence of GTP, but the Bmax was markedly increased in the presence of the guanine nucleotide, in agreement with observations made in brain (Fastbom and Fredholm, 1990; Parkinson and Fredholm, 1992). The binding in the absence of GTP is decreased because endogenous adenosine binds quasi-irreversibly to the receptor. Therefore, in the experiments to determine if there are changes with sciatic nerve ligation, banding was performed both
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Fig. 5. Heat hypersensitivity in A1RC/C (black circles, nZ9). C/K (triangels, nZ11) and K/K (open circles, nZ11) mice after partial nerve injury. ANOVA indicated significant overall difference among the three groups (F(2,512)Z7, P!0.01). Individual comparisons are made with paired or unpaired unpaired t-test, *P!0.05 and **P!0.01 compared to time 0 and †P!0.05 compare to A1RC/C group.
in the presence and in the absence of GTP. The results, presented in Fig. 7, show that there are no clear changes in A1 receptors upon sciatic lesion. 3.6. Effects of i.t. R-PIA on neuropathic pain-like behavior in mice and rats after partial sciatic nerve injury The experiment was conducted 7–14 days after photochemically-induced partial sciatic nerve injury when animals were exhibiting stable pain-related behaviors. i.t. R-PIA effectively alleviated mechanical and cold hypersensitivity in nerve injured C57/BL/6 mice already at a dose of 0.1 nmol i.t. and the effect was further increased at higher doses (Fig. 8A and B). A similar effect was observed in rats, although higher doses were required (1–10 nmol i.t.) (Fig. 8C and D). No motor effect was observed after i.t. R-PIA administration at any of the doses employed.
4. Discussion 4.1. Hyperalgesa in A1RK/K mice In the tail flick test we consistently observed a shortened latency in A1RK/K mice, indicating hyperalgesia to heat stimulation. These mice, however, did not exhibit increased response to innocuous or noxious mechanical and cold stimulation under basal condition. Thus, it is likely that adenosine, via A1R, exerts a tonic inhibitory effect on noxious heat input, but not on input mediating mechanical or cold stimulation, in normal mice. Release of adenosine in response to noxious stimulation occurred in the spinal cord (Cahill et al., 1993a; Sweeney et al., 1989) and adenosine deaminase
Fig. 6. Cold hypersensitivity in A1RC/C (nZ9), C/K (nZ11) and K/K mice (nZ11) after partial sciatic nerve ischemic injury. The data are expressed as medianGMAD. *P!0.05 and **P!0.01 compared to normal value (time 0) with Wilcoxon signed-rank test. †P!0.05 compared to A1RC/C mice with Mann–Whitney U-test.
and particularly adenosine kinase inhibitors induced antinociception, suggesting that released adenosine serves to inhibit noxious input in rodents (Keil and DeLander, 1992, 1996; Kowaluk, 1998; Poon and Sawynok, 1998). Further evidence for an ongoing release of adenosine can be observed after i.t. administration of adenosine receptor antagonists. Thus, several antagonists, with limited receptor selectivity, induce hyperalgesia or a pain-like behavioral response (Jurna, 1984; Keil and DeLander, 1996; Nagaoka et al., 1993; Sawynok et al., 1986). Our results imply that the adenosine A1R in the spinal cord is an important link in such an action. Detailed morphological studies have localized A1Rs in a subpopulation of superficial dorsal horn interneurons.
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et al., 2001). Interestingly, two previous studies have shown that hyperalgesia after systemic or i.t. administration of theophylline is blocked by NMDA receptor antagonists (Nagaoka et al., 1993; Paalzow, 1994). 4.2. A1R is important for the spinal, but not the supraspinal, antinociceptive effect of morphine
Fig. 7. Binding of [3H]-DPCPX (1 nM) to dorsal spinal cord at different times after unilateral ligation of the sciatic nerve. Binding experiments were performed in the presence and absence of 1 mM GTP. Mean of 4–8 determinations. The standard deviations were close to 10%.
They do not express numerous markers, including glutamic acid decarboxylase (GAD), glycine and nitric oxide synthase (Schulte et al., 2003), suggesting that these neurons are not inhibitory interneurons. Adenosine caused postsynaptic inhibition in neurons in substantia gelatinosa through membrane hypopolarization and depression of excitatory postsynaptic currents (De Koninck and Henry, 1992; Li and Perl, 1994; Salter et al., 1993), suggesting that these A1R positive neurons are likely to be excitatory interneurons containing glutamate. Thus, it is probable that the hyperalgesia observed in A1RK/K mice may be due to a reduced inhibition of glutamatergic neuronal activity, especially as inhibition of glutamatergic excitatory neurotransmission is a well known feature of A1R stimulation (see Dunwiddie and Masino, 2001; Johansson
It has been reported that morphine releases adenosine from brain slices and that some effects of morphine in brain are mediated by adenosine (Fredholm and Vernet, 1978; Phillis et al., 1980). Extensive studies, many done by Sawynok and coworkers, have shown that this is also the case for spinal morphine (see Sawynok and Liu, 2003). Thus, morphine induced a calcium-dependent release of adenosine from dorsal spinal cord in vivo and in vitro (Cahill et al., 1993b; Sweeney et al., 1987, 1989) and antinociceptive effect of i.t. morphine is blocked by adenosine receptor antagonists (Delander and Hopkins, 1986; Sawynok et al., 1989). In the present study we found that the effect of a moderate dose of i.t. morphine was significantly reduced in A1RK/K mice whereas that of a high dose of morphine was unchanged. Since A1R deletion did not alter DAMGO binding in the spinal cord, it is unlikely that this reduction in the effect of i.t. morphine is due to changes in the level of m-opioid receptors. This suggests that the spinal antinociceptive effect of morphine is linked to the activation of A1Rs in mice, supporting earlier findings of morphineinduced spinal release of adenosine. It also suggests that i.t. morphine also possesses adenosine-independent effects that
Fig. 8. Effect of i.t. R-PIA on mechanical (A, C) and cold (B, D) hypersensitivity in mice (A, B) and rats (C, D) after partial sciatic nerve injury. The data are expressed as medianGMAD and 7–9 animals were included in each group. *P!0.05 and **P!0.01 compared to control value at time 0 with Wilcoxon signed-ranks test.
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are able to fully compensate the loss of A1R at high doses. The effect of systemic morphine was unchanged in the A1RK/K mice. Since systemic morphine acts through spinal and supraspinal sites, our results suggest that A1R plays little or no role in the supraspinal antinociceptive effect of morphine. 4.3. Carrageenan-induced inflammation No difference was seen among A1RC/C, C/K and K/K mice in paw edema after carrageenan-induced localized inflammation, indicating that A1R may not be involved in localized non-neurogenic inflammatory response in the periphery. This is in contrast to the findings that deletion of A3Rs reduces carrageenan-induced inflammatory edema and plasma extravasation (Wu et al., 2002). Adenosine possesses anti-inflammatory properties mainly mediated by the A2ARs rather than the A1Rs (Cronstein, 1994; Montesinos et al., 2002). Carrageenan inflammation in our present paradigm produced bilateral mechanical hypersensitivity, which has been observed in several strains of mice (Wu et al., 2002; Xu et al., 1997). This is likely an indication of central integration of inflammatory sensitization (Koltzenburg et al., 1999). A1R depletion does not affect mechanical hypersensitivity, but A1RK/K mice exhibited significant heat hyperalgesia whereas the wild-type and heterozygotes did not. This would suggest a selective inhibitory effect by A1R on input from noxious heat stimulation after inflammation similar to that observed previously in normal A1RK/K mice. Such inhibition may be exerted at the spinal level or in the periphery since activation of A1R in the periphery has been shown to produce antinociception in inflamed tissue (Sawynok and Poon, 1999; Sawynok and Liu, 2003). 4.4. Physiological role of the A1 receptor in nerve injured mice A1RK/K mice differ little from wild-type mice in mechanical allodynia following partial nerve injury. Thus, A1Rs are physiologically involved in modulation of mechanical input after nerve injury, which involves activation of large diameter Ab-fibers (Field et al., 1999; Ossipov et al. 1999). Nevertheless R-PIA is active in alleviating mechanical allodynia in nerve injured mice, suggesting that A1Rs are present. A1RK/K mice exhibited more marked heat hyperalgesia than A1RC/C mice after nerve injury. This agrees with what is seen in normal and inflamed rats, supporting a role for A1R in capsaicin-sensitive C-fiber input (Field et al., 1999; Ossipov et al. 1999). Interestingly, there was a marked increase in cold allodynia in A1RK/K as well as A1RC/K mice, suggesting that cold input after nerve injury is particularly sensitive to adenosinergic control. The characteristics of cold afferent input in mice under normal conditions and after nerve injury are unknown and studies in
rats have suggested that cold input and cold allodynia may be mediated by C-fibers (Hao et al., 1996; Leem et al., 1993). Other studies have suggested that cold is transmitted via a special population of superficial dorsal horn neurons in a manner that is different from other sensory modalities (Craig and Hunsley, 1991; Han et al., 1998). It would therefore be of interest to determine the relationship between the A1R containing neurons and those neurons involved in cold transmission. Our results support an ongoing release of adenosine and tonic activation of A1R after nerve injury as implied by studies in which adenosine kinase inhibitors were shown to reduce pain-like behaviors in rat models of neuropathic pain (Lavand’homme and Eisenach, 1999; Suzuki et al., 2001; Zhu et al., 2001). Furthermore, spontaneous GTP-g-S binding is elevated in dorsal spinal cord in rats with spinal nerve ligation, suggesting an altered interaction between A1R and G proteins in neuropathic states (Bantel et al., 2002). 4.5. Activation of A1 receptors alleviates neuropathic pain-like behavioral responses in rats and mice Peripheral nerve injury induces complex plastic changes in the levels of neurotransmitters, as well as their receptors, in DRG cells and in the spinal cord (Ho¨kfelt et al., 1994). Some of these plastic changes have been shown to be related to the potency and efficacy of analgesics (Ho¨kfelt et al., 1994). Little is known about the changes in A1Rs after peripheral nerve injury, although earlier work has shown that dorsal rhizotomy does not markedly alter A1R binding (Choca et al., 1988). Bantel et al. recently reported that in rats after spinal nerve ligation there is no change in R-PIA stimulated GTP-g-S binding in the spinal cord, indicating that nerve injury does not reduce signal transduction through the A1R. Our present results showing that axotomy did not reduce the level of DPCPX binding in rats support this conclusion. In accordance with the unaltered level of A1R in the spinal cord after peripheral nerve injury, spinal administration of adenosine analogues probably acting on the A1R (Johansson et al., 2001) alleviated neuropathic pain-like behaviors in rats after partial peripheral nerve injury or spinal cord injury (Cui et al., 1997; Lee and Yaksh, 1996; Von Heijne et al., 2000). In both rat and mouse, the antinociceptive effect of R-PIA in nerve injured animal occurs at lower doses than motor effects. These observations indicate that drugs directly, or indirectly, stimulating A1Rs may be beneficial in neuropathic pain.
Acknowledgements The present study was supported by the Swedish Science Council (02553, 07913, 12168), the Clinical Research Center at the Karolinska University Hospital-Huddinge and the Research Funds of the Karolinska Institutet.
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