The α2A adrenoceptor and the sympathetic postganglionic neuron contribute to the development of neuropathic heat hyperalgesia in mice

Pain 85 (2000) 345±358 www.elsevier.nl/locate/pain

The a2A adrenoceptor and the sympathetic postganglionic neuron contribute to the development of neuropathic heat hyperalgesia in mice Wade S. Kingery a, b,*, Tian Z. Guo c, d, M. Frances Davies c, d, Lee Limbird e, Mervyn Maze c, d a

b

Department of Functional Restoration, Stanford University, Stanford, CA, USA Department of Physical Medicine and Rehabilitation, Veterans Affairs, Palo Alto Health Care System, Palo Alto, CA 94304, USA c Department of Anesthesia, Stanford University, Stanford, CA, USA d Department of Anesthesiology Services, Veterans Affairs, Palo Alto Health Care System, Palo Alto, CA 94304, USA e Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA Received 4 May 1999; received in revised form 26 July 1999; accepted 19 November 1999

Abstract We have addressed the role of the sympathetic nervous system in the development and maintenance of neuropathic pain. Using a new neuropathic mouse model, we examined the development of hyperalgesia in transgenic mice lacking functional a2A adrenoceptors and in sympathectomized wild-type mice, to determine if sympathetic±sensory coupling generates hyperalgesia. The development of neuropathic heat hyperalgesia required the presence of both the a2A adrenoceptor and the sympathetic postganglionic neuron (SPGN), but the development of mechanical hyperalgesia did not require either the a2A adrenoceptor or the SPGN, indicating different mechanisms of sensitization. These results suggest that the development of neuropathic heat hyperalgesia, but not mechanical hyperalgesia, requires sympathetic±sensory coupling in the peripheral nervous system. Nerve injury enhanced the analgesic ef®cacy of the a2 adrenoceptor agonist dexmedetomidine, and paradoxically also induced an analgesic response to a2 adrenoceptor antagonists. The a2 agonist-evoked analgesia to mechanical stimuli was mediated by activating central a2A adrenoceptors, possibly at the spinal level. The peripherally restricted a2 antagonist L659,066 evoked analgesia for heat, but not for mechanical stimuli, ®ndings which support the hypothesis that the peripheral a2 adrenoceptor plays a role in both the development and the maintenance of neuropathic heat hyperalgesia. The a2 antagonist-evoked analgesia for heat stimuli was mediated by blocking peripheral and probably central a2 adrenoceptors, while the analgesia for mechanical stimuli was mediated by blocking central a2A adrenoceptors. Intradermal injections with an a2 agonist or antagonist had no effect on nociceptive thresholds, indicating that sympathetic±sensory coupling at the level of the cutaneous nociceptor did not contribute to the maintenance of neuropathic hyperalgesia. Published for the International Association for the Study of Pain by Elsevier Science B.V. Keywords: Neuropathic pain; Sympathetic nervous system; Adrenosensitivity; a adrenergic; Dexmedetomidine; Atipamezole

1. Introduction Injury to a peripheral nerve in humans can result in hyperalgesia and pain precipitated or exacerbated by increased activity of sympathetic postganglionic neurons (SPGN), and by the cutaneous injection of epinephrine or norepinephrine (Walker and Nulsen, 1948; Chabal et al., 1992; Torebjork et al., 1995; Choi and Rowbotham, 1997). This clinical condition has been referred to as sympathetically maintained pain, and can be seen in complex regional pain syndromes (CRPS) and other types of neuropathic pain. * Corresponding author. VAPAHCS, Physical Medicine and Rehabilitation Service (117), 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel.: 11-650-493-5000 ext. 64768; fax: 11-650-852-3470. E-mail address: [email protected] (W.S. Kingery)

Fig. 1 illustrates several potential sites of sympathetic± sensory coupling in the peripheral nervous system. Sympathetic activity or local applications of adrenergic agonists do not normally excite primary afferent nociceptors or cause hyperalgesia (Janig et al., 1996), but after partial nerve injury SPGN stimulation and norepinephrine injection activates the undamaged cutaneous primary afferents (Sato and Perl, 1991; O'Halloran and Perl, 1997). Adrenergic excitation of primary afferents can also occur at the dorsal root ganglion and at the axonal site of injury, and this excitation can be blocked by a2 antagonists (Devor, 1994; Michaelis et al., 1996). Furthermore, after peripheral nerve injury there is extensive sprouting of SPGN terminals into the neuroma and dorsal root ganglia, providing a source of adrenergic ligand (Small et al., 1990; McLachlan et al., 1993; Ramer et al., 1997). In addition, both sympathectomy and adrener-

0304-3959/00/$20.00 Published for the International Association for the Study of Pain by Elsevier Science B.V. PII: S 0304-395 9(99)00286-9

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Fig. 1. Three possible sites of sympathetic±sensory coupling in the peripheral nervous system: the L4/5 dorsal root ganglion, the tibial neuroma, and the peroneal cutaneous nociceptor. Chemical sympathectomy with 6OHDA, which blocked the development of heat hyperalgesia in the tibial sectioned wild-type mice, destroys the terminals of the postganglionic sympathetic neurons and prevents coupling at all three peripheral sites. Similarly, D79N mice with mutant nonfunctioning a2A adrenoceptors failed to develop heat hyperalgesia. The loss of a2A adrenoceptor function may also block coupling at any peripheral site. NE, norepinephrine; PG, prostaglandin; Ach, acetylcholine.

gic antagonists have been reported to reduce autotomy behavior, hyperalgesia, and allodynia in various neuropathic pain models (Wall et al., 1979; Coderre et al., 1984; Kim and Chung, 1991; Neil et al., 1991; Kim et al., 1993, 1998; Choi et al., 1994; Colado et al., 1994; Kinnman and Levine, 1995; Tracey et al., 1995; Stone et al., 1997; Malmberg and Basbaum, 1998). Collectively, this clinical and preclinical evidence demonstrates a novel coupling of the sympathetic±sensory nervous systems after peripheral nerve injury. The most parsimonious explanation for nerve injury-evoked sympathetic±sensory coupling is the direct coupling hypothesis, which proposes that norepinephrine released from SPGNs acts on the a adrenoceptors on the membrane of the sensory afferent, resulting in impulse activity and nociceptor sensitization which causes neuropathic pain and hyperalgesia (Devor, 1983). The indirect coupling hypothesis invokes a more convoluted mechanism of sympathetic±sensory interaction, proposing that after injury SPGNs release norepinephrine, which binds to a2 adrenoceptors on the SPGNs, triggering the release of prostaglandins from the SPGN terminals, which then act on the primary afferents, evoking impulse activity, nociceptor sensitization, pain and hyperalgesia (Levine et al., 1986). The effects of localized subcutaneous injections of a2 agonists, a2 antagonists, and cyclo-oxygenase inhibitors on hyperalgesia in neuropathic rats support this hypothesis (Tracey et al., 1995). It is clear that sympathetic±sensory coupling can occur after nerve injury, but further studies are needed to implicate, unequivocally, the a2 adrenoceptor and the SPGN in the development and maintenance of neuropathic hyperalgesia and pain. In the current study we used mutant mice (D79N) with nonfunctioning a2A adrenoceptors (MacMillan et al., 1996), chemical sympathectomy with 6-hydroxydopamine (6-OHDA) (Thoenen, 1972), and systemic and

intradermal adrenergic pharmacologic interventions. The speci®c aims of this investigation were to test for (1) possible effects of sympathetic±sensory coupling on neuropathic hyperalgesia, (2) possible sites of coupling, and (3) possible mechanisms of coupling. In addition, we recently observed a three- to six-fold leftward shift in the analgesic ef®cacy of systemic dexmedetomidine for mechanical and heat nociception in neuropathic rats (Poree et al., 1998). Since this was the ®rst report of enhanced analgesic potency for systemic a2 agonists in a neuropathic model, further investigation was required. In the current study we evaluated the effect of nerve injury on the analgesic ef®cacy of systemic a2 agonists and antagonists and identi®ed the a2 adrenoceptor subtype mediating these effects. 2. Methods 2.1. Animals These experiments were reviewed and approved by our institute's Animal Care and Use Committee and followed the guidelines of the IASP (Zimmermann, 1983). Adult (20± 30 g) male D79N mice, which have a point mutation in the a2A adrenoceptor (MacMillan et al., 1996), and their wildtype (WT) controls, all on a hybrid C57BL/6 and 129Sv background, were used for these experiments. To determine if other strains of tibial nerve sectioned mice would develop hyperalgesia we used ICR CD1 and 129svJ male mice (20± 30 g) in one experiment. Mice were housed in a temperature and humidity controlled environment and were maintained on a 12 h light/dark cycle. Food and water were available ad libitum. 2.2. Surgery The mice were deeply anesthetized with halothane and underwent a right limb tibial nerve transection just distal to the sciatic trifurcation, with a tight 3-0 silk ligation of the proximal nerve stump. Control mice underwent sham surgery, with the right tibial nerve exposed, but not ligated or sectioned. 2.3. Test procedures Mechanical nociceptive withdrawal responses were measured with calibrated von Frey ®bers (North Coast Medical, San Jose, CA), applied over the dorsum of the right hindpaw, between the second and third metatarsal. Each ®ber was applied three consecutive times, pushing down on the hindpaw until the mouse withdrew its paw or the ®ber bowed. Four graduated ®bers were used sequentially (10, 23, 57, and 85 g), for a total of 12 consecutive ®ber applications. The withdrawal threshold was the smallest ®ber size which evoked at least two withdrawal responses during three consecutive applications with the

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same ®ber. Each ®ber was applied for approximately 1 s and the interstimulus interval was approximately 5 s. Heat nociceptive thresholds were determined from the mean of three consecutive withdrawal thresholds to a Peltier device (4 £ 4 cm surface, CP1: 4-127-06L, Melcor, Trenton, NJ) applied to the hindpaw. A linear ramped temperature (18C/s, starting at 408C and with a cut-off of 528C) was used as previously described (Kingery et al., 1994, 1998). The interstimulus interval was approximately 20 s. The examiner controlled the Peltier using a foot pedal switch. Heat thresholds were tested over the medial dorsum surface of the hindpaw by holding the mouse above the Peltier with the hindpaw extended behind the mouse, so that the dorsum of the hindpaw just rested on the Peltier surface. To ensure there was contact between the hindpaw skin and the Peltier, the investigator lightly pressed on the lateral edge of the paw with the tip of his ®nger. The rota-rod treadmill (IITC Life Science Instruments, Woodland Hills, CA) had a constant speed (10 rev./min) rotating drum, and the mice were timed on the treadmill. If the mouse fell off an automatic timer was triggered by a plate beneath the drum, which gave a fall latency. The cutoff latency for this test was 60 s. The rota-rod latency was the mean of three consecutive fall latencies. Animals were tested weekly or biweekly, and before baseline measurements were taken the mice were trained with two sessions of Peltier, von Frey, and rota-rod testing. The testing procedure always followed the same sequence, ®rst measuring the von Frey thresholds, then the Peltier thresholds, then the rota-rod latencies. The testing room was dimly lit and the room temperature was maintained between 24 and 268C. The mice were placed in a ¯exible clear vinyl cone with openings for the nose and limbs. They were then gently held during the nociceptive testing and the testing was performed only when the mice were quietly resting in the investigator's hand. The investigator performing the measurements was blinded to treatment. 2.4. Neurochemical analysis The mice were killed by cervical dislocation, the entire hindpaw skin rapidly removed, then the tissue was frozen in liquid nitrogen and ground with mortar and pestle. The skin was then homogenized with a polytron in 2 ml of 2% perchloric acid, centrifuged at 2000 rev./min for 5 min at 48C, then 400 ml of supernatant was twice ®ltered with a Millipore Ultrafree-MC ®lter while centrifuged at 14 000 rev./min for 30 min at 48C. Norepinephrine was then separated by reverse-phase chromatography on an ESA column (catecholamine R-80) maintained at 248C with a column heater. The mobile phase (Cat-A-Phase, ESA, Medford, MA) was delivered at a ¯ow rate of 1.0 ml/min using a Beckman 118 Solvent Module. Samples were injected with a Bio-Rad model AS-100 HPLC Automatic Sampling System and norepinephrine was detected coulometrically (ESA, Model 5011). Retention time, peak area, and concen-

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tration of norepinephrine were measured by comparison with known standards and were determined with the Dynamax MacIntegrator software system (Rainin Instrument Co.). The detection limit on our assay was 1 pg/sample. 2.5. Drugs All testing was performed between 20 and 30 min after drug administration. When drugs were co-administered, the antagonist was given 5 min before the agonist. Drugs were usually administered by the intraperitoneal route (volume of 10 ml/kg), except for several experiments using subcutaneous injections into the medial dorsum of the hindpaw (volume of 3 ml). Dexmedetomidine, a selective a2 adrenoceptor agonist, and atipamezole, a selective a2 adrenoceptor antagonist, were supplied by Orion Corp. (Turku, Finland). L-659,066, a peripherally restricted a2 adrenoceptor antagonist (Clineschmidt et al., 1988), was supplied by Merck Sharp and Dohme Labs (Rahway, NJ). Yohimbine, prazosin, indomethacin, and 6-OHDA were obtained from Sigma (St. Louis, MO). All drugs were dissolved in 0.9% saline, except for prazosin (sterile water) and indomethacin (40% hydroxypropyl-b-cyclodextrin). Control animals received the appropriate vehicle (saline, sterile water, or 40% hydroxypropyl-b-cyclodextrin). 2.6. Statistical analysis All data are presented as the mean ^ SD (standard error of the mean), and differences are considered signi®cant at a P-value less than 0.05. A two-way repeated measures analysis of variance (ANOVA) was performed on the threshold values over time when comparing groups of mice. Heat thresholds and rota-rod latencies were analyzed with a one-way repeated measures ANOVA and a Fisher PLSD was used to test for contrasts. Mechanical thresholds, which are nonparametric data, were analyzed using a Friedman ANOVA by ranks and a Wilcoxon signed-rank test for contrasts. The 50% effective doses (ED50s) and 95% con®dence intervals (CI) were analyzed from a log-linear regression of the means on the slope of the dose±response curve (Tallarida and Murray, 1987). All ED50s are presented with 95% CIs. Tables comparing analgesic effects of various adrenergic agonists and antagonists present the mean threshold changes as the percentage of the maximum possible effect (%MPE); %MPE ˆ ([drug threshold 2 saline threshold]/[cut-off threshold 2 saline threshold] £ 100). The cut-off thresholds were 528C for heat and 85 g for mechanical nociception. 2.7. Study design 2.7.1. Nociceptive threshold testing after tibial nerve section or sham surgery in WT and D79N mice Three groups of mice were measured for baseline nociceptive thresholds. The right tibial nerve was then ligated and transected in one group of WT mice and in one group of

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D79N mice. Sham surgery was performed in another group of WT mice. Postoperatively, the mice were repeatedly tested for nociceptive thresholds over the next 70 days to investigate the temporal development of neuropathic heat and mechanical hyperalgesia in the WT and D79N mice. An additional group of WT mice had simultaneous ligation and transection of both the tibial and peroneal nerves in the right limb. These mice also had repeated nociceptive threshold testing to determine if the peroneal nerve mediated the neuropathic hyperalgesia observed after tibial transection. To determine if other strains of mice would develop hyperalgesia after tibial transection we performed tibial transections as described above in ICR CD1 and 129svJ male mice (20±30 g) and performed nociceptive threshold testing every week for 7 weeks. 2.7.2. Nociceptive threshold testing after tibial section in sympathectomized or control WT mice Initially, we needed to de®ne the time course for norepinephrine depletion in the hindpaw skin after systemic 6OHDA, since this treatment spares the PGSN ganglia, but selectively destroys their terminals, which then slowly regenerate (Thoenen, 1972). Wild-type mice underwent a series of intraperitoneal injections with either 6-OHDA (50 mg/kg per day for 2 days, then 100 mg/kg per day for the next 3 days) or saline. Hindpaw skin was collected the day after the saline injection series, and at days 8, 30 and 49 after the 6-OHDA treatments. Hindpaw cutaneous norepinephrine content was measured as described in Section 2.4. After the time course of norepinephrine depletion in the hindpaw skin was established, a behavioral study was performed. After baseline nociceptive threshold measurements, one group of mice was treated with a 5 day series of intraperitoneal 6-OHDA injections, while a second group received saline injections. Nociceptive thresholds were retested after norepinephrine depletion, and then the right tibial nerve was ligated and transected in both groups. Nociceptive thresholds were tested in both groups on a weekly basis over the next 35 days. 2.7.3. Analgesic effect of dexmedetomidine after tibial nerve section or sham surgery in WT mice Two groups of WT mice were tested for baseline nociceptive thresholds. The right tibial nerve was ligated and transected in one group and sham surgery was performed in the other. At 29±36 days postoperatively the dose response to systemic dexmedetomidine was measured for right hindpaw nociceptive thresholds and for rota-rod latencies. The left hindpaw and the left forepaw nociceptive thresholds were also tested in the neuropathic mice, thereby examining dexmedetomidine's analgesic effects in limbs unaffected by the right tibial lesion. 2.7.4. Analgesic effect of dexmedetomidine after tibial nerve section or sham surgery in D79N mice Two groups of D79N mice were tested for baseline noci-

ceptive thresholds. The right tibial nerve was ligated and transected in one group and sham surgery was performed in the other. At 29±36 days postoperatively the dose response to systemic dexmedetomidine was measured for right hindpaw nociceptive thresholds and for rota-rod latencies. 2.7.5. Analgesic effect of a 2 antagonists, alone, and in combination with dexmedetomidine, after tibial nerve section or sham surgery in WT and D79N mice Four groups of mice were measured for baseline nociceptive thresholds. The right tibial nerve was then ligated and transected in one group of WT mice and in one group of D79N mice. Sham surgery was performed in another group of WT mice and another group of D79N mice. Postoperatively, the mice were tested weekly over the next 28 days to con®rm the development of hyperalgesia. Between 29±42 days postoperatively, the analgesic effects of dexmedetomidine and various doses of adrenergic antagonists, administered alone and in combination with dexmedetomidine, were compared to saline injections. These agents were usually administered by intraperitoneal injection on a biweekly schedule, but in some studies the drugs were injected intradermally into the right hindpaw. 2.7.6. Iso¯urane sedation effect on nociceptive thresholds in WT neuropathic mice Iso¯urane anesthesia was used to test the hypothesis that dexmedetomidine's sedative effect may actually be raising nociceptive thresholds. Baseline Peltier and von Frey withdrawal thresholds were performed in WT tibial sectioned mice (n ˆ 10, 24 days after surgery), then iso¯urane (1.1% concentration) and oxygen (30% concentration) were administered using a small mask over the nose. After 5 min of iso¯urane inhalation, each animal demonstrated a loss of righting re¯ex, indicating deep sedation. While under continuous iso¯urane anesthesia, each animal was then immediately retested for Peltier and von Frey withdrawal thresholds. 3. Results 3.1. After tibial section the WT mice developed heat and mechanical hyperalgesia Within 14 days after surgery the heat thresholds were lower in the WT tibial sectioned than the WT sham-operated mice (49.7 ^ 0.4 versus 51.5 ^ 0.38C, P , 0:05), and this heat hyperalgesia in the WT sectioned mice persisted for at least 70 days (Fig. 2A). Heat hyperalgesia failed to develop in the sham-operated WT mice. Mechanical hyperalgesia developed within 21 days of tibial section in WT (55 ^ 6 versus 76 ^ 5 g in WT sham-operated mice, P , 0:05), and this hyperalgesia persisted for at least 70 days postoperatively (Fig. 2B). Tibial nerve transection was also performed in ICR CD1 (n ˆ 10) and 129SvJ (n ˆ 12) male mice with

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identical results (data not shown). There was no autotomy behavior in the tibial transection model. Repeated testing with large von Frey ®bers did not result in any obvious trauma to the dorsum of the hindpaw, and the mechanical thresholds did not signi®cantly change with repeated testing in the sham-operated mice (Fig. 2B). The baseline withdrawal thresholds in the WT mice (77 ^ 3 g) were similar to von Frey thresholds reported using these methods in normal rats (Kingery et al., 1993; Ma and Woolf, 1996; Rueff et al., 1996; Walker et al., 1996; Kolhekar et al., 1997). After tibial transection all mice demonstrated intact ¯exor withdrawal responses to nociceptive stimuli over the dorsum of the hindpaw. WT mice who had their tibial and

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peroneal nerves transected (n ˆ 6) did not withdraw to Peltier and von Frey nociceptive stimuli applied over the medial dorsum site. This indicates that after tibial section alone, it is the peroneal nerve mediating the nociceptive thresholds over the medial dorsum of the hindpaw. 3.2. After tibial section the D79N mice developed mechanical, but not heat hyperalgesia Fig. 2A,B illustrates that there were no differences in the baseline heat (51.1 ^ 0.2 versus 51.0 ^ 0.18C) and mechanical (77 ^ 3 versus 80 ^ 3 g) nociceptive thresholds of the WT and D79N mice. The D79N mice failed to develop heat hyperalgesia after tibial section, but mechanical hyperalgesia developed within 21 days after section (56 ^ 5 versus 76 ^ 5 g for sham-operated mice, P , 0:05). The mechanical hyperalgesia in the D79N mice persisted for at least 70 days and was the same as the hyperalgesia seen in the WT neuropathic mice. 3.3. After tibial section chemically sympathectomized WT mice developed mechanical, but not heat hyperalgesia Chemical sympathectomy with 6-OHDA depleted norepinephrine in the hindpaw skin of the treated mouse (Fig. 3A). Control mice had 63 ^ 10 pg of norepinephrine per mg wet weight of hindpaw skin. Eight days after 6-OHDA treatment there was no detectable norepinephrine (assay sensitivity was 5 pg/mg wet weight of skin). At 30 days after 6OHDA the skin norepinephrine content was increasing (22 ^ 5 pg/mg) and after 49 days the norepinephrine content had returned to control levels (67 ^ 10 pg/mg). Fig. 3B,C illustrates that 6-OHDA sympathetomized WT mice failed to develop heat hyperalgesia after tibial section, but mechanical hyperalgesia developed within 7 days after tibial section in the 6-OHDA mice (55 ^ 6 versus 77 ^ 3 g before section, P , 0:05). The mechanical hyperalgesia in the 6-OHDA mice persisted for at least 35 days and developed earlier than the hyperalgesia seen in the saline mice (21 days after tibial section, 57 ^ 6 versus 77 ^ 3 g before section, P , 0:05). The 6-OHDA sympathectomy had no signi®cant effect on baseline heat and mechanical thresholds. 3.4. a 2 agonist-evoked analgesia was enhanced in the neuropathic WT mice, and is absent in the D79N mice

Fig. 2. The temporal development of heat (A) and mechanical (B) hyperalgesia after tibial nerve ligation and transection. Three groups of mice (n ˆ 15 in each group) were used in this experiment: sham-operated wild-type mice (WT/Sham), tibial transected mutant mice (D79N/Section), and tibial transected wild-type mice (WT/Section). Relative to the thresholds of sham-operated WT mice, heat hyperalgesia gradually developed over 14 days after tibial section in the WT, but not the D79N mice. Mechanical hyperalgesia gradually developed in over 21 days after tibial section in both the WT and D79N mice. Relative to baseline thresholds, sham-operated WT mice failed to develop heat or mechanical hyperalgesia.

Systemic dexmedetomidine (0.1±200 mg/kg i.p.) had no dose±response analgesic effect on hindpaw heat and mechanical nociceptive thresholds in sham-operated WT mice (Fig. 4A,B, Table 1). Dexmedetomidine (200 mg/kg i.p.) had no effect on thresholds or latencies in sham-operated WT mice when compared to saline injection. After the development of heat and mechanical hyperalgesia (29±36 days after tibial section) dexmedetomidine had a dose-dependent anti-hyperalgesic effect, returning nociceptive thresholds to normal baselines (Fig. 4A,B). Dexme-

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Neither analgesic nor sedative effects were observed with systemic dexmedetomidine (0.1±200 mg/kg i.p.) in shamoperated or neuropathic D79N mice (Fig. 5, Table 1). Dexmedetomidine (200 mg/kg i.p.) had no effect on thresholds or latencies in neuropathic D79N mice when compared

Fig. 3. (A) Eight days after chemical sympathectomy with 6-OHDA the norepinephrine content of the hindpaw skin was below detection thresholds, after 30 days the norepinephrine content had started to increase, and by 49 days the content had returned to control levels (n ˆ 6±10 per group). (B) Heat hyperalgesia failed to develop after tibial transection in the WT mice who had undergone chemical sympathectomy with 6-OHDA immediately before nerve injury. (C) Mechanical hyperalgesia developed earlier after tibial section in the 6-OHDA sympathectomized WT mice (day 7) than in the saline treated WT mice (day 21), and lasted for at least 35 days.

detomidine's analgesic effect was limited to the neuropathic hindpaw, with no analgesia observed in the contralateral hindpaw or contralateral forepaw with dexmedetomidine doses up to 100 mg/kg i.p. Dexmedetomidine (0.1±200 mg/kg i.p.) had marked sedative effects on the rota-rod latency test in the sham-operated and tibial sectioned WT mice (Fig. 4C). Both groups of mice fell off the rotating cylinder within seconds at 100 mg/kg. There were no signi®cant differences between the sham-operated and sectioned WT mice for the dexmedetomidine dose responses.

Fig. 4. (A) The a2 agonist dexmedetomidine (0.1±200 mg/kg, i.p.) had no effect on heat nociceptive thresholds in sham-operated wild-type mice (WT/Sham, n ˆ 14), but in wild-type mice made hyperalgesic after tibial section (WT/Section, n ˆ 14) there was a dose-dependent analgesic effect. The ED50 for the heat analgesia in the neuropathic paw was 28 mg/kg (8± 100). (B) Dexmedetomidine had no effect on mechanical nociceptive thresholds in the WT/Sham mice, but in the hyperalgesic WT/Section mice (29±36 days after surgery) there was a dose-dependent analgesic effect. The ED50 and 95% CI for mechanical analgesia was 30 mg/kg (12±71). (C) The sedative effects (reduction of rota-rod latencies) with dexmedetomidine were the same in WT/Sham and in hyperalgesic WT/ Section mice (repeated-measures ANOVA comparing the latencies between groups at the same doses). The ED50s and 95% CIs for WT/ Sham and hyperalgesic WT/Section mice were 21 mg/kg (11±38) and 30 mg/kg (12±98), respectively.

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Table 1 Analgesic response to a2 adrenoceptor agonists and antagonists a Drugs

DEX ATI YOH L659 DEX 1 ATI DEX 1 YOH DEX 1 L659 INDO SAL DEX ATI

Dose (i.p.)

80 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 80 mg 1 2 mg/kg 80 mg 1 2 mg/kg 80 mg 1 2 mg/kg 4 mg/kg Dose (i.d.) 0.9%/3 ml vol 10 mg/kg 100 mg/kg

WT/Section

WT/Sham

Heat

Mech

11 11 11 1 11 11 111 2

11 11 11 2 11 11 11 2

2 2 2

2 2 2

Heat

D79N/Section Mech

Heat

Mech

2 2 2 2

2 2 2 2

2 2 2

2 2 2

2

2

2

2

a

WT/Section, tibial sectioned wild-type mice; WT/Sham, sham-operated wild-type mice; D79N/Section, tibial sectioned mutant mice; Heat, heat nociception; Mech, mechanical nociception; DEX, dexmedetomidine; ATI, atipamezole; YOH, yohimbine; L659, L659,066; INDO, indomethacin; SAL, saline; i.p., intraperitoneal; i.d., intradermal; vol, volume.

to saline injection. These results indicate that a2 agonistinduced analgesia to mechanical stimuli is mediated by the a2A adrenoceptor, possibly at the spinal level.

3.6. Combinations of dexmedetomidine with various a 2 antagonists retained analgesic ef®cacy in neuropathic WT mice

3.5. Nerve injury induced an analgesic response to a 2 antagonists in the WT, but not the D79N mice

Dexmedetomidine alone, or in combination with the peripherally restricted antagonist L659,066, caused profound sedation within 20 min. Combinations of dexmedetomidine and atipamezole or yohimbine had no apparent sedative effects, indicating an effective block of the supraspinal a2A adrenoceptor by the antagonist. It was not possible to antagonize the analgesic effects of dexmedetomidine with a2 antagonists in the neuropathic WT mice (Fig. 8, Table 1), presumably due to the analgesic effects of the antagonists themselves. The combination of dexmedetomidine and L659,066 had the greatest analgesic effect for heat nociception and this effect was signi®cantly greater than the analgesia seen with dexmedetomidine alone (91 ^ 3 versus 77 ^ 5% MPE, P , 0:05), evidence of a possible peripheral site of action for the heat analgesia observed with L659,066. The combination of dexmedetomidine and L659,066 also had a greater analgesic effect on heat nociception than a 10 mg/kg dose of L659,066 alone (91 ^ 3 versus 31 ^ 7% MPE, P , 0:001), suggesting a central nervous system site of activity for heat analgesia with dexmedetomidine. The combination of dexmedetomidine and L659,066 had the same analgesic effect as dexmedetomidine alone for mechanical stimuli (61 ^ 28 versus 71 ^ 30% MPE). Since L659,066 had no analgesic effect for mechanical stimuli when administered alone (Fig. 7B), these data indicate that the analgesic effects of dexmedetomidine on mechanical nociception are mediated in the central nervous system of the neuropathic mice.

Fig. 6A and Table 1 illustrate that the systemically administered a2 antagonists atipamezole, yohimbine, and the peripherally restricted L659,066 all had analgesic effects on heat nociception in WT neuropathic mice, but not in WT sham-operated or neuropathic D79N mice. This analgesia was dose-dependent in the WT neuropathic mice (Fig. 6B). The systemically acting antagonists had a greater analgesic effect than the peripherally restricted a2 antagonist at the ceiling dose of 10 mg/kg (58 ^ 11% MPE with atipamezole and 54 ^ 10% MPE with yohimbine versus 31 ^ 7% MPE with L659,066, P , 0:05). These data suggest that blocking peripheral and possibly the central a2 adrenoceptor induced analgesia to noxious heat stimuli applied to the neuropathic hindpaw. Similarly, the systemically acting a2 antagonists atipamezole and yohimbine had analgesic effects on mechanical nociception in WT neuropathic mice, but not in WT sham-operated or neuropathic D79N mice (Fig. 7A, Table 1). However, L659,066 had no effect on mechanical nociception in WT neuropathic mice. Again, there was a dosedependent effect with the systemically acting antagonists (Fig. 7B), with the 10 mg/kg dose evoking an MPE of 70 ^ 24% with atipamezole and 80 ^ 23% with yohimbine. These data indicate that blocking the central a2A adrenoceptor subtype evoked analgesia to noxious mechanical stimuli in the neuropathic WT mouse.

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3.8. Iso¯urane sedation did not affect nociceptive thresholds in neuropathic WT mice Iso¯urane, at a concentration that induced loss of righting re¯ex, failed to alter heat or mechanical nociceptive thresholds (Fig. 10). 4. Discussion Studies with knock-out and transgenic mice offer a promising approach for delineating the neurobiology of chronic pain states (Mogil and Grisel, 1998). To utilize such mice requires the development of neuropathic hyperalgesic models. Tight ligation and transection of the tibial nerve reliably evoked long lasting heat and mechanical hyperalgesia over the medial dorsum of the hindpaw in the WT (B6, 129Sv) hybrid mice and in two other strains. The peroneal nerve mediates this neuropathic hyperalgesia, which may be the result of central sensitization. In support of this argument, it has been reported that tibial section can

Fig. 5. Systemic dexmedetomidine (0.1±200 mg/kg, i.p.) had no effect on heat nociceptive thresholds (A), mechanical nociceptive thresholds (B), or rota-rod latencies (C) in either sham-operated mutant mice (D79N/Sham, n ˆ 15) or hyperalgesic tibial sectioned mutant mice (D79N/Section, n ˆ 15).

3.7. Intradermal injection of an a 2 agonist or antagonist into the hyperalgesic skin did not affect nociceptive thresholds in the neuropathic WT mice Intradermal injection of dexmedetomidine (10 mg/kg) or atipamezole (100 mg/kg) over the medial dorsum of the neuropathic hindpaw had no effect on heat or mechanical nociceptive thresholds in the WT mice (Fig. 9A, Table 1). Systemic indomethacin was also administered as a test for the possible role for prostaglandins in the maintenance of neuropathic hyperalgesia. Indomethacin had no effect on nociceptive thresholds in neuropathic WT or D79N mice, or in sham-operated WT mice (Fig. 9B, Table 1).

Fig. 6. (A) High-dose (10 mg/kg, i.p.) a2 antagonists evoked heat analgesia in hyperalgesic wild-type tibial sectioned mice (WT/Section, n ˆ 13±34), but not in wild-type sham-operated mice (WT/Sham, n ˆ 12±24) or in mutant tibial sectioned mice (D79N, n ˆ 13±16). (B) In the hyperalgesic WT/Section mice all the a2 antagonists, including the peripherally restricted L659,066, evoked a dose-dependent analgesia to noxious heat (n ˆ 12±34). Drug effects were compared to vehicle effects using a oneway repeated measures ANOVA and a Fisher PLSD test for contrasts. *P , 0:05, **P , 0:01, ***P , 0:001.

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Fig. 7. (A) High doses (10 mg/kg, i.p.) of the systemic a2 antagonists atipamezole and yohimbine, but not the peripherally restricted a2 antagonist L659,066, evoked mechanical analgesia in hyperalgesic wild-type tibial sectioned mice (WT/Section, n ˆ 13±34). None of the a2 antagonists evoked mechanical analgesia in the wild-type sham-operated mice (WT/ Sham, n ˆ 12±24) or in mutant tibial sectioned mice (D79N, n ˆ 13±16). (B) In the hyperalgesic WT/Section mice only the systemic a2 antagonists atipamezole and yohimbine evoked a dose-dependent analgesia to noxious mechanical stimuli (n ˆ 12±34). Drug effects were compared to vehicle effects using a one-way repeated measures ANOVA and a Fisher PLSD test for contrasts for heat stimuli. *P , 0:05, **P , 0:01, ***P , 0:001.

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coupling hypothesis would presume that the PGSNs release norepinephrine which binds to the a2A adrenoceptors on the primary afferent, directly sensitizing the afferent. The indirect coupling hypothesis would posit that the PGSNs release norepinephrine which binds to the a2A adrenoceptors on the PGSNs, causing the release of prostaglandins which sensitize the primary afferents (Martin et al., 1987; Gonzales et al., 1989, 1991; Taiwo and Levine, 1989; Pitchford and Levine, 1991; Sherbourne et al., 1992). Immunohistochemical evidence of a2A adrenoceptors expressed by primary afferents, primarily capsaicin sensitive C-®bers, provides a potential anatomic substrate for direct sympathetic±sensory coupling (Stone et al., 1998). In addition, the heat nociceptive thresholds, measured with our Peltier method, are mediated by capsaicin sensitive C-®bers (Kingery et al., 1998), as is the heat hyperalgesia observed in various neuropathic rat models (Shir and Seltzer, 1990; Meller et al., 1992; Kim et al., 1995; Kinnman and Levine, 1995). Furthermore, after peripheral nerve injury in rats, there is a 10- to 20-fold increase in a2A adrenoceptor labeling in the dorsal root ganglia, and it has been proposed that stimulation of these abundant adrenoceptors leads to the activation or sensitization of the primary afferent neuron (Perl, 1994; Birder and Perl, 1996). On the other hand, there is also electrophysiologic evidence from D79N mice indicating that the a2A adrenoceptor is the primary subtype mediating autoinhibition in the SPGNs (Lakhlani et al., 1997), data which could also support the argument for the indirect sympathetic±sensory coupling mechanism. The development of mechanical hyperalgesia did not require the presence of either the a2A adrenoceptor or the SPGN. This ®nding is in agreement with previous reports that sympathectomy prior to nerve injury eliminates or delays the onset of heat hyperalgesia, but not mechanical

enhance trans-synaptic transmission of peroneal nociceptive input, increasing excitation-dependent gene expression in spinal afferents (Sugimoto et al., 1993). Regardless of the mechanism of its generation, our data indicate that this novel neuropathic mouse model could be of value in future transgenic studies. 4.1. Different mechanisms for the development of heat and mechanical hyperalgesia The development of heat hyperalgesia required the presence of both the a2A adrenoceptor and the SPGN. These behavioral data support a role for sympathetic± sensory coupling in the generation of neuropathic heat hyperalgesia. Fig. 1 illustrates possible sites and mechanisms in the peripheral nervous system for sympathetic± sensory coupling in this neuropathic model. The direct

Fig. 8. Dexmedetomidine (Dex, 80 mg/kg, i.p.) alone or in combination with low doses (2 mg/kg, i.p.) of atipamezole (Ati), yohimbine (Yoh), and L659,066 (L659) evoked both heat and mechanical analgesia in hyperalgesic wild-type tibial sectioned mice (WT/Section, n ˆ 12±13). Drug effects were compared to vehicle effects using a one-way repeated measures ANOVA and a Fisher PLSD test for contrasts for mechanical thresholds. Drug effects were compared to vehicle effects using a one-way repeated measures ANOVA and a Fisher PLSD test for contrasts for heat stimuli, and a Friedman ANOVA by ranks and a Wilcoxon signed-rank test for contrasts for mechanical stimuli. *P , 0:05, **P , 0:01, ***P , 0:001.

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responsiveness is rarely observed in intact cutaneous Ad nociceptors after partial nerve injury (Bossut and Perl, 1995). Thus, several lines of evidence suggest that C-®bers mediate neuropathic heat hyperalgesia and frequently develop sympathetic±sensory coupling after nerve injury, while myelinated ®bers mediate neuropathic mechanical hyperalgesia but only rarely demonstrate sympathetic± sensory interactions in the intact axons after partial nerve injury. 4.2. Nerve injury enhanced a 2 agonist-evoked analgesia, which is mediated by the a 2A adrenoceptor subtype for mechanical nociceptive stimuli

Fig. 9. (A) Intradermal injection of saline (n ˆ 46), dexmedetomidine (Dex, n ˆ 13), or atipamezole (Ati, n ˆ 11) into the hyperalgesic skin of wild-type tibial sectioned mice had no effect on heat and mechanical hyperalgesia. (B) The cyclo-oxygenase inhibitor indomethacin (4 mg/kg, i.p.) also had no effect on heat or mechanical nociceptive thresholds in wildtype tibial sectioned mice (WT/Section, n ˆ 14), wild-type sham-operated mice (WT/Sham, n ˆ 15) or in mutant tibial sectioned mice (D79N, n ˆ 16). Drug effects were compared to vehicle effects using a one-way repeated measures ANOVA and a Fisher PLSD test for contrasts for heat stimuli, and a Friedman ANOVA by ranks and a Wilcoxon signed-rank test for contrasts for mechanical stimuli. Saline effects were compared to preinjection baselines. *P , 0:05, **P , 0:01, ***P , 0:001.

hyperalgesia or allodynia (Neil et al., 1991; Shir and Seltzer, 1991; Kinnman and Levine, 1995). Collectively, these studies suggest that different mechanisms could be responsible for the development of heat hyperalgesia versus mechanical hyperalgesia in neuropathic pain states. Different types of primary afferent ®bers may mediate heat and mechanical hyperalgesia, and these afferents may also differ in their ability to develop adrenergic responsiveness after nerve injury. Neonatal capsaicin treatment can semi-selectively destroy C-®bers and spare small myelinated Ad afferents (Buck and Burks, 1986). Capsaicin treated rats fail to develop neuropathic heat hyperalgesia but can develop mechanical hyperalgesia after nerve injuries, indicating that C-®bers mediate heat, but perhaps not mechanical hyperalgesia (Shir and Seltzer, 1990; Kinnman and Levine, 1995). Furthermore, after partial nerve injury the intact cutaneous C-®ber polymodal nociceptors frequently develop a novel responsiveness to sympathetic stimulation which is mediated by the a2 adrenoceptor (Sato and Perl, 1991; O'Halloran and Perl, 1997). Conversely, adrenergic

There was an enhancement of dexmedetomidine analgesic potency after tibial section in WT mice, but no change in sedative ef®cacy (Fig. 4, Table 1). We have previously reported a similar enhancement of dexmedetomidine analgesic potency in spinal nerve ligated rats (Poree et al., 1998). These data from different species and neuropathic models demonstrate that nerve injuries can enhance a2 adrenoceptor mediated analgesia. Several lines of evidence indicate that the ability of systemic dexmedetomidine to increase the nociceptive thresholds in the neuropathic mice is due to the analgesic

Fig. 10. Iso¯urane anesthesia (1.1% concentration for 5 min) caused profound sedation but had no effect on heat (A) or mechanical (B) nociceptive thresholds in neuropathic wild-type mice (n ˆ 10, day 24 after tibial ligation and section). *P , 0:05, **P , 0:01, ***P , 0:001.

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rather than the sedative effects. Unlike supraspinally mediated nociceptive assays such as the hot plate or formalin tests, which require complex behavioral responses such as licking or jumping, the withdrawal re¯exes used in this study are spinally mediated and are observed after spinal cord transection and in decerebrated animals (Pertovaara et al., 1991; Xu et al., 1992a,b). Furthermore, nociceptive thresholds in sham-operated WT mice were unchanged by doses of systemic dexmedetomidine that were severely sedating on the rota-rod assay (Fig. 4). In addition, highdose (100 mg/kg) dexmedetomidine completely reversed hyperalgesia in the neuropathic paw of the WT tibial sectioned mice (Fig. 4), but had no effect on nociceptive thresholds in the forepaw of the same mice. The effect of sedation on nociceptive thresholds would not be restricted to the neuropathic paw. Finally, we have demonstrated that iso¯urane anesthesia, at a concentration and exposure time that induces profound sedation as demonstrated by a loss of the righting re¯ex, has no effect on nociceptive thresholds in neuropathic WT mice (Fig. 10), con®rming that nociceptive thresholds mediated by ¯exor re¯exes are not in¯uenced by sedation. The mechanism of this enhanced analgesic ef®cacy is unknown. Normally, the primary site of a2 agonist analgesic action is at the spinal cord (Fig. 1), where a2 agonists exert both pre- and postsynaptic inhibitory actions (Kingery et al., 1997). In the neuropathic WT mice, dexmedetomidine analgesia was observed only in the neuropathic limb, indicating a segmental restriction on this effect of nerve injury. After sciatic section, the spinal ¯exor re¯exes demonstrate an enhanced inhibitory response to intrathecal clonidine, pointing to a spinal locus for the enhanced a2 analgesic activity seen in neuropathic states (Xu et al., 1992a,b, 1993). Nerve injuries can also cause dramatic changes in the coupling of adrenoceptors and calcium channels in the dorsal root ganglion neurons (Abdulla and Smith, 1997). There is also an upregulation of a2A adrenoceptors on the intact neurons after partial nerve injury (Birder and Perl, 1996). Pre- or postsynaptic upregulation of adrenoceptors or changes in adrenoceptor±ion channel coupling may occur after nerve injury, changes which may account for an enhancement of a2 adrenoceptor mediated spinal analgesia. Transgenic studies have indicated that the a2A adrenoceptor subtype mediates a2 agonist-evoked sedation and analgesia for acute nociception (Hunter et al., 1997; Lakhlani et al., 1997; Stone et al., 1997). No mechanical analgesia or sedative effects were observed with dexmedetomidine in the neuropathic or sham-operated D79N mice in our study, indicating that there is no phenotypic change in the adrenoceptor subtype mediating analgesia or sedation in the neuropathic pain state (Fig. 5, Table 1). 4.3. Nerve injury induced an analgesic response to a 2 antagonists Analgesia to heat stimuli is mediated by blocking periph-

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eral and possibly central a2 adrenoceptors, while analgesia to mechanical stimuli is mediated by blocking central a2A adrenoceptors. Prior electrophysiologic and behavioral investigations have indicated that intrathecally administered a2 adrenoceptor antagonists do not modify acute nociceptive responses in normal rats (Takano and Yaksh, 1992; Diaz et al., 1997). Similarly, we found that the systemic administration of high doses of a2 antagonists had no effect on nociceptive thresholds in sham-operated WT mice. After nerve injury in WT mice, systemically administered a2 antagonists had dose-dependent analgesic effects for both heat and mechanical nociceptive stimuli and these analgesic effects were absent in the neuropathic D79N mice. There is a paucity of published data on the analgesic effects of a2 antagonists on neuropathic hyperalgesia and pain. In neuropathic rats systemically administered phentolamine and idazoxan evoked analgesia for mechanical allodynia and hyperalgesia (Kim et al., 1993; Kayser et al., 1995), but intrathecal yohimbine was ineffective for neuropathic mechanical allodynia (Yaksh et al., 1995). Clinically, the combined a1/a2 antagonist phentolamine has been used primarily in CRPS patients with mixed analgesic results (Kingery, 1997). The peripherally restricted a2 antagonist L659,066 had an analgesic effect for heat but not mechanical nociception (Figs. 6 and 7, Table 1). These results support the hypothesis that sympathetic±sensory coupling in the peripheral nervous system not only contributes to the development but also to the maintenance of neuropathic heat hyperalgesia, and this hyperalgesia can be partially reversed by blocking the peripheral a2 adrenoceptor (Fig. 1). The difference in the analgesic effects of the peripherally restricted antagonist compared to the systemically acting antagonists indicate that for heat nociception blocking the peripheral, and possibly the central a2 adrenoceptors can induce analgesia, while for mechanical nociception only blocking the central nervous system a2 adrenoceptor can generate analgesia (Table 1). Furthermore, the mechanical analgesic effects of the a2 antagonists are absent in the D79N neuropathic mice, indicating that the central analgesic effect is mediated via the a2A adrenoceptor. 4.4. Combinations of dexmedetomidine with various a 2 antagonists retained analgesic ef®cacy in neuropathic WT mice, and indicate a central site of a 2 agonist analgesia for mechanical, and probably heat stimuli Dexmedetomidine analgesia was not antagonized by the systemic administration of a2 antagonists, presumably due to the intrinsic analgesic activity of the antagonists. Intriguingly, dexmedetomidine and the peripherally restricted antagonist L659,066 had the largest analgesic effect for heat of any drug combination, greater than the analgesia seen with either drug alone (Figs. 6 and 8, Table 1). This synergistic analgesia may re¯ect the central effects of a2 adrenoceptor activation with dexmedetomidine and the

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peripheral analgesic effects of a2 adrenoceptor blockade with L659,066. The combination of dexmedetomidine and L659,066 evoked analgesia for mechanical nociception that did not differ from the analgesia seen with dexmedetomidine alone (Figs. 7 and 8, Table 1). Since L659,066 had no effect on mechanical hyperalgesia, dexmedetomidine analgesia for mechanical stimuli was centrally mediated in these neuropathic mice. 4.5. There was no evidence of cutaneous or prostanoid mediated sympathetic±sensory coupling in neuropathic WT mice Intradermal injection with either dexmedetomidine or atipamezole had no effect on neuropathic hyperalgesic thresholds, nor did the systemic administration of the cyclo-oxygenase inhibitor indomethacin (Fig. 9, Table 1). These data are in contrast to studies in neuropathic rats showing analgesic effects after subcutaneous injection of a2 adrenoceptor antagonists and algogesic effects with subcutaneous injection of norepinephrine (Tracey et al., 1995), but in agreement with other reports that intradermal a2 adrenoceptor antagonists and norepinephrine have no effect on nociceptive thresholds in neuropathic rats (Vallin and Kingery, 1991; Moon et al., 1999; Ringkamp et al., 1999). Our results do not support the hypothesis that nerve injury can evoke indirect sympathetic±sensory coupling in the cutaneous tissues, mediated by the release of prostaglandins from the SPGN terminals. In conclusion, we have introduced a new model of neuropathic hyperalgesia in the mouse and used it to show that the a2A adrenoceptor and the SPGN play a critical role in the development of heat, but not mechanical hyperalgesia. Furthermore, these neuropathic mice developed enhanced analgesic responses to a2 agonists and antagonists, mediated by the a2A adrenoceptor subtype. The site of analgesic activity for the a2 agonist was within the central nervous system. The loci of analgesic action for the a2 antagonists were in peripheral and probably central nervous systems for heat stimuli, but for mechanical stimuli there was only central analgesic activity. Overall, these data support a role for sympathetic±sensory coupling in the peripheral nervous system in the development and maintenance of heat hyperalgesia in this neuropathic model. No cutaneous site of adrenergic analgesic activity or prostanoid mediation of neuropathic hyperalgesia was observed, suggesting that neither direct nor indirect sympathetic±sensory coupling at the level of the cutaneous nociceptor contributed to the maintenance of hyperalgesia. Acknowledgements This research was supported by NIH grant GM-30232 and a Department of Veterans Affairs Merit Review grant.

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