Allosteric Adenosine Modulation to Reduce Allodynia

Background: Adenosine and adenosine agonists reduce hypersensitivity following inflammation and peripheral nerve injury models of chronic pain. Because inhibitors of adenosine reuptake or metabolism are also effective at reducing hypersensitivity, it is likely that there is a tonic release of spinal adenosine in these models. One approach to avoid adverse effects from direct agonists is to enhance the effect of the endogenous ligand by administering a positive allosteric modulator of its receptor.

Methods: Rats with mechanical hypersensitivity after spinal nerve ligation received intrathecal injections of adenosine, the allosteric adenosine receptor modulator T62, or their combination, or received systemic T62 alone or with intrathecal injection of a specific A1 adenosine antagonist.

Results: Both adenosine and T62 reduced hypersensitivity alone, with 50% maximal doses (ED50) of 14 +/- 5.9 and 3.7 +/- 0.8 [mu]g, respectively. They interacted in an additive manner as determined by isobolography. T62 also reduced mechanical hypersensitivity after systemic administration (15 mg/kg), and this effect was blocked by intrathecal injection of 9 [mu]g of the A1-specific adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine.     

Spinal adenosine receptor activation reduces hypersensitivity after surgery by a different mechanism than after nerve injury

BACKGROUND: Intrathecal adenosine has antinociceptive effects under conditions of hypersensitivity. T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophen) is an allosteric adenosine receptor modulator that enhances adenosine binding to the A1 receptor. Intrathecal T62 reduces hypersensitivity to mechanical stimuli in a rat model of neuropathic pain by a circuit that totally relies on activation of alpha2 adrenoceptors. Here, the authors tested whether this same dependence was present in the acute setting of hypersensitivity after surgery. METHODS: Intrathecal catheters were inserted in male Sprague-Dawley rats. An incision of the plantar aspect of the hind paw resulted 24 h later in hypersensitivity, as measured by applying von Frey filaments to the paw. At this time, rats received intrathecal T62, clonidine, or the combination in a blinded, isobolographic design. The effect of the alpha2-adrenoceptor antagonist idazoxan on T62 was also tested. RESULTS: Intrathecal T62 produced a dose-dependent antihypersensitivity effect, with no effect on ambulation or activity level. Clonidine also produced a dose-dependent antihypersensitivity effect. The ED40 (95% confidence interval) for T62 was 0.77 (0.63-0.91) microg, and that for clonidine was 1.23 (0.56-1.9) microg. Isobolographic analysis indicated synergism between T62 and clonidine. Intrathecal pretreatment with idazoxan only partially inhibited the antihypersensitivity effect of T62. CONCLUSIONS: Intrathecal T62 is effective for postoperative hypersensitivity. The synergy of T62 with clonidine and its only partial antagonism by idazoxan suggest that T62 does not rely entirely on activation of alpha2 adrenoceptors. These results indicate that, after surgery, T62 acts via a mechanism different from that of spinal nerve ligation, a model of chronic neuropathic pain.     

Intrathecal morphine reduces allodynia after peripheral nerve injury in rats via activation of a spinal A1 adenosine receptor

BACKGROUND: The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine. METHODS: Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined. RESULTS: Intrathecal morphine (0.25-30 microg) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 microg. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 microg. CONCLUSIONS: This study is in accord with two recent reports that support short-term efficacy of intrathecal morphine to reverse hypersensitivity to mechanical stimuli in animal models of neuropathic pain. Despite previous studies demonstrating that morphine releases less adenosine after peripheral nerve injury, the current study suggests that the antihypersensitivity effect of morphine in these conditions is totally reliant on A1 adenosine receptor activation.     

Intrathecal adenosine interacts with a spinal noradrenergic system to produce antinociception in nerve-injured rats.

BACKGROUND: Adenosine analogs produce antinociception in animal models of acute pain, reduce hypersensitivity in models of inflammatory and nerve-injury pain, and stimulate neurotransmitter release in the brain. Adenosine itself is entering clinical trials for analgesia, and the current study examined the effect, mechanisms of action, and interaction with noradrenergic systems of intrathecal adenosine in a rat model of neuropathic pain. METHODS: The left L5 and L6 spinal nerve roots were ligated and, 1 week later, an intrathecal catheter was inserted in male rats. Withdrawal threshold to mechanical stimulation of the left hind paw was determined before and after surgery, confirming mechanical hypersensitivity. The effects of intrathecal adenosine, clonidine, and their combination on withdrawal threshold were determined, and reversal of the effects of adenosine by adenosine and alpha2-adrenergic antagonists and by destruction of noradrenergic nerve terminals was tested. Finally, spinal cord slices were perfused in vitro with the adenosine agonist 5'-N-ethylcarboxamide adenosine, and norepinephrine release was measured. RESULTS: Intrathecal adenosine and clonidine reduced hypersensitivity and interacted in an additive manner. The effects of adenosine were blocked by intrathecal injection of A1 but not A2 adenosine receptor antagonists, by an alpha2-adrenergic antagonist, and by destruction of spinal noradrenergic nerve terminals. Perfusion of spinal cord slices with 5'-N-ethylcarboxamide adenosine resulted in a concentration-dependent increase in norepinephrine release. CONCLUSION: These data support clinical examination of intrathecal adenosine alone and with clonidine in the treatment of chronic pain states that include a component of mechanical hypersensitivity and suggest that, after nerve injury, adenosine acts to reduce hypersensitivity through spinal norepinephrine release.     

Spinal Adenosine Receptor Activation Reduces Hypersensitivity after Surgery by a Different Mechanism Than after Nerve Injury

Background: Intrathecal adenosine has antinociceptive effects under conditions of hypersensitivity. T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophen) is an allosteric adenosine receptor modulator that enhances adenosine binding to the A1 receptor. Intrathecal T62 reduces hypersensitivity to mechanical stimuli in a rat model of neuropathic pain by a circuit that totally relies on activation of [alpha]2 adrenoceptors. Here, the authors tested whether this same dependence was present in the acute setting of hypersensitivity after surgery.

Methods: Intrathecal catheters were inserted in male Sprague-Dawley rats. An incision of the plantar aspect of the hind paw resulted 24 h later in hypersensitivity, as measured by applying von Frey filaments to the paw. At this time, rats received intrathecal T62, clonidine, or the combination in a blinded, isobolographic design. The effect of the [alpha]2-adrenoceptor antagonist idazoxan on T62 was also tested.

Results: Intrathecal T62 produced a dose-dependent antihypersensitivity effect, with no effect on ambulation or activity level. Clonidine also produced a dose-dependent antihypersensitivity effect. The ED40 (95% confidence interval) for T62 was 0.77 (0.63-0.91) [mu]g, and that for clonidine was 1.23 (0.56-1.9) [mu]g. Isobolographic analysis indicated synergism between T62 and clonidine. Intrathecal pretreatment with idazoxan only partially inhibited the antihypersensitivity effect of T62.     

Repeated dosing with oral allosteric modulator of adenosine A1 receptor produces tolerance in rats with neuropathic pain

BACKGROUND: The positive allosteric adenosine receptor modulator, T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophene), has been shown to reduce mechanical allodynia in a rat model of neuropathic pain. However, whether chronic oral T62 retains efficacy in this pain model has not been examined. Therefore, the authors studied antiallodynic effects of chronic oral T62 in spinal nerved-ligated rats, as well as motor and sedative behavioral effects. METHODS: Oral T62, 100 mg/kg, or oral oil was applied daily to spinal nerve-ligated rats for 4 weeks, with rat weights examined daily. Sedation, placing and ambulation scores, and withdrawal threshold were observed for 3 h daily for the first 2 weeks and then once a week. At the end of observation, the animals were killed, and the spinal tissues were collected for radioligand binding. In addition, withdrawal thresholds were also observed in rats with 5 days of treatment with 50 mg/kg oral T62. Furthermore, the effects of intrathecal adenosine on rats with oral T62 or oil treatment were compared. RESULTS: Chronic oral T62, at 100 mg/kg, initially returned the withdrawal threshold to mechanical testing to preinjury levels, with minor or no sedative or motor effects. Tolerance was observed, with a 60% loss of most possible effects in antiallodynia within 5 days of daily administration. Similarly, tolerance also occurred with chronic oral T62 at 50 mg/kg but did not alter the effect of intrathecal adenosine. Furthermore, 4 weeks of exposure to 100 mg/kg T62 resulted in a small reduction in spinal cord A1 receptor number. CONCLUSION: The results imply that chronically administered A1 adenosine modulators lose efficacy over time, partly as a result of receptor down-regulation.     

Intrathecal Morphine Reduces Allodynia after Peripheral Nerve Injury in Rats via Activation of a Spinal A1 Adenosine Receptor

Background: The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine.

Methods: Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined.

Results: Intrathecal morphine (0.25-30 [mu]g) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 [mu]g. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 [mu]g.     

Repeated Dosing with Oral Allosteric Modulator of Adenosine A1 Receptor Produces Tolerance in Rats with Neuropathic Pain

Background: The positive allosteric adenosine receptor modulator, T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophene), has been shown to reduce mechanical allodynia in a rat model of neuropathic pain. However, whether chronic oral T62 retains efficacy in this pain model has not been examined. Therefore, the authors studied antiallodynic effects of chronic oral T62 in spinal nerved-ligated rats, as well as motor and sedative behavioral effects.

Methods: Oral T62, 100 mg/kg, or oral oil was applied daily to spinal nerve-ligated rats for 4 weeks, with rat weights examined daily. Sedation, placing and ambulation scores, and withdrawal threshold were observed for 3 h daily for the first 2 weeks and then once a week. At the end of observation, the animals were killed, and the spinal tissues were collected for radioligand binding. In addition, withdrawal thresholds were also observed in rats with 5 days of treatment with 50 mg/kg oral T62. Furthermore, the effects of intrathecal adenosine on rats with oral T62 or oil treatment were compared.

Results: Chronic oral T62, at 100 mg/kg, initially returned the withdrawal threshold to mechanical testing to preinjury levels, with minor or no sedative or motor effects. Tolerance was observed, with a 60% loss of most possible effects in antiallodynia within 5 days of daily administration. Similarly, tolerance also occurred with chronic oral T62 at 50 mg/kg but did not alter the effect of intrathecal adenosine. Furthermore, 4 weeks of exposure to 100 mg/kg T62 resulted in a small reduction in spinal cord A1 receptor number.     

Intrathecal Adenosine Interacts with a Spinal Noradrenergic System to Produce Antinociception in Nerve-injured Rats

Background: Adenosine analogs produce antinociception in animal models of acute pain, reduce hypersensitivity in models of inflammatory and nerve-injury pain, and stimulate neurotransmitter release in the brain. Adenosine itself is entering clinical trials for analgesia, and the current study examined the effect, mechanisms of action, and interaction with noradrenergic systems of intrathecal adenosine in a rat model of neuropathic pain.

Methods: The left L5 and L6 spinal nerve roots were ligated and, 1 week later, an intrathecal catheter was inserted in male rats. Withdrawal threshold to mechanical stimulation of the left hind paw was determined before and after surgery, confirming mechanical hypersensitivity. The effects of intrathecal adenosine, clonidine, and their combination on withdrawal threshold were determined, and reversal of the effects of adenosine by adenosine and [alpha]2-adrenergic antagonists and by destruction of noradrenergic nerve terminals was tested. Finally, spinal cord slices were perfused in vitro with the adenosine agonist 5'-N-ethylcarboxamide adenosine, and norepinephrine release was measured.

Results: Intrathecal adenosine and clonidine reduced hypersensitivity and interacted in an additive manner. The effects of adenosine were blocked by intrathecal injection of A1 but not A2 adenosine receptor antagonists, by an [alpha]2-adrenergic antagonist, and by destruction of spinal noradrenergic nerve terminals. Perfusion of spinal cord slices with 5'-N-ethylcarboxamide adenosine resulted in a concentration-dependent increase in norepinephrine release.     

Production of paradoxical sensory hypersensitivity by alpha 2-adrenoreceptor agonists

BACKGROUND: Administration of opioid receptor agonists is followed by paradoxical sensory hypersensitivity. This hypersensitivity has been suggested to contribute to the antinociceptive tolerance observed with opioids. The authors hypothesized that alpha 2-adrenoreceptor agonists, which also produce antinociceptive tolerance, would produce sensory hypersensitivity. METHODS: alpha 2-Adrenoreceptor agonists were administered to male Sprague-Dawley rats as a single subcutaneous injection, a continuous subcutaneous infusion, a single intrathecal injection, or a continuous intrathecal infusion. Thermal sensitivity was determined using latency to withdrawal of the hind paw from radiant heat. Tactile sensitivity was determined using withdrawal threshold to von Frey filaments. Spinal dynorphin content was measured by enzyme immunoassay. RESULTS: Single systemic or intrathecal injections of clonidine or dexmedetomidine produced antinociception followed by delayed thermal and tactile hypersensitivity. Six-day systemic or intrathecal infusion of clonidine produced tactile and thermal hypersensitivity observed even during clonidine infusion. Sensory hypersensitivity was prevented by coadministration of the alpha 2-adrenoreceptor-selective antagonist idazoxan or the N-methyl-D-aspartate receptor-selective antagonist MK-801. Six-day infusion of intrathecal clonidine increased dynorphin content in dorsal lumbar spinal cord. MK-801 and dynorphin antiserum reversed clonidine-induced sensory hypersensitivity. CONCLUSIONS: alpha 2-Adrenoreceptor agonists produce sensory hypersensitivity that may be analogous to that produced by opioids. Sensory hypersensitivity was prevented by idazoxan, demonstrating that it is mediated by alpha 2 receptors. Clonidine infusion increased spinal dynorphin content. Sensory hypersensitivity was prevented or reversed by MK-801 and dynorphin antiserum, implicating N-methyl-D-aspartate receptors and spinal dynorphin in its production. Clinicians should be mindful of the possibility of drug-induced hyperalgesia in patients treated with alpha 2-adrenoreceptor agonists.     

Adenosine reduces glutamate release in rat spinal synaptosomes

BACKGROUND: A1 adenosine receptor activation reduces hypersensitivity in animal models of chronic pain, but intrathecal adenosine does not produce analgesia to acute noxious stimuli. Here, the authors test whether increased inhibition by adenosine of glutamate release from afferents after injury accounts for this difference. METHODS: Synaptosomes were prepared from the dorsal half of the lumbar spinal cord of normal rats or those with spinal nerve ligation. Glutamate release evoked by the TRPV-1 receptor agonist, capsaicin, was measured. Adenosine with or without adenosine A1 and A2 receptor antagonists was applied to determine the efficacy and mechanism of adenosine to reduce capsaicin-evoked glutamate release. RESULTS: Capsaicin produced a concentration-dependent glutamate release similarly in normal and nerve-injured rats. Capsaicin-evoked glutamate release was inhibited by adenosine or R-PIA (R-N6-(2- phenylisopropyl)-adenosine) in a concentration-dependent manner, with a threshold of 10 nm in both normal and nerve-ligated synaptosomes. Blockade of capsaicin-evoked glutamate release by adenosine was reversed similarly in synaptosomes from normal and spinal nerve-ligated animals by an A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) but not by an A2 adenosine receptor antagonist DMPX (3'7-dimethyl-1-proparaglyxanthine). Capsaicin-evoked glutamate release, as well as its inhibition by adenosine, did not differ between synaptosomes prepared from tissue ipsilateral and contralateral to spinal nerve ligation. CONCLUSION: These observations confirm previous neurophysiologic studies that presynaptic adenosine A1 receptor activation inhibits glutamate release from primary afferents. This effect is unaltered after peripheral nerve injury and thereby is unlikely to account for the enhanced analgesic efficacy of intrathecal adenosine in this setting.     

Intrathecal adenosine following spinal nerve ligation in rat: short residence time in cerebrospinal fluid and no change in A(1) receptor binding.

BACKGROUND: Intrathecal adenosine produces a remarkably prolonged effect to relieve mechanical hypersensitivity after peripheral nerve injury in animals. The purpose of the current study was to investigate whether this reflected an alteration in kinetics of adenosine in cerebrospinal fluid or in the number of spinal A1 adenosine receptors after nerve injury. METHODS: Male rats were anesthetized, and the left L5 and L6 spinal nerves were ligated. Two weeks later, a lumbar intrathecal catheter and intrathecal space microdialysis catheter were inserted. Adenosine, 20 microg, was injected intrathecally in these and in normal rats, and microdialysates of the intrathecal space were obtained. Radioligand binding studies of adenosine A1 receptors were determined in spinal cord tissue from other normal and spinal nerve-ligated rats. RESULTS: Adenosine disappeared from rat cerebrospinal fluid within 30 min after intrathecal injection, with no difference between normal and spinal nerve-ligated animals. A1 adenosine receptor binding sites in the spinal cord were increased after spinal nerve ligation. This increase disappeared when adenosine deaminase was added to the membrane homogenates, suggestive of decreased endogenous adenosine in the membranes of nerve-ligated animals. CONCLUSION: These data show that prolonged alleviation of hypersensitivity observed with intrathecal adenosine in this animal model of neuropathic pain is not due to prolonged residence in cerebrospinal fluid, although pharmacokinetics in tissues are unknown. Similarly, there is no evidence for up-regulation in spinal A1 adenosine receptors after spinal nerve ligation, and the adenosine deaminase experiment is consistent with a depletion of adenosine in spinal cord tissue after spinal nerve ligation.     

Spinal cannabinoid receptor type 2 activation reduces hypersensitivity and spinal cord glial activation after paw incision

BACKGROUND: Cannabinoids bind to cannabinoid receptors type 1 and 2 and produce analgesia in several pain models, but central side effects from cannabinoid 1 receptors limit their clinical use. Cannabinoid 2 receptors reduce inflammatory responses in the periphery by acting on immune cells, and they are present on glia in the central nervous system. This study tested whether spinal cannabinoid activation would induce analgesia, glial inhibition, and central side effects in a postoperative model or incisional pain. METHODS: Rats underwent paw incision surgery, with intrathecal injections of cannabinoid agonists and antagonists and assessment of withdrawal thresholds and behavioral side effects. Spinal glial activation was determined by immunohistochemistry. RESULTS: Intrathecal administration CP55940 reduced postoperative hypersensitivity (91 +/- 9% maximum possible effect; P < 0.05), and this was prevented by intrathecal administration of both cannabinoid 1 receptor (AM281) and cannabinoid 2 receptor (AM630) antagonists. CP55940 also caused several behavioral side effects, and these were prevented by the cannabinoid 1 receptor but not by the cannabinoid 2 receptor antagonist. Intrathecal injection of the cannabinoid 2 receptor agonist JWH015 reversed postoperative hypersensitivity (89 +/- 5% maximum possible effect; P < 0.05), and this was reversed by the cannabinoid 2 but not by the cannabinoid 1 receptor antagonist. JWH015, which did not induce behavioral side effects, reduced paw incision induced microglial and astrocytic activation in spinal cord (P < 0.05). CONCLUSIONS: These data indicate that intrathecal administration of cannabinoid receptor agonists may provide postoperative analgesia while reducing spinal glial activation, and that selective cannabinoid 2 receptor agonists may do so without central side effects.     

Production of Paradoxical Sensory Hypersensitivity by α2-Adrenoreceptor Agonists

Background: Administration of opioid receptor agonists is followed by paradoxical sensory hypersensitivity. This hypersensitivity has been suggested to contribute to the antinociceptive tolerance observed with opioids. The authors hypothesized that [alpha]2-adrenoreceptor agonists, which also produce antinociceptive tolerance, would produce sensory hypersensitivity.

Methods: [alpha]2-Adrenoreceptor agonists were administered to male Sprague-Dawley rats as a single subcutaneous injection, a continuous subcutaneous infusion, a single intrathecal injection, or a continuous intrathecal infusion. Thermal sensitivity was determined using latency to withdrawal of the hind paw from radiant heat. Tactile sensitivity was determined using withdrawal threshold to von Frey filaments. Spinal dynorphin content was measured by enzyme immunoassay.

Results: Single systemic or intrathecal injections of clonidine or dexmedetomidine produced antinociception followed by delayed thermal and tactile hypersensitivity. Six-day systemic or intrathecal infusion of clonidine produced tactile and thermal hypersensitivity observed even during clonidine infusion. Sensory hypersensitivity was prevented by coadministration of the [alpha]2-adrenoreceptor-selective antagonist idazoxan or the N-methyl-d-aspartate receptor-selective antagonist MK-801. Six-day infusion of intrathecal clonidine increased dynorphin content in dorsal lumbar spinal cord. MK-801 and dynorphin antiserum reversed clonidine-induced sensory hypersensitivity.     

Intrathecal Adenosine: Interactions with Spinal Clonidine and Neostigmine in Rat Models of Acute Nociception and Postoperative Hypersensitivity

Background: Spinal adenosine receptor agonists exert antinociception in animal models of acute and chronic pain, but adenosine itself has not been examined. The authors tested the antinociceptive and antihypersensitivity interactions of intrathecal adenosine and its interactions with intrathecal clonidine and neostigmine in rat models of acute thermal nociception and postoperative hypersensitivity.

Methods: Rats were prepared with lumbar intrathecal catheters. Responses to acute noxious stimulation were evaluated by latency to paw withdrawal from a radiant heat source focused on the hind paw. Postoperative hypersensitivity was measured after an incision in the rat hind paw by application of von Frey filaments to the heel adjacent to the wound. An isobolographic design was used to distinguish between additive and synergistic drug interactions.

Results: Spinal administration of clonidine and neostigmine, but not adenosine, produced dose-dependent antinociception to noxious thermal stimulation. Addition of adenosine enhanced the antinociceptive effect of clonidine but not neostigmine. In contrast, each of these three agents alone reversed postoperative hypersensitivity. Pretreatment with the [Greek small letter alpha]-adrenergic antagonist phentolamine completely reversed adenosine's antihypersensitivity action. Adenosine interacted synergistically with neostigmine and additively with clonidine in reducing postoperative hypersensitivity.     

Intrathecal adenosine: interactions with spinal clonidine and neostigmine in rat models of acute nociception and postoperative hypersensitivity.

BACKGROUND: Spinal adenosine receptor agonists exert antinociception in animal models of acute and chronic pain, but adenosine itself has not been examined. The authors tested the antinociceptive and antihypersensitivity interactions of intrathecal adenosine and its interactions with intrathecal clonidine and neostigmine in rat models of acute thermal nociception and postoperative hypersensitivity. METHODS: Rats were prepared with lumbar intrathecal catheters. Responses to acute noxious stimulation were evaluated by latency to paw withdrawal from a radiant heat source focused on the hind paw. Postoperative hypersensitivity was measured after an incision in the rat hind paw by application of von Frey filaments to the heel adjacent to the wound. An isobolographic design was used to distinguish between additive and synergistic drug interactions. RESULTS: Spinal administration of clonidine and neostigmine, but not adenosine, produced dose-dependent antinociception to noxious thermal stimulation. Addition of adenosine enhanced the antinociceptive effect of clonidine but not neostigmine. In contrast, each of these three agents alone reversed postoperative hypersensitivity. Pretreatment with the alpha-adrenergic antagonist phentolamine completely reversed adenosine's antihypersensitivity action. Adenosine interacted synergistically with neostigmine and additively with clonidine in reducing postoperative hypersensitivity. CONCLUSIONS: These data indicate that intrathecal adenosine by itself has no antinociceptive properties to acute noxious thermal stimulation in rats, but enhances clonidine's antinociception. In contrast, intrathecal adenosine is active against postoperative hypersensitivity by an adrenergic mechanism. Different interactions between adenosine, clonidine, and neostigmine in acute nociception and postoperative hypersensitivity models are consistent with altered central processing of sensory information after peripheral injury.     

Systemic ketamine inhibits hypersensitivity after surgery via descending inhibitory pathways in rats

PURPOSE: Systemic ketamine suppresses several types of chronic pain. Although ketamine is used as a general anesthetic agent, the analgesic effect of systemic ketamine for early-stage postoperative pain is not clear. We investigated the efficacy and mechanism of systemic ketamine in a rat model of postoperative pain. METHODS: An incision was made in the plantar aspect of the left hind paw in male Wistar rats. Mechanical hypersensitivity was measured using calibrated von Frey filaments. The anti-hypersensitivity effect of systemic or intrathecal administration of ketamine was determined every hour after making the incision. We examined the effects of intrathecal pretreatment with yohimbine, an alpha2-adrenoceptor antagonist, and methysergide, a serotonergic receptor antagonist, on the anti-hypersensitivity effect of ketamine. We also examined the effect of systemic ketamine on the c-fos immunoreactivity in the spinal cord. RESULTS: Systemic administration of ketamine at doses from 3 to 30 mg.kg(-1) produced anti-hypersensitivity effects in a dose-dependent manner. Intrathecal administration of ketamine had no effect. There was no significant difference between effects of pre- and post-incisional administration. Intrathecal pretreatment with yohimbine (10 microg) or methysergide (15 microg) completely reversed the anti-hypersensitivity effects of systemic ketamine. Systemic ketamine reduced fos expression in laminae I-II in the dorsal horn of the lumbar spinal cord ipsilateral to the paw incision. CONCLUSIONS: The results suggest that systemic administration of ketamine perioperatively suppresses early-stage postoperative pain via monoaminergic descending inhibitory pathways.     

Adenosine Reduces Glutamate Release in Rat Spinal Synaptosomes

Background: A1 adenosine receptor activation reduces hypersensitivity in animal models of chronic pain, but intrathecal adenosine does not produce analgesia to acute noxious stimuli. Here, the authors test whether increased inhibition by adenosine of glutamate release from afferents after injury accounts for this difference.

Methods: Synaptosomes were prepared from the dorsal half of the lumbar spinal cord of normal rats or those with spinal nerve ligation. Glutamate release evoked by the TRPV-1 receptor agonist, capsaicin, was measured. Adenosine with or without adenosine A1 and A2 receptor antagonists was applied to determine the efficacy and mechanism of adenosine to reduce capsaicin-evoked glutamate release.

Results: Capsaicin produced a concentration-dependent glutamate release similarly in normal and nerve-injured rats. Capsaicin-evoked glutamate release was inhibited by adenosine or R-PIA (R-N6-(2- phenylisopropyl)-adenosine) in a concentration-dependent manner, with a threshold of 10 nm in both normal and nerve-ligated synaptosomes. Blockade of capsaicin-evoked glutamate release by adenosine was reversed similarly in synaptosomes from normal and spinal nerve-ligated animals by an A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) but not by an A2 adenosine receptor antagonist DMPX (3"7-dimethyl-1-proparaglyxanthine). Capsaicin-evoked glutamate release, as well as its inhibition by adenosine, did not differ between synaptosomes prepared from tissue ipsilateral and contralateral to spinal nerve ligation.     

Spinal Microglial and Perivascular Cell Cannabinoid Receptor Type 2 Activation Reduces Behavioral Hypersensitivity without Tolerance after Peripheral Nerve Injury

Background: Cannabinoids induce analgesia by acting on cannabinoid receptor (CBR) types 1 and/or 2. However, central nervous system side effects and antinociceptive tolerance from CBR1 limit their clinical use. CBR2 exist on spinal glia and perivascular cells, suggesting an immunoregulatory role of these receptors in the central nervous system. Previously, the authors showed that spinal CBR2 activation reduces paw incision hypersensitivity and glial activation. This study tested whether CBR2 are expressed in glia and whether their activation would induce antinociception, glial inhibition, central side effects, and antinociceptive tolerance in a neuropathic rodent pain model.

Methods: Rats underwent L5 spinal nerve transection or sham surgery, and CBR2 expression and cell localization were assessed by immunohistochemistry. Animals received intrathecal injections of CBR agonists and antagonists, and mechanical withdrawal thresholds and behavioral side effects were assessed.

Results: Peripheral nerve transection induced hypersensitivity, increased expression of CR3/CD11b and CBR2, and reduced ED2/CD163 expression in the spinal cord. The CBR2 were localized to microglia and perivascular cells. Intrathecal JWH015 reduced peripheral nerve injury hypersensitivity and CR3/CD11b expression and increased ED2/CD163 expression in a dose-dependent fashion. These effects were prevented by intrathecal administration of the CBR2 antagonist (AM630) but not the CBR1 antagonist (AM281). JWH015 did not cause behavioral side effects. Chronic intrathecal JWH015 treatment did not induce antinociceptive tolerance.     

Spinal Cannabinoid Receptor Type 2 Activation Reduces Hypersensitivity and Spinal Cord Glial Activation after Paw Incision

Background: Cannabinoids bind to cannabinoid receptors type 1 and 2 and produce analgesia in several pain models, but central side effects from cannabinoid 1 receptors limit their clinical use. Cannabinoid 2 receptors reduce inflammatory responses in the periphery by acting on immune cells, and they are present on glia in the central nervous system. This study tested whether spinal cannabinoid activation would induce analgesia, glial inhibition, and central side effects in a postoperative model or incisional pain.

Methods: Rats underwent paw incision surgery, with intrathecal injections of cannabinoid agonists and antagonists and assessment of withdrawal thresholds and behavioral side effects. Spinal glial activation was determined by immunohistochemistry.

Results: Intrathecal administration CP55940 reduced postoperative hypersensitivity (91 +/- 9% maximum possible effect; P < 0.05), and this was prevented by intrathecal administration of both cannabinoid 1 receptor (AM281) and cannabinoid 2 receptor (AM630) antagonists. CP55940 also caused several behavioral side effects, and these were prevented by the cannabinoid 1 receptor but not by the cannabinoid 2 receptor antagonist. Intrathecal injection of the cannabinoid 2 receptor agonist JWH015 reversed postoperative hypersensitivity (89 +/- 5% maximum possible effect; P < 0.05), and this was reversed by the cannabinoid 2 but not by the cannabinoid 1 receptor antagonist. JWH015, which did not induce behavioral side effects, reduced paw incision induced microglial and astrocytic activation in spinal cord (P < 0.05).