The effect of ABT-702, a novel adenosine kinase inhibitor, on the responses of spinal neurones following carrageenan inflammation and peripheral nerve injury.

1. Adenosine (ADO) receptor activation modulates sensory transmission in the dorsal horn. Little is known about the circumstances underlying release of the purine. The present study was conducted to investigate the effect of a novel and potent non-nucleoside adenosine kinase (AK) inhibitor, ABT-702, on the responses of dorsal horn neurones to selected peripheral stimuli. ABT-702 is orally effective to reduce behavioural signs of nociception in models of acute, inflammatory, and neuropathic pain. 2. Electrophysiological recordings were made from wide dynamic range (WDR) neurones in halothane-anaesthetized rats. ABT-702 was given subcutaneously following either carrageenan inflammation or peripheral nerve injury (L5/L6 spinal nerve ligation). Comparisons were made between carrageenan and uninjected control animals, and similarly between spinal nerve ligated (SNL) and sham operated animals. 3. ABT-702 produced inhibition of the postdischarge, wind-up and C-fibre evoked responses in both carrageenan and nerve-injured animals. Furthermore, the mechanical and thermal evoked responses were similarly reduced in SNL rats. Overall, ABT-702 produced a significantly greater inhibition of these responses in SNL rats as compared to sham controls. Similarly ABT-702 tended to produce greater effects after carrageenan inflammation, however this did not reach significance. 4. Protection of endogenous adenosine by ABT-702 therefore produces a marked inhibition of the noxious evoked neuronal activity in inflamed and neuropathic rats. Our results demonstrate a plasticity in the endogenous adenosine-mediated inhibitory system following SNL and provide a possible basis for the use of this compound for the treatment of neuropathic and other persistent pain states.

Electrophysiologic evidence for increased endogenous gabaergic but not glycinergic inhibitory tone in the rat spinal nerve ligation model of neuropathy.

BACKGROUND: Changes in the inhibitory activity mediated by gamma-aminobutyric acid (GABA) and glycine, acting at spinal GABAA receptors and strychnine-sensitive glycine receptors, are of interest in the development of neuropathic pain. There is anatomic evidence for changes in these transmitter systems after nerve injuries, and blocking either GABAA or glycine receptors has been shown to produce allodynia-like behavior in awake normal animals. METHODS: In this study, the possible changes in GABAergic and glycinergic inhibitory activity in the spinal nerve ligation model of neuropathic pain were studied by comparing the effects of the GABAA-receptor antagonist bicuculline and the glycine-receptor antagonist strychnine in neuropathic rats to their effects in sham-operated and nonoperated control rats. RESULTS: Bicuculline produced a dose-related facilitation of the Adelta-fiber-evoked activity in all study groups and increased C-fiber-mediated activity in the spinal nerve ligation group but not in either of the control groups. There were no differences in the effect of bicuculline on low threshold responses between the study groups. The glycine receptor antagonist strychnine did not have a statistically significant effect on any of the parameters studied in any of the control groups. CONCLUSIONS: These results support the idea of an increased GABAergic inhibitory tone in the spinal cord of neuropathic rats, possibly as compensation for increased excitability after nerve injury.

Role of Ca2+-permeable non-NMDA glutamate receptors in spinal nociceptive transmission.

The functional role of Ca2+-permeable non-NMDA receptors in spinal nociceptive processing was investigated using joro spider toxin (JSTx), a selective blocker of these receptors. JSTx 0.25 and 1 microg administered spinally produced a significant facilitation of the C-fibre evoked response and post-discharge, but not the A-fibre response, of dorsal horn neurones recorded in adult rats. This may result from a block of Ca2+-permeable AMPA receptors located on GABAergic interneurones. At higher doses, this facilitation of responses was lost, suggesting additional Ca2+-permeable non-NMDA receptors, possibly kainate receptors, in excitatory spinal pathways. Thus, functional Ca2+-permeable AMPA receptors are present within the dorsal horn, predominantly within inhibitory pathways, and play a role distinct from other excitatory amino acid receptors in spinal nociceptive processing.

Effects of spinally administered P2X receptor agonists and antagonists on the responses of dorsal horn neurones recorded in normal, carrageenan-inflamed and neuropathic rats.

1. The function and role of P2X receptors in the spinal transmission of nociception was investigated using the selective P2X receptor agonists, alpha,beta-methylene ATP (alpha,beta-me ATP) and beta, gamma-methylene-L-ATP (beta,gamma-me-L-ATP) and the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. 2. Intrathecal administration of 5 and 50 microg of beta,gamma-me-L-ATP produced a significant facilitation of the C-fibre evoked response and a tendency towards increased excitability of the post-discharge, but not Abeta-fibre evoked response of dorsal horn neurones recorded in normal animals. Administration of similar doses of alpha,beta-me ATP did not produce an overall change in the response of the neuronal population. 3. Peripheral administration of 20 microg of these agonists into the paw of the rat evoked firing in the dorsal horn neurones. 4. Intrathecal administration of the antagonists, suramin (50 and 500 microg) and PPADS (5, 50 and 500 microg), to normal animals and to animals with a model of neuropathy induced by spinal nerve ligation did not alter the evoked neuronal responses. In contrast, intrathecal administration of 500 microg of suramin to animals 3 h after the induction of carrageenan inflammation produced a significant inhibition of the C-fibre evoked response of the neurones. Similar inhibitions were also seen following high doses of intrathecal PPADS, although this did not reach significance. 5. These results suggest that spinal P2X receptors may play a role in the modulation of spinal nociceptive transmission following the development of inflammation, but that these receptors play at most a minor role in spinal nociceptive processing in normal and neuropathic animals.

The role of non-N-methyl-D-aspartate ionotropic glutamate receptors in the spinal transmission of nociception in normal animals and animals with carrageenan inflammation.

The role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors in spinal nociceptive transmission in both normal animals and animals with carrageenan inflammation was investigated using the AMPA/kainate receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX) and the selective GluR5 kainate receptor antagonist LY382884 [3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8, 8a-deca-hydroisoquinoline-3-carboxylic acid]. In normal animals, spinal administration of 100 microg of LY382884 produced a significant inhibition of both the C-fibre-evoked response and post-discharge of dorsal horn neurons, with the wind-up of the neurons being reduced by both 50 and 100 microg of LY382884. The spinal actions of LY382884 were enhanced following 3 h of carrageenan inflammation, such that doses of 20 microg and above were able to produce significant inhibitions of the noxious-evoked response of the neurons. Spinal administration of NBQX in normal animals (5-50 microg) inhibited the C-fibre-evoked response of the dorsal horn neurons, but only 50 microg of NBQX was able to inhibit the wind-up and post-discharge of the neurons. Following 3 h of carrageenan inflammation, the ability of NBQX to inhibit the wind-up and post-discharge of the neurons was markedly enhanced. These data suggest that both AMPA and kainate GluR5 receptors play an enhanced role in spinal nociceptive processing following the development of peripheral inflammation, as antagonists at both receptors are more effective against nociceptive responses, including wind-up under these inflammatory conditions.

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Inflammation alters the effects of mGlu receptor agonists on spinal nociceptive neurones.

Several types of metabotropic glutamate receptor are known to be located in the spinal cord. This study examined the effects of the metabotropic glutamate receptor agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) and (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3S)-ACPD) on the electrically evoked responses of dorsal horn neurones recorded in normal animals and in animals 3 h after the induction of carrageenan inflammation. The group I and II agonist (1S,3R)-ACPD produced facilitations of the noxious evoked neuronal responses in normal animals, but inhibited these responses following carrageenan inflammation. The group II agonist (1S,3S)-ACPD also produced inhibitions in the carrageenan animals, in contrast to the mixed effects seen in normal animals. The group I agonist (S)-3,5-DHPG produced mixed effects (inhibitions and facilitations) in both normal and carrageenan animals. This in vivo study shows that the effects of metabotropic glutamate receptor agonists are more complex than in vitro studies have suggested to date.

Gabapentin, ineffective in normal rats, markedly reduces C-fibre evoked responses after inflammation.

Gabapentin (Neurontin) is a novel anticonvulsant with an as yet unknown mechanism of action. This electrophysiological study investigated the potential antinociceptive actions of systemically administered gabapentin in normal animals and after inflammation induced by the injection of carrageenan. Gabapentin facilitated the noxious evoked responses of dorsal horn neurones recorded in normal animals. In complete contrast, gabapentin strongly and dose-dependently inhibited the C-fibre evoked response and post-discharge, but not the A beta-fibre evoked response, of neurones recorded in animals 3 h after the injection of carrageenan. This unique and selective profile of gabapentin may provide a novel treatment for clinical inflammatory pain states.

Amplification of spinal nociceptive transmission depends on the generation of nitric oxide in normal and carrageenan rats.

It has been proposed that nitric oxide (NO) is involved in the spinal transmission of nociceptive information, particularly following the development of peripheral inflammation. In this electrophysiological study the ability of the nitric oxide synthase inhibitor 7-nitro indazole (7-NI), which does not block endothelial nitric oxide in vivo, to inhibit the electrically evoked responses of dorsal horn neurones recorded in both normal animals and in animals 3 h after the injection of carrageenan into the ipsilateral hind paw, was investigated. In both normal and carrageenan inflamed animals, 7-NI (1-100 micrograms), administered intrathecally, strongly inhibited the NMDA receptor mediated wind-up and post-discharge of the neurones, having relatively little effect on the acute C- or A beta-fibre evoked activity of the neurones. This inhibitory action of 7-NI on the noxious evoked responses of the neurones was completely blocked by the prior intrathecal administration of 500 micrograms of L-arginine. Inflammation did not alter the effects of 7-NI since there was no difference in the dose-response curve between the normal and carrageenan animals. In normal animals, stimuli of sufficient duration/intensity to enable the activation of NMDA receptors to occur, shown in this study by the occurrence of wind-up, also lead to the generation of nitric oxide, which then participates in nociceptive transmission. These effects appear to be independent of the vascular effects of NO. Inflammation-induced changes could facilitate activation of spinal NMDA receptors, such that nitric oxide is now generated by stimuli previously sub-threshold for this event. Previous studies, reporting a unique role of NO in nociceptive transmission following the development of peripheral inflammation, may have resulted from inadequate stimuli in the normal animal.

Inhibitory action of nociceptin on spinal dorsal horn neurones of the rat, in vivo.

Intrathecally administered nociceptin (5, 50, 225 micrograms) dose-relatedly inhibited the C-fibre evoked wind-up and post-discharge of dorsal horn neurones, but not the baseline C-fibre evoked responses. Spinal naloxone 50 micrograms, but not 10 micrograms, reversed the effects of nociceptin. Thus the antinociceptive role of nociceptin in the spinal cord differs from that of classical opioids.

Enhanced alpha-2 adrenergic controls and spinal morphine potency in inflammation.

The antinociceptive potency of spinal morphine is enhanced in rats after carrageenan-induced inflammation. This electrophysiological study examines whether changes in alpha-2 adrenergic systems are responsible. Dorsal horn nociceptive neurones were recorded under halothane anaesthesia in normal animals and animals with carrageenan inflammation. There was a mild increase in potency of the selective alpha-2 adrenoceptor agonist dexmedetomidine following carrageenan (ED50 = 1 microgram). Intrathecal idazoxan (100 micrograms), an alpha-2 antagonist, produced a significant facilitation of the C-fibre evoked response in carrageenan-treated but not normal animals. However since neither idazoxan (100 micrograms) or atipamezole (50 micrograms, another antagonist) influenced the potency of spinal morphine, the increased alpha-2 adrenergic activity in inflammation does not contribute to the enhanced potency of spinal morphine.

Cholecystokinin as a factor in the enhanced potency of spinal morphine following carrageenin inflammation.

1. Cholecystokinin (CCK) has been shown to diminish opioid analgesia. Here we investigate whether changes in the physiological levels of spinal CCK are responsible for the enhanced potency of spinal morphine in animals following carrageenin inflammation, as compared with normal animals. 2. Single dorsal horn nociceptive neurones were recorded in intact halothane-anaesthetized rats in the presence and absence of carrageenin-induced inflammation and comparisons were made between the two groups of animals. Inflammation was induced by the injection of 100 microliters of 2% lambda-carrageenin into the hind paw. 3. The inhibitory effect of intrathecal morphine on the C-fibre-evoked responses of the neurones was enhanced in the carrageenin-treated animals such that the effects of 0.25 microgram and 10 micrograms of morphine in normal animals were comparable to those of 0.01 microgram and 2.5 micrograms in the carrageenin animals. The effect of 0.2 mg kg-1 of the CCKB antagonist, L-365,260, on the antinociceptive potency of intrathecal morphine was examined in both groups of animals. In normal animals, L-365,260 produced a significant enhancement in the effect of morphine indicating a tonic CCK modulation in these animals, but it had no effect on the inhibitions produced by either dose of morphine in the carrageenin animals. 4. The inhibition of the C-fibre-evoked response produced by intrathecal morphine in the presence of 1 microgram of CCK was examined in both groups of animals. CCK attenuated the effects of morphine only in animals with carrageenin inflammation, having no effect on the action of morphine in normal animals. 5. The effects of both CCK and L-365,260 were therefore dependent on the inflammatory state of the animal, with each drug being active in opposite situations.6. We propose that in normal animals, morphine may produce a maximal stimulation of the release of CCK such that exogenous CCK is unable to reduce further the analgesic effects under these conditions.However, the differential effects of the agonist and antagonist in the normal and inflamed rats points to a role of CCK in the enhanced opiate actions. This enhancement of the potency of spinal morphine in inflammation is best explained by a reduction in spinal CCK release by morphine in this state.

Dextromethorphan and levorphanol on dorsal horn nociceptive neurones in the rat.

Intrathecal administration of dextromethorphan and levorphanol and intravenous injection of dextromethorphan were tested on the electrophysiological response of deep multireceptive dorsal horn neurones to peripheral stimuli. Both blockade of C-fibre input to the cells and wind-up, the increase in C-fibre firing with repeated stimulus, were recorded. Intrathecal injection of levorphanol (0.25-100 micrograms) had a typical opioid effect, blocking the C-fibre input. Its affect on wind-up was dose-dependent, paralleled precisely the blocking effect on the C-fibre input and both effects were reversed by naloxone. Unlike levorphanol and other opiates, intrathecal administration of dextromethorphan (50-500 micrograms) blocked the C-fibre input and A beta response in parallel and was not reversed by naloxone. Wind-up was reduced by a maximum of 56% at the largest dose tested. Intravenous injection of dextromethorphan (5 mg/kg) also produced a reduction in wind-up but not in the C-fibre response.