Microinjection of an adenosine A1 agonist into the medial pontine reticular formation increases tail flick latency to thermal stimulation

BACKGROUND: Both pain and the pharmacologic management of pain can cause the undesirable effect of sleep disruption. One goal of basic and clinical neuroscience is to facilitate rational drug development by identifying the brain regions and neurochemical modulators of sleep and pain. Adenosine is thought to be an endogenous sleep promoting substance and adenosinergic compounds can contribute to pain management. In the pontine brain stem adenosine promotes sleep but the effects of pontine adenosine on pain have not been studied. This study tested the hypothesis that an adenosine agonist would cause antinociception when microinjected into pontine reticular formation regions that regulate sleep. METHODS: The tail flick latency (TFL) test quantified the time in seconds for an animal to move its tail away from a thermal stimulus created by a beam of light. TFL measures were used to evaluate the antinociceptive effects of the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA). Pontine microinjection of SPA (0.1 microg/0.25 microl, 0.88 mm) was followed by TFL measures as a function of time after drug delivery and across the sleep-wake cycle. RESULTS: Compared with saline (control), pontine administration of the adenosine agonist significantly increased latency to tail withdrawal (P < 0.0001). The increase in antinociceptive behavior evoked by the adenosine agonist SPA was blocked by pretreatment with the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.75 ng/0.25 microl, 10 microm). CONCLUSIONS: These preclinical data encourage additional research on the cellular mechanisms by which adenosine in the pontine reticular formation contributes to the supraspinal modulation of pain.     

Dialysis Delivery of an Adenosine A1 Receptor Agonist to the Pontine Reticular Formation Decreases Acetylcholine Release and Increases Anesthesia Recovery Time

Background: Adenosine modulates cell excitability, acetylcholine release, nociception, and sleep. Pontine cholinergic neurotransmission contributes to the generation and maintenance of electroencephalographic and behavioral arousal. Adenosine A1 receptors inhibit arousal-promoting, pontine cholinergic neurons, and adenosine enhances sleep. No previous studies have determined whether pontine adenosine also modulates recovery from anesthesia. Therefore, the current study tested the hypotheses that dialysis delivery of the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA) into the pontine reticular formation would decrease acetylcholine release and increase the time needed for recovery from halothane anesthesia.

Methods: A microdialysis probe was positioned in the pontine reticular formation of halothane-anesthetized cats. Probes were perfused with Ringer's solution (control) followed by the adenosine A1 receptor agonist SPA (0.088 or 8.8 mm). Dependent measures included acetylcholine release and a numeric assessment of recovery from anesthesia. An intensive, within-subjects design and analysis of variance evaluated SPA's main effect on acetylcholine release and anesthetic recovery. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 [mu]m) was coadministered with SPA to test for antagonist blocking of SPA's effects.

Results: SPA significantly (P < 0.0001) decreased acetylcholine release in the pontine reticular formation and significantly (P < 0.0001) delayed recovery from anesthesia. Coadministration of SPA and DPCPX caused no decrease in acetylcholine release or delay in postanesthetic recovery. Dialysis delivery of SPA into the cerebellar cortex confirmed that the SPA effects were site-specific to the pontine reticular formation.     

Effects of Radolmidine, A Novel α2-Adrenergic Agonist Compared with Dexmedetomidine in Different Pain Models in the Rat

Background: Intrathecally administered [alpha]2-adrenoceptor agonists produce effective antinociception, but sedation is an important adverse effect. Radolmidine is a novel [alpha]2-adrenoceptor agonist with a different pharmacokinetic profile compared with the well-researched dexmedetomidine. This study determined the antinociceptive and sedative effects of radolmidine in different models of acute and chronic pain. Dexmedetomidine and saline served as controls.

Methods: Male Sprague-Dawley rats were studied in acute pain (tail flick), carrageenan inflammation, and the spinal nerve ligation model of neuropathic pain. Mechanical allodynia was assessed with von Frey filaments, cold allodynia with the acetone test, and thermal hyperalgesia with the paw flick test. Locomotor activity-vigilance was assessed in a dark field. Dexmedetomidine and radolmidine were administered intrathecally in doses of 0.25 [mu]g, 2.5 [mu]g, 5 [mu]g, and 10 [mu]g.

Results: In the tail flick test, radolmidine showed a dose-dependent antinociceptive effect, being equipotent compared with dexmedetomidine. In carrageenan inflammation, intrathecal doses of 2.5 [mu]g or 5 [mu]g of dexmedetomidine/radolmidine produced significant antinociception compared with saline (P < 0.01). The two drugs were equianalgesic. In the neuropathic pain model, an intrathecal dose of 5 [mu]g dexmedetomidine-radolmidine had a significant antiallodynic effect compared with saline (P < 0.01). The two drugs were equipotent. Intrathecal administration of both dexmedetomidine and radolmidine dose dependently decreased spontaneous locomotor acitivity-vigilance, but this effect was significantly smaller after intrathecal administration of radolmidine than after intrathecal dexmedetomidine.     

Supraspinal Antinociceptive Effects of μ and δ Agonists Involve Modulation of Adenosine Uptake

Background: The modulation of extracellular adenosine concentration by opioids provides evidence that the antinociceptive effects of these compounds involve endogenous adenosine. The aim of this study was to determine whether there is a relation between the inhibition of brain synaptosome adenosine uptake by opioid agonists and the analgesic effects of these compounds.

Methods: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a [mu] agonist, SNC80 as a [delta] agonist, or U69593 as a [kappa] agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes.

Results: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not.     

5-HT spinal antinociception involves mu opioid receptors: cross tolerance and antagonist studies

The antinociceptive effects of intrathecal 5-HT, fentanyl, ICI197067 and U50488H were assessed by electrical current nociceptive threshold and tail flick latency measurements. Equieffective doses of these agonists were then given intrathecally with a range of doses of naloxone or the highly selective mu opioid antagonist, beta- funaltrexamine. Antagonist dose-response curves were plotted. Other rats were made tolerant to either fentanyl or 5-HT by intrathecal injections of these drugs seven times daily and the antinociceptive effects of intrathecal fentanyl and 5-HT were assessed in each group. All intrathecal drugs caused spinally mediated antinociception in both tests. The antinociceptive effects of intrathecal 5-HT assessed by the electrical test (ECT) but not by tail flick latency (TFL) were suppressed by both opioid antagonists at doses similar to those required to suppress all of the effects of intrathecal fentanyl. The ED50 values were 0.22 (fentanyl, ECT), 0.25 (fentanyl, TFL) and 0.18 (5- HT, ECT) mumol kg-1 for naloxone and for beta-funaltrexamine 2.2 fmol (5-HT, ECT), the same order as that required to produce similar suppression of the antinociceptive effects of fentanyl (46 amol: fentanyl, ECT; 4.6 fmol: fentanyl, TFL) and very different from the ED50 for beta-FNA suppression of the antinociceptive effects of the kappa opioid, U50488H (5.88 pmol). Cross tolerance in both directions was demonstrated between intrathecal fentanyl and 5-HT in the electrical test but not in the tail flick test. We conclude that intrathecal 5-HT caused spinally mediated antinociceptive effects revealed by electrical current and tail flick latency tests. The antinociceptive effects in the electrical test involved spinal cord mu opioid receptors.      

Dialysis delivery of an adenosine A1 receptor agonist to the pontine reticular formation decreases acetylcholine release and increases anesthesia recovery time

BACKGROUND: Adenosine modulates cell excitability, acetylcholine release, nociception, and sleep. Pontine cholinergic neurotransmission contributes to the generation and maintenance of electroencephalographic and behavioral arousal. Adenosine A(1) receptors inhibit arousal-promoting, pontine cholinergic neurons, and adenosine enhances sleep. No previous studies have determined whether pontine adenosine also modulates recovery from anesthesia. Therefore, the current study tested the hypotheses that dialysis delivery of the adenosine A(1) receptor agonist N6-p-sulfophenyladenosine (SPA) into the pontine reticular formation would decrease acetylcholine release and increase the time needed for recovery from halothane anesthesia. METHODS: A microdialysis probe was positioned in the pontine reticular formation of halothane-anesthetized cats. Probes were perfused with Ringer's solution (control) followed by the adenosine A(1) receptor agonist SPA (0.088 or 8.8 mm). Dependent measures included acetylcholine release and a numeric assessment of recovery from anesthesia. An intensive, within-subjects design and analysis of variance evaluated SPA's main effect on acetylcholine release and anesthetic recovery. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 microm) was coadministered with SPA to test for antagonist blocking of SPA's effects. RESULTS: SPA significantly (P < 0.0001) decreased acetylcholine release in the pontine reticular formation and significantly (P < 0.0001) delayed recovery from anesthesia. Coadministration of SPA and DPCPX caused no decrease in acetylcholine release or delay in postanesthetic recovery. Dialysis delivery of SPA into the cerebellar cortex confirmed that the SPA effects were site-specific to the pontine reticular formation. CONCLUSION: The results provide a novel extension of the sleep-promoting effects of adenosine by showing that pontine delivery of an adenosine A(1) receptor agonist delays resumption of wakefulness following halothane anesthesia. This extension is consistent with a potentially larger relevance of the current findings for efforts to specify neurons and molecules causing physiologic and behavioral traits comprising anesthetic states. These data support the conclusion that adenosine A(1) receptors in medial regions of the pontine reticular formation, known to modulate sleep, also contribute to the generation and/or maintenance of halothane anesthesia.     

Brain Stem Opioidergic and GABAergic Neurons Mediate the Antinociceptive Effect of Nitrous Oxide in Fischer Rats

Background: Recent studies have revealed that N2O exerts its antinociceptive effect by inducing opioid peptide release in the brain stem, thereby activating the descending noradrenergic inhibitory neurons, which modulate pain processing in the spinal cord. However, the precise neuronal pathways that mediate these events remain to be determined.

Methods: Using immunohistochemical and behavioral techniques in adult male Fischer rats, the authors studied the involvement of brain stem opioidergic and [gamma]-aminobutyric acid-mediated (GABAergic) neurons in the N2O-induced antinociceptive effect using discrete microinjections of an opioid receptor antagonist or GABAergic activator into the periaqueductal gray area and pontine noradrenergic nuclei. They used c-Fos expression as an immunohistochemical mark of neuronal activation induced by N2O and the plantar test as the behavioral paradigm for nociception.

Results: Microinjection of either naloxone (an opioid receptor antagonist) or muscimol (a [gamma]-aminobutyric acid receptor type A agonist) into the ventrolateral periaqueductal gray area inhibited N2O-induced c-Fos expression in the spinal cord and pontine noradrenergic nuclei, particularly in the A7. Microinjection of either naloxone or muscimol into the A7 nuclei also inhibited N2O-induced c-Fos expression in the spinal cord and the N2O-induced antinociceptive effect by the plantar test.     

Differential Presynaptic Effects of Opioid Agonists on Aδ- and C-afferent Glutamatergic Transmission to the Spinal Dorsal Horn

Background: Although intrathecal administration of opioids produces antinociceptive effects in the spinal cord, it has not been established whether intrathecal opioid application more effectively terminates C fiber-mediated pain than A fiber-mediated pain. Here, the authors focus on the differences in opioid actions on A[delta]- and C-afferent responses.

Methods: Using the whole cell patch clamp technique, the authors investigated the presynaptic inhibitory actions of [mu]-, [delta]-, and [kappa]-opioid receptor agonists on primary afferent-evoked excitatory postsynaptic currents (EPSCs) in substantia gelatinosa neurons of adult rat spinal cord slices.

Results: The [mu] agonist DAMGO (0.1, 1 [mu]m) reduced the amplitude of glutamatergic monosynaptic A[delta]- or C fiber-evoked EPSCs. C fiber-evoked EPSCs were inhibited to a greater extent than A[delta] fiber-evoked EPSCs. The [delta] agonist DPDPE (1, 10 [mu]m) produced modest inhibition of A[delta]- or C fiber-evoked EPSCs. In contrast, the [kappa] agonist U69593 (1 [mu]m) did not affect the amplitude of either A[delta] or C fiber-evoked EPSCs.     

Pharmacologic Interaction between Cannabinoid and either Clonidine or Neostigmine in the Rat Formalin Test

Background: Although spinal cannabinoid receptor agonist (WIN 55,212-2) has been shown to encounter various models of pain, the role of two subtypes of cannabinoid receptor for the antinociceptive effect of cannabinoids has not been investigated at the spinal level. Spinal [alpha]2 receptor agonist (clonidine) and cholinesterase inhibitor (neostigmine) are also active in the modulation of nociception. The authors examined the properties of drug interaction after coadministration of WIN 55,212-2-clonidine, and intrathecal WIN 55,212-2-neostigmine, and further clarified the role of cannabinoid 1 and 2 receptors in cannabinoid-induced antinociception at the spinal level.

Methods: Catheters were inserted into the intrathecal space of male Sprague-Dawley rats, and 50 [mu]l of 5% formalin solution was injected into the hind paw to evoke the pain. Isobolographic analysis was used for evaluation of pharmacologic interaction.

Results: Intrathecal 55,212-2, clonidine, and neostigmine dose-dependently suppressed the flinching observed during phase 1 and 2 in the formalin test. Isobolographic analysis revealed a synergistic interaction after intrathecal delivery of WIN 55,212-2-clonidine or WIN 55,212-2-neostigmine mixture in both phases. The antinociceptive effect of WIN 55,212-2 was antagonized by cannabinoid 1 receptor antagonist (AM 251) but not by cannabinoid 2 receptor antagonist (AM 630). No antinociceptive effect was seen after intrathecal administration of cannabinoid 2 receptor agonist (JWH 133).     

Supraspinal antinociceptive effects of mu and delta agonists involve modulation of adenosine uptake

BACKGROUND: The modulation of extracellular adenosine concentration by opioids provides evidence that the antinociceptive effects of these compounds involve endogenous adenosine. The aim of this study was to determine whether there is a relation between the inhibition of brain synaptosome adenosine uptake by opioid agonists and the analgesic effects of these compounds. METHODS: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a mu agonist, SNC80 as a delta agonist, or U69593 as a kappa agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes. RESULTS: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not. CONCLUSION: The antinociceptive effects obtained with mu or delta (but not kappa) agonists administered supraspinally were indicative of the involvement of modulation of adenosine uptake.     

Antihyperalgesic and Side Effects of Intrathecal Clonidine and Tizanidine in a Rat Model of Neuropathic Pain

Background: Although intrathecal clonidine produces pronounced analgesia, antinociceptive doses of intrathecal clonidine produce several side effects, including hypotension, bradycardia, and sedation. Intrathecal tizanidine, another [alpha]2-adrenergic agonist, has provided antinociception without producing pronounced hemodynamic changes in animal studies. However, it has been unclear whether antihyperalgesic doses of intrathecal clonidine and tizanidine produce hypotension and bradycardia in a neuropathic pain state. This study was designed to evaluate the antihyperalgesic effects and side effects of intrathecal clonidine and tizanidine in a rat model of neuropathic pain.

Methods: Male Sprague-Dawley rats were chronically implanted with lumbar intrathecal catheters, and the sciatic nerve was loosely ligated. After 21-28 days after surgery, the rats received intrathecal clonidine (0.3, 1.0, and 3.0 [mu]g) and tizanidine (1.0, 2.0, and 5.0 [mu]g), and the antihyperalgesic effects of thermal and mechanical stimuli were examined. In addition, the changes in blood pressure and heart rate, sedation level, and other side effects after intrathecal administration of drugs were recorded.

Results: The administration of 3.0 [mu]g intrathecal clonidine or 5.0 [mu]g tizanidine significantly reversed both thermal and mechanical hyperalgesia. The administration of 3.0 [mu]g intrathecal clonidine, but not 5.0 [mu]g tizanidine, significantly decreased mean blood pressure and heart rate and produced urinary voiding. A greater sedative effect was produced by 3.0 [mu]g intrathecal clonidine than by 5.0 [mu]g tizanidine.     

Corticotropin-releasing Factor Mediates the Antinociceptive Action of Nitrous Oxide in Rats

Background: Exposure to nitrous oxide activates brainstem noradrenergic nuclei and descending inhibitory pathways, which produce the acute antinociceptive action of nitrous oxide. Because corticotropin-releasing factor (CRF) can produce activation of noradrenergic neurons in the locus ceruleus, the authors sought to determine whether it might be responsible for the antinociceptive action of nitrous oxide.

Methods: Male Sprague-Dawley rats (250-300 g) were exposed for 60 min to room air or 25, 50 or 70% nitrous oxide in oxygen. Brain sections including the hypothalamus were immunostained for both c-Fos (a marker of neuronal activation) and CRF and the percentage of CRF-positive neurons expressing c-Fos was determined. The functional consequences of changes in CRF were investigated by assessing the effect of intracerebroventricular administration of a CRF antagonist ([alpha]-helical CRF9-41, 20 [mu]g/10 [mu]l) on both activation of locus ceruleus noradrenergic neurons and the antinociception (with the tail-flick latency test) produced by nitrous oxide.

Results: Inhalation of nitrous oxide induced a dose-dependent increase in c-Fos expression in CRF-positive neurons in the paraventricular nucleus of the hypothalamus. Intracerebroventricular administration of CRF antagonist inhibited nitrous oxide-induced c-Fos expression in the locus ceruleus and the antinociceptive effect of nitrous oxide.     

Spinal Glucocorticoid Receptors Contribute to the Development of Morphine Tolerance in Rats

Background: Opioid analgesic tolerance is a pharmacologic phenomenon involving the mechanisms of cellular adaptation. Central glucocorticoid receptors (GRs) have been implicated in the cellular mechanism of neuronal plasticity that has many cellular steps in common with the mechanism of opioid tolerance. In a rat model of morphine tolerance, the authors examined the hypothesis that spinal GRs would play a significant role in the development of tolerance to the antinociceptive effect of morphine.

Methods: In experiment 1, each group of rats received the GR antagonist RU38486 (0.5 or 1 [mu]g), the mineralocorticoid receptor antagonist spironolactone (3 [mu]g), or a vehicle, given intrathecally with morphine (10 [mu]g) twice daily for 6 days. In experiment 2, four groups of rats were used, and each group received intrathecally 10 [mu]g morphine plus 5 [mu]mol GR antisense oligodeoxynucleotide, sense oligodeoxynucleotide, mixed-base oligodeoxynucleotide, or vehicle. Western blotting was used to examine the expression of GRs within the spinal cord dorsal horn. In experiment 3, the GR agonist dexamethasone (4 [mu]g) was given intrathecally twice daily in combination with 10 [mu]g morphine. For all experiments, the development of morphine antinociceptive tolerance was assessed using the tail-flick test.

Results: The development of tolerance to the antinociceptive effect of morphine was substantially attenuated when the GR antagonist RU38486 (1 > 0.5 [mu]g > vehicle) but not spironolactone was coadministered with morphine for 6 days. A single treatment with RU38486 did not affect morphine antinociception, nor did it reverse morphine tolerance on day 7. A similar reduction of morphine tolerance was observed in those rats treated with a GR antisense oligodeoxynucleotide but not a sense or mixed-base oligodeoxynucleotide. The administration of the GR antisense oligodeoxynucleotide also prevented GR up-regulation within the spinal cord dorsal horn. Moreover, the GR agonist dexamethasone facilitated the development of morphine tolerance.     

Antinociceptive Effect of Morphine, but not μ Opioid Receptor Number, Is Attenuated in the Spinal Cord of Diabetic Rats

Background: The mechanisms of decreased analgesic potency of [mu] opioids in diabetic neuropathic pain are not fully known. The authors recently found that G protein activation stimulated by the [mu] opioid agonist is significantly reduced in the spinal cord dorsal horn in diabetes. In the current study, they determined potential changes in the number and binding affinity of [mu] opioid receptors in the spinal cord in diabetic rats.

Methods: Rats were rendered diabetic with an intraperitoneal injection of streptozotocin. The nociceptive withdrawal threshold was measured before and after intrathecal injection of morphine by applying a noxious pressure stimulus to the hind paw. The [mu] opioid receptor was determined with immunocytochemistry labeling and a specific [mu] opioid receptor radioligand, [3H]-(d-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin ([3H]-DAMGO), in the dorsal spinal cord obtained from age-matched normal and diabetic rats 4 weeks after streptozotocin treatment.

Results: The antinociceptive effect of intrathecal morphine (2-10 [mu]g) was significantly reduced in diabetic rats, with an ED50 about twofold higher than that in normal rats. However, both the dissociation constant (3.99 +/- 0.22 vs. 4.01 +/- 0.23 nm) and the maximal specific binding (352.78 +/- 37.26 vs. 346.88 +/- 35.23 fmol/mg protein) of [3H]-DAMGO spinal membrane bindings were not significantly different between normal and diabetic rats. The [mu] opioid receptor immunoreactivity in the spinal cord dorsal horn also was similar in normal and diabetic rats.     

Role of Adenosine Receptors in Spinal G-Protein Activation after Peripheral Nerve Injury

Background: Spinally injected adenosine induces antinociception in animal models of neuropathic but not acute pain. The reasons for this discrepancy remain unclear. Adenosine receptors are coupled to G proteins, and increased efficiency of adenosine-induced G-protein activity in neuropathic pain could contribute to the antinociceptive effect of adenosine. In this study the authors used [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP[gamma]S) autoradiography in rat spinal cord sections to test this possibility.

Methods: The spinal cords of normal animals and those that underwent left L5 and L6 spinal nerve ligation (SNL) were removed and immediately frozen. Horizontal spinal cord sections were cut and mounted on chrom-alum gelatin-subbed slides. Sections were incubated with guanosine diphosphate, [35S]GTP[gamma]S, the adenosine A1 agonist R-N6-phenylisopropyladenosine, and various other drugs, apposed to films, and analyzed.

Results: Baseline and R-N6-phenylisopropyladenosine-stimulated [35S]GTP[gamma]S binding was predominantly localized to the superficial dorsal horns of both normal and SNL animals. This binding was significantly increased in SNL compared with normal animals. In contrast, no difference in R-N6-phenylisopropyladenosine-stimulated [35S]GTP[gamma]S binding was observed between SNL and normal animals. Blockade of adenosine A1 receptors by 1,3-dipropyl-8-cyclopentylxanthine, or adenosine destruction by added adenosine deaminase, reduced the increased basal activity in SNL to baseline levels of normal dorsal horns, whereas atropine and naloxone had no effect.     

Antinociceptive actions of intrathecal xylazine: interactions with spinal cord opioid pathways

We have studied rats with chronically implanted subarachnoid catheters. Xylazine, an alpha 2 adrenoceptor agonist, was injected intrathecally and nociceptive thresholds measured at two skin sites: the tail and the neck. Intrathecal xylazine (dose range 24.3-389 nmol) produced increases in electrical thresholds for nociception in the tail without any change in the neck; this observation suggested that the antinociceptive action of this drug was confined to the caudal part of the spinal cord responsible for tail innervation. The magnitude of this effect was dose-dependent. Tail flick latency also increased in these rats and the antinociceptive effects were antagonized in a dose- dependent manner by the selective alpha 2 adrenoceptor antagonist idazoxan (dose range 6.7-540 nmol). Intrathecal idazoxan also suppressed the increase in tail flick latency caused by the mu opioid agonist fentanyl (0.74 nmol) given intrathecally. This effect was also dose-dependent. The idazoxan dose-response curve for this suppression of fentanyl antinociception assessed with tail flick latency was the same as that for suppression of xylazine. In contrast, the antinociceptive effects of intrathecal xylazine were not affected by concurrent administration of opioid or GABAA antagonists. We conclude that intrathecal xylazine produced spinally mediated antinociceptive effects by combination with spinal cord alpha 2 adrenoceptors and that neither opioid nor GABA-containing propriospinal neurones were involved in the mediation of this effect. However, alpha 2 adrenoceptors in the spinal cord appear to be involved with antinociception produced by intrathecal fentanyl.      

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.     

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.     

Spinal Endogenous Acetylcholine Contributes to the Analgesic Effect of Systemic Morphine in Rats

Background: Systemic morphine is known to cause increased release of acetylcholine in the spinal cord. Intrathecal injection of the cholinergic receptor agonists or acetylcholinesterase inhibitors produces antinociception in both animals and humans. In the present study, we explored the functional importance of spinal endogenous acetylcholine in the analgesic action produced by intravenous morphine.

Methods: Rats were implanted with intravenous and intrathecal catheters. The antinociceptive effect of morphine was determined by the paw-withdrawal latency in response to a radiant heat stimulus after intrathecal treatment with atropine (a muscarinic receptor antagonist), mecamylamine (a nicotinic receptor antagonist), or cholinergic neurotoxins (ethylcholine mustard aziridinium ion [AF64A] and hemicholinium-3).

Results: Intravenous injection of 2.5 mg/kg morphine increased significantly the paw-withdrawal latency. Intrathecal pretreatment with 30 [mu]g atropine (n = 7) or 50 [mu]g mecamylamine (n = 6) both attenuated significantly the antinociceptive effect of morphine. The inhibitory effect of atropine on the effect of morphine was greater than that of mecamylamine. Furthermore, the antinociceptive effect of morphine was significantly reduced in rats pretreated with intrathecal AF64A (n = 7) or hemicholinium-3 (n = 6) to inhibit the high-affinity choline transporter and acetylcholine synthesis. We found that intrathecal AF64A reduced significantly the [3H]hemicholinium-3 binding sites but did not affect its affinity in the dorsal spinal cord.     

The Mechanical Antihyperalgesic Effect of Intrathecally Administered MPV-2426, a Novel α2-Adrenoceptor Agonist, in a Rat Model of Postoperative Pain

Background: MPV-2426 is a novel [alpha]2-adrenoceptor agonist developed for spinal pain therapy. It has proved to be effective in physiologic and neuropathic conditions. In the current study its effectiveness on mechanical hyperalgesia was assessed in a rat model of postoperative pain.

Methods: Rats with intrathecal catheters were anesthetized with pentobarbital, and a 1-cm incision was made in the plantar aspect of the foot and closed. During postoperative days 1 and 2 the antihyperalgesic effects induced by intrathecal MPV-2426, clonidine, and dexmedetomidine were determined by assessing the hind limb withdrawal threshold to calibrated von Frey hairs applied to the skin of the hind paw adjacent to the wound.

Results: MPV-2426 administered into the lumbar spinal cord produced a dose-dependent (0.3-10 [mu]g) attenuation of the mechanical hyperalgesia, and this antihyperalgesic effect was completely reversed by yohimbine (1 mg/kg, subcutaneous), an [alpha]2-adrenoceptor antagonist. Dexmedetomidine (1-3 [mu]g) produced an equipotent antihyperalgesic effect, whereas the effect of clonidine (1-10 [mu]g) was markedly weaker. MPV-2426 (10 [mu]g in 20 [mu]l) administered adjacent to the wound did not produce any effect. Preoperative treatment with an antihyperalgesic dose of MPV-2426 did not prevent the development of hyperalgesia.