Capsaicin-induced pain and sensitisation in the postpartum period.

BACKGROUND: Recovery from Caesarean delivery in women and surgical nerve injury in animals after delivery is more rapid than expected, an effect reversed in animals by spinal injection of an oxytocin receptor antagonist. We hypothesised that endogenous modulation of acute pain is altered postpartum. METHODS: Endogenous inhibition of acute pain in a conditioned pain modulation paradigm or endogenous sensitisation by topical capsaicin was tested in women who were breastfeeding 10-14 days after Caesarean delivery and age-matched controls (n=80 total: 20 per group and 20 per test). The study was powered to detect a difference in area of hyperalgesia after capsaicin of 33%. Capsaicin-evoked pain was recorded in women, and capsaicin-evoked mechanical hypersensitivity was measured in rats 48 h after delivery and in age-matched female and male animals. RESULTS: There was no effect of the postpartum period in the endogenous sensitisation assay in women, and the conditioned pain modulation assay failed to produce analgesia in either group. Postpartum women, however, reported less intense pain than controls at the end of topical capsaicin exposure (1.3 [1.4] vs 2.0 [2.0] on 0-10 verbal scale), and acute hypersensitivity after capsaicin was less in postpartum than control rats (withdrawal threshold 25 [15] vs 3.6 [1] g). CONCLUSIONS: These results agree with a recent report that oxytocin may desensitise the transient receptor potential for vanilloid-1 channel, although other explanations, including hormone effects, are possible. These results do not, however, support the inhibition of capsaicin-evoked spinal sensitisation in the postpartum period. CLINICAL TRIAL REGISTRATION: NCT01843517.

The puerperium alters spinal cord plasticity following peripheral nerve injury.

Tissue and nerve damage can result in chronic pain. Yet, chronic pain after cesarean delivery is remarkably rare in women and hypersensitivity from peripheral nerve injury in rats resolves rapidly if the injury occurs in the puerperium. Little is known regarding the mechanisms of this protection except for a reliance on central nervous system oxytocin signaling. Here we show that the density of inhibitory noradrenergic fibers in the spinal cord is greater when nerve injury is performed in rats during the puerperium, whereas the expression of the excitatory regulators dynorphin A and neuregulin-1 in the spinal cord is reduced. The puerperium did not alter spinal cord microgial and astrocyte activation. Astrocyte activation, as measured by glial fibrillary acidic protein (GFAP) expression, was not evident in female rats with injury, regardless of delivery status suggesting sex differences in spinal astrocyte activation after injury. These results suggest a change in the descending inhibitory/facilitating balance on spinal nociception neurotransmission during the puerperium, as mechanisms for its protective effect against injury-induced hypersensitivity.

Opioid-independent mechanisms supporting offset analgesia and temporal sharpening of nociceptive information.

The mechanisms supporting temporal processing of pain remain poorly understood. To determine the involvement of opioid mechanisms in temporal processing of pain, responses to dynamic noxious thermal stimuli and offset analgesia were assessed after administration of naloxone, a µ-opioid antagonist, and on a separate day, during and after intravenous administration of remifentanil, a µ-opioid agonist, in 19 healthy human volunteers. Multiple end points were sampled from real-time computerized visual analog scale ratings (VAS, 1 to 10) to assess thermal sensitivity, magnitude and duration of offset analgesia, and painful after sensations. It was hypothesized that the magnitude of offset analgesia would be reduced by direct opioid antagonism and during states of acute opioid-induced hypersensitivity (OIH), as well as diminished by the presence of exogenous opioids. Surprisingly, the magnitude of offset analgesia was not altered after naloxone administration, during remifentanil infusion, or after the termination of remifentanil infusion. Because thermal hyperalgesia was observed after both drugs, 8 of the original 19 subjects returned for an additional session without drug administration. Thermal hyperalgesia and increased magnitude of offset analgesia were observed across conditions of remifentanil, naloxone, and no drug within this subset analysis, indicating that repeated heat testing induced thermal hyperalgesia, which potentiated the magnitude of offset analgesia. Thus, it is concluded that the mechanisms subserving temporal processing of nociceptive information are largely opioid-independent, but that offset analgesia may be potentiated by heat-induced thermal hyperalgesia in a proportion of individuals.

Inhibitory M2 muscarinic receptors are upregulated in both axotomized and intact small diameter dorsal root ganglion cells after peripheral nerve injury.

Acetylcholine reduces nociceptive input in part by activating inhibitory M2 muscarinic receptors on primary sensory neurons, and acetylcholinesterase inhibitors and muscarinic agonists produce analgesia in humans and animals. M2 muscarinic receptors are upregulated in animals with diabetic neuropathy, but their level of expression and function after peripheral nerve injury has not been previously examined. This study tested, using intracellular Ca(2+) response to membrane depolarization, the effect of the M2 muscarinic receptor agonist bethanechol on individual dorsal root ganglion cells from normal and L5-6 spinal nerve-ligated rats, followed by M2 muscarinic receptor immunostaining. We also examined functional transient receptor potential for vanilloids-1 activity by determining intracellular Ca(2+) response evoked by capsaicin in M2 muscarinic receptor immunoreactive cells. In normal dorsal root ganglion cells, bethanechol inhibited the Ca(2+) response in a concentration-related fashion, and this inhibition was blocked by the M2 muscarinic receptor antagonist gallamine. Cells expressing M2 muscarinic receptors by immunostaining were significantly inhibited by bethanechol, whereas those lacking positive staining were not. The proportion of studied dorsal root ganglion neurons with positive M2 muscarinic receptor staining increased significantly in the injured ipsilateral L5-6 and the uninjured ipsilateral L4 ganglia, but not in the contralateral dorsal root ganglion neurons compared with normals. In contrast, the proportion of neurons responding to capsaicin significantly decreased in the injured ipsilateral L5-6 dorsal root ganglion cells. These results suggest that inhibitory M2 muscarinic receptors are upregulated in small- and medium-sized axotomized dorsal root ganglion neurons and their uninjured neighbors following nerve injury, and may represent an appropriate target for analgesia in this setting.

alpha2-adrenoceptors inhibit the intracellular Ca2+ response to electrical stimulation in normal and injured sensory neurons, with increased inhibition of calcitonin gene-related peptide expressing neurons after injury.

Nerve injury resulting in chronic pain is associated with novel excitatory effects of norepinephrine on injured peripheral nerve terminals and their cell bodies, due to actions on alpha2-adrenoceptors. Paradoxically, alpha2-adrenoceptor agonists administered near peripheral terminals or their cell bodies results in analgesia, not pain. This study tested, using intracellular Ca2+ response to stimulation, the effects of alpha2-adrenoceptor agonists on injured sensory neurons and classified their neuronal phenotype. Dorsal root ganglion cells from normal and spinal nerve-ligated rats were dissociated and activated twice with electrical field stimulation, while measuring Fura-2 fluorescence. Cells were perfused between stimulations with vehicle or alpha2-adrenoceptor agonists alone or with antagonists. Cells were considered inhibited if the ratio of their peak Ca2+ response to the second stimulus divided by the first was less than the 2.5th percentile for vehicle controls. alpha2-, But not alpha1-adrenoceptor agonists inhibited the Ca2+ response in a concentration related fashion, and this inhibition was blocked by alpha2-adrenoceptor antagonists. Clonidine inhibited a similar percentage of cells in the normal and spinal nerve-ligated group. In both groups, the large majority of clonidine-inhibited cells stained for isolectin B4. Spinal nerve ligation resulted in a 4-10-fold increase in the percentage of clonidine inhibited cells which immunostained for calcitonin gene-related peptide. These data are consistent with the known inhibition of Ca2+ currents by alpha2-adrenoceptors and suggest that, at the level of intracellular Ca2+, the key determinant of neurotransmitter release, alpha2-adrenoceptors are inhibitory after nerve injury, not excitatory. There is a shift in phenotype of sensory neurons which are inhibited by clonidine after nerve injury, which may explain clonidine's increased potency in the treatment of neuropathic compared with acute pain.

Intraplantar injection of a cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance P, calcitonin gene-related peptide, and dynorphin in the dorsal horn of rats with nerve injury or inflammation.

We previously reported that partial sciatic nerve ligation (PSNL) dramatically up-regulates cyclooxygenase 2 (COX2) in injured sciatic nerve, and local injection of the COX inhibitor, ketorolac, reverses tactile allodynia and suppresses increased phosphorylation of the transcription factor cAMP responsive element binding protein [Eur J Neurosci 15 (2002) 1037]. These findings suggest that peripheral prostaglandins (PGs) are over-produced and contribute to the central plasticity and the maintenance of neuropathic pain after nerve injury. PGs, particularly PGE2, are well known to facilitate the release of the pro-nociceptive neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) from primary sensory afferents. Thus, suppressing peripheral PG over-production may inhibit the release of these two neuropeptides from primary afferents and thereby increase the content of these neuropeptides remaining in afferent terminals in the dorsal horn. In this study we tested this hypothesis by examining the immunoreactivities of SP and CGRP in the dorsal horn of PSNL rats intraplantarly injected with saline and ketorolac. Four weeks after PSNL, SP- and CGRP-immunoreactivities (IR) in the ipsilateral dorsal horn were not significantly different from the contralateral side. Five days following intraplantar injection of ketorolac, CGRP- and SP-IR in the ipsilateral and contralateral dorsal horn were dramatically reduced compared with saline-injected PSNL rats. Local ketorolac also suppressed PSNL-induced increase in dynorphin-IR in dorsal horn neurons. Since abundant production of PGs during inflammation is well documented, we further examined the effect of intraplantar ketorolac on neuropeptide expression in the dorsal horn following carrageenan inflammation. We observed that co-administration of ketorolac with carrageenan in the hindpaw also reduced SP- and dynorphin-IR in the ipsilateral and contralateral dorsal horn. These findings are in contrast to our hypothesis, suggesting that peripherally over-produced PGs following nerve injury and inflammation possibly contribute to the production of SP and CGRP in primary sensory neurons, to the up-regulation of dynorphin in the dorsal horn neurons, and finally to the mechanisms of neuropathic and inflammation pain.

Cyclooxygenase 2 in infiltrating inflammatory cells in injured nerve is universally up-regulated following various types of peripheral nerve injury.

We previously reported the up-regulation of cyclooxygenase 2 (COX2) in injured sciatic nerve of rats with partial sciatic nerve ligation (PSNL) and the reversal of PSNL-elicited tactile allodynia by local injection of the COX inhibitor ketorolac [Eur J Neurosci 15 (2002) 1037]. We further asked whether COX2 up-regulation in injured nerve is a universal phenomenon following various types of nerve injury. In the current study, we observed that abundant COX2 immunoreactive (IR) cell profiles appeared in injured nerves of rats following spinal nerve ligation (SNL), chronic constriction injury (CCI) and complete sciatic nerve transection. Most COX2-IR cells were identified as infiltrating macrophages. Partial injury induced greater COX2 up-regulation than complete injury. COX2 up-regulation reached a peak at 2-4 weeks, evidently declined by 3 months and disappeared by 7 months postlesion. These findings suggest that up-regulation of COX2 in injured nerve is a common event during the initial several months after nerve injury. We observed that local ketorolac-elicited anti-allodynia was closely associated with the abundance of COX2-IR cells in injured nerve, varying with the type of injury and time after injury. The anti-allodynia lasted the longest when local ketorolac was given 2-4 weeks after PSNL, CCI and SNL. The duration of local ketorolac's anti-allodynia was the longest in CCI rats, which also exhibited the most abundant COX2 up-regulation. Local ketorolac's anti-allodynia lasted much shorter when given 2-3 months after lesion. Local ketorolac failed to induce anti-allodynia 7 months after lesion, a time when COX2-IR cells completely disappeared from the injured nerve except a few cells at the injury site. Our data strongly suggest that during the initial several months after nerve injury, peripherally over-produced prostaglandins play an important role in the maintenance of neuropathic pain.

Perioperative administration of the alpha2-adrenoceptor agonist clonidine at the site of nerve injury reduces the development of mechanical hypersensitivity and modulates local cytokine expression.

The development of chronic pain after surgery is not rare. Nerve injury from complete or partial nerve section during surgery leads to macrophage recruitment and release of pro-inflammatory cytokines, leading in turn to sensitization. Macrophages also express alpha2-adrenoceptors, and we previously demonstrated a prolonged reduction in hypersensitivity following peri-neural injection of the alpha2-adrenoceptor agonist, clonidine, in rats with chronic nerve injury. The current study tested whether peri-neural clonidine at the time of injury could also prevent development of hypersensitivity. Rats underwent partial ligation of one sciatic nerve, and peri-neural saline, clonidine or a combination of clonidine and the alpha2A-adrenceptor-preferring antagonist, BRL44408, were administered before wound closure and, in some animals, also 24 and 48 h later. The single clonidine injection reduced hypersensitivity for only 5 h, whereas repeated injection for three days reduced hypersensitivity for 28 days. Peri-neural clonidine reduced the increase in tissue content of the proinflammatory cytokines IL-1beta and particularly TNFalpha in sciatic nerve, DRG and spinal cord while increasing concentrations of the anti-inflammatory cytokine TGF-beta1. Clonidine's effects on behavior and TNFalpha content were blocked by BRL44408. We conclude that peri-neural administration of clonidine at the site and time of injury reduces the degree of hypersensitivity in part by altering the balance of pro- and anti-inflammatory cytokines through activation of alpha2A-adrenoceptors. These results support testing of whether clonidine, as an adjuvant in continuous peripheral nerve blocks in settings of known major nerve injury, such as limb amputation, might prevent the development of chronic pain.

Morphine-induced spinal release of adenosine is reduced in neuropathic rats.

BACKGROUND: Spinally administered opioids show decreased potency and efficacy in the treatment of neuropathic pain. As reported previously, morphine stimulates spinal opioid receptors to effect adenosine release, which acts at adenosine receptors to produce analgesia. The authors hypothesized that morphine induces less adenosine release in neuropathic compared with normal rats, explaining its reduced potency and efficacy. METHODS: Sprague-Dawley rats (200-250 g) were divided into three groups: no surgery (n = 52), sham surgery (n = 20), or left L5 and L6 spinal nerve ligation (n = 64). Two weeks after surgery, mechanical hypersensitivity of the left hind paw was verified. For each experiment, a crude synaptosomal P2 suspension was prepared by homogenizing cervical and lumbar dorsal spinal cord halves from four rats, followed by differential centrifugation, and aliquots incubated with morphine sulfate from 10(-8) to 10(-4) m alone or in presence of 10(-5) m dipyridamole. Extrasynaptosomal concentrations of adenosine were analyzed by high-pressure liquid chromatography. RESULTS: Synaptosomal release of adenosine in the absence of morphine was similar between groups. Morphine produced a concentration-dependent adenosine release, which was less in synaptosomes from dorsal lumbar spinal cord in spinal nerve ligation compared with normal or sham animals. This reduction was removed by adding dipyridamole. CONCLUSION: Morphine normally stimulates spinal release of adenosine, a potent antihypersensitivity compound. Because this effect of morphine is diminished in spinal nerve ligation animals, one explanation for decreased efficacy and potency of opioids in the treatment of neuropathic pain may be a dipyridamole-sensitive disruption in the opioid-adenosine link in the spinal cord.

Pharmacology of opioid and nonopioid analgesics in chronic pain states.

Chronic pain represents a mixture of pathophysiologic mechanisms, a complex assortment of spontaneous and elicited pain states, and a somewhat unpredictable response to analgesics. Opioids remain the mainstay of treatment of moderate to severe chronic pain, although there is little systematic examination to guide drug selection. Cyclooxygenase inhibitors play primarily an adjunctive role in chronic pain treatment. Agents with little activity in the treatment of acute pain, such as antidepressants, antiepileptics, and i.v. administered local anesthetics, are initiated in many patients and have significant long-term efficacy in some patients with chronic pain. The N-methyl-D-aspartate antagonist ketamine and the alpha(2)-adrenergic agonist clonidine exhibit activity in patients with acute or chronic pain and reduce opioid consumption, but are often poorly tolerated due to side effects. Topical treatment with capsaicin or lidocaine exhibits efficacy in a subset of patients, and invasive intrathecal treatment with opioids as well as clonidine, neostigmine, and adenosine may have advantages in some patients. Several laboratory models have been developed to mimic chronic pain states found in humans. Nerve injury has been induced in rats by a variety of means, resulting in mechanical allodynia and thermal hyperalgesia. A number of arthritic states have also been produced by means of chronic joint inflammation in rats. The pharmacology of these neuropathic and arthritic pain models generally resembles that found in the respective human conditions. Additional models of chronic pain, particularly visceral pain, have been developed; however, the pharmacology of these models is not well established at this time.

alpha2A-adrenoceptor stimulation reduces capsaicin-induced glutamate release from spinal cord synaptosomes.

Glutamate (Glu) is involved in excitatory neurotransmission and nociception and plays an essential role in relaying noxious stimuli in the spinal cord. Intrathecal or epidural injection of alpha2-adrenergic agonists produces potent antinociceptive effects, alters spinal neurotransmitter release, and effectively treats acute nociceptive and chronic neuropathic pain. Although it is generally believed that alpha2-adrenergic receptor stimulation reduces excitatory neurotransmitter release from peripheral afferents, the subtype of receptor causing this effect and its specificity to nociceptive neurotransmission have been inadequately studied. We therefore examined the pharmacology of adrenergic agents to inhibit Glu release in spinal cord from stimulation with capsaicin, a specific agonist for receptors on nociceptive afferents. Capsaicin evoked Glu release in synaptosomes from normal rat dorsal spinal cord in a concentration-dependent manner. Glu release from 30 microM capsaicin was inhibited by adrenergic agonists with a relative potency of clonidine = dexmedetomidine > norepinephrine > ST91 >> phenylephrine = 0, consistent with an action on alpha2A/D subtype receptors. Also consistent with this interpretation was the observation that inhibition of capsaicin-induced Glu release by clonidine or dexmedetomidine was blocked by the alpha2A/D antagonist BRL44408 but not by the alpha2B/C-preferring antagonist ARC239. Similar results were obtained in perfused spinal cord slices. These data suggest that capsaicin-evoked Glu release, likely reflecting stimulation of C fiber terminals, can be inhibited by activation of the alpha2A/D subtype, and this action of adrenergic agonists may reflect in part their efficacy in the treatment of acute pain.

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 microg, 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 microg of the A1-specific adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. CONCLUSIONS: These results add to previous studies that suggest ongoing spinal release of adenosine, which is antiallodynic, in this animal model of neuropathic pain. Positive allosteric modulation of the adenosine receptor reduces hypersensitivity by a spinal mechanism involving A1 adenosine receptor stimulation. Although obvious adverse effects were not observed in this investigation, further study is required to determine the feasibility of the use of such modulators in the treatment of chronic pain associated with hyperalgesia and allodynia.

An obligatory role for spinal cholinergic neurons in the antiallodynic effects of clonidine after peripheral nerve injury.

BACKGROUND: Indirect evidence supports a role of spinal cholinergic neurons in tonically reducing response to noxious mechanical stimulation and in effecting analgesia from alpha2-adrenergic agonists. This study directly assessed the role of cholinergic neurons in regulating the level of mechanical allodynia and in participating in the antiallodynic effect of the clinically used alpha2-adrenergic agonist, clonidine, in an animal model of neuropathic pain. METHODS: Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Rats received a single intrathecal injection of saline or one of three different doses of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64-A; 2, 5, and 15 nmol). Seven days later, allodynia was assessed before and after intrathecal injection of 15 microg clonidine. The spinal cord was removed, and spinal cord acetylcholine content, cholinergic neuron number and distribution, and alpha2-adrenergic receptor expression were determined. RESULTS: AF64-A administration reduced both the number of cholinergic cells and the acetylcholine content of the lumbar dorsal spinal cord by 20-50% but did not affect level of mechanical allodynia. AF64-A did, however, completely block the anti-allodynic effect of clonidine. AF64-A did not reduce alpha2-adrenergic ligand binding in dorsal lumbar cord. CONCLUSIONS: These data suggest that spinal cholinergic tone does not affect the level of mechanical allodynia after peripheral nerve injury. There is a quantitative reliance on spinal cholinergic neurons in the allodynia relieving properties of intrathecal clonidine, and this reliance does not depend on alpha2-adrenergic receptors colocalized on spinal cholinergic interneurons.

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Morphine-induced spinal cholinergic activation: in vivo imaging with positron emission tomography.

Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.

P75-expressing elements are necessary for anti-allodynic effects of spinal clonidine and neostigmine.

Cells expressing nerve growth factor are implicated in development of hypersensitivity following nerve injury and cholinergic neurons are implicated in reduction of such hypersensitivity by alpha2-adrenergic agonists. Intrathecal injection of the cell toxin, saporin, linked to an antibody to the low-affinity nerve growth factor, p75 (192-IgG saporin), an agent which destroys cholinergic neurons in the brain, was used in the current study to further elucidate these mechanisms. Mechanical hypersensitivity was established in rats by ligation of the L5 and L6 spinal nerves. Animals were pretreated with intrathecal saline or 192-IgG saporin, and one week later received intrathecal clonidine or neostigmine. Spinal cords were removed for acetylcholine and norepinephrine analysis and for cholinergic and p75 immunohistochemistry. Treatment with 192-IgG saporin had no effect on mechanical hypersensitivity following spinal nerve ligation, but blocked the anti-hypersensitivity effects of intrathecal clonidine and neostigmine. Destruction of p75-expressing fibers in the superficial dorsal horn by 192-IgG saporin was not accompanied by changes in acetylcholine or norepinephrine content or by reduction in cholinergic neuronal number in the spinal cord dorsal horn. Unlike in the brain, 192-IgG saporin does not destroy cholinergic neurons in the spinal cord dorsal horn and cannot be used as a tool for this purpose. P75-expressing elements are not necessary for the maintenance of mechanical hyperalgesia in this model of neuropathic pain, but their destruction disrupts the targets or circuitry activated by alpha2-adrenergic and cholinergic agents to reduce hypersensitivity.

Intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine attenuate nerve injury-induced allodynia through noradrenergic activation in rats.

Spinal norepinephrine release and activation of spinal alpha(2)-adrenergic receptors represent important components of descending control of nociception. Recent studies have shown that nitric oxide is capable of stimulating neuronal norepinephrine release in the presence of thiol-containing compounds such as L-cysteine. In the present study, we tested a hypothesis in a rodent model of neuropathic pain that intrathecal injection of the nitric oxide donor S-nitroso-N-acetylpenicillamine and L-cysteine produces an antiallodynic action mediated by the spinal alpha(2)-adrenergic receptors. Allodynia was induced in rats by ligation of the left lumbar L5/L6 spinal nerves. Mechanical allodynia was quantified by application of von Frey filaments to the left hindpaw. Intrathecal injection of 20-100microg of S-nitroso-N-acetylpenicillamine in the presence of 200microg of L-cysteine, but not D-cysteine, dose-dependently attenuated the allodynia. Intrathecal injection of a combination of 100microg of S-nitroso-N-acetylpenicillamine and 50-200microg of L-cysteine also inhibited the allodynia in a dose-dependent manner. Pretreatment with a nitric oxide scavenger, carboxy-PTIO, or depletion of norepinephrine with a specific neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, prevented the antiallodynic action of intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine. Furthermore, the antiallodynic effect produced by intrathecal injection of a combination of S-nitroso-N-acetylpenicillamine and L-cysteine was abolished by pretreatment with intrathecal injection of a non-specific alpha-adrenergic receptor antagonist, phentolamine, or an alpha(2) receptor antagonist, idazoxan. This study provides the first functional evidence that spinal nitric oxide interacts with the thiol-containing compounds to produce an antiallodynic effect in neuropathic pain. We propose that such an action is mediated by endogenous norepinephrine and spinal alpha(2)-adrenergic receptors.

Formation of 6-nitro-norepinephrine from nitric oxide and norepinephrine in the spinal cord and its role in spinal analgesia.

Spinally released norepinephrine is thought to produce analgesia in part by stimulating alpha(2)-adrenergic receptors, which in turn leads to nitric oxide synthesis. Also, nitric oxide is known to react with norepinephrine in vivo in the brain to form 6-nitro-norepinephrine, which inhibits neuronal norepinephrine reuptake. In the present study, we tested the hypothesis that formation of 6-nitro-norepinephrine occurs in the spinal cord and that intrathecal administration of 6-nitro-norepinephrine produces analgesia by stimulating norepinephrine release. 6-Nitro-norepinephrine was present in rat spinal cord tissue and microdialysates of the dorsal horn and intrathecal space. Intrathecal norepinephrine injection increased 6-nitro-norepinephrine. 6-Nitro-norepinephrine also stimulated norepinephrine release in dorsal spinal cord in vitro. Intrathecal injection of 6-nitro-norepinephrine produced antinociception and interacted additively with norepinephrine for antinociception. Spinal noradrenergic nerve destruction increased antinociception from intrathecally injected norepinephrine, but decreased antinociception from 6-nitro-norepinephrine. These results suggest a functional interaction between spinal nitric oxide and norepinephrine in analgesia, mediated in part by formation of 6-nitro-norepinephrine. Stimulation of auto-inhibitory alpha(2)-adrenergic receptors at noradrenergic synapses decreases norepinephrine release. Paradoxically, alpha(2)-adrenergic agonist injection increases and alpha(2)-adrenergic antagonist injection decreases norepinephrine release in the spinal cord. 6-Nitro-norepinephrine may be an important regulator of spinal norepinephrine release and could explain the positive feedback on norepinephrine release after activation of spinal alpha(2)-adrenergic receptors.