Persistent Nociception Research Articles

Involvement of peripheral NMDA and non-NMDA receptors in development of persistent firing of spinal wide-dynamic-range neurons induced by subcutaneous bee venom injection in the cat

To study the roles of peripheral excitatory amino acids receptor subtypes N-methyl- d-aspartate (NMDA) and non-NMDA receptors in persistent nociception, extracellular single unit recording technique was used to assess the effects of a single dose NMDA and non-NMDA receptor antagonists, AP 5 (5-aminophosphonovaleric acid) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) or DNQX (6,7-dinitroquinoxaline-2,3-dione), on s.c. bee venom-induced increase in firing of wide-dynamic-range (WDR) neurons in the spinal dorsal horn of the urethane–chloralose anesthetized cats. Subcutaneous bee venom injection into the cutaneous receptive field resulted in a single phase of increased firing of WDR neurons over the background activity for more than 1 h. Local pre-administration of AP 5 (200 μg/100 μl) or CNQX (8.3 μg/100 μl) into the bee venom injection site produced 94% (1.01±0.96 spikes/s, n=5) or 76% (2.97±0.58 spikes/s, n=4) suppression of the increased neuronal firing when compared with local saline (16.32±4.55 spikes/s, n=10) or dimethyl sulfoxide (DMSO) (12.37±6.36 spikes/s, n=4) pre-treated group, respectively. Local post-administration of the same dose of AP 5 produced a similar result to the pre-treatment group with a 67% inhibition of the mean firing rate, however, the same treatment with CNQX and even a higher dose of DNQX (100 μg/100 μl) did not produce any inhibition of the neuronal firing induced by s.c. bee venom injection (DNQX vs. DMSO: 23.91±0.25 vs. 22.14±0.04 spikes/s, P=0.0298, n=5). In the control experiments, local pre-administration of the same dose of AP 5 or CNQX into a region on the contralateral hindpaw symmetrical to the bee venom injection site produced no significant influence on the increased firing of the WDR neurons [contralateral AP 5 vs. saline: 14.17±6.27 spikes/s ( n=5) vs. 16.32±4.55 spikes/s ( n=10), P>0.05; contralateral CNQX vs. DMSO: 12.85±6.38 spikes/s ( n=4) vs. 12.37±6.36 spikes/s ( n=4), P>0.05], implicating that the suppressive action of local AP 5 or CNQX was not the result of systemic effects. The present results suggest that activation of the peripheral NMDA receptors is involved in both induction and maintenance, while activation of non-NMDA receptors is only involved in induction, but not in the maintenance of persistent firing of the dorsal horn WDR neurons induced by s.c. bee venom injection.

Differential effects of neurotoxic destruction of descending noradrenergic pathways on acute and persistent nociceptive processing

Although many pharmacological studies indicate that bulbospinal noradrenergic projections contribute to antinociception, lesions of the major brainstem noradrenergic cell groups have provided conflicting evidence. Here we used a new immunotoxin, anti-dopamine β-hydroxylase-saporin, to re-examine the contribution of noradrenergic pathways to nociception and to morphine analgesia. We treated rats intrathecally by lumbar puncture with the immunotoxin and examined dopamine β-hydroxylase (D βH) immunoreactivity seven and 14 days after treatment. There was no change in D βH staining at 7 days; however, 14 days after treatment we demonstrated significant destruction of noradrenergic neurons in the locus coeruleus and in the A5 and A7 cell groups. There was a concomitant loss of noradrenergic axons in the dorsal and ventral horns of the lumbosacral and cervical cord. Consistent with the lack of anatomical changes, we found no difference in nociceptive responses in the hot-plate, tail-flick or formalin tests one week post-toxin. On day 14 we examined the behavioral response to injection of formalin into the hindpaw and found that responses during the second phase of pain behavior were significantly reduced. There was no change during the first phase. Formalin-evoked fos expression in the spinal cord was also reduced. We also evaluated morphine analgesia in the formalin test and found that toxin-treated animals exhibited enhanced morphine analgesia. These results establish the utility of using this immunotoxin to selectively destroy subpopulations of noradrenergic cell groups and provide evidence that acute and persistent nociception are differentially regulated by descending noradrenergic pathways.

Inflammation-induced up-regulation of protein kinase C γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing

Activation of various second messengers contributes to long-term changes in the excitability of dorsal horn neurons and to persistent pain conditions produced by injury. Here, we compared the time-course of decreased mechanical nociceptive thresholds and the density of protein kinase C γ immunoreactivity in the dorsal horn after injections of complete Freund's adjuvant in the plantar surface of the rat hindpaw. Complete Freund's adjuvant significantly increased paw diameter and mechanical sensitivity ipsilateral to the inflammation. The changes peaked one day post-injury, but endured at least two weeks. In these rats, we recorded a 75–100% increase in protein kinase C γ immunoreactivity in the ipsilateral superficial dorsal horn of the L4 and L5 segments at all time-points. Electron microscopy revealed that the up-regulation was associated with a significant translocation of protein kinase C γ immunoreactivity to the plasma membrane. In double-label cytochemical studies, we found that about 20% of the protein kinase C γ-immunoreactive neurons, which are concentrated in inner lamina II, contain glutamate decarboxylase-67 messenger RNA, but none stain for parvalbumin or nitric oxide synthase. These results indicate that persistent changes in protein kinase C γ immunoreactivity parallel the time-course of mechanical allodynia and suggest that protein kinase C γ contributes to the maintenance of the allodynia produced by peripheral inflammation. The minimal expression of protein kinase C γ in presumed inhibitory neurons suggests that protein kinase C γ-mediated regulation of excitatory interneurons underlies the changes in spinal cord activity during persistent nociception.

Mechanisms of chronicity?

Most of us experience acute episodes of back pain. Clinical evaluation helps us segregate patients into those with simple backache, nerve root pain and possible serious spinal pathology. The vast majority are in the first group. The prognosis of simple backache is good, with most being better within a couple of weeks and 90% better within 6 weeks. However, a small proportion have persistent problems and it is these which require difficult management decisions and lead to the medical and societal costs of backache. This article focuses on the reasons why simple backache may fail to resolve and some patients develop chronic persistent problems. In this context, I will restrict myself to mechanical disorders and not deal with inflammatory conditions such as ankylosing spondylitis, metabolic bone disease or neoplasms. Some patients have persistent nociception, e.g. there may be a chronic herniated disc, a spondylolisthesis or spinal stenosis syndrome. There are specific and well identified clinical features enabling us to recognise these conditions. The vast majority have so-called non-specific back pain. They may show pain and tenderness over the facet joints or the sacroiliac joints. However, there is no clear evidence that pathology in

The differential contribution of capsaicin-sensitive afferents to behavioral and cardiovascular measures of brief and persistent nociception and to Fos expression in the formalin test

Intraplantar injection of dilute formalin evokes brief (Phase 1) and persistent (Phase 2) increases in primary afferent activity, pain behavior, and cardiovascular responses, and induces spinal cord Fos-like immunoreactivity (Fos-LI). Although previous studies demonstrated that the destruction of small diameter primary afferents with neonatal capsaicin treatment decrease formalin-evoked nociception, these studies only evaluated behavioral responses, and did not distinguish between Phase 1 and 2. To address these questions, we simultaneously evaluated formalin-evoked pain behavior (flinching of the afflicted paw), cardiovascular responses (heart rate and mean arterial pressure), and lumbar spinal cord Fos expression in control rats and in rats treated with capsaicin (100 mg/kg) one day postpartum. We found that neonatal capsaicin-treated rats, compared to controls, exhibited similar cardiovascular res and slightly less flinching behavior during Phase 1. During Phase 2, however, capsaicin-treated rats exhibited 59% less flinching and 45% smaller heart rate responses. Also, in capsaicin-treated rats, we counted 59% fewer Fos-labeled neurons in the spinal cord. These results indicate that capsaicin-sensitive afferents contribute to formalin-evoked behavioral and cardiovascular responses and to spinal cord neuronal responses. The differential effect of neonatal capsaicin on nociception during Phase 1 and Phase 2 suggests that sensitization mechanisms during Phase 1 do not contribute to the magnitude of nociceptive responses during Phase 2.

Comparison of nociceptive effects produced by intrathecal administration of mGluR agonists.

The present study examined the mGluR subtypes involved in (1S, 3R)-ACPD-induced spontaneous nociceptive behaviours (SNB) by administering the following selective agonists by the intrathecal (i.t.) route: (RS)-DHPG, trans-ADA (Group I; mGluR1/5 and mGluR5, respectively), (1S, 3S)-ACPD, (2R, 4R)-APDC (Group II), and L-AP4 (Group III). (RS)-DHPG administration induced SNB that were of significantly greater intensity and longer duration than those induced by an equal dose of (1S, 3R)-ACPD. No other agonists produced SNB, except (1S, 3S)-ACPD, which may be attributable to a nonselective action at mGluR1. Intrathecal treatment with the mGluR antagonist (+)-MCPG or the NMDA antagonist D-AP5 prior to (RS)-DHPG administration dose-dependently reduced SNB. It is suggested that a possible interaction between NMDA and mGluR1 is a critical event in the maintenance of persistent nociception.

Dual effect of serotonin on formalin-induced nociception in the rat spinal cord

To examine the role of the descending serotonergic system in the regulation of spinal nociceptive processing, the effects of serotonin (5-HT) and selective ligands for 5-HT receptor subtypes on persistent nociception were investigated. Formalin (5% formaldehyde) injected into the plantar region of the rat hindpaw induced two phases of aversive responses such as licking and biting. Intrathecal administration of selective 5-HT 3 receptor antagonists, granisetron (0.1–100 pmol/rat) and ondansetron (1–1000 pmol/rat), reduced the second phase of the formalin-induced aversive responses without affecting the first one. The antinociceptive effect of granisetron (100 pmol/rat) was abolished when 5-HT was depleted from the lumbar cord by pretreatment with 5,7-dihydroxytryptamine (5,7-DHT). In the 5,7-DHT-treated rats, intrathecal administration of 1-(m-chlorophenyl)-biguanide, a selective 5-HT 3 receptor agonist, facilitated the aversive responses in the second phase whereas that of 8-OH-DPAT, a selective 5-HT 1A receptor agonist, suppressed them. Intrathecal administration of 5-HT showed a dual effect on the second phase of the aversive responses in the 5,7-DHT-treated rats; 5-HT inhibited the aversive responses when administered at a low dose (0.1 nmol/rat) but facilitated them at a high dose (1 nmol/rat). In addition, the inhibitory and facilitatory effects of intrathecal 5-HT were blocked by its co-administration with NAN190, a 5-HT 1A receptor antagonist, and granisetron, respectively. These results suggest that 5-HT suppresses formalin-induced nociception in the spinal cord via the 5-HT 1A receptor and facilitates it via the 5-HT 3 receptor.

Altered c-fos expression in the parabrachial nucleus in a rodent model of CFA-induced peripheral inflammation.

Increases in the expression of immediate early genes have been shown to occur in the lumbar spinal cord dorsal horn after peripheral inflammation. Given that the pontine parabrachial nucleus has been implicated in nociceptive as well as antinociceptive processes and is reciprocally connected with the spinal cord dorsal horn, it seems likely that peripheral inflammation will cause alterations in immediate early gene expression in this nucleus. To test this hypothesis we examined cFos-like immunoreactivity in a rodent complete Freund's adjuvant-induced peripheral inflammatory model of persistent nociception. Unilateral hind paw injections of complete Freund's adjuvant produced inflammation, hyperalgesia of the affected limb, and alterations in open field behaviors. Immunocytochemical analysis demonstrated a bilateral increase in cFos-like immunoreactivity in the lateral and Kolliker-Fuse subdivisions of the parabrachial nucleus at 6 and 24 hours postinjection and an ipsilateral decrease below basal levels in the Kolliker-Fuse subdivision at 96 hours postinjection when compared to saline controls. Taken together, these results suggest that select parabrachial neurons are activated by noxious somatic inflammation. These active parabrachial neurons are likely to participate in ascending nociceptive and/or descending antinociceptive pathways.

Basal release of Met-enkephalin and neurotensin in the ventrolateral periaqueductal gray matter of the rat: a microdialysis study of antinociceptive circuits

The periaqueductal gray (PAG) contains neural circuits that participate in descending antinociception. Anatomical and electrophysiological evidence suggests that these circuits might employ opioid peptides and GABA in series to remove a tonic inhibition of descending PAG output neurons. The present studies examined the release of the antinociceptive peptides Met-enkephalin and neurotensin in the ventrolateral PAG, and investigated the interaction between GABA and Met-enkephalin release. In awake and freely moving rats the ventrolateral PAG was dialysed using 25 ga. concentric probes. Basal release of peptide in 12 min or 40 min fractions was determined using radioimmunoassays. To establish how the ventrolateral PAG responds to nociception, dialysis was performed following unilateral hindpaw inflammation using Complete Freund's Adjuvant. Twenty-four hours after inflammation was induced, neurotensin release was increased 133% and Met-enkephalin release was increased 353% compared to control animals. Seven days after inflammation was induced, neurotensin release declined precipitously, while basal Met-enkephalin release remained elevated 313% above controls. Thus, unlike enkephalin, increased basal neurotensin release is not sustained with persistent tonic nociception. In addition, we confirmed in normal animals that the ventrolateral PAG is induced to release Met-enkephalin by systemic morphine. A 43% increase in basal Met-enkephalin release was observed immediately following a 12 mg/kg i.p. morphine injection. Morphine should have the opposite effect (inhibit peptide release) if it acts directly on the enkephalinergic neurons. Thus, we examined the hypothesis that GABAergic interneurons in the PAG mediated morphine-stimulated enkephalin release. When the GABAantagonist bicuculline (0.25 μM to 25 μM) was co-infused with the dialysis medium, Met-enkephalin release increased in a dose-dependent fashion and peaked 68% above pre-infusion levels. These data elucidate the reciprocal inhibitory relationship between GABA and enkephalin in the ventrolateral PAG. We hypothesize that, when nociception induces Met-enkephalin release within this region, the tonic GABAergic inhibition is overcome, resulting in greater sensitivity of PAG enkephalinergic neurons. Ultimately, this enhanced enkephalin release should result in greater excitability of the descending PAG output neurons that are responsible for antinociception.

Conditioned place preference paradigm: a novel approach for analgesic drug assessment against chronic pain

In response to concerns over the clinical relevance of analgesic testing paradigms which involve acute nociceptive stimuli, the present research examined the utility of the conditioned place preference (CPP) paradigm as a novel approach for determination of analgesic drug efficacy against chronic nociception. Rats display preferences for environments that have been previously paired with positively reinforcing drugs; whether place preference to the negatively reinforcing effects of analgesic drugs in an animal model of chronic pain occurs is yet unknown. The present research sought to determine whether animals experiencing chronic pain would display a place preference for an environment paired with analgesic drug treatment. Persistent inflammatory nociception was induced by unilateral injections of complete Freund's adjuvant (0.1 ml) into the rat hind paw. Place preference to the opiate agonist morphine, the N- methyl- d-aspartate (NMDA) receptor antagonist MK-801 and the non-steroidal anti-inflammatory drug (NSAID) indomethacin was examined in 3 separate experiments. Rats received 8 counterbalanced conditioning trials (4 drug, 4 no-drug) of 60 min each with various drug doses (morphine: 3.0 and 10.0 mg/kg; indomethacin: 2.5 and 5.0 mg/kg; MK-801: 0.03, 0.1 and 0.3 mg/kg i.p.) or vehicle serving as the reinforcing stimuli in a 3 compartment (2 stimuli, 1 neutral) place preference apparatus. In general, morphine place preference was observed in both inflamed and non-inflamed groups; inflamed groups exhibited enhanced morphine place preference than non-inflamed groups. MK-801 produced a low-dose place preference in inflamed animals; higher doses of MK-801 produced a place aversion in both inflamed and non-inflamed groups. Indomethacin failed to produced place preference in either inflamed or non-inflamed groups. These data demonstrate that the negatively reinforcing properties of analgesic drugs can be assessed via the CPP paradigm. In addition, this paradigm offers greater clinical relevance as animals determine drug efficacy without the involvement of high-intensity, phasic nociceptive stimulation.

Intracellular messengers contributing to persistent nociception and hyperalgesia induced by L-glutamate and substance P in the rat formalin pain model.

The contribution of the intracellular messengers nitric oxide, arachidonic acid and protein kinase C to persistent nociception in response to tissue injury in rats was examined following the subcutaneous injection of formalin into the hindpaw. Formalin injury-induced nociceptive behaviours were reduced by intrathecal pretreatment with inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester, L-NAME), arachidonic acid (dexamethasone) or protein kinase C [protein kinase C (19-26) and 1-95-(isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride, H-7]. Each of these agents affected the tonic, but not the acute, phase of the formalin response. Furthermore, none of these agents affected mechanical or thermal flexion reflex thresholds in rats not injected with formalin. Conversely, formalin-induced nociceptive responses were enhanced by stimulators of nitric oxide (sodium nitroprusside), arachidonic acid metabolism (arachidonic acid) or protein kinase C [(+/-)-1-oleoyl-2-acetyl-glycerol], and were slightly reduced by inositol trisphosphate. Mechanical flexion reflexes were also reduced by arachidonic acid, while thermal flexion reflexes were reduced after treatment with sodium nitroprusside, arachidonic acid or [(+/-)-1-oleoyl-2-acetyl-glycerol]. The enhancement of formalin nociceptive behaviours (hyperalgesia) in rats treated with L-glutamate or substance P was reversed by pretreatment with inhibitors of nitric oxide (L-NAME), arachidonic acid (dexamethasone) or protein kinase C (H-7). The results suggest that central sensitization and persistent nociception following formalin-induced tissue injury, and the hyperalgesia in the formalin test induced by L-glutamate and substance P, are dependent on the intracellular messengers nitric oxide, arachidonic acid and protein kinase C.

Specific contribution of lumbar spinal mechanisms to persistent nociceptive responses in the formalin test

To determine whether a lumbar spinal mechanism mediates the late phase of the nociceptive response in the formalin test, we compared the effects of intrathecal (i.t.) lidocaine given pre- or post-formalin at the lumbar or thoracic level. Pre- but not post-formalin administration of lidocaine to the lumbar level significantly attenuated late phase nociceptive responses. In contrast, neither pre- nor post-formalin administration of lidocaine to the mid-thoracic level altered nociceptive responses. In rats chronically spinalized at the mid-thoracic level, nociceptive responses to formalin were observed which were blocked by i.t. pre-treatment with lidocaine at the lumbar level. These results suggest that a specific lumbar spinal mechanism is involved in the initiation of persistent nociception in the formalin test.

Specific contribution of lumbar spinal mechanisms to persistent nociceptive responses in the formalin test

To determine whether a lumbar spinal mechanism mediates the late phase of the nociceptive response in the formalin test, we compared the effects of intrathecal (i.t.) lidocaine given pre- or post-formalin at the lumbar or thoracic level. Pre- but not post-formalin administration of lidocaine to the lumbar level significantly attenuated late phase nociceptive responses. In contrast, neither pre- nor post-formalin administration of lidocaine to the mid-thoracic level altered nociceptive responses. In rats chronically spinalized at the mid-thoracic level, nociceptive responses to formalin were observed which were blocked by i.t. pre-treatment with lidocaine at the lumbar level. These results suggest that a specific lumbar spinal mechanism is involved in the initiation of persistent nociception in the formalin test.

The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats.

Increased pain sensitivity (hyperalgesia) and persistent nociception following peripheral tissue injury depends both on an increase in the sensitivity of primary afferent nociceptors at the site of injury (peripheral sensitization), and on an increase in the excitability of neurons in the central nervous system (central sensitization). We will review evidence that central sensitization, and the persistent nociception it leads to, are dependent on an action of glutamate and aspartate at excitatory amino acid (EAA) receptors. Additional evidence will be presented implicating a role of various intracellular second messengers that are coupled to EAA receptors (nitric oxide, arachidonic acid, and protein kinase C) to central sensitization and persistent nociception following tissue injury. Finally, we will examine the evidence for a contribution of molecular events, including noxious stimulus-induced expression of immediate-early genes such as c-fos to persistent nociception.

The contribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury

The contribution of excitatory amino acids (EAAs) to the development of central sensitization and persistent nociception in response to tissue injury in rats was examined following the subcutaneous injection of formalin into the hindpaw. Formalin-induced nociceptive behaviors were enhanced by intrathecal pretreatment with the EAAs L-glutamate and L-aspartate. An enhancement of the formalin nociceptive response was also produced by intrathecal pretreatment with the receptor-selective EAA agonists NMDA and trans-(+/- )-1-amino-1,3-cyclopentane dicarboxylic acid (ACPD), but not (R,S)-alpha-amino-3-hydroxy-5-methylisozazole-4-propionic acid hydrobromide (AMPA). The effect of NMDA was enhanced by a combined administration with AMPA or APCD. Formalin nociceptive responses were dose-dependently reduced by intrathecal pretreatment with the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid (APV) and (+)-MK-801 hydrogen maleate, but not the selective AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione or the selective metabotropic EAA receptor antagonist 2-amino-3-phosphonopropionic acid. The results suggest that EAAs acting at the NMDA receptor contribute to central sensitization and persistent nociception following subcutaneous formalin injection.

The role of NMDA receptor-operated calcium channels in persistent nociception after formalin-induced tissue injury

The contribution of intracellular calcium to central sensitization and persistent nociception in response to tissue injury in rats was examined following the subcutaneous injection of formalin into the hindpaw. Formalin injury-induced nociceptive behaviors were enhanced by intrathecal pretreatment with the calcium ionophore A23187 or the calcium channel agonist Bay-K8644. Conversely, formalin nociceptive responses were reduced by intrathecal pretreatment with the calcium chelator Quin 2 or the calcium channel antagonists verapamil and nifedipine. Each of these agents affected the tonic, but not the acute, phase of the formalin response. The enhancement in formalin nociceptive behavior in rats treated with L-aspartate or L-glutamate was reversed by combined pretreatment with the noncompetitive NMDA antagonist MK-801, but not by nifedipine or the non-NMDA excitatory amino acid antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione. In rats not treated with excitatory amino acids, the analgesic effect of MK-801 was also significantly greater than that produced by nifedipine. Furthermore, combining nifedipine with MK-801 did not produce a significantly greater analgesic effect than MK-801 alone. The results suggest that central sensitization and persistent nociception following formalin-induced tissue injury are dependent on the influx of calcium through predominantly NMDA receptor-operated (and to a lesser extent voltage-gated) calcium channels.

Contribution of protein kinase C to central sensitization and persistent pain following tissue injury.

This paper provides evidence that central sensitization and persistent nociception following formalin-induced tissue injury in rats is dependent on the production of protein kinase C. Persistent nociceptive behavior in rats induced by subcutaneous formalin injection was significantly reduced by intrathecal pretreatment with a phospholipase C inhibitor (neomycin), and an inhibitor of protein kinase C (W-7), and was significantly enhanced by a phorbol ester (phorbol 12-myristate 13-acetate, PMA) and a stimulator of protein kinase C (SC-10). It is expected that noxious inputs associated with tissue injury produce a release of aspartate and glutamate within the spinal dorsal horn which by acting at ionotropic (NMDA) and metabotropic excitatory amino acid receptors produce an increase in intracellular messengers such as calcium and diacylglycerol which stimulate protein kinase C.