Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors.

In vivo analysis of protein function in nociceptor subpopulations using antisense oligonucleotides and short interfering RNAs is limited by their non-selective cellular uptake. To address the need for selective transfection methods, we covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors. Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA were found to have: a) less PKCε in dorsal root ganglia (DRG), b) reduced PKCε expression in IB4(+) but not IB4(-) DRG neurons, and c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)-nociceptors. These results establish that IB4-streptavidin can be used to study protein function in a defined subpopulation of nociceptive C-fiber afferents.

The symptom phenotype of oncology outpatients remains relatively stable from prior to through 1 week following chemotherapy.

Some oncology outpatients experience a higher number of and more severe symptoms during chemotherapy (CTX). However, little is known about whether this high risk phenotype persists over time. Latent transition analysis (LTA) was used to examine the probability that patients remained in the same symptom class when assessed prior to the administration of and following their next dose of CTX. For the patients whose class membership remained consistent, differences in demographic and clinical characteristics, and quality of life (QOL) were evaluated. The Memorial Symptom Assessment Scale (MSAS) was used to evaluate symptom burden. LTA was used to identify subgroups of patients with distinct symptom experiences based on the occurrence of the MSAS symptoms. Of the 906 patients evaluated, 83.9% were classified in the same symptom occurrence class at both assessments. Of these 760 patients, 25.0% were classified as Low-Low, 44.1% as Moderate-Moderate and 30.9% as High-High. Compared to the Low-Low class, the other two classes were younger, more likely to be women and to report child care responsibilities, and had a lower functional status and a higher comorbidity scores. The two higher classes reported lower QOL scores. The use of LTA could assist clinicians to identify higher risk patients and initiate more aggressive interventions.

Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation.

Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat.

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine.

Antihyperalgesic effect of tetrodotoxin in rat models of persistent muscle pain.

Persistent muscle pain is a common and disabling symptom for which available treatments have limited efficacy. Since tetrodotoxin (TTX) displays a marked antinociceptive effect in models of persistent cutaneous pain, we tested its local antinociceptive effect in rat models of muscle pain induced by inflammation, ergonomic injury and chemotherapy-induced neuropathy. While local injection of TTX (0.03-1 µg) into the gastrocnemius muscle did not affect the mechanical nociceptive threshold in naïve rats, exposure to the inflammogen carrageenan produced a marked muscle mechanical hyperalgesia, which was dose-dependently inhibited by TTX. This antihyperalgesic effect was still significant at 24h. TTX also displayed a robust antinociceptive effect on eccentric exercise-induced mechanical hyperalgesia in the gastrocnemius muscle, a model of ergonomic pain. Finally, TTX produced a small but significant inhibition of neuropathic muscle pain induced by systemic administration of the cancer chemotherapeutic agent oxaliplatin. These results indicate that TTX-sensitive sodium currents in nociceptors play a central role in diverse states of skeletal muscle nociceptive sensitization, supporting the suggestion that therapeutic interventions based on TTX may prove useful in the treatment of muscle pain.

Homocysteine-induced attenuation of vascular endothelium-dependent hyperalgesia in the rat.

We have recently demonstrated a role of the vascular endothelium in peripheral pain mechanism by disrupting endothelial cell function using intravascular administration of octoxynol-9, a non-selective membrane active agent. As an independent test of the role of endothelial cells in pain mechanisms, we evaluated the effect of homocysteine, an agent that damages endothelial cell function. Mechanical stimulus-induced enhancement of endothelin-1 hyperalgesia in the gastrocnemius muscle of the rat was first prevented then enhanced by intravenous administration of homocysteine, but was only inhibited by its precursor, methionine. Both homocysteine and methionine significantly attenuated mechanical hyperalgesia in two models of ergonomic muscle pain, induced by exposure to vibration, and by eccentric exercise, and cutaneous mechanical hyperalgesia in an ischemia-reperfusion injury model of Complex Regional Pain Syndrome type I, all previously shown responsive to octoxynol-9. This study provides independent support for a role of the endothelial cell in pain syndromes thought to have a vascular basis, and suggests that substances that are endothelial cell toxins can enhance vascular pain.

Impact of surgical excision of lesions on pain in a rat model of endometriosis.

BACKGROUND: Chronic pain is the most common and disabling feature of endometriosis. Surgical excision of endometriosis lesions provides relief but pain relapse is common. Studies in a preclinical model of endometriosis might help to unravel the role of the ectopic lesions as the source of pain. Thus, we evaluated the impact of lesion excision on mechanical hyperalgesia in a preclinical model of endometriosis pain. METHODS: Endometriosis was induced by implanting autologous uterine tissue onto the gastrocnemius muscle. Surgical excision or aspiration drainage of the cystic lesion was performed at different times post-implant and mechanical nociceptive thresholds were assessed at the site of the lesion. RESULTS: Lesions at 2, 8 and 16 weeks post-implant produced mechanical hyperalgesia of similar magnitude (n = 6/group). Excision of lesions (n = 6/group) produced a longer inhibition, with a magnitude and time course depending upon the timing of excision. Excision at 2 and 8 weeks produced a rapid onset marked attenuation of hyperalgesia, which returned to pre-excision values by post-surgical week 3. In contrast, excision of the lesion at 16 weeks produced a peak of inhibition of hyperalgesia 2 weeks post-excision, but then the inhibition was sustained. Aspiration of fluid from cysts in the lesions briefly attenuated mechanical hyperalgesia (n = 6/group). CONCLUSIONS: In this preclinical model, we demonstrate that endometriosis pain is alleviated by surgical excision of the ectopic lesion or drainage of its cysts, providing support for the clinical observation that endometriosis pain is dependent upon the ongoing presence of the lesions.

Automated identification of social interaction criteria in Drosophila melanogaster.

The study of social behaviour within groups has relied on fixed definitions of an 'interaction'. Criteria used in these definitions often involve a subjectively defined cut-off value for proximity, orientation and time (e.g. courtship, aggression and social interaction networks) and the same numerical values for these criteria are applied to all of the treatment groups within an experiment. One universal definition of an interaction could misidentify interactions within groups that differ in life histories, study treatments and/or genetic mutations. Here, we present an automated method for determining the values of interaction criteria using a pre-defined rule set rather than pre-defined values. We use this approach and show changing social behaviours in different manipulations of Drosophila melanogaster. We also show that chemosensory cues are an important modality of social spacing and interaction. This method will allow a more robust analysis of the properties of interacting groups, while helping us understand how specific groups regulate their social interaction space.

Emerging pathogen in immunocompromised hosts: Exophiala dermatitidis mycosis in graft-versus-host disease.

Infection with the dematiaceous environmental fungus Exophiala, an emerging pathogen in immunocompromised individuals, poses a diagnostic and therapeutic challenge. Herein, we report the first Exophiala dermatitidis fungemia case, to our knowledge, in an allogeneic hematopoietic stem cell transplant patient with graft-versus-host disease, expanding the clinical setting where Exophiala species mycosis should be suspected.

NOP receptor mediates anti-analgesia induced by agonist-antagonist opioids.

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ∼90min after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J-113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia.

Ectopic endometrium-derived leptin produces estrogen-dependent chronic pain in a rat model of endometriosis.

Endometriosis pain is a very common and extremely disabling condition whose mechanism is still poorly understood. While increased levels of leptin have been reported in patients with endometriosis, their contribution to endometriosis pain has not been explored. Using a rodent model of endometriosis we provide evidence for an estrogen-dependent contribution of leptin in endometriosis-induced pain. Rats implanted with autologous uterine tissue onto the gastrocnemius muscle developed endometriosis-like lesions and local chronic pain. Compared to eutopic uterine tissue, leptin mRNA and protein were up-regulated in the endometriosis-like lesions. Intramuscular injection of recombinant leptin in naive rats produced dose-dependent local mechanical hyperalgesia and nociceptor sensitization to mechanical stimulation. Ovariectomy attenuated the mechanical hyperalgesia induced by recombinant leptin, in rats treated with vehicle compared to those treated with 17ß-estradiol replacement, at 1 and 24 h after leptin injection. Finally, intralesional injections of a pegylated leptin receptor (Ob-R) antagonist or of an inhibitor of Janus kinase2, which transduces the Ob-R signal, markedly attenuated pain in the endometriosis model. Taken together these data support the hypothesis that leptin, generated in ectopic endometrial lesions produces mechanical hyperalgesia by acting on nociceptors innervating the lesion. This sensitivity to leptin is dependent on estrogen levels. Thus, interventions targeting leptin signaling, especially in combination with interventions that lower estrogen levels, might be useful for the treatment of endometriosis pain.

Role of endothelial cells in antihyperalgesia induced by a triptan and ß-blocker.

While blood vessels have long been implicated in diverse pain syndromes (e.g., migraine headache, angina pectoris, vasculitis, and Raynaud's syndrome), underlying mechanisms remain to be elucidated. Recent evidence supports a contribution of the vascular endothelium in endothelin-1-induced hyperalgesia, and its enhancement by repeated mechanical stimulation; a phenomenon referred to as stimulus-induced enhancement of (endothelin) hyperalgesia (SIEH). SIEH is thought to be mediated by release of ATP from endothelial cells, to act on P2X3 receptors on nociceptors. In the present study we evaluated the ability of another vasoactive hyperalgesic agent, epinephrine, to induce endothelial cell-dependent hyperalgesia and SIEH. We found that epinephrine also produces hyperalgesia and SIEH. Both P2X3 receptor antagonists, A317491 and octoxynol-9, which attenuate endothelial cell function, eliminated SIEH without affecting epinephrine hyperalgesia. We further evaluated the hypothesis that members of two important classes of drugs used to treat migraine headache, whose receptors are present in endothelial cells - the triptans and ß blockers - have a vascular component to their anti-hyperalgesic action. For this, we tested the effect of ICI-118,551, a ß2-adrenergic receptor antagonist and sumatriptan, an agonist at 5-HT1B and 5-HT1D receptors, on nociceptive effects of endothelin and epinephrine. ICI-118,551 inhibited endothelin SIEH, and attenuated epinephrine hyperalgesia and SIEH. Sumatriptan inhibited epinephrine SIEH and inhibited endothelin hyperalgesia and SIEH, while having no effect on epinephrine hyperalgesia or the hyperalgesia induced by a prototypical direct-acting inflammatory mediator, prostaglandin E2. These results support the suggestion that triptans and ß-blockers interact with the endothelial cell component of the blood vessel to produce anti-hyperalgesia.

Independent contributions of alcohol and stress axis hormones to painful peripheral neuropathy.

Painful small-fiber peripheral neuropathy is a debilitating complication of chronic alcohol abuse. Evidence from previous studies suggests that neuroendocrine mechanisms, in combination with other, as yet unidentified actions of alcohol, are required to produce this neuropathic pain syndrome. In addition to neurotoxic effects of alcohol, in the setting of alcohol abuse neuroendocrine stress axes release glucocorticoids and catecholamines. Since receptors for these stress hormones are located on nociceptors, at which they can act to cause neuronal dysfunction, we tested the hypothesis that alcohol and stress hormones act on the nociceptor, independently, to produce neuropathic pain. We used a rat model, which allows the distinction of the effects of alcohol from those produced by neuroendocrine stress axis mediators. We now demonstrate that topical application of alcohol and exposure to unpredictable sound stress, each alone, has no effect on the nociceptive threshold. However, when animals that had previous exposure to alcohol were subsequently exposed to stress, they rapidly developed mechanical hyperalgesia. Conversely, sound stress followed by topical alcohol exposure also produced mechanical hyperalgesia. The contribution of stress hormones was prevented by spinal intrathecal administration of oligodeoxynucleotides antisense to ß(2)-adrenergic or glucocorticoid receptor mRNA, which attenuates receptor level in nociceptors, as well as by adrenal medullectomy. These experiments establish an independent role of alcohol and stress hormones on the primary afferent nociceptor in the induction of painful peripheral neuropathy.

Ectopic uterine tissue as a chronic pain generator.

While chronic pain is a main symptom in endometriosis, the underlying mechanisms and effective therapy remain elusive. We developed an animal model enabling the exploration of ectopic endometrium as a source of endometriosis pain. Rats were surgically implanted with autologous uterus in the gastrocnemius muscle. Within two weeks, visual inspection revealed the presence of a reddish-brown fluid-filled cystic structure at the implant site. Histology demonstrated cystic glandular structures with stromal invasion of the muscle. Immunohistochemical studies of these lesions revealed the presence of markers for nociceptor nerve fibers and neuronal sprouting. Fourteen days after surgery rats exhibited persistent mechanical hyperalgesia at the site of the ectopic endometrial lesion. Intralesional, but not contralateral, injection of progesterone was dose-dependently antihyperalgesic. Systemic administration of leuprolide also produced antihyperalgesia. In vivo electrophysiological recordings from sensory neurons innervating the lesion revealed a significant increase in their response to sustained mechanical stimulation. These results are consistent with clinical and pathological findings observed in patients with endometriosis, compatible with the ectopic endometrium as a source of pain. This model of endometriosis allows mechanistic exploration at the lesion site facilitating our understanding of endometriosis pain.

Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain.

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E(2), epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

GDNF induces mechanical hyperalgesia in muscle by reducing I(BK) in isolectin B4-positive nociceptors.

We have assessed the mechanism underlying glial cell-derived neurotrophic factor (GDNF)-induced mechanical hyperalgesia in the gastrocnemius muscle, using patch clamp electrophysiology, in vivo electrophysiology and behavioral studies. Cultured isolectin B4-positive (IB4+) dorsal root ganglion neurons that innervated this muscle were held under current clamp; the majority developed an increase in action potential duration (a factor of increase of 2.29±0.24, compared to 1.13±0.17 in control, P<0.01) in response to GDNF (200 ng/ml) by 15 min after application. They also demonstrated a depolarization of resting membrane potential, but without significant changes in rheobase, action potential peak, or after-hyperpolarization. Large-conductance voltage- and calcium-activated potassium (BK) channels, which have recently been shown to play a role in the repolarization of IB4+ nociceptors, were inhibited under voltage clamp, as indicated by a significant reduction in the iberiotoxin-sensitive current. In vivo single-fiber recording from muscle afferents revealed that injection of iberiotoxin into their peripheral nociceptive field caused an increase in nociceptor firing in response to a 60s suprathreshold stimulus (an increase from 392.2±119.8 spikes to 596.1±170.8 spikes, P<0.05). This was observed in the absence of changes in the mechanical threshold. Finally, injection of iberiotoxin into the gastrocnemius muscle produced dose-dependent mechanical hyperalgesia. These data support the suggestion that GDNF induces nociceptor sensitization and mechanical hyperalgesia, at least in part, by inhibiting BK current in IB4+ nociceptors.

Stress enhances muscle nociceptor activity in the rat.

Chronic widespread pain, such as observed in irritable bowel (IBS) and fibromyalgia (FMS) syndrome, are markedly affected by stress. While such forms of stress-induced hyperalgesia are generally considered manifestations of "central sensitization," recent studies in patients with IBS and FMS suggest an additional, peripheral contribution. To examine the effect of stress on muscle nociceptor function, we evaluated activity in nociceptors innervating the gastrocnemius muscle in an animal model of chronic widespread pain, water avoidance stress, in the rat. This stressor, which produces mechanical hyperalgesia in skeletal muscle produced a significant decrease (∼34%) in mechanical threshold of muscle nociceptors and a marked, ∼two-fold increase in the number of action potentials produced by a prolonged (60 s) fixed intensity suprathreshold 10 g stimulus. Stress also induced an increase in conduction velocity from 1.25 m/s to 2.09 m/s, and increased variability in neuronal activity. Given that these changes, each of at least moderate magnitude, would be expected to enhance nociceptor activity, it is likely that, taken together, they contribute to the enhanced nociception observed in this model of stress-induced chronic widespread pain.

Mechanical stimulation enhances endothelin-1 hyperalgesia.

When comparing a cumulative dose-response curve for endothelin-1 (ET-1)-induced mechanical hyperalgesia to the effect of individual doses (1 ng, 10 ng, 100 ng, and 1 µg) administered in separate groups of rats, a marked difference was observed in the peak magnitude of hyperalgesia. Hyperalgesia was measured as decrease in the threshold for mechanically-induced withdrawal of the hind paw. The cumulative dosing protocol produced markedly greater maximum hyperalgesia. To determine whether this was due to the cumulative dosing protocol or to the repeated exposure to the mechanical test stimulus, we evaluated the impact of repeated testing on ET-1-induced mechanical hyperalgesia. While ET-1-induced mechanical hyperalgesia was dose- and time-dependent, repeated testing of nociceptive threshold, at 5 min intervals, following a single dose of ET-1, produced further decrease in nociceptive threshold. This mechanical stimulation-induced enhancement of ET-1 hyperalgesia lasted only 3-4 h, while the hyperalgesia lasted in excess of 5 days. The stimulation-enhanced hyperalgesia also occurred after a second injection of ET-1, administered 24 h after the initial dose. That this phenomenon is unique to ET-1 is suggested by the observation that while five additional, direct-acting hyperalgesic agents-prostaglandin E2 (PGE2), nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα)-induced robust mechanical hyperalgesia, none produced mechanical stimulation-enhanced hyperalgesia.

Mu and delta opioid receptors on nociceptors attenuate mechanical hyperalgesia in rat.

Sensitization to mechanical stimuli is important in most pain syndromes. We evaluated the populations of nociceptors mediating mechanical hyperalgesia and those mediating mu-opioid receptor (MOR) and delta-opioid receptor (DOR) agonist-induced inhibition of hyperalgesia, in the rat. We found that: (1) intradermal injection of both the endogenous ligand for the Ret receptor, glia-derived growth factor (GDNF), and the ligand for the tropomyosin receptor kinase A (TrkA) receptor, nerve growth factor (NGF)-which are present on distinct populations of nociceptors-both produce mechanical hyperalgesia; (2) DOR agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC) but not MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibit GDNF-induced hyperalgesia; (3) both DAMGO and SNC inhibit NGF hyperalgesia, even in rats pretreated with isolectin B4 (IB4)-saporin, a toxin that destroys IB4-binding neurons; (4) co-administration of low doses of DAMGO and SNC produce enhanced analgesia, and; (5) repeated administration of DAMGO produces cross-tolerance to the analgesic effect of SNC. These findings demonstrate that, most nociceptors have a role in mechanical hyperalgesia, only the DOR agonist inhibits GDNF hyperalgesia, and MOR and DOR are co-localized on a functionally important population of TrkA-positive nociceptors.

Hyperalgesic priming is restricted to isolectin B4-positive nociceptors.

We have previously described a rat model for the contribution of neuroplastic changes in nociceptors to the transition from acute to chronic pain. In this model a prior injury activates protein kinase C epsilon (PKCepsilon), inducing a chronic state characterized by marked prolongation of the hyperalgesia induced by inflammatory cytokines, prototypically prostaglandin E(2) (PGE(2)), referred to as hyperalgesic priming. In this study we evaluated the population of nociceptors involved in priming, by lesioning isolectin B4-positive (IB4(+)) nociceptors with intrathecal administration of a selective neurotoxin, IB4-saporin. To confirm that the remaining, TrkA(+)/IB4(-), nociceptors are still functional, we evaluated if nerve growth factor (NGF) induced hyperalgesia. While pretreatment with IB4-saporin eliminated the acute mechanical hyperalgesia induced by glia-derived neurotrophic factor (GDNF), NGF and PsiepsilonRACK, a highly selective activator of PKCepsilon, induced robust hyperalgesia. After injection of NGF, GDNF or PsiepsilonRACK, at a time at which hyperalgesia induced by PGE(2) is markedly prolonged (hyperalgesic priming) in control rats, in IB4-saporin-pretreated rats PGE(2) failed to produce this prolonged hyperalgesia. Thus, while PKCepsilon is present in most dorsal root ganglion neurons, where it can contribute to acute mechanical hyperalgesia, priming is restricted to IB4(+)-nociceptors, including those that are TrkA(+). While PKCepsilon activation can induce acute hyperalgesia in the IB4(+) population, it fails to induce priming. We suggest that hyperalgesic priming occurs only in IB4(+) nociceptors, and that in the peripheral terminals of nociceptors separate intracellular pools of PKCepsilon mediate nociceptor sensitization and the induction of hyperalgesic priming.