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.

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.

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.

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.

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.

Nociceptor subpopulations involved in hyperalgesic priming.

We have previously developed a model in the rat for the transition from acute to chronic pain, hyperalgesic priming, in which a long-lasting neuroplastic change in signaling pathways mediates a prolongation of proinflammatory cytokine-induced nociceptor sensitization and mechanical hyperalgesia, induced at the site of a previous inflammatory insult. Induction of priming is mediated by activation of protein kinase C(epsilon) (PKC(epsilon)) in the peripheral terminal of the primary afferent nociceptor. Given that hyperalgesic mediator-induced PKC(epsilon) translocation occurs in isolectin B4 (IB4)(+)-nonpeptidergic but not in receptor tyrosine kinase (TrkA)(+)-peptidergic nociceptors, we tested the hypothesis that hyperalgesic priming was restricted to the IB4(+) subpopulation of nociceptors. After recovery from nerve growth factor (NGF)- and GDNF-induced hyperalgesia, a proinflammatory cytokine, prostaglandin E(2) (PGE(2)) induced, PKC(epsilon)-dependent, markedly prolonged hyperalgesia, two features that define the development of the primed state. Thus, hyperalgesic priming occurs in both the IB4(+)-nonpeptidergic and TrkA(+)-peptidergic subpopulations of nociceptive afferents. Of note, however, while attenuation of PKC(epsilon) prevented NGF-induced priming, the hyperalgesia induced by NGF is PKC(epsilon) independent. We propose that separate intracellular pools of PKC(epsilon), in the peripheral terminals of nociceptors, mediate nociceptor sensitization and the induction of hyperalgesic priming.

A novel technique to perform direct intraganglionar injections in rats.

The present work describes a simple method for direct drug administration into the dorsal root ganglion (DRG) in anesthetized rats. This technique does not involve surgery, is easy to learn and allows behavioral testing within minutes after the injection. Based on landmarks that target the L5 DRG, an orifice was created with a guide needle through which a specially designed needle was inserted for solution injection. Its introduction into the ganglia was ensured by the triggering of an ipsilateral hindpaw reflex. The precision of the technique was checked by injections of the biological dye Pontamine Sky Blue (PSB) or C14-labeled arginine. There was no leakage of the dye to the surrounding tissues after a single 4 microl or three successive 2.5 microl injections (at 30-min intervals). Moreover, identical effects were observed with prostaglandin E2 (PGE2), morphine or glibenclamide injected intraplantarly or in the DRG, thus confirming the precision of the method and suggesting that the ganglion cells and peripheral nociceptors may display similar receptor population.