Luiz F. Ferrari

Early-life stress produces muscle hyperalgesia and nociceptor sensitization in the adult rat

Summary Neonatal stress induces a persistent decrease in skeletal muscle, but not cutaneous, mechanical nociceptive threshold and a PKCε‐dependent hyperalgesic priming in muscle as well as skin. ABSTRACT Chronic pain in adults has been associated with early‐life stress. To examine the pronociceptive effect of early‐life stress, we evaluated cutaneous and muscle nociception and activity in muscle nociceptors in an animal model of neonatal stress, limited bedding, in the rat. In this neonatal limited bedding (NLB) model, litters are exposed to limited bedding between postnatal days 2 and 9, and controls to standard bedding. In adult NLB‐treated rats, mechanical nociceptive threshold in skeletal muscle was significantly lower (∼22%) than in controls. Furthermore, administration of prostaglandin E2 in skin as well as muscle produced markedly prolonged hyperalgesia, an effect prevented by spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cε (PKCε), a second messenger in nociceptors that has been implicated in the induction and maintenance of chronic pain. In electrophysiological studies, mechanical threshold of muscle nociceptors was reduced by ∼31% and conduction velocity significantly increased (∼28%). These findings indicate that neonatal stress induces a persistent hyperalgesia and nociceptor sensitization manifest in the adult and that the second messenger PKCε may be a target against which therapies might be directed to treat a chronic pain syndrome that is associated with early‐life traumatic stress.

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.

15d-Prostaglandin J2 Inhibits Inflammatory Hypernociception: Involvement of Peripheral Opioid Receptor

The 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptors γ (PPAR-γ) and is now recognized as a potent anti-inflammatory mediator. However, information regarding the influence of 15d-PGJ2 on inflammatory pain is still unknown. In this study, we evaluated the effect of 15d-PGJ2 upon inflammatory hypernociception and the mechanisms involved in this effect. We observed that intraplantar administration of 15d-PGJ2 (30–300 ng/paw) inhibits the mechanical hypernociception induced by both carrageenan (100 μg/paw) and the directly acting hypernociceptive mediator, prostaglandin E2 (PGE2). Moreover, 15d-PGJ2 [100 ng/temporomandibular joint (TMJ)] inhibits formalin-induced TMJ hypernociception. On the other hand, the direct administration of 15d-PGJ2 into the dorsal root ganglion was ineffective in blocking PGE2-induced hypernociception. In addition, the 15d-PGJ2 antinociceptive effect was enhanced by the increase of macrophage population in paw tissue due to local injection of thioglycollate, suggesting the involvement of these cells on the 15d-PGJ2-antinociceptive effect. Moreover, the antinociceptive effect of 15d-PGJ2 was also blocked by naloxone and by the PPAR-γ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662), suggesting the involvement of peripheral opioids and PPAR-γ receptor in the process. Similar to opioids, the 15d-PGJ2 antinociceptive action depends on the nitric oxide/cGMP/protein kinase G \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{PKG}){/}\mathrm{K}_{\mathrm{ATP}}^{+}\) \end{document} channel pathway because it was prevented by the pretreatment with the inhibitors of nitric-oxide synthase (NG-monomethyl-l-arginine acetate), guanylate cyclase]1H-(1,2,4)-oxadiazolo(4,2-α)quinoxalin-1-one[, PKG [indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycone (KT5823)], or with the ATP-sensitive potassium channel blocker glibenclamide. Taken together, these results demonstrate for the first time that 15d-PGJ2 inhibits inflammatory hypernociception via PPAR-γ activation. This effect seems to be dependent on endogenous opioids and local macrophages.

Role of Nociceptor αCaMKII in Transition from Acute to Chronic Pain (Hyperalgesic Priming) in Male and Female Rats

We have previously shown that activation of protein kinase Cε (PKCε) in male rats induces a chronic, long-lasting change in nociceptors such that a subsequent exposure to proinflammatory mediators produces markedly prolonged mechanical hyperalgesia. This neuroplastic change, hyperalgesic priming, is dependent on activation of cytoplasmic polyadenylation element-binding protein (CPEB), downstream of PKCε, and consequent translation of mRNAs in the peripheral terminal of the nociceptor. Since α calmodulin-dependent protein kinase II (αCaMKII), a molecule implicated in neuroplasticity, is a target of CPEB and can also affect CPEB function, we investigated its role in the transition from acute to chronic pain. Priming induced by direct activation of PKCε can be prevented by inhibition of αCaMKII. In addition, direct activation of αCaMKII induces priming, which was not prevented by pretreatment with PKCε antisense, suggesting that αCaMKII is downstream of PKCε in the induction of priming. Activation of ryanodine receptors (RyRs), which can lead to activation of αCaMKII, also induced priming, in a calcium- and αCaMKII-dependent manner. Similarly, inhibition of the RyR and a calcium buffer prevented induction of priming by PKCε. Unlike activation of PKCε, ryanodine and αCaMKII induced priming in female as well as male rats. Our results demonstrate a contribution of αCaMKII to induction of hyperalgesic priming, a phenomenon implicated in the transition from acute to chronic pain.

Role of Drp1, a key mitochondrial fission protein, in neuropathic pain.

While oxidative stress has been implicated in small-fiber painful peripheral neuropathies, antioxidants are only partially effective to treat patients. We have tested the hypothesis that Drp1 (dynamin-related protein 1), a GTPase that catalyzes the process of mitochondrial fission, which is a mechanism central for the effect and production of reactive oxygen species (ROS), plays a central role in these neuropathic pain syndromes. Intrathecal administration of oligodeoxynucleotide antisense against Drp1 produced a decrease in its expression in peripheral nerve and markedly attenuated neuropathic mechanical hyperalgesia caused by HIV/AIDS antiretroviral [ddC (2′,3′-dideoxycytidine)] and anticancer (oxaliplatin) chemotherapy in male Sprague Dawley rats. To confirm the role of Drp1 in these models of neuropathic pain, as well as to demonstrate its contribution at the site of sensory transduction, we injected a highly selective Drp1 inhibitor, mdivi-1, at the site of nociceptive testing on the dorsum of the rats hindpaw. mdivi-1 attenuated both forms of neuropathic pain. To evaluate the role of Drp1 in hyperalgesia induced by ROS, we demonstrated that intradermal hydrogen peroxide produced dose-dependent hyperalgesia that was inhibited by mdivi-1. Finally, mechanical hyperalgesia induced by diverse pronociceptive mediators involved in inflammatory and neuropathic pain—tumor necrosis factor α, glial-derived neurotrophic factor, and nitric oxide—was also inhibited by mdivi-1. These studies provide support for a substantial role of mitochondrial fission in preclinical models of inflammatory and neuropathic pain.

Peripheral Administration of Translation Inhibitors Reverses Increased Hyperalgesia in a Model of Chronic Pain in the Rat

UNLABELLED Chronic pain is extremely difficult to manage, in part due to lack of progress in reversing the underlying pathophysiology. Since translation of messenger ribonucleic acids (mRNAs) in the peripheral terminal of the nociceptor plays a role in the transition from acute to chronic pain, we tested the hypothesis that transient inhibition of translation in the peripheral terminal of the nociceptor could reverse hyperalgesic priming, a model of transition from acute to chronic pain. We report that injection of translation inhibitors rapamycin and cordycepin, which inhibit translation by different mechanisms, at the peripheral terminal of the primed nociceptor produces reversal of priming in the rat that outlasted the duration of action of these drugs to prevent the development of priming. These data support the suggestion that interruption of translation in the nociceptor can reverse a preclinical model of at least 1 form of chronic pain. PERSPECTIVE This study provides evidence that ongoing protein translation in the sensory neuron terminals is involved in pain chronification, and local treatment that transiently interrupts this translation may be a useful therapy to chronic pain.

Neural mechanisms of pain and alcohol dependence

An association between chronic pain conditions and alcohol dependence has been revealed in numerous studies with episodes of alcohol abuse antedating chronic pain in some people and alcohol dependence emerging after the onset of chronic pain in others. Alcohol dependence and chronic pain share common neural circuits giving rise to the possibility that chronic pain states could significantly affect alcohol use patterns and that alcohol dependence could influence pain sensitivity. The reward and emotional pathways that regulate drug/alcohol addiction also mediate chronic pain. For example, pain-evoked activation of brain learning and brain reward circuitry may modulate cortical processing of pain and central sensitization mediated by mesocorticolimbic circuitry. Imbalance and reorganization of amygdala-mPFC interactions may not only be important for persistent pain, but also for disorders characterized by the abnormal persistence of emotional-affective states such as drug and alcohol addiction. Further studies are necessary to understand how these neural circuits are regulated in comorbid conditions of alcoholism and chronic pain. In addition, long term alcohol use could induce pain symptoms and may exacerbate chronic pain arising from other sources. While prior studies have established a role of neuroendocrine stress axis mediators in alcohol abuse and neurotoxic effects, these studies have not explored the distinction between the individual impact of alcohol and stress hormones. Future studies should explore the mechanisms mediating the contribution of alcohol and stress axis hormones on pain, an important question in our understanding of the neurobiology of alcohol abuse and chronic 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.

Alcohol consumption enhances antiretroviral painful peripheral neuropathy by mitochondrial mechanisms

A major dose‐limiting side effect of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) chemotherapies, such as the nucleoside reverse transcriptase inhibitors (NRTIs), is a small‐fiber painful peripheral neuropathy, mediated by its mitochondrial toxicity. Co‐morbid conditions may also contribute to this dose‐limiting effect of HIV/AIDS treatment. Alcohol abuse, which alone also produces painful neuropathy, is one of the most important co‐morbid risk factors for peripheral neuropathy in patients with HIV/AIDS. Despite the prevalence of this problem and its serious impact on the quality of life and continued therapy in HIV/AIDS patients, the mechanisms by which alcohol abuse exacerbates highly active antiretroviral therapy (HAART)‐induced neuropathic pain has not been demonstrated. In this study, performed in rats, we investigated the cellular mechanism by which consumed alcohol impacts antiretroviral‐induced neuropathic pain. NRTI 2′,3′‐dideoxycytidine (ddC; 50 mg/kg) neuropathy was mitochondrial‐dependent and PKCε‐independent, and alcohol‐induced painful neuropathy was PKCε‐dependent and mitochondrial‐independent. At low doses, ddC (5 mg/kg) and alcohol (6.5% ethanol diet for 1 week), which alone do not affect nociception, together produce profound mechanical hyperalgesia. This hyperalgesia is mitochondrial‐dependent but PKCε‐independent. These experiments, which provide the first model for studying the impact of co‐morbidity in painful neuropathy, support the clinical impression that alcohol consumption enhances HIV/AIDS therapy neuropathy, and provide evidence for a role of mitochondrial mechanisms underlying this interaction.

Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain

The primary afferent nociceptor was used as a model system to study mechanisms of pain induced by chronic opioid administration. Repeated intradermal injection of the selective mu-opioid receptor (MOR) agonist DAMGO induced mechanical hyperalgesia and marked prolongation of prostaglandin E2 (PGE2) hyperalgesia, a key feature of hyperalgesic priming. However, in contrast to prior studies of priming induced by receptor-mediated (i.e., TNFα, NGF, or IL-6 receptor) or direct activation of protein kinase Cε (PKCε), the pronociceptive effects of PGE2 in DAMGO-treated rats demonstrated the following: (1) rapid induction (4 h compared with 3 d); (2) protein kinase A (PKA), rather than PKCε, dependence; (3) prolongation of hyperalgesia induced by an activator of PKA, 8-bromo cAMP; (4) failure to be reversed by a protein translation inhibitor; (5) priming in females as well as in males; and (6) lack of dependence on the isolectin B4-positive nociceptor. These studies demonstrate a novel form of hyperalgesic priming induced by repeated administration of an agonist at the Gi-protein-coupled MOR to the peripheral terminal of the nociceptor. SIGNIFICANCE STATEMENT The current study demonstrates the molecular mechanisms involved in the sensitization of nociceptors produced by repeated activation of mu-opioid receptors and contributes to our understanding of the painful condition observed in patients submitted to chronic use of opioids.