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An accelerating basic science literature is providing key insights into the mechanisms by which spinal neuropeptide Y (NPY) inhibits chronic pain. A key target of pain inhibition is the G i -coupled neuropeptide Y1 receptor (Y1). Y1 is located in key sites of pain transmission, including the peptidergic subpopulation of primary afferent neurons and a dense subpopulation of small, excitatory, glutamatergic/somatostatinergic interneurons (Y1-INs) that are densely expressed in the dorsal horn, particularly in superficial lamina I-II. Selective ablation of spinal Y1-INs with an NPY-conjugated saporin neurotoxin attenuates the development of peripheral nerve injury-induced mechanical and cold hypersensitivity. Conversely, conditional knockdown of NPY expression or intrathecal administration of Y1 antagonists reinstates hypersensitivity in models of chronic latent pain sensitization. These and other results indicate that spinal NPY release and the consequent inhibition of pain facilitatory Y1-INs represent an important mechanism of endogenous analgesia. This mechanism can be mimicked with exogenous pharmacological approaches (e.g. intrathecal administration of Y1 agonists) to inhibit mechanical and thermal hypersensitivity and spinal neuron activity in rodent models of neuropathic, inflammatory, and postoperative pain. Pharmacological activation of Y1 also inhibits mechanical- and histamine-induced itch. These immunohistochemical, pharmacological, and cell type-directed lesioning data, in combination with recent transcriptomic findings, point to Y1-INs as a promising therapeutic target for the development of spinally directed NPY-Y1 agonists to treat both chronic pain and itch., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Central neuropathic pain develops in greater than 75% of individuals suffering a spinal cord injury (SCI). Increasingly, sex is recognized as an important biological variable in the development and treatment of peripheral neuropathic pain, but much less is known about the role of sex in central neuropathic pain and its pharmacological inhibition. To test the hypothesis that efficacy of analgesic therapies differs between males and females in SCI, we used a mouse model of SCI pain to determine the analgesic efficacy of pioglitazone (PIO), U.S. Food and Drug Administration-approved drug for the treatment of diabetes, and azithromycin (AZM), a commonly prescribed macrolide antibiotic with immunomodulatory properties. Male and female mice received moderate-severe T9 contusion SCI (75-kdyn). A robust heat hyperalgesia developed similarly between male and female mice by 4 weeks post-injury and lasted throughout the duration of the study (14 weeks). Three months after SCI, mice were treated with PIO (10 mg/kg, intraperitoneal) or AZM (160 mg/kg, oral). We observed a sex-specific effect of PIO with significant antihyperalgesic effects in females, but not males. In contrast, AZM was effective in both sexes. Our data support the use of PIO and AZM as novel therapies for SCI pain and highlight the importance of considering sex as a biological variable in clinical and experimental SCI pain research.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (IV) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP- AMPAR antagonist IEM-1460 (50 μM) inhibited AMPAR-evoked Ca 2+ transients 21d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MOR CA ). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MOR CA inverse agonist naltrexone (NTX, 1 μg, i.t.) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 μM) increased the peak amplitude of AMPAR-evoked Ca 2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca 2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca 2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain., (Copyright © 2019. Published by Elsevier Inc.)

Central noradrenergic centers such as the locus coeruleus (LC) are traditionally viewed as pain inhibitory; however, complex interactions among brainstem pathways and their receptors modulate both inhibition and facilitation of pain. In addition to the well-described role of descending pontospinal pathways that inhibit spinal nociceptive transmission, an emerging body of research now indicates that noradrenergic neurons in the LC and their terminals in the dorsal reticular nucleus (DRt), medial prefrontal cortex (mPFC), spinal dorsal horn, and spinal trigeminal nucleus caudalis participate in the development and maintenance of allodynia and hyperalgesia after nerve injury. With time after injury, we argue that the balance of LC function shifts from pain inhibition to pain facilitation. Thus, the pain-inhibitory actions of antidepressant drugs achieved with elevated noradrenaline concentrations in the dorsal horn may be countered or even superseded by simultaneous activation of supraspinal facilitating systems dependent on α 1 -adrenoreceptors in the DRt and mPFC as well as α 2 -adrenoreceptors in the LC. Indeed, these opposing actions may account in part for the limited treatment efficacy of tricyclic antidepressants and noradrenaline reuptake inhibitors such as duloxetine for the treatment of chronic pain. We propose that the traditional view of the LC as a pain-inhibitory structure be modified to account for its capacity as a pain facilitator. Future studies are needed to determine the neurobiology of ascending and descending pathways and the pharmacology of receptors underlying LC-mediated pain inhibition and facilitation. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Nociception is maintained by genome-wide regulation of transcription in the dorsal root ganglia—spinal cord network. Hence, transcription factors constitute a promising class of targets for breakthrough pharmacological interventions to treat chronic pain. DNA decoys are oligonucleotides and specific inhibitors of transcription factor activities. A methodological series of in vivo–in vitro screening cycles was performed with decoy/transcription factor couples to identify targets capable of producing a robust and long-lasting inhibition of established chronic pain. Decoys were injected intrathecally and their efficacy was tested in the spared nerve injury and chronic constriction injury models of chronic pain in rats using repetitive von Frey testing., Results: Results demonstrated that a one-time administration of decoys binding to the Kruppel-like transcription factors (KLFs) 6, 9, and 15 produces a significant and weeks–month long reduction in mechanical hypersensitivity compared to controls. In the spared nerve injury model, decoy efficacy was correlated to its capacity to bind KLF15 and KLF9 at a specific ratio, while in the chronic constriction injury model, efficacy was correlated to the combined binding capacity to KLF6 and KLF9. AYX2, an 18-bp DNA decoy binding KLF6, KLF9, and KLF15, was optimized for clinical development, and it demonstrated significant efficacy in these models., Conclusions: These data highlight KLF6, KLF9, and KLF15 as transcription factors required for the maintenance of chronic pain and illustrate the potential therapeutic benefits of AYX2 for the treatment of chronic pain.

Latent sensitization is a rodent model of chronic pain that reproduces both its episodic nature and its sensitivity to stress. It is triggered by a wide variety of injuries ranging from injection of inflammatory agents to nerve damage. It follows a characteristic time course in which a hyperalgesic phase is followed by a phase of remission. The hyperalgesic phase lasts between a few days to several months, depending on the triggering injury. Injection of μ-opioid receptor inverse agonists (e.g., naloxone or naltrexone) during the remission phase induces reinstatement of hyperalgesia. This indicates that the remission phase does not represent a return to the normal state, but rather an altered state in which hyperalgesia is masked by constitutive activity of opioid receptors. Importantly, stress also triggers reinstatement. Here we describe in detail procedures for inducing and following latent sensitization in its different phases in rats and mice., (Copyright © 2015 John Wiley & Sons, Inc.)

The persistence of pain after surgery increases the recovery interval from surgery to a normal quality of life. AYX1 is a DNA-decoy drug candidate designed to prevent post-surgical pain following a single intrathecal injection. Tissue injury causes a transient activation of the transcription factor EGR1 in the dorsal root ganglia-dorsal horn network, which then triggers changes in gene expression that induce neuronal hypersensitivity. AYX1 is a potent, specific inhibitor of EGR1 activity that mimics the genomic EGR1-binding sequence. Administered in the peri-operative period, AYX1 dose dependently prevents mechanical hypersensitivity in models of acute incisional (plantar), inflammatory (CFA), and chronic neuropathic pain (SNI) in rats. Furthermore, in a knee surgery model evaluating functional measures of postoperative pain, AYX1 improved weight-bearing incapacitance and spontaneous rearing compared to control. These data illustrate the potential clinical therapeutic benefits of AYX1 for preventing the transition of acute to chronic post-surgical pain., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

We discuss and present new data regarding the physiological and molecular mechanisms of nuclear receptor activation in pain control, with a particular emphasis on non-genomic effects of ligands at peroxisome proliferator-activated receptor (PPAR), GPR30, and classical estrogen receptors. PPARalpha agonists rapidly reduce both acute and chronic pain in a number of pain assays. These effects precede transcriptional anti-inflammatory actions, and are mediated in part by IK(ca) and BK(ca) channels on DRG neurons. In contrast to the peripheral site of action of PPARalpha ligands, the dorsal horn supports the expression of PPARgamma. Intrathecal administration of PPARgamma ligands rapidly (< or =5 min) attenuated mechanical and thermal hypersensitivity associated with nerve injury in a dose-dependent manner that could be blocked with PPARgamma antagonists. By contrast, a PPARgamma antagonist itself rapidly increased the mechanical allodynia associated with nerve injury. These data suggest that ligand-dependent, non-genomic activation of spinal PPARgamma decreases behavioral signs of inflammatory and neuropathic pain. We also report that the GPR30 is expressed on cultured sensory neurons, that activation of the receptor elicits signaling to increase calcium accumulation. This signaling may contribute to increased neuronal sensitivity as treatment with the GPR30 agonist induces hyperalgesia. Finally, application of the membrane-impermeable 17beta-E(2)-BSA rapidly (within 15 min) enhanced BK-stimulated inositol phosphate (IP) accumulation and PGE(2)-mediated cAMP accumulation in trigeminal ganglion cultures. We conclude that nuclear receptor ligands may operate through rapid, non-genomic mechanisms to modulate inflammatory and neuropathic pain.

Dramatically up-regulated in the dorsal horn of the mammalian spinal cord following inflammation or nerve injury, neuropeptide Y (NPY) is poised to regulate the transmission of sensory signals. We found that doxycycline-induced conditional in vivo (Npytet/tet) knockdown of NPY produced rapid, reversible, and repeatable increases in the intensity and duration of tactile and thermal hypersensitivity. Remarkably, when allowed to resolve for several weeks, behavioral hypersensitivity could be dramatically reinstated with NPY knockdown or intrathecal administration of Y1 or Y2 receptor antagonists. In addition, Y2 antagonism increased dorsal horn expression of Fos and phosphorylated form of extracellular signal-related kinase. Taken together, these data establish spinal NPY receptor systems as an endogenous braking mechanism that exerts a tonic, long-lasting, broad-spectrum inhibitory control of spinal nociceptive transmission, thus impeding the transition from acute to chronic pain. NPY and its receptors appear to be part of a mechanism. whereby mammals naturally recover from the hyperalgesia associated with inflammation or nerve injury. [ABSTRACT FROM AUTHOR]

Peripheral inflammation produces a long-lasting latent sensitization of spinal nociceptive neurons, that is, masked by tonic inhibitory controls. We explored mechanisms of latent sensitization with an established four-step approach: (1) induction of inflammation; (2) allow pain hypersensitivity to resolve; (3) interrogate latent sensitization with a channel blocker, mutant mouse, or receptor antagonist; and (4) disrupt compensatory inhibition with a receptor antagonist so as to reinstate pain hypersensitivity. We found that the neuropeptide Y Y1 receptor antagonist BIBO3304 reinstated pain hypersensitivity, indicative of an unmasking of latent sensitization. BIBO3304-evoked reinstatement was not observed in AC1 knockout mice and was prevented with intrathecal co-administration of a pharmacological blocker to the N-methyl-D-aspartate receptor (NMDAR), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), transient receptor potential cation channel A1 (TRPA1), channel V1 (TRPV1), or exchange protein activated by cAMP (Epac1 or Epac2). A PKA activator evoked both pain reinstatement and touch-evoked pERK expression in dorsal horn; the former was prevented with intrathecal co-administration of a TRPA1 or TRPV1 blocker. An Epac activator also evoked pain reinstatement and pERK expression. We conclude that PKA and Epac are sufficient to maintain long-lasting latent sensitization of dorsal horn neurons that is kept in remission by the NPY-Y1 receptor system. Furthermore, we have identified and characterized 2 novel molecular signaling pathways in the dorsal horn that drive latent sensitization in the setting of chronic inflammatory pain: NMDAR→AC1→PKA→TRPA1/V1 and NMDAR→AC1→Epac1/2. New treatments for chronic inflammatory pain might either increase endogenous NPY analgesia or inhibit AC1, PKA, or Epac. [ABSTRACT FROM AUTHOR]

Abstract: We review previously published data, and present some new data, indicating that spinal application of neuropeptide Y (NPY) reduces behavioral and neurophysiological signs of acute and chronic pain. In models of acute pain, early behavioral studies showed that spinal (intrathecal) administration of NPY and Y2 receptor agonists decrease thermal nociception. Subsequent neurophysiological studies indicated that Y2-mediated inhibition of excitatory neurotransmitter release from primary afferent terminals in the substantia gelatinosa may contribute to the antinociceptive actions of NPY. As with acute pain, NPY reduced behavioral signs of inflammatory pain such as mechanical allodynia and thermal hyperalgesia; however, receptor antagonist studies indicate an important contribution of spinal Y1 rather than Y2 receptors. Interestingly, Y1 agonists suppress inhibitory synaptic events in dorsal horn neurons (indeed, well known μ-opioid analgesic drugs produce similar cellular actions). To resolve the behavioral and neurophysiological data, we propose that NPY/Y1 inhibits the spinal release of inhibitory neurotransmitters (GABA and glycine) onto inhibitory neurons, e.g. disinhibition of pain inhibition, resulting in hyporeflexia. The above mechanisms of Y1- and Y2-mediated analgesia may also operate in the setting of peripheral nerve injury, and new data indicate that NPY dose-dependently inhibits behavioral signs of neuropathic pain. Indeed, neurophysiological studies indicate that Y2-mediated inhibition of Ca2+ channel currents in dorsal root ganglion neurons is actually increased after axotomy. We conclude that spinal delivery of Y1 agonists may be of use in the treatment of chronic inflammatory pain, and that the use of Y1 and Y2 agonists in neuropathic pain warrants further consideration. [Copyright &y& Elsevier]

Tissue injury produces a delicate balance between latent pain sensitization (LS) and compensatory endogenous opioid receptor analgesia that continues for months, even after re-establishment of normal pain thresholds. To evaluate the contribution of mu (MOR), delta (DOR), and/or kappa (KOR) opioid receptors to the silencing of chronic postoperative pain, we performed plantar incision at the hindpaw, waited 21 days for the resolution of hyperalgesia, and then intrathecally injected subtype-selective ligands. We found that the MOR-selective inhibitor CTOP (1–1000 ng) dose-dependently reinstated mechanical hyperalgesia. Two DOR-selective inhibitors naltrindole (1–10 μg) and TIPP[Ψ] (1–20 μg) reinstated mechanical hyperalgesia, but only at the highest dose that also produced itching, licking, and tail biting. Both the prototypical KOR-selective inhibitors nor-BNI (0.1–10 μg) and the newer KOR inhibitor with more canonical pharmocodynamic effects, LY2456302 (0.1–10 μg), reinstated mechanical hyperalgesia. Furthermore, LY2456302 (10 μg) increased the expression of phosphorylated signal-regulated kinase (pERK), a marker of central sensitization, in dorsal horn neurons but not glia. Sex studies revealed that LY2456302 (0.3 μg) reinstated hyperalgesia and pERK expression to a greater degree in female as compared to male mice. Our results suggest that spinal MOR and KOR, but not DOR, maintain LS within a state of remission to reduce the intensity and duration of postoperative pain, and that endogenous KOR but not MOR analgesia is greater in female mice. • Surgery produces a long-lasting kappa opioid receptor analgesia. • KOR analgesia that prevents the transition from acute to chronic postoperative pain. • KOR analgesia is, particularly strong in females. [ABSTRACT FROM AUTHOR]