Effects of Target-Controlled Infusion of High-Dose Naloxone on Pain and Hyperalgesia in a Human Thermal Injury Model: A Study Protocol

Mu-opioid-receptor antagonists have been extensively studied in experimental research as pharmacological tools uncovering mechanisms of pain modulation by the endogenous opioid system. In rodents, administration of high doses of mu-opioid-receptor antagonists after the resolution of an inflammatory injury has demonstrated reinstatement of nociceptive hypersensitivity indicating unmasking of latent sensitization. In a recent human study, pain hypersensitivity assessed as secondary hyperalgesia area (SHA), was reinstated 7 days after a mild thermal injury, in 4 out of 12 subjects after a naloxone infusion. The aims of the present study are first, to replicate our previous findings in a larger-sized study; second, to examine if high sensitizers (subjects presenting with large SHA after a thermal injury) develop a higher degree of hypersensitivity after naloxone challenge than low sensitizers (subjects presenting with restricted SHA after a thermal injury); and third to examine a dose–response relationship between 3 stable naloxone concentrations controlled by target-controlled infusion, and the unmasking of latent sensitization. Healthy participants (n = 80) underwent a screening day (day 0) with induction of a thermal skin injury (47°C, 420 seconds, 12.5 cm2). Assessment of SHA was performed 1 and 2 hours after the injury. Using an enriched design, only participants belonging to the upper quartile of SHA (Q4, high sensitizers; n = 20) and the lower quartile of SHA (Q1, low sensitizers; n = 20) continued the study, comprising 4 consecutive days—days 1 to 4. Thermal skin injuries were repeated on day 1 and day 3, whereas day 2 and day 4 (7 days after day 1 and day 3, respectively) were target-controlled infusion days in which the subjects were randomly allocated to receive either naloxone (3.25 mg/kg, 4 mg/mL) or placebo (normal saline) intravenous. The primary outcome was SHA assessed by weighted-pin instrument (128 mN) 0, 1, 2, and 165 to 169 hours after the thermal injury (day 1–4). The secondary outcomes were pin-prick pain thresholds assessed by weighted-pin instrument (8–512 mN) at primary and secondary hyperalgesia areas (days 1–4). The naloxone-induced unmasking of latent sensitization is an interesting model for exploring the transition from acute to chronic pain. The results from the present study may provide valuable information regarding future research in persistent postsurgical pain states

Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Bath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn

Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145

The final publication is available at Elsevier via http://doi.org/10.1016/j.bbamem.2016.05.020 © 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity. (C) 2016 Elsevier B.V. All rights reserved.This study was supported by operating grants by NSERC to Scott Taylor (155283-2012) and Michael Palmer (250265-2013)

Facilitation of Neuropathic Pain by the NPY Y1 Receptor-Expressing Subpopulation of Excitatory Interneurons in the Dorsal Horn

Endogenous neuropeptide Y (NPY) exerts long-lasting spinal inhibitory control of neuropathic pain, but its mechanism of action is complicated by the expression of its receptors at multiple sites in the dorsal horn: NPY Y1 receptors (Y1Rs) on post-synaptic neurons and both Y1Rs and Y2Rs at the central terminals of primary afferents. We found that Y1R-expressing spinal neurons contain multiple markers of excitatory but not inhibitory interneurons in the rat superficial dorsal horn. To test the relevance of this spinal population to the development and/or maintenance of acute and neuropathic pain, we selectively ablated Y1R-expressing interneurons with intrathecal administration of an NPY-conjugated saporin ribosomal neurotoxin that spares the central terminals of primary afferents. NPY-saporin decreased spinal Y1R immunoreactivity but did not change the primary afferent terminal markers isolectin B4 or calcitonin-gene-related peptide immunoreactivity. In the spared nerve injury (SNI) model of neuropathic pain, NPY-saporin decreased mechanical and cold hypersensitivity, but disrupted neither normal mechanical or thermal thresholds, motor coordination, nor locomotor activity. We conclude that Y1R-expressing excitatory dorsal horn interneurons facilitate neuropathic pain hypersensitivity. Furthermore, this neuronal population remains sensitive to intrathecal NPY after nerve injury. This neuroanatomical and behavioral characterization of Y1R-expressing excitatory interneurons provides compelling evidence for the development of spinally-directed Y1R agonists to reduce chronic neuropathic pain

Sex differences in a mouse model of multiple sclerosis: neuropathic pain behavior in females but not males and protection from neurological deficits during proestrus

BACKGROUND: Multiple sclerosis (MS), a demyelinating disease of the central nervous system, is one of the most prevalent neurological disorders in the industrialized world. This disease afflicts more than two million people worldwide, over two thirds of which are women. MS is typically diagnosed between the ages of 20–40 and can produce debilitating neurological impairments including muscle spasticity, muscle paralysis, and chronic pain. Despite the large sex disparity in MS prevalence, clinical and basic research investigations of how sex and estrous cycle impact development, duration, and severity of neurological impairments and pain symptoms are limited. To help address these questions, we evaluated behavioral signs of sensory and motor functions in one of the most widely characterized animal models of MS, the experimental autoimmune encephalomyelitis (EAE) model. METHODS: C57BL/6 male and female mice received flank injection of complete Freund’s adjuvant (CFA) or CFA plus myelin oligodendrocyte glycoprotein 35-55 (MOG(35-55)) to induce EAE. Experiment 1 evaluated sex differences of EAE-induced neurological motor deficits and neuropathic pain-like behavior over 3 weeks, while experiment 2 evaluated the effect of estrous phase in female mice on the same behavioral measures for 3 months. EAE-induced neurological motor deficits including gait analysis and forelimb grip strength were assessed. Neuropathic pain-like behaviors evaluated included sensitivity to mechanical, cold, and heat stimulations. Estrous cycle was determined daily via vaginal lavage. RESULTS: MOG(35-55)-induced EAE produced neurological impairments (i.e., motor dysfunction) including mild paralysis and decreases in grip strength in both females and males. MOG(35-55) produced behavioral signs of neuropathic pain—mechanical and cold hypersensitivity—in females, but not males. MOG(35-55) did not change cutaneous heat sensitivity in either sex. Administration of CFA or CFA + MOG(35-55) prolonged the time spent in diestrus for 2 weeks, after which normal cycling returned. MOG(35-55) produced fewer neurological motor deficits when mice were in proestrus relative to non-proestrus phases. CONCLUSIONS: We conclude that female mice are superior to males for the study of neuropathic pain-like behaviors associated with MOG(35-55)-induced EAE. Further, proestrus may be protective against EAE-induced neurological deficits, thus necessitating further investigation into the impact that estrous cycle exerts on MS symptoms

Intrathecal Administration of AYX2 DNA Decoy Produces a Long-Term Pain Treatment in Rat Models of Chronic Pain by Inhibiting the KLF6, KLF9, and KLF15 Transcription Factors

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

Pharmacology, Pharmacokinetics, and Metabolism of the DNA-Decoy AYX1 for the Prevention of Acute and Chronic Post-Surgical Pain

Background: AYX1 is an unmodified DNA-decoy designed to reduce acute post-surgical pain and its chronification with a single intrathecal dose at the time of surgery. AYX1 inhibits the transcription factor early growth response protein 1, which is transiently induced at the time of injury and triggers gene regulation in the dorsal root ganglia and spinal cord that leads to long-term sensitization and pain. This work characterizes the AYX1 dose-response profile in rats and the link to AYX1 pharmacokinetics and metabolism in the cerebrospinal fluid, dorsal root ganglia, and spinal cord. Results: The effects of ascending dose-levels of AYX1 on mechanical hypersensitivity were measured in the spared nerve injury model of chronic pain and in a plantar incision model of acute post-surgical pain. AYX1 dose-response profile shows that efficacy rapidly increases from a minimum effective dose of ∼ 0.5 mg to a peak maximum effective dose of ∼ 1 mg. With further dose escalation, the efficacy paradoxically appears to decrease by ∼ 30% and then returns to full efficacy at the maximum feasible dose of ∼ 4 mg. The reduction of efficacy is associated to doses triggering a near-saturation of AYX1 metabolism by nucleases in the cerebrospinal fluid and a paradoxical reduction of AYX1 exposure during the period of early growth response protein 1 induction. This effect is overcome at higher doses that compensate for the effect of metabolism. Discussion: AYX1 is a competitive antagonist of early growth response protein 1, which is consistent with the overall increased efficacy observed as dose-levels initially escalate. Chemically, AYX1 is unprotected against degradation by nucleases. The sensitivity to nucleases is reflected in a paradoxical reduction of efficacy in the dose-response curve. Conclusions: These findings point to the importance of the nuclease environment of the cerebrospinal fluid to the research and development of AYX1 and other intrathecal nucleotide-based therapeutics

Latent Sensitization in a Mouse Model of Ocular Neuropathic Pain

Purpose: Chronic ocular pain is poorly understood and difficult to manage. We developed a murine model of corneal surface injury (CSI)–induced chronic ocular neuropathic pain. The study focuses on changes in corneal nerve morphology and associated short- and long-term pain-like behavior after CSI. Methods: CSI was induced in mice by local application of an alkali solution (0.75 N NaOH). Corneal nerve architecture, morphology, density, and length were studied. Eye-wiping was evaluated before and after CSI in response to hypertonic saline (2 M NaCl). Naltrexone (NTX) or Naloxone-methiodide (NLX-me), opioid receptor antagonists, were given subcutaneously (s.c., 3 mg/kg) or topically (eye drop, 100 μM), and then an eye-wiping test was performed. Results: CSI caused partial corneal deinnervation followed by gradual reinnervation. Regenerated nerves displayed increased tortuosity, beading, and branching. CSI enhanced hypertonic saline-induced eye-wiping behavior compared to baseline or sham-injury (P \u3c 0.01). This hypersensitivity peaked at 10 days and subsided 14 days after CSI. Administration of NTX, or NLX-me, a selective peripheral opioid antagonist, reinstated eye-wiping behavior in the injury group, but not in the sham groups (P \u3c 0.05). Conclusions: This study introduces a model of chronic ocular pain and corneal neuropathy following CSI. CSI induces central and peripheral opioid receptor-dependent latent sensitization (LS) that is unmasked by systemic or topical administration of opioid antagonists. Translational Relevance: This model of chronic ocular pain establishes LS as a new inhibitory mechanism in the oculotrigeminal system and may be used for potential diagnostic and therapeutic interventions for ocular neuropathy

Chemical-potential standard for atomic Bose-Einstein condensates

When subject to an external time periodic perturbation of frequency $f$, a Josephson-coupled two-state Bose-Einstein condensate responds with a constant chemical potential difference $\Delta\mu=khf$, where $h$ is Planck's constant and $k$ is an integer. We propose an experimental procedure to produce ac-driven atomic Josephson devices that may be used to define a standard of chemical potential. We investigate how to circumvent some of the specific problems derived from the present lack of advanced atom circuit technology. We include the effect of dissipation due to quasiparticles, which is essential to help the system relax towards the exact Shapiro resonance, and set limits to the range of values which the various physical quantities must have in order to achieve a stable and accurate chemical potential difference between the macroscopic condensates.Comment: 13 pages, 4 figure