Renee R. Donahue

Constitutive μ-Opioid Receptor Activity Leads to Long-Term Endogenous Analgesia and Dependence

Pain and Dependence The properties and functions of µ-opioid receptors have been studied intensively with respect to the binding of endogenous or exogenous ligands. However, much less is known about the constitutive, ligand-independent, activation of opioid receptors. Working in mice, Corder et al. (p. 1394) observed the prolonged constitutive activation of µ-opioid receptors in the spinal dorsal horn after transient peripheral inflammation. The results suggest that constitutive activation of µ-opioid receptors depresses nociception—the perception of pain—for long periods of time and induces cellular and physical dependence on endogenous opioid signaling. Transient inflammation can lead to prolonged activation of pain-relieving opioid receptors in the spinal cord. Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced μ-opioid receptor (MOR) constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3′,5′-monophosphate overshoot and hyperalgesia) that required N-methyl-d-aspartate receptor activation of adenylyl cyclase type 1. Thus, MORCA initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MORCA suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Tonic inhibition of chronic pain by neuropeptide Y

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.

Activation of peroxisome proliferator-activated receptor γ in brain inhibits inflammatory pain, dorsal horn expression of Fos, and local edema

Systemic administration of thiazolidinediones reduces peripheral inflammation in vivo, presumably by acting at peroxisome proliferator-activated receptor gamma (PPARgamma) in peripheral tissues. Based on a rapidly growing body of literature indicating the CNS as a functional target of PPARgamma actions, we postulated that brain PPARgamma modulates peripheral edema and the processing of inflammatory pain signals in the dorsal horn of the spinal cord. To test this in the plantar carrageenan model of inflammatory pain, we measured paw edema, heat hyperalgesia, and dorsal horn expression of the immediate-early gene c-fos after intracerebroventricular (ICV) administration of PPARgamma ligands or vehicle. We found that ICV rosiglitazone (0.5-50 microg) or 15d-PGJ(2) (50-200 microg), but not vehicle, dose-dependently reduced paw thickness, paw volume and behavioral withdrawal responses to noxious heat. These anti-inflammatory and anti-hyperalgesia effects result from direct actions in the brain and not diffusion to other sites, because intraperitoneal and intrathecal administration of rosiglitazone (50 microg) and 15d-PGJ(2) (200 microg) had no effect. PPARgamma agonists changed neither overt behavior nor motor coordination, indicating that non-specific behavioral effects do not contribute to PPAR ligand-induced anti-hyperalgesia. ICV administration of structurally dissimilar PPARgamma antagonists (either GW9662 or BADGE) reversed the anti-inflammatory and anti-hyperalgesic actions of both rosiglitazone and 15d-PGJ(2). To evaluate the effects of PPARgamma agonists on a classic marker of noxious stimulus-evoked gene expression, we quantified Fos protein expression in the dorsal horn. The number of carrageenan-induced Fos-like immunoreactive profiles was less in rosiglitazone-treated rats as compared to vehicle controls. We conclude that pharmacological activation of PPARgamma in the brain rapidly inhibits local edema and the spinal transmission of noxious inflammatory signals.

PPARγ activation blocks development and reduces established neuropathic pain in rats

Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a new pharmacotherapeutic target for chronic pain. When oral (3-30 mg/kg/day in chow for 7 wk) or twice-daily intraperitoneal (1-10 mg/kg/day for 2 wk) administration began before spared nerve injury (SNI), pioglitazone, a PPARγ agonist, dose-dependently prevented multiple behavioral signs of somatosensory hypersensitivity. The highest dose of intraperitoneal pioglitazone did not produce ataxia or reductions in transient mechanical and heat nociception, indicating that inhibitory effects on hypersensitivity were not secondary to adverse drug-induced behaviors or antinociception. Inhibitory effects on hypersensitivity persisted at least one week beyond cessation of pioglitazone administration, suggestive of long-lasting effects on gene expression. Blockade of PPARγ with GW9662, an irreversible and selective PPARγ antagonist, dose-dependently reduced the inhibitory effect of pioglitazone on hypersensitivity, indicating a PPARγ-dependent action. Remarkably, a single preemptive injection of pioglitazone 15 min before SNI attenuated hypersensitivity for at least 2 weeks; this was enhanced with a second injection delivered 12 h after SNI. Pioglitazone injections beginning after SNI also reduced hypersensitivity, albeit to a lesser degree than preemptive treatment. Intraperitoneal pioglitazone significantly reduced the nerve injury-induced up-regulation of cd11b, GFAP, and p-p38 in the dorsal horn, indicating a mechanism of action involving spinal microglia and/or astrocyte activation. Oral pioglitazone significantly reduced touch stimulus-evoked phospho-extracellular signal-related kinase (p-ERK) in lamina I-II, indicating a mechanism of action involving inhibition of central sensitization. We conclude that pioglitazone reduces spinal glial and stimulus-evoked p-ERK activation and that PPARγ activation blocks the development of and reduces established neuropathic pain.

Endogenous Opioid-Masked Latent Pain Sensitization: Studies from Mouse to Human

Following the resolution of a severe inflammatory injury in rodents, administration of mu-opioid receptor inverse agonists leads to reinstatement of pain hypersensitivity. The mechanisms underlying this form of latent pain sensitization (LS) likely contribute to the development of chronic pain, but LS has not yet been demonstrated in humans. Using a C57BL/6 mouse model of cutaneous mild heat injury (MHI) we demonstrated a dose-dependent reinstatement of pain sensitization, assessed as primary (P < 0.001) and secondary hyperalgesia (P < 0.001) by naloxone (0.3–10 mg/kg), 168 hrs after the induction of MHI. Forward-translating the dose data to a human MHI model (n = 12) we could show that LS does indeed occur after naloxone 2 mg/kg, 168 hrs after a MHI. Our previous unsuccessful efforts to demonstrate unmasking of LS in humans are thus likely explained by an insufficient naloxone dose (0.021 mg/kg). However, while LS was consistently demonstrated in 21/24 mice, LS was only seen in 4/12 subjects. This difference is likely due to selection bias since the C57BL/6 mouse strain exhibits markedly enhanced pain sensitivity in assays of acute thermal nociception. Future exploratory studies in humans should prioritize inclusion of “high-sensitizers” prone to develop LS and use post-surgical models to elucidate markers of vulnerability to chronic postsurgical pain. Trial Registration EudraCT 2012-005663-27

Ranolazine Attenuates Behavioral Signs of Neuropathic Pain

Ranolazine modulates the cardiac voltage-gated sodium channel (NaV 1.5) and is approved by the FDA in the treatment of ischemic heart disease. Ranolazine also targets neuronal (NaV 1.7, 1.8) isoforms that are implicated in neuropathic pain. Therefore, we determined the analgesic efficacy of ranolazine in a preclinical animal model of neuropathic pain. Both intraperitoneal and oral administration of ranolazine dose-dependently inhibited the mechanical and cold allodynia associated with spared nerve injury, without producing ataxia or other behavioral side effects. These data warrant clinical investigation of the potential use of ranolazine in the treatment of neuropathic pain.

Mechanical Conflict System: A Novel Operant Method for the Assessment of Nociceptive Behavior.

A new operant test for preclinical pain research, termed the Mechanical Conflict System (MCS), is presented. Rats were given a choice either to remain in a brightly lit compartment or to escape to a dark compartment by crossing an array of height-adjustable nociceptive probes. Latency to escape the light compartment was evaluated with varying probe heights (0, .5, 1, 2, 3, and 4 mm above compartment floor) in rats with neuropathic pain induced by constriction nerve injury (CCI) and in naive control rats. Escape responses in CCI rats were assessed following intraperitoneal administration of pregabalin (10 and 30 mg/kg), morphine (2.5 and 5 mg/kg), and the tachykinin NK1 receptor antagonist, RP 67580 (1 and 10 mg/kg). Results indicate that escape latency increased as a function of probe height in both naive and CCI rats. Pregabalin (10 and 30 mg/kg) and morphine (5 mg/kg), but not RP 67580, decreased latency to escape in CCI rats suggesting an antinociceptive effect. In contrast, morphine (10 mg/kg) but not pregabalin (30 mg/kg) increased escape latency in naive rats suggesting a possible anxiolytic action of morphine in response to light-induced fear. No order effects following multiple test sessions were observed. We conclude that the MCS is a valid method to assess behavioral signs of affective pain in rodents.

Pioglitazone rapidly reduces neuropathic pain through astrocyte and nongenomic PPARγ mechanisms

Abstract Repeated administration of peroxisome proliferator–activated receptor gamma (PPAR&ggr;) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPAR&ggr; is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal (i.t.) injection of pioglitazone, a PPAR&ggr; agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid antihyperalgesic actions of PPAR&ggr; activation, we administered pioglitazone to rats with spared nerve injury and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 minutes of injection, consistent with a nongenomic mechanism. Systemic or i.t. administration of GW9662, a PPAR&ggr; antagonist, inhibited the antihyperalgesic actions of intraperitoneal or i.t. pioglitazone, suggesting a spinal PPAR&ggr;-dependent mechanism. To further address the contribution of nongenomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When coadministered intrathecally, anisomycin did not change pioglitazone antihyperalgesia at an early 7.5-minute time point, further supporting a rapid nongenomic mechanism. At later time points, anisomycin reduced pioglitazone antihyperalgesia, suggesting delayed recruitment of genomic mechanisms. Pioglitazone reduction of spared nerve injury–induced increases in GFAP expression occurred more rapidly than expected, within 60 minutes. We are the first to show that activation of spinal PPAR&ggr; rapidly reduces neuropathic pain independent of canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation and through both genomic and nongenomic PPAR&ggr; mechanisms.

Single intrathecal administration of the transcription factor decoy AYX1 prevents acute and chronic pain after incisional, inflammatory, or neuropathic injury

Summary AYX1 is a DNA decoy designed to specifically inhibit, in the spinal cord and dorsal root ganglia network, the trauma‐induced transcription factor EGR1 responsible for long‐term neuronal hyper‐excitability. A single intrathecal administration of AYX1 around the time of surgery or trauma prevents acute and chronic pain in animal models of inflammatory, incisional, bone, and neuropathic pain. ABSTRACT 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.

Pioglitazone inhibits the development of hyperalgesia and sensitization of spinal nociresponsive neurons in type 2 diabetes

UNLABELLED Thiazolidinedione drugs (TZDs) such as pioglitazone are approved by the U.S. Food and Drug Administration for the treatment of insulin resistance in type 2 diabetes. However, whether TZDs reduce painful diabetic neuropathy (PDN) remains unknown. Therefore, we tested the hypothesis that chronic administration of pioglitazone would reduce PDN in Zucker Diabetic Fatty (ZDF(fa/fa) [ZDF]) rats. Compared with Zucker Lean (ZL(fa/+)) controls, ZDF rats developed: (1) increased blood glucose, hemoglobin A1c, methylglyoxal, and insulin levels; (2) mechanical and thermal hyperalgesia in the hind paw; (3) increased avoidance of noxious mechanical probes in a mechanical conflict avoidance behavioral assay, to our knowledge, the first report of a measure of affective-motivational pain-like behavior in ZDF rats; and (4) exaggerated lumbar dorsal horn immunohistochemical expression of pressure-evoked phosphorylated extracellular signal-regulated kinase. Seven weeks of pioglitazone (30 mg/kg/d in food) reduced blood glucose, hemoglobin A1c, hyperalgesia, and phosphorylated extracellular signal-regulated kinase expression in ZDF. To our knowledge, this is the first report to reveal hyperalgesia and spinal sensitization in the same ZDF animals, both evoked by a noxious mechanical stimulus that reflects pressure pain frequently associated with clinical PDN. Because pioglitazone provides the combined benefit of reducing hyperglycemia, hyperalgesia, and central sensitization, we suggest that TZDs represent an attractive pharmacotherapy in patients with type 2 diabetes-associated pain. PERSPECTIVE To our knowledge, this is the first preclinical report to show that: (1) ZDF rats exhibit hyperalgesia and affective-motivational pain concurrent with central sensitization; and (2) pioglitazone reduces hyperalgesia and spinal sensitization to noxious mechanical stimulation within the same subjects. Further studies are needed to determine the anti-PDN effect of TZDs in humans.