Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal μ-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the μ-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the μ-receptor antibody staining specificity. We found that the antibody specifically labeled μ-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of μ/δ/κ-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of μ-receptor expression in the DRG was not possible. However, we demonstrate that the μ-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, μ-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies' specificity, preferably using knockout animals.
The development of opioid addiction in subjects with established chronic pain is an area that is poorly understood. It is critically important to clearly understand the neurobiology associated with propensity toward conversion to addiction under conditions of chronic pain. To pose the question whether the presence of chronic pain influences motivation to self-administer opioids for reward, we applied a combination of rodent models of chronic mechanical hyperalgesia and opioid self-administration. We studied fentanyl self-administration in mice under three conditions that induce chronic mechanical hyperalgesia: inflammation, peripheral nerve injury, and repeated chemotherapeutic injections. Responding for fentanyl was compared among these conditions and their respective standard controls (naïve condition, vehicle injection or sham surgery). Acquisition of fentanyl self-administration behavior was reduced or absent in all three conditions of chronic hyperalgesia relative to control mice with normal sensory thresholds. To control for potential impairment in ability to learn the lever-pressing behavior or perform the associated motor tasks, all three groups were evaluated for acquisition of food-maintained responding. In contrast to the opioid, chronic hyperalgesia did not interfere with the reinforcing effect of food. These studies indicate that the establishment of chronic hyperalgesia is associated with reduced or ablated motivation to seek opioid reward in mice.
A large number of experimental and clinical studies have confirmed that brief remifentanil exposure can enhance pain sensitivity presenting as opioid-induced hyperalgesia (OIH). N-methyl-D-aspartate (NMDA) receptor antagonists have been reported to inhibit morphine analgesic tolerance in many studies. Recently, we found that glycogen synthase kinase-3β (GSK-3β) modulated NMDA receptor trafficking in a rat model of remifentanil-induced postoperative hyperalgesia. In the current study, it was demonstrated that GSK-3β inhibition prevented remifentanil-induced hyperalgesia via regulating the expression and function of spinal NMDA receptors in vivo and in vitro. We firstly investigated the effects of TDZD-8, a selective GSK-3β inhibitor, on thermal and mechanical hyperalgesia using a rat model of remifentanil-induced hyperalgesia. GSK-3β activity as well as NMDA receptor subunits (NR1, NR2A and NR2B) expression and trafficking in spinal cord L4-L5 segments were measured by Western blot analysis. Furthermore, the effects of GSK-3β inhibition on NMDA-induced current amplitude and frequency were studied in spinal cord slices by whole-cell patch-clamp recording. We found that remifentanil infusion at 1 μg·kg-1·min-1 and 2 μg·kg-1·min-1 caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate of the spinal cord dorsal horn and increased GSK-3β activity in spinal cord dorsal horn. GSK-3β inhibitor TDZD-8 significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was prevented with the application of TDZD-8. These results suggest that inhibition of GSK-3β can significantly ameliorate remifentanil-induced hyperalgesia via modulating the expression and function of NMDA receptors, which present useful insights into the mechanistic action of GSK-3β inhibitor as potential anti-hyperalgesic agents for treating OIH.
Multimodal analgesia is designed to optimize pain relief by coadministering drugs with distinct mechanisms of action or by combining multiple pharmacologies within a single molecule. In clinical settings, combinations of monoamine reuptake inhibitors and opioid receptor agonists have been explored and one currently available analgesic, tapentadol, functions as both a µ-opioid receptor agonist and a norepinephrine transporter inhibitor. However, it is unclear whether the combination of selective norepinephrine reuptake inhibition and µ-receptor agonism achieves an optimal antinociceptive synergy. In this study, we assessed the pharmacodynamic interactions between morphine and monoamine reuptake inhibitors that possess different affinities and selectivities for norepinephrine and serotonin transporters. Using the rat formalin model, in conjunction with measurements of ex vivo transporter occupancy, we show that neither the norepinephrine-selective inhibitor, esreboxetine, nor the serotonin-selective reuptake inhibitor, fluoxetine, produce antinociceptive synergy with morphine. Atomoxetine, a monoamine reuptake inhibitor that achieves higher levels of norepinephrine than serotonin transporter occupancy, exhibited robust antinociceptive synergy with morphine. Similarly, a fixed-dose combination of esreboxetine and fluoxetine which achieves comparable levels of transporter occupancy potentiated the antinociceptive response to morphine. By contrast, duloxetine, a monoamine reuptake inhibitor that achieves higher serotonin than norepinephrine transporter occupancy, failed to potentiate the antinociceptive response to morphine. However, when duloxetine was coadministered with the 5-HT3 receptor antagonist, ondansetron, potentiation of the antinociceptive response to morphine was revealed. These results support the notion that inhibition of both serotonin and norepinephrine transporters is required for monoamine reuptake inhibitor and opioid-mediated antinociceptive synergy; yet, excess serotonin, acting via 5-HT3 receptors, may reduce the potential for synergistic interactions. Thus, in the rat formalin model, the balance between norepinephrine and serotonin transporter inhibition influences the degree of antinociceptive synergy observed between monoamine reuptake inhibitors and morphine.
Lipoxins and resolvins have anti-inflammatory and pro-resolving actions and accumulating evidence indicates that these lipid mediators also attenuate pain-like behavior in a number of experimental models of inflammation and tissue injury-induced pain. The present study was undertaken to assess if spinal administration of lipoxin A4 (LXA4) or 17 (R)-resolvin D1 (17(R)-RvD1) attenuates mechanical hypersensitivity in the carrageenan model of peripheral inflammation in the rat. Given the emerging role of spinal cytokines in the generation and maintenance of inflammatory pain we measured cytokine levels in the cerebrospinal fluid (CSF) after LXA4 or 17(R)-RvD1 administration, and the ability of these lipid metabolites to prevent stimuli-induced release of cytokines from cultured primary spinal astrocytes. We found that intrathecal bolus injection of LXA4 and17(R)-RvD1 attenuated inflammation-induced mechanical hypersensitivity without reducing the local inflammation. Furthermore, both LXA4 and 17(R)-RvD1 reduced carrageenan-induced tumor necrosis factor (TNF) release in the CSF, while only 17(R)-RvD1attenuated LPS and IFN-γ-induced TNF release in astrocyte cell culture. In conclusion, this study demonstrates that lipoxins and resolvins potently suppress inflammation-induced mechanical hypersensitivity, possibly by attenuating cytokine release from spinal astrocytes. The inhibitory effect of lipoxins and resolvins on spinal nociceptive processing puts them in an intriguing position in the search for novel pain therapeutics.
Despite the subjective nature of pain experience with cognitive and affective dimensions, preclinical pain research has largely focused on its sensory dimension. Here, we examined the relationship between learning/memory and nociceptive behavior in rats with combined learning impairment and persistent nociception. Learning impairment was induced by bilateral hippocampal injection of a mixed Aβ solution, whereas persistent nociception produced in these rats by complete Freund’s adjuvant-induced ankle inflammation. Those rats with learning impairment showed a diminished development of thermal hyperalgesia and mechanical allodynia and a shorter time course of nociceptive behavior without alteration of their baseline nociceptive threshold. In rats with pre-established hyperalgesia and allodynia due to ankle inflammation, bilateral intra-hippocampal injection of cycloheximide (a protein synthesis inhibitor) promoted the earlier recovery of nociceptive behavior. Moreover, expression of Aβ, NR1 subunit of the N-methyl-D-aspartate receptor, and protein kinase Cγ was upregulated, whereas the choline acetyl transferase expression was downregulated, in the hippocampus, thalamus, amygdala, and/or spinal cord of rats with combined learning impairment and persistent nociception. The data indicate that learning impairment could disrupt the response to a state of persistent nociception, suggesting an important role for cognitive maladaptation in the mechanisms of chronic pain. These results also suggest that a preclinical model of combined learning impairment and persistent nociception may be useful to explore the brain mechanisms underlying the transition from acute to chronic pain.
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. PPARα 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 IKca and BKca channels on DRG neurons. In contrast to the peripheral site of action of PPARα ligands, the dorsal horn supports the expression of PPARγ. Intrathecal administration of PPARγ ligands rapidly (≤5 minutes) attenuated mechanical and thermal hypersensitivity associated with nerve injury in a dose-dependent manner that could be blocked with PPARγ antagonists. By contrast, a PPARγ antagonist itself rapidly increased the mechanical allodynia associated with nerve injury. These data suggest that ligand-dependent, non-genomic activation of spinal PPARγ 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 and PKCε translocation, and that this signaling may contribute to increased neuronal sensitivity as treatment with the GPR30 agonist induces hyperalgesia. Finally, application of the membrane-impermeable 17ß-E2-BSA rapidly (within 15 min) enhanced BK-stimulated inositol phosphate (IP) accumulation and PGE2-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.
Development of secondary hyperalgesia following a cutaneous injury is a centrally mediated, robust phenomenon. The pathophysiological role of endogenous opioid signalling to the development of hyperalgesia is unclear. Recent animal studies, carried out after the resolution of inflammatory pain, have demonstrated reinstatement of tactile hypersensitivity following administration of μ-opioid-receptor-antagonists. In the present study in humans, we analyzed the effect of naloxone when given after the resolution of secondary hyperalgesia following a first-degree burn injury.
Twenty-two healthy volunteers were included in this placebo-controlled, randomized, double-blind, cross-over study. Following baseline assessment of thermal and mechanical thresholds, a first-degree burn injury (BI; 47°C, 7 minutes, thermode area 12.5 cm2) was induced on the lower leg. Secondary hyperalgesia areas around the BI-area, and separately produced by brief thermal sensitization on the contralateral thigh (BTS; 45°C, 3 minutes, area 12.5 cm2), were assessed using a polyamide monofilament at pre-BI and 1, 2, and 3 hours post-BI. At 72 hrs, BI and BTS secondary hyperalgesia areas were assessed prior to start of a 30 minutes intravenous infusion of naloxone (total dose 21 microg/kg) or placebo. Fifteen minutes after start of the infusion, BI and BTS secondary hyperalgesia areas were reassessed, along with mechanical and thermal thresholds.
Secondary hyperalgesia areas were demonstrable in all volunteers 1–3 hrs post-BI, but were not demonstrable at 72 hrs post-burn in 73–86% of the subjects. Neither magnitude of secondary hyperalgesia areas nor the mechanical and thermal thresholds were associated with naloxone-treated compared to placebo-treated subjects.
Naloxone (21 microg/kg) did not reinstate secondary hyperalgesia when administered 72 hours after a first-degree burn injury and did not increase BTS-generated hyperalgesia. The negative results may be due to the low dose of naloxone or insufficient tissue injury to generate latent sensitization.
Changes in brain structure and cortical function are associated with many chronic pain conditions including low back pain and fibromyalgia. The magnitude of these changes correlates with the duration and/or the intensity of chronic pain. Most studies report changes in common areas involved in pain modulation, including the prefrontal cortex (PFC), and pain-related pathological changes in the PFC can be reversed with effective treatment. While the mechanisms underlying these changes are unknown, they must be dynamically regulated. Epigenetic modulation of gene expression in response to experience and environment is reversible and dynamic. Epigenetic modulation by DNA methylation is associated with abnormal behavior and pathological gene expression in the central nervous system. DNA methylation might also be involved in mediating the pathologies associated with chronic pain in the brain. We therefore tested a) whether alterations in DNA methylation are found in the brain long after chronic neuropathic pain is induced in the periphery using the spared nerve injury modal and b) whether these injury-associated changes are reversible by interventions that reverse the pathologies associated with chronic pain. Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by decreased global methylation in the PFC and the amygdala but not in the visual cortex or the thalamus. Environmental enrichment attenuated nerve injury-induced hypersensitivity and reversed the changes in global PFC methylation. Furthermore, global PFC methylation correlated with mechanical and thermal sensitivity in neuropathic mice. In summary, induction of chronic pain by peripheral nerve injury is associated with epigenetic changes in the brain. These changes are detected long after the original injury, at a long distance from the site of injury and are reversible with environmental manipulation. Changes in brain structure and cortical function that are associated with chronic pain conditions may therefore be mediated by epigenetic mechanisms.
Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.
The European ban on battery cages has forced a change towards the use of non-cage or furnished cage systems, but unexpectedly this has been associated with an increased prevalence of keel bone fractures in laying hens. Bone fractures are acutely painful in mammals, but the effect of fractures on bird welfare is unclear. We recently reported that keel bone fractures have an effect on bird mobility. One possible explanation for this is that flying becomes mechanically impaired. However it is also possible that if birds have a capacity to feel pain, then ongoing pain resulting from the fracture could contribute to decreased mobility. The aim was to provide proof of concept that administration of appropriate analgesic drugs improves mobility in birds with keel fracture; thereby contributing to the debate about the capacity of birds to experience pain and whether fractures are associated with pain in laying hens. In hens with keel fractures, butorphanol decreased the latency to land from perches compared with latencies recorded for these hens following saline (mean (SEM) landing time (seconds) birds with keel fractures treated with butorphanol and saline from the 50, 100 and 150 cm perch heights respectively 1.7 (0.3), 2.2 (0.3), p = 0.05, 50 cm; 12.5 (6.6), 16.9 (6.7), p = 0.03, 100 cm; 20.6 (7.4), 26.3 (7.6), p = 0.02 150 cm). Mobility indices were largely unchanged in birds without keel fractures following butorphanol. Critically, butorphanol can be considered analgesic in our study because it improved the ability of birds to perform a complex behaviour that requires both motivation and higher cognitive processing. This is the first study to provide a solid evidential base that birds with keel fractures experience pain, a finding that has significant implications for the welfare of laying hens that are housed in non-cage or furnished caged systems.
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.
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.
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.
Pain; AMPA receptor; Central sensitization; Calcium imaging
Ranolazine modulates the cardiac voltage-gated sodium channel (Nav 1.5) and is FDA-approved for 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 pre-clinical animal model of neuropathic pain. Both i.p. 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.
spared nerve injury; hyperalgesia; allodynia; rat; Nav 1.7; Nav 1.8
Current theories of neuropathic hypersensitivity include an imbalance of supraspinal inhibition and facilitation. Our overall hypothesis is that the locus coeruleus (LC), classically interpreted as a source of pain inhibition, may paradoxically result in facilitation after tibial and common peroneal nerve transection (spared sural nerve injury – SNI). We first tested the hypothesis that non-noxious tactile hindpaw stimulation of the spared sural innervation territory increases neuronal activity in the LC in male rats. We observed a bilateral increase in the stimulus-evoked expression of transcription factors Fos and phosphorylated CREB (pCREB) in LC after SNI but not sham surgery; these markers of neuronal activity correlated with the intensity of tactile allodynia. We next tested the hypothesis that noradrenergic neurons contribute to the development of neuropathic pain. To selectively destroy these neurons, we delivered anti-dopamine-β-hydroxylase saporin (anti-DβH-saporin) into the intracerebroventricular space two weeks before SNI. We found that anti-DβH-saporin, but not an IgG-saporin control, reduced behavioural signs of tactile allodynia, mechanical hyperalgesia, and cold allodynia from 3-28 d after SNI. Our final experiment tested the hypothesis that the LC contributes to the maintenance of neuropathic pain. We performed SNI, waited two weeks for maximal allodynia and hyperalgesia to develop, and then administered the local anaesthetic lidocaine (4%) directly into the LC parenchyma. Lidocaine reduced all behavioural signs of neuropathic pain in a reversible manner, suggesting that the LC contributes to pain facilitation. We conclude that, in addition to its well-known inhibition of acute and inflammatory pain, the LC facilitates the development and maintenance of neuropathic pain in the SNI model. Further studies are needed to determine the facilitatory pathways emanating from the LC.
Nerve injury increases the spinal cord expression and/or activity of voltage- and ligand-gated ion channels, peptide receptors, and neuro-immune factors that then drive dorsal horn neuron hyperexcitability. The intensity and duration of this central sensitization is determined by the net activity of local excitatory and inhibitory neurotransmitter systems, together with ongoing/evoked primary afferent activity and descending supraspinal control. Spinal endogenous inhibitory systems serve as opposing compensatory influences, and are gaining recognition for their powerful capacity to restrain allodynia and hyperalgesia. These include numerous G-protein coupled receptors (mu and delta opioid, α2-adrenergic, purinergic A1, neuropeptide Y Y1 and Y2, cannabinoid CB1 and CB2, muscarinic M2, GABAB, metabotropic glutamate type II-III, somatostatin) and perhaps nuclear receptors (PPARγ). Excessive down-regulation or defective compensatory up-regulation of these systems may contribute to the maintenance of neuropathic pain. An increasing number of pharmacotherapeutic strategies for neuropathic pain are emerging that mimic and enhance inhibitory neurotransmission in the dorsal horn.
We first demonstrate the transcription, expression, and DNA binding properties of the PPARγ subtype of the peroxisome proliferator-activated nuclear receptor family to the spinal cord with real time PCR, western blot, and electrophoretic mobility shift assay. To test the hypothesis that activation of spinal PPARγ decreases nerve injury-induced allodynia, we intrathecally administered PPARγ agonists and/or antagonists in rats following transection of the tibial and common peroneal branches of the sciatic nerve. Single injection of either a natural (15-deoxy-prostaglandin J2, 15d-PGJ2) or synthetic (rosiglitazone) PPARγ agonist dose-dependently decreased mechanical and cold hypersensitivity. These effects were maximal at a dose of 100μg and peaked at ~60 min after injection, a rapid time course suggestive of transcription-independent mechanisms of action. Concurrent administration of a PPARγ antagonist (bisphenol A diglycidyl ether, BADGE) reversed the effects of 15d-PGJ2 and rosiglitazone, further indicating a receptor-mediated effect. In animals without nerve injury, rosiglitazone did not alter motor coordination, von Frey threshold, or withdrawal response to a cool stimulus. Intraperitoneal and intracerebroventricular administration of PPARγ agonists (100μg) did not decrease mechanical and cold hypersensitivity, arguing against effects subsequent to diffusion from the intrathecal space. We conclude that ligand-induced activation of spinal PPARγ rapidly reverses nerve injury-induced mechanical allodynia. New or currently-available drugs targeted at spinal PPARγ may yield important therapeutic effects for the management of neuropathic pain.
PPARγ receptor agonists such as rosiglitazone and pioglitazone remain FDA approved as insulin sensitizers. We demonstrate PPARγ expression in the spinal cord, and report that activation of these receptors inhibits allodynia. BBB-permeant PPARγ agonists may yield important therapeutic effects for the management of neuropathic pain.
Brain microinjection studies in the rat using local anesthetics suggest that the rostral ventral medulla (RVM) contributes to the facilitation of neuropathic pain. However, these studies were restricted to a single model of neuropathic pain (the spinal nerve ligation model) and to just two stimulus modalities (non-noxious tactile stimulus and heat). Also, few neurotransmitter systems have been shown to modulate descending facilitation. After either partial sciatic nerve ligation (PSNL) or spared nerve injury (SNI), we found that unilateral or bilateral microinjection of lidocaine into the RVM reduced not only mechanical allodynia (decreased threshold to von Frey hairs and/or an automated device) and mechanical hyperalgesia (increased paw lifting in response to a noxious pin), but also cold hypersensitivity (increased lifting in response to the hindpaw application of a drop of acetone). Application of a drop of water did not elicit paw withdrawal, indicating that the acetone test is indeed a measure of cold hypersensitivity. We found significant neuropeptide Y Y1-like immunoreactivity within, and lateral to, the midline RVM. Intra-RVM injection of neuropeptide Y (NPY) dose-dependently inhibited the mechanical and cold hypersensitivity associated with PSNL or SNI, an effect that could be blocked by the Y1 receptor antagonist BIBO 3304. We conclude that medullary facilitation spans multiple behavioral signs of allodynia and hyperalgesia in multiple models of neuropathic pain. Furthermore, NPY inhibits behavioural signs of neuropathic pain, possibly by acting at Y1 receptors in the RVM.
descending facilitation; allodynia; hyperalgesia; spared nerve injury; lidocaine; rat
Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by inhibiting the activation of NF-κB, AP-1, and C/EBPβ and that gemfibrozil, a prescribed drug for humans, may further find its therapeutic use in neuroinflammatory diseases.