The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats.

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

The glial cell's role in antinociceptive differential effects of oxytocin upon female and male rats.

BACKGROUND: Sex plays a crucial role in pain processing and response to analgesic drugs. Indeed, spinal glia seems to be significant in the sexual dimorphism observed in the above effects. Recently, studies have associated oxytocin with antinociceptive effects, but these have been mainly performed in male animals; consequently, the influence of sex has been poorly explored. METHODS: Using a model of spinal nociception that produces pain through activation of the spinal glia, that is, intrathecal (i.t.) lipopolysaccharide (LPS) injection, we analysed the changes in the analgesic response to i.t. oxytocin in female and male rats by behavioural (punctate mechanical hypersensitivity), electrophysiological (unitary extracellular recordings of wide dynamic range [WDR] cells) and molecular biology (real-time PCR of proinflammatory genes) experiments. RESULTS: We found that LPS-induced hypersensitivity was longer in female (>96 h) than in male (≈4 h) rats. Besides, spinal oxytocin preferentially prevents the LPS-induced hypersensitivity in male rather than female rats. Indeed, LPS increases the spinal neuronal-evoked activity associated with the activation of peripheral Aδ- and C-fibres and post-discharge in males, whereas only C-fibre discharge was enhanced in females. The electrophysiological data correlate with the fact that spinal oxytocin only prevented TNF-α and IL-1ß synthesis in male rats. CONCLUSIONS: Therefore, these data suggest that oxytocin-mediated analgesia depends on a sexual dimorphism involving activation of the spinal glia. These results reinforced the idea that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men. SIGNIFICANCE STATEMENT: Oxytocin is a molecule that emerges as a potent analgesic in preclinical and clinical studies. We investigated the contribution of glia to the response of oxytocin-induced analgesia and how sex influences in this response show that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men.

Interaction of NHE1 and TRPA1 Activity in DRG Neurons Isolated from Adult Rats and its Role in Inflammatory Nociception.

Transient receptor potential ankyrin 1 (TRPA1) channel is expressed in a subset of nociceptive neurons. This channel integrates several nociceptive signals. Particularly, it is modulated by intracellular pH (pHi). Na+/H+ exchanger 1 (NHE1) contributes to the maintenance of pHi in nociceptors. However, it is currently unknown whether the interaction between TRPA1 and NHE1 contributes to the nociceptive processing. Thus, the purpose of this study was to assess the functional interaction between NHE1 and TRPA1 in small dorsal root ganglion (DRG) neurons from primary culture obtained from adult rats. Moreover, we also evaluated their possible interaction in acute and inflammatory pain. Zoniporide (selective NHE1 inhibitor) reduced pHi and increased intracellular calcium in a concentration-dependent fashion in DRG neurons. Zoniporide and allyl isothiocyanate (AITC, TRPA1 agonist) increased calcium transients in the same DRG neuron, whereas that A-967079 (TRPA1 antagonist) prevented the effect of zoniporide in DRG neurons. Repeated AITC induced TRPA1 desensitization and this effect was prevented by zoniporide. Both NHE1 and TRPA1 were localized at the membrane surface of DRG neurons in culture. Local peripheral zoniporide enhanced AITC-induced pronociception and this effect was prevented by A-967079. Likewise, zoniporide potentiated Complete Freund's Adjuvant (CFA)-induced hypersensitivity, effect which was prevented by A-967079 in vivo. CFA paw injection increased TRPA1 and decresed NHE1 protein expression in DRG. These results suggest a functional interaction between NHE1 and TRPA1 in DRG neurons in vitro. Moreover, data suggest that this interaction participates in acute and inflamatory pain conditions in vivo.

Sex-dependent pronociceptive role of spinal α5 -GABAA receptor and its epigenetic regulation in neuropathic rodents.

Extrasynaptic α5 -subunit containing GABAA (α5 -GABAA ) receptors participate in chronic pain. Previously, we reported a sex difference in the action of α5 -GABAA receptors in dysfunctional pain. However, the underlying mechanisms remain unknown. The aim of this study was to examine this sexual dimorphism in neuropathic rodents and the mechanisms involved. Female and male Wistar rats or ICR mice were subjected to nerve injury followed by α5 -GABAA receptor inverse agonist intrathecal administration, L-655,708. The drug produced an antiallodynic effect in nerve-injured female rats and mice, and a lower effect in males. We hypothesized that changes in α5 -GABAA receptor, probably influenced by hormonal and epigenetic status, might underlie this sex difference. Thus, we performed qPCR and western blot. Nerve injury increased α5 -GABAA mRNA and protein in female dorsal root ganglia (DRG) and decreased them in DRG and spinal cord of males. To investigate the hormonal influence over α5 -GABAA receptor actions, we performed nerve injury to ovariectomized rats and reconstituted them with 17ß-estradiol (E2). Ovariectomy abrogated L-655,708 antiallodynic effect and E2 restored it. Ovariectomy decreased α5 -GABAA receptor and estrogen receptor α protein in DRG of neuropathic female rats, while E2 enhanced them. Since DNA methylation might contribute to α5 -GABAA receptor down-regulation in males, we examined CpG island DNA methylation of α5 -GABAA receptor coding gene through pyrosequencing. Nerve injury increased methylation in male, but not female rats. Pharmacological inhibition of DNA methyltransferases increased α5 -GABAA receptor and enabled L-655,708 antinociceptive effect in male rats. These results suggest that α5 -GABAA receptor is a suitable target to treat chronic pain in females.

Blockade of spinal α5-GABAA receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats.

The role of spinal α5 subunit-containing GABAA (α5-GABAA) receptors in chronic pain is controversial. The purpose of this study was to investigate the participation of spinal α5-GABAA receptors in the reserpine-induced pain model. Reserpine administration induced tactile allodynia and muscle hyperalgesia in female and male rats. Intrathecal injection of L-655,708 and TB 21007 (7 days after the last reserpine injection) decreased tactile allodynia and, at a lesser extent, muscle hyperalgesia in female rats. The effects of these drugs produced a lower antiallodynic and antihyperalgesic effect in male than in female rats. Contrariwise, these drugs produced tactile allodynia and muscle hyperalgesia in naïve rats and these effects were lower in naïve male than female rats. Intrathecal L-838,417 prevented or reversed L-655,708-induced antiallodynia in reserpine-treated female rats. Repeated treatment with α5-GABAA receptor small interfering RNA (siRNA), but not scramble siRNA, reduced reserpine-induced allodynia in female rats. Accordingly, α5-GABAA receptor siRNA induced nociceptive hypersensitivity in naïve female rats. Reserpine enhanced α5-GABAA receptors expression in spinal cord and dorsal root ganglia (DRG), while it increased CD11b (OX-42) and glial fibrillary acidic protein (GFAP) fluorescence intensity in the lumbar spinal cord. In contrast, reserpine diminished K+-Cl- co-transporter 2 (KCC2) protein in the lumbar spinal cord. Data suggest that spinal α5-GABAA receptors play a sex-dependent proallodynic effect in reserpine-treated rats. In contrast, these receptors have a sex-dependent antiallodynic role in naïve rats.

α5GABAA receptors play a pronociceptive role and avoid the rate-dependent depression of the Hoffmann reflex in diabetic neuropathic pain and reduce primary afferent excitability.

Diabetic neuropathy is an incapacitating complication in diabetic patients. The cellular and molecular mechanisms involved in this pathology are poorly understood. Previous studies have suggested that the loss of spinal GABAergic inhibition participate in painful diabetic neuropathy. However, the role of extrasynaptic α5 subunit-containing GABAA (α5GABAA) receptors in this process is not known. The purpose of this study was to investigate the role of α5GABAA receptors in diabetes-induced tactile allodynia, loss of rate-dependent depression (RDD) of the Hoffmann reflex (HR), and modulation of primary afferent excitability. Intraperitoneal administration of streptozotocin induced tactile allodynia. Intrathecal injection of α5GABAA receptor inverse agonist, L-655,708, produced tactile allodynia in naive rats, whereas it reduced allodynia in diabetic rats. In healthy rats, electrical stimulation of the tibial nerve at 5 Hz induced RDD of the HR, although intrathecal treatment with L-655,708 (15 nmol) abolished RDD of the HR. Streptozotocin induced the loss of RDD of the HR, while intrathecal L-655,708 (15 nmol) restored RDD of the HR. L-655,708 (15 nmol) increased tonic excitability of the primary afferents without affecting the phasic excitability produced by the primary afferent depolarization. α5GABAA receptors were immunolocalized in superficial laminae of the dorsal horn and L4 to L6 dorsal root ganglion. Streptozotocin increased mean fluorescence intensity and percentage of neurons expressing α5GABAA receptors in dorsal horn and L4 to L6 dorsal root ganglia in 10-week diabetic rats. Our results suggest that spinal α5GABAA receptors modulate the HR, play an antinociceptive and pronociceptive role in healthy and diabetic rats, respectively, and are tonically active in primary afferents.

ATF2, but not ATF3, participates in the maintenance of nerve injury-induced tactile allodynia and thermal hyperalgesia.

Transcription factors are proteins that modulate the transcriptional rate of target genes in the nucleus in response to extracellular or cytoplasmic signals. Activating transcription factors 2 (ATF2) and 3 (ATF3) respond to environmental signals and maintain cellular homeostasis. There is evidence that inflammation and nerve injury modulate ATF2 and ATF3 expression. However, the function of these transcription factors in pain is unknown. The purpose of this study was to investigate the contribution of ATF2 and ATF3 to nerve injury-induced neuropathic pain. L5/6 spinal nerve ligation induced tactile allodynia and thermal hyperalgesia. Moreover, nerve damage enhanced ATF2 and ATF3 protein expression in injured L5/6 dorsal root ganglia and spinal cord but not in uninjured L4 dorsal root ganglia. Nerve damage also enhanced ATF2 immunoreactivity in dorsal root ganglia and spinal cord 7 to 21 days post-injury. Repeated intrathecal post-treatment with a small-interfering RNA targeted against ATF2 (ATF2 siRNA) or anti-ATF2 antibody partially reversed tactile allodynia and thermal hyperalgesia. In contrast, ATF3 siRNA or anti-ATF3 antibody did not modify nociceptive behaviors. ATF2 immunoreactivity was found in dorsal root ganglia and spinal cord co-labeling with NeuN mainly in non-peptidergic (IB4+) but also in peptidergic (CGRP+) neurons. ATF2 was found mainly in small- and medium-sized neurons. These results suggest that ATF2, but not ATF3, is found in strategic sites related to spinal nociceptive processing and participates in the maintenance of neuropathic pain in rats.

Ultra-Low Doses of Naltrexone Enhance the Antiallodynic Effect of Pregabalin or Gabapentin in Neuropathic Rats.

Preclinical Research Treatment of neuropathic pain is an area of largely unmet medical need. Pregabalin and gabapentin are anticonvulsants widely used for the treatment of neuropathic pain. Unfortunately, these drugs are only effective in 50-60% of the treated patients. In addition, both drugs have substantial side effects. Several studies have reported that ultralow doses of opioid receptor antagonists can induce analgesia and enhance the analgesic effect of opioids in rodents and humans. The objective of the present study was to assess the antiallodynic synergistic interaction between gabapentinoids and naltrexone in rats. Oral administration of pregabalin (ED50 = 2.79 ± 0.16 mg/kg) or gabapentin (ED50 = 21.04 ± 2.87 mg/kg) as well as intrathecal naltrexone (ED50 = 0.11 ± 0.02 ng) reduced in a dose-dependent manner tactile allodynia in rats. Maximal antiallodynic effects (∼100%) were reached with 30 mg/kg of pregabalin, 300 mg/kg of gabapentin or 0.5 ng of naltrexone. Co-administration of pregabalin or gabapentin and naltrexone in a fixed-dose ratio (1:1) remarkably reduced spinal nerve ligation-induced tactile allodynia showing a synergistic interaction. The data indicate that combinations of pregabalin or gabapentin and ultra-low doses of naltrexone are able to reduce tactile allodynia in neuropathic rats with lower doses that those used when drugs are given individually and with an improved side effects profile. Drug Dev Res 78 : 371-380, 2017. © 2017 Wiley Periodicals, Inc.