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The purpose of this study was to examine the changes in cold block of unmyelinated C-fibers in the tibial nerve by pre-conditioning with heating and to develop a safe method for thermal block of C-fiber conduction. In 7 cats under α-chloralose anesthesia, C-fiber evoked potentials elicited by electrical stimulation were recorded on the tibial nerve during block of axonal conduction induced by exposing a small segment (9 mm) of the nerve to cooling (from 35 °C to ≤5 °C) or heating (45 °C). Before heating partial, reproducible, and reversible cold block was first detected at a threshold cold block temperature of 15 °C and complete cold block occurred at a temperature of ≤5 °C. After heating the nerve at 45 °C for 5-35 minutes the threshold cold block temperature significantly (p<0.05) increased from 15° C to 25 °C and the complete cold block temperature significantly (p<0.05) increased from ≤5 °C to 15°C on average. The increased cold block temperatures persisted for the duration of the experiments (30-100 minutes) while the amplitude of the C-fiber evoked potential measured at 35 °C recovered significantly (p<0.05) to about 80% of control. This study discovered a novel thermal method to block mammalian C-fibers at an elevated temperature (15-25 °C), providing the opportunity to develop a thermal nerve block technology to suppress chronic pain of peripheral origin. The interaction between heating and cooling effects on C-fiber conduction indicates a possible interaction between different temperature sensitive channels known to be present in the mammalian C-fibers.
Learn More >Natural or synthetic ligands for peroxisome proliferator-activated receptor gamma (PPAR-γ) represent an interesting tool for pharmacological interventions to treat inflammatory conditions. In particular, PPAR-γ activation prevents pain and inflammation in the temporomandibular joint (TMJ) by decreasing cytokine release and stimulating the synthesis of endogenous opioids. The goal of this study was to clarify whether PPAR-γ activation induces macrophage polarization, inhibiting inflammatory cytokine release and leukocyte recruitment. In addition, we investigated the involvement of heme oxygenase 1 (HO-1) in downstream events after PPAR-γ activation. Our results demonstrate that PPAR-γ activation ablates cytokine release by Bone Marrow-Derived Macrophages (BMDM) in vitro. 15d-PGJ induces the PPAR-γ heterodimer activation from rat macrophages, with macrophage polarization from M1-like cells toward M2-like cells. This response is mediated through HO-1. PPAR-γ activation diminished neutrophil migration induced by carrageenan, which was also HO-1 dependent. Ca/calmodulin expression did not change after PPAR-γ activation indicating that is not required for the activation of the intracellular L-arginine/NO/cGMP/K channel pathway. In summary, the anti-inflammatory actions induced by PPAR-γ activation involve macrophage polarization. HO-1 expression is increased and HO-1 activity is required for the suppression of neutrophil migration.
Learn More >Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating μ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.
Learn More >Recently the voltage-gated sodium (Nav) channels began to be studied as possible targets for analgesic drugs. In addition, specific Nav 1.8 blockers are currently being used to treat some types of chronic pain pathologies such as neuropathies and fibromyalgia. Nav 1.8 fibers convey nociceptive information to brain structures belonging to the limbic system, which is involved in the pathophysiology of major depressive disorders. From this, using a model of chronic social defeat stress (SDS) and intrathecal injections of Nav 1.8 antisense, this study investigated the possible involvement of Nav 1.8 nociceptive fibers in SDS- induced hyperalgesia in C57/BL mice. Our results showed that SDS induced a depressive-like behavior of social avoidance and increased the sensitivity to mechanical (electronic von Frey test) and chemical (capsaicin test) nociceptive stimuli. We also showed that intrathecal injection of Nav 1.8 antisense reversed the SDS-induced hyperalgesia as demonstrated by both, mechanical and chemical nociceptive tests. We confirmed the antisense efficacy and specificity in a separate no-defeated cohort through real-time PCR, which showed a significant reduction of Nav 1.8 mRNA and no reduction of Nav 1.7 and Nav 1.9 in the L4, L5 and L6 dorsal root ganglia (DRG). The present study advances the understanding of SDS-induced hyperalgesia, which seems to be dependent on Nav 1.8 nociceptive fibers.
Learn More >Voltage gated sodium channel NaV1.7 is a genetically validated target for pain. Identification of NaV1.7 inhibitors with all the desired properties to develop as an oral therapeutic for pain has been a major challenge. Herein, we report systematic SAR studies carried out to identify novel sulfonamide derivatives as potent, selective and state-dependent NaV1.7 inhibitors for pain. Scaffold hopping from benzoxazine to chroman and indane bicyclic system followed by thiazole replacement on sulfonamide led to identification of lead molecules with significant improvement in solubility, selectivity over NaV1.5 and CYP2C9 inhibition. The lead molecules 13, 29, 32, 43 and 51 have shown favorable PK profile across different species and robust efficacy in veratridine and formalin induced inflammatory pain models in mice. Compound 51 has also shown significant effect in CCI induced neuropathic pain model. Profile of 51 has indicated that it has the potential for further evaluation as a therapeutic for pain.
Learn More >Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here a drug-design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II-VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CA II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.
Learn More >Pain is one of the most common reasons to seek medical attention and chronic pain is a worldwide epidemic. There are currently no relevant biomarkers for the diagnosis of chronic pain, and new therapeutic strategies for chronic pain treatment are desperately needed. The chronic constriction injury (CCI) of the sciatic nerve is a widely used preclinical model of pathological neuropathic pain. Over the past decade, investigators have come to appreciate the many contributions of noncoding RNA including microRNA (miRNA), and other long and short noncoding (nc) RNAs. The development and/or maintenance of chronic pain could be controlled epigenetically through ncRNAs. Here we seek to characterize CNS tissues in a mouse model of neuropathic pain as this may serve to elucidate potential biomarkers relevant to pathological pain in humans. Male C57BL6/J mice (6 CCI and 6 sham procedure) underwent surgery for sciatic nerve ligation with chromic gut sutures. Following 7 days, mechanical allodynia was quantified using the von Frey assay. Mice were then euthanized for collection of spinal cord and sciatic nerve. cDNA was synthesized to 627 unique mature miRNAs from the total RNA. In the CCI mice that displayed mechanical allodynia, 11 and 125 miRNAs were differentially expressed (i.e., greater than 1.5-fold increase or decrease; P < 0.05) in the spinal cord and sciatic nerve, respectively, as compared to sham controls. Among those differentially expressed miRNAs in the sciatic nerve of CCI mice, the following passed the more stringent Bonfferoni correction: miR-138-3p, miR-138-5p and miR-676-3p, reduced and miR-142-5p, increased. Our data support miRNAs as promising therapeutic targets for the treatment of pathological pain.
Learn More >Single cell sequencing has provided unprecedented information about the transcriptomic diversity of somatosensory systems. Here we describe a simple and versatile in situ hybridization (ISH) based approach for mapping this information back to the tissue. We illustrate the power of this approach by demonstrating that ISH localization with just eight probes is sufficient to distinguish all major classes of neurons in sections of the trigeminal ganglion. To further simplify the approach and make transcriptomic class assignment and cell segmentation automatic we developed a machine learning approach for analyzing images from multiprobe ISH experiments. We demonstrate the power of in situ class assignment by examining the expression patterns of voltage gated sodium channels that play roles in distinct somatosensory processes and pain. Specifically, this analysis resolves intrinsic problems with single cell sequencing related to the sparseness of data leading to ambiguity about gene expression patterns. We also used the multiplex in situ approach to study the projection fields of the different neuronal classes. Our results demonstrate that the surface of the eye and meninges are targeted by broad arrays of neural classes despite their very different sensory properties but exhibit idiotypic patterns of innervation at a quantitative level. Very surprisingly, itch related neurons extensively innervated the meninges, indicating that these transcriptomic cell classes are not simply labeled lines for triggering itch. Together, these results substantiate the importance of a sensory neuron's peripheral and central connections as well as its transcriptomic class in determining its role in sensation.
Learn More >Recent studies have drawn the attention to the link between Alcohol Use Disorder (AUD) and the presence of pain. Indeed, the correct management of pain in patients with a previous history of AUD has been reported to decrease the risk of relapse in alcohol drinking, suggesting that in this prone population, pain may increase the vulnerability to relapse. Previous data in male rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA) and increases intake of high doses of heroine. Due to the relevant role of MORs in alcohol effects, we hypothesize that pain may also alter alcohol reinforcing properties and therefore affect alcohol relapse in male rats. Our microdialysis studies show that the presence of inflammatory pain blunted the increase of extracellular dopamine levels in the Nucleus Accumbens induced by 1.5g/kg of ethanol (s.c.). Moreover, we also revealed that the administration of 52 nmol of ethanol into the VTA failed to induce place preference only in inflammatory pain-suffering animals, and a higher dose (70nmol) was necessary to reverse this effect. Finally, we evaluated the effect of inflammatory pain on the alcohol deprivation effect (ADE) in long-term ethanol-experienced male rats. After four cycles of free ethanol intake and abstinence periods, inflammatory pain induced ADE without affecting its magnitude. These intriguing data reveals the impact of pain on neurochemical and behavioral effects following alcohol administration but also underscore the necessity of finding an appropriate paradigm to determine the long-term behavioral consequences.
Learn More >Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently their activity has been related to the block of N-type voltage-gated Ca channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing post-inflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intra-colonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 ml 50% EtOH). Visceral hypersensitivity was assessed by measuring the viscero-motor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the post-inflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in DRG neurons isolated from either control or DNBS-treated rats. The effect on Ca current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.
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