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The κ-opioid receptor (KOR) system has been implicated in the regulation of many behaviors including pain. While there are numerous studies suggesting KOR regulation of pain being mediated spinally, there have been reports of pain-like behaviors regulated by central KOR signaling. In particular, oxytocin-induced analgesia appears to be mediated by KOR receptors within the ventrolateral periaqueductal gray (vlPAG). We recently found that activation of dopamine (DA) neurons within the vlPAG is antinociceptive. In this study, we sought to determine the impact of KOR signaling on -GABAergic inputs onto vlPAG DA neurons, and the mechanism through which KOR impacts these inputs. We found that activation of KOR reduced GABAergic transmission onto vlPAG DA neurons. In addition, our data suggest this effect is mediated presynaptically via the G protein βγ-subunit. They raise the possibility that KOR activation disinhibits -vlPAG DA neurons, which could lead to altered regulation of pain-related behaviors.
Neuropathic pain represents one of the most common complications associated with diabetes mellitus (DM) that impacts quality of life. Accumulating studies have highlighted the involvement of microRNAs (miRNAs) in DM. Thus, the current study aimed to investigate the roles of microRNA-155 (miR-155) in diabetic peripheral neuropathy (DPN). In vitro DPN models were established using rat Schwann cells (SCs) by treatment with 5.5 mM glucose. Gain- or loss-of-function studies were conducted to determine the effect of miR-155 on Nrf2, cellular function, reactive oxygen species, and inflammation. Rat DNP models were established by streptozotocin injection and damage of sciatic nerve. Next, miR-155 antagomir or agomir was employed to investigate the effects associated with miR-155 on motor and sciatic nerve conduction velocity (MNCV, SNCV), angiogenesis and inflammatory response in vivo. Nrf2 was identified to be a target of miR-155 by dual-luciferase reporter gene assay. Silencing of miR-155 or restoration of Nrf2 promoted cell proliferation, inhibited apoptosis and alleviated inflammation in vitro. miR-155 antagomir-induced inhibition increased MNCV and SNCV, strengthened angiogenesis and alleviated inflammation in DPN rats. Additionally, the effects exerted by miR-155 were reversed when Nrf2 was restored both in vitro and in vivo. Taken together, the key findings of our study provide evidence indicating that miR-155 targeted and suppressed Nrf2 in DPN. miR-155 silencing was found to alleviate sciatic nerve injury in DPN, highlighting its potential as a therapeutic target for DPN.
Spinal cord stimulation (SCS) is used clinically to limit chronic pain but questions remain on the identity of axonal populations recruited. We developed an adult mouse spinal cord preparation to assess recruitment following delivery of clinically-analogous stimuli determined by downscaling a finite element model of clinical SCS. Analogous electric field distributions were generated with 300 µm x 300 µm electrodes positioned 200 µm above the dorsal column (DC) with stimulation between 50-200 µA. We compared axonal recruitment using electrodes of comparable size and stimulus amplitudes – when contacting the caudal thoracic DC, and at 200 or 600 mm above. Antidromic responses recorded from the DC, the adjacent Lissauer tract (LT), and in dorsal roots (DRs) were found to be amplitude and site-dependent. Responses in the DC included a unique component not seen in DRs, having the lowest SCS recruitment amplitude and fastest conduction velocity. At 200 mm above, mean cathodic SCS recruitment threshold for axons in DRs and LT were 2.6 and 4.4 times higher, respectively, than DC threshold. SCS recruited primary afferents in all (up to 9) caudal segments sampled. While A and C fibers could be recruited at nearby segments, only A fiber recruitment and synaptically-mediated dorsal root reflexes were observed in more distant segments. In sum, clinically-analogous SCS led to multisegmental recruitment of several somatosensory-encoding axonal populations. Most striking is the possibility that the lowest threshold recruitment of a non-primary afferent population in the DC are postsynaptic dorsal column tract cells (PSDCs) projecting to gracile nuclei.
The specific mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated neuropathic pain at the spinal cord level remain elusive. The aim of the present study was to explore the role of crosstalk between Cdk5/p35 and extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in mediating spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury (CCI). Here, we quantified pain behaviour after CCI; detected the localization of p35, Cdk5, phosphorylated ERK1/2 (pERK1/2), phosphorylated peroxisome proliferator-activated receptor γ (pPPARγ), neuronal nuclei (NeuN, a neuronal marker), glial fibrillary acidic protein (GFAP, an activated astrocyte marker) and ionized calcium binding adaptor molecule 1 (IBA1, a microglial marker) in the dorsal horn (DH) using immunofluorescence; measured the protein levels of Cdk5, p35, pERK1/2, pPPARγ and GFAP using Western blot analysis; and gauged the enzyme activity of Cdk5/p35 kinase by a Cdk5/p35 kinase activity assay kit. Tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 levels were measured by ELISA. Ligation of the right sciatic nerve induced mechanical allodynia; thermal hyperalgesia; and the time-dependent upregulation of p35, pERK1/2 and GFAP and downregulation of pPPARγ. p35 colocalized with Cdk5, pERK1/2, pPPARγ, neurons and astrocytes but not microglia. Meanwhile, intrathecal injection of the Cdk5 inhibitor roscovitine, the mitogen-activated ERK kinase (MEK) inhibitor U0126 and the PPARγ agonist pioglitazone prevented or reversed behavioural allodynia, increased pPPARγ expression, inhibited astrocyte activation, and alleviated proinflammatory cytokine (TNFα, IL-1β, and IL-6) release from activated astrocytes. Furthermore, crosstalk between the Cdk5/p35 and ERK1/2 pathways was observed with CCI. Blockade of either Cdk5/p35 or ERK1/2 inhibited Cdk5 activity. These findings indicate that spinal crosstalk between the Cdk5/p35 and ERK1/2 pathways mediates astrocyte activity via the PPARγ pathway in CCI rats and that targeting this crosstalk could be an effective strategy to attenuate CCI and astrocyte-derived neuroinflammation. This article is protected by copyright. All rights reserved.
Injury associated pain involves subjective perception and emotional experience. The anterior cingulate cortex (ACC) is a key area involved in the affective component of pain processing. However, the neuroimmune mechanisms underlying enhanced ACC excitability following peripheral nerve injury are still not fully understood. Our previous work has shown that tumor necrosis factor-alpha (TNF-α) overexpression leads to peripheral afferent hyperexcitability and synaptic transmission potentiation in spinal cord. Here, we aimed to reveal the potential role of ACC TNF-α in ACC hyperexcitability and neuropathic pain. c-Fos, a widely used neuronal activity marker, was induced especially in contralateral ACC early [postoperative (PO) 1 h] and later (PO day 7 and 10) during the development of neuropathic pain. Spared nerve injury (SNI) elevated TNF-α level in contralateral ACC from PO day 5 to 14, delayed relative to decreased ipsilateral paw withdrawal threshold apparent from PO day 1 to 14. Microinjection of anti-TNF-α antibody into the ACC completely eliminated c-Fos overexpression and greatly attenuated pain aversion and mechanical allodynia induced by SNI, suggesting an important role of ACC TNF-α in the pain aversiveness and pain maintenance. Furthermore, modulating ACC pyramidal neurons via a Gi-coupled human M4 muscarinic receptor (hM4Di) or a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD), greatly changed the ACC TNF-α level and the mechanical paw withdrawal threshold. The positive interactions between TNF-α and ACC neurons might modulate the cytokine microenvironment thus contribute to the neuropathic pain.
Membrane metallo-endopeptidase (MME), also known as neprilysin (NEP), has been of interest for its role in neurodegeneration and pain due to its ability to degrade β-amyloid and substance-P, respectively. In addition to its role in the central nervous system, MME has been reported to be expressed in the peripheral system, specifically in the inner and outer border of myelinating fibers, in the Schmidt-Lantermann cleft and in the paranodes. Recently, mutations of this gene have been associated with Charcot-Marie-Tooth Type 2 (CMT2). Peripheral nerve morphometry in mice lacking MME previously showed minor abnormalities in aged animals in comparison to CMT2 patients. We found that MME expression was dysregulated after nerve injury in a Neuregulin-1 dependent fashion. We therefore explored the hypothesis that MME may have a role in remyelination. In the naïve state in adulthood we did not find any impairment in myelination in MME KO mice. After nerve injury the morphological outcome in MME KO mice was indistinguishable from WT littermates in terms of axon regeneration and remyelination. We did not find any difference in functional motor recovery. There was a significant difference in sensory function, with MME KO mice starting to recover response to mechanical stimuli earlier than WT. The epidermal reinnnervation, however, was unchanged and this altered sensitivity may relate to its known function in cleaving the peptide substance-P, known to sensitise nociceptors. In conclusion, although MME expression is dysregulated after nerve injury in a NRG1-dependent manner this gene is dispensable for axon regeneration and remyelination after injury.
AMP activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates mechanistic target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling, two signaling pathways that are linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators as well as indirectly through the upregulation of upstream kinases such as liver kinase B1 (LKB1) which is a mechanism of action of metformin. Metformin's anti-hyperalgesic effects have been shown to occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin as well as other structurally distinct AMPK activators narciclasine, ZLN-024 and MK8722 to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. We found that metformin was the only AMPK activator to have sex specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming while direct AMPK activators, ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree. However, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they are able to drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment. SIGNIFICANCE STATEMENT: Non-opioid treatments for post-surgical pain are needed. Our work focused on whether direct or indirect AMPK activators would show greater efficacy for inhibiting incisional pain and also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for post-surgical pain because they inhibit acute pain caused by incision and also prevent long-term neuronal plasticity that is involved in persistent post-surgical pain. Our work points to the natural product, indirect AMPK activator, narciclasine, as an excellent starting point for development of therapeutics.
Vincristine, a widely used antineoplastic agent, is known to be neurotoxic and to lead to chemotherapy-induced peripheral neuropathy (CIPN), which is characterized by nerve damage. Growing evidence suggests that disruption of intracellular calcium homeostasis in peripheral neurons contributes largely to the pathological conditions of CIPN. Our previous study showed that forced expression of a peripheral nerve injury-induced small heat shock protein (Hsp), Hsp27, accelerates axon regeneration and functional recovery. In the current study, we examined whether neuronal expression of human Hsp27 (hHsp27) can prevent the inhibitory effects of vincristine in two mouse models of peripheral nerve injury, namely, sciatic nerve crush and CIPN.
Botulinum neurotoxin type A, an FDA-approved prophylactic drug for chronic migraine, is thought to achieve its therapeutic effect through blocking activation of unmyelinated meningeal nociceptors and their downstream communications with myelinated nociceptors and potentially the vasculature and immune cells. Prior investigations to determine botulinum neurotoxin type A effects on meningeal nociceptors were carried out in male rats and tested with stimuli that act outside the blood brain barrier. Here, we sought to explore the effects of extracranial injections of botulinum neurotoxin type A on activation of meningeal nociceptors by cortical spreading depression, an event which occurs inside the blood brain barrier, in female rats.