Animal Studies

In addition to analgesia, opioids produce hyperalgesia (opioid-induced hyperalgesia [OIH]) and neuroplasticity characterized by prolongation of inflammatory mediator-induced hyperalgesia (hyperalgesic priming). We evaluated the hypothesis that hyperalgesia and priming induced by opioids, are mediated by similar nociceptor mechanisms. In male rats, we first evaluated the role of nociceptor toll-like receptor 4 (TLR4), in OIH and priming induced by systemic low dose morphine (LDM, 0.03 mg/kg). Intrathecal oligodeoxynucleotide antisense to TLR4 mRNA (TLR4 AS-ODN) prevented OIH and prolongation of PGE hyperalgesia (priming) induced by LDM. In contrast, high dose morphine (HDM, 3 mg/kg) increased nociceptive threshold (analgesia) and induced priming, neither of which were attenuated by TLR4 AS-ODN. Protein kinase C epsilon (PKCε) AS-ODN also prevented LDM-induced hyperalgesia and priming, while analgesia and priming induced by HDM were unaffected. Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4-positive [IB4] and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups. In conclusion, while OIH and priming induced by LDM share receptor and second messenger mechanisms in common, action at TLR4 and signaling via PKCε, HDM-induced analgesia and priming are neither TLR4 nor PKCε dependent. OIH produced by LDM is mediated by both IB4 and peptidergic nociceptors, while priming is not dependent on the same population. In contrast, priming induced by HDM is mediated by both IB4 and peptidergic nociceptors. Implications for the use of low dose opioids combined with non-opioid analgesics and in treatment of opioid use disorder are discussed.Opioid-induced hyperalgesia (OIH) and priming are common side effects of opioid agonists, e.g., morphine, which acts at mu-opioid receptors (MORs). We demonstrate that OIH and priming induced by systemic low dose morphine (LDM) share action at TLR4 and signaling via PKCε, in common, while systemic high dose morphine (HDM)-induced analgesia and priming are neither TLR4 nor PKCε dependent. OIH produced by systemic LDM is mediated by IB4 and peptidergic nociceptors, while priming is dependent on a different class of nociceptors. Priming induced by systemic HDM is, however, mediated by both IB4 and peptidergic nociceptors. Our findings may provide useful information for use of low dose opioids combined with non-opioid analgesics to treat pain and opioid use disorders.

Expressional changes of pain-associated genes in primary sensory neurons of dorsal root ganglion (DRG) are critical for neuropathic pain genesis. DNA methyltransferase (DNMT)-triggered DNA methylation silences gene expression. We show here that DNMT1, a canonical maintenance methyltransferase, acts as the DNMT and is required for neuropathic pain genesis likely through repressing at least DRG gene expression in male mice. Peripheral nerve injury upregulated DNMT1 expression in the injured DRG through the transcription factor cAMP response element binding protein-triggered transcriptional activation of gene. Blocking this upregulation prevented nerve injury-induced DNA methylation within the promoter and 5'-untranslated region of gene, rescued expression and total Kv current, attenuated hyperexcitability in the injured DRG neurons, and alleviated nerve injury-induced pain hypersensitivities. Given that is a key player in neuropathic pain, our findings suggest that DRG DNMT1 may be a potential target for neuropathic pain management.In the present study, we reported that DNMT1, a canonical DNA maintenance methyltransferase, is upregulated via the activation of the transcription factor CREB in the injured DRG after peripheral nerve injury. This upregulation was responsible for nerve injury-induced DNA methylation within the promoter and 5'-untranslated region of the gene, reductions in expression and Kv current and increases in neuronal excitability in the injured DRG. Since pharmacological inhibition or genetic knockdown of DRG DNMT1 alleviated nerve injury-induced pain hypersensitivities, DRG DNMT1 contributes to neuropathic pain genesis partially through repression of DRG gene expression.

Accumulating evidence indicates that phosphorylated serum- and glucocorticoid-regulated kinase 1 (SGK1) is associated with spinal nociceptive sensitization by modulating glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined whether spinal SGK1 signaling contributes to the development of morphine analgesic tolerance. Chronic morphine administration markedly induced phosphorylation of SGK1 in the spinal dorsal horn neurons. Intrathecal injection of SGK1 inhibitor GSK-650394 reduced the development of morphine tolerance with a significant leftward shift in morphine dose-effect curve. Furthermore, spinal inhibition of SGK1 suppressed morphine-induced phosphorylation of nuclear factor kappa B (NF-κB) p65 and upregulation of NMDAR NR1 and NR2B expression in the spinal dorsal horn. In contrast, intrathecal administration of NMDAR antagonist MK-801 had no effect on the phosphorylation of SGK1 in morphine-treated rats. In addition, morphine-induced upregulation of NR2B, but not NR1, was significantly abolished by intrathecal pretreatment with PDTC, a specific NF-κB activation inhibitor. Finally, spinal delivery of SGK1 small interfering RNA exhibited similar inhibitory effects on morphine-induced tolerance, phosphorylation of NF-κB p65, as well as upregulation of NR1 and NR2B expression. Our findings demonstrate that spinal SGK1 contributes to the development of morphine tolerance by enhancing NF-κB p65/NMDAR signaling. Interfering spinal SGK1 signaling pathway could be a potential strategy for prevention of morphine tolerance in chronic pain management.

We previously demonstrated that sodium channel 1.7 (Nav1.7) in trigeminal ganglion (TG) was a critical factor in temporomandibular joint (TMJ) inflammation-induced hypernociception, but the mechanism underlying inflammation-induced upregulation of Nav1.7 remained unclear. Glial-neuron interaction plays a critical role in pain process and connexin 43 (Cx43), a gap junction protein expressed in satellite glial cells (SGCs) has been shown to play an important role in several pain models. In the present study, we investigate the role of Cx43 in TMJ inflammation-induced hypernociception and its possible impact on neuronal Nav1.7. We induced TMJ inflammation in rats by injecting complete Freund's adjuvant (CFA) into TMJ and observed a decrease in head withdraw threshold after 24 hours. Electron microscopy showed morphological alterations of SGCs in TMJ-inflamed rats. The expression of Cx43, glial fibrillary acidic protein (GFAP), and Nav1.7 increased greatly compared with controls. In addition, pretreatment with Cx43 blockers in TMJ-inflamed rats could alleviate mechanical hypernociception, inhibit SGCs activation and IL-1βrelease, and thus block the upregulation of Nav1.7. These findings indicate that the propagation of SGCs activation via Cx43 plays a critical role in Nav1.7-involved mechanical hypernociception induced by TMJ inflammation.

Cutaneous TRPV1 neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1 neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1 neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1 neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1 neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Voltage-gated sodium (Na) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.