Accepted

Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system. Approximately half of the patients with MS experience severe pain; however, currently available therapeutics provide only insufficient relief. The mechanisms underlying the generation of neuropathic pain in patients with MS are not fully understood. Recently, we found that neutrophil elastase from accumulated neutrophils in the dorsal root ganglion (DRG) sensitizes DRG neurons and induces mechanical allodynia in a mouse model of experimental autoimmune encephalomyelitis (EAE). However, the mechanism underlying neutrophil accumulation in the DRG after myelin oligodendrocyte glycoprotein (MOG, immunogenic peptide) immunization remains unclear. Here, we found that C-X-C motif ligand 1 (CXCL1) was upregulated in DRG neurons after MOG immunization. Increased expression of CXCL1 protein was also observed in primary cultured DRG neurons treated with MOG, which was mediated through toll-like receptor 4 (TLR4). Gene silencing of TLR4 or CXCL1 in DRG neurons significantly attenuated neutrophil accumulation in the DRG and mechanical allodynia during the preclinical phase of EAE (around day 5 after immunization). Our results thus suggest that a TLR4-CXCL1 pathway in DRG neurons triggers neutrophil recruitment in the DRG and subsequent mechanical allodynia in response to MOG.

The primary somatosensory cortex (S1) comprises a number of functionally distinct regions, reflecting the diversity of somatosensory receptor submodalities innervating the body. In particular, two spatially and functionally distinct nociceptive regions have been described in primate S1 (Vierck et al., 2013; Whitsel et al., 2018). One region is located mostly in Brodmann cytoarchitectonic area 1, where a subset of neurons exhibit functional characteristics associated with myelinated Aδ nociceptors and perception of 1/sharp, discriminative pain. The second region is located at the transition between S1 and primary motor cortex (M1) in area 3a, where neurons exhibit functional characteristics associated with unmyelinated C nociceptors and perception of 2/slow, burning pain. To test the hypothesis that in rats the transitional zone (TZ) – which is a dysgranular region at the transition between M1 and S1 – is the functional equivalent of the nociresponsive region of area 3a in primates, extracellular spike discharge activity was recorded from TZ neurons in rats under general isoflurane anesthesia. Thermonoxious stimuli were applied by lowering the contralateral forepaw or hindpaw into a 48-51˚C heated water bath for 5-10s. Neurons in TZ were found to be minimally affected by non-noxious somatosensory stimuli, but highly responsive to thermonoxious skin stimuli in a slow temporal summation manner closely resembling that of nociresponsive neurons in primate area 3a. Selective inactivation of TZ by topical lidocaine application suppressed or delayed the nociceptive withdrawal reflex, suggesting that TZ exerts a tonic facilitatory influence over spinal cord neurons producing this reflex. In conclusion, TZ appears to be a rat homolog of the nociresponsive part of monkey area 3a. A possibility is considered that this region might be primarily engaged in autonomic aspects of nociception.

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.