Animal Studies

Dorsal root ganglion (DRG) sensory neuron subtypes defined by their in vivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.

Trigeminal neuropathic pain (TGN) is an attacking, abrupt, electric-shock headache involving abnormal cortical activity. The neural mechanism underlying TGN remains elusive. In this study, we explored the role of microglia in the primary somatosensory barrel cortex (S1BF), which is a critical region for TGN, of a mouse model of TGN that displayed significant pain-related behaviors. Using electrophysiological recordings, we found robust neuronal hyperactivity in glutamatergic neurons of S1BF (Glu). Chemogenetic inhibition of Glu neurons significantly relieved mechanical allodynia in TGN mice. In naïve mice, chemogenetic activation of Glu neurons induced pain sensitization. In addition, we found that microglia in the S1BF (microglia) were significantly activated, with density and morphology changes. Intraperitoneal administration of minocycline, a microglia inhibitor, attenuated pain sensitization, and decreased Glu neuronal activity. Together, these findings demonstrate the putative importance of microglia as a key regulator in TGN through actions on Glu neuronal adaptation.

Fibromyalgia (FM) is characterized by chronic widespread pain. The pathogenesis of FM remains unclear. No specific biomarkers are available. Animal models of FM may provide an opportunity to explore potential biomarkers in a relative homogenous disease condition. Here, we probed the metabolomics profiles of serum and urine in a mouse model of FM induced by intermittent cold stress (ICS). We focused on the role of acid-sensing ion channel 3 (ASIC3) in the metabolomics profiling because ICS treatment induced chronic widespread muscle pain lasting for 1 month in wild-type (Asic3) but not Asic3-knockout (Asic3) mice. Serum and urine samples were collected from both genotypes at different ICS stages, including before ICS (basal level) and post-ICS at days 10 (middle phase, P10) and 40 (recovery phase, P40). Control naïve mice and ICS-induced FM mice differed in H-NMR- and LC-MS-based metabolomics profiling. On pathway analysis, the leading regulated pathways in Asic3 mice were taurine and hypotaurine, cysteine and methionine, glycerophospholipid, and ascorbate and aldarate metabolisms, and the major pathways in Asic3 mice involved amino acid-related metabolism. Finally, we developed an algorithm for the impactful metabolites in the FM model including cis-aconitate, kynurenate, taurine, pyroglutamic acid, pyrrolidonecarboxylic acid, and 4-methoxyphenylacetic acid in urine as well as carnitine, deoxycholic acid, lysoPC(16:0), lysoPC(20:3), oleoyl-L-carnitine, and trimethylamine N-oxide in serum. Asic3 mice were impaired in only muscle allodynia development but not other pain symptoms in the ICS model, so the ASIC3-dependent metabolomics changes could be useful for developing diagnostic biomarkers specific to chronic widespread muscle pain, the core symptom of FM. Further pharmacological validations are needed to validate these metabolomics changes as potential biomarkers for FM diagnosis and/or treatment responses.

Tumor necrosis factor receptor 2 (TNFR2) is a transmembrane receptor that is linked to immune modulation and tissue regeneration. Here, we show that TNFR2 essentially promotes long-term pain resolution independently of sex. Genetic deletion of TNFR2 resulted in impaired neuronal regeneration and chronic nonresolving pain after chronic constriction injury (CCI). Further, pharmacological activation of TNFR2 using the TNFR2 agonist EHD2-sc-mTNF in mice with chronic neuropathic pain promoted long-lasting pain recovery. TNFR2 agonist treatment reduced neuronal injury, alleviated peripheral and central inflammation, and promoted repolarization of central nervous system (CNS)-infiltrating myeloid cells into an antiinflammatory/reparative phenotype. Depletion of regulatory T cells (Tregs) delayed spontaneous pain recovery and abolished the therapeutic effect of EHD2-sc-mTNF This study therefore reveals a function of TNFR2 in neuropathic pain recovery and demonstrates that both TNFR2 signaling and Tregs are essential for pain recovery after CCI. Therefore, therapeutic strategies based on the concept of enhancing TNFR2 signaling could be developed into a nonopioid therapy for the treatment of chronic neuropathic pain.

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.