Pain is a subjective experience derived from complex interactions among biological, environmental, and psychosocial pathways. Sex differences in pain sensitivity and chronic pain prevalence are well established. However, the molecular basis underlying these sex dimorphisms are poorly understood particularly with regard to the role of the peripheral nervous system. Here we sought to identify shared and distinct gene networks functioning in the peripheral nervous systems that may contribute to sex differences of pain in rats after nerve injury. We performed RNA-seq on dorsal root ganglia following chronic constriction injury of the sciatic nerve in male and female rats. Analysis from paired naive and injured tissues showed that 1513 genes were differentially expressed between sexes. Genes which facilitated synaptic transmission in naïve and injured females did not show increased expression in males. Appreciating sex-related gene expression differences and similarities in neuropathic pain models may help to improve the translational relevance to clinical populations and efficacy of clinical trials of this major health issue.

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using Pirt^EGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

Chronic pain after spine surgery (CPSS) is often characterized by intractable low back pain and/or radiating leg pain, and has been reported in 8–40% of patients that received lumbar spine surgery. We conducted a literature search of PubMed, MEDLINE/OVID with a focus on studies about the etiology and treatments of CPSS and low back pain. Our aim was to provide a narrative review that would help us better understand the pathogenesis and current treatment options for CPSS. This knowledge will aid in the development of optimal strategies for managing postoperative pain symptoms and potentially curing the underlying etiologies. Firstly, we reviewed recent advances in the mechanistic study of CPSS, illustrated both structural (e.g., fibrosis and scaring) and non-structural factors (e.g., inflammation, neuronal sensitization, glial activation, psychological factor) causing CPSS, and highlighted those having not been given sufficient attention as the etiology of CPSS. Secondly, we summarized clinical evidence and therapeutic perspectives of CPSS. We also presented new insights about the treatments and etiology of CPSS, in order to raise awareness of medical staff in the identification and management of this complex painful disease. Finally, we discussed potential new targets for clinical interventions of CPSS and future perspectives of mechanistic and translational research. CPSS patients often have a mixed etiology. By reviewing recent findings, the authors advocate that clinicians shall comprehensively evaluate each case to formulate a patient-specific and multi-modal pain treatment, and importantly, consider an early intraoperative intervention that may decrease the risk or even prevent the onset of CPSS. CPSS remains difficult to treat. This review broadens our understanding of clinical therapies and underlying mechanisms of CPSS, and provides new insights which will aid in the development of novel mechanism-based therapies for not only managing the established pain symptoms but also preventing the development of CPSS.

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated MrgC agonist treatments and examined the underlying mechanisms. In animal behavior studies conducted in male rats at 4 to 5 weeks after an L5 spinal nerve ligation (SNL), the ability of dipeptide MrgC agonist JHU58 (0.1 mM, 10 μL, i.t.) to inhibit mechanical and heat hypersensitivity decreased after 3 days of treatment with a tolerance-inducing dose (0.5 mM, 10 μL, i.t., twice/day). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to β-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, pretreatment with TAK-243 (0.2 mM, 5 μL, i.t.), a small-molecule inhibitor of the ubiquitin-activating enzyme, during tolerance induction attenuated the development of tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Interestingly, morphine analgesia was also decreased in SNL rats that had become tolerant to JHU58, suggesting a cross-tolerance. Furthermore, i.t. pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia. These findings suggest that tolerance can develop to MrgC agonist–induced pain inhibition after repeated i.t. administrations. This tolerance development to JHU58 may involve increased coupling of MrgC to β-arrestin-2 and ubiquitin-mediated receptor degradation.