Neuropathic Pain: Mechanisms, Sex Differences, and Potential Therapies for a Global Problem.

The study of chronic pain continues to generate ever-increasing numbers of publications, but safe and efficacious treatments for chronic pain remain elusive. Recognition of sex-specific mechanisms underlying chronic pain has resulted in a surge of studies that include both sexes. A predominant focus has been on identifying sex differences, yet many newly identified cellular mechanisms and alterations in gene expression are conserved between the sexes. Here we review sex differences and similarities in cellular and molecular signals that drive the generation and resolution of neuropathic pain. The mix of differences and similarities reflects degeneracy in peripheral and central signaling processes by which neurons, immune cells, and glia codependently drive pain hypersensitivity. Recent findings identifying critical signaling nodes foreshadow the development of rationally designed, broadly applicable analgesic strategies. However, the paucity of effective, safe pain treatments compels targeted therapies as well to increase therapeutic options that help reduce the global burden of suffering.

Sex-Dependent Mechanisms of Chronic Pain: A Focus on Microglia and P2X4R.

For over two decades, purinergic signaling in microglia has persisted in the spotlight as a major pathomechanism of chronic pain. Of the many purinoreceptors, the P2X4R of the ionotropic family, has a well-described causal role underlying chronic neuropathic pain. This review will briefly examine microglial P2X4R signaling in the spinal cord as it relates to chronic pain through a historical lens, followed by a more in-depth examination of recent work, which has revealed major sex differences. We also discuss the generalizability of sex differences in microglial and P2X4R signaling in other pain conditions as well as in nonspinal regions. Finally, we speculate on remaining gaps in the literature as well as what can be done to address them with the ultimate goal of using our collective knowledge to treat chronic pain effectively and in both sexes. SIGNIFICANCE STATEMENT: Effective treatments are lacking for chronic pain sufferers, and this may be explained by the vast sex differences underlying chronic pain mechanisms. In this minireview, we focus on the roles of microglia and P2X4R in chronic pain, with specific attention to the circumstances under which these pathomechanisms differ between males and females. By delineating the ways in which pain occurs differently between the sexes, we can start developing successful therapies for all.

Conserved transcriptional programming across sex and species after peripheral nerve injury predicts treatments for neuropathic pain.

BACKGROUND AND PURPOSE: Chronic pain is a devastating problem affecting one in five individuals around the globe, with neuropathic pain the most debilitating and poorly treated type of chronic pain. Advances in transcriptomics have contributed to cataloguing diverse cellular pathways and transcriptomic alterations in response to peripheral nerve injury but have focused on phenomenology and classifying transcriptomic responses. EXPERIMENTAL APPROACH: To identifying new types of pain-relieving agents, we compared transcriptional reprogramming changes in the dorsal spinal cord after peripheral nerve injury cross-sex and cross-species, and imputed commonalities, as well as differences in cellular pathways and gene regulation. KEY RESULTS: We identified 93 transcripts in the dorsal horn that were increased by peripheral nerve injury in male and female mice and rats. Following gene ontology and transcription factor analyses, we constructed a pain interactome for the proteins encoded by the differentially expressed genes, discovering new, conserved signalling nodes. We investigated the interactome with the Drug-Gene database to predict FDA-approved medications that may modulate key nodes within the network. The top hit from the analysis was fostamatinib, the molecular target of which is the non-receptor spleen associated tyrosine kinase (Syk), which our analysis had identified as a key node in the interactome. We found that intrathecally administrating the active metabolite of fostamatinib, R406 and another Syk inhibitor P505-15, significantly reversed pain hypersensitivity in both sexes. CONCLUSIONS AND IMPLICATIONS: Thus, we have identified and shown the efficacy of an agent that could not have been previously predicted to have analgesic properties.

Regulation of neuropathic pain by microglial Orai1 channels.

Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.

In utero exposure to low doses of genistein and di-(2-ethylhexyl) phthalate (DEHP) alters innate immune cells in neonatal and adult rat testes.

BACKGROUND: Although humans are exposed to mixtures of endocrine disruptor chemicals, few studies have examined their toxicity on male reproduction. We previously found that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di(2-ethylhexyl) phthalate (DEHP) altered gene expression in adult rat testes. OBJECTIVES: Our goal was to investigate the effects of fetal exposure to GEN-DEHP mixtures at two doses relevant to humans on testicular function and transcriptome in neonatal and adult rats. MATERIALS AND METHODS: Pregnant SD rats were gavaged with vehicle, GEN or DEHP, alone or mixed at 0.1 and 10 mg/kg/day, from gestation day 14 to birth. Fertility, steroid levels, and testis morphology were examined in neonatal and adult rats. Testicular transcriptomes were examined by gene array and functional pathway analyses. Cell-specific genes/proteins were determined by quantitative real-time PCR and immunohistochemistry. RESULTS: GEN-DEHP mixtures increased the rates of infertility and abnormal testes in adult rats. Gene array analysis identified more genes exclusively altered by the mixtures than individual compounds. Altered top canonical pathways included urogenital/reproductive developmental and inflammatory processes. GEN-DEHP mixtures increased innate immune cells and macrophages markers at both doses and ages, more strongly and consistently than DEHP or GEN alone. Genes exclusively increased by the mixture in adult testis related to innate immune cells and macrophages included Kitlg, Rps6ka3 (Rsk2), Nr3c1, Nqo1, Lif, Fyn, Ptprj (Dep-1), Gpr116, Pfn2, and Ptgr1. DISCUSSION AND CONCLUSION: These findings demonstrate that GEN-DEHP mixtures at doses relevant to human induce adverse testicular phenotypes, concurrent with age-dependent and non-monotonic changes in testicular transcriptomes. The involvement of innate immune cells such as macrophages suggests immediate and delayed inflammatory responses which may contribute to testicular dysfunction. Moreover, these effects are complex and likely involve multiple interactions between immune and non-immune testicular cell types that will entail further studies.