Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

Abstract

Background The channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood. Methods Wild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice. Results Global or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG. Conclusions The present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

The emerging role of Panx1 as a potential therapeutic target for chronic pain

“…Panx1 is a large-pore ion channel in the plasma membrane of dorsal root ganglion (DRG) neurons that allows for the release of Adenosine triphosphate (ATP). This commentary elaborates on the findings of Xing et al [ 3 ], who investigated the role of Panx1 in peripheral pain sensitization following inflammatory stimuli. We examine the role of Panx1 in chronic pain, critique Xing et al’s [ 3 ] adjuvant selection and power analysis, and discuss the challenges of translating findings from animal models to human conditions.…”

“…Current research also suggests that the Wnt/β-catenin signaling pathway, which regulates neuronal-related gene expression and cell proliferation [ 8 ], is upregulated in neuropathic pain [ 9 ]. Notably, Xing et al [ 3 ] determined that the Wnt/β-catenin pathway may modulate the activity of Panx1, so targeting the interaction of these elements is an attractive strategy for the treatment of pain.…”

“…In their study, Xing et al [ 3 ] used complete Freund’s adjuvant (CFA), an inflammatory agent consisting of heat-killed mycobacteria, to induce hypersensitivity in the mouse hind paw. While CFA is an effective immune stimulator in animals, its clinical applicability is limited because it cannot be used in humans to induce chronic pain due to its potency.…”

“…Finally, Xing et al [ 3 ] employed the von Frey test to assess allodynia, a behavioral measure of pain avoidance, in conjunction with electrophysiology to quantify the neuronal excitability of the Panx1 mutants. The utilization of two complementary methods strengthens the validity of their results, but this study still faces the challenge of demonstrating the applicability of these findings to humans.…”

“…Due to the challenges in investigating treatments for chronic pain in human subjects, additional efforts are needed to adapt animal models to the human condition and ensure high-quality, reproducible studies. Xing et al [ 3 ] provide promising insights into how Panx1 facilitates inflammatory pain through experiments on pain sensitivity and neuronal excitability. Few studies have demonstrated the translational progress of Panx1 as the target for inflammation and disease control [ 11 – 13 ], so further research characterizing the interactions of pharmacological treatments on Panx1, the Wnt/β-catenin signaling pathway, and DRG may elucidate promising avenues for the treatment of chronic pain in the military population.…”