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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.
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.
摘要：
背景：通道形成蛋白Pannexin1（Panx1）在慢性疼痛的人类研究和动物模型中都有牵连，但潜在的机制仍未完全理解。
方法：野生型（WT，n=24),全局Panx1KO(n=24)，神经元特异性Panx1KO（n=20），在阿尔伯特·爱因斯坦医学院的这项研究中使用了胶质细胞特异性的Panx1KO（n=20）小鼠。在完全弗氏佐剂(CFA)注射(7、14和21天)后,使用vonFrey测试来量化这些小鼠的疼痛敏感性。qRT-PCR用于测量Panx1、Panx2、Panx3、Cx43、Calhm1和β-联蛋白的mRNA水平。激光扫描共聚焦显微镜成像，Sholl分析，和电生理学用于评估Panx1对神经2a和背根神经节神经元（DRGNs）中神经元兴奋性和形态的影响，其中操纵了Panx1的表达或功能。采用溴化乙锭（EtBr）染料吸收测定和钙成像来研究Panx1在三磷酸腺苷（ATP）敏感性中的作用。应用β-半乳糖苷酶（β-gal）染色确定转基因小鼠的三叉神经节（TG）和DRG中Panx1的相对细胞表达水平。
结果：整体或神经元特异性Panx1缺失显着降低CFA刺激后的疼痛阈值（7、14和21d；P<0.01vs.WT组)，表明Panx1与疼痛敏感性呈正相关。在Neuro2a中，与WT组相比，整体Panx1缺失显着降低了神经突延伸和内向电流（P<0.05），揭示了Panx1增强了神经发生和兴奋性。同样，神经生长因子（NGF）治疗5d后，整体Panx1缺失显着抑制Wnt/β-catenin依赖性DRG神经发生（P<0.01vs.WT组)。此外，Panx1通道增强CFA注射后DRG神经元对ATP的反应（P<0.01vs.Panx1KO组）。此外，CFA治疗7d后，ATP释放增加了DRGNs及其周围卫星胶质细胞的Ca2反应（P<0.01vs.Panx1KO组)，这表明神经胶质中的Panx1也会影响过度的神经元兴奋性。有趣的是,发现神经元特异性Panx1缺失显着降低培养的DRGN的分化，如神经突生长迟缓(P<0.05vs.Panx1KO组；P<0.01vs.WT组或GFAP-Cre组)，Wnt/β-catenin信号激活减弱(P<0.01vs.WT,Panx1KO和GFAP-Cre组)，细胞兴奋性降低(P<0.01vs.GFAP-Cre组）和对ATP刺激的反应（P<0.01vs.WT组)。β-gal染色的分析表明，DRG中神经元中Panx1的细胞表达水平显着高于TG（增加2.5倍）。
结论：本研究表明，神经元Panx1通过细胞自主作用神经元兴奋性，在炎性疼痛的背景下是外周敏感性的主要驱动因素。这种对神经元Panx1的过度兴奋依赖性与炎性口面疼痛形成对比，类似的研究揭示了神经胶质Panx1的重要作用。神经元和非神经元TG和DRG细胞中Panx1表达的明显差异可能是这些细胞类型在两种疼痛模型中的不同影响的原因。
方法：野生型（WT，n=24),全局Panx1KO(n=24)，神经元特异性Panx1KO（n=20），在阿尔伯特·爱因斯坦医学院的这项研究中使用了胶质细胞特异性的Panx1KO（n=20）小鼠。在完全弗氏佐剂(CFA)注射(7、14和21天)后,使用vonFrey测试来量化这些小鼠的疼痛敏感性。qRT-PCR用于测量Panx1、Panx2、Panx3、Cx43、Calhm1和β-联蛋白的mRNA水平。激光扫描共聚焦显微镜成像，Sholl分析，和电生理学用于评估Panx1对神经2a和背根神经节神经元（DRGNs）中神经元兴奋性和形态的影响，其中操纵了Panx1的表达或功能。采用溴化乙锭（EtBr）染料吸收测定和钙成像来研究Panx1在三磷酸腺苷（ATP）敏感性中的作用。应用β-半乳糖苷酶（β-gal）染色确定转基因小鼠的三叉神经节（TG）和DRG中Panx1的相对细胞表达水平。
结果：整体或神经元特异性Panx1缺失显着降低CFA刺激后的疼痛阈值（7、14和21d；P<0.01vs.WT组)，表明Panx1与疼痛敏感性呈正相关。在Neuro2a中，与WT组相比，整体Panx1缺失显着降低了神经突延伸和内向电流（P<0.05），揭示了Panx1增强了神经发生和兴奋性。同样，神经生长因子（NGF）治疗5d后，整体Panx1缺失显着抑制Wnt/β-catenin依赖性DRG神经发生（P<0.01vs.WT组)。此外，Panx1通道增强CFA注射后DRG神经元对ATP的反应（P<0.01vs.Panx1KO组）。此外，CFA治疗7d后，ATP释放增加了DRGNs及其周围卫星胶质细胞的Ca2反应（P<0.01vs.Panx1KO组)，这表明神经胶质中的Panx1也会影响过度的神经元兴奋性。有趣的是,发现神经元特异性Panx1缺失显着降低培养的DRGN的分化，如神经突生长迟缓(P<0.05vs.Panx1KO组；P<0.01vs.WT组或GFAP-Cre组)，Wnt/β-catenin信号激活减弱(P<0.01vs.WT,Panx1KO和GFAP-Cre组)，细胞兴奋性降低(P<0.01vs.GFAP-Cre组）和对ATP刺激的反应（P<0.01vs.WT组)。β-gal染色的分析表明，DRG中神经元中Panx1的细胞表达水平显着高于TG（增加2.5倍）。
结论：本研究表明，神经元Panx1通过细胞自主作用神经元兴奋性，在炎性疼痛的背景下是外周敏感性的主要驱动因素。这种对神经元Panx1的过度兴奋依赖性与炎性口面疼痛形成对比，类似的研究揭示了神经胶质Panx1的重要作用。神经元和非神经元TG和DRG细胞中Panx1表达的明显差异可能是这些细胞类型在两种疼痛模型中的不同影响的原因。
