Background According to our previous study, microglia P2X4 receptors (P2X4Rs) play a pivotal role in the central sensitization of chronic migraine (CM). However, the molecular mechanism that underlies the crosstalk between microglia P2X4Rs and neurons of the trigeminal nucleus caudalis (TNC) is not fully understood. Therefore, the aim of this study is to examine the exact P2X4Rs signalling pathway in the development of central sensitization in a CM animal model. Methods We used an animal model with recurrent intermittent administration of nitroglycerin (NTG), which closely mimics CM. NTG-induced basal mechanical and thermal hypersensitivity were evaluated using a von Frey filament test and an increasing-temperature hot plate apparatus (IITC). We detected P2X4Rs, brain-derived neurotrophic factor (BDNF) and phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK) expression profiles in the TNC. We investigated the effects of a P2X4R inhibitor (5-BDBD) and an agonist (IVM) on NTG-induced hyperalgesia and neurochemical changes as well as on the expression of p-p38-MAPK and BDNF. We also detected the effects of a tropomyosin-related kinase B (TrkB) inhibitor (ANA-12) on the CM animal model in vivo. Then, we evaluated the effect of 5-BDBD and SB203580 (a p38-MAPK inhibitors) on the release and synthesis of BDNF in BV2 microglia cells treated with 50 μM adenosine triphosphate (ATP). Results Chronic intermittent administration of NTG resulted in chronic mechanical and thermal hyperalgesia, accompanied by the upregulation of P2X4Rs and BDNF expression. 5-BDBD or ANA-12 prevented hyperalgesia induced by NTG, which was associated with a significant inhibition of the NTG-induced increase in phosphorylated extracellular regulated protein kinases (p-ERK) and calcitonin gene related peptide (CGRP) release in the TNC. Repeated administration of IVM produced sustained hyperalgesia and significantly increased the levels of p-ERK and CGRP release in the TNC. Activating P2X4Rs with ATP triggered BDNF release and increased BDNF synthesis in BV2 microglia, and these results were then reduced by 5-BDBD or SB203580. Conclusions Our results indicated that the P2X4R contributes to the central sensitization of CM by releasing BDNF and promoting TNC neuronal hyper-excitability. Blocking microglia P2X4R-BDNF signalling may have an effect on the prevention of migraine chronification.

中文翻译：

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小胶质细胞 P2X4R-BDNF 信号有助于复发性硝酸甘油诱导的慢性偏头痛模型中的中枢敏化

背景 根据我们之前的研究，小胶质细胞 P2X4 受体 (P2X4Rs) 在慢性偏头痛 (CM) 的中枢致敏中起关键作用。然而，小胶质细胞 P2X4Rs 与三叉神经核 (TNC) 神经元之间串扰的分子机制尚不完全清楚。因此，本研究的目的是检查在 CM 动物模型中中枢致敏发展中确切的 P2X4Rs 信号通路。方法 我们使用了一种动物模型，它反复间歇性地给予硝酸甘油 (NTG)，它与 ​​CM 非常相似。使用冯弗雷灯丝试验和升温热板装置 (IITC) 评估 NTG 诱导的基础机械和热超敏反应。我们检测到 P2X4Rs，TNC 中的脑源性神经营养因子 (BDNF) 和磷酸化 p38 丝裂原活化蛋白激酶 (p-p38-MAPK) 表达谱。我们研究了 P2X4R 抑制剂 (5-BDBD) 和激动剂 (IVM) 对 NTG 诱导的痛觉过敏和神经化学变化以及 p-p38-MAPK 和 BDNF 表达的影响。我们还检测了原肌球蛋白相关激酶 B (TrkB) 抑制剂 (ANA-12) 对体内 CM 动物模型的影响。然后，我们评估了 5-BDBD 和 SB203580（一种 p38-MAPK 抑制剂）对用 50 μM 三磷酸腺苷 (ATP) 处理的 BV2 小胶质细胞中 BDNF 的释放和合成的影响。结果 NTG 的长期间歇给药导致慢性机械和热痛觉过敏，伴随着 P2X4Rs 和 BDNF 表达的上调。5-BDBD 或 ANA-12 可防止 NTG 诱导的痛觉过敏，这与 NTG 诱导的 TNC 中磷酸化细胞外调节蛋白激酶 (p-ERK) 和降钙素基因相关肽 (CGRP) 释放增加的显着抑制有关。重复施用 IVM 会产生持续的痛觉过敏，并显着增加 TNC 中 p-ERK 和 CGRP 的释放水平。用 ATP 激活 P2X4Rs 触发 BDNF 释放并增加 BV2 小胶质细胞中 BDNF 的合成，然后这些结果被 5-BDBD 或 SB203580 降低。结论 我们的结果表明 P2X4R 通过释放 BDNF 和促进 TNC 神经元过度兴奋来促进 CM 的中枢敏化。阻断小胶质细胞 P2X4R-BDNF 信号可能对预防偏头痛慢性化有影响。这与 TNC 中 NTG 诱导的磷酸化细胞外调节蛋白激酶 (p-ERK) 和降钙素基因相关肽 (CGRP) 释放增加的显着抑制有关。重复施用 IVM 会产生持续的痛觉过敏，并显着增加 TNC 中 p-ERK 和 CGRP 的释放水平。用 ATP 激活 P2X4Rs 触发 BDNF 释放并增加 BV2 小胶质细胞中 BDNF 的合成，然后这些结果被 5-BDBD 或 SB203580 降低。结论 我们的结果表明 P2X4R 通过释放 BDNF 和促进 TNC 神经元过度兴奋来促进 CM 的中枢敏化。阻断小胶质细胞 P2X4R-BDNF 信号可能对预防偏头痛慢性化有影响。这与 TNC 中 NTG 诱导的磷酸化细胞外调节蛋白激酶 (p-ERK) 和降钙素基因相关肽 (CGRP) 释放增加的显着抑制有关。重复施用 IVM 会产生持续的痛觉过敏，并显着增加 TNC 中 p-ERK 和 CGRP 的释放水平。用 ATP 激活 P2X4Rs 触发 BDNF 释放并增加 BV2 小胶质细胞中 BDNF 的合成，然后这些结果被 5-BDBD 或 SB203580 降低。结论 我们的结果表明 P2X4R 通过释放 BDNF 和促进 TNC 神经元过度兴奋来促进 CM 的中枢敏化。阻断小胶质细胞 P2X4R-BDNF 信号可能对预防偏头痛慢性化有影响。