Microglia P2X4R-BDNF signalling contributes to central sensitization in a recurrent nitroglycerin-induced chronic migraine model

Abstract

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.