P2X4-receptor participates in EAAT3 regulation via BDNF-TrkB signaling in a model of trigeminal allodynia

Abstract

ObjectivePrevious studies of neuropathic pain have suggested that the P2X4 purinoceptor (P2X4R) in spinal microglia is essential for maintaining allodynia following nerve injury. However, little is known about its role in inflammatory soup-induced trigeminal allodynia, which closely mimics chronic migraine status. Here, we determined the contributions of P2X4R and related signaling pathways in an inflammatory soup-induced trigeminal allodynia model.MethodsP2X4R gene and protein levels in the trigeminal nucleus caudalis were analyzed following repeated dural inflammatory soup infusions. p38, brain-derived neurotrophic factor, excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide protein levels in the trigeminal nucleus caudalis, as well as trigeminal sensitivity, were assessed among the different groups. Immunofluorescence staining was used to detect protein localization and expression in the trigeminal nucleus caudalis.ResultsRepeated inflammatory dural stimulation induced trigeminal hyperalgesia and the upregulation of P2X4R. Immunofluorescence revealed that P2X4R was expressed in trigeminal nucleus caudalis microglial cells. Blockage of P2X4R produced an anti-nociceptive effect, which was associated with an inhibition of inflammatory soup-induced increases in p38, brain-derived neurotrophic factor, excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide protein levels. The tyrosine receptor kinase B antagonist ANA-12 reversed trigeminal allodynia and the upregulation of excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide, whereas the agonist 7,8-dihydroxyflavone exacerbated these effects. Double immunostaining indicated that p38 and brain-derived neurotrophic factor were mainly expressed in microglial cells, whereas excitatory amino acid transporter 3 was primarily expressed in trigeminal nucleus caudalis neurons.ConclusionsThese data indicate that microglial P2X4R is involved in the regulation of excitatory amino acid transporter 3 via brain-derived neurotrophic factor-tyrosine receptor kinase B signaling following repeated inflammatory dural stimulation. Microglial P2X4R activation and microglia–neuron interactions in the trigeminal nucleus caudalis may play a role in the pathogenesis of migraine chronicity, and the modulation of P2X4R activation might be a potential therapeutic strategy.

Effect of Electroacupuncture on Hyperalgesia and Vasoactive Neurotransmitters in a Rat Model of Conscious Recurrent Migraine

“…DES induced elevations in c-Fos expression in the TG. C-Fos expression is regarded as a biomarker of pain-related behavior, reflecting the degree of hyperalgesia and the level of activation of the trigeminovascular system (TS) [29, 48–50]. To investigate the effects of EA on hyperalgesia, we performed immunofluorescence analyses and western blotting to examine the number of c-Fos-positive cells and levels of c-Fos protein in the TG (Figure 3).…”

The effect of orofacial complete Freund’s adjuvant treatment on the expression of migraine-related molecules

“…In another study, activated microglial cells were detected in the ipsilateral TNC and in the cervical dorsal horn 72 h after orofacial CFA treatment of rats [29]. In this context, the activated microglial cells might also be involved to the mechanism of central sensitization and nociception [30][31][32]. It is proved that an antagonist of P2X4 microglia receptor blocked the NTG-induced c-Fos and CGRP release in the TNC, subsequently the hyperalgesia [33].…”

Up‐regulation of astrocyte excitatory amino acid transporter 2 alleviates central sensitization in a rat model of chronic migraine

“…Rats were first anesthetized with 10% chloral hydrate (4 ml/kg, intraperitoneal), then 0.01 mg/kg buprenorphine was injected subcutaneously for analgesia. In addition, 1 ml of bupivacaine (5 mg/1 ml) was applied as a local preventive anesthetic under the surgical area of the skin as described in our previous studies (He et al, 2019; Liu et al, 2018), and placed in a stereotactic instrument (ST‐51603; Stoelting Co.). The surgical area was first disinfected with iodide, and an incision was made on the midline of each rat's head using a surgical blade to completely expose the skull.…”

“…Schematic representation of the brain region for trigeminal nucleus caudalis (TNC). The white dotted frame represents the TNC regions (Liu et al, 2018) and the white solid line rectangular box in the TNC areas were selected for the analysis of astrocytes. Nuclei were labeled with DAPI (blue), and astrocytes were labeled with GFAP (red)…”