Investigation of the purinergic receptor P2RX4 within the spinal dorsal horn in chronic pain state
Introduction: Long-term and intense noxious stimulation related to chronic inflammation lead to central sensitization within the superficial spinal dorsal horn. Interleukins play a pivotal role in pain processing, the activation of nociceptive primary afferents and the consecutive release of ATP induce considerable increase of interleukin-1βeta contributing to the hyperexcitability of the neural circuit. Accumulating evidence suggests that the P2X4 receptor (P2RX4) may be one of the major key mediators that are involved in the cytokine secretion. Methods: Inflammatory pain was evoked by unilateral intraplantar injection of complete Freund adjuvant (CFA) in male Wistar rats and Black 6 mice. The mechanical nociceptive responsiveness of control and CFA-treated animals were tested daily by von Frey test. Changes in the gene- and the protein expression of P2RX4 after CFA injection were measured with PCR and Western blot analysis. The cellular localization of P2RX4 was investigated by immunohistochemical stainings and evaluated by IMARIS software.Results: After CFA injection the mechanical withdrawal threshold of the treated animals was significantly reduced. We found almost a two-fold increase in mRNA and protein levels in the dorsal horn of CFA-injected animals. Single immunoperoxidase reactions revealed a substantial receptoral expression within the lamina I-II of the spinal gray matter following CFA injection which was further validated by Western blot analysis. Abundant expression of P2RX4 was observed on excitatory but not inhibitory neuronal axon terminals in CFA model. Conclusion: P2RX4 probably exerts its effect on spinal pain processing via excitatory interneurons, modulating the nociceptive signals towards projection neurons.