P2X7R-mediated autophagic impairment contributes to central sensitization in a chronic migraine model with recurrent nitroglycerin stimulation in mice

Background: Central sensitization is an important pathophysiological mechanism of chronic migraine (CM). According to our previous studies, microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to the central sensitization. The P2X7 Receptor (P2X7R) is a purinergic receptor expressed in microglia and participates in central sensitization in chronic pain, but its role in CM is unclear. Numerous studies have shown that P2X7R regulates the level of Autophagy and that Autophagy affects the microglial activation and inflammation. Recently, Autophagy has been shown to be involved in neuropathic pain, but there is no information about Autophagy in CM. Therefore, the current study investigated the role of P2X7R in CM and its underlying mechanism, focusing on Autophagy regulation.

Methods: The CM model was established by repeated intraperitoneal injection of nitroglycerin (NTG) in mice. A Von Frey filament and radiant heat were used to assess the mechanical and thermal hypersensitivity. Western blotting and immunofluorescence assays were performed to detect the expression of P2X7R, autophagy-related proteins, and the cellular localization of P2X7R. To determine the role of P2X7R and Autophagy in CM, we detected the effects of the Autophagy inducer, rapamycin (RAPA) and P2X7R antagonist, Brilliant Blue G (BBG), on pain behavior and the expression of Calcitonin gene-related peptide (CGRP) and c-fos. In addition, the effect of RAPA and BBG on microglial activation and subsequent inflammation were investigated.

Results: The expression of P2X7R was increased and was mainly colocalized with microglia in the TNC following recurrent NTG administration. The autophagic flux was blocked in CM, which was characterized by upregulated LC3-II, and accumulated Autophagy substrate protein, p62. RAPA significantly improved the basal rather than acute hyperalgesia. BBG alleviated both basal and acute hyperalgesia. BBG activated the level of autophagic flux. RAPA and BBG inhibited the activation of microglia, limited the inflammatory response, and reduced the expression of CGRP and c-fos.

Conclusions: Our results demonstrate the dysfunction of the autophagic process in CM. Activated Autophagy may have a preventive effect on migraine chronification. P2X7R contributes to central sensitization through mediating Autophagy regulation and might become a potential target for CM.