Excessive use of headache treatments often leads to the development, progression and exacerbation of primary headache, which is defined as medication overuse headache (MOH). A significant pathophysiological mechanism of MOH is central sensitization. Recent evidence suggests that central sensitization in chronic headache is a result of inflammatory responses mediated by microglial activation in the trigeminal nucleus caudalis (TNC). However, it is unknown whether microglial activation has an impact on the central sensitization of MOH. Accordingly, the goal of this research was to determine how microglial activation and the P2X7R/NLRP3 inflammasome signaling pathway in the TNC contribute to the pathogenesis of MOH.