Methods
Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), plasma P2X7R and P-gp, number of extra-cellular vesicles (EV), serum ATP and EV-ATP levels. Brain microvessel gene expression and EV mtDNA copy numbers were measured by RT2 PCR array and digital PCR, respectively. A RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed animals, which were decreased 15-50-fold in BBG-treated CIE-exposed animals. Plasma P-gp levels and serum P2X7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2X7R decreased P2X7R shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2X7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2X7R inhibition or receptor knockout. These observations suggested that P2X7R signaling plays a critical role in ethanol-induced brain injury. Increased eATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2X7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2X7R signaling in CIE-induced brain injury.