Background and purpose. Traumatic brain injury (TBI) is an acute brain lesion considered as one of the leading causes of mortality and disability worldwide. After TBI, innate immunity is rapidly activated in response to damage-associated molecular patterns, such as ATP release, recognized by P2X7 purinergic receptors (P2X7R). The P2X7R-NLRP3 inflammasome axis has been identified as one of the main actors in neuroinflammation. Therefore, this study aimed to validate P2X7R as therapeutic target in TBI. Experimental approach. P2X7R was validated through genetic and pharmacological approaches. Six non-nucleotide purine derivatives were evaluated as P2X7R antagonists. Compounds that prevented LPS+ATP-induced IL-1β release from primary glial cultures were investigated in the closed-head injury TBI model in vivo. Finally, we evaluated sP2X7R plasmatic levels in a cohort of TBI patients. Key results. p2x7 -/- mice showed an exaggerated inflammatory response 24 h post-TBI compared to control mice. However, animals treated with the selective P2X7R antagonist JNJ-47965567 (30 mg/kg i.p.) 30 min post-TBI showed improved neurological and inflammatory parameters. The purine derivative ITH15004 was the most potent compound reducing IL-1β production in vitro. When administered in vivo 30 min post-TBI, ITH15004 (1 mg/kg i.p.) improved both neurobehavioral and inflammatory markers at 24 h. In TBI patients, enhanced levels of circulating sP2X7R correlated with the lesion severity 72 h post-TBI and with unfavourable outcomes 24 h post-TBI. Conclusion and implications. These results highlight the importance of P2X7R in the acute phase of TBI and present ITH15004 as a new pharmacological tool to counteract P2X7R-dependent neuroinflammation in vivo.

Background: Post-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit. Methods: WT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Results: We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. Conclusions: These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.