Abstract WP317: Phagocytic Potentials of Purinergic Receptor P2x4 Blockade After Ischemic Stroke

Abstract

Backgrounds: We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides neuroprotection by reducing acute excessive inflammation and thus can be potential drug targets to treat ischemic stroke. However, the mechanism how P2X4R blockade provide long term benefits are not known. Here we hypothesize that P2X4R blockade increase phagocytic uptake of damaged or dead tissue and facilitate swift recovery. Methods: We used both 1μm size fluorescent phagocytic bead (Polyscience Inc) and pHrodo Red zymosan A Bioparticles conjugate (Thermofisher) to study in vitro and ex-vivo phagocytic uptake by LPS (200 μg/ml for 2 hours) primed Bone Marrow Derived Macrophages (BMDMs) from P2X4R KO and littermate WT mice. We also used flow sorted Ly6C hi and low monocytes for phagocytic uptake study obtained from perilesional ipsilateral mouse brain tissue of P2X4R KO and littermate WT mice after 3 or 7 days of stroke. The later mice were subjected to 60 mins of transient middle cerebral artery occlusion. Results: Both phagocytic beads and pHrodo bioparticle uptake study suggest significantly higher uptake (1.5 and 2-fold P<0.05 vs WT student t test n=4 mice/group x 2 technical replication) by BMDMs obtained from P2X4R KO mice. FACS sorted infiltrated Ly6Chi inflammatory monocytes from global P2X4R KO mice brain after 3 days of stroke show significantly (*P>0.05 vs WT) high phagocytic bead uptake as compared to WT control. Further these monocytes also showed elevated expression of CD36, a marker for scavenger receptor at 7 days after stroke. Conclusions: This study suggest that P2X4R blockade provide swift and sustained neuroprotection by increasing phagocytic uptake of damage tissue after ischemic stroke.