Neuroprotection via Carbon Monoxide Depends on the Circadian Regulation of CD36-Mediated Microglial Erythrophagocytosis in Hemorrhagic Stroke

Abstract

The molecular basis for circadian dependency in stroke due to subarachnoid hemorrhagic stroke (SAH) remains unclear. We reasoned that microglial erythrophagocytosis, crucial for SAH response, follows a circadian pattern involving carbon monoxide (CO) and CD36 surface expression. The microglial BV-2 cell line and primary microglia (PMG) under a clocked medium change were exposed to blood ± CO (250 ppm, 1 h) in vitro. Circadian dependency and the involvement of CD36 were analyzed in PMG isolated from control mice and CD36−/− mice and by RNA interference targeting Per-2. In vivo investigations, including phagocytosis, vasospasm, microglia activation and spatial memory, were conducted in an SAH model using control and CD36−/− mice at different zeitgeber times (ZT). In vitro, the surface expression of CD36 and its dependency on CO and phagocytosis occurred with changed circadian gene expression. CD36−/− PMG exhibited altered circadian gene expression, phagocytosis and impaired responsiveness to CO. In vivo, control mice with SAH demonstrated circadian dependency in microglia activation, erythrophagocytosis and CO-mediated protection at ZT2, in contrast to CD36−/− mice. Our study indicates that circadian rhythmicity modulates microglial activation and subsequent CD36-dependent phagocytosis. CO altered circadian-dependent neuroprotection and CD36 induction, determining the functional outcome in a hemorrhagic stroke model. This study emphasizes how circadian rhythmicity influences neuronal damage after neurovascular events.