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

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.

Circadian dependency of microglial heme oxygenase-1 expression and inflammation determine neuronal injury in hemorrhagic stroke.

BACKGROUND: The heme oxygenase-1 (HO-1) enzyme pathway is of crucial importance in the removal of toxic blood components and regulation of neuroinflammation following hemorrhagic stroke. Although a circadian pattern dependency in the incidence and severity of hemorrhagic stroke exists, it is unknown whether the activity of the HO-1 system in the context of hemorrhagic injury also exhibits circadian dependency. We hypothesized that the circadian regulation of microglial HO-1 would determine the extent of neuroinflammation and neuronal injury in a murine model of subarachnoid hemorrhage (SAH). METHODS: In vitro expression patterns of HO-1 and circadian rhythm genes were analyzed in the microglial BV-2 cell line and primary microglia (PMG) using Western blot and qPCR. PMG isolated from Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice were used to evaluate the role of microglial HO-1. We further investigated the in vivo relevance in a murine subarachnoid hemorrhage (SAH) model using Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice with myeloid cell HO-1 deficiency, inducing SAH at different zeitgeber (ZT) times and analyzing the expression of HO-1 and the circadian control gene Period-2 (Per-2), respectively. Furthermore, we measured the inflammatory cytokine Monocyte Chemoattractant Protein-1 (MCP-1) in the cerebrospinal fluid of SAH patients in correlation with clinical outcome. RESULTS: HO-1 baseline expression and response to CO with blood exposure depended on ZT. In vitro expression of circadian control genes was de-synchronized in LyzM-Cre-Hmox1fl/fl PMG and did not respond to exogenous CO exposure. We found that circadian rhythm plays a crucial role in brain damage after SAH. At ZT2, we observed less phagocytic function, more vasospasm and increased microglial activation. CO reduced mortality at ZT12 in HO-1 deficient mice and reduced the difference between ZT2 and ZT12 in the inflammatory response. Induction of MCP-1 in the CSF from SAH patients was time-dependent and correlated with the expression of circadian control genes, SAH severity, functional impairment and delirium. CONCLUSIONS: Our data point towards a crucial role for the HO-1 enzyme system and circadian control in neuronal injury after a hemorrhagic stroke.