Abstract: Microglial erythrophagocytosis is crucial in injury response to hemorrhagic stroke. We hypothesized that regulation of microglial erythrophagocytosis via HO-1/CO depends on a pathway involving reactive oxygen species (ROS) and CD36 surface-expression. The microglial BV-2 cell line and primary microglia (PMG) were incubated +/−blood and +/−CO-exposure. PMG isolated from tissue-specific HO-1-deficient (LyzM-Cre-Hmox1 fl/fl) and CD36 −/− mice or siRNA against AMPK (AMP-activated protein kinase) were used to test our hypothesis. In a murine subarachnoid hemorrhage (SAH) model, we compared neuronal injury in wild-type and CD36 −/− mice. Readouts included vasospasm, microglia activation, neuronal apoptosis, and spatial memory. We observed increased microglial HO-1-expression after blood-exposure. A burst in ROS-production was seen after CO-exposure, which led to increased amounts of phosphorylated AMPK with subsequently enhanced CD36 surface-expression. Naïve PMG from LyzM-Cre-Hmox1 fl/fl mice showed reduced ROS-production and CD36 surface-expression and failed to respond to CO with increased CD36 surface-expression. Lack of HO-1 and CD36 resulted in reduced erythrophagocytosis that could not be rescued with CO. Erythrophagocytosis was enhanced in BV-2 cells in the presence of exogenous CO, which was abolished in cells treated with siRNA to AMPK. CD36 −/− mice subjected to SAH showed enhanced neuronal cell death, which resulted in impaired spatial memory function. We demonstrate that microglial phagocytic function partly depends on a pathway involving HO-1 with changes in ROS-production, phosphorylated AMPK, and surface expression of CD36. CD36 was identified as a crucial component in blood clearance after hemorrhage that ultimately determines neuronal outcome. These results demand further investigations studying the potential neuroprotective properties of CO

Abstract: Circadian rhythm gene expression in cerebral pacemaker regions is regulated by a transcriptional-translational feedback loop across the 24-h day-night cycle. In preclinical models of subarachnoid hemorrhage (SAH), cyclic gene expression is disrupted. Stabilization of circadian rhythm gene expression attenuates susceptibility to ischemic damage in both neuronal and myocardial tissues. In this clinical observational study, circadian rhythm gene Period-2 (Per2) mRNA expression levels were determined from blood leukocytes and cerebrospinal fluid (CSF) cells via real-time PCR on days 1, 7 and 14 after aneurysm rupture in 49 patients with spontaneous SAH. CSF Per2 expression was markedly suppressed immediately after SAH and remained suppressed over the course of two weeks of ICU treatment. Short-term mortality as well as occurrence of delirium was associated with greater extent of Per2 suppression on day 1 after SAH. Patients that developed delayed cerebral ischemia exhibited comparatively lower Per2 expression levels on day 7 after SAH, while presence of vasospasm remained unaffected. However, Per2 expression did not differ in patient groups with favourable or non-favourable functional neurological outcome (modified Rankin Scales 1-3 vs. 4-6). While our findings suggest a potential protective effect of stable circadian rhythm gene expression on the extent of ischemic damage, this effect was confined to the early disease course and was not reflected in patients' functional neurological outcome