(1) Background: A detailed understanding of the pathophysiology of hemorrhagic stroke is still missing. We hypothesized that expression of heme oxygenase-1 (HO-1) in microglia functions as a protective signaling pathway. (2) Methods: Hippocampal HT22 neuronal cells were exposed to heme-containing blood components and cell death was determined. We evaluated HO-1-induction and cytokine release by wildtype compared to tissue-specific HO-1-deficient (LyzM-Cre.Hmox1 fl/fl) primary microglia (PMG). In a study involving 46 patients with subarachnoid hemorrhage (SAH), relative HO-1 mRNA level in the cerebrospinal fluid were correlated with hematoma size and functional outcome. (3) Results: Neuronal cell death was induced by exposure to whole blood and hemoglobin. HO-1 was induced in microglia following blood exposure. Neuronal cells were protected from cell death by microglia cell medium conditioned with blood. This was associated with a HO-1-dependent increase in monocyte chemotactic protein-1 (MCP-1) production. HO-1 mRNA level in the cerebrospinal fluid of SAH-patients correlated positively with hematoma size. High HO-1 mRNA level in relation to hematoma size were associated with improved functional outcome at hospital discharge. (4) Conclusions: Microglial HO-1 induction with endogenous CO production functions as a crucial signaling pathway in blood-induced inflammation, determining microglial MCP-1 production and the extent of neuronal cell death. These results give further insight into the pathophysiology of neuronal damage after SAH and the function of HO-1 in humans.

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.

Background Red blood cell-induced cerebral inflammation and toxicity has been shown to be attenuated by induction of the heme-catalyzing enzyme, hemoxygenase-1 (HO-1), in animal models of subarachnoid hemorrhage (SAH). Although inflammatory mechanisms leading to secondary neuronal injury in SAH are becoming increasingly well understood, markers of cerebral inflammation have so far not been implemented in clinical prediction models of SAH. Methods In this biomarker observational study, HO-1 messenger ribonucleic acid (mRNA) expression levels were determined in cerebrospinal fluid (CSF) and blood of 66 patients with aneurysmal SAH on days 1, 7, and 14 after the SAH event. HO-1 mRNA expression was determined via real time polymerase chain reaction (PCR), and relative expression changes were quantified in comparison with expression levels in nonhemorrhagic control CSF. Subarachnoid blood burden, as well as presence of vasospasm and delayed cerebral ischemia (DCI), were recorded. Short and long-term clinical outcomes were assessed using the Modified Rankin Scale at discharge and 1 year after the SAH event. Results CSF HO-1 expression levels showed a significant increase over the 14-day observation period (p < 0.001, F = 22.53) and correlated with intracranial hematoma burden (ρ = 0.349, p = 0.025). In multivariate analyses, CSF HO-1 expression levels did not reach significance as independent predictors of outcome. Vasospasm on computed tomographic angiography was associated with lower CSF HO-1 expression levels on day 7 after SAH (n = 53, p = 0.010), whereas patients with DCI showed higher CSF HO-1 expression levels on day 14 after SAH (n = 21, p = 0.009). Conclusions HO-1 expression in CSF in patients with SAH follows a distinct temporal induction pattern and is dependent on intracranial hematoma burden. CSF HO-1 expression was unable to predict functional outcome. Associations of early low HO-1 expression with vasospasm and late elevated HO-1 expression with DCI may point to detrimental effects of late HO-1 induction, warranting the need for further investigation in a larger study population.