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Background and Purpose Subarachnoid hemorrhage (SAH) is associated with a temporal pattern of stroke incidence. We hypothesized that natural oscillations in gene expression controlling circadian rhythm impact the severity of neuronal injury. We moreover predict that heme oxygenase-1 (HO-1/Hmox1) and its product carbon monoxide (CO) contribute to restoration of rhythm and neuroprotection. Methods Murine SAH model where blood was injected at various time points of the circadian cycle. Readouts included circadian clock gene expression, locomotor activity, vasospasm, neuroinflammatory markers, and apoptosis. Additionally, cerebrospinal fluid and peripheral blood leukocytes from SAH patients and controls were analyzed for ‘clock gene’ expression. Results Significant elevations in the clock genes Per-1, Per-2 and NPAS-2 were observed in the hippocampus, cortex and suprachiasmatic nucleus (SCN) in mice subjected to SAH at zeitgeber time (ZT) 12 when compared to ZT2. Clock gene expression amplitude correlated with basal expression of HO-1, which was also significantly greater at ZT12. SAH animals showed a significant reduction in cerebral vasospasm, neuronal apoptosis, and microglia activation at ZT12 compared to ZT2. In animals with myeloid-specific HO-1 deletion (Lyz-Cre-Hmox1fl/fl) Per-1, Per-2, and NPAS-2 expression was reduced in the SCN, which correlated with increased injury. Treatment with low dose CO rescued Lyz-Cre-Hmox1fl/fl mice, restored Per-1, Per-2 and NPAS-2 expression and reduced neuronal apoptosis. Conclusions Clock gene expression regulates, in part, the severity of SAH and requires myeloid HO-1 activity to clear the erythrocyte burden and inhibit neuronal apoptosis. Exposure to CO rescues the loss of HO-1 and thus merits further investigation in patients with SAH.

BackgroundSubarachnoid hemorrhage (SAH) is a devastating disease that affects over 30,000 Americans per year. Previous animal studies have explored the therapeutic effects of deferoxamine (DFX) via its iron-chelating properties after SAH, but none have assessed the necessity of microglial/macrophage heme oxygenase-1 (HO-1 or Hmox1) in DFX neuroprotection, nor has the efficacy of an intracerebroventricular (ICV) administration route been fully examined. We explored the therapeutic efficacy of systemic and ICV DFX in a SAH mouse model and its effect on microglial/macrophage HO-1.MethodsWild-type (WT) mice were split into the following treatment groups: SAH sham + vehicle, SAH + vehicle, SAH + intraperitoneal (IP) DFX, and SAH + ICV DFX. For each experimental group, neuronal damage, cognitive outcome, vasospasm, cerebral and hematogenous myeloid cell populations, cerebral IL-6 concentration, and mitochondrial superoxide anion production were measured. HO-1 co-localization to microglia was measured using confocal images. Trans-wells with WT or HO-1−/− microglia and hippocampal neurons were treated with vehicle, red blood cells (RBCs), or RBCs with DFX; neuronal damage, TNF-α concentration, and microglial HO-1 expression were measured. HO-1 conditional knockouts were used to study myeloid, neuronal, and astrocyte HO-1 involvement in DFX-induced neuroprotection and cognitive recovery.ResultsDFX treatment after SAH decreased cortical damage and improved cognitive outcome after SAH yet had no effect on vasospasm; ICV DFX was most neuroprotective. ICV DFX treatment after SAH decreased cerebral IL-6 concentration and trended towards decreased mitochondrial superoxide anion production. ICV DFX treatment after SAH effected an increase in HO-1 co-localization to microglia. DFX treatment of WT microglia with RBCs in the trans-wells showed decreased neuronal damage; this effect was abolished in HO-1−/− microglia. ICV DFX after SAH decreased neuronal damage and improved cognition in Hmox1fl/fl control and NesCre:Hmox1fl/fl mice, but not LyzMCre:Hmox1fl/fl mice.ConclusionsDFX neuroprotection is independent of vasospasm. ICV DFX treatment provides superior neuroprotection in a mouse model of SAH. Mechanisms of DFX neuroprotection after SAH may involve microglial/macrophage HO-1 expression. Monitoring patient HO-1 expression during DFX treatment for hemorrhagic stroke may help clinicians identify patients that are more likely to respond to treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0709-1) contains supplementary material, which is available to authorized users.