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.

Temporal Expression Pattern of Hemoxygenase-1 Expression and Its Association with Vasospasm and Delayed Cerebral Ischemia After Aneurysmal Subarachnoid Hemorrhage.

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.

Expression of HO1 and PER2 can predict the incidence of delirium in trauma patients with concomitant brain injury.

Intensive care unit (ICU)-acquired delirium is associated with adverse outcome in trauma patients with concomitant traumatic brain injury (TBI), but diagnosis remains challenging. Quantifying circadian disruption by analyzing expression of the circadian gene period circadian regulator 2 (PER2) and heme oxygenase 1 (HO1), which determines heme turnover, may prove to be potential diagnostic tools. Expression of PER2 and HO1 was quantified using qPCR from blood samples 1 day and 7 days after trauma. Association analysis was performed comparing mRNA expression levels with parameters of trauma (ISS-injury severity score), delirium, acute kidney injury (AKI) and length of ICU stay. 48 polytraumatized patients were included (equal distribution of TBI versus non-TBI) corrected for ISS, age and gender using a matched pairs approach. Expression levels of PER2 and HO1 were independent of age (PER2: P = 0.935; HO1: P = 0.988), while expression levels were significantly correlated with trauma severity (PER2: P = 0.009; HO1: P < 0.001) and longer ICU length of stay (PER2: P = 0.018; HO1: P < 0.001). High expression levels increased the odds of delirium occurrence (PER2: OR = 4.32 [1.14-13.87]; HO1: OR = 4.50 [1.23-14.42]). Patients with TBI showed a trend towards elevated PER2 (OR = 3.00 [0.84-9.33], P = 0.125), but not towards delirium occurrence (P = 0.556). TBI patients were less likely to develop AKI compared to non-TBI (P = 0.022). Expression levels of PER2 and HO1 correlate with the incidence of delirium in an age-independent manner and may potentially improve diagnostic algorithms when used as delirium biomarkers.Trial registration: German Clinical Trials Register (Trial-ID DRKS00008981; Universal Trial Number U1111-1172-6077; Jan. 18, 2018).

Patients with Subarachnoid Hemorrhage Exhibit Disturbed Expression Patterns of the Circadian Rhythm Gene Period-2.

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.