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

Luise Henrich; Iva Kiessling; Matti Steimer; Sibylle Frase; Sandra Kaiser; Nils Schallner

doi:10.1186/s12950-023-00371-w

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

J Inflamm (Lond). 2023 Dec 16;20(1):43. doi: 10.1186/s12950-023-00371-w.

Authors

Luise Henrich^#^{1

2}, Iva Kiessling^#^{1

2}, Matti Steimer^{1

2}, Sibylle Frase^{2

3}, Sandra Kaiser^#^{1

2}, Nils Schallner^#^{4

5}

Affiliations

¹ Department of Anesthesiology & Critical Care, Medical Center, University of Freiburg, Hugstetter Str. 55, Freiburg, 79106, Germany.
² Faculty of Medicine, University of Freiburg, Freiburg, Germany.
³ Department of Neurology and Neuroscience, Medical Center, University of Freiburg, Freiburg, Germany.
⁴ Department of Anesthesiology & Critical Care, Medical Center, University of Freiburg, Hugstetter Str. 55, Freiburg, 79106, Germany. nils.schallner@uniklinik-freiburg.de.
⁵ Faculty of Medicine, University of Freiburg, Freiburg, Germany. nils.schallner@uniklinik-freiburg.de.

^# Contributed equally.

Abstract

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 Hmox1^fl/fl and LyzM-Cre-Hmox1^fl/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 Hmox1^fl/fl and LyzM-Cre-Hmox1^fl/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-Hmox1^fl/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.

Keywords: Brain Hemorrhage; Carbon Monoxide; Circadian rhythm; Heme Oxygenase; Microglia; Neuroinflammation; Phagocytosis.