Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1
Nils Schallner, Rambhau Pandit, Robert LeBlanc III, Ajith J. Thomas, Christopher S. Ogilvy, Brian S. Zuckerbraun, David Gallo, Leo E. Otterbein, Khalid A. Hanafy
Nils Schallner, Rambhau Pandit, Robert LeBlanc III, Ajith J. Thomas, Christopher S. Ogilvy, Brian S. Zuckerbraun, David Gallo, Leo E. Otterbein, Khalid A. Hanafy
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Research Article Neuroscience

Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1

Abstract

Subarachnoid hemorrhage (SAH) carries a 50% mortality rate. The extravasated erythrocytes that surround the brain contain heme, which, when released from damaged red blood cells, functions as a potent danger molecule that induces sterile tissue injury and organ dysfunction. Free heme is metabolized by heme oxygenase (HO), resulting in the generation of carbon monoxide (CO), a bioactive gas with potent immunomodulatory capabilities. Here, using a murine model of SAH, we demonstrated that expression of the inducible HO isoform (HO-1, encoded by Hmox1) in microglia is necessary to attenuate neuronal cell death, vasospasm, impaired cognitive function, and clearance of cerebral blood burden. Initiation of CO inhalation after SAH rescued the absence of microglial HO-1 and reduced injury by enhancing erythrophagocytosis. Evaluation of correlative human data revealed that patients with SAH have markedly higher HO-1 activity in cerebrospinal fluid (CSF) compared with that in patients with unruptured cerebral aneurysms. Furthermore, cisternal hematoma volume correlated with HO-1 activity and cytokine expression in the CSF of these patients. Collectively, we found that microglial HO-1 and the generation of CO are essential for effective elimination of blood and heme after SAH that otherwise leads to neuronal injury and cognitive dysfunction. Administration of CO may have potential as a therapeutic modality in patients with ruptured cerebral aneurysms.

Authors

Nils Schallner, Rambhau Pandit, Robert LeBlanc III, Ajith J. Thomas, Christopher S. Ogilvy, Brian S. Zuckerbraun, David Gallo, Leo E. Otterbein, Khalid A. Hanafy

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Figure 6

Importance of microglial HO-1 for erythrophagocytosis and neuronal survival in vitro.

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Importance of microglial HO-1 for erythrophagocytosis and neuronal survi...
(A) Quantification of the percentage of microglia positive for fluorescence-labeled (pHrodo) red blood cells by flow cytometric analysis. *P = 0.0015 for Hmox1fl/fl red blood cells versus LyzM-Cre Hmox1fl/fl red blood cells (2-tailed Student’s t test; n = 3). (B) Mean fluorescent signal in microglia after exposure to fluorescence-labeled red blood cells. *P = 0.0274 for Hmox1fl/fl red blood cells versus LyzM-Cre Hmox1fl/fl red blood cells (2-tailed Student’s t test; n = 3). (C) Representative (n = 3) light microscopic images (original magnification, ×40) of Hmox1fl/fl and LyzM-Cre Hmox1fl/fl microglia after exposure to red blood cells. (D) Quantification of red blood cells per microglia, analyzed by light microscopy. *P < 0.0001 for Hmox1fl/fl red blood cells versus LyzM-Cre Hmox1fl/fl red blood cells (2-tailed Student’s t test; n = 3). (E) Quantification of the percentage of red blood cell–positive microglia by light microscopy. *P < 0.0001 for Hmox1fl/fl red blood cells versus LyzM-Cre Hmox1fl/fl red blood cells (2-tailed Student’s t test; n = 3). (F) Representative images (original magnification, ×20; n = 3) from TUNEL staining (red) of neuronal cells cocultured with Hmox1fl/fl or LyzM-Cre Hmox1fl/fl microglia with or without red blood cells. Cell nuclei were counterstained with Hoechst 33258 (blue). (G) Quantification of TUNEL-positive neuronal cells per microscopic view. *P < 0.0001 for Hmox1fl/fl red blood cells versus LyzM-Cre Hmox1fl/fl red blood cells (2-tailed Student’s t test; n = 3). (H) Quantification of TUNEL-positive neuronal cells per microscopic view. Neuronal cells were cocultured with WT PMG treated with the indicated pharmacological inhibitors of phagocytosis. *P < 0.0001 for red blood cells without inhibitor versus red blood cells plus cytochalasin and versus red blood cells plus dynasore (1-way ANOVA; n = 3).