473 Role of Neutrophils and Neutrophil Extracellular Traps in Subarachnoid Hemorrhage in Mice

Abstract

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (aSAH) continues to be a devastating disease. The role of neutrophils and neutrophil extracellular traps (NETs) have been investigated in other cerebrovascular disorders. Recent evidence suggest that neutrophils might play a role in outcomes after aSAH. METHODS: Subarachnoid hemorrhage model: The endovascular perforation model was utilized to induce SAH in mice. Flow cytometry: Flow cytometry technique was utilized to quantify neutrophils. Neutrophils were identified as CD45+Cd11b+Ly6G+ cells. NETosis assay: Neutrophils were assayed for their NETosis potential using a previously described method. The ratio of PMA (SYTOX+) to DMSO (SYTOX+) defined the NETosis induction. RNAseq: RNA sequencing was performed on neutrophils isolated from skull and femur bone marrow. Immunohistochemistry: NETs were assessed using classical IHC techniques and in vivo microscopy. Neurobehavior score: Mice were assessed daily for their behavior score using previously reported techniques. RESULTS: Neutrophils infiltrate brain within 24 hours and normalize by day 5. RNAseq demonstrates segregation of skull and femur bone marrow neutrophils following SAH. SAH induces a NETosis phenotype in skull neutrophils. NETs form starting day 1 after injury and persist until at least day 7. Neutrophil depletion and NETs inhibition attenuates early brain injury after SAH. Patients developing DCI have significantly higher blood markers of NETs. CONCLUSIONS: Neutrophils infiltrate the brain within the first 24 hours with NETs forming as early as day 1. Depletion of neutrophils and NET inhibition mitigated early brain injury following SAH. Blood markers of NETs are elevated in DCI patients and may be used as predictors of DCI. This highlights NETs as potential therapeutic target for patients with SAH and paves the way for future studies.