The Evolution Of NETosis Biomarkers And Their Relationship With Peripheral Immune Cells In A Preclinical Model Of Ischemic Stroke

Introduction: Reperfusion resistance is one major challenge encountered during the thrombolytic treatment of acute ischemic stroke. Neutrophil extracellular traps formation (NETosis) facilitates thrombosis and contributes to reperfusion resistance. However, the plasma biomarker profile of NETs following stroke is poorly understood. Hypothesis: Understanding the precise evolution of NETosis biomarkers and their relationship with peripheral immune cells may provide new insight into the therapeutic options of acute ischemia stroke. Methods: Three-month-old C57BL/6 mice were subjected to middle cerebral artery occlusion for 60 minutes followed by reperfusion (Ischemia-Reperfusion, IR). Peripheral blood was collected on day 1, 2, 3, 5, and 7 after IR and whole blood cell counts were assessed. The concentrations of NETosis biomarkers [Citrullinated histone 3 (CitH3), Neutrophil Elastase (NE), Myeloperoxidase (MPO), and deoxyribonucleic acid (DNA)] in plasma were measured. Pearson correlation analysis was used to determine the relationship between the levels of NETosis biomarkers, blood cell counts and the ratios of neutrophils, lymphocytes, and platelets. Results: Compared to control (Ctrl) mice, the IR-subjected mice exhibited a drastic increase in plasma CitH3 and NE on day 1 (p<0.05) while DNA and MPO reached their peak levels on day 3. IR-subjected mice also showed a significant increase in peripheral neutrophils and decline in peripheral leukocytes, lymphocytes, and monocytes on day 1 and day 2 (p<0.05). The ratios of neutrophil to lymphocyte, neutrophil to leukocyte, and lymphocyte to monocyte dramatically increased on day 1 (p<0.05). Plasma NE, CitH3 and MPO were positively correlated with peripheral neutrophil and the ratio of neutrophil to lymphocyte, but inversely correlated with peripheral lymphocyte (p<0.05). Conclusions: There are time dependent changes in plasma NETosis biomarkers and their relationship with peripheral immune cells post-stroke may inform effectiveness of potential treatments to prevent the expansion of cerebral infarction and functional deterioration. However, further investigation is needed to confirm this hypothesis and its clinical implication.