Microthrombosis plays an important role in secondary brain injury after experimental subarachnoid hemorrhage (SAH), but the specific mechanism of microthrombosis remains unclear. The purpose of this study was to investigate the role of neutrophil extracellular traps (NETs) in microthrombosis after SAH. SAH was induced in male C57BL/6 mice using an endovascular perforation technique. The marker protein of NETs, citrullinated histone H3 (CitH3), was significantly elevated in the cerebral cortex after SAH, and was co-labeled with microthrombi. Both depletion of neutrophils by anti-Ly6G antibody and DNase I treatment significantly reduced the formation of NETs and microthrombi, and ameliorated neurological deficits, brain edema, BBB disruption, and neuronal injury at 24 h after SAH induction. Cerebral hypoperfusion in the first hours after SAH is a major determinant of poor neurological outcome; in this study, we found that DNase I treatment significantly improved the restoration of early cortical perfusion after SAH. In addition, DNase I treatment also significantly attenuated cerebrospinal fluid (CSF) flow after SAH, which was associated with the diffusion barrier caused by microthrombi in the paravascular space after SAH. In conclusion, NETs are associated with early microthrombosis after SAH; they may be a novel therapeutic target for early brain injury (EBI) after SAH. Supplementary Information The online version contains supplementary material available at 10.1007/s12975-022-01074-9.

OBJECTIVETissue plasminogen activator (tPA) fibrinolysis did not improve functional outcomes of patients with intraventricular hemorrhage (IVH), largely because of the unsatisfactory clot clearance. The presence of neutrophil extracellular traps (NETs) within the clot has been confirmed to impair tPA fibrinolysis, but the mechanism has been unclear. The authors hypothesized that cell-free DNA (cfDNA), the main framework of NETs, might be the important reason for the fibrinolysis resistance, and they validated the hypothesis, hoping to provide a new target to promote intraventricular fibrinolysis.METHODSFirst, cfDNA was detected in IVH clots by immunofluorescence staining in a rat model of IVH. Second, after blood (with or without exogenous cfDNA) intraventricular injection, IVH rats were given intraventricular infusion of 2 μl of saline, tPA, or tPA + DNase1 randomly. Then, the ventricular volume, animal behavior, and reactive astrocyte proliferation were assessed. Third, the IVH clots were collected for fibrinolysis assay in vitro. Finally, the effects of exogenous cfDNA in IVH were evaluated.RESULTSThe presence of cfDNA in clots was observed as early as 1 hour after IVH. Compared with the whole-blood model, blood + cfDNA caused more severe ventricular dilation (day 7: blood 32.47 ± 2.096 mm3 vs blood + DNA 40.09 ± 2.787 mm3, p < 0.05), increased fibrinolysis resistance to tPA (day 7: tPA + DNA 26.04 ± 1.318 mm3 vs tPA 22.15 ± 1.706 mm3, p < 0.05), and further deteriorated the functional defects in rats (blood vs blood + DNA, p < 0.05). Degradation of cfDNA by DNase1 further enhanced the fibrinolysis effects on relieving the ventricular dilation (day 7: tPA + DNase1 11.67 ± 2.023 mm3 vs tPA, p < 0.05), improving the functional outcome (tPA vs tPA + DNase1, p < 0.05) and reducing periventricular astrocyte proliferation.CONCLUSIONScfDNA impaired tPA fibrinolysis for IVH, and degradation of cfDNA may be a new target to improve this condition.

Microthrombosis plays an important role in secondary brain injury after experimental subarachnoid hemorrhage (SAH), but the specific mechanism of microthrombosis remains unclear. The purpose of this study was to investigate the role of neutrophil extracellular traps (NETs) in microthrombosis after SAH. SAH was induced in male C57BL/6 mice using an endovascular perforation technique. The marker protein of NETs, citrullinated histone H3 (CitH3), was significantly elevated in the cerebral cortex after SAH, and was co-labeled with microthrombi. Both depletion of neutrophils by anti-Ly6G antibody and DNase I treatment significantly reduced the formation of NETs and microthrombi, ameliorated neurological deficits, brain edema, BBB disruption, and neuronal injury at 24 h after SAH induction. Cerebral hypoperfusion in the first hours after SAH is a major determinant of poor neurological outcome, in this study, we found that DNase I treatment significantly improved the restoration of early cortical perfusion after SAH. In addition, DNase I treatment also significantly attenuated cerebrospinal fluid (CFS) flow and glymphatic system dysfunction after SAH, which was associated with the diffusion barrier caused by microthrombi in the paravascular space after SAH. In conclusion, NETs is associated with early microthrombosis after SAH, it may be a novel therapeutic target for early brain injury (EBI) after SAH.