The Role of Neutrophil Extracellular Traps in Early Microthrombosis and Brain Injury After Subarachnoid Hemorrhage in Mice

Abstract

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.

Perivascular Macrophages Mediate Microvasospasms After Experimental Subarachnoid Hemorrhage

“…29 Similarly, inhibiting NETs may reduce microthrombosis and therefore attenuate early brain injury after SAH. 30 Despite its apparent advantages, the current study has also some limitations. Clodronate had to be applied by intrathecal injection; however, the application of vehicle alone already increased the number of CD206 + cells compared with naive mice.…”

“…29 Similarly, inhibiting NETs may reduce microthrombosis and therefore attenuate early brain injury after SAH. 30…”

Neutrophils and Neutrophil Extracellular Traps Cause Vascular Occlusion and Delayed Cerebral Ischemia After Subarachnoid Hemorrhage in Mice

“…NETs have been shown to be key players in causing/ promoting systemic inflammation, 57,58 neuroinflammation and microglia activation after CNS injury, 59,60 vascular instability of intracranial aneurysms, 61 blood-brain barrier dysfunction after CNS injury, 20,62 and coagulation. 25,[63][64][65] In relation to DCI after SAH, inflammation, neuroinflammation, and coagulation are all known factors contributing to DCI and poor outcome. 46,53,66,67 Although these pathophysiological events were not the focus of this study, preventing NETs after SAH via PAD4 inhibition or neutrophil depletion may attenuate or prevent these deleterious events, thereby lessening their contributions to DCI.…”

“…20,22,24 In a recent experimental article, degrading NETs, using DNAse, was shown to improve outcomes after SAH. 25…”

“…20,22,24 In a recent experimental article, degrading NETs, using DNAse, was shown to improve outcomes after SAH. 25 Thus, we hypothesized that neutrophils contribute to DCI and worsen outcomes after SAH via cerebrovascular occlusion induced by NETs and that early prevention of neutrophil NETosis can improve outcomes. To test our hypothesis, we used a mouse model of SAH to examine (1) if depleting neutrophils can reduce NETs, improve cerebral perfusion, and prevent DCI and (2) if preventing NETs or degrading them after SAH is therapeutically beneficial.…”