PAD4 Inhibitor Therapy Prevents Nets and Improves Neurological Deficits Resulting from Subarachnoid Hemorrhage

Introduction: Neutrophil extracellular traps (NETs) are critical mediators of the immune response, often contributing to pathology in neovascular events by acting through the peptidyl arginine deiminase 4 (PAD4) molecular pathway. In subarachnoid hemorrhage (SAH), NETs can hinder recovery by exacerbating inflammation and inducing vasoconstriction, ultimately obstructing cerebrovascular healing. This study aims to elucidate the role of NETs in SAH and evaluate whether their inhibition could serve as a therapeutic intervention. We hypothesize that SAH-induced mice treated with the PAD4 inhibitor, GSK484, will exhibit reduced deficits compared to sham and vehicle groups.

Methods: SAH was induced in C57BL/6 male mice (n=22) via endovascular perforation, and female mice (n=24) were injected with blood to simulate SAH. Mice were administered either GSK106 (vehicle) or GSK484 (treatment). Daily neurobehavioral exams were conducted, scored from 0 to 24, with higher scores indicating better outcomes. In-vivo MRI evaluated cerebral blood flow and infarct volume 1 and 5 days post-SAH. Behavioral data were analyzed using a two-way ANOVA, and MRI data were examined with two-way ANOVA and post hoc Tukey’s test. NET formations were quantified post-SAH in both male and female groups.

Results: Results showed significant recovery in the treatment group. In 72% of males (16/22) and 62% of females (15/24), the GSK484-treated SAH group had a neuroscore of 23 (similar to the sham group), which was significantly higher than the SAH-only group. Infarct volume was reduced by 22% in treated males and 60% in treated females. NET formation was also significantly higher in the vehicle group compared to GSK484-treated mice, indicating a positive correlation between PAD4 pathway activation and NET formation.

Conclusion : These findings suggest that PAD4 inhibition with GSK484 enhances recovery in murine SAH models, supporting its potential as a viable therapeutic intervention. Future research will explore anti-neutrophil therapies currently under development to further elucidate their role in SAH treatment.