Mechanistic characterization and inhibition of PAD4-dependent NETosis following experimental neonatal hypoxic-ischemic brain injury.

Neonatal hypoxia-ischemia (HI) remains a major global cause of neonatal morbidity and mortality. To develop effective therapeutic strategies, a deeper understanding of the acute inflammatory processes following HI is required. As the role of peripheral immune cell infiltration is poorly understood we used single nuclei RNA sequencing to investigate acute sequalae post-HI. This revealed a distinct neutrophil population characterized by increased expression of markers associated with neutrophil extracellular trap (NET) formation. The study evaluated the therapeutic potential of inhibiting NETosis formation using the peptidylarginine-deiminase-4 (PAD4) inhibitor Cl-amidine. We demonstrated that while oxygen-glucose deprivation (OGD) induces NET-formation and matrix-metalloproteinase-9 release in neutrophils, this is significantly reduced by the PAD4 inhibitor Cl-amidine. Second, in a co-culture model, inhibition of NETosis during OGD conditions improved neuronal survival. Third, Cl-amidine treatment reduced brain tissue loss and decreased expression levels of NETosis-related proteins 48 h post-HI in vivo. Moreover, behavioral testing performed six weeks after HI revealed improved motor function in the treatment group. This study highlights the detrimental role of neutrophil activation and NETosis in exacerbating brain injury following neonatal HI. Targeting NET formation in the acute phase after HI may represent a promising therapeutic approach to improve both immediate and long-term neurological outcomes.