The Role of Neutrophils and NETosis in Experimental Neonatal Hypoxic-Ischemic Brain Injury

Abstract

Hypoxic-ischemic (HI) injury, which arises from a reduction in cerebral blood flow and oxygen availability shortly after birth, triggers a profound neuroinflammatory response characterized by activation of microglia and astrocytes, leading to neuronal and endothelial cell damage. Recent evidence indicates that microglia contribute to the recruitment of neutrophils into the brain, suggesting a crucial role for these immune cells in the early stages of injury progression. <br/> This study aimed to investigate the role of neutrophil extracellular trap NET formation (NETosis) in the pathophysiology of HI brain injury and to explore the therapeutic potential of NETosis inhibition. Single-nucleus RNA sequencing of cortical tissue after HI identified a distinct neutrophil population expressing NETosis-related marker genes. To determine whether hypoxia and nutrient deprivation can directly induce NET formation, an oxygen-glucose deprivation (OGD) model was employed. OGD induced NETosis in the reoxygenation phase, indicated by increased citrullinated histone H3 (H3Cit) and matrix metalloproteinase-9 (MMP9) release, both of which were reduced by the peptidylarginine deiminase 4 (PAD4) inhibitor Cl-amidine. To assess the contribution of NETosis to neuronal cell damage in a co-culture system, PC12 cells were used as a neuronal-like model. NETosis exacerbated the loss of cell viability in PC12 cultures following OGD stress. Treatment with Cl-amidine during neutrophil co-culture significantly reduced PC12 cell apoptosis, indicating that NETosis contributes to neuronal injury under HI conditions. To translate these findings in an <em>in vivo</em> model, Cl-amidine was administered in a neonatal rat model of HI using postnatal day 7 (P7) Wistar rats. Treatment with Cl-amidine reduced brain area loss in a dose-dependent manner and significantly decreased cortical expression of NETosis-associated proteins 48 h after HI compared to Vehicle controls. Behavioral testing six weeks post-injury revealed improved locomotor outcomes in Cl-amidine-treated animals, however cognitive function at 3 month after HI could not be improved. <br/> The second part of this study examined caffeine, a known neuroprotective compound in neonatal HI, for its effects on neutrophil activation and NETosis. In OGD/Reoxygenation experiments, caffeine inhibited NETosis in a dose-dependent manner. Transcriptomic and protein analyses revealed reduction of protein kinase C (PKC) pathway-related genes after caffeine treatment. Reduced pERK, PAD4 and H3Cit expression suggested that caffeine-mediated NETosis inhibition occurs <em>via</em> suppression of PKC-ERK signaling. <br/> Overall, these findings provide new insights into the contribution of neutrophils and NETosis to neonatal HI brain injury and identified NETosis inhibition as a promising therapeutic approach for neonatal neuroprotection after HI.