Neutrophil Extracellular Traps Induce Brain Edema Around Intracerebral Hematoma via ERK-Mediated Regulation of MMP9 and AQP4

Perihematomal edema (PHE) significantly aggravates secondary brain injury in patients with intracerebral hemorrhage (ICH), yet its detailed mechanisms remain elusive. Neutrophil extracellular traps (NETs) are known to exacerbate neurological deficits and worsen outcomes after stroke. This study explores the potential role of NETs in the pathogenesis of brain edema following ICH. The rat ICH model was created, immunofluorescence and Western blot were used to examine neutrophil accumulation, NET markers citrullinated histone H3 (CitH3) and myeloperoxidase (MPO), tight junction proteins (ZO-1 and Occludin), Aquaporin-4 (AQP4), matrix metalloproteinase-9 (MMP-9), and ERK phosphorylation (p-ERK) in brain tissues surrounding the hematoma. TUNEL staining and behavioral tests were employed to evaluate neuronal apoptosis and neurological dysfunction, while blood-brain barrier (BBB) permeability and brain edema were also measured by Evans blue and brain water content. Furthermore, the molecular mechanisms related to NETs-induced PHE were investigated using NETs, ERK, MMP-9 and AQP4 regulators, respectively. Ly6G⁺ neutrophils surrounding the hematoma developed NETs within 3 days post-ICH. NETs decreased tight junction proteins, destroyed BBB integrity, promoted brain edema, increased neuronal apoptosis, and exacerbated neurological deficits. Conversely, inhibition of NETs mitigated PHE, reduced neuronal apoptosis, and improved neurological functions. Mechanistically, NET-induced PHE was originated from impairment of BBB tight junction via ERK/MMP9 pathway, coupled with ERK-mediated AQP4 downregulation in perihematomal regions. These findings elucidated the effects of NETs on PHE, which offered promising insights for targeting NETs to relieve brain edema and secondary brain injury post-ICH.