# Study on the Function and Mechanism of Neutrophil Extracellular Traps in Regulating Necroptosis Following Traumatic Brain Injury

## TL;DR

This study explores how neutrophil extracellular traps (NETs) contribute to brain injury after trauma by activating a cell death pathway called necroptosis.

## Contribution

The paper reveals that NETs exacerbate traumatic brain injury by activating the necroptosis pathway, offering a potential therapeutic target.

## Key findings

- NETs are formed after TBI, marked by increased PAD4 and MPO levels.
- Inhibiting NETs or necroptosis reduces neuronal death and brain swelling.
- Combined inhibition of NETs and necroptosis does not provide additional benefits.

## Abstract

Traumatic brain injury (TBI) remains a major global public health challenge with high morbidity and mortality, and secondary injury characterized by neuroinflammation, brain edema, and neuronal cell death is a critical determinant of patient prognosis. Neutrophil extracellular traps (NETs) and necroptosis are involved in TBI pathology, but their crosstalk remains unclear. Here, we used NETs inhibitors (Cl‐amidine and DNase I) and the necroptosis inhibitor Necrostatin‐1 (Nec‐1) to investigate the roles of NETs and necroptosis in neuronal injury following TBI.

Male C57BL/6J mice were used to establish a TBI model via controlled cortical impact (CCI). Cl‐amidine, DNase I, and Necrostatin‐1 were administered to explore the mechanism by which NETs regulate necroptosis and exacerbate TBI‐induced secondary injury. The modified neurological severity score (mNSS) assessment, brain edema measurement, enzyme‐linked immunosorbent assay (ELISA), Western blotting, immunofluorescence staining, and TUNEL staining were performed in this study. Mice were sacrificed at 1, 3, 5, and 7 days post‐TBI, with Day 3 post‐TBI designated as the key time point for primary analyses due to the peak expression of NETs markers: myeloperoxidase (MPO) and peptidyl arginine deiminase 4 (PAD4).

Our results showed that TBI induced a time‐dependent upregulation of MPO and PAD4 in the ipsilateral cortex. Inhibition of NETs or blockade of necroptosis significantly reduced neuronal apoptosis, alleviated brain edema, improved mNSS scores, preserved blood–brain barrier integrity, and decreased levels of pro‐inflammatory cytokines (TNF‐α, IL‐1β). Western blot analysis revealed that TBI markedly upregulated the expression of RIP1, RIP3, MLKL, and their phosphorylated forms, while NETs inhibition downregulated these necroptosis‐related proteins. Notably, combined inhibition of NETs and necroptosis did not exert synergistic protective effects on TBI‐induced brain injury.

NETs exacerbate TBI‐induced secondary brain injury partially by activating the necroptosis pathway. Inhibition of NETs exerts neuroprotective effects. Targeting NETs may serve as a promising therapeutic strategy to improve prognosis in TBI patients.

TBI induces NETs formation, as evidenced by the upregulation of PAD4 and MPO. NETs activate the necroptosis pathway via phosphorylation of RIP1‐RIP3‐MLKL, thereby exacerbating secondary brain injury (neuronal death, BBB disruption, and neuroinflammation). Inhibition of NETs or necroptosis significantly alleviates these pathological changes, but combined inhibition shows no synergistic effect. These findings demonstrate that NETs aggravate TBI‐induced secondary injury partially through the necroptosis pathway.