Neutrophil Extracellular Traps Activate Meningeal Fibroblast to Aggravate Subarachnoid Fibrosis in Kaolin-Induced Hydrocephalus in Rats

Background and objectives: Subarachnoid fibrosis is the key pathology of hydrocephalus, but its underlying mechanisms remains poorly understood. In the present study, we aim to verify the hypothesis that neutrophil extracellular traps (NETs), released by neutrophils infiltrated into the subarachnoid space following hemorrhage and Infection, might be a crucial culprits in promoting subarachnoid fibrosis in hydrocephalus.

Methods: Firstly, NETs in cerebrospinal fluid (CSF) specimens from patients and subarachnoid fibrosis of kaolin-induced hydrocephalus rat model were detected by assay kit and immunofluorescence, respectively. Secondly, kaolin-induced hydrocephalus rats were treated by peptidylarginine deiminase 4 (PAD4) inhibitor and DNase I. NETs, subarachnoid fibrosis, reactive gliosis, proliferation and differentiation of meningeal fibroblasts were detected by immunofluorescence and Western Blot (WB), ventricular volumes were evaluated by magnetic resonance imaging (MRI). Finally, primary meningeal fibroblasts were stimulated with NET, and their proliferation and differentiation were measured by flow cytometer, WB and immunofluorescence, respectively.

Results: Combining with CSF specimens from patients and hydrocephalus rat model, we found that infiltrating neutrophils release NETs in the subarachnoid space after subarachnoid hemorrhage and hydrocephalus. Further, combining with in vivo animal experiments and in vitro experiments, we demonstrated that NETs aggravate subarachnoid fibrosis to promote hydrocephalus via stimulating the proliferation and differentiation of meningeal fibroblasts. What's more, both inhibiting NETs production with PAD4 inhibitor and degrading NETs with DNase I significantly prevent the development of hydrocephalus by attenuating subarachnoid fibrosis.

Conclusion: NETs aggravate subarachnoid fibrosis to promote hydrocephalus via stimulating the proliferation and differentiation of meningeal fibroblasts, and restricting NETs significantly prevents the development of hydrocephalus through attenuating subarachnoid fibrosis. It indicates that NETs might be a promising potential target for clinical hydrocephalus treatment.

DNase I; fibroblast; fibrosis; hydrocephalus; neutrophil extracellular traps.