Investigation of Neutrophil Extracellular Traps as Potential Mediators in the Pathogenesis of Non-Acute Subdural Hematomas: A Pilot Study

Abstract

Non-acute subdural hematomas (NASHs) are a cause of significant morbidity and mortality, particularly with recurrences. Although recurrence is believed to involve a disordered neuroinflammatory cascade involving vascular endothelial growth factor (VEGF), this pathway has yet to be completely elucidated. Neutrophil extracellular traps (NETs) are key factors that promote inflammation/apoptosis and can be induced by VEGF. We investigated whether NETs are present in NASH membranes, quantified NET concentrations, and examined whether NET and VEGF levels are correlated in NASHs. Samples from patients undergoing NASH evacuation were collected during surgery and postoperatively at 24 and 48 h. Fluid samples and NASH membranes were analyzed for levels of VEGF, NETs, and platelet activation. NASH samples contained numerous neutrophils positive for NET formation. Myeloperoxidase-DNA complexes (a marker of NETs) remained elevated 48 h postoperatively (1.06 ± 0.22 day 0, 0.72 ± 0.23 day 1, and 0.83 ± 0.33 day 2). VEGF was also elevated in NASHs (7.08 ± 0.98 ng/mL day 0, 3.40 ± 0.68 ng/mL day 1, and 6.05 ± 1.8 ng/mL day 2). VEGF levels were significantly correlated with myeloperoxidase-DNA levels. These results show that NETs are increasing in NASH, a finding that was previously unknown. The strong correlation between NET and VEGF levels indicates that VEGF may be an important mediator of NET-related inflammation in NASH.

Keywords: VEGF; neutrophil extracellular trap; recurrence; subdural hematoma; vascular endothelial growth factor.

Figure 1

NETs and platelets are present in NASH outer membranes. The outer membranes of NASHs were collected, fixed, and embedded in OCT. Membranes were cryosectioned and stained for platelets (CD41, white), neutrophils (MPO, red), and H3Cit, a marker of NETosis (green). Nucleated cells were stained with DAPI (blue). The image shown is representative of three independent samples.

Figure 2

NETosis is observed in fluid from NASHs. NASH samples were collected from patients intraoperatively (day 1) and from subdural drains on the indicated days. Cellular components were removed as described in the Methods section. NETosis was measured by the presence of MPO-DNA complexes (A). As a comparison, NET, PF4, and VEGF levels were examined in 10 healthy donors. Platelet activation was measured by a commercially available ELISA for PF4 (B). VEGF was measured in the fluid from NASH using an ELISA (C). As a comparison, PF4 and VEGF levels were examined in 10 healthy donors. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 3

NET formation correlates with VEGF levels. Spearman’s rank correlation coefficient matrix of NET levels compared with PF4 and VEGF using day 1 CDSH fluid (A). MPO-DNA complexes significantly (p < 0.0001) correlate with VEGF levels in fluid from NASH ((B), p < 0.0001).

Figure 4

NASH fluid increases NETosis in healthy neutrophils compared with healthy plasma. Neutrophils were isolated from healthy donors using CD15+ selection. Neutrophils were incubated with buffer (resting), healthy plasma, or fluid from NASH samples for one hour. After one hour, neutrophils were resuspended in M199 media and allowed to undergo NETosis. Cell-free media was collected, and NET formation was measured by cell-free DNA release (N = 5 per group). ** p ≤ 0.01. ns= not significant.