The role of cell-free DNA in fibrinolysis for intraventricular hemorrhage

Abstract

OBJECTIVETissue plasminogen activator (tPA) fibrinolysis did not improve functional outcomes of patients with intraventricular hemorrhage (IVH), largely because of the unsatisfactory clot clearance. The presence of neutrophil extracellular traps (NETs) within the clot has been confirmed to impair tPA fibrinolysis, but the mechanism has been unclear. The authors hypothesized that cell-free DNA (cfDNA), the main framework of NETs, might be the important reason for the fibrinolysis resistance, and they validated the hypothesis, hoping to provide a new target to promote intraventricular fibrinolysis.METHODSFirst, cfDNA was detected in IVH clots by immunofluorescence staining in a rat model of IVH. Second, after blood (with or without exogenous cfDNA) intraventricular injection, IVH rats were given intraventricular infusion of 2 μl of saline, tPA, or tPA + DNase1 randomly. Then, the ventricular volume, animal behavior, and reactive astrocyte proliferation were assessed. Third, the IVH clots were collected for fibrinolysis assay in vitro. Finally, the effects of exogenous cfDNA in IVH were evaluated.RESULTSThe presence of cfDNA in clots was observed as early as 1 hour after IVH. Compared with the whole-blood model, blood + cfDNA caused more severe ventricular dilation (day 7: blood 32.47 ± 2.096 mm3 vs blood + DNA 40.09 ± 2.787 mm3, p < 0.05), increased fibrinolysis resistance to tPA (day 7: tPA + DNA 26.04 ± 1.318 mm3 vs tPA 22.15 ± 1.706 mm3, p < 0.05), and further deteriorated the functional defects in rats (blood vs blood + DNA, p < 0.05). Degradation of cfDNA by DNase1 further enhanced the fibrinolysis effects on relieving the ventricular dilation (day 7: tPA + DNase1 11.67 ± 2.023 mm3 vs tPA, p < 0.05), improving the functional outcome (tPA vs tPA + DNase1, p < 0.05) and reducing periventricular astrocyte proliferation.CONCLUSIONScfDNA impaired tPA fibrinolysis for IVH, and degradation of cfDNA may be a new target to improve this condition.

Neutrophil Extracellular Traps (NETs): A New Therapeutic Target for Neuroinflammation and Microthrombosis After Subarachnoid Hemorrhage?

“…Furthermore, Middleton et al reported that NETs contribute to COVID-19-associated lung injury and microthrombosis, and lung autopsy confirmed microthrombi containing NETs with neutrophil-platelet infiltration [3]. We reported that in a rat model of intracerebral hemorrhage and intraventricular hemorrhage, where large numbers of NETs are formed in and around the hematoma, the use of tPA in combination with recombinant deoxyribonuclease1 (DNase1) to break down NETs accelerates hematoma clearance and reduces hydrocephalus, whereas cell-free DNA, the main skeletal structure of NETs, caused delayed hematoma clearance and aggravated brain damage [4,5]. In neuroinflammation after subarachnoid hemorrhage (SAH), neutrophils play an important and complex role [6].…”

Neutrophil extracellular trap-mediated impairment of meningeal lymphatic drainage exacerbates secondary hydrocephalus after intraventricular hemorrhage

“…Previous studies have demonstrated the occurrence of NET formation in arterial thrombi following acute cerebral ischemia. In our previous study, significant neutrophil infiltration and NET formation were observed following IVH 38 . Subsequently, in our study on SAH, we observed the colocalization of NETs with fibrinogen-positive microthrombi 29 .…”

“…injection of 300 U (2 µL) DNase I 1 h after IVH induction. Subsequent injections of DNase I (150 U/μL in 2 µL) were administered at 12-h intervals until the predetermined time points 38 . For IF staining, Western blot (WB) analysis, RT‒qPCR, and enzyme-linked immunosorbent assay (ELISA), the meninges were collected 24 h after IVH.…”

Neutrophil Extracellular Traps Exacerbate Secondary Injury via Promoting Neuroinflammation and Blood–Spinal Cord Barrier Disruption in Spinal Cord Injury

“…SYTOX Orange staining was performed as previous described ( 21 ). Sections were stained with SYTOX Orange (Molecular Probes, Inc.) at a concentration of 5 μM for 10 min.…”