Table 1.

Summary of included studies on experimental models. POC: prospective observational cohort; INT: interventional; PTS: photothrombotic stroke; t MCAO: transient middle cerebral artery occlusion; p MCAO: surgical permanent right MCA occlusion; NIT: Neonatal NET-inhibitory factor; SCR: inactive scrambled peptide control; RhDNAse I: Recombinant Human DNAse I; RRT: righting reflex time; FFP: fresh frozen plasma; NS: normal saline.

Authors	Study	Population	Mechanism of Injury	NET Markers	Methods	Experiments	Outcome	Results
Wu (2023) [29]	INT	C57BL/6 mice	PTS	cfDNA	Quant-iT™ PicoGreen® assay (Invitrogen, MA, USA)	Quantification cfDNA at 24 h after MCAO in: -mice injected with CD21 -mice injected with CD21 + pretreated with Compound C	Neuroprotective effect of CD21	CD21 significantly reduced levels of NETs in blood and ischemic brain tissues in PTS mice (p < 0.01); effect reversed by pretreatment with Compound C (p < 0.01).
Denorme (2022) [18]	INT	C57BL/6J mice 10–12-week-old male or female; 18-month-old male C57BL/6J mice	tMCAO	CitH3 MPO-DNA	Citrullinated Histone H3 ELISA Kit (Cayman Chemical) In-house made ELISA kit (C: anti-MPO antibody D: anti-DNA antibody)	(A) Quantification of MPO-DNA complex at 24 h after tMCAO in mice: -pretreated with BoxA (n° = 6) -pretreated with vehicle (n° = 5) (B) Quantification of MPO-DNA complex at 24 h after tMCAO in mice with DM I and older than 18 months: -injected with nNIF (n° = 10) -injected with SCR (n° = 9) (C) Quantification of MPO-DNA complex at 24 h after tMCAO in mice: -pretreated with GSK-199 (n° = 9) -pretreated with DNase I (n° = 9) -pretreated with vehicle (n° = 10)	Differences in NET markers levels, functional outcome assessed by modified Bederson test and Grip strength test and mortality on day 7 post t MCAO	nNIF mice reported better functional outcome (p < 0.01) and increased survival at 7 days (p = 0.0097). Treatment with nNIF BoxA DNAse I or GMKS significantly reduced MPO-DNA levels (p < 0.01).
Kim (2020) [26]	INT	Male Sprague-Dawley rats (8 weeks old, 230–250 g body weight)	pMCAO	cfDNA NE-DNA	Quant-iT™ PicoGreen® dsDNA assay kit (Invitrogen, Carlsbad, CA, USA)	(A) Quantification of cfDNA in pMCAO mice: -treated with ATP (n° = 4) -treated with BzATP (n° = 6) -treated with PMA (missing number) -treated with ATP + A438079 (missing number) (B) Quantification of NE-DNA complex in PMCAO mice: -treated with ATP (n° = 4) -treated with ATP + A438079 (missing number) -treated with ATP + Cl-Amidine (missing number)	Release of NET markers after administration of ATP	Significant increase of circulating cfDNA upon injection of ATP (p < 0.05), BzATP (p < 0.01) or PMA (p < 0.01), reversed by A438079 (P2X7 antagonist) (p < 0.01). Significant increase of the release of NE-DNA induced by ATP (p < 0.01), reversed by A438079 (P2X7 antagonist) or CL-Amidine (p < 0.01).
De Meyer (2012) [27]	INT	Wild-type C57BL/6 8–10-week-old males	tMCAO	cfDNA Histone -DNA	fluorometric fluoroscopy (Fluoroskan; Thermo Fisher Scientific, Waltham, MA, USA) with fluorochrome Sytox Green Cell death detection ELISA or Cell Death Detection ELISA plus (Roche, Indianapolis, IN, USA)	(A) Quantification of cfDNA and histone-DNA complex in: -tMCAO mice at 24 h from the event (n° = 9) -sham mice (n° = 9) (B) Functional outcome assessment in: -mice treated with RhDNAse I (n° = 14) -mice treated with vehicle (n° = 15)	Early differences in NET markers levels, functional outcome assessed by modified Bederson test, grip test and corner test	Increase in cfDNA and histone-DNA complexes in AIS group baseline and after 24 h from stroke (p< 0.05). Better neurological outcome after treatment with RhDNAse I (p < 0.05).
Kmet’ova (2022) [28]	POC	young adult CD1 mice (n° = 44)	Blunt TBI	cfDNA *	QIAamp® DNABlood MiniKit (Qiagen, Hilden, Germany) + QubitTM (Thermo Fisher Scientific, Waltham, MA, USA) + fluorometry	Quantification of cfDNA in: -TBI mice at 3 h from the event (n° = 34) -healthy control (n° = 10)	Early levels of cfDNA after TBI and its correlation with worse neurological function evaluated by static rods test at 1 month	Significant cfDNA increase in TBI after 1 h and 2 h (p < 0.05), Higher cfDNA in mice failing behavioural test (p = 0.014).
Wang (2014) [31]	POC	Male C57BL/6J mice (8–10 weeks old, weighed 22–26 g)	TBI from repeated blasts	cfDNA	fluorescence with SYBR Green I Nucleic Acid Gel Stain (Invitrogen Corporation, Grand Island, NY, USA)	Quantification of cfDNA at 2 h, 6 h, 24 h, 72 d in: -TBI mice (n° = 10) -sham mice (n° = 10)	cfDNA trend after repeated blast exposures and correlation with functional outcome assessed by RRT	Circulating cfDNA peaked 2 h after blast exposure (p <0.001); significant linear correlation between RRT and cfDNA (p < 0.005).
Sillesen (2013) [24]	INT	female Yorkshire swine (40–50 kg) (n° =12)	Blunt TBI + severe haemorrhagic shock	Nucleosomes DNAse I	Cell death detection ELISA plus, (Roche Applied Sciences, Indianapolis, IN, USA) ELISA (Bluegene Biotech, Shangai, China)	Quantification of nucleosomes and DNAse I in: -mice receiving FFP (n° = 6) -mice receiving NS (n° = 6)	Early differences in NET markers levels, correlation of circulating nucleosome and DNAse I with lesion size and brain swelling	Reduction in circulating nucleosome and DNAse I depletion after FFP; circulating nucleosome levels significantly correlated with lesion size (p = 0.002) and brain swelling (p < 0.001), circulating DNAse I correlated with brain swelling (p = 0.036) but not with lesion size (p = 0.124).

* = De Meyer et al. referred to cfDNA as extracellular DNA (ecDNA); Kim et al. referred to cfDNA as double-strand DNA (dsDNA).