Stroke affects 15 million people per year due to a thrombus producing an abrupt decrease in blood flow supply to the brain that leads to the initiation of an inflammatory cascade and that, ultimately, will drive to cell death if the blood flow is not restored. Only two treatment regimens are currently approved by the FDA and the EMA: pharmacological thrombolysis using t-PA (tissue plasminogen activator) and mechanical thrombectomy. Unfortunately, both treatments have, as limitations, a low rate of arterial recanalization and the phenomenon of hemorrhagic transformation (HT), among others (1, 2). Indeed, recanalization of the occluded artery is limited to a small proportion of patients (3): specifically, due to highly restricted criteria, t-PA can only be administrated to less than 10% of stroke patients and less than 50% of t-PA-treated patients have successful recanalization (4). In this context, the research in fibrinolytic but also neuroprotective agents in stroke has been extensive with no translational results from medical research to clinical practice up to now.

Stroke elicits an extensive inflammatory response in brain with the recruitment of circulating leukocytes (5). Among these, neutrophils are the most abundant white blood cells that are rapidly recruited to the site of injury providing an effective immune response. Neutrophils are known to release their intracellular content in a web-like structure called Neutrophil Extracellular Traps (NETs). NETosis is a complex cellular process characterized by the release of DNA decorated with granular proteins (MPO or elastase) and histones in response to microbial infection (6). The release of these traps is dependent on the citrullination of histones by the peptidyl arginine deiminase type IV (PAD) enzyme and decondensation of chromatin (7). In addition to their bactericidal effect, NETs have also been documented in sterile inflammation, atherosclerosis, or ischemia/reperfusion injury (8, 9). Additionally, NET formation has been related to platelet TLR4 (Toll-like receptor 4) activation, but its role in the setting of thrombotic stroke is unknown (10).

Importantly, it has been widely described that stroke thrombi are mainly composed by fibrin, platelets, red blood cells, and NETs (11, 12) Since Fuchs and cols. described for the first time the relationship between NETs and thrombosis in a platelet-dependent manner (13), many researchers including us have been trying to understand how platelets and NETs interact to lead to thrombus formation (14–16), (16). Indeed, our group recently described that inhibition of NETs through different pharmacological approaches leads to an effective lysis of platelet-rich thrombi in a platelet TLR4-dependent manner (17). However, it remains unknown whether NETs play a crucial role in fibrin-rich thrombi, as well as in tissue damage in the acute phase of stroke.

Therefore, in this study, we decided to elucidate 1) the role of NETs in fibrin-rich thrombosis, and 2) whether NET formation affects outcome in acute stroke.

Stroke is caused primarily by a thrombotic vascular occlusion that initiates a cascade of events that ultimately lead to neuronal cell death. Recent data suggest that the efficacy of either thrombectomy or intravenous thrombolysis is affected by thrombi composition; more specifically it has been found that fibrin-rich clots were associated with less favourable clinical outcomes (28). Since NETs have a predominant role in thrombosis (12) and since we have previously demonstrated that these extracellular traps play a key role in platelet-rich thrombosis in cerebral ischemia (17), we next want to explore their role in high-fibrin rich thrombosis. Our results demonstrate for the first time that NETs are not a main component in stroke thrombi with high fibrin content ( Figure 1 ). This is in accordance with previous results showing that fibrin content in thrombus is associated with a decrease in neutrophil accumulation and NET formation (29).

At the same time, on the one hand, we have demonstrated that DNase-I was not able to effectively reperfuse the MCA, at least within the first 3 hours after the thromboembolic occlusion, whereas tPA did ( Figure 1 ). However, we cannot discard the possible recanalization within the next following 20 hours previous to the sacrifice, issue that should be explore in future studies. This is in agreement with previous results showing that lower levels of NETs in thrombi produced by fibrin makes the administration of DNase ineffective (24). On the other hand, despite non-reperfusion, a novel and important observation of our study is that the late administration of DNase-I had a protective effect shown as an improvement in infarct volume and in stroke outcome ( Figure 2 ). At the mechanistic level, this could be due to a direct protective effect of DNase-I and/or to a decrease in NET-dependent microthrombotic “no-reflow” phenomena that could contribute to ischemic injury.

In this context, DNAse-I, by favouring the degradation of NETs, could have direct protective properties since NETs have been implicated in brain damage: specifically, neutrophils, as the first group of cells infiltrating the damaged brain tissue, produce NETosis in brain parenchyma and peripheral blood that aggravate inflammation and subsequent brain damage after stroke (30, 31). NETs are also associated with severity and mortality in patients (32). In fact, we have demonstrated that NET degradation has a protective effect as measured by the peripheral levels of IL-10 ( Figure 3 ) which is in accordance with what others have observe not only in stroke but in sepsis models (33, 34). However, the fact that we didn’t observe any significant difference among treatments in other cytokines should be deeply explore in future studies.

Furthermore, hyperglycemia, which increases NETs formation, also exacerbates ischaemic brain damage (35). Finally, we and others have shown that drugs that prevent NET formation protect against ischemic damage in several stroke models ( Figure 4 ) (16, 36). However, not only neutrophils released their content to extracellular space but also macrophages (METs), eosinophils (ETs) or even mast cells released their intracellular content in a ETosis cell-death way (37–39). In this context, even though we have target NETs with neutrophil specific antibodies, we cannot exclude the detrimental role of other form of ETosis in the acute phase of stroke, a fact that should be explore in future studies.

Secondly, DNAse-I, by preventing the formation of microthrombosis and “no-reflow” phenomena, may have also neuroprotective effects. It is well known that clot removal and vessel recanalization do not always go along with tissue reperfusion, a phenomenon called “no-reflow” that occurs at both the cardiac and cerebral levels (40). Only about 50% of t-PA treated patients have successful reperfusion. It is likely that multiple mechanisms might contribute to the microvascular no-reflow phenomenon, including endothelial cell dysfunction, microthrombosis, neutrophil accumulation and NETs formation, among others (41–43). In this context, several studies have shown the existence of microthrombi-induced occlusions in small-calibre vessels adjacent to the infarcted area (41, 44). Microvascular obstruction of 20-30% of capillaries in the core and penumbra of the infarct has been shown to be produced by neutrophils adhering to distal capillary segments; furthermore, removal of circulating neutrophils using an anti-Ly6G antibody restores microvascular perfusion without increasing the rate of hemorrhagic complications (41). Of note, the involvement of NETs in microthrombosis phenomena has been demonstrated (43). Therefore, NETs could be participating in the formation of small thrombi generated after a large vessel occlusion, in such a way that both DNase-I (by favouring the degradation of NETs) and Cl-amidine (by inhibiting the formation of NETs) could be beneficial in resolving the microthrombosis phenomena associated with stroke, a fact that deserves to be studied in more detail.

Interestingly, we have also demonstrated the deleterious role of platelet TLR4 in brain ischemia caused by non-platelet-rich thrombosis ( Figure 5 ). We had previously demonstrated that TLR4 in platelets is determinant for the photothrombotic formation of a stable platelet-rich thrombus which, in turn, depended on NETs (16); now, we demonstrate that platelet TLR4 is also determinant in brain damage produced by occlusions/thrombi in which NETs do not play a relevant role. These data support the role of NETs-induced microthrombosis in ischemic damage, since platelet TLR4 ultimate triggers the NETosis process (10, 17).

DNAse-I treatment did not affect the blood-brain barrier since, as opposed to t-PA, its delayed administration produced neither cerebral edema nor HT ( Figure 1 ). This finding supports that DNase-I could be a safe therapy, deprived of the severe side effects associated with tPA-induced fibrinolysis.

Our study has some limitations. One is that the experiments have been performed in young animals, without the co-morbidities present in many stroke patients. In addition, we included only male mice to compare our results with the previous work done in this stroke model. Therefore, co-morbidities, sex-related differences and mechanisms involved in neuroprotection and no-reflow after stroke deserve further investigations.

Overall, our results support the important role of NETs in cerebrovascular disease and point to the involvement of the platelet TLR4 receptor in this process, thus providing new avenues for the treatment of the acute phase of stroke through the inhibition and/or degradation of the extracellular traps released by the neutrophil.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Ethics Committee on Animal Welfare of University Complutense (PROEX number 016/18).

CP-M, VD-L, AG-C, MM, and IL designed the research studies. CP-M, VD-L, AG-C, and MIC conducted the experiments and/or acquired the data. CP-M, VD-L, AG-C, MIC, MM, and IL contributed to the analysis and/or interpretation of the results. CP-M, MM, and IL wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019-106581RB-I00 (MÁM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MÁM), from Fundación La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.