Daytime DNase-I Administration Protects Mice From Ischemic Stroke Without Inducing Bleeding or tPA-Induced Hemorrhagic Transformation, Even With Aspirin Pretreatment

Abstract

Background: Acute ischemic stroke treatment typically involves tissue-type plasminogen activator (tPA) or tenecteplase, but about 50% of patients do not achieve successful reperfusion. The causes of tPA resistance, influenced by thrombus composition and timing, are not fully clear. Neutrophil extracellular traps (NETs), associated with poor outcomes and reperfusion resistance, contribute to thrombosis. DNase-I, which degrades neutrophil extracellular traps, could improve thrombolytic efficacy. However, more studies are needed to understand the impact of DNase-I in tPA-sensitive stroke models, the safety of coadministering DNase-I and tPA regarding hemorrhagic transformation (HT), optimal timing for use, and effects on aspirin-treated animals.

Methods: We used in situ thromboembolic stroke, a tPA-sensitive model, where late tPA administration causes HT. Middle cerebral artery occlusion was induced at different zeitgeber times (ZT) to study the optimal timing for administration. DNase-I, tPA, and aspirin were administered at various times to evaluate their effects.

Results: DNase-I reduced infarct volume and improved functional outcomes 24 hours post-middle cerebral artery occlusion by decreasing plasma and cortical neutrophil extracellular trap levels. DNase-I caused no bleeding or impact on HT induced by late tPA. Its protective effect was only seen when given during the daytime (rodent inactive phase; ZT4-7), not overnight (active phase; ZT13-16). Chronic aspirin pretreatment increased tPA-induced HT but did not change the protective effects of DNase-I, with or without tPA.

Conclusions: Our study demonstrates that daytime (inactive phase) DNase-I administration is a safe and effective treatment for experimental stroke. This is particularly important given the 2 ongoing clinical trials for stroke patients.

Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05203224 and NCT05880524.

Keywords: aspirin; extracellular traps; ischemic stroke; thrombosis; tissue-type plasminogen activator.

Figure 1.

Daytime DNase-I administration is protective after stroke by reducing neutrophil extracellular traps (NETs). A, Experimental design. Effects of treatments on infarct volume and neurobehavioral tests 24 hours post-middle cerebral artery occlusion (MCAO). B and E, Cortical NETs and plasma elastase measured 24 and 4 hours, respectively, post-MCAO. C, Representative images of cortical NETs. D, Circadian effects on infarct volume and neurobehavioral tests 24 hours post-MCAO. F, DNase/tissue-type plasminogen activator (tPA) effects, with/without aspirin pretreatment. Data are mean±SEM (n=6–8).

Figure 2.

DNase-I administration does not cause bleeding or increase tissue-type plasminogen activator (tPA)-induced hemorrhagic transformation. A, Staining of brain sections from all groups with/without aspirin pretreatment. B, Effects of treatments on hemorrhage volume 24 hours post-magnetic resonance imaging (MCAO). C, Cerebral perfusion and reperfusion rates measured during 4 hours post-MCAO. D, Effects of treatments on hemorrhage volume in reperfused/nonreperfused animals. E, Effects of treatments on hemorrhage volume with/without aspirin pretreatment. Data are mean±SEM (n=6–8).