Delayed DNase-I Administration but Not Gasdermin-D Inhibition Induces Hemorrhagic Transformation After Transient Focal Cerebral Ischemia in Mice

Abstract

olymorphonuclear neutrophils are early invaders of the ischemic brain and exacerbate brain damage. 1,2 Neutrophil-released extracellular DNA traps significantly contribute to ischemic damage. 3 Hence, 3 randomized controlled trials currently assess the effects of intravenously administered DNase-I, which degrades neutrophil-released extracellular DNA traps, in patients with stroke (URL: https://www.clinicaltrials. gov; Unique identifier: NCT05203224, NCT05880524, NCT04785066). To explore the roles of neutrophilreleased extracellular DNA traps, we exposed 10-to 12-week-old male C57BL6/j mice (Envigo, Darmstadt, Germany) anesthetized with 1.5% isoflurane (30% O 2 , remainder N 2 O) to 30 minutes intraluminal middle cerebral artery occlusion (MCAO). 3 Rectal temperature was maintained at 37 °C. Laser Doppler flow was recorded above the middle cerebral artery territory. Buprenorphine (0.1 mg/kg SC; Reckitt Benckiser, Slough, United Kingdom) was injected before MCAO for analgesia, followed by carprofen (4 mg/kg per day SC; Bayer, Leverkusen, Germany) at 0, 24, and 48 hours post-MCAO as anti-inflammatory drug. Immediately or 72 hours post-MCAO, vehicle or DNase-I (10 µg; 11284932001; Roche, Basel, Switzerland) was IV administered. In the high-dose, but not low-dose group, vehicle or DNase-I (50 µg) was simultaneously applied IP. This IP DNase-I administration was repeated 3× at 12-hour intervals in the high-dose group. In groups with immediate highdose DNase-I delivery, vehicle or recombinant tPA ([tissue-type plasminogen activator]; 10 mg/kg IV, 10% as bolus, 90% as 30-minute infusion; Actilyse, Boehringer-Ingelheim, Ingelheim, Germany) was administered 10 minutes after DNase-I. In additional studies, vehicle or the gasdermin-D inhibitor LDC7559 (10 mg/ kg IP; HY-111674; MedChemExpress, Monmouth Junction, NJ), which prevents neutrophil extracellular DNA trap release, 3 was injected at 72 hours post-MCAO. At 120 hours post-MCAO, mice were deeply anesthetized and transcardially perfused with PBS, followed by 4% paraformaldehyde. From 20-µm-thick brain sections collected at 1-mm intervals, cresyl violet stainings were performed, which were analyzed with ImageJ (National Institutes of Health, Bethesda, MD). Infarct volume was measured by subtracting healthy brain tissue from both hemispheres. Brain edema was examined as a percent volume increase in the ischemic compared with the contralateral hemisphere. The incidence and area of hemorrhages were evaluated by diaminobenzidine staining (D5905; Sigma-Aldrich, Deisenhofen, Germany) in sections obtained +1.0, ±0.0, −1.0, and −2.0 mm rostral to bregma. Experiments were performed with government approval (LANUV, Recklinghausen, Germany) in accordance with EU Directive 2010/63/ EU. Groups were strictly randomized and experimenters