Experimental subjects and study design

Male C57BL6/J mice (n = 30, Charles River Laboratories) with an age of 12–15 weeks and a weight of 28–33 g were used. All animals were kept in an enriched environment with unlimited access to food and water. During the entire experimental phase, animals were continuously monitored for their well-being. All experimental procedures were conducted in the same order and manner across all experimental groups to avoid confounders and achieve high level standardization.

At the beginning of the experimental procedure (Fig. 1), ischemic stroke was induced in all animals by occlusion of the dMCAO as described in Schumm et al. 2021. In brief, animals were anaesthetized by intraperitoneal injection of ketamine/xylazine (80 mg/kg; 16 mg/kg), a left sided cranial window was prepared, and the left distal MCA was completely and permanently electrocoagulated. Paracetamol enriched drinking water (300 mg/kg) and local Xylocain gel was used as analgesia.

Schematic representation of the study design including the temporal course of the experiment and the different experimental groups. KCl—potassium chloride application location; ECoG—electrophysiological recording location in the penumbra region; ROI—region of interest used for laser speckle imaging analysis.

24 h after stroke induction all animals were subjected to magnetic resonance imaging (MRI; 7 T rodent scanner (Pharmascan70/16US, Bruker Biospin, USA) T2-weighted 2D turbo spin-echo sequence) as described before^41,42.

The MRI was followed by a three-hour electrocorticographic (ECoG) recording of SDs. Simultaneously, regional CBF was measured by laser speckle imaging (LSI). Animals were randomly assigned into three different experimental groups: In group 1 (n = 10; control group) the spontaneous occurrence of SDs was measured without further interventions. In group 2 (n = 10; KCl group) SDs were repetitively induced every 15–20 min through KCl application. In group 3 (n = 10; ketamine group) SDs were repetitively induced every 15–20 min by KCl while the animals received ketamine.

A second MRI was performed in all animals 48 h after stroke induction to evaluate lesion progression. After the second MRI, all animals were killed by decapitation under deep anaesthesia with intraperitoneal injection of ketamine/xylazine (80 mg/kg; 16 mg/kg).