2.9. In vivo ischaemia‐reperfusion injury

Male wild‐type C57Bl/6 mice (12‐ to 15‐week‐old) were anaesthetized intraperitoneally with 80 mg/kg pentobarbital. They were placed in supine position on a heating pad (36.5‐37.5°C). After tracheostomy, artificial ventilation was established using a 19G cannula connected to a MiniVent type 845 animal ventilator (Harvard Apparatus). Flow rate was 1.0 L/min with stroke volume 200 µL at 130 strokes/min. An expiratory tube was submerged in water to apply 2 cmH₂O positive end‐expiratory pressure. Electrocardiogram (one lead) was recorded continuously until the end of the experiment with PowerLab/4SP system using LabChart 7 software (ADInstruments). An incision was made at the fourth intercostal space. Equal volumes of saline containing the indicated numbers of ExoDiff, ExoPr0 or PBS were injected via a jugular vein 5 min prior to heart ischaemia. The assignment of animals to groups was randomized, and the investigator was blinded to the treatments. Myocardial IR injury was induced by ligation of the left anterior descending artery with a silk suture for 40 min followed by 2 h reperfusion. 38 Myocardial ischaemia was confirmed by changes in electrocardiogram and blanching of myocardium distal to the suture.

The hearts excised post‐reperfusion were cannulated and washed with saline. The previously blocked artery was re‐closed with the suture and the heart was perfused with 1% Evan's blue dye in saline (w/v) to determine the area at risk using standard methods. 38 Consequently, hearts were frozen, sectioned and stained with 1% triphenyl tetrazolium chloride in phosphate buffer (w/v; pH7.4) at 37°C for 15 min. Heart sections were fixed with 4.0% formaldehyde solution in water (v/v) overnight. The sections were scanned with CanoScan LiDE 220 scanner (Cannon) and analysed. The area at risk (Evan's Blue‐negative), the infarct size (not stained with Evan's Blue and triphenyl tetrazolium chloride) and the non‐risk area (Evan's Blue‐positive) were determined using ImageJ (National Institutes of Health). The infarct size was measured as a percentage of the area at risk. Hearts with <30% risk area were excluded from analysis.