Introcan

Wistar rats were anesthetized by an intraperitoneal injection of thiobutabarbital sodium salt hydrate 150 mg/kg (Inactin^®; Sigma‐Aldrich GmbH, Buchs, Switzerland). After tracheotomy, an intravenous cannula (Introcan, 20G × 1¼, B Braun, REF 4252110 B, Sempach, Switzerland) was inserted into the right carotid artery and 7–10 mL blood was removed into a syringe containing 1 mL napsagatran (1 mmol/L in 0.9% NaCl, 1% DMSO), a direct thrombin inhibitor (Ro 46‐6240 from Hoffmann‐La Roche, Basel, Switzerland). The blood was centrifuged for 5 min at 650 g and platelet‐rich plasma (PRP) was stored at 37°C. Platelet aggregation was assessed by light transmission aggregometry (LTA) with a four‐channel Chronolog Lumi‐Aggregometer 490‐4D (Probe & Go Labordiagnostica GmbH, Osburg, Germany) with the AggroLink software package. Aggregation was started by addition of 10 μL of ADP solution into 240 μL PRP at 520 × 10⁶ platelets/mL and monitored for up to 8 min. IC₅₀ values were calculated using XLfit software (IDBS, Guildford, UK).

After the loss of their pedal withdrawal reflex, the rats were intubated with a 16 gauge tube (B Braun Introcan; Oss, The Netherlands) for ventilation. They were placed on a heating pad to maintain body temperature at 37℃. Following intubation, the rats were ventilated using 40% oxygen (UNO micro ventilator-03; UNO, Zevenaar, The Netherlands), their fur was removed locally and their skin was disinfected with 70% v/v ethanol. All operations were performed by the same surgeon during daytime using freshly autoclaved instruments. Cardiac I/R was induced as described previously.^{2 (link)} In brief, a left thoracotomy was performed between the third and fourth ribs. To prevent the lungs from collapsing, positive end expiratory pressure (PEEP) of 2 mbar was maintained using a microventilator. During surgery, the effects of anaesthesia were assessed by closely monitoring the HR, heart rhythm and signs of hypercapnia such as changes in respiratory rate and depth caused by carbon dioxide accumulation. To induce MI, the left anterior descending artery was ligated using a 6-0 prolene suture. After 40 min of ischaemia, the suture was removed to allow reperfusion and the thorax was closed using two sutures and maximal lung pressure. On average, total surgery time was 65 min. Welfare of the animals was assessed daily until termination.