Mouse infarct size quantification

At the end of follow up, mice were re-anesthetized and re-intubated, and the LAD coronary artery was re-occluded by ligating the suture in the same position as the original infarction⁵⁴. Animals were then killed and rapidly 1 ml of 1% (w per v) Evans Blue dye was infused i.v. to delineate AAR: myocardium lacking blood flow, that is, negative for blue dye staining. The heart was then harvested, LV was isolated, cut into transverse slices (5–7 1-mm thick slices per LV) and both sides were imaged. Sections post-Evans blue staining present two different areas: one palish negative for Evans blue perfusion, delineating AAR, and another blueish (positive Evans blue) area indicating remote tissue. In order to differentiate infarcted from viable tissue, same slices were incubated in triphenyltetrazolium chloride (TTC, 1% (w per v) diluted in PBS) at 37 °C for 15 min in constant shacking. The slices were then re-photographed and weighed. Post TTC incubation, Evans blue staining clears out and slices present two areas: one necrotic (palish negative to TTC staining) and one reddish alive (positive to TTC staining). Regions negative for Evans Blue staining (AAR) and for TTC (infarcted myocardium) were quantified using ImageJ (NIH, Bethesda, MD, USA) by blinded observer. Percentage values for AAR and infarcted myocardium were corrected to mg independently for each slice. Absolute AAR and infarct size were determined as the mg:mg ratio of AAR:LV and infarcted myocardium:AAR, respectively. Animals exceeding 80% of IS were excluded assuming absence of reperfusion.