In Vivo quantification of area-at-risk following transient ischemia in mouse heart using multi-parametric resonance imaging

Abstract

Background: Animal models of cardiac ischemia/reperfusion (IR) injury mimic the clinical scenario where a period of ischemia, i.e. restriction of the blood flow, is followed by restoration of blood flow in the myocardium resulting in reperfusion injury. As histological methods used to quantify the area-at-risk (AAR) of the myocardium are not ideal, requiring mice to be euthanized in early time points, the measurement of the AAR, which includes the area-of-necrosis (AON) and the salvage myocardium, is one of the main goals to reach in cardiac magnetic resonance imaging (MRI). Therefore this project aims to quantify the AAR using non-invasive multi-parametric MRI technique. Methods: Left coronary artery occlusion has been done in mice and MR experiments were carried out at 9.4 Tesla following the injection of gadolinium (Gd) and microparticles of iron oxide (MPIO) contrast agents. Gd allows to assess necrotic tissue (AON) (Rehwald et al, 2002) and it is hypothesized that MPIO, conjugated to a specific antibody, are able to target vascular adhesion molecules (VCAM-1) expressed in endothelial cells after inflammation (IR injury) (McAteer et al, 2007 & Akhtar et al, 2010). Several experiments were then performed in healthy mice (n=6) and in diseased mice subjected to 30 min (n=7) and 45 min (n=5) of ischemia. Additionally, in two of seven 30 min ischemic mice the MPIO contrast agent was conjugated to an irrelevant antibody. These mice were subjected to 24 h of reperfusion. Furthermore, the MR experiments were repeated in 30 min ischemic mice subjected to 16 h of reperfusion (n=2) in order to test whether VCAM-1 expression is more pronounced at shorter reperfusion times. The quantitative analysis was done considering both ventricular septum and skeletal muscle as regions of interest (ROI) and using different threshold values. Results: After 30 min of ischemia and 24h of reperfusion the area of reversible injury in the myocardium was higher than after 45 min of ischemia and 24 h of reperfusion. Furthermore, after 24 h of reperfusion, the reversible injury was higher in inferior myocardium and not in the territory supplied by the left coronary artery. MPIO marked with an irrelevant antibody was not able to trace VCAM-1. After 16 h of reperfusion, it was obtained excellent consistency between Post-MPIO results and histology. Therefore, quantitative analysis proved that, after 16 h of reperfusion, the entire myocardium was affected. LGE (Late Gadolinium Enhanced) data analysis proved that, after 24 h of reperfusion, diseased mice had the most affected region (irreversible injury) in anterior myocardium, i.e. where the ischemia develops. Conclusions: Due to some limitations of the project, it was not possible so far to conclusively assess the AAR of the myocardium with this technique. However, this project was particularly important to validate the MPIO contrast binding in heart and to prove that 30 min of ischemia followed by 16 h of reperfusion is a suitable time point to acquire MR images in mice using MPIO contrast agent. In addition, this work also proved that ventricular septum does not remain unaffected after IR injury and MPIO results, after 24 h of reperfusion, are not affected by the presence of the Gd, contrary to the MPIO results obtained after 16 h of reperfusion.

Date of Award	2012
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Jürgen Schneider (Supervisor)