Advances in Imaging Technology for Cardiac Research

Introduction: While stem cell (SC)-based therapy following heart injury holds promise, clinical success is compromised by poor SC recruitment to the heart following infusion. One solution is to identify strategies that may increase SC capture in cardiac microvessels. We have routinely used intravital microscopy (IVM), a method of live in vivo imaging of the microcirculation, to monitor cell trafficking. To do so in the heart would help facilitate research aimed at improving SC delivery. However, IVM of the beating mouse heart remains challenging due to difficulties with organ access and impractical degrees of tissue motion. The study presents a method that allows for cardiac IVM using a 3D-printed stabilizing device. Methods: Anaesthetized mice were artificially ventilated using a small rodent ventilator which delivered medical oxygen. PEEP was applied by immersing the ventilator outlet in 4cm of ddH2O. A chest wall incision, expanded with retractors, facilitated the adhesive positioning of a specially designed tissue stabilizer onto the heart surface. This limited respiratory and cardiac contractile motion in a small area without compromising overall heart function. Imaging was performed through a small hole in the centre of the stabilizer. Preliminary studies were performed in the myocardial ischemia-reperfusion injury model. This injury was established by occlusion of the left anterior descending artery (LAD). The vessel was occluded for 45 minutes using a small piece of plastic tubing within a loop which was placed under the LAD. Identification of vascular dynamics (e.g. cell recruitment, monitoring of vascular no-reflow) took place at various points during reperfusion. Administration of FITC-BSA allowed for visualisation of the microvasculature. Endogenous neutrophils were labelled by intra-arterial administration of 6mg eFluor-660 labelled anti-Gr-1 antibody. Results: Infusion of FITC-BSA permitted visualization of the coronary microcirculation. Vascular integrity was not disturbed and flow was visible as a result of application of the stabiliser. Endogenous (labelled) neutrophils were easily identified circulating in the cardiac microcirculation. Following cardiac IR, we identified significantly enhanced neutrophil recruitment as early as 60 minutes postreperfusion, when compared to sham-operated control animals. Furthermore, using FITC-BSA visualization of the coronary microcirculation, we identified areas of vascular no-reflow following cardiac IR. Finally, systemically administered SCs (fluorescently labelled HSCs) could be identified trafficking through the cardiac microvasculature following cardiac IR injury. Conclusion: We present a model for intravital imaging of the mouse beating heart in vivo. This method may be useful in monitoring cell dynamics and microvascular disturbances in the heart at the single cell level.