Cardiac Intravital Microscopy Enables Quantitative Mapping of Capillary-Level Blood Flow in the Murine Heart

Introduction: Coronary microvascular disease (CMD) is increasingly appreciated as a contributor to adverse cardiovascular outcomes, but underlying mechanisms of CMD remain poorly understood. Investigation of CMD in animal models is limited by a lack of techniques to quantify flow in the heart. We assessed the hypothesis that intravital microscopy (IVM) can enable in vivo imaging of red blood cell (RBC) flow profiles in the coronary microcirculation at cellular resolution. Methods: C57BL/6 mice (n=20) were used to develop cardiac IVM protocols. Cardiomyocyte membrane staining was achieved using intravenous injection of a voltage dye (di-2-anepeq). RBC labeling was done by ex-vivo staining of cells with Ter119 followed by transfusion. IVM was performed through a thoracotomy with animals anesthetized and ventilated. Confocal and two-photon microscopy was done using a custom tissue stabilizer and an Olympus FV1000MPE microscope synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols. Images were analyzed in MATLAB to extract myocyte contractile properties, capillary diameter, and capillary flow properties. Results: Using cardiac and respiratory gating, images with cellular resolution could be measured in the beating heart at all phases of the cardiac cycle. This enabled quantification of cardiomyocyte contractile function and capillary diameter and widefield imaging by stitching of several images (Fig. 1A). Tracking of RBCs over serial images allowed quantification of RBC velocity across multiple capillaries simultaneously (Fig. 1B). Conclusion: Cardiac IVM can provide quantification of cardiac function and blood flow at the single cell scale and will provide an exciting tool for investigating CMD in models of heart disease.