Detection and quantification methods of monocyte homing in coronary vasculature with an imaging cryomicrotome

Cellular imaging modalities are important for revealing the behavior and role of monocytes in response to neovascularization progression in coronary artery disease. In this study we aimed to develop methods for high-resolution three-dimensional (3D) imaging and quantification of monocytes relative to the entire coronary artery network using a novel episcopic imaging modality. In a series of ex vivo experiments, human umbilical vein endothelial cells and CD14+ monocytes were labeled with fluorescent live cell tracker probes and infused into the coronary artery network of excised rat hearts by a Langendorff perfusion method. Coronary arteries were subsequently infused with fluorescent vascular cast material and processed with an imaging cryomicrotome, whereby each heart was consecutively cut (5 μm slice thickness) and block face imaged at appropriate excitation and emission wavelengths. The resulting image stacks yielded 3D reconstructions of the vascular network and the location of cells administered. Successful detection and quantification of single cells and cell clusters were achieved relative to the coronary network using customized particle detection software. These methods were then applied to an in vivo rabbit model of chronic myocardial ischemia in which autologous monocytes were isolated from peripheral blood, labeled with a fluorescent live cell tracker probe and re-infused into the host animal. The processed 3D image stacks revealed homing of monocytes to the ischemic myocardial tissue. Monocytes detected in the ischemic tissue were predominantly concentrated in the mid-myocardium. Vessel segmentation identified coronary collateral connections relative to monocyte localization. This study established a novel imaging platform to efficiently determine the localization of monocytes in relation to the coronary microvascular network. These techniques are invaluable for investigating the role of monocyte populations in the progression of coronary neovascularization in animal models of chronic and sub-acute myocardial ischemia.