Intravital 2-photon imaging of leukocyte trafficking in beating heart

Abstract

Two-photon intravital microscopy has substantially broadened our understanding of tissue-and organ-specific differences in the regulation of inflammatory responses. However, little is known about the dynamic regulation of leukocyte recruitment into inflamed heart tissue, largely due to technical difficulties inherent in imaging moving tissue. Here, we report a method for imaging beating murine hearts using intravital 2-photon microscopy. Using this method, we visualized neutrophil trafficking at baseline and during inflammation. Ischemia reperfusion injury induced by transplantation or transient coronary artery ligation led to recruitment of neutrophils to the heart, their extravasation from coronary veins, and infiltration of the myocardium where they formed large clusters. Grafting hearts containing mutant ICAM-1, a ligand important for neutrophil recruitment, reduced the crawling velocities of neutrophils within vessels, and markedly inhibited their extravasation. Similar impairment was seen with the inhibition of Mac-1, a receptor for ICAM-1. Blockade of LFA-1, another ICAM-1 receptor, prevented neutrophil adherence to endothelium and extravasation in heart grafts. As inflammatory responses in the heart are of great relevance to public health, this imaging approach holds promise for studying cardiac-specific mechanisms of leukocyte recruitment and identifying novel therapeutic targets for treating heart disease.

Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis

“…Various techniques (tissue glue, suction ring, compression) have been success fully used for physically restraining tissue movement (Looney et al, 2011;Lee et al, 2012a,b;Li et al, 2012b;Vinegoni et al, 2012;Jung et al, 2013;Chèvre et al, 2014). As described above, it is important to ensure that placing the stabilizer does not induce any tissue damage.…”

Optical Imaging Innovations for Atherosclerosis Research

“…Image acquisition triggered on respiratory/ heart cycles, mechanical stabilization, and strong fluorescent markers enabled recording of good quality time-lapse movies of the arterial wall and leukocyte interactions with the vessel wall, 17 even in 3D ( Figure 1B). 20 Using a comparable layout, Li et al 21 managed to track single leukocytes recruited to damaged cardiac tissue in living mice after coronary ischemia reperfusion, both in transplanted and in native hearts, ≤120-μm depth. More recently, an intravital live cell-triggered imaging system has been developed by Ley et al 22 to reveal monocyte patrolling and macrophage migration in atherosclerotic arteries, implementing cardiac triggered acquisition, as well as frame selection and image registration algorithms, to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice.…”

Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments

“…With respect to the location and motility: B cell migration has been extensively studied in germinal centers to understand details of the maturation process, possibly involving a recycling process in two spatially distinct regions, the dark and light zones: [13,61,24,2,6,63,7]. More complex motility of leukocytes has been studied using 2pM IVM in a beating heart [34].…”