Intravital 2-photon imaging of leukocyte trafficking in beating heart
Wenjun Li, … , Mark J. Miller, Daniel Kreisel
Wenjun Li, … , Mark J. Miller, Daniel Kreisel
Published June 18, 2012
Citation Information: J Clin Invest. 2012;122(7):2499-2508. https://doi.org/10.1172/JCI62970.
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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.

Authors

Wenjun Li, Ruben G. Nava, Alejandro C. Bribriesco, Bernd H. Zinselmeyer, Jessica H. Spahn, Andrew E. Gelman, Alexander S. Krupnick, Mark J. Miller, Daniel Kreisel

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Figure 7

Intravital 2P imaging of the native murine heart.

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Intravital 2P imaging of the native murine heart. B6 LysM-GFP hearts wer...
B6 LysM-GFP hearts were imaged in their natural intrathoracic position. All mice tolerated imaging for at least 1 hour, with the majority of imaging periods lasting more than 3 hours. (A, Supplemental Video 9) Sequential video-rate frames of 2 neutrophils (yellow arrowhead with yellow track and white arrowhead with white track) moving through a coronary artery. The measured speed of the neutrophil marked in yellow is 1875.2 μm/s. (B) Representative images of native hearts in the steady state depicting capillaries (red, left panel) and a vein (red, right panel). Only few neutrophils (green) are visible. (C, E, G) 2P microscopy of beating native hearts after ischemia/reperfusion induced through transient ligation of the left coronary artery. (C) Intravascular rolling behavior of neutrophils (white arrowheads) was observed in coronary veins (white track). (D) Analysis of rolling velocities in native hearts subjected to ischemia/reperfusion injury. (E) Crawling behavior (yellow arrowhead) was associated with cell flattening along the endothelium. A second crawling neutrophil is indicated by a white arrowhead and white track. (F) Analysis of crawling velocities in native hearts subjected to ischemia/reperfusion injury. Symbols represent individual cells, horizontal bars depict mean, and error bars represent SEM. (G, Supplemental Video 10) Dynamic neutrophil clusters (yellow arrowheads) in myocardial tissue of native hearts that have been subjected to ischemia/reperfusion injury. Blood vessels (red) were labeled by intravenous injection of nontargeted 655-nm Q-dots. Relative time is displayed in h:min:s. Scale bars: 60 μm. Data are representative of 4 independent experiments.