Intravital 2-photon imaging of leukocyte trafficking in beating heart
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
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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Technical Advance Cardiology

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 4

2P imaging of intravascular neutrophil dynamics in heterotopic heart transplants.

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2P imaging of intravascular neutrophil dynamics in heterotopic heart tra...
(A, Supplemental Video 3) WT heart graft with extensive neutrophil arrest inside blood vessels (white outline) and intravascular cluster formation (white arrowheads). n = 4 mice. Comparable results were obtained in untreated and isotype control–treated recipients of WT cardiac grafts. (B, Supplemental Video 4) Neutrophil trafficking in ICAM-1–mutant (denoted as ICAM-1) donor hearts (n = 2 mice), (C, Supplemental Video 5) after treatment of recipient mice with anti-LFA 1 (n = 4 mice), and (D, Supplemental Video 6) after treatment with anti-Mac 1 antibodies (n = 4 mice). Scale bars: 60 μm. (E) A comparison of cell-rolling velocities in each group. Rolling velocities were lower in ICAM-1–mutant grafts and anti–Mac-1–treated recipient mice compared with WT mice, while velocities after treatment with anti–LFA-1 were comparable to those in WT. WT, n = 37; ICAM-1–mutant, n = 61; anti–LFA-1, n = 56; anti–Mac-1, n = 47. *P < 0.05; **P < 0.01; ***P < 0.0001. (F) Intraluminal crawling velocities. ICAM-1–mutant grafts, and anti–Mac-1–treated recipients had significantly lower crawling velocities when compared with WT mice. Due to the low number of adherent neutrophils, crawling activity could not be analyzed after blockade of LFA-1. WT, n = 260; ICAM-1–mutant, n = 317; anti–LFA-1, n = NA; anti–Mac-1, n = 481. *P < 0.05; ***P < 0.0001. For parts E and F, symbols represent individual cells, and horizontal bars depict means. (G) Intravascular neutrophil clusters. Cluster frequency increased in ICAM-1–mutant hearts and with anti–Mac-1 antibody treatment and decreased when LFA-1 was blocked. *P < 0.05; **P < 0.01. Symbols represent intravascular neutrophil clusters. (H) Neutrophil cluster size. ICAM-1–mutant grafts had larger neutrophil clusters compared with WT hearts. **P < 0.01; ***P < 0.0001. For G and H, horizontal bars denote means, and error bars denote SEM.