Immunology and Infection
Published: April 2nd, 2013
Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.
Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil β2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-α-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.
1. Preparation of Intravital Microscope (Figure 1A)
Using a detailed intravital microscopy analysis, heterotypic platelet-neutrophil interactions on the activated endothelium were visualized by infusion of fluorescently-labeled antibodies against a platelet (CD42c) or neutrophil marker (Gr-1) into live mice.
In a model of TNF-α-induced venular inflammation, most rolling neutrophils were stably adhered to the endothelium presumably by interaction of activated β2 integrins with ICAM-1 during the recording period (3-4.5 hr after injecti.......
Here we describe a detailed protocol for real-time fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombosis. Previously, similar fluorescence microscopic approaches were reported to study the molecular mechanism of thrombus formation and vascular inflammation.8,12 Since the heterotypic cell-cell interaction could be important for vaso-occlusion at the injury site, this technology will be a valuabl.......
This work was supported in part by grants from National Institutes of Health (P30 HL101302 and RO1 HL109439 to J.C.) and American Heart Association (SDG 5270005 to J.C.). A. Barazia was supported by a T32HL007829 NIH training grant.....
|Name of Reagent/Material
|0.9% NaCl Saline
|Intramedic Tubing (PE 90)
|Intramedic Tubing (PE 10)
|Dylight 488- labeled rat anti-mouse CD42b antibody
|Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody
|NESLAB EX water bath/circulator
|Olympus BX61W microscope
|ROE-200 stage controller
|C9300 high-speed camera
|Video Scope International
|Photonic Instruments, Inc.
|Intelligent Imaging Innovations
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