Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Summary

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.

Abstract

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.

Protocol

1. Preparation of Intravital Microscope (Figure 1A)

1. Prepare superfusion buffer (125 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, and 17 mM NaHCO₃, pH 7.4).
2. Turn on a circulatory water bath to maintain temperature of buffer and a thermo-controlled blanket at 37 °C. Aerate buffer with nitrogen gas (5% CO₂ balanced with nitrogen).
3. Turn on the microscope system (Sutter Lambda DG-4 high speed wavelength changer, workstation computer, Olympus BX61W microscope, MPC-200 multi-manipulator, ROE-200 stage controller, high speed camera, and intensifier).
4. For the vascular inflammation model....

Results

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 injection of TNF-α.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Fisher Scientific	7647-14-5
KCl	Sigma-Aldrich	7447-40-7
CaCl2 2H2O	Sigma-Aldrich	10035-04-8
MgCl2 6H2O	Fisher Scientific	7791-18-6
NaHCO3	Fisher Scientific	144-55-8
0.9% NaCl Saline	Hospira	0409-4888-10
Ketamine	Hospira	0409-2051-05
Xylazine	Lloyd
Intramedic Tubing (PE 90)	BD Diagnostics	427421
Intramedic Tubing (PE 10)	BD Diagnostics	427401
Murine TNF-α	R&D Systems	410-MT
Dylight 488- labeled rat anti-mouse CD42b antibody	Emfret Analytics	X488
Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody	BioLegend	108418
NESLAB EX water bath/circulator	Thermo-Scientific
Olympus BX61W microscope	Olympus
TH4-100 Power	Olympus
Lambda DG-4	Sutter
MPC-200 multi-manipulator	Sutter
R–-200 stage controller	Sutter
C9300 high-speed camera	Hamamatsu
Intensifier	Video Scope International
Ablation Laser	Photonic Instruments, Inc.
SlideBook 5.0	Intelligent Imaging Innovations