Study of Platelet Behavior in Vivo after Endothelial Stimulation with Laser Irradiation Using Fluorescence Intravital Videomicroscopy and PEGylated Liposome Staining

Abstract

Platelets contain an array of potent proinflammatory mediators, and therefore they are regarded as mediator and effector cells in inflammation. Knowing the role of platelets during these processes is crucial and the analysis of their behavior in situ and the associated mechanisms is consequently particularly important. However, conventional in vitro staining techniques induce modification of the characteristics of platelets. This study aimed to evaluate platelet behavior in vivo after endothelial stimulation (without endothelial denudation or exposure of basal lamina and/or collagen) with an argon laser, using video intravital microscopy in combination with a new an innovative platelet staining technique based on polyethyleneglycol (PEG) liposomes. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters. Platelets were stained by 5,6-CF-encapsulated PEGylated liposomes injected intravenously. The skin microcirculation was observed with an intravital microscope (using ×25, ×40, and ×80 magnifications) fitted with a xenon light source, an epifluorescence assembly, and an ultra-high sensitivity video camera for fluorescence imaging. Platelet activation without endothelial denudation or exposure of basal lamina and/or collagen was obtained with an argon laser emitting at 514.5 nm with the following parameters: 20 mW, 300 ms, 120 J/cm 2. The 80-μm laser beam was focused on a vessel and its position was controlled with the microscope. Thanks to the spatial resolution of the intravital microscopic imaging system, the platelets were seen rolling individually on the endothelium. After laser stimulation, platelets were activated and three phases were observed: recruitment, adhesion and detachment. The observation of these three phases was time dependent and the kinetics of the process were quantified. The recruitment reached a maximum after 90 ± 20 s. The adhesion phase lasted for 110 ± 25 s. At last, detachment of all platelets was observed. This detachment started 200 ± 20 s after irradiation and was completed in less than 2 min. This study confirms that laser irradiation used with optimal parameters can induce platelet activation without thrombus formation. Platelets can adhere only transiently on stimulated endothelium. This phenomenon may therefore represent a defense mechanism, by which platelets would accumulate in the vicinity of an injury, making them available for immediate response. At last, this study has clearly demonstrated the advantages of our new and innovative platelet staining method using PEGylated liposomes, which are (i) in situ labeling, (ii) use of a hydrophilic marker located in an aqueous compartment within the platelet, and (iii) labeling of platelets allowing observation during the whole experiment.