In vivo visualization of platelet/endothelium cell interaction in muscle flaps

Increasing evidence underlines the substantial pathophysiological impact of platelets on the development of ischemia/reperfusion injury (I/R) in flaps. Methods for studying dynamic platelet mechanisms in flaps in vivo are not available. The aim of this study was to develop a model enabling quantitative analysis of platelet kinetics and platelet-endothelium cell interaction within the microcirculation of muscle flaps in vivo. Balb/c mice (n = 16) were anesthetized, and an epigastric muscle flap was prepared. Autologous platelets were separated from blood donor animals (n = 16) and labeled ex vivo by means of rhodamine-6-G. After I/R (90 minutes' clamping, 10 minutes' reperfusion), the platelets were administered intra-arterially (i.a.). Microhemodynamics and kinetics of platelets were investigated by intravital fluorescence microscopy. I/R of muscle flaps induced disturbances in microcirculation. The number of rolling platelets, as well as platelets adhering to the inner vessel wall of venules, was increased in the ischemia group. Using intravital fluorescence microscopy, platelet kinetics were analyzed directly in flap microcirculation in vivo for the first time. Since platelet/endothelial cell interaction is a key event in the pathophysiology after microsurgical procedures, this model will help to understand basic molecular mechanisms of platelet behavior during I/R.