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TRP X27 - In vivo Visualization of Monocytes in Thrombus Formation

Funding period: 01.01.2019 – 31.08.2020

Project Summary

The CTH’s intravital microscopy platform offers researchers the opportunity to study their target structures in real-time in vivo. To be up-to-date in intravital microscopy technique, the CTH was able to extend this platform with a spinning disc confocal microscope financed by a DFG equipment grant. The development of new imaging approaches and methods for this microscope will be done based on a project focussing on the role of monocyte-derived factor VII in thrombus formation. This project became possible, because the new microscope offers the opportunity to image four different colours (excitation lasers 405 nm, 488 nm, 561 nm, 640 nm/ emission filters 450/50 nm, 525/50 nm, 600/50 nm, 685/50 nm), whereas the old epifluorescence microscope was able to depict only two colours. Imaging of four colours enables us to combine reporter mouse lines with the application of labelled antibodies or dyes. In this way, we can illustrate more components of the coagulation system at the same time. As the microscope operates with a spinning disc illumination, it achieves a maximum frame rate of 100 frames per second. This is extremely beneficial regarding the high speed of our target cells moving in the vasculature. Because we are interested in an expression pattern of molecules on cells and cell-to-cell interaction, the microscope is not only equipped with high-resolution cameras, but can also be used with objectives up to 60-fold magnification. Altogether, this state of the art technique will provide a much more detailed in vivo image concerning temporal and spatial resolution. In addition, thrombus growth can be followed by 3D-reconstruction generated by a Piezo Z-stage.

Within this project, we will examine the role of monocytes and monocytes-derived factor VII in arterial (carotid artery ligation model for 30min) and venous thrombosis (vena cava flow reduction model for 6h). A monocyte-specific reporter mouse line will be bread to a monocyte-specific Cre deleter and factor VII flox mouse to achieve factor VII-deficient fluorescent monocytes. Examination of these animals in arterial and venous thrombosis models with the EPCR and TF antibodies will illuminate the influence of factor VII derived from monocytes and the role of the interaction of EPCR with factor VII in thrombus formation in vivo. By the use of the spinning disc confocal microscope, we will be able to follow not only the monocytes during thrombus formation, but we will also be able to depict the expression pattern of TF and EPCR. Compared to the former epifluorescence system, much more information regarding monocyte-endothelial cell or monocyte-platelet interaction can be taken out of these experiments thanks to the modern cameras and microscope set-up. The high-resolution high-speed images will reveal new mechanisms in thrombus formation in real-time.

Principal Investigator