Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events

Naoki Honkura,Mark Richards,Lena Claesson-Welsh,Miguel Sáinz-Jaspeado,Christer Betsholtz,Bàrbara Laviña

doi:10.1038/s41467-018-04929-8

Naoki Honkura, Mark Richards + Show 4 more

Open Access

https://doi.org/10.1038/s41467-018-04929-8

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Abstract

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature.

Highlights

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli
Postcapillary venules have been considered the main sites of leakage in most organs[11,12] but in the central nervous system (CNS), a greater abundance of endothelial tight junctions (TJs) contributes to restrictions in blood–tissue exchange imposed by the blood–brain barrier[13]
To follow vascular dynamics over time, we established a setup for non-invasive intravital time-lapse imaging using Multi-photon laser scanning microscopy (MPLSM)

Summary

Results

Experimental setup for in vivo imaging of vascular dynamics. To follow vascular dynamics over time, we established a setup for non-invasive intravital time-lapse imaging using MPLSM. Imaging was performed on the ventral aspect of the C57BL/6 mouse ear dermal vasculature by fixing to a glass slide (Fig. 1a). Vessels were visualized by systemic administration of fluorescent tracers via tail vein cannulation. A sub-micron glass capillary was used a b

Objective

Discussion

Methods