Abstract
Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism.
Highlights
Blood vessels are highly organized structures consisting of various cell types including endothelial cells in the intima, smooth muscle cells (SMCs) in the media, and fibroblasts in the adventitia
We describe two models of intravital fluorescence resonance energy transfer (FRET)/Cyclic guanosine monophosphate (cGMP) imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber
Ratiometric FRET-based biosensors are advantageous over cpEGFPbased biosensors, because not a single fluorescence intensity (e.g., EGFP) but the ratio between two simultaneously recorded fluorescence intensities (e.g., CFP/YFP) is taken to estimate ligand binding to the sensor
Summary
Blood vessels are highly organized structures consisting of various cell types including endothelial cells in the intima, smooth muscle cells (SMCs) in the media, and fibroblasts in the adventitia. These cells receive signals from surrounding tissue, blood, and the nervous system, and they interact with each other in multiple ways. Intravital microscopy (IVM) is a powerful tool to study signaling processes in vascular cells while still in their natural environment. In combination with motion compensation, IVM can achieve spatiotemporal resolutions similar to microscopy under in vitro conditions (Lee et al, 2014)
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