title: Correlative intravital imaging of cGMP signals and vasodilation in mice.
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 Ca(2+), 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.
authors.affiliation: Interfakultäres Institut für Biochemie, University of Tübingen Tübingen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Thunemann
authors.affiliation: Institut für Physiologie, Universität zu Lübeck Lübeck, Germany.
authors.forename: Kjestine
authors.initials: K
authors.lastname: Schmidt
authors.affiliation: Institut für Physiologie, Universität zu Lübeck Lübeck, Germany.
authors.forename: Cor
authors.initials: C
authors.lastname: de Wit
authors.affiliation: Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School Boston, MA, USA.
authors.forename: Xiaoxing
authors.initials: X
authors.lastname: Han
authors.affiliation: Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School Boston, MA, USA.
authors.forename: Rakesh K
authors.initials: RK
authors.lastname: Jain
authors.affiliation: Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School Boston, MA, USA.
authors.forename: Dai
authors.initials: D
authors.lastname: Fukumura
authors.affiliation: Interfakultäres Institut für Biochemie, University of Tübingen Tübingen, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Feil
keywords: biosensor; cremaster; cyclic GMP; dorsal skinfold chamber; fluorescence resonance energy transfer; intravital imaging; microcirculation; multiphoton microscopy