Intravital Macrozoom Imaging and Automated Analysis of Endothelial Cell Calcium Signals Coincident with Arteriolar Dilation in Cx40^BAC-GCaMP2 Transgenic Mice

Calcium signaling is integral to endothelium-dependent vasodilation. Our goal was to develop methods enabling automated analyses for accurately and objectively determining the dynamic relationship between EC Ca²⁺ responses and arteriolar diameter in vivo. Methods: User-friendly software (DiaFluor) written in LabView was applied to images acquired at 15fps with a custom macrozoom intravital microscope to evaluate changes in EC Ca²⁺ concomitant with arteriolar diameter. Transgenic Cx40^BAC-GCaMP2 mice expressing a fluorescent Ca²⁺ indicator molecule in arteriolar ECs enabled resolution of EC Ca²⁺ signaling in response to ACh microiontophoresis (500nA, 100-1000msec pulse) from a micropipette (1μm tip) positioned adjacent to an arteriole in the superfused cremaster muscle preparation. Results: A 100-msec pulse of ACh (1M) had little effect on EC Ca²⁺ or arteriolar diameter. As pulse duration increased, vasodilation increased with fluorescence intensity (p<0.01). Based upon fluorescence responses (F/F_o), the effective diffusion distance of ACh along arterioles increased from ∼100μm (250msec pulse) to ∼200μm (1000msec pulse) with a peak velocity of ∼150μm/sec. Conclusions: The novel imaging and software presented here are the first to enable automated simultaneous evaluation of EC Ca²⁺ signaling and endothelium-dependent vasodilation in vivo.