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Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.

ABSTRACT: ?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 Ca(2+) responses and arteriolar diameter in vivo.?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 Ca(2+) concomitant with arteriolar diameter. Transgenic Cx40(BAC) -GCaMP2 mice expressing a fluorescent Ca(2+) indicator molecule in arteriolar ECs enabled resolution of EC Ca(2+) signaling in response to ACh microiontophoresis (500nA, 100-1000msec pulse) from a micropipette (1?m tip) positioned adjacent to an arteriole in the superfused cremaster muscle preparation.?A 100-msec pulse of ACh (1M) had little effect on EC Ca(2+) 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 ?100?m (250msec pulse) to ?200?m (1000msec pulse) with a peak velocity of ?150?m/sec.The novel imaging and software presented here are the first to enable automated simultaneous evaluation of EC Ca(2+) signaling and endothelium-dependent vasodilation in vivo.

SUBMITTER: Bagher P

PROVIDER: S-EPMC3082596 | biostudies-other | 2011 May

REPOSITORIES: biostudies-other

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Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.

Bagher Pooneh P Davis Michael J MJ Segal Steven S SS

Microcirculation (New York, N.Y. : 1994) 20110501 4

<h4>Objective</h4> 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 Ca(2+) responses and arteriolar diameter in vivo.<h4>Methods</h4> 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 Ca(2+) concomitant with arteriolar diamete ...[more]

PMID: 21418383

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Project description:Acetylcholine evokes endothelium-dependent vasodilation subsequent to a rise in intracellular calcium. Despite widespread application in human and animal studies, calcium responses to intravascular ACh have not been visualized in vivo. Microiontophoresis of ACh in tissue adjacent to an arteriole activates abluminal muscarinic receptors on endothelial cells within a "local" region of diffusion, but it is unknown whether ACh released in such fashion gains access to the flow stream resulting in further actions downstream. To test this hypothesis and provide new insight into calcium signaling in vivo, we studied the cremaster muscle microcirculation of anesthetized male Cx40(BAC)-GCaMP2 transgenic mice (n = 22; 5-9 mo; 33 ± 1 g) expressing the fluorescent calcium sensor GCaMP2 selectively in arteriolar endothelial cells. Submaximal ACh stimuli were delivered using microiontophoresis (1-μm pipette tip, 500 nA). With stimulus duration <500 ms or with the micropipette positioned within one vessel diameter (∼30 μm) away from an arteriole, endothelial cell calcium fluorescence was restricted to the region of ACh diffusion (<200 μm). In contrast, with the micropipette tip positioned immediately adjacent to an arteriole or within its lumen, calcium fluorescence encompassed entire networks downstream. The velocity of downstream calcium signaling in response to ACh increased with centerline velocity of fluorescent tracer microbeads (r(2) > 0.99; range: <1 mm/s to >10 mm/s). Diverting arteriolar blood flow into a side branch redirected downstream fluorescence responses to ACh; occluding flow abolished responses. Blocking luminal muscarinic receptors (intravascular glycopyrrolate; 6 μg/kg) inhibited downstream responses reversibly. Through visualizing the actions of a "local" ACh stimulus on endothelial cell calcium fluorescence in vivo, the present findings illustrate that transmural diffusion and convection of an agonist can activate entire networks of arteriolar endothelial cells concomitant with its delivery in the flow stream.

Dataset Information

Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.

Publications

Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.

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