title: Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.
abstract: OBJECTIVE
  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.
  
  
  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 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 (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 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 ∼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 Ca(2+) signaling and endothelium-dependent vasodilation in vivo.
authors.affiliation: Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212, USA.
authors.forename: Pooneh
authors.initials: P
authors.lastname: Bagher
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davis
authors.forename: Steven S
authors.initials: SS
authors.lastname: Segal
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001160:Arterioles / Q000378:metabolism / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D003952:Diagnostic Imaging / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D007478:Iontophoresis; D051379:Mice; D008822:Mice, Transgenic; D008853:Microscopy; D012984:Software; D014664:Vasodilation
chemical_list: D000109:Acetylcholine