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Optimization of Vascular Casting for Three-Dimensional Fluorescence Cryo-Imaging of Collateral Vessels in the Ischemic Rat Hindlimb

Published online by Cambridge University Press:  23 February 2017

Janina C.V. Schwarz
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Monique G.J.T.B. van Lier
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Erik N.T.P. Bakker
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Judith de Vos
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Jos A.E. Spaan
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Ed VanBavel
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Maria Siebes*
Affiliation:
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
*
*Corresponding author.m.siebes@amc.uva.nl
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Abstract

Development of collateral vessels, arteriogenesis, may protect against tissue ischemia, however, quantitative data on this process remain scarce. We have developed a technique for replicating the entire arterial network of ischemic rat hindlimbs in three dimensions (3D) based on vascular casting and automated sequential cryo-imaging. Various dilutions of Batson’s No. 17 with methyl methacrylate were evaluated in healthy rats, with further protocol optimization in ischemic rats. Penetration of the resin into the vascular network greatly depended on dilution; the total length of casted vessels below 75 µm was 13-fold higher at 50% dilution compared with the 10% dilution. Dilutions of 25–30%, with transient clamping of the healthy iliac artery, were optimal for imaging the arterial network in unilateral ischemia. This protocol completely filled the lumina of small arterioles and collateral vessels. These appeared as thin anastomoses in healthy legs and increasingly larger vessels during ligation (median diameter 1 week: 63 µm, 4 weeks: 127 µm). The presented combination of quality casts with high-resolution cryo-imaging enables automated, detailed 3D analysis of collateral adaptation, which furthermore can be combined with co-registered 3D distributions of fluorescent molecular imaging markers reflecting biological activity or perfusion.

Type
Biological Applications
Copyright
© Microscopy Society of America 2017 

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