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The Application of Iodine‐Enhanced Micro‐CT Scanning Protocols to Analyze Multiple Tissue Types and Organ Systems in Mouse Embryos with Short‐term Gestational Hypoxia
Author(s) -
Fisher Rebecca E.,
Chapman Gavin,
Dunwoodie Sally L.,
Kusumi Kenro
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.780.2
Subject(s) - embryo , soft tissue , biology , x ray microtomography , skeletal muscle , staining , anatomy , pathology , andrology , biomedical engineering , medicine , microbiology and biotechnology , radiology
We have previously demonstrated that mutations in Hes7 and Mesp2 genes, coupled with short‐term gestational hypoxia, significantly increase the range and severity of vertebral defects in a mouse model (Sparrow et al., 2012). In addition, disruptions of the Notch pathway result not only in vertebral, but also muscle defects (Fisher et al., 2012). These studies utilized skeletal preparations to document the range of vertebral defects, while micro‐dissections were conducted to investigate muscle defects. These techniques are relatively labor‐intensive and not amenable to analyses of multiple tissue types and organ systems within each specimen. In order to facilitate the analysis of multiple tissues in whole embryos, we investigated the utility of iodine‐enhanced microCT scanning protocols. A series of experiments were conducted to determine the optimal percentage concentration and duration of staining for 17.5‐day‐old mouse embryos. Embryos were fixed in 4% paraformaldehyde and subsequently stained with various concentrations of Lugol's iodine solution (25%, 50%, 75%, 100%), for different lengths of time (2, 5, 10 days). Images were acquired using a SKYSCAN 1172 micro‐CT scanner, with an X‐ray source at 70kVp and 142 μA, or 55kVp and 181 μA, using a 0.5 mm aluminum filter. The iodine staining protocols did result in visualization of a diversity of soft‐tissue structures including skeletal muscle, blood vessels, and internal organs. However, the specific parameters of the staining protocols greatly impacted the quality of the images, with low concentrations (25%) resulting in poor resolution of soft tissue and high concentrations (100%) resulting in tissue deformation. Results also indicate that the duration of staining as well as the delivery of the stain (one dose versus a step‐wise progression) may affect outcomes. Despite these challenges, this study suggests that iodine‐enhanced micro‐CT scanning protocols can be utilized to conduct whole embryo analyses of defects related to hypoxic conditions during development. Support or Funding Information Funding for this project was provided by the American Association of Anatomists and the University of Arizona.