Eur J Anat, 24 (3): 211-220 (2020)

Late administration of caffeine affects cardiac maturation in chick embryos: a combined two and threedimensional morphogenetic and gene analyses

Nichapha Chandee^1,2, Nutmethee Kruepunga¹, Thanyarat Leckchaoum¹, Pakpoom Thintharua¹, Wattana Weerachatyanukul¹, Somluk Asuvapongpatana¹

¹Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand, ²School of Allied Health Science, Walailak University, Nakhon Si Thammarat, Thailand

ABSTRACT Cardiac malformations are very prevalent and can be caused both by defective genes and envi-ronmental teratogens. Among the latter, caffeine causes malformations when exposed during early cardiac development, whereas its later effects are still unclear. We exposed three-day incubated (D3) chick embryos to 2 mg caffeine and analyzed them at D5, D7 and D9. The embryos were serially sec-tioned and analyzed two-dimensionally.Alternatively, the sections of D9 embryos were reconstructed three-dimensionally using Amira® software and analyzed volumetrically. The expres-sion of genes involved in endothelial-mesenchymal transformation (EMT) was studied by real-time PCR. Interestingly, caffeine treatment at D3 em-bryos did not induce cardiac malformations, but did delay growth, in particular that of the ventricles and ventricular trabeculae. Furthermore, it affected EMT in the endocardial cushion and atrioventricu-lar valves. Gene-expression analysis revealed that caffeine had a progressively deleterious effect on the expressions of GATA4, MMP2, SNAIL1, TWIST1, and VIMENTIN. The effect of late caf-feine administration on the chicken embryos would provide suggestive evident towards a possible heart developmental defect in humans, particularly heavy caffeine consumers during pregnancy.

Keywords: Chick embryo â?? Cardiac development â?? Cardiac maturation â?? Ventricular defect â?? Caffeine â?? 3D analysis