Immunorectivity in the cardiac tissue of the crocodile embryos using conventional antibodies

According to phylogeny the crocodile has the most perfect heart of all the reptiles, having a completed septation of the ventricles similar to the human heart. In the basic features it is thus very similar to human. However, the question of presence or nature of the crocodilian cardiac conduction system is not yet clarified. To be able to embark upon the study of this issue, we attempted to identify working antibodies to crocodile tissue from commercially available antibodies. We used the antibodies, which are known to react with mouse, rat, human or chicken, as antibodies tested specifically for crocodile tissues are currently not available.

Experiments were carried out on a series of crocodile embryonic hearts of Crocodylus siamensis at different stages of development. We dissected the thorax and opened the chest cavity to gain the access to the heart. The hearts were isolated and fixed for 24 h at 4°C in 4% paraformaldehyde in PBS and then processed into OCT medium for serial sectioning on a cryomicrotome. We first performed H&E / Alcian Blue staining in histological sections at 100 μm intervals (guide series). Then we selected slices containing the tissues of interest, where we focused primarily on the atria, the interventricular septum, the ventricles, and the outflow tract. We tested the following antibodies for immunostaining: α-Smooth Muscle Actin (SMA), α-Sarcomeric Actin (SA), Collagen type I, Periostin, Connexin 40, HCN4 and CD57 (HNK -1). In parallel with crocodile tissue we processed control chick and/or mouse embryonic heart samples, which served as a positive control.

Immunohistochemistry using SMA, SA, Periostin and CD57 antibodies in crocodile tissues produced expected results similar to control samples. Interestingly, SMA staining persisted in the crocodile ventricles even after septation, while it was absent in older chick or mouse hearts. The other antibodies produced either non-specific staining, or there was no immuto study the nature of myocardial atrio-ventricular continuity and development of annulus fibrosus in the crocodilian heart.

We would like to thank the Protivin ZOO (Ing. Prochazka) for permission to collect the damaged Crocodylus siamensis eggs.

This study was supported by MSMT PRVOUK-P35/LF1/5.