Femtosecond laser photodisruption of vitelline vessels of avian embryos as a technique to study embryonic vascular remodeling.

Abstract

During cardiogenesis, congenital heart defects (CHDs), generally start as local tissue abnormalities without underlying genetic causes, suggesting abnormal hemodynamics may be an important source. Due to the scarcity of experimental techniques that permits the formation of minimally-invasive and well-controlled cardiac perturbations, experimental investigation of embryonic development of CHD via in-vivo models is difficult. In this study, in order to investigate the relationship between abnormal mechanical signaling and embryonic CHD development, a previously developed laser-based technique was adopted to alter chicken embryonic cardiovascular development. The technique incorporates two-photon fluorescence microscopy to visualize deep tissue while femtosecond-pulsed laser photodisruption is used to ablate targeted tissue. Vitelline vessel remodeling under abnormal hemodynamics was the prime concern of the study. In order to alter the hemodynamics, blood flowing inside 50-300 µm diameter Hamburger-Hamilton 24 embryonic vessels was selectively ablated. Red blood cells in the blood and endothelial cells of the vessel walls were damaged as a result of ablation. Cellular injuries led to micro-occlusions in the vessels. Several micro-occlusions formed stable clots, resulting in a complete cessation of blood flow in the targeted vessels. By measuring blood velocities in the surrounding vessels via line scanning technique, the subsequent redistribution of blood flow in the immediate upstream and downstream vessels was revealed. The network was analyzed after 24 h, and it was found to be degraded. Degradation of the entire network can be attributed to the abnormalities in hemodynamics within the vessels. For studying embryonic development of heart defects under disturbed flow conditions, the present study can be extended to clot a blood vessel inside the embryo or a vitelline vessel in the vicinity of the heart. These results demonstrate that, laser-based noninvasive tools should be considered as powerful techniques to analyze hemodynamic signals encountered in embryonic development of CHD.