Articles by Akshay Shekhar in JoVE
An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications Huseyin C. Yalcin1,2, Akshay Shekhar1, Ajinkya A. Rane1, Jonathan T. Butcher1 1Department of Biomedical Engineering, Cornell University, 2Current Address: Mechanical Engineering Department, Dogus University In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.
Other articles by Akshay Shekhar on PubMed
Two-photon Microscopy-guided Femtosecond-laser Photoablation of Avian Cardiogenesis: Noninvasive Creation of Localized Heart Defects American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2010 | Pubmed ID: 20709864 Embryonic heart formation is driven by complex feedback between genetic and hemodynamic stimuli. Clinical congenital heart defects (CHD), however, often manifest as localized microtissue malformations with no underlying genetic mutation, suggesting that altered hemodynamics during embryonic development may play a role. An investigation of this relationship has been impaired by a lack of experimental tools that can create locally targeted cardiac perturbations. Here we have developed noninvasive optical techniques that can modulate avian cardiogenesis to dissect relationships between alterations in mechanical signaling and CHD. We used two-photon excited fluorescence microscopy to monitor cushion and ventricular dynamics and femtosecond pulsed laser photoablation to target micrometer-sized volumes inside the beating chick hearts. We selectively photoablated a small (âˆ¼100 Î¼m radius) region of the superior atrioventricular (AV) cushion in Hamburger-Hamilton 24 chick embryos. We quantified via ultrasound that the disruption causes AV regurgitation, which resulted in a venous pooling of blood and severe arterial constriction. At 48 h postablation, quantitative X-ray microcomputed tomography imaging demonstrated stunted ventricular growth and pronounced left atrial dilation. A histological analysis demonstrated that the laser ablation produced defects localized to the superior AV cushion: a small quasispherical region of cushion tissue was completely obliterated, and the area adjacent to the myocardial wall was less cellularized. Both cushions and myocardium were significantly smaller than sham-operated controls. Our results highlight that two-photon excited fluorescence coupled with femtosecond pulsed laser photoablation should be considered a powerful tool for studying hemodynamic signaling in cardiac morphogenesis through the creation of localized microscale defects that may mimic clinical CHD.
Hemodynamic Patterning of the Avian Atrioventricular Valve Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jan, 2011 | Pubmed ID: 21181939 In this study, we develop an innovative approach to rigorously quantify the evolving hemodynamic environment of the atrioventricular (AV) canal of avian embryos. Ultrasound generated velocity profiles were imported into Micro-Computed Tomography generated anatomically precise cardiac geometries between Hamburger-Hamilton (HH) stages 17 and 30. Computational fluid dynamic simulations were then conducted and iterated until results mimicked in vivo observations. Blood flow in tubular hearts (HH17) was laminar with parallel streamlines, but strong vortices developed simultaneous with expansion of the cushions and septal walls. For all investigated stages, highest wall shear stresses (WSS) are localized to AV canal valve-forming regions. Peak WSS increased from 19.34 dynes/cm(2) at HH17 to 287.18 dynes/cm(2) at HH30, but spatiotemporally averaged WSS became 3.62 dynes/cm(2) for HH17 to 9.11 dynes/cm(2) for HH30. Hemodynamic changes often preceded and correlated with morphological changes. These results establish a quantitative baseline supporting future hemodynamic analyses and interpretations.