The overall goal of this Ex Ovo banding procedure is to create a reproducible, nonlethal alteration in cardiac hemodynamics to study the resulting genetic consequences. This method can help answer key questions in the cardiovascular development field, such as the development of cardiac valves, and associated disease states. The power of this system is that it can be used to study genetic pathways that respond to changing hemodynamics in the developing heart and valves and also in help finding a tipping point at which rescuing a heart does not have any long-term deleterious effects.
So demonstrating the procedure will be Lorain Junor, a technician from my laboratory, and Mr.Vinal Menon, a PhD student in the laboratory, who will be demonstrating the handling of the eggs and the use of the ultrasound machine. Incubate 80 to 90 fertilized, Bovan chicken eggs with their blunt ends up on plastic egg trays in a humidified rocker incubator set to 40 degrees Celsius. Let them develop for approximately 72 hours to HH 17.
Once at Stage 17, pour about 20 milliliters of warm Tyrode's Buffer, supplemented with sodium bicarbonate into a 100 millimeter by 26 millimeter dish. Next, remove an egg from the incubator and sterilize it with 70%ethanol. Then, gently crack the shell using a scalpel handle and carefully release its contents into the dish.
Now, check the embryo under a dissection microscope. Discard the embryo if it appears abnormal, such as being at the wrong developmental stage, being improperly oriented on the yolk, or if there is any bleeding. Be sure to look for the appropriate folding and bending of the vasculature.
Keep some embryos not subjected to OFT Banding at 40 degrees Celsius as control embryos. In preparation, tweeze out individual one centimeter long threads from a single 11 to zero nylon surgical suture and form them into loose knots. UV sterilize all the preformed knots three times at 1, 200 by 100 micro joules.
Under a dissection microscope, visually ensure that the heart of the embryo is beating at a normal rate, and if not, discard the embryo. Just before the surgery, pipet five to six milliliters of warm Tyrode's Buffer onto the embryo's surface. Then, pass one free end of a preformed knot under the OFT.
Position the suture at the OVJ or any other desired location and pass the free end into the knot to constrict the OFT, including the membranes surrounding the heart. Then, wet the surface of the yolk with Tyrode's Buffer, as before. Transfer all the manipulated embryos to the developmental incubator, along with the non-banded control embryos, and let them develop for a set amount of time.
Later, excise the embryo from the yolk using straight scissors. Then, dissect the heart with fine forceps and proceed with downstream studies, such as examination of the cardiac morphology. The parse constriction caused by the banding intervention leads to an increase in blood flow at the OVJ.
This hemodynamic parameter is conveniently assessed using 2D ultrasound imaging. After removing a single embryo from the incubator for ultrasound imaging, place its dish on a heating pad set to 40 degrees Celsius that is on an adjustable stand. Then, slowly and carefully fill the dish to the brim with warm Tyrode's Buffer.
Do not disturb the yolk. If the yolk is compromised, discard the embryo. Now, orient the embryo to the ultrasound probe by adjusting the stand.
Orient the embryo such that the central axis of the embryo is perpendicular to the ultrasound probe. With the ultrasound machine operating in B mode, obtain a 2D image of the beating heart. Then adjust the position of the embryo so that the OFT, ventricle, and OVJ are clearly visible in the ultrasound.
Next, switch to the pulse wave mode, set the desired pulse repetition frequency, and collect velocity data exactly at the OVJ. If the heart rate of the embryo decreases during imaging, velocity data obtained should not be used for analysis. All embryos used for velocity measurements should preferably not be used for any other experiments after ultrasound imaging.
Embryos were developed to HH 17 and a suture was placed at the OVJ as described. 48 hours later, the embryos were examined with 2D ultrasound to measure changes in blood flow velocity at the OVJ. As expected, the velocity at the OVJ is higher in the banded embryo relative to the control.
Once mastered, this technique can be done in less than five minutes, from cracking the egg to banding of the embryonic heart, if it is performed properly. While attempting this procedure, it's important to remember to have the band already created and to be careful in not damaging surrounding tissue when placing the band around the heart. Following this procedure, other methods like real-time PCR, Immunohistochemistry, and Insitu Hybridization can be performed to answer additional questions, like how are key genes and proteins affected by the altered hemodynamics.
