The overall goal of this procedure is to measure the heart rates of mouse embryos in utero at E 18.5 using a simple M mode ultrasound. This is accomplished by first securely placing the pregnant female on the platform with continuous anesthesia. Second, the location of the embryos is identified along the epidermis of the female's abdomen.
The third step is to measure the heart rate of each embryo using a 30 megahertz transducer. Then the embryos tails are collected for genotyping. Ultimately, results can show how the heart rate of each embryo is determined in utero by measuring the time taken in milliseconds per cardiac cycle.
The main advantage of this technique over the existing method like video microscopy, is that it's a simple method designed for both specialists and non-specialists, and it provides highly accurate heart rate measurements. Proper preparation of the system and material is critical and overly long procedure can affect the physiologic and hemodynamic characteristics of the fetuses. Start the ultrasound imaging system.
Connect the scan head that corresponds to the 30 megahertz transducer and connect the physiology controller unit on the controller unit. Choose the cardiac measurement program. Next, fill the nose piece of the scan head with the ionized water.
Avoid air bubbles as they interfere with imaging resolution. Place the scan head with the handle site up on its holder near the imaging platform on the disinfected working area, completely Fill the bottle of ultrasound gel. Avoid forming large bubbles.
Place the bottle upside down in its pre warming container set to 37 degrees Celsius. Verify the levels of oxygen and iso fluorine in the tubing system for anesthesia. One experiment with about eight embryos will use 15 to 20 liters of oxygen and should not take more than an hour to perform.
Position the bottles of ophthalmic balm, depilatory cream, and electrode gel near the imaging platform. Check that the infrared heat lamp is working. It should be set at the correct distance and intensity so that mice placed below it can maintain their body temperature and a constant heart rate.
Anesthetize the pregnant female in a chamber with a continuous supply of 2%isof, fluorine, and oxygen until it becomes non-responsive. Place the mouse on the imaging platform in a supine position. Adjust the tubing for continuous inhalation of 1.5%iso fluorine and oxygen, and fix the inhalation tube with tape.
Sufficient anesthesia should be confirmed during the procedure by the relaxed posture of the mouse and the absence of any response to tail and toe pinches. Place electrode gel only on the top left and bottom right electrode pads for the right four leg and left hind leg. For electrocardiography, secure the four paws to each pad using tape to prevent dryness of the eyes.
Apply one drop of ophthalmic balm into each eye. Next, shave the abdomen from chest to bladder with a hair clipper. Be careful not to cut the nipples, then apply depilatory cream for two minutes and carefully wipe it off with gauze to remove any remaining hair.
Lastly, insert the thermometer pre lubricated with electrode gel into the female's rectum. Maintain the body temperature and heart rate by adjusting the infrared heat lamp. If the body temperature drops below 36.5 degrees Celsius, stop, make lamp adjustments and wait a couple of minutes before proceeding.
This will require patience after preparing the ma dam with all instruments at the ready, gently pressed down on the naked abdomen to locate the embryos slowly and lightly. Spread them out to have most of the embryos in a single layer under the abdominal surface in each uterine horn, the sacks are connected linearly. Try to respect this order when spreading.
In some strains, a few embryos are located beneath the others. Exclude these embryos from the analysis. Mark each embryo on the naked abdomen of the female with a permanent marker with their anterior, posterior, and dorsal ventral directions.
Number the embryos in the right and left horns starting from the cervix. Then sketch their locations on a piece of paper to track them. Place a small amount of prewarm ultrasound gel on the naked abdomen and spread it evenly.
Avoid bubble formation. Add about five milliliters of gel on the specific area to image Long anesthetization hair loss and ultrasonic gel. Although prewarm can lead to hypothermia and subsequently affect females body temperature.
If the body temperature drops below 36.5 degrees Celsius or the heart rate drops below 400 beats per minute, at any time during the procedure, stop, move the heating lamp closer and wait a couple of minutes to allow the vital signs to readjust. Hold the probe in contact with the thick gel layer and gradually move the probe toward the skin while looking for the beating heart. Then adjust the angle of the probe to have both ventricles in their largest size in the imaging plane.
Begin acquiring images with the forearm resting on the station. Place the transducer on the ultrasound gel to obtain a live image on the viewing screen. Maintain the ridge of the transducer facing upward and click the skin head orientation marker.
Starting the cervix, move to the closest embryo and visualize the beating heart at the center of the screen in the focal zone. Gently move the probe up and down and also sideways without losing contact with the gel. To obtain the desired image plane, acquire the live recording.
Obtain the scout image and resume recording for at least five seconds before proceeding to the next embryo. Once all the embryos are analyzed, press browse on the keyboard. To view the list of the recordings, play each recorded M mode tracing and measure the spacing between at least six adjacent peaks to obtain the average heart rate.
Do not move the mouse, but rather perform the surgical procedure on the imaging platform in order to keep track of the numbered embryos using clean surgical scissors and forceps longitudinally in sizes, the skin and muscle layers of the abdomen. Locate the sacks in each uterine horn and match the numbers. Cut open the oak sack to expose the embryo in the order used above.
If not in a linear arrangement, see the paper sketch of the embryo positions cut about four millimeters of each tail. For genotyping, as genomic DNA is very efficiently extracted from embryonic tails when finished, euthanize all the animals according to your animal care committee. Using the described method, the impact of the Syrian protease furin in endothelial cells was assessed.
Measuring the heart rates of mouse embryos at E 18.5 in utero revealed that homozygous but not heterozygous echo embryos lacking furin suffered from tachycardia Once mastered. This technique can be done in approximately one hour for one liter of six to eight embryos.
