Developmental Biology
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High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo
Summary November 8th, 2018
Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.
Transcript
This method is very important to answer key questions in the areas of Oncology, Neuro-Biology, Cardiology and Fetal Development, and of course also it's important in the area of reproductive research. The main advantages of this technique are it is non-invasive, painless, easy, fast and allows the follow-up of fetuses at relevant gestational time points throughout pregnancy. At least a half hour before the imaging procedure, turn on the ultrasound system, the heated platform, and the gel warmer.
Open a new study or new series in an existing study in the browser. Enter all the required information in the study info window. Click OK, and confirm that the B-mode imaging window appears.
When the ultrasound is ready, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal's eyes. Place one drop of electro gel onto each of the 4 copper sections of the heated platform, and secure the paws to the electro gel coated sections of the platform. Remove the hair from the abdomen of the animal with depilatory cream and a cotton swab, washing the cream from the depilated skin after one minute with a water-soaked compress.
Then apply pre-warmed ultrasound gel onto the exposed area. One reason for low signal intensity can be the low amount of gel placed between the mouse and the ultrasound beam. In our experience, a rather large amount of gel is necessary for a good signal quality.
When the animal is ready, place the transducer over the bladder for use as a reference point, and move the heating platform table to the left and right sides of the abdomen to trace the fetal implantations in 2D B-mode, adjusting the transducer or platform as necessary until the first implantation is visible on the screen, at its largest size. Select Image Label, and enter a name for the image file. Adjust the position of the image, until the blood flow within the arteria umbilicalis of the placenta is in view, and store a single frame or cineloop for placental measurements.
For color Doppler imaging to determine the direction of the blood flow, press the Color button, and use the trackball to move the color box to the appropriate position, clicking Update to adjust the size of the box as necessary. When the box is the correct size, click Select and store the image as experimentally appropriate. For pulsed wave Doppler quantification of the blood flow through the vessels, locate the region of interest in the color Doppler acquisition mode and click Pulse Wave.
A dashed line will appear. Move the line to the blood vessel of interest and use the Doppler angle knob to adjust the angle of the line to align with the blood flow. For pulsed wave Doppler measurements, it is important to watch the angle between the direction of the blood flow and the ultrasound beam.
Too high angles, or different angles between animals in the same experiment may lead to inaccurate velocity measurements. Then, click Update and store cineloop of the appearing Doppler lines in the pulsed wave Doppler acquisition window. To review the data, click Study Management, scroll to the thumbnail image of interest, and double click Update, then click Study Management again, and click Close to close the browser window, to finish the data acquisition and save a recorded series.
To export the data, connect a hard disk, click Export To, to mark one or more series of data, and select the storage space for copying the data onto the hard disk. Here, representative ultrasound images acquired in the B-mode at gestational days 5, 8, 10 and 12, and their corresponding implantation area measurement results are shown, revealing a significant reduction in the implantation area in anti-CD122 treated mast cell-deficient mice from gestational day 10 onwards. B-mode imaging facilitates the imaging and measurement of individual parts of an implantation.
For example, anti-CD122 treated mast cell-deficient animals demonstrate a significant reduction in the placental area, thickness and diameter compared to untreated mice at gestational days 10 and 12. In contrast, the placental area and diameter are similar between the groups at gestational day 14, and the placental thickness, a significant increase in the anti-CD122 treated transgenic animals compared to untreated mice at gestational day 14. Pulsed wave Doppler imaging of the arteria uterina for measurement of the peak systolic velocity and diastolic velocity, and calculated resistance index, reveal similar values between the treated and untreated animals.
The resistance index of the arteria umbilicalis in anti-CD122 treated mast cell deficient animals, however, is significantly increased compared to untreated mice. While attempting this procedure, it is important to keep in mind, that you should do this every other day rather than every day, to avoid the stress, and the risk of repetitive narcotization of the animals. Following this procedure, other measurements, like 3D measurement, visualization and quantification of tissue movement over time, blood pressure measurements, and also ultrasound guided injections can be performed.
After it's development, this method paved the way for physicians and researchers in the area of Obstetric and pre-natal care, because it's possible to follow up fetal development and to recognize possible abnormalities during pregnancy.
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