High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Podsumowanie

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.

Streszczenie

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies.

The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

Wprowadzenie

Ultrasound is sound waves with frequencies above the audible range of the human ear, higher than about 20 kHz¹. Animals like bats, wales, dolphins^2,3, mice⁴, rats⁵, and mouse lemurs⁶ all use ultrasound for orientation or communication. Humans take advantage of ultrasound for several technical and medical applications. An ultrasound device is able to create the sound wave and distribute and represent the signal. If ultrasound encounters an obstacle, the sound is reflected, absorbed or can go through it. The application of ultrasound as an imaging method, called sonography, is used for the analysis of organic tissues in human or veterinary medicine like the heart (echocardiography)^7,8, lung⁹, thyroid gland¹⁰, kidneys¹¹, and urinary and reproductive tracts^12,13; detecting gallstones¹⁴ and tumors¹⁵; and evaluating perfusion of blood vessels or organs^16,17. Ultrasound is a standard method in prenatal care during pregnancy, and fetal developmental disabilities or impairments can be recognized early. Specifically, the growth of a fetus is closely monitored at regular intervals to recognize a possible IUGR. Finally, fetal blood flow can be monitored, as this can point out growth restrictions^18,19,20,21.

A major advantage of ultrasound imaging compared to other methods like radiography is the sound's harmlessness of the tissues to be analyzed. This easy and fast method is non-invasive, painless, and can be used a number of times. The initial outlay of an ultrasound device is expensive; however, the consumable materials needed are cheap. The ultrasound system used in this manuscript is suitable for a range of animal models (i.e., mice and fish) While for humans an ultrasound device requires a frequency of 3-15 mHz, a frequency of 15-70 mHz is required for mice.

The present manuscript describes a protocol for the use of B-mode, color doppler mode, and pulse-wave doppler mode. The description includes preparation of the mice as well as performance, data acquisition, and storage. This method has been successfully applied to different mouse strains at all gestational days and can be used to investigate fetal and placental development as well as maternal and fetal blood parameters. Here, all applications are explained based on our studies employing pregnant MC/NK-deficient and control mice.

Protokół

All methods described here have been approved by the “Landesverwaltungsamt Sachsen Anhalt: 42502-2-1296UniMD.”

1. Experimental Procedure

1. Mate 6 to 8-week-old female MC-deficient C57BL/6J-Cpa3^Cre/+ (Cpa3^Cre/+) mice and MC-sufficient C57BL/6J-Cpa3^+/+ (colony controls; Cpa3^+/+) with BALB/c males.
2. Define the gestation day (gd) 0 after confirmation of the vaginal plug and treat the females immediately after plug confirmation.
   NOTE: A plug is the sperm of the male in the vaginal orifice of the female.
   1. Inject 250 µL of PBS intraperitoneally in control Cpa3^+/+ females.
   2. Inject 250 µL of anti-CD122 (0.25 mg) intraperitoneally in MC-deficient Cpa3^Cre/+ females.
      Note: An injection of 0.25 mg of anti-CD122 depletes peripheral NKs and uNKs in MC-deficient Cpa3^Cre/+ females as described previously²².
3. Wait until gd5.
   NOTE: At gd5, there is the earliest possibility for implantation analysis.
   1. Proceed with steps 2-5 for the ultrasound analysis.
4. Perform the ultrasound imaging at gd5, 8, 10, 12, and 14.

2. Preparation of the Ultrasound System

1. Turn on the system (Figure 1A; main power on the back and computer standby at the left site), the heated platform (Figure 1B; at the control pad), and the gel warmer (Figure 1C).
   NOTE: Ultrasound gel needs to warm up for approximately 0.5 h.
2. Ensure that the isoflurane unit is filling sufficiently (Figure 1D).
3. Open a New Study or New Series in an existing study in the browser. Fill in all the required information (owner, study name, series name, animal data) in the Study Info window. Click Ok.
4. After clicking Ok, ensure that the B-mode imaging window appears and the imaging in B-mode begins automatically.

3. Mouse Handling

1. Anesthetization of the mouse
   1. Place the mouse in the knockdown box (Figure 1E), close the box, open the isoflurane tube to the knockdown box, and turn on the isoflurane (concentration 3.5%).
   2. When the mouse is anesthetized, lower (to concentration 1.5%) and redirect the isoflurane flow by opening the tube in the direction of the heating platform and close the flow to the knockdown box.
      NOTE: To reach sufficient anesthesia, wait an additional 10 s after the mouse is no longer moving.
   3. Transfer the mouse quickly from the knockout box to the heating platform (Figure 1F) in a dorsal position, and gently position its nose in the anesthesia nose tube located on the top of the platform.
2. Fixation, depilation, and preparation of the mouse for measuring
   1. Place eye protection cream in each eye of the mouse to prevent dry eyes.
   2. Place one drop of electrode gel on each of the four copper areas on the heated platform (Figure 1F).
   3. Tap the paws with surgical tape on the electrode gel-coated areas of the heating platform.
   4. Check ECG [optimal value = 450-550 beats/min (BPM)] and respiratory physiology at all times.
      NOTE: By using a rectal probe, body temperature measurement is possible, but not necessary.
   5. Place depilatory cream at the abdomen of the mouse, rub the cream with a cotton swab and wait around 1 min. Remove the cream with a water-soaked compress. Repeat this step if not all hairs are gone.
   6. Apply the pre-warmed ultrasound gel on the depilated skin.

4. Measurements and Acquisition of Images and Videos

1. Hold the transducer (Figure 1G) in the hand or clamp it in the holding device (Figure 1H; holding device is recommended).
2. Identify the bladder with the transducer and use it as reference point. Move the transducer to the left and right sites of the abdomen to trace implantations.
3. B-mode for visualization of anatomical structures in 2D grayscale image
   1. Move the transducer or heating platform table where the mouse is fixated until the first implantation is visible on the screen at its largest size.
      1. Select Image Label and enter a name, or Frame Store (storage without name) to store single frames, or Cine Store to store a cineloop for whole implantation measurements.
   2. Move the transducer or table to bring the placenta to a position where blood flow in the UmA is visible. Store a single frame or cineloop (see step 4.3.1.1) for placental measurements.
      NOTE: Placental measurements are possible from gd10 onwards.
   3. Continue with all implantations using the same method.
4. Color doppler mode to visualize and determine the direction of blood flow
   1. Press the Color button.
   2. Move the Color Box (in this area, the signal is visible) to the required position by using the trackball. If necessary, change the size of the box by pressing Update and move the trackball (to the right side/upward = bigger; to the left side/downward = smaller). When the box has the right size, press Select.
   3. Store single frames or cineloops as described in step 4.3.1.1.
5. Pulse-wave (PW) doppler mode to quantify blood flow through the vessels in the Arteria uterina (uterine artery, UA) and UmA
   1. Locate the region of interest in the color doppler acquisition.
      NOTE: The UA is located caudal to the bladder, and the UmA is located between the fetus and placenta.
   2. Press PW, and a dashed line will appear. Move this line to the blood vessel of interest and adjust the angle of the line using the “Doppler Angle” knob in line with the blood flow. Press Update.
      NOTE: The angle between the direction of the blood flow and the transducer must be consistent in all animals, especially when using angles of greater than 60° (here, 70° for UAs and 45° for UmAs were used).
   3. Store a cineloop of the appearing doppler lines in the PW doppler acquisition window.

5. Reviewing and Finishing Data Acquisition and Saving a Series

1. To review data, press Study Management. Scroll to the thumbnail image of interest and double-click Update.
2. Press first Study Management then Close in the browser window to finish data acquisition and save a recorded series.
   NOTE: After closing a series, it is not possible to store frames or cineloops in this series anymore.

6. Mouse Handling Following Acquisition of Data

1. Remove the gel from the anaesthetized animal with the help of dry compresses.
2. Remove the surgical tape carefully from the paws.
3. Close the isoflurane tube (concentration 0%).
4. Proceed with following ultrasound analysis at gd5, 8, 10, and 12.
   1. Place the animal alone in a cage for a minimum of 5 min so it has time to wake up and orientate.
   2. Place the mouse back in the original cage.
      NOTE: Do not turn off the isoflurane before removing the gel and surgical tape, as mice wake up very quickly (around 20 s) after turning off the isoflurane.
5. Proceed with the following ultrasound analysis at gd14.
   1. Sacrifice the female before it wakes up by cervical dislocation. Open the animal, remove the uterus, separate the fetuses and placentas, and measure fetal and placental weights.

7. Copying and Importing the Data

1. Mark one or more series by clicking Export To and choose the storage space to copy data onto a hard disk.
2. Open the software at a computer and click Copy From and select the study/series from the hard disk to import a study/series into the software.
3. Analyze the data with the software.

Wyniki

Individual components of the ultrasound system used in this manuscript are shown in Figure 1. Figure 2 shows representative ultrasound images acquired in B-mode at gd5, 8, 10, and 12 (B) and corresponding implantation area measurement results (A), demonstrating a significant reduced implantation area of anti-CD122-treated Cpa3^Cre/+ mice from gd10 onwards.

Dyskusje

Using our ultrasound system, we demonstrated fetal growth restriction in MC/NK-deficient mothers from gd10 on. Furthermore, at gd10 and 12, we observed reduced placental dimensions, and at gd14 the absence or reversion of end diastolic flow in the UmAs of some fetuses of uMC/uNK-deficient mice. This sign of poor vascularization was associated with a significant resistance index of the arteries indicating IUGR. Results confirm the important role of uMCs and uNKs in pregnancy and fetal well-being and in understanding the c...

Materiały

Name	Company	Catalog Number	Comments
LEAF anti-Maus CD122 (IL-2Rb)	BioLegend	123204	Klon TM-β1; 500 µg
Vevo 2100 System	FujiFilm VisualSonics Inc.		Transducer MS550D-0421
Vevo LAB Software	FujiFilm VisualSonics Inc.
Isoflurane	Baxter	PZN: 6497131
Electrode gel	Parker	12_8
Surgical tape	3M Transpore	1527-1
Eye cream	Bayer	PZN: 1578675
Cotton tipped applicators	Raucotupf	11969	100 pieces
Depilatory cream	Reckitt Benckiser	2077626
Compresses	Nobamed Paul Danz AG	856110	10 x 10 cm
Ultrasound gel	Gello GmbH	246000