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

Summary

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.

Abstract

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.

Introduction

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 applicati....

Protocol

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 v.......

Discussion

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.......

Materials

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