Embryonic echocardiography for assessment of congenital and functional cardiac defects

Abstract

Cardiac function and morphology by mouse fetal echocardiography can be assessed by scanning the uterus extracted from the abdominal cavity (trans-uterine ultrasound) or the womb (trans-abdominal ultrasound). Advantages of trans-abdominal ultrasound include (1) non-invasive longitudinal analysis at different stages, reducing animal use; and (2) maintenance of natural environment, diminishing perturbations on functional parameters, which are more frequent in trans-uterine conditions. Here we describe both approaches, explaining how to identify congenital cardiac defects and defining the correlation between echocardiography findings and histological analysis. For complete details on the use and execution of this protocol, please refer to (Menendez-Montes et al., 2016) and (Menendez-Montes et al., 2021).

Keywords: Developmental biology; Health Sciences; Model Organisms.

Graphical abstract

Figure 1

Mouse breeding and plug check (A) Schematic of breeding strategy. (B) Representative image showing normal aspect of C57B6/N female genitalia. (C) View of C57B6/N female genitalia with a positive vaginal mating plug (black arrowhead on inset).

Figure 2

Fetal echocardiography by trans-uterine ultrasound (A) Oxygen flow meter showing appropriate opening direction (white arrow) to administer oxygen to the anesthetic circuit up to around 0.4–0.6 mL (white square), the usual oxygen range used in mice. (B) Isoflurane vaporizer in closed position (OFF) and white flow indicating head′s vaporizer opening direction to dispense anesthesia. (C) Pregnant female placed in the warmed platform (white arrowhead), showing mouse head inside the anesthetic nose adapter (black arrowhead) and the paws attached to the electrode pads (ECG pads, white lines). (D) The skin of the caudal part of the abdomen is grasped and lifted to pull it upwards to open the abdominal cavity. A small incision is performed to localize the embryo closer to the ribs on the left lateral side (left uterine horn). (E) Embryo outside the abdomen inside the uterus. (F) Individual embryo inside the uterus but outside the abdomen covered with warmed gel and with the echography transductor on top ready to begin a direct trans-uterine ultrasound.

Figure 3

Fetal echocardiography by trans-abdominal ultrasound (A) Pregnant female placed in the warmed platform with a scheme of the urinary bladder (UB) location at the point of uterine bifurcation, left and right uterine horns (LH, RH) and embryos from E1-E4 in each side. (B) Pregnant female abdomen covered with abundant warmed gel and the transductor positioned to visualize the embryos of the left uterine horn. (C) Representative echocardiography view of an embryo in cranio-caudal position, showing the head (recognized by visualization of the eye) and the heart with both ventricles (white square).

Figure 4

Basic echocardiographic analysis in a long axis view using the 2D mode (A) Heart echocardiography view when the probe is positioned longitudinal to the embryo. (B) Optimized long axis view of the heart visualizing the four chambers and the interventricular septum (IVS) by slight angulation of previous longitudinal position. (C) Color Doppler echocardiography through the atrioventricular valves. Red color indicates flow towards the upper part of the screen and blue towards the lower part. Black arrows indicate the direction of the blood flow (from the atria into the ventricles). (D) Pulse wave Doppler obtained positioning the sample in a semilunar valve and showing a regular rhythm (upper image) and an arrhythmia (lower image). In this image, the heart rate can be obtained positioning the heart rate ultrasound caliper in three consecutives waves (white lines). (E) Color Doppler echocardiography view placing the area of interest in the IVS to confirm its integrity and lack of communication between ventricular chambers in a normal embryo. Black arrows indicate the direction of the blood flow (from the ventricles to the big vessels, (Aorta and Pulmonary artery)). (F) Color Doppler echocardiography view placing the area of interest in the IVS to identify pathological septal defects in an abnormal embryo with ventricular communication (VSD, white arrowhead). A flow (blue area) crossing the IVS can be identified. Red color indicates flow towards the upper part of the screen and blue towards the lower part. A, atria; V, ventricle; IVS, interventricular septum; VSD, ventricular septal defect.

Figure 5

Basic echocardiographic parameters in a short axis view using the M-mode (A) Short axis view of the heart visualized when the probe is positioned in a frontal plane to the embryo, and the M-mode line (yellow line) crossing the middle of the ventricles. (B) Representative M-mode image of a short axis view showing the measures required to perform structural and functional assessment of the heart. V, ventricle; RVPW, right ventricular wall thickness; IVS; interventricular septum and LVPW, left ventricular wall thickness (in diastole); RVIDd, right ventricular internal diameter; LVIDd, left ventricular internal diameter (d, in diastole); RVIDs; right ventricular internal diameter; LVIDs, left ventricular internal diameter (s, in systole). The yellow lines shown in the right follow the endocardium and the epicardium′s motion as a reference to trace the detailed measures.

Figure 6

Midterm embryo collection, dissection and preparation of thoracic region for paraffin inclusion (A) View of the whole uterus containing embryos at E14.5 in cold-PBS over a Petri dish. (B) Excision of the first decidua located in the more distal position relative to the uterus bifurcation on the left horn of the uterus. (C) Isolated decidua at E14.5, containing the embryo and the placenta. (D) View of an E14.5 embryo, still connected to the placenta through the umbilical cord. (E) Fragmented E14.5 embryo to isolate the trunk, allowing a better fixation of the thoracic region (white arrowhead). (F) Trunks containing the heart are incubated in P12 wells filled with ice-cold- 4% PFA for fixation.

Figure 7

Histological analysis of midterm hearts: ventricular dimensions and correlation with echocardiography findings (A) Representative H&E staining of a 4-chamber cardiac section of a E14.5 embryo to perform morphological measurements. Scale bar represents 300 μm (100 μm on insets). (B) Section shown in A with the area to determine compact myocardium thickness highlighted in yellow. Inset shows a detail of the three length measures. (C) Section shown in A with measure lines to calculate IVS thickness length at the middle point of the septum. (D) Section shown in A with measure lines of minor and major chamber axis, from the epicardium to the septum endocardium at the midplane of the chamber and from the valvular region to the apex on each ventricle, respectively. Inset indicates a detail on how measurements should be taken up to the septum and not to the trabeculations. (E) Representative H&E staining of control (left), HIF1 KO (middle) and VHL KO (right) E14.5 cardiac sections in a 4-chamber view. Scale bar represents 200 μm. Note that 2D echocardiography (middle) and Doppler-color images (bottom) are comparable with the phenotype observed by classical histology analysis (top panels), revealing a VSD in the VHL KO (arrowhead in right top and bottom panels). (F and G) Quantification of right and left ventricular compact myocardium and IVS thickness (F) and relative right and left ventricle sphericity (G) of a representative control (WT), HIF1a KO and VHL KO E14.5 embryos. RV: right ventricle, LV: left ventricle, IVS: interventricular septum.