This protocol outlines cutting edge methods for generating high resolution imaging data for diagnosing congenital heart defects. We conduct functional assessments with non-invasive fetal echocardiography combined with necropsy followed by episcopal confocal microscopy histopathology to generate serial 2D image stacks and 3D reconstructions for comprehensive CHD diagnosis. This method can elucidate the detailed anatomical changes not only in congenital heart diseases, but also can be used to investigate structural birth defects in other organs including cranial facial defects, limb and skeletal anomalies, as well as defects of the brain and other visceral organs.
Demonstrating these procedures will be Meghan Holbrook, Carla Guzman, Peizhao Zhang, and Benjamin Glennon, all researchers from our laboratory. After preparing the mouse for the procedure, warm up the ultrasound gel to a body temperature and apply it generously over the imaging area. Then place the transducer on the abdomen oriented in a horizontal plane and identify the bladder on the screen.
Once the bladder is identified, scan cranially from the bladder, look for the fetus, and determine the gestational age by measuring the crown to rump length. Next, use the color doppler to analyze blood flow from the heart. To allow fixative penetration into the internal organs, make incisions in the lateral thorax and abdomen using forceps or dissecting scissors and allow the sample to fix for at least 24 hours in a cold room.
Then set up the software to save the images with a name, including the sample's identification, the microscope magnification, and the content of the picture. Pin the sample through its wrists and ankles. Before cutting the skin along the median axis towards the tail, lift the skin in the middle of the neck using forceps and cut the skin toward both the armpits with the scissors.
Then cut the skin from the umbilicus to the legs. After embedding the samples, adjust the laser projection position to the center of the specimen on the screen and adjust the zoom knob to 20x. To optimize the resolution, set the gain to 1, 250V.
Maximize the blue area using the focus knob, and then reset the gain to approximately 750V for imaging. Next, switch the cutting method to auto. Set the thickness to around 50 micrometers and run the slides.
Stop cutting when the lungs and airway are in view. Choose a cutting thickness between eight to 10 micrometers and stop the live view open. Open Microtome Communicator to start imaging.
Ensure the temporary storage folder is empty before collecting images. Stop the image collection when no hard structure is visible. Output the temporary stored files into one TIFF image series.
For 3D image reconstruction, drag and drop the image files into the image processing software. Once the pop-up window appears, select the links or the files to copy into the database. Once the file is opened, click on the 2D 3D reconstruction tools menu from the toolbar and select 3D MPR.
For pixel X resolution, and pixel Y resolution, input the image resolution by giving the zoom used during ECM imaging. For the slice interval, input the slice thickness used to cut during ECM imaging. Next, use the WWWL tool to adjust the window width and window level.
Click and drag the tool on the image upward to decrease image brightness and downward to increase it. Click and drag the tool on the image rightward to decrease image contrast and left word to increase it. Drag the images into the desired positions using the pan tool.
Use the zoom tool to enlarge or shrink the image and the rotate tool to rotate the image as desired. Now, click and drag the colored axis of the first panel. Notice how rotating this axis changes the orientation of the other two panels.
Rotate the three panels axes until the three panels represent coronal, sagittal, and transverse views of the sample. Once all three panels are appropriately positioned, oriented, and brightened, click on the panel representing the coronal view. Then click on Movie Export on the right side of the menu bar.
Click on batch and drag the from and to sliders to encompass the entire region of interest. For interval, select the same as thickness option and save the video indicating the views orientation. The echo shows an intact ventricular septum without an intraventricular shunt and normal related great arteries in the normal control, which was confirmed by ECM.
The frontal view demonstrates communication between LA, RA, LV, and RV in both echo and ECM, suggesting atrioventricular septal defect. The color flow demonstrates the flow across LV and RV indicating ventricular septal defect. Echo and ECM demonstrated a double outlet of right ventricle with a ventricular septal defect between LV and RV with side by side great arteries.
Ultrasound diagnosis of persistent truncus arteriosus demonstrates communication between LV and RV with a single outflow tract overriding both ventricles, also confirmed by ECM at the E14.5 stage. The sections collected from ECM histology can be further processed for immunostaining, hematoxylin and eosin staining and even nucleic acid extraction for genotyping and transcriptional profiling or protein extraction for proteomic analysis. The images from three-dimensional reconstruction can be further processed for assessments of quantitative perimeters such as area or volume.
Accurate phenotyping to identify defects in the cardiovascular anatomy is essential for establishing genetically engineered and mutant mouse models of congenital heart disease to elucidate the pathomechanisms of congenital heart disease. This can lead to potential opportunities that develop therapies and intervention to improve the clinical outcome in patients with congenital heart disease.
