Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

The overall goal of this echocardiographic examination combined with histological investigations is to examine cardiac function and morphology in different transgenic mouse models under baseline and pathophysiological conditions. To begin, while lightly holding a mouse by it's tail, use a standard laboratory balance to determine the body weight of the animal. After anesthetizing the mouse according to the text protocol lightly squeeze a rear foot and observe if the leg retracts.

Use a commercial rodent shaver to shave the left side of the thorax and the left armpit. Position the animal on a warm pad set at 40 to 42 degrees Celsius in a shallow left sided position, with the head at 12 o'clock and the tail at 6 o'clock. Then use tape to fix the left arm, left leg and tail.

Apply pre-warmed echocardiography gel onto the shaved chest and the head of the transducer. Next place the echocardiograph transducer parasternal left, directing it to the right side of the neck to obtain a two-dimensional parasternal long access view on the level of the papillary muscle. Then turn the transducer 90 degrees clockwise to obtain a short access view.

Establish a minimal depth setting and a zoom to maximize image quality and frame rate. Set the sweep speed to the maximum. Record 2-D guided M-mode images in short access view.

Record at least three series of three heartbeat semiloops for each animal. After obtaining successful recordings, remove the tape from the limbs and tail. Wipe off the echocardiogrpahy gel from the mouse thorax heating pad and transducer.

Leave the mouse under observation on the heating pad, covered with tissue to avoid unnecessary light exposure and heat loss until it wakes up. Then put the animal back in the cage. Analyze recorded M-mode images from short access view to determine left ventricular or LV dimensions and function.

Using the identification of the tissue blood interface on the stored images, measure the thickness of the LV anterior wall in systole and diastole, the LV internal end-systolic and end-diastolic diameters, and the LV posterior wall thickness in systole and diastole. Measure the diastolic dimensions at the time of the apparent maximal LV diastolic dimensions and the LV end-systolic dimensions at the end of the most anterior systolic excursion of the LV posterior wall. Tap the touchscreen on the analyze icon and then on the LVAWd icon.

Position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in diastole. Next position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain LV diastolic anterior wall thickness. Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal and diastolic diameter.

Position the caliper on the interface between the LV posterior wall and the pericardium to obtain the LV diastolic posterior wall thickness. For LV systolic dimensions, tap the touchscreen on the LVAWs icon and position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in systole. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV systolic anterior wall thickness.

Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal and systolic diameter. Position the caliper on the interface between the LV posterior wall and the pericardium to obtain the LV systolic posterior wall thickness. After sacrificing the animals according to the text protocol measure their body weights.

Then use a 70%alcohol swab to disinfect the chest and abdomen. Make a transverse incision in the skin, one centimeter distal to the sternum. Then using blunt forceps, remove the skin from the thorax moving in the direction of the head.

With fine forceps, hold the sternum lightly and open the diaphragm by inserting the blunt end of fine scissors. Cut the ribcage on both sides parallel to the sternum. Then move the sternum in the direction of the head.

Locate the heart in the thorax. With fine forceps, hold the vascular trunk of the heart and use fine scissors to cut below the organ. Then measure the heart weight and establish a heart-to-body weight ratio.

Transfer the heart into two milliliters of 10%neutral buffered Formalin solution in a 15 milliliter tube and fix the organ at four degrees Celsius overnight. To perform Wheat Germ Agglutinin or WGA staining, place the previously prepared slides in Tetramethylrhodamine conjugated WGA and incubate the samples at room temperature in a humid chamber for sixty minutes. Use PBS to wash the samples three times for five minutes each.

Then use fluorescence mounting medium contained DAPI to mount the sections. Store them at four degrees Celsius in the dark before analysis. These representative echocardiographic recordings demonstrate the usefulness of echocardiography to identify cardiac phenotypes in genetically modified mice.

The difference between a mouse with normal cardiac function and an animal with a dilated left ventricle or LV and reduced LV function can easily be identified. Shown here is a comparison of cardiomyocyte diameter measurements in animals without and with cardiac hypertrophy using H&E stained paraffin heart sections and measurements of longitudinal sections of heart tissue. This figure illustrates the measurement of cardiomyocyte transverse diameters using WGA stained paraffin sections from hearts of control mice and animals with cardiac hypertrophy.

These panels depict the utility of PECAM1 Immunohistochemistry of cardiac sections to determine the vessel density of normal heart tissue and hearts with increased angiogenesis.