Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Summary

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.

Abstract

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Introduction

A large variety of genetically modified mouse models are available, and the number of pharmacological studies in mice is high^1,2. Echocardiography and MRI are commonly used approaches for the phenotypic characterization of cardiac function and morphology in these mouse models³. The aim of the presented protocol is to analyze cardiac function and morphology in adult mice. It combines echocardiographic, histological, and immunohistochemical measurements. Echocardiographic examination is widely used in mice^{4,5,6,7,8,9,10,11,12}. Pachon et al.¹¹ identified 205 studies published in Circulation, Circulation Research, American Journal of Physiology - Heart and Circulatory Physiology, and Cardiovascular Research between 2012 and 2015 that used echocardiographic examination in animals.

Echocardiography is used to identify cardiac phenotypes in genetically modified mice^{5,6,13,14,15,16,17,18,19,20,21,22}, as well as to analyze cardiac function in chronic overload-induced hypertrophy, myocardial ischemia, and cardiomyopathy models in mice (reviewed in¹²). Improved echocardiography equipment allows for the the standard measure of left-ventricular (LV) systolic and diastolic dimensions, tissue Doppler imaging, myocardial contrast echography, and the assessment of LV regional function and coronary reserve¹². Ideally, echocardiographic examination should be performed in conscious mice to avoid the negative effects of anesthesia on contractile function, autonomic reflex control, and heart rate¹¹. Nevertheless, this approach is limited by the requirement to train the animals; difficulties in keeping the body temperature stable; movement artifacts; stress; very high cardiac frequencies; and the requirement for at least two investigators to perform the experiment, especially if a large number of animals are under investigation. Interestingly, a recent study reported no differences in echocardiographic parameters in trained and untrained animals¹⁹. We perform echocardiographic measurements in anesthetized mice. Different anesthesia protocols will be discussed below.

Although standard resolution echocardiography (>10 MHz) is sufficient to measure LV systolic and diastolic dimensions and cardiac function in adult mice, the method is limited in its description of underlying structural phenomena. Thus, we combine the in vivo measurements with histological and immunohistological analyses to measure, for example, cardiomyocyte diameter and vessel density. Other histological and immunohistological investigations, such as the determination of proliferation, examination of apoptosis, infarct size measurements, determination of fibrosis, and specific marker expression, can also be performed on the same type of processed tissue but are not the subject of this protocol. The combination of in vivo echocardiographic examination with histological analyses provides additional insights into underlying structural alterations. In an additional step, we can complete these measurements with molecular and ultra-structural investigations. Histological analyses not only complete the echocardiographic examination but also become indispensible when the resolution of echocardiography is not sufficient. This is especially the case in models of genetically modified mice that are embryonic lethal^23,24.

Protocol

The experiments described here were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines, and policies. They have been approved by the French ethics committee (Comité Institutionnel d'Ethique Pour l'Animal de Laboratoire; number NCE/2012-106).

1. Echocardiography

1. Determine the body weight of the mouse using a standard laboratory balance while holding it lightly by the tail to ensure proper positioning.
2. Anesthetize the animal by the intraperitoneal (i.p.) injection of 50 mg/kg pentobarbital^25,26.
   NOTE: Any other kind of anesthesia can be used if the same protocol is used throughout the study. Advantages and disadvantages will be discussed below.
3. Put the mouse back in its own cage and wait until it is unresponsive, it shows steady breathing, and rear foot reflexes are absent. To test this, squeeze a foot slightly and observe whether the leg still retracts.
4. Shave the left side of the thorax and the left armpit using a commercial rodent shaver.
   NOTE: The use of a dedicated rodent shaver allows for the complete removal of fine mouse hair to avoid interference in the echocardiographic measurement. Commercial hair removal creams or solutions should be avoided, as they are usually perfumed, which will disturb the animal after it awakens. Avoid excessive shaving, as it increases heat loss.
5. Put the sleeping animal on a warm pad set to 40 - 42 °C in a shallow left-sided position, with the head at 12 o'clock and the tail at 6 o'clock. Fix the left arm, left leg, and tail with tape.
6. Apply pre-warmed echocardiography gel onto the shaved chest and the head of the transducer.
7. Place the transducer parasternal-left, directing it to the right side of the neck to obtain a two-dimensional (2D) parasternal long-axis view on the level of the papillary muscle. Turn the transducer 90° clockwise to obtain a short-axis view at papilary muscle level. Use a minimal depth setting and a zoom to maximize image quality and frame rate. Set the sweep speed to the maximum.
   1. To obtain these settings, different zoom and depth setting options may be used, depending on the machine and software. Record 2D-guided M-mode images in short-axis view²⁷. Refer to Figure 1A and B²⁷.
      NOTE: Take care to avoid applying excessive pressure to the chest, as this may cause bradycardia.
8. Record at least 3 series of 3 heart beat cine loops for each animal.
   NOTE: For the echocardiograph software used in this study, press the "Acquire/Save" button only once. This methodology is specific to the echocardiograph with this particular software. Other software packages may be used with different machines.
9. After successful recordings, wipe off the echocardiography gel from the mouse thorax, heating pad, and transducer. Remove the tape from the limbs and tail.
10. Leave the mouse under observation on the heating pad, covered with tissue to avoid unnecessary light exposure and heat loss, until it wakes up.
11. Put the animal back in its cage.
12. Analyze recorded M-mode images from parasternal short-axis view to determine left ventricular (LV) dimensions and function. Measure the thickness of the LV anterior wall in systole and diastole (LVAWs and LVAWd), the LV internal end-systolic and end-diastolic diameters (LVIDs and LVIDd), and the LV posterior wall thickness (LVPW) in systole and diastole (LVPWs and LVPWd) using the identification of the tissue-blood interface on the stored images.
13. Measure the diastolic dimensions at the time of the apparent maximal LV diastolic dimensions and LV end-systolic dimensions at the time 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.
14. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV diastolic anterior wall thickness; the software will directly switch to the LV internal end-diastolic measurement.
15. Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal end-diastolic diameter; the software will switch to the LV posterior wall thickness measurement.
16. 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 touch screen on the "LVAWs" icon and position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in systole.
17. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV systolic anterior wall thickness. Repeat the process as described above for the LV internal end-systolic diameter and the LV systolic posterior wall thickness. Use the leading-edge convention adopted by the American Society of Echocardiography to trace the endocardial and epicardial borders^13,27.
    NOTE: LV contractile function parameters will be automatically calculated using the previous measurements. The LV fractional shortening (FS) is defined as FS (%) = [(LVIDd - LVIDs)/LVIDd] x 100. The LV ejection fraction (EF) is calculated with the modified Teicholz formula, where EF (%) = [(LVIDd³ - LVIDs³)/LVIDd³] x 100¹². Refer to Figure 1A and B.
18. Store the data on compact discs or USB memory sticks and make backup copies.
19. Import, analyze, and export the data using the appropriate software.
    NOTE: After these baseline measurements, the experiment can be paused. If performing a direct comparison between knockout animals and wild-type littermates, proceed with the histological analyses⁶. For Cre-ERT2; lox/lox mouse lines, continue the following day with tamoxifen induction by i.p. injection, as described^5,24. If inducing experimental myocardial infarction by the ligation of the left coronary artery^5,28, the surgery could be performed directly after the echocardiographic measurements, when the mice are under anesthesia. Otherwise, a minimum delay of one week between two rounds of anesthesia should be maintained to limit the rate of post-operative lethality.

2. Preparation of Heart Samples for Histological Evaluation

1. Sacrifice the animals by cervical dislocation. Measure their body weights. Disinfect the chest and abdomen using a 70% alcohol swab.
2. Make a transverse incision in the skin 1 cm distal to the sternum. Using blunt forceps, remove the skin from the thorax, moving in the direction of the head. Hold the sternum lightly with fine forceps and open the diaphragm by inserting the blunt end of fine scissors.
3. Cut the rib cage on both sides parallel to the sternum. Move the sternum in the direction of the head. Locate the heart in the thorax. Hold the vascular trunk of the heart with the fine forceps and cut below using fine scissors.
4. Open the chest and excise the entire heart out of the thorax, measure the heart weight, and establish a heart-to-body weight ratio^{4,5,6,23,29,30}.
5. Fix the heart in 2 mL of 10% neutral buffered formalin solution in 15-mL tubes overnight at 4 °C.
   CAUTION: Danger! Work with formalin solutions must be done in a chemical fume hood; wear gloves and safety glasses.
   NOTE: As the buffer composition for formalin solutions varies with different suppliers, use the same supplier throughout the study.
6. The next day, cut the hearts in the transverse plane, in the middle, and transfer them into cassettes for paraffin embedding, which is performed in the pathology laboratory using an automated embedding apparatus.
7. Perform sectioning.
   1. Section paraffin blocks at a thickness of 3 µm using a microtome and float them in a 40 °C water bath containing distilled water.
   2. Transfer the sections onto slides. Allow the slides to dry overnight in a 37 °C incubator and store them at 4 °C until ready for use.
      NOTE: The protocol can be paused here until the user is ready for staining (step 3).
8. Deparaffinize and rehydrate the tissue slides.
   1. Place the slides in staining jars with glass inserts in a 55 °C oven for 10 min to melt the paraffin.
   2. Deparaffinize the slides in two changes of 200 mL of xylene or xylene substitute for 5 min each.
      CAUTION: Highly flammable and toxic! Work in a chemical fume hood; wear gloves and safety glasses.
   3. Transfer the slides to 200 mL of 100% alcohol. Make two changes for 3 min each and transfer once through 200 mL of 95% alcohol for 3 min.
      CAUTION: Highly flammable! Keep away from sources of ignition; no smoking.
   4. Rinse twice in 200 mL of phosphate-buffered saline solution (PBS) for 5 min each and continue with step 3, 4, or 5.

3. Hematoxylin and Eosin Staining

1. Rinse the slides with their sections in distilled water.
2. Stain the nuclei with hematoxylin solution for 8 min.
3. Rinse in running tap water for 10 min.
4. Stain with eosin solution for 2 min.
5. Dehydrate three times for 2 min in 100% ethanol (EtOH). Clear three times for 2 min in xylene or xylene substitute. Mount in a xylene-based mounting medium.
6. Photograph the slides and measure the cardiomyocyte diameter at the level of the nucleus in longitudinal sections of the interventricular septum. Measure at least 100 cells per section and three sections per heart.

4. WGA Staining

1. Incubate the slides obtained from step 2.7 with tetramethylrhodamine (or other fluorescent dyes)-conjugated WGA (1:100 in PBS) for 60 min at room temperature in a humid chamber.
2. Wash three times with PBS for 5 min each.
3. Mount with fluorescence mounting media containing DAPI. Store at 4 °C in the dark before analysis.
   CAUTION: Wear eye protection and compatible chemical-resistant gloves.
4. Photograph the slides.
   1. Use a microscope equipped with fluorescence epi-illumination and filter sets for DAPI and tetramethylrhodamine (refer to the Table of Materials). Set the aperture to the maximum and the brightness to auto-exposure.
   2. Acquire separate images at 400x magnification for the blue and the red channels. Open the images in ImageJ. Adjust the brightness and contrast if necessary (Image > Adjust > Brightness/contrast).
   3. Set each image to 8-bit (Image > Type > 8-bit). Overlay the DAPI and WGA images. Use the blue channel for DAPI and the red channel for WGA; set the green and gray channels to none (Image > Color > Merge channels)³¹.
5. Determine the cardiomyocyte diameters at the level of the nucleus in transverse sections of the interventricular septum.
   1. Define the scale of the images in ImageJ. For this purpose, photograph an object of known size (e.g., a hemocytometer chamber at the same magnification as the heart sections; step 4.4).
   2. Use the straight-line tool to draw a line from beginning to end of the known structure (Analyze > Set scale).
      NOTE: The distance in pixels will be displayed automatically; the known distance and unit of length will have to be entered. Proceed with cardiomyocyte measurements at the level of the nucleus.
   3. Draw a straight line from the WGA-positive membrane through the DAPI-positive nucleus to the opposite site of the WGA-positive cell membrane (Analyze > Measure). In the results window, make sure that the length values have a meaningful number of decimal places (Analyze > Set measurements > Decimal places).
   4. Measure at least 100 cells per section and three sections per heart. Export the results to Excel (Click on Results > File > Save as > .csv)^6,31.

5. Pecam-1 Immunostaining

1. Perform antigen unmasking.
   1. Add 1,600 mL of sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) to a pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point; simply rest it on top.
      CAUTION: Hot!
   2. Once the sodium citrate buffer is boiling, transfer the slides from step 2.5 to the pressure cooker. Secure the pressure cooker lid. As soon as the cooker has reached full pressure, wait for 7 min. When 7 min have elapsed, turn off the hotplate and place the pressure cooker in an empty sink. Activate the pressure release valve and run cold water over the cooker. Once it has de-pressurized, open the lid and run cold water into the cooker for 5 min. Place the slides in 200 mL of PBS.
      NOTE: Alternatively, microwave antigen unmasking could be used, although the risk of overheating is increased.
2. Use 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity for 5 min. Rinse the slides for three times for 2 min each in 200 mL of PBS.
   CAUTION: Flammable and toxic!
3. Incubate the slides for 15 min in diluted normal blocking serum (5% normal goat serum in PBS) that also contains an avidin block (4 drops in 1 mL).
4. Carefully tap the liquid from the sections and incubate them with Pecam-1 antibody from rabbit, diluted 1:50 in PBS containing 2.5% normal goat serum and 4 drops of biotin block per mL. Incubate the slides overnight in a humid chamber at 4 °C.
5. Wash the slides three times for 5 min each in 200 mL of PBS. Incubate the sections with biotinylated goat anti-rabbit IgG antibody diluted 1:200 in PBS containing 2.5% normal goat serum for 1 h at room temperature. Wash the slides three times for 5 min each in 200 mL of PBS.
6. Incubate the sections with an avidin/biotin-based peroxidase system for 20 min (reagent A and reagent B need to be combined 30 mins prior to use). Wash the slides three times for 5 min each in 200 mL of PBS.
7. Dissolve 1 3,3'-diaminobenzidine (DAB) and 1 urea hydrogen peroxide tablet in 5 mL of double-distilled water. Incubate the sections with DAB solution for approximately 3 min, carefully monitoring color development. Stop the color reaction by gently washing the slides in 200 mL of PBS.
   CAUTION: Carcinogenic; wear chemical-resistant gloves!
8. Counterstain the nuclei for 6 min with hematoxylin. Rinse in running tap water for 2 min. Dehydrate three times for 2 min each in 200 mL of 100% alcohol. Clear three times for 2 min each in 200 mL of xylene or a xylene substitute. Mount in a xylene-based mounting medium.
9. Photograph the slides (at least ten fields at 40x magnification from the interventricular septum of each heart) and measure the Pecam-1 area density using the freely available ImageJ software³¹. Use the color deconvolution³² plugin for DAB and hematoxylin and adjust the image contrast to the same level.
   NOTE: In case the brown and purple/blue color have significant spectral overlap, which might cause difficulties with color deconvolution, one could try different brands of hematoxylin solution to obtain clear, light-blue nuclear staining. Alternatively, immunofluorescence or manual counting of the capillaries could be performed.

Results

In Figure 1, 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 (Figure 1A) and an animal with a dilated left ventricle and reduced LV function (Figure 1B) can easily be identified. Figure 2 shows the comparison ...

Discussion

Different methods have been developed to evaluate cardiac structure and function in mice, including echocardiography, contrast-enhanced MRI, micro CT, and PET scan. Due to its cost-effectiveness and simplicity, echocardiography is the most widely used technique for functional analysis in mice¹¹. In general, because of the small size of the heart and the high frequency of the heart rate in mice, transducers with a frequency >10 MHz should be used, although successful measurements have been repo...

Materials

Name	Company	Catalog Number	Comments
Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine	Life Technologies, Molecular Probes	W849
Biotinylated Goat Anti-Rabbit IgG Antibody	Vectorlabs	BA-1000
Avidin/Biotin Blocking Kit	Vectorlabs	SP-2001
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)	Vectorlabs	PK-6100
VECTASHIELD Antifade Mounting Medium with DAPI	Vectorlabs	H-1200
SIGMAFAST 3,3'-Diaminobenzidine tablets	Sigma	D4168
Hydrogen peroxide solution	Sigma	H1009
Anti-Pecam-1 (CD31) antibody	Abcam	ab28364
Ultrasound transmission gel, Gel Aquasonic 100	Parker
Linear ultrasound probe, L15-7io	Philips Healthcare
Echocardiograph, IE33 xMATRIX	Philips Healthcare
Microscope, Leica DMi8	Leica
Fluorescence Filterset DAPI	Leica	11525304
Filterset TxR	Leica	11525310
Digital Camera, SPOT RT3 Color Slider	Spot Imaging
Imaging Software, SPOT 5.2 Advanced and Basic Software	Spot Imaging
Imaging Computer	Dell
Fine Scissors	Fine Science Tools	14028-10
Large Scissors	Fine Science Tools	14501-14
Scalpel blades	Fine Science Tools	10023-00
Graefe Forceps	Fine Science Tools	11650-10
Rodent shaver	Harvard Apparatus	34-0243
cassettes for paraffin embedding	Sakura	4155F
neutral buffered Formalin	Sakura	8727
Xylene	Sakura	8733
Paraffine TEK III	Sakura	4511
automated embedding apparatus, Tissue-Tek VIP	Sakura	6032
paraffin-embedding station Tissue-Tek TEC 5	Sakura	5229
microtome blades,Accu-Edge S35	Sakura	4685
microscopy slides, Tissue-Tek	Sakura	9533
cover slips, Tissue-Tek	Sakura	9582
Mounting medium Tissue-Tek	Sakura	1408
slide boxes	Sakura	3958
eosine solution	Sakura	8703
hematoxyline solution	Sakura	8711
microtome, RM2125RT	Leica	720-1880 (VWR)
water bath, Leica HI1210	Leica	720-0113(VWR)
Ethanol	VWR	ACRO444220050
15 ml tubes	VWR	734-0451
staining glass dish	VWR	MARI4220004
staining jars	VWR	MARI4200005
Incubator	Binder	9010-0012
DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3′-Diaminobenzidine tablets	Sigma	D4293
PBS (10X)	Thermo Fisher Scientific	70011044