We thank Manon Laprise for training in echocardiography and Ann Chamberland for taking the photos shown in Figures 1 and 2. This work was supported by Canadian Institutes of Health Research grant MOP 44363 and Canada Chair 950-216684.

All procedures shown in this protocol have been approved by the IRCM Animal Care Committee.
1. Ultrasound System and Station Preparation
<ol>
	<li>Start the ultrasound imaging system and connect the scanhead as well as the physiology controller unit according to the manufacturer&#39;s instruction (Figure 1). Choose Cardiac Measurement Program and the scanhead that corresponds to the 30 MHz transducer.</li>
	<li>Fill the nosepiece of the scanhead with deionized water. Avoid air bubbles as they interfere with imaging resolution. Place the scanhead with the handle side up on its holder near the imaging platform (Figure 1A).</li>
	<li>Disinfect the imaging platform and working area.</li>
	<li>Fill completely the bottle of ultrasound gel to avoid the formation of bubbles and place it upside down in its prewarming container set to 37 &#176;C (Figure 1B).</li>
	<li>Verify the levels of oxygen and isoflurane and the tubing system for anesthesia. One experiment with ~8 embryos would use approximately 15-20 L oxygen.</li>
	<li>Place the bottles of ophthalmic balm, depilatory cream and electrode gel near the imaging platform (Figure 1B).</li>
	<li>Verify that the infrared heat lamp functions. During training, set the heat level, the position of the lamp and the distance of the lamp to mice in order to maintain constant body temperature and heart rate. 
	Note: Proper preparation of the system and material is critical. Lengthy procedures may affect the physiologic and hemodynamic characteristics of fetuses as long anesthesia depresses cardiac function by lowering heart rate, blood pressure and blood-oxygenation level. The processing time for ~8 fetuses (average litter size in C57BL/6) should be approximately 1 hr. Other mouse strains may provide a higher number of fetuses, possibly with a larger female size. Training is essential to optimize the processing time.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Anesthetize the pregnant female in a chamber with continuous supply of 2% isoflurane in oxygen (200 ml/min) until it becomes nonresponsive.</li>
	<li>Place the mouse on the imaging platform in a supine position, adjust the tubing for continuous inhalation of 1.5% isoflurane in oxygen (200 ml/min), and fix the inhalation tube with tape (Figure 2A). Sufficient anesthesia should be confirmed during the procedure by the relaxed posture of the mouse and the absence of any response to tail and toe pinches.</li>
	<li>Place electrode gel only on the left top and right bottom electrode pads (right fore leg and left hind leg, also called lead II position) for electrocardiography. Lead II position provides better defined upright positive P-wave and QRS complex to determine heart rate. Secure the four paws to each pad using tape (Figure 2A). To prevent dryness of the eyes, apply 1 drop of ophthalmic balm into each eye.</li>
	<li>Shave the abdomen from chest to bladder with a hair clipper. Then apply depilatory cream for 2 min and wipe it off with gauze and/or cotton swab to carefully remove any remaining hair. Be careful not to cut the nipples.</li>
	<li>Insert the thermometer prelubricated with electrode gel into the female&#39;s rectum to monitor the body temperature (should be maintained at 37&#177;0.5 &#176;C by adjusting an infrared heat lamp placed above the platform). Heart rate should be 450&#177;50 beats/min (bpm). Both temperature and heart rate are displayed on the physiology controller unit (Figure 1B). 
	Note: Long anesthetization, hair loss and ultrasonic gel (although prewarmed) can lead to hypothermia, which can affect the female&#39;s heart rate. If the body temperature drops below 36.5 &#176;C, stop the procedure and modify the position of the heating lamp to adjust the temperature and the heart rate. Wait a couple of minutes before proceeding again.</li>
</ol>
3. Embryo Identification
<ol>
	<li>Gently press down on the naked abdomen to locate the embryos. Slowly and lightly spread them out to have most of embryos in a single layer under the abdominal surface. In each uterine horn, sacs are linearly connected to each other. Try to respect this order when spreading.</li>
	<li>Mark each embryo on the naked abdomen of the female with a permanent marker with their anterior/posterior and dorsal/ventral directions. Knowing the orientation will facilitate locating the heart with the probe. Number the embryos in the right and left horns starting from the cervix (1, 2, 3&#8230; and 1&#39;, 2&#39;, 3&#39;&#8230; respectively; Figure 2B). 
	Note: (i) Avoid spreading the embryos with excessive force. Sketch their locations on a piece of paper to track them (Figure 2C). (ii) C57BL/6 females have ~8 fetuses per litter. However, 0-2 embryos in each litter are located beneath the others, making their imaging impossible. Exclude these embryos from the analysis and assess more litters if needed.</li>
</ol>
4. Heart Rate Measurement
<ol>
	<li>Place a small amount of prewarmed ultrasound gel on the naked abdomen and spread it evenly. Avoid bubble formation. Add a larger amount of gel (~ 5 ml) on the specific area to image.</li>
	<li>Hold the probe in contact with the thick gel layer (10 mm) and gradually move the probe toward the skin while looking for the beating heart. Once the beating heart is visualized on the screen, adjust the angle of the probe to have both ventricles in their largest size in the imaging plane (Figure 2D).</li>
	<li>Begin acquiring image. With the forearm resting on the station, place the transducer on the ultrasound gel to obtain a live image on the viewing screen (Figure 2D). Maintaining the ridge of the transducer on the top and clicking the scanhead orientation marker (in the left top corner of the image) allow the coordination of the hand movement and area visualized on the screen.</li>
	<li>Starting from the cervix, move to the closest embryo marked (1 or 1&#39; in right or left uterine horn, respectively) to visualize the beating heart on the screen. Because focal depth is fixed, gently move the probe up/down and sideways without losing contact with gel to obtain the desired image plane.</li>
	<li>Position the beating heart in the center of the screen within the region indicated by the yellow dashed line representing the focal zone for better data acquisition.</li>
	<li>Acquire the live recording. Obtain the scout image and resume recording (Figure 3A). Once a minimum of 10 sec of stable recording is obtained, stop recording and save.</li>
	<li>Proceed to the next embryo.</li>
	<li>Once all the embryos are analyzed, press &#34;Browse&#34; on the keyboard to view the list of the recordings. Play each recorded M-mode tracing and perform multiple measurements (at least 5 per tracing) of the spacing between adjacent peaks (time/flow cycle) to obtain the average heart rate (Figure 3B). 
	Note: Some protocols suggest using a stationary stand for the scanhead to avoid shaking. However, it restrains the angle of analysis, making the observation of lateral embryos difficult. It also slows down the analysis, as the stand has to be adjusted for each embryo. Holding the scanhead while settling the forearm on the station efficiently minimizes shaking. Training with 5-10 pregnant females is recommended to optimize the outcome.</li>
</ol>
5. Genotyping
<ol>
	<li>Using clean surgical scissors and forceps, incise longitudinally the skin and muscle layer of the abdomen. Locate the sacs in each uterus horn and match the numbers. Cut open the yolk sac to expose the embryo in the order used above. If not in a linear arrangement, the sketch of the embryo positions on a piece of paper will be essential once the marks on the skin are no longer visible.</li>
	<li>Cut only ~4 mm of tail for genotyping as genomic DNA is very efficiently extracted from embryonic tails. 
	Note: (i) The surgical procedure may have to be performed in a separate location from the imaging room. However, it is important not to move the mouse and have the surgical procedure on the imaging platform in order to keep track of the numbered embryos. Consult the animal facility manager to optimize the surgical procedure. (ii) The pregnant female rapidly dies due to blood loss as the fetuses are removed for genotyping after imaging. After tail cut, the fetuses are placed 3 min on ice for anesthesia by hypothermia and then decapitated with scissors, as recommended by our Animal Care Committee.</li>
</ol>

<ol>
	<li>Hoffman, J. I., Kaplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+congenital+heart+disease.">The incidence of congenital heart disease.</a> J. Am. Coll. Cardiol. 39, 1890-1900 (2002).</li><li>Miller, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+brain+development+in+newborns+with+congenital+heart+disease.">Abnormal brain development in newborns with congenital heart disease.</a> N. Engl. J. Med. 357, 1928-1938 (2007).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Wessels, A., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+anatomy+of+the+heart:+a+tale+of+mice+and.">Developmental anatomy of the heart: a tale of mice and.</a> 15, 165-176 (2003).</li><li>Yu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ENU+induced+mutations+causing+congenital+cardiovascular+anomalies.">ENU induced mutations causing congenital cardiovascular anomalies.</a> Development. 131, 6211-6223 (2004).</li><li>Seidah, N. G., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+therapeutic+targeting+of+the+proprotein+convertases.">The biology and therapeutic targeting of the proprotein convertases.</a> Nat. Rev. Drug Discov. 11, 367-383 (2012).</li><li>Roebroek, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Roebroek, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+redundancy+of+the+proprotein+convertase+furin+in+mouse+liver.">Limited redundancy of the proprotein convertase furin in mouse liver.</a> J. Biol. Chem. 279, 53442-53450 (2004).</li><li>Kisanuki, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2-Cre+transgenic+mice:+a+new+model+for+endothelial+cell-lineage+analysis+in+vivo.">Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.</a> Dev. Biol. 230, 230-242 (2001).</li><li>Kim, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+endothelial+furin+leads+to+cardiac+malformation+and+early+postnatal+death.">Loss of endothelial furin leads to cardiac malformation and early postnatal death.</a> Mol. Cell Biol. 32, 3382-3391 (2012).</li></ol>

No conflict of interest declared.

M-mode echocardiography is an effective and simple method to measure in utero heart rates of mouse embryos. Commercially available transducers provide sufficient resolution to visualize small beating hearts. Thus, they allow a highly accurate heart rate measurement, as compared to other methods such as pulse measurement, and can substitute high resolution video microscopy. However, current tools do not allow the simultaneous analysis of all embryos, implying a tedious procedure to visualize each of the embryos. ...

CHD is the most common noninfectious cause of death at birth1. Multiple surgeries are often needed to correct the structural defects in subjects whose quality of life may remain compromised1. Children with CHD frequently develop neurological disorders even if they have not undergone surgery, indicating important in utero consequences on development2,3. Both genetic and environmental factors, such as exposure to viruses or chemicals (alcohol) during pregnancy, cause CHD. Studying the genetic contributors is still at its early phase, but growing rapidly. To identify these contributors and understand their role in heart development, phenotyping mutant mice with a simple and powerful tool will be highly beneficial.
Mouse is indeed an animal model of choice to study CHD, and most of the human cases can be reproduced in mice4,5. Consequently, fetal mouse cardiac phenotyping has become increasingly important to investigate the etiology of human CHD and requires adequate tools. Although histological studies on fixed specimens are invaluable, real-time imaging of live animals is crucial to understand the physiology of the heart. Video microscopy offers live imaging. However, it requires laparotomy to expose embryos, thereby compromising their physiologic and hemodynamic state. Recently, echocardiography has become the standard imaging technique for cardiac assessments in the clinic as well as in mice.
Mouse fetal echocardiography is conducted using standard clinical ultrasound systems as well as ultra-high frequency ultrasound systems. The latter provide 30 MHz or higher frequency transducers that generate two-dimensional images and allow the assessment of early embryonic stages. These transducers have a relatively poor penetration depth (~13 mm), which is, however, sufficient to obtain adequate imaging planes and determine fundamental heart parameters, such as heart rate, left and right ventricular internal diameter at diastole and systole and septum and wall thickness, without performing laparotomy.
In our study, we have used an ultra-high frequency ultrasound system to assess heart rates of mouse embryos at embryonic day E18.5. We chose a 30 MHz transducer that provides a field view of 20 mm x 20 mm, which is ideal given the size of the fetuses, with a focal length of 12.7 mm. However, a higher frequency transducer may be chosen to analyze earlier developmental stages. The selected M-mode allows the visualization of tissues in motion thanks to a high temporal resolution of 1,000 frames/sec. The full procedure is simple and should be performed as quickly as possible to avoid any perturbation of the physiologic and hemodynamic states of the fetus. The analysis of about 8 embryos requires approximately 1 hr.

<table border="0" cellpadding="0" cellspacing="0" width="591">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">Isoflurane</td>
			<td style="width:160px;">Pharmaceutical Partners of Canada Inc.</td>
			<td style="width:104px;">CP0406v2</td>
			<td style="width:163px;">1-chloro-2,2,2-trifluoroethyl difluoromethyl ether</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ultrasound gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>Aquasonic Clear</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Electrode gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>Spectra 360</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ophthalmic gel</td>
			<td>Novartis</td>
			<td>Tear-Gel</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Depilatory cream</td>
			<td>Church &#38; Dwight Co., Inc.</td>
			<td>Nair</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hair clipper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gauze/cotton swap</td>
			<td>Q-tips</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Permanent marker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">High-Resolution In vivo Ultrasound Imaging System</td>
			<td style="width:160px;">Visual Sonics</td>
			<td style="width:104px;">Vevo770</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">30 MHz Transducer</td>
			<td>Visual Sonics</td>
			<td>RMV707B</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">Imaging platform and physiology controller unit</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Anesthetic System</td>
			<td>Cyprane North America Inc.</td>
			<td>312462</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Infrared heating lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero measurement of heart rate in mouse by noninvasive m-mode echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

The above method was used to assess the impact of the presence or absence of the serine protease furin in endothelial cells on heart rates of mouse embryos at E18.5 in utero. Furin belongs to the family of proprotein convertases (PCs) that cleave protein precursors after basic residues. Furin and its substrates are secretory proteins and cleavage can occur in the Golgi apparatus, endosomes or at the cell surface. Key substrates of furin include TGFb, and TGFb-like factors, such as the bone morphogenetic proteins...

Watch this Scientific Journal Video about In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography at JoVE.com

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

in-utero-measurement-heart-rate-mouse-noninvasive-m-mode

Research

JoVE Journal

Medicine

11.3K Aufrufe.  Institut de Recherches Cliniques de Montréal.  Ultrahochfrequenz-Ultraschall ist ein leistungsstarkes Live-Imaging-Tool, um Herzrhythmusstörungen bei Kleintieren zu untersuchen. Seine Nichtinvasivität ermöglicht die Aufrechterhaltung der physiologischen Zustand der Embryonen. Hier zeigen wir die Verwendung von M-Mode-Ultraschall, um die Herzfrequenz von Embryonen bei E18.5 in utero zu messen. 

Serie: In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ventricular (RV) function has strong diagnostic and prognostic power in various cardiovascular disorders. Due to its complex morphology and function, assessment of the RV by conventional two-dimensional echocardiography is limited: the everyday clinical practice usually relies on simple linear dimensions and functional measures. Three-dimensional (3D) echocardiography overcame these limitations by providing volumetric quantification of the RV free of geometrical assumptions. Here, we offer a step-by-step guide to obtain and analyze 3D echocardiographic data of the RV using the leading commercially available software. We will quantify 3D RV volumes and ejection fraction. Several technical aspects may help to improve the quality of RV acquisition and analysis as well, which we present in a practical manner. We review the current opportunities and the limiting factors of this method and also highlight the potential applications of 3D RV assessment in current clinical practice.

Here, we provide a step-by-step acquisition and analysis protocol for the 3D volumetric assessment of the right ventricle, mainly focusing on the practical aspects that maximize the feasibility of this technique.

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...