This work was supported in part by: Fundaci&#243;n S&#233;neca, Agencia de Ciencia y Tecnolog&#237;a, Regi&#243;n de Murcia, Spain (JT) (Grant number: 11935/PI/09) and the University of Reading, United Kingdom (AG, GB) (Central Funding). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Echocardiography Committee of the Specialty of Cardiology, American College of Veterinary Internal Medicine.</a> Journal of Veterinary Internal Medicine. 7, 247-252 (1993).</li><li>Lang, R. M., 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=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> European Heart Journal Cardiovascular Imaging. 16, 233-270 (2015).</li><li>Talavera, 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=An+Upgrade+on+the+Rabbit+Model+of+Anthracycline-Induced+Cardiomyopathy:+Shorter+Protocol,+Reduced+Mortality,+and+Higher+Incidence+of+Overt+Dilated+Cardiomyopathy.">An Upgrade on the Rabbit Model of Anthracycline-Induced Cardiomyopathy: Shorter Protocol, Reduced Mortality, and Higher Incidence of Overt Dilated Cardiomyopathy.</a> BioMed Research International. 2015, 465342 (2015).</li><li>Borkowski, R., Karas, A. 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The authors have nothing to disclose.

We have described a protocol for the echocardiographic examination of cardiac function parameters in the rabbit, representing a large preclinical model1,2,3. The step by step methodology described herein should be considered guidance, which with a complementary study of the basic principles of echocardiography, and a basic knowledge of ultrasound imaging, will help the researcher to obtain, through practice and complementary and expert guidance, good quality data in a relative short period of time.
There are several critical steps to increase the value and reproducibility of the results whilst using the echocardiography protocol described here. First, ensure the skin of the thorax is hair free and clean; for this we recommend cleaning the skin with ethanol to remove excess of natural skin grease before applying ultrasound gel. Next, whilst it is possible to image the chest in a supine position, the lungs tend to inflate and reduce an already difficult to image chest wall with poor echogenicity, thus, a left or right recumbent position of the rabbit and the application of the transducer to the chest through the cut-out notch of a purpose built imaging table is the best way to improve overall imaging quality. Then, the researcher operating the ultrasound system should spend some time creating cardiac imaging presets with optimized imaging settings, which are essential to improve overall imaging quality in all views and will also shorten your imaging time at future imaging sessions. Some of the most important control settings to master are total gain and time-gain compensation, given the poor imaging of the chest of the rabbit (see step 2.4.2). It is also important to be systematic and always perform the echocardiographic examination in an orderly fashion. For this, getting into the habit of acquiring all the imaging views and imaging planes in the same sequence will avoid missing important information whilst performing the study. Furthermore, during imaging analysis it is recommended to perform all measurements in at least three consecutive cardiac cycles in the acquired images for each modality. Finally, the blinding of the observer during imaging as well as during the offline analysis is important to avoid bias and increase the value of the results for translational medicine. Taking into account all of the above considerations, together with the application of the principles of imaging and analysis according to current guidelines7,8, will ensure the reproducibility of the research using longitudinal evaluation of cardiac function via echocardiography in a large animal model such as the rabbit.
Given the variability in body size and fat composition at different ages of the rabbits and the particular experimental settings, some variations of the technique will be required, such as subtle movements of the transducer (e.g., sweeping, rotation) relative to the intercostal space, in order to achieve the desired imaging planes. Therefore, the protocol described here must be interpreted as a starting point that should be adapted to the particular objectives of the research program involving this technique.
Whilst clinical echocardiography systems are widely available in most research centers, there are some limitations to the technique described herein. Indeed, the quality of the images obtained from echocardiographic studies depends to a large extent on the sophistication and technology of the ultrasound machine, the skills and expertise of the operator, and the individual patient characteristics. The minimum technical characteristics that the ultrasound equipment must meet were described in the introduction. Thus, inadequate equipment (e.g., a linear array transducer) constitutes a fundamental limitation for the use of the echocardiographic technique in the rabbit model. In addition, the echocardiographic technique and its results are strongly influenced by the operator. Therefore, an operator without enough experience and practical training could dramatically limit the obtaining of standardized images of appropriate quality. Similarly, inexperienced operators could also make mistakes in obtaining measurements even if they are performed on echocardiographic images of excellent technical quality. Furthermore, as mentioned above, some of the limitations are inherent to the rabbit model, such as age and, more specifically, by the size and body fat composition of the rabbits studied via echocardiography. In our experience, young rabbits weighing up to 2.5 kg have low subcutaneous and intra-thoracic fatty deposits. This phenotypic stage provides the best acoustic windows and offers crisper and sharper echocardiographic images and very few artefacts. As the size and body fat composition increase, the quality and accuracy of the echocardiographic study becomes limited, and the skills of the operator will ultimately play a fundamental role in achieving the best possible imaging under these circumstances.
We currently use echocardiography for longitudinal evaluation of cardiac function in a rabbit model of cardiomyopathy induced by anthracyclines and to test stem cell therapies for this condition9,12,13. The technique described here could also be used in other preclinical studies involving ischemia or valvular heart disease.
Another cardiovascular imaging technique is cardiac magnetic resonance (CMR), whose main advantage is better endocardial-myocardial definition, which translates into a more accurate estimation of LV volumes and systolic function14. However, CMR is limited by its high cost and lack of portability and therefore its limited availability in most research centers. Similarly, CMR has relative poor performance for the analysis of diastolic function, thus making echocardiography a better overall choice for longitudinal evaluation of systolic and diastolic function of the heart15.
In our experience, the anesthetic regime used in the protocol described herein is safe and achieves reproducible results without significant depression of myocardial function attributable to the anesthesia9. However, it is important to standardize the anesthetic regime in each laboratory to ensure reproducible results for your particular experimental settings. After inducing anesthesia, in experienced hands the echocardiographic examination can be completed within 15 min.

Longitudinal evaluation of cardiac function in large animal models is a robust research methodology commonly used for the assessment of the effects of novel therapies for treating ischemic and non-ischemic cardiomyopathy. Amongst the several cardiovascular imaging techniques available for preclinical research, echocardiography has been used extensively because of its non-invasive and portable characteristics. In experienced hands, echocardiography is also a very reproducible imaging technique to study cardiac anatomy as well as systolic and diastolic function of the heart.
Large preclinical animal models such as pigs, dogs and rabbits, are paramount for preclinical translational research1,2,3. Indeed, the potential benefit of novel therapies such as cardiac regenerative medicine in the setting of cardiomyopathy requires extensive hypothesis testing in large preclinical models before they can be considered for human use2,4. Compared to other large preclinical models, the rabbit model offers some advantages, including its low maintenance cost, which is comparable to that of mice and rats. However, in contrast to mice and rats, the Ca+2 transport system and cardiac electrophysiology are similar in rabbits as those of humans, and those of other large animal models such as dogs and pigs, thus increasing the translational potential of the rabbit model1,5. Therefore, the rabbit, as a large experimental preclinical model, has an exceptional balance of cost and reproducibility for preclinical translational research.
The rabbit has the additional benefit of its amenability for echocardiographic imaging using clinical ultrasound units routinely used in human and veterinary patients, thus taking advantage of the superiority of harmonic imaging and state-of-the-art technology. For this, sector transducers (also known as phase array) of relatively high frequency (up to 12 MHz), such as those used in neonatal/pediatric cardiology, are preferred. Echocardiographic examination in the rabbit preclinical model allows the complete evaluation of systolic and diastolic function using multiple views and different modes available in modern echocardiographic units (e.g. continuous wave Doppler (CWD), pulsed-wave Doppler (PWD), and Tissue Doppler imaging (TDI)).
Echocardiography is an operator-dependent technique and therefore requires extensive training and core knowledge of the technique in accord with international guidelines. Part of this training can be facilitated with the visualization of videos explaining in detail how different echocardiographic views can be obtained. The achievement of high competency in echocardiographic imaging, as well as development of a standardized protocol and correct technique, are essential to minimize the influence of the operator and to generate reliable quantitative data, as required in rigorous scientific research.
Some considerations are necessary regarding the system and laboratory setup used for echocardiography in rabbits and other large animal models. For a standard transthoracic echocardiographic evaluation of cardiac function, the ultrasound system must include the following modalities: bi-dimensional mode (B-mode or 2D), motion mode (M-mode), color Doppler, as well as CWD, PWD and TDI. Moreover, the machine should have full cardiac analysis and measurement software installed, as well as sufficient internal hard drive space to store enough high quality digital still images and video loops for offline analysis. Some systems use linear array transducers; however, for the best imaging of the heart, phased array sector transducers with a small scan head diameter are preferred, because these allow an easier passage of the ultrasound waves through the narrow intercostal spaces. For rabbits, we use relatively high frequency transducers (up to 12 MHz). The position of the animal for imaging is of utmost importance to acquire good quality images. Thus, both right and left lateral recumbent positions are recommended to obtain all standard imaging planes during an echocardiographic examination. For this, a table with a notch that coincides with the cardiac area of the chest is advisable (Figure 1A). This notched table facilitates the access with the transducer to the area of the chest that will be scanned, and therefore allows free mobility of the hand of the operator whist maintaining the best scanning position of the animal. Positioning the animal in a lateral recumbent position results in a fall of the heart towards the transducer and elevation of the lungs, as well as widening the access window of the ultrasound beam through the intercostal spaces, thus improving overall imaging quality (Figure 1A). The echocardiographic examination should be performed in a blinded fashion and following the guidelines of the Echocardiography Committee of the American College of Veterinary Internal Medicine and the American Society of Echocardiography/European Association for Cardiovascular Imaging6,7,8.
Part of our scientific team is associated with the Cardiology Service of a Veterinary Teaching Hospital that attends daily to veterinary patients (e.g. dogs and cats), for which it has the relevant training and accreditation in veterinary cardiology and echocardiography, and its different imaging modalities, as well as extensive experience in imaging different sizes of animal patients and thoracic conformations with this technique. In addition, we commonly use echocardiography for longitudinal evaluation of cardiac function in a rabbit model of cardiomyopathy induced by anthracyclines9. Here, we describe a step by step echocardiography protocol for evaluation of cardiac function using a clinical ultrasound unit in a large preclinical model such as the rabbit. This protocol is adapted for current international guidelines8, and includes practical recommendations based on our own experiences in clinical and experimental settings.

<table><tbody><tr><td>Bluesensor</td><td>Medicotest</td><td>13BY1062</td><td>Disposable adhesive ECG lectrodes</td></tr><tr><td>Domtor (Medetomidine)</td><td>Esteve</td><td>CN 570686.3</td><td>Veterinary prescription is necessary</td></tr><tr><td>HD11 XE Ultrasound System</td><td>Philips</td><td>10670267</td><td>Echocardiography system.</td></tr><tr><td>Heating Pad</td><td>Solac</td><td>CT8632</td><td></td></tr><tr><td>Imalgene (Ketamine)</td><td>Merial</td><td>RN 9767</td><td>Veterinary prescription is necessary</td></tr><tr><td>Omnifix-F 1 ml syringe</td><td>Braun</td><td>9161406V</td><td></td></tr><tr><td>S12-4</td><td>Philips</td><td>B01YgG</td><td>4-12 MHz phase array transducer</td></tr><tr><td>Ultrasound Transmision Gel (Aquasone)</td><td>Parker laboratories Inc.</td><td>N 01-08</td><td></td></tr></tbody></table>

transthoracic echocardiographic examination in the rabbit model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Parasternal long axis view of the heart
Figure 5A shows an imaging plane of the right PSLAX view where the 4 chambers of the heart are clearly distinguished. You can identify in this view the right ventricle (RV), tricuspid valve (TV), IVS, LV, FW, as well as the mitral valve (MV). When the apex is clearly visible on the left side of the image in this view and the LV is not foreshortened, it is possible to estimate accurately the LV volume using the biplane method of disks (modified Simpson&#39;s rule) as shown in Figure 5B,C8, which for accuracy should be combined with a similar measurement of the LV volume in the AP4C view, especially if the rabbit model used presents with wall motion abnormalities. Figure 5D shows the other imaging plane of the right PSLAX where the LVOT and the Aorta (Ao) also come into view. The location for placement of the calipers for accurate measurement of the LVOT is also shown in Figure 5D.&#160;
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59457/59457fig5.jpg" /> 
Figure 5. Imaging planes obtained in a PSLAX view of the heart. (A) Imaging plane demonstrating the 4 chambers of the heart. (B) End diastolic and (C) end systolic images, demonstrating Simpson&#39;s method for analysis of the LV. (D) Imaging plane where the LVOT and aorta come into view in the PSLAX view of the heart. LV = Left ventricle; RV = right ventricle; IVS = interventricular septum; Ao = aorta; LVOT = left ventricular outflow track; LA = Left atrium; RA = right atrium; MV = mitral valve; TV = tricuspid valve; FW = free wall of the LV; PC = pericardium. <a href="https://www.jove.com/files/ftp_upload/59457/59457fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Parasternal short axis view of the heart
In Figure 6A, a right PSSAX view of the heart at the level of the papillary muscles and chordae tendineae plane is shown. It is possible identify in this view the RV, IVS, LV, and FW, as well as the anterolateral (AL) and posteromedial (PM) papillary muscles (Figure 6A). In this view, the area trace tool is used to measure the circumferential area in end-diastole (CAd) (Figure 6B), and in end-systole (CAs) (Figure 6C), which allows the calculation of the total circumferential shortening area (CSA) by using the formula:
CSA=CAd-CAs/CAd&#215;100.
An example of an M-mode trace in the PSSAX at the papillary muscles level is shown in Figure 6D, where the placement of calipers, leading edge to leading edge, for the different measurements of the structures of the LV is also demonstrated. These measurements provide useful information regarding size of the LV structures. Thus, measuring the LV end-diastolic diameter (LVDd) and LV end-systolic diameter (LVDs) from three consecutive heart beats allows the calculation of the LV shortening fraction (%SF), using the formula:
SF%= LVDd-LVDs/LVDd
as well as the LV systolic and diastolic volumes (LVVd, LVVs), using the Teichholz formula:
(7&#215;(LVD)3)/(2.4+LVD)
The LV ejection fraction (LVEF (%)) is subsequently calculated according the formula LVEF=(LVVd-LVVs)/(LVVd&#215;100).
An M-mode trace at the level of the MV plane in PSSAX view is shown in Figure 6E, where the location of the calipers for measurement of the E-point to septal separation (EPSS) of the mitral valve is also shown. An example of a PSSAX view of the heart at the AoV plane level is shown in Figure 6F, where the location of the calipers for measurement of the Aortic root diameter (AoD), as well as the left atrial dimension (LAD) are demonstrated.
An example of the PV outflow analysis using both color Doppler and pulsed wave Doppler is shown in Figure 6G. Note the blue colored outflow through the PV with color Doppler, which indicates that the flow observed is moving away from the transducer. Examples of how to quantitate the pre-ejection period of the PV (PEP PV), as well as the PV outflow using the volume time integral (VTI), are shown in Figure 6H.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59457/59457fig6.jpg" /> 
Figure 6. Imaging planes obtained in the PSSAX view. (A) Representative image of a PSSAX view at the papillary muscles plane. (B) End diastolic and (C) end systolic tracing of the endocardial border to measure the total CSA. (D) M-mode trace obtained in a PSSAX view at the level of the papillary muscles. (E) An example of M-mode trace obtained in a PSSAX view at the level of the MV. (F) Representative 2D image of a PSSAX vie in the plane of the AV. (G) Color Doppler-guided PWD tracing of the PV outflow. (H) Demonstration of a VTI tracing using the PWD signal obtained from the PV outflow. LV = Left ventricle; RV = right ventricle; IVS = interventricular septum; FW = free wall of the LV; AL = anterolateral papillary muscle; PM = posteromedial papillary muscle; LVDd = left ventricular diameter at end-diastole; LVDs = left ventricular diameter at end-systole; PC = pericardium; EPSS = E-point to septal separation; AoD = aortic root diameter; LAD = left atrial dimension; MV = mitral valve; TV = tricuspid valve; PEP PV = pre-ejection period of the pulmonary valve; ET PV = ejection time of the pulmonary valve; VTI PV = volume time integral of the pulmonary valve. <a href="https://www.jove.com/files/ftp_upload/59457/59457fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Apical 4 chambers view
An example of MV inflow using color Doppler in an AP4C view is shown in Figure 7A. Note the predominant red color of the MV inflow indicating that the flow is moving towards the transducer. Thus, a useful mnemonic to describe and learn how blood flows across the structures of the heart is the acronym BART (Blue Away, Red Towards the transducer). Using PWD, the MV inflow spectrum can be assessed as shown in Figure 7B, where the early (E) and late (A) filling waves during diastole are easily differentiated. Examples of myocardial tissue velocities of the MV annulus as assessed by TDI at both the lateral and septal walls are shown in Figure 7C and Figure 7D, respectively. The systolic component is denoted by the S wave, whilst the E&#39; and A&#39; waves correspond with myocardial movement of the mitral valve annulus during early filling (E&#39;) and late filling (A&#39;) components of diastole.
Apical 5 chambers view
Figure 7E shows an example of color Doppler positioned at the LVOT in an apical 5 chambers view. Note that, in line with the BART mnemonic described above, the blue color observed indicates that blood flow is moving away from the transducer. Figure 7F shows an example of how to quantitate the AoV outflow using PWD signal to evaluate the VTI of the AoV, systolic ejection time (ET) and pre-ejection period of the AoV (PEP AoV).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59457/59457fig7.jpg" /> 
Figure 7. The AP4C and AP5C views. (A) An example of color Doppler in an AP4C view. (B) Representative image of the PWD signal of the MV inflow in an AP4C, where E wave corresponds with early diastolic filling and A corresponds with atrial contraction component during diastole. (C-D) Representative images of myocardial velocity signals obtained from the lateral (C) and septal (D) segments of the MV annulus using TDI in an AP4C view. S corresponds with systole, whilst E&#39; corresponds with early filling phase and A&#39; with late filling phase during diastole. (E) An example of color Doppler signal obtained from the AoV in an AP5C view. (F) Demonstration of a VTI tracing using the PWD signal obtained from the AoV outflow. AoV = Aortic valve; VTI = volume time integral; PEP = pre-ejection period; ET = ejection time.&#160;<a href="https://www.jove.com/files/ftp_upload/59457/59457fig7large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transthoracic Echocardiographic Examination in the Rabbit Model at JoVE.com

Transthoracic Echocardiographic Examination in the Rabbit Model

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

transthoracic-echocardiographic-examination-in-the-rabbit-model

Nanyang Technological University

Research

JoVE Journal

Medicine

12.4K Views.  University of Reading. Deze video is bedoeld om stap voor stap een protocol aan te tonen voor een echocardiografisch onderzoek in een groot diermodel zoals het konijn. Dit wordt bereikt door eerst het dier te verdoven en vervolgens de kist zorgvuldig voor te bereiden op de procedure. Vervolgens, na het plaatsen van het konijn op een thermische deken en het verifiëren dat een ECG-signaal correct wordt weergegeven, plaats het dier in een juiste ligfietspositie om de beeldvormingsvlakken te verkrijgen in zowel een pa...

Video: Transthoracic Echocardiographic Examination in the Rabbit Model

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 ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

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, ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs

One of the main causes of out-of-hospital cardiac arrest is acute myocardial infarction (AMI). After successful resuscitation from cardiac arrest, approximately 70% of patients die before hospital discharge due to post-resuscitation myocardial and cerebral dysfunction. In experimental models, myocardial dysfunction after cardiac arrest, characterized by an impairment in both left ventricular (LV) systolic and diastolic function, has been described as reversible but very little data are available in cardiac arrest models associated with AMI in pigs. Transthoracic echocardiography is the first-line diagnostic test for the assessment of myocardial dysfunction, structural changes and/or AMI extension. In this pig model of ischemic cardiac arrest, echocardiography was done at baseline and 2-4 and 96 hours after resuscitation. In the acute phase, the examinations are done in anesthetized, mechanically ventilated pigs (weight 39.8 &#177; 0.6 kg) and ECG is recorded continuously. Mono- and bi-dimensional, Doppler and tissue Doppler recordings are acquired. Aortic and left atrium diameter, end-systolic and end-diastolic left ventricular wall thicknesses, end-diastolic and end-systolic diameters and shortening fraction (SF) are measured. Apical 2-, 3-, 4-, and 5-chamber views are acquired, LV volumes and ejection fraction are calculated. Segmental wall motion analysis is done to detect the localization and estimate the extent of myocardial infarction. Pulsed Wave Doppler echocardiography is used to record trans-mitral flow velocities from a 4-apical chamber view and trans-aortic flow from a 5-chamber view to calculate LV cardiac output (CO) and stroke volume (SV). Tissue Doppler Imaging (TDI) of LV lateral and septal mitral anulus is recorded (TDI septal and lateral s&#39;, e&#39;, a&#39; velocities). All the recordings and measurements are done according to the recommendations of the American and European Societies of Echocardiography Guidelines.

Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest.

One of the main causes of out-of-hospital cardiac arrest is acute myocardial infarction (AMI). After successful resuscitation from cardiac arrest, ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.

Transthoracic Echocardiographic Examination in the Rabbit Model

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