This work was supported by NIH grants HL111121 (JDM) and TR000954 (JDM).

<ol>
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The authors have nothing to disclose.

Induction of anesthesia
Proper induction and maintenance of anesthesia is critical for the accurate assessment of changes in heart valve and cardiac function in mice. Given the rapid induction of anesthesia elicited by isoflurane and the relatively long wash-out time of this anesthetic following deep anesthesia, we do not use a stand-alone anesthesia chamber for induction. Instead, as noted in detail above, animals are guided directly to the anesthesia cone, which allows for rapi...

Aging is associated with progressive increases in cardiovascular calcification1. Hemodynamically significant aortic valve stenosis affects 3% of the population over the age of 652, and patients with even moderate aortic valve stenosis (peak velocity of 3-4 m/s) have a 5 year event-free survival of less than 40%3. Presently, there are no effective treatments to slow the progression of aortic valve calcification, and surgical aortic valve replacement is the only available treatment for advanced aortic valve stenosis4.
Studies aimed at gaining a deeper understanding of the mechanisms that contribute to the initiation and progression of aortic valve calcification are a key first step in moving towards pharmacological and non-surgical methods to manage aortic valve stenosis5,6. Genetically-altered mice have played a major role in developing our understanding of the mechanisms that contribute to a variety of diseases and are now coming to the forefront of mechanistic studies aimed at understanding the biology of aortic valve stenosis6,7,8. Unlike other cardiovascular diseases such as atherosclerosis and heart failure-where standard protocols for evaluating vascular and ventricular function are for the most part well-established-there are unique challenges associated with in vivo phenotyping of heart valve function in mice. While recent reviews have provided thorough discussions regarding the advantages and disadvantages to numerous imaging and invasive modalities used to assess valve function in rodents9,10,11, to date, we are not aware of a publication that provides a comprehensive, step-by-step protocol for phenotyping heart valve function in mice.
The purpose of this manuscript is to describe the methods and protocols to phenotype heart valve function in mice. All methods and procedures have been approved by the Mayo Clinic Institutional Animal Care and Use Committee. Key components of this protocol include the depth of anesthesia, the evaluation of cardiac function, and the evaluation of heart valve function. We hope this report will not only serve to guide investigators interested in pursuing research in the field of heart valve disease, but will also start a national and international dialogue related to protocol standardization to ensure data reproducibility and validity in this rapidly-growing field. Importantly, successful imaging using high-resolution ultrasound systems requires a working knowledge of the principles of sonography (and terminology commonly used in sonography), an understanding of the fundamental principles of cardiac physiology, and significant experience with sonography to allow for accurate and time-efficient assessment of cardiac function in rodents.

<table border="0" cellpadding="0" cellspacing="0" width="1906">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">High resolution ultrasound machine</td>
			<td style="width:453px;">VisualSonics, Fujifilm</td>
			<td style="width:293px;">Vevo 2100&#160;</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:693px;">Isoflurane diffuser (capable of delivering 1 % to 1.5 % isoflurane mixed with 1 L/min 100% O2</td>
			<td>VisualSonics, Fujifilm</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:693px;">Transducers for small mice (550D) or larger mice (400)</td>
			<td>MicroScan, VisualSonics, Fujifilm</td>
			<td>MS 550D, MS 400</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Animal platform</td>
			<td>VisualSonics, Fujifilm</td>
			<td>11503</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Advanced physiological monitoring unit</td>
			<td>VisualSonics, Fujifilm</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Isoflurane</td>
			<td>Terrell</td>
			<td>NDC 66794-019-10</td>
			<td></td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:693px;">Nose cone and tubing connected to isoflurane diffuser and 100% O2</td>
			<td>Custom Engineered in-house</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Hair razor</td>
			<td>Andis Super AGR+ vet pack clipper</td>
			<td>AD65340</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Ultrasound gel</td>
			<td>Parker Laboratories</td>
			<td>REF 01-08</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Electrode gel&#160;</td>
			<td>Parker Laboratories</td>
			<td>REF 15-25</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Adhesive tapes</td>
			<td>Fisher Laboratories</td>
			<td>1590120B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

echocardiographic approaches and protocols for comprehensive phenotypic characterization of valvular heart disease in mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

Examples of images that are routinely obtained from animal cardiac ultrasound imaging are included in this manuscript. An illustration of transducer placement on the animal&#39;s chest is provided to give the reader a clear understanding of where the transducer is positioned to obtain the images as described. A photograph of the ultrasound laboratory set-up is also included to emphasize the importance of the proper equipment, particularly the ultrasound transducer to be used and the metho...

Watch this Scientific Journal Video about Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice at JoVE.com

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

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15.8K Views.  Mayo Clinic. This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice. 

Video: Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Implantation of Total Artificial Heart in Congenital Heart Disease

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital heart disease (CHD), the malformed heart presents a challenge to TAH implantation. 
In the case presented here, a 17 year-old patient with congenital transposition of the great arteries (CCTGA) experienced progressively worsening HF due to his congenital condition. He was hospitalized multiple times and received an implantable cardioverter defibrillator (ICD). However, his condition soon deteriorated to end-stage HF with multisystem organ failure. 
Due to the patient's grave clinical condition and the presence of complex cardiac lesions, the decision was made to proceed with a TAH. The abnormal arrangement of the patient's ventricles and great arteries required modifications to the TAH during implantation.
With the TAH in place, the patient was able to return home and regain strength and physical well-being while awaiting a donor heart. He was successfully bridged to heart transplantation 5 months after receiving the device. This report highlights the TAH is feasible even in patients with structurally abnormal hearts, with technical modification.

This is a case report of a patient with congenitally corrected transposition of the great arteries (CCTGA) who received a total artificial heart (TAH) as a bridge to heart transplant. The TAH was successfully implanted with modifications to accommodate the patient's congenitally malformed heart.

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

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&#160;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.

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.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

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 ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

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.

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 ...