We thank Fred Roberts for exemplary technical support and also appreciate the help from the histology core in Beth Israel Hospital. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by a Department of Medicine Sundry Fund.

NOTE: All procedures were performed in mice in accordance with American Veterinary Medical Association (AVMA) guidelines and approved Institutional Animal Care and Use Committees (IACUC) protocols.
1. Study Design
<ol>
	<li>Use 8-10 week old male C57BL/6 mice (BW~25 g) in the study.</li>
	<li>Randomize the mice (n = 11) into two groups, the study group selected for aortic banding (n = 8), and the control group (n = 3) to undergo sham operation via thoracotomy.</li>
	<li>Prepare the animal for imaging by removing hair from the chest using depilatory cream that is medical grade.</li>
	<li>Perform a first ultrasound (section 2) 24 hr prior to aortic banding to determine baseline parameters at Day -1, between a range of 1% and 2.5% isoflurane (mixed with 100% O2 via nosecone) induced anesthesia.</li>
	<li>Choose a medically approved anesthetic agent (i.e. isoflurane) and monitor the degree of anesthesia (2-3% to induce, and 1.0% to maintain). 
	NOTE: Proper anesthesia is crucial in the maintenance of heart beat at normal physiological rates (about 500 beats/min).</li>
	<li>Confirm the depth of anesthesia by loss of motion from the animal in response to a pedal-withdrawal reflex. Use paralube vet ointment on the eyes to prevent dryness while under anesthesia.</li>
	<li>Perform surgery at Day 0 20,21.</li>
	<li>For aortic banding, ligate the aorta using a 7-0 silk suture around a tapered 26 G needle placed on the arch. 
	NOTE: Details regarding the experimental protocol, including the surgical aortic banding procedures, have been described previously 20,21.</li>
	<li>Perform post-surgery ultrasound imaging (section 2) at Day(s) 2, 6 and 13.</li>
	<li>Euthanize the mice on Day 14 and harvest the hearts for histological assessment. Euthanize the animals using an overdose of pentobarbital followed by removal of a vital organ such as the heart. Arrest the hearts in diastole and fix with formalin. Use the procedure of the heart harvesting that has been described previously 22.</li>
	<li>Fix all heart tissues with buffered 10% formalin solution. For trichrome staining, embed tissues in paraffin before sectioning. Use the details of trichrome staining that have been well illustrated previously 14,23.</li>
	<li>Analyze the data using offline software (section 3).</li>
</ol>
2. Imaging Protocol
<ol>
	<li>Long and short axis images of septal coronary artery (SCA) (B- Mode)
	<ol>
		<li>Using MS550D probe with center frequency of 40 MHz connected to the active-port, set the application preset to &#8220;cardiac imaging&#8221;.</li>
		<li>With the animal supine on the heated platform, and under anesthesia controlled via nose cone, position the probe using the rail system to obtain the parasternal long axis view (PSLAX) (Figure 1A). Always ensure that the animal is kept warm on the prewarmed platform and body temperature is maintained at physiologic levels. </li>
		<li>Rotate the probe (with notch pointing caudally) clockwise such that the probe angle is 15&#176; to the left parasternal line (long-axis view) (Figure 1B).</li>
		<li>Adjust the probe angle by tilting slightly along y axis of the probe to obtain a full-length longitudinal view of the SCA in the center of the screen (Figure 1B).</li>
		<li>Once the proper landmarks (aortic valve and pulmonary artery) are viewed, cine store the image using the highest frame-rate possible.</li>
		<li>By using the &#8220;xy&#8221; axes micro-manipulators (Figure 1D), adjust the probe position to obtain the clearest image of the SCA.</li>
		<li>Rotate the probe 90&#176; (with notch pointing caudally) clockwise such that notched end of the probe is to the left of midline (short-axis) (Figure 1C).</li>
	</ol>
	</li>
	<li>Long and short axis images of SCA (Color-Doppler Mode)
	<ol>
		<li>Once a B Mode image is captured or cine-stored, click the color Doppler key on the keyboard to turn on color Doppler acoustic window (Figure 2). 
		NOTE: This helps to isolate coronary artery (white arrow indicates SCA) either in the long (Figure 2A) or in the short axis (Figure 2C). Red color is as seen in real time and is indicative of the direction of flow (away from the aortic valve).</li>
		<li>Ensure that the focus depth (indicated by a yellow arrowhead on right of the image screen), lies in the center of coronary artery.</li>
		<li>Ensure that the data is recorded, using the cine-store key, at the highest possible frame rate (&#62;100 frames/sec).</li>
	</ol>
	</li>
	<li>PW Doppler Imaging of SCA (Pulsed-Wave or PW Mode)
	<ol>
		<li>While in color-Doppler mode, click on the PW key to bring up a yellow-indicator line on the coronary artery (Figure 2, shown in red).</li>
		<li>Place the yellow PW line in the middle of the coronary artery in view, at an angle that parallels the directionality of the flow. Note that velocity measurements are highly dependent on the angle of image acquisition.</li>
		<li>Adjust the angle of flow (PW angle key) and sample volume (SV key) such that the PW angle key is 60&#176; or less and sample volume captures flow right in the center of the SCA.</li>
		<li>Use cine store to capture the wave forms that indicate the velocity of the coronary flow at peak systole (S) and diastole (D) (Figures 3A and 3B), using 1% and 2.5% isoflurane.</li>
	</ol>
	</li>
</ol>
3. Data Calculation and Analysis
<ol>
	<li>Select the velocity time integral (VTI) tool to obtain the peak systolic and diastolic velocities from the images shown in Figures 3A and 3B.</li>
	<li>Calculate the coronary flow reserve index (CFR) as the ratio of hyperemic (2.5% isoflurane) peak diastolic flow velocity to baseline (1% isoflurane) peak diastolic flow velocity.</li>
	<li>Calculate the S/D ratio as the peak systolic coronary flow velocity/ peak diastolic coronary flow velocity. Determine the ratio at baseline (1% isoflurane) and at hyperemia (2.5% isoflurane).</li>
	<li>For standard cardiac function parameters such as FS, FAC, LVM, refer to the manuals from the manufacturer to perform data analysis using proprietary software or refer to Cheng&#8217;s JoVE paper 2.</li>
</ol>

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The authors report no disclosures.

In this ultrasound based study, non-invasive assessment of coronary flow was reproducibly performed in real time, over days, in live experimental mice; furthermore, the protocol demonstrated the potential to detect coronary artery dysfunction that was present at an early stage and was associated with deficiency in myocardial perfusion. This method could ultimately be leveraged as a clinical tool for cardiovascular risk stratification and/or assessing response to therapeutic intervention.
First...

Clinical aortic stenosis (AS) is well known to promote a progressive increase in left ventricular (LV) afterload. To compensate for this chronically rising hemodynamic load, LV hypertrophy (LVH) ensues as an adaptive response1,2. The development of LVH is often associated with abnormalities in coronary microcirculation. It is thought that microvascular dysfunction contributes to chronic ischemia in these patients 5. In addition to coronary flow 3,4, coronary flow reserve (CFR) represents functional change of coronary arteries 1,3 and is defined as the ratio of maximal flow velocity in hyperemia to baseline flow velocity or resting flow velocity4,6,7. CFR is decreased during LV remodeling 1-3,5-9 and is used as an index of the extent of functional severity of coronary dysfunction 1,10,17. It is known to be impaired in many forms of dilated cardiomyopathy 10 and also coronary stenosis6. CFR is also a prognostic marker for poor clinical outcomes 12.
LV remodeling in the setting of cardiac dysfunction such as ischemia or LVH is also accompanied by extensive fibrosis, changes in coronary microcirculation and thickening of coronary arteries 1,2. As a result of these changes in coronary physiology, there is likely remodeling of the coronary arteries. This helps mitigate the effects of low oxygen diffusion and LV diastolic dysfunction that could result in susceptibility to myocardial ischemia 1,2,13.
Genetically modified mice are now a widely prevalent investigational tool for mimicking human disease conditions such as coronary atherosclerosis 5,7,10,12,17. Particularly the pressure overload model in mice has been widely studied 14,17. The trans-aortic constriction model (TAC) has been shown to be associated with extensive fibrosis, and coronary stenosis resulting, in part, from medial thickening of coronary arteries and with accompanying changes in coronary flow patterns 1,11,17,19 similar to what is seen in the setting of LVH in humans. While it is known that prolonged pressure overload leads to decompensated heart failure in about 4-8 weeks, the effects on coronary flow dynamics and flow reserve in these models, early in the process of disease progression, and at different stages after banding, are yet to be clearly delineated.
Numerous strains of mice are currently available for research use, including well-characterized LDLR-/- or ApoE-/- mice 10-12, and these have prompted development of sensitive techniques for assessing cardiovascular function and morphology in living mice 11-15. Such techniques include MRI, PET, contrast CT, high frequency ultrasound, and electron beam tomography 2,9,17,19, all of which provide promising alternatives to invasive methods such as cardiac catheterizations and coronary angiography 12. However, in mice with very small size of the coronary arteries and high heart rates (HR), imaging of coronary circulation still constitutes a technical challenge for many currently available techniques 4,12. Interestingly, there has been an exponential rise in technical advances in the field of transthoracic Doppler echocardiography (TTDE), including the development of high-frequency array scan heads with center frequencies from 15 to 50 MHz allowing axial resolutions of approximately 30-100 &#956;m, at depths of 8-40 mm, and frame rates greater than 400 frames-captured/sec. In turn, TTDE-based techniques have emerged as a potentially powerful tool for imaging larger 2 or even smaller vessels such as coronary arteries 5,12.
Another critical advance that has allowed investigators to conduct diagnostic imaging studies of the vasculature in small animals is the carefully controlled use of anesthetics that maintain the heart and respiratory rate of the animals during imaging 11. Controlled anesthesia maintenance is particularly important for studies related to vasodilation in mice, and the effect of anesthesia also needs to be further explored in this context 10,11. In humans, on the other hand, TTDE-derived CFR measurements have become a more commonly used tool for evaluation of stenosed and non-obstructed epicardial coronary arteries, predominantly in left anterior descending (LAD) coronary artery 5,16. However, the prognostic role of CFR and coronary flow changes in asymptomatic patients or mice with preserved LV systolic function at rest has been much less explored 16. Therefore, the aim of the study was to first establish a clear step-by-step protocol, to evaluate changes in coronary flow using TTDE in a pressure overload mouse model; second, this study examined the prognostic significance of CFR and coronary flow changes in response to pressure overload stress in these mice. We hypothesized that TTDE based assessment of CFR and coronary flow may be useful in the early detection of coronary dysfunction that may precede LV dysfunction.

<table><tbody><tr><td>Depilatory cream</td><td>Miltex, Inc.</td><td>Surgi-Prep</td><td>Apply 24 hours prior to imaging</td></tr><tr><td>Isoflurane</td><td>Baxter International Inc.</td><td>NDC 10019-773-40</td><td>2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute</td></tr><tr><td>High Frequency Ultrasound</td><td>FUJIFILM VisualSonics, Inc.</td><td>Vevo 2100</td><td></td></tr><tr><td>High-frequency Mechanical Transducer</td><td>FUJIFILM VisualSonics, Inc.</td><td>MS250, MS550D, MS400</td><td></td></tr></tbody></table>

ultrasound based assessment of coronary artery flow and coronary flow reserve using the pressure overload model in mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Of the 11 mice that were studied (banded, n=8 and sham, n = 3), adequate and reproducible images were obtained by a single observer at several time-points: at baseline (D-1), D2, D6 and D13. Also, the flow velocity at the constrictive site was measured as 2225 &#177; 110.9 mm/s, compared with 277.5 &#177; 10.51 mm/s in the sham mice on the day after the surgery (p&#60;0.05). The increase in velocity was the verification of the successful establishment of the pressure overload model. The SCA flow velocity, also referred t...

Watch this Scientific Journal Video about Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice at JoVE.com

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

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Research

JoVE Journal

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14.9K Views.  Brigham and Women's Hospital, Harvard Medical School. Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Video: Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

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 ...

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 ...

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.

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 ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

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 ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

Exploring HR-Dependent Cardiac Performance Using an Atrial-Pacing Protocol

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.

Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function&#160;in mouse models of heart failure.

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

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