Animal experiments were performed in accordance with the European Directive (2010/63/UE) and the Italian law (D.Lvo 26/2014), and it followed principles of laboratory animal care. The Local Ethical Approval Panel approved the study.
1. Imaging Procedure
<ol>
	<li>Place the mouse in an anesthesia induction chamber filled with 2.5% isoflurane in 1 L/min pure oxygen. Verify the depth of anesthesia by unresponsiveness to toe pinch.</li>
	<li>Place the animal on a temperature controlled board in a supine position. Moisten the animal&#39;s eyes with eye salve, in order to avoid them running dry. Supply anesthetic gas flow (1.5% isoflurane) by placing the mouse&#39;s nose in the dedicated nose cone. If necessary, adjust the percentage of isoflurane from case to case, depending on the animal under study. Fix the board temperature at 40 &#176;C.</li>
	<li>Coat the four limbs of the animal with conductive paste and tape them on the ECG electrodes embedded in the board. Measure body temperature with a rectal probe lubricated with petroleum jelly. Check that all the physiological measurements (ECG and respiration signal as well as temperature) are correctly acquired and displayed.</li>
	<li>Remove hair chemically from the abdomen with depilatory cream and coat it with acoustic coupling gel.</li>
	<li>Place the (13-24 MHz) US probe in the mechanical arm.</li>
	<li>Fix the US probe parallel to the animal and adjust its position in order to obtain longitudinal images of the abdominal aorta with the region of interest located in the focal zone.</li>
	<li>Collect anatomical information.
	<ol>
		<li>Click on the button allowing the high frame rate ECG-gated acquisition9, choose a frame rate acquisition equal to 700 fps and start the acquisition. Note: In this way anatomical images of the vessel related to a single cardiac cycle can be acquired.</li>
	</ol>
	</li>
	<li>Collect flow velocity information.
	<ol>
		<li>Using the same scan projection, click on the PW-Doppler button, place the sample volume in the centre of the vessel and acquire images ensuring that the cine loop is not shorter than 3 s. Obtain these data maintaining the angle correction as small as possible, adjusting it from case to case on the basis of the US projection obtained.</li>
	</ol>
	</li>
	<li>Remove the animal from the temperature controlled board and wait for complete recovery. 
	NOTE: In our experience this takes about 10 min. Do not leave and animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
2. Post-Processing
<ol>
	<li>Export B-mode and PW-Doppler images as DICOM files and save them on a personal computer. Transform PW-Doppler DICOM files to .tiff images.</li>
	<li>Process B-mode images.
	<ol>
		<li>Import the corresponding DICOM file using the dedicated Graphical User Interface (GUI).</li>
		<li>To initialize contours, draw a line close to the far wall of the vessel (single click for starting it and double click for ending it) and double-click close to the near wall. A line parallel to that close to the far wall will appear automatically. Apply the algorithm on a single frame by pressing the &#34;ANALYSE&#34; button.</li>
		<li>Check the result. If the edges have been correctly identified (i.e., the evolution of the initialized points have detected both the posterior and the anterior wall), apply the algorithm on the whole cine-loop by clicking on the &#34;GO&#34; button. If the edges are not correctly identified, clear them by clicking on &#34;Clear Contour&#34; and initialize them again by repeating point 2.2.2. 
		NOTE: The algorithm is based on edge detection and contour tracking techniques and has been previously described in detail10.</li>
		<li>Obtain the final result by pressing on the &#34;RECORD&#34; button and save the corresponding .mat file which contains instantaneous diameter values related to a single cardiac cycle.</li>
	</ol>
	</li>
	<li>Open the GUI for the lnD-V loop implementation.</li>
	<li>Click on the &#34;VELOCITY&#34; button in order to start PW-Doppler image processing which leads to a single-beat mean velocity curve.
	<ol>
		<li>Identify the PW-Doppler trace and locate the line corresponding to a velocity value equal to zero by pressing the &#34;WHITE LINE&#34; button.</li>
		<li>Perform velocity calibration and time calibration by using the &#34;VELOCITY&#34; and &#34;TIME&#34; buttons (in the Calibration panel), respectively. Pressing on these buttons allows one to draw a line whose length corresponds to the calibration factor inserted.</li>
		<li>Manually select a ROI containing the physiological signals by using the &#34;ROI PHYSIO&#34; button.</li>
		<li>Manually select a ROI containing the PW-Doppler trace by pressing the &#34;ROI SIGNAL&#34; button.</li>
		<li>Click on the &#34;ANALYSE&#34; button and check if the envelope is identified. If the result is not satisfactory, change the threshold (by typing the new value in the &#34;Velocity Threshold&#34; editable text field) and press the &#34;ANALYSE&#34; button again. Tune the threshold from case to case, depending on the quality of the images. Press the &#34;ELABORATION&#34; button.</li>
		<li>Locate the R-peaks of the ECG signal and divide the velocity envelope signal accordingly by clicking the &#34;UPDATE&#34; button. Choose beats that are not corrupted by noise or located in the inspiration phase by clicking on the &#34;CHOOSE BEATS&#34; button. In this way, obtain a single-beat mean velocity waveform.</li>
	</ol>
	</li>
	<li>Use the Fast Fourier Transform method to interpolate the selected beats in the frequency domain and make them all composed of the same number of points, as detailed in reference11. Perform this operation automatically by simply pressing the return key on the PC keyboard once the beats have been selected. If the &#34;MEAN VELOCITY&#34; checkbox is checked the single beat mean velocity signal is achieved by dividing the maximum velocity curve by two, hypothesizing a parabolic velocity profile12. Press the &#34;OK&#34; button.</li>
	<li>Click on the &#34;DIAMETER&#34; button. Interpolate the single-beat diameter waveform in the time domain in order to obtain a single-beat diameter signal with the same sample frequency as the single-beat velocity signal by pressing the &#34;INTERPOLATE&#34; button. Click on the &#34;OK&#34; button. 
	NOTE: In order to have the single-beat diameter and mean velocity curves with the same sample frequency and the same number of data points, they are interpolated in the frequency domain.</li>
	<li>Select the &#34;Second derivative&#34; approach as the alignment method (above the graph displaying the diameter and velocity waveforms) and click on the &#34;UPDATE&#34; button. The two curves will be automatically time-aligned using the second derivative method14.</li>
	<li>Build the final lnD-V loop by plotting the natural logarithm of the single-beat diameter values against the single-beat mean velocity values. This is automatically done when the two waveforms are aligned. Note: The points included between the 5% and the 90% of the maximum value of the single-beat mean velocity curve are automatically located and a linear interpolation on these points is applied to evaluate the slope of the linear part of the loop.</li>
	<li>Calculate PWV according to the following equation7 
	<img alt="Equation 1" src="/files/ftp_upload/54362/54362eq1.jpg" /></li>
</ol>

<ol>
	<li>Zaragoza, C., 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=Animal+Models+of+Cardiovascular+Diseases.">Animal Models of Cardiovascular Diseases.</a> J Biomed Biotechnol. 2011, 497-841 (2011).</li><li>Laurent, S., 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=Expert+consensus+document+on+arterial+stiffness:+methodological+issues+and+clinical+applications.">Expert consensus document on arterial stiffness: methodological issues and clinical applications.</a> Eur Heart J. 27, 2588-2605 (2006).</li><li>Wang, Y. X., 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=Increased+aortic+stiffness+assessed+by+pulse+wave+velocity+in+apolipoprotein+E-deficient+mice.">Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice.</a> Am. J. Physiol. Heart Circ. Physiol. 278, 428-434 (2000).</li><li>Mitchell, G. F., Pfeffer, M. A., Finn, P. V., Pfeffer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+techniques+for+measuring+pulse-wave+velocity+in+the+rat.">Comparison of techniques for measuring pulse-wave velocity in the rat.</a> J Appl Physiol. 82 (1), 203-210 (1997).</li><li>Parczyk, M., Herold, V., Klug, G., Bauer, W. R., Rommel, E., Jakob, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+in+vivo+transit+time+measurements+of+aortic+pulse+wave+velocity+in+mice+with+high-field+CMR+at+17.6+Tesla.">Regional in vivo transit time measurements of aortic pulse wave velocity in mice with high-field CMR at 17.6 Tesla.</a> J Cardiovasc Magn Reson. 12, 72 (2010).</li><li>Hartley, C. J., Taffet, G. E., Michael, L. H., Pham, T. T., Entman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+pulse-wave+velocity+in+mice.">Noninvasive determination of pulse-wave velocity in mice.</a> Am J Physiol. 273 (1), 494-500 (1997).</li><li>Feng, J., Khir, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+wave+speed+and+wave+separation+in+the+arteries+using+diameter+and+velocity.">Determination of wave speed and wave separation in the arteries using diameter and velocity.</a> J Biomech. 43 (3), 455-462 (2010).</li><li>Borlotti, A., Khir, A. W., Rietzschel, E. R., De Buyzere, M. L., Vermeersch, S., Segers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+local+pulse+wave+velocity+and+wave+intensity:+changes+with+age+and+gender+in+the+carotid+and+femoral+arteries+of+healthy+human.">Noninvasive determination of local pulse wave velocity and wave intensity: changes with age and gender in the carotid and femoral arteries of healthy human.</a> J Appl Physiol. 113 (5), 727-735 (2012).</li><li>Ch&#233;rin, E., 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=Ultrahigh+frame+rate+retrospective+ultrasound+microimaging+and+blood+flow+visualization+in+mice+in+vivo.">Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo.</a> Ultrasound Med Biol. 32 (5), 683-691 (2006).</li><li>Gemignani, V., Faita, F., Ghiadoni, L., Poggianti, E., Demi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+real-time+measurement+of+the+brachial+artery+diameter+in+B-mode+ultrasound+images.">A system for real-time measurement of the brachial artery diameter in B-mode ultrasound images.</a> IEEE Trans Med Imaging. 26 (3), 393-404 (2006).</li><li>Di Lascio, N., Stea, F., Kusmic, C., Sicari, R., Faita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+assessment+of+pulse+wave+velocity+in+mice+by+means+of+ultrasound+images.">Non-invasive assessment of pulse wave velocity in mice by means of ultrasound images.</a> Atherosclerosis. 237 (1), 31-37 (2014).</li><li>Nichols, W. W., O'Rourke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> McDonald's Blood Flow in Arteries: Theoretical, Experimental, and Clinical Principles. , 215-358 (1998).</li><li>Williams, R., 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=Noninvasive+ultrasonic+measurement+of+regional+and+local+pulse+wave+velocity+in+mice.">Noninvasive ultrasonic measurement of regional and local pulse wave velocity in mice.</a> Ultrasound Med Biol. 33 (9), 1368-1375 (2007).</li><li>Penny, D. 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=Aortic+wave+intensity+of+ventricular-vascular+interaction+during+incremental+dobutamine+infusion+in+adult+sheep.">Aortic wave intensity of ventricular-vascular interaction during incremental dobutamine infusion in adult sheep.</a> Am J Physiol Heart Circ Physiol. 294, 481-489 (2008).</li><li>Segers, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wave+reflection+leads+to+over-+and+underestimation+of+local+wave+speed+by+the+PU-+and+QA-loop+methods:+theoretical+basis+and+solution+to+the+problem.">Wave reflection leads to over- and underestimation of local wave speed by the PU- and QA-loop methods: theoretical basis and solution to the problem.</a> Physiol Meas. 35 (5), 847-861 (2014).</li></ol>

The authors have nothing to disclose.

In this study, an image processing algorithm based on the lnD-V loop for PWV assessment in mice has been described in detail. The proposed approach is based on the processing of US images only and, thus, could represent a valid alternative to existing techniques6,13 for the evaluation of arterial stiffness in mouse models. In fact, conversely to invasive methods6 which are based on the acquisition of intra-arterial pressure signals and require the animal to be sacrificed, this technique is completely non-invasive and, thus, can be particularly suitable in the case of longitudinal studies. In addition, it provides an evaluation of arterial stiffness which is local and not regional. Although the proposed method is known to be influenced by the reflected wave15, it may provide a more precise PWV assessment with respect to other US-based approaches for local PWV assessment, such as that based on the flow-area technique13 which requires the acquisition of color-flow data in cross-sectional projection. The PWV value shown in the &#34;Representative Results&#34; section, equal to 1.69 m/sec, is in line with what is reported in reference11: in fact, in this study, abdominal aorta PWV was equal to 1.91 &#177;0.44 m/sec in adult mice and 2.71 &#177;0.63 m/sec in old animals.
In order to minimize errors in PWV assessments, great attention has to be paid when acquiring US images. In particular, B-mode images should be acquired with very clear anterior and posterior walls in order to achieve a diameter curve which is not corrupted by noise. With regard to PW-Doppler images, the angle correction should be minimized. The value of 60 degrees may represent, in most of cases, a good compromise between a good long axis view and a good approximation of velocity components. Furthermore, in the case of longitudinal studies with repeated measurements, care should be taken in imaging the vessel in the same manner, i.e., with the same scan projection.
The main limitation of the presented approach concerns the fact that the images required for the diameter and mean velocity waveform assessments are not acquired simultaneously. This lack of simultaneity can represent a source of error for PWV assessment and make the measurement less precise. In addition, a real simultaneous acquisition would guarantee a better time alignment between the two curves and avoid problems linked to heart rate variability. From a practical point of view, another limitation could be the lack of availability of the high frame rate ECG-gated modality. This problem can be partially overcome by acquiring images in B-mode modality and adjusting the acquisition parameters in order to achieve the highest temporal resolution. In this case, the diameter signal should be divided by the ECG base and processed in the same manner as the velocity signal, in order to obtain a single beat diameter waveform. However, in some conditions, the obtained temporal resolution will not be appropriate to achieve a valid diameter signal. Future modifications of the image processing chain aimed to overcome these limitations would improve the technique and lead to a more precise assessment of local arterial stiffness.
Future applications of the presented approach would concern other arterial districts. Effectively, thanks to the fact that it requires the acquisition of B-mode and PW-Doppler images only, this method can be easily applied to other arterial sites, such as the carotid artery, thus providing a multi-site arterial stiffness assessment. A higher frequency probe should be chosen for more superficial arteries such as the common carotid; in any case, the choice of the probe should ensure the correct visualization of the artery for the animals in the study. In conclusion, the described system might offer a simple way to assess functional properties of different arteries in mouse models.

Mouse models are increasingly employed for the investigation of cardiovascular disease (CVD) and particularly used in longitudinal studies which allow the characterization of different phases of disease development1. Elastic properties of large arteries are related to different pathological conditions; from a technical point of view, arterial stiffness can be assessed by measuring pulse wave velocity (PWV), which represents the speed with which the pulse wave travels in a conduit vessel2. Because of its clinical significance, it is increasingly measured even in preclinical small animal models3.
Different techniques are available for assessing PWV in mice. Invasive approaches are based on the use of catheter-tip pressure transducers. PWV is assessed by acquiring pressure signals at two different arterial sites and dividing the distance between the two measurement sites by the time shift between the signals4. The main disadvantage related to these kinds of techniques is that they require animal sacrifice for the evaluation of the distance between the two measurement sites and, thus, cannot be used in longitudinal studies. To overcome this limitation, non-invasive approaches, based on different imaging techniques, have been developed. Previous studies have reported PWV assessments in mice obtained by applying the transit time method on velocity-encoded magnetic resonance imaging data5 and Pulsed-Doppler signals6. However, the PWV value obtained with these methods is a regional evaluation of arterial stiffness. In fact, it represents an average value, accounting for different arteries in terms of size and elastic properties. In addition, these kinds of evaluations require the assessment of the distance between the two measurements sites which is a source of error that could influence the final result.
PWV can be assessed using the diameter-velocity (lnD-V) loop7. This method is based on the simultaneous evaluation of diameter and flow velocity values in a selected vessel. According to this approach, the lnD-V loop is obtained by plotting natural logarithm diameter values vs mean velocity values and PWV is estimated by calculating the slope of the linear part of the obtained loop corresponding to the early systolic phase. With regard to the practical implementation of this method, previous works have already reported results about its application in an in vitro set-up system7 and its use for the assessment of both carotid and femoral PWV in humans8.
The principal aim of the present study is to provide a detailed description of an image processing algorithm that provides a non-invasive arterial PWV measurement in mice using US images only. The proposed approach allows the evaluation of local arterial stiffness by means of the processing of both B-mode and Pulsed-Wave Doppler (PW-Doppler) images and can be applied on arteries of key importance, such as the abdominal aorta.

<table><tbody><tr><td>VEVO2100</td><td>FUJIFILM VisualSonics Inc, Toronto, Canada</td><td></td><td>micro-ultrasound equipment</td></tr><tr><td>MS250 Ultrasound Probe</td><td>FUJIFILM VisualSonics Inc, Toronto, Canada</td><td></td><td>micro-ultrasound probe</td></tr><tr><td>EKV Software</td><td>FUJIFILM VisualSonics Inc, Toronto, Canada</td><td></td><td>Software</td></tr><tr><td>Matlab R2015a&amp;nbsp;</td><td>MathWorks Inc, Natick, MA, USA</td><td></td><td>Software</td></tr><tr><td>Conductive Paste</td><td>Chosen by the operator</td><td></td><td>Laboratory material</td></tr><tr><td>Petroleum Jelly</td><td>Chosen by the operator</td><td></td><td>Laboratory material</td></tr><tr><td>Depilatory Cream</td><td>Chosen by the operator</td><td></td><td>Laboratory material</td></tr><tr><td>Acoustic Coupling Gel&amp;nbsp;</td><td>Chosen by the operator</td><td></td><td>Laboratory material</td></tr><tr><td>Developed Matlab Software</td><td></td><td></td><td>The authors are willing to collaborate with those researchers who are interested in the software and to make the software available under their supervision</td></tr></tbody></table>

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.

The proposed approach has been applied to mice abdominal aorta in a previous study11. The following figures show the results of the application of the described approach on real mice images. These data are from a single animal (wild type mice, 13 weeks old, strain: C57BL6, weight: 33 g) In particular, Figure 1 represents the result of the analysis of the US images. Edge detection and contour tracking techniques applied to B-mode images acquired with the high frame rate ECG-gated modality provide the diameter waveform; on the other hand, the identification of the PW-Doppler signal envelope leads to the single-beat mean velocity curve assessment. The evaluation of the single-beat mean velocity waveforms includes the average of data from different cardiac cycles. For the data shown, the standard deviation of the velocity curves (calculated as the average of the standard deviation obtained at each time point) is 0.0137 m/sec.
Single-beat diameter and mean velocity waveforms are interpolated in both the frequency and time domain and then time aligned (Figure 2A). The lnD-V loop is obtained by plotting the natural logarithm diameter values vs. the mean velocity measurements, as shown in Figure 2B. PWV is assessed by calculating the slope of the linear part of the loop, which is known to correspond to the early systolic phase. This portion is automatically identified as that corresponding to the upslope of the mean velocity curve. These figures indicate that the image processing operations required for the implementation of the proposed technique leads to a final lnD-V loop which is similar to that obtained in humans using a similar approach7. This suggests that this technique could represent a valid alternative for non-invasive PWV assessment in mice.
<img alt="Figure 1" src="/files/ftp_upload/54362/54362fig1.jpg" /> 
Figure 1: Processing of B-mode and PW-Doppler Images. B-Mode images (a) are processed using edge detection and contour tracking techniques. PW-Doppler images (b) are processed for the identification of the envelope signal from which the single-beat mean velocity waveform is obtained. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54362/54362fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54362/54362fig2.jpg" /> 
Figure 2: Implementation of lnD-V Loop for PWV Calculation. Diameter and mean velocity waveforms obtained from B-mode and PW-Doppler image processing. (a). The lnD-V loop is obtained by plotting the natural logarithm of the diameter values against the mean velocity values (b). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54362/54362fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ultrasound-based Pulse Wave Velocity Evaluation in Mice at JoVE.com

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

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 ...

ultrasound-based-pulse-wave-velocity-evaluation-in-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

13.2K Views.  Scuola Superiore Sant'Anna.  Arteriële stijfheid is een belangrijke factor bij hart-en vaatziekten en pulsgolfsnelheid (PWV) kan als een surrogaat index voor arteriële stijfheid worden beschouwd. Dit protocol beschrijft een beeld processing algoritme voor het berekenen van PWV in muizen op basis van echografie beeldverwerking die van toepassing zijn op verschillende arteriële sites is. 

Video: Ultrasound-based Pulse Wave Velocity Evaluation in Mice

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 ...

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 ...

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

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, ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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.

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 ...

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 ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

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