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. Mo...

<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>

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 req...

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 border="0" cellpadding="0" cellspacing="0" width="772">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">VEVO2100</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td style="width:177px;">micro-ultrasound equipment</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">MS250 Ultrasound Probe</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td>micro-ultrasound probe</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:216px;">EKV Software</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matlab R2015a&#160;</td>
			<td style="width:260px;">MathWorks Inc, Natick, MA, USA</td>
			<td style="width:119px;"></td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conductive Paste</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petroleum Jelly</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Depilatory Cream</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acoustic Coupling Gel&#160;</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developed Matlab Software</td>
			<td style="width:260px;"></td>
			<td style="width:119px;"></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 wit...

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 ...

The overall goal of this procedure is to provide an image processing algorithm for assessing arterial stiffness in mice based on the ultrasound imaging of different arterial sites. Arterial stiffness is associated with cardiovascular disease and is increasing used as therapeutic target due to its prognostic value. After confirming the appropriate levels of sedation by toe pinch, place the animal on a 40 degree Celsius temperature controlled board in the supine position and moisten the animals eyes with ointment.Coat the four limbs of the animal with conductive paste and tape the limbs onto the ECG electrodes embedded in the board. Measure the body temperature with a rectal probe lubricated with petroleum jelly and confirm that all of the physiological measurements are correctly displayed. Next, use depilatory cream to chemically remove the hair from the abdomen and coat the exposed skin with acoustic coupling gel.Place a 13 to 24 mega hertz ultrasound probe in the mechanical arm and fix the probe parallel to the animal. Adjust the probe so that the desired longitudinal images of the abdominal aorta located in the focal zone can be obtained and select high frame rate ECG gate at acquisition with a frame rate acquisition of 700 frames per second. Start the acquisition, then using the same scan projection select pulse wave doppler and move the sample volume to the center of the vessel.Obtain images with the cineloop of at least three seconds, maintaining a small an angle correction as possible. After all of the images have been acquired, remove the animal from the board with monitoring until full recovery. For post processing analysis of the ultrasound data, first export the B mode and post wave doppler images as dicom files.After saving the files, convert the post wave doppler dicom files to tif images Next using the dedicated graphical user interface, initialize the contours by drawing a line close to the far wall of the vessel and double click close to the near wall. A line parallel to the drawn line will automatically appear. Click analyze to apply the algorithm on a single frame.If the edges have been correctly identified click go"to apply the algorithm to the whole cineloop. Then click record"and save the corresponding mat file containing the instantaneous diameter values related to a single cardiac cycle. For the diameter velocity loop implementation, open the dedicated graphical user interface.To begin the pulse wave doppler image processing, click velocity"generating a single beat mean velocity curve. After identifying the post wave doppler trace, select white line"to locate the line corresponding to a velocity value equal to zero. Then, in the calibration panel, click velocity"to draw lines with lengths that correspond to the calibration factor.Repeat the operation for the time calibration clicking time. Using the region of interest physio button, manually select a region of interest containing the physiological signals, then click region of interest signal to manually select a region of interest containing the post wave doppler trace. Select analyze"to check if the envelope is identified.If the result is not satisfactory, enter the new value in the velocity threshold editable text field and analyze the envelope again. Next, click elaboration"and check for errors in our peaks detection of the ECG signal. Change the minimum peaks distance value, and then click update"for a correct identification of our peaks.Click choose beats, select all of the beats that are uncorrupted by noise or located in the inspiration phase. Press the return button on the keyboard to re-sample selected beats and to obtain a single beat velocity wave form, then select the mean velocity check box to achieve a single beat mean velocity signal and click okay. Now click diameter"and press the interpolate button on the keyboard to obtain a single beat diameter signal with the same sample frequency as the single beat velocity signal.Click okay, then select the second derivative approach as the alignment method and click update. The two curves will be automatically time aligned using the second derivative method. The natural logarithm of the single beat diameter values are automatically plotted against the single beat mean velocity values.And the corresponding pulse wave value is assessed. In this image, edge detection and contour tracking techniques were applied to B mode images acquired with a high frame rate, ECG gated modality to provide the diameter wave form. Identification of the post wave doppler signal envelope and the average of data from different cardiac cycle leads to the single beat mean velocity curve assessment.Single beat diameter and mean velocity wave forms are interpolated in both the frequency and time domain and then time aligned. A diameter velocity loop is obtained by plotting the natural logarithm diameter values against the mean velocity measurements, allowing assessment of the pulse wave velocity value. This is possible by calculating the slope of the linear part of the loop, which is known to correspond to the early systolic phase as it happens in humans using a similar approach.to assess local arterial stiffness, noninvasively.

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

Research

JoVE Journal

Medicine

12.8K Views.  Scuola Superiore Sant'Anna. The overall goal of this procedure is to provide an image processing algorithm for assessing arterial stiffness in mice based on the ultrasound imaging of different arterial sites. Arterial stiffness is associated with cardiovascular disease and is increasing used as therapeutic target due to its prognostic value. After confirming the appropriate levels of sedation by toe pinch, place the animal on a 40 degree Celsius temperature controlled board in the supine position and moisten the animals...