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 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 ...

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

Research

JoVE Journal

Medicine

12.9K Views.  Scuola Superiore Sant'Anna. 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.

Video: Ultrasound-based Pulse Wave Velocity Evaluation in Mice

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 ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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 ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively by the measurement of carotid to femoral pulse wave velocity (cf-PWV) (Class I; Level of Evidence A). In literature, numerous community-based or disease-specific studies have reported that higher cf-PWV is associated with increased cardiovascular risk. Here, we discuss strategies to evaluate arterial stiffness with cf-PWV. Following the well-defined steps detailed here, e.g., proper position operator, distance measurement, and tonometer position, we will obtain a standard cf-PWV value to evaluate arterial stiffness. In this paper, a detailed stepwise method to record a good quality PWV and pulse wave analysis (PWA) using a non-invasive tonometry-based device will be discussed.

Measuring the Carotid to Femoral Pulse Wave Velocity Cf-PWV to Evaluate Arterial Stiffness

This protocol describes a method to standardize the measurements of carotid-femoral pulse wave velocity to evaluate arterial stiffness.

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...