This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, which is funded by the Ministry of Education (2021R1I1A3040346, 2020R1A4A1019475, 2021R1C1C1003481, and HI19C0760). This study was also supported by 2018 Research Grant (PoINT) from Kangwon National University.

NOTE: The protocol is largely composed of three stages: (1) model fabrication, (2) MRI scan and parameter selection, and (3) data analysis. Figure 1 is a flow diagram showing the overall process of the protocol.
1. Model fabrication
<ol>
	<li>Aortic root model
	<ol>
		<li>As shown in Figure 2, determine the parameter values of the aortic root, such as valve base diameter and sinus radius. For this experiment, the values were DA = 32.24 mm, DO = 26 mm, LB = 8.84 mm, LA = 26 mm, rmin = 16.64 mm, rmax = 21.32 mm.</li>
		<li>Run the 3D modeling software by clicking Sketch &#62; Tools Sketch Tools &#62; Sketch Picture. 
		NOTE: Solidwork is used for 3D modeling in the experiment.</li>
		<li>To make a sinus model, sketch circles corresponding to rmax and rmin using the circle tool. Draw a curved line of the sinus using the free curve function11, click Loft Tool and select the sketch area for loft.</li>
		<li>Sketch additional circles on the top and bottom of the current model, click Extrude Tool, and select the circles. Set the options as 20 mm downward and 30 mm upward. Make a hexahedron model of size 100 mm x 100 mm x 76 mm in the same way.</li>
		<li>Click Combine Tool from Insert &#62; Features &#62; Combine. Select Subtract in the property manager. Select the hexahedron model and the sinus model. Fabricate the final design as an acrylic model with a 5-axis CNC machine as per manufacturer&#39;s instruction.</li>
	</ol>
	</li>
	<li>Valve frame
	<ol>
		<li>Run 3D modeling software and open a new sketch. Draw a square of size 100 mm x 100 mm and a circle of 25 mm in the center for the valve base, manually. Click the Extrude Tool and adjust the height of the valve base to 5 mm.</li>
		<li>Extrude the circle with a height of 23.5 mm and a thickness of 3 mm thick. Divide the model into 12 uniform pieces using Line Tool so that each piece has 30&#176;. Select three pieces with 120&#176; intervals and extrude with a height of 16.5 mm to make three pillars.</li>
		<li>Click Fillet Tool and select the pillars. Adjust the fillet radius at the top and bottom as 4 mm and 10 mm, respectively. Save it in an STL file format.</li>
		<li>3D-print the valve frame. Set the infill density to 100% and use acrylonitrile butadiene styrene as fill material. See Figure 3 for the shape and dimensions of the aortic valve frame.</li>
	</ol>
	</li>
	<li>Aortic regurgitation model using expanded polytetrafluoroethylene (ePTFE)
	<ol>
		<li>Run the 3D modeling software and open a new sketch. Draw a horizontal line of 23.24 mm and a vertical line of 15 mm with reference to Figure 4A. 
		NOTE: The geometric parameters of the valve&#39;s base, height, and leaflet free-edge length were chosen according to a previous study12.</li>
		<li>Click 3 Point Arc Tool from the arc command manager and set two points on each end of the horizontal line and the last point on the end of the vertical line. Extrude the sketch with a thickness of 5 mm. Export the model with STL file format and 3D print it.</li>
		<li>Overlap the ePTFE membrane in two layers and draw three leaflet borders at intervals of 2 mm using the printed leaflet. Suture along the drawn lines and side borders at 1 mm intervals with a polyamide suture with a diameter of 0.1 mm. Suture the ePTFE valve from top to bottom on the frame at 1 mm intervals.</li>
		<li>Cut the outer side of the membrane and suture it with each other. Perform the following three modifications to obtain three different models.
		<ol>
			<li>Dilatation model: Reduce the ratio of the designed leaflet parameters to 90%.</li>
			<li>Perforation model: Make a circular hole with a diameter of 2 mm using scissors in the center of one leaflet.</li>
			<li>Prolapse: Fix the two commissures of the valve at a hole with a low post height. 
			NOTE: Figure 4 shows the materials and fabrication method of ePTFE valve. Figure 5 shows the characteristics of each AR-type.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. MRI scan and parameter selection
<ol>
	<li>Prepare the experimental system consisting of an AR model, aortic sinus model, a heart simulation pump, and MRI.</li>
	<li>Set the experiment models in the MRI room and connect the pump, reservoir, and models using a 25 mm (inner diameter) silicone tube. Use a 10 cm long cable tie to fasten the connection parts to prevent possible leakage.</li>
	<li>Use a motor-controlled piston pump to simulate the aortic blood flow waveforms to generate a physiological flow waveform through the flow circuit system. Use water as the working fluid and attach one-way valves to the inlet and outlet to prevent backflow. Details of the flow pump can be found in the previous study23.</li>
	<li>Locate the model within the field of view (FOV) of the MRI. Perform a scout scan to observe phantom images in the coronal, axial, and sagittal views in MRI operating console monitor. This image is used as a guide to position the following image sequences.</li>
	<li>Locate the 2D image plane in the center of the aorta model. Run a variable velocity-encoding parameter (VENC) 2D phase-contrast imaging to select the most appropriate VENC value for 4D flow MRI.</li>
	<li>Set VENC to a 10% higher value in 4D flow MRI to minimize possible velocity aliasing7. Enter the desired spatial resolution and the temporal resolution on the MRI console. The spatial and temporal resolution for the aortic flow is recommended to be 2-3 mm and 20-40 ms, respectively7. Table 2 shows the MRI scan parameters.</li>
	<li>Acquire data for both with and without flow using the 3 types of AR valves and the without valve.</li>
</ol>
3. Data analysis
<ol>
	<li>Data sorting and correction
	<ol>
		<li>Copy raw data files from the scanner to proceed with the data analysis. Sort the dicom files according to the header named series description using the Dicom sort software. Click Sort Images in Dicom sort software to sort three-directional phase images and magnitude images in separate folders.</li>
		<li>Load magnitude image into the ITK-snap software. Click Brush in the ITK-snap and manually paint the internal fluid region of the phantom using the brush tool. Save segmented image.</li>
		<li>(Optional) Load both phase image data obtained with the flow on and off using MATLAB. Subtract the data with the flow by the data without flow to remove background errors. Repeat this for every direction and cardiac cycle.</li>
		<li>Calculate the velocity of 5D matrix phase data (row x column x slice x direction x time) using a vendor-specific pixel-to-velocity equation. In general, the maximum intensity of the pixel corresponds to the selected VENC value.</li>
	</ol>
	</li>
	<li>Visualization
	<ol>
		<li>Load the 5D matrix velocity from step 3.1.4 into flow visualization analysis software. 
		NOTE: Input velocity matrix may vary according to the analysis software. Ensight users should follow Ensight gold case format guide13.</li>
		<li>Click the Isosurface Part, change the data type from isosurface to isovolume for 3D analysis by clicking the Isovolume button. Drag the speed data in the variables command manager, add it to the isovolume to check the velocity distribution of the model.</li>
		<li>Click Particle Trace Emitters Tool in the main menu. Check Advanced Option for a more accurate analysis. Select the desired visualization such as Streamlines or Pathlines in creation.</li>
		<li>For this experiment, set the following value: Emit From Option = Part, Part ID = 2, No. of Emitters = 10000, Direction = +/-. Create and check the results over time.</li>
		<li>Right-click the Particle Trace model and click the Color by. Select the velocity component to color the streamline with the velocity.</li>
	</ol>
	</li>
	<li>Quantification
	<ol>
		<li>Load the velocity data (step 3.1.4) and segmented image (step 3.1.2) onto MATLAB. Set the velocity outside of the segmentation region to zero. This can be easily performed by elementwise multiplying the segmented matrix data and the velocity matrix data.</li>
		<li>Check if the velocity data has phase-wrapping using the Imshow function of MATLAB. Inversion of the velocity direction indicates phase-wrapping.</li>
		<li>Slice the desired plane of the matrix data. Sum all velocity data within the plane and multiply spatial resolution to calculate the flow rate through the plane. Sum all flow rates throughout the cardiac cycle and multiply the temporal resolution to calculate the stroke volume.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Four-dimensional flow MRI has recently been verified by various ex vivo and in vivo studies as an application for clinical routine use14. As the 4D flow MRI obtains 3D velocity information over the entire cardiac cycle, one strong application is a direct quantification of the valvular regurgitant volume, which conventional 2D Doppler echocardiography is not capable of quantifying15. In vitro experiments using 4D Flow MRI can provide the 3D flow velocity and related hemodynamic parameters which can be used for investigating the relationship between cardiovascular disease and hemodynamics. However, despite its promising capability, no systematic studies on this application have been reported yet. This is possibly due to the lack of well-controlled in vitro experiments that mimic the regurgitation of the tri-leaflet valves.
Recent developments in in vitro studies have provided more accurate and realistic experimental methods to access the pre- and post-valvular hemodynamics16,17. Coupled with an optical image-based particle image velocimetry (PIV), accurate measurement and quantification of the flow around the valve was possible in previous in vitro studies18. However, accurate 3D flow fields, especially for the post-valvular flow, were limited owing to the opaque model and refraction. On the other hand, 3D velocity measurements using MRI were also limited, as metal components cannot be used19,20.
Hence in this study, a protocol to build a flow experimental platform that is MR compatible and highly modifiable to reproduce various clinical scenarios of valvular diseases is introduced. The ePTFE membrane is used to mimic the tricuspid valve without metal components as it has been widely used as a valve and vascular graft material due to its high tensile strength and chemical resistance17,21,22. Based on ePTFE films, three different origins of the AR have been reproduced (dilatation, perforation, and prolapse) as well as a model without a valve for comparison. The next important step in this flow experimental protocol is MR imaging and quantification. A motor-controlled piston pump that can simulate the aortic blood flow waveforms is used to generate a physiological flow waveform through the flow circuit system. Details of the flow pump can be found in the previous study23. As this study also aims to validate the accuracy of the 4D flow MRI in flow quantification, all the imaging parameters are selected based on the previous study which summarizes the parameters that can be used in the clinical routine24. As the MRI system includes inherent errors due to imperfections such as eddy currents and nonlinearity of the magnetic field25, the background correction strategy is applied prior to the actual data quantification as described in step 3.1.3.
The hand-made aortic regurgitation model suggested in this study showed similar hemodynamic characteristics of regurgitant jet according to model classification as previous studies reported26,27. The closed shape was symmetrical, and a straight jet occurred at the center of the valve in the dilatation model. A posteriorly directed eccentric jet appears owing to cusp damage in the perforation model. Partial prolapse of the valve shows a jet whose direction was bent from the culprit cup owing to limited mobility. The aortic regurgitation volume directly measured using the 4D flow MRI was overestimated in the without valve and dilatation model, while it was largely underestimated in the prolapse model when compared with the ground truth. However, when the regurgitant fraction was calculated, the largest bias was only 11% in the prolapse model. This strongly indicates that not only the regurgitant flow but also the normal aortic jet was affected by the MR scan. At the current stage, individual scan parameters were not optimized for each AR model. A future systemic parameter study may improve the accuracy of regurgitant volume measurement. Alternatively, the use of regurgitant fraction is more robust as it cancels out the inherent errors in 4D flow MRI but also is clinically more relevant than simply measuring the absolute regurgitant volume.
In conclusion, this study suggests an MR compatible in vitro flow experimental model that is highly modifiable to simulate various types of AR. Also, the accuracy of AR volume measurement using 4D flow MRI was compared. The limitation of this study is that the motion of the aortic valve was not simulated, which can affect the actual development of the regurgitant jet. In addition, the partial volume effect and temporal averaging nature of the 4D-flow MRI may limit the accuracy of the flow measurement, especially considering the high dynamic range of velocity within the jet and surroundings. Therefore, further systematic parameter study is required.

Aortic regurgitation (AR) refers to the backward flow from the aorta into the left ventricle during the diastolic phase of the ventricle. AR is typically classified into aortic dilatation, cups prolapse, cups perforation, cups retraction, and others1. Chronic AR may cause the volume overload of the LV mainly due to hypertrophy and dilatation, and eventually causes its decompensation2. Acute AR is mainly caused by infectious endocarditis, aortic dissection, and traumatic rupture, which leads to hemodynamic emergencies2.
Current clinical standards for AR diagnosis are mainly based on transthoracic echocardiography (TTE) or transesophageal echocardiography (TEE)3. Despite the advantages of real-time imaging and short exam time, the accuracy of echocardiography is highly operator-dependent. Especially for the regurgitant volume measurement, direct measurement of the regurgitant volume is limited as the regurgitant jet shifts out of the two-dimensional (2D) measurement plane due to the motion of the aortic valve. Indirect estimation using proximal iso-velocity surface area (PISA) methods are often used, but assumptions such as circular orifice area often limit the accurate measurement4.
Recent medical guidelines5 also recommend cardiac MR (CMR), especially for moderate or severe AR patients to compensate for the limitation of echocardiography by measuring the mass and global function of the LV. Structural parameters such as aortic leaflets and LV size, and flow parameters such as jet width, vena contracta width, and regurgitant volume can also be comprehensively considered in AR diagnosis6. However, aortic regurgitation volume estimated with the LV global function may fail especially for patients with other heart valvular diseases or shunt.
Alternatively, 4D flow MRI has been considered as a promising technique that can directly measure the regurgitant volume with time-resolved velocity information within the volume of interest7. The motion of the valve according to the time can be easily tracked and compensated when measuring the regurgitant flow volume8,9. Also, an arbitrary plane perpendicular to the regurgitant jet can be retrospectively positioned, which increases the accuracy of the measurement10. However, as the 4D flow MRI inherently obtains the spatiotemporally averaged information, the accuracy of this technique still warrants validation by using well-controlled in vitro flow experiments.
This study aims to (i) develop MRI compatible in vitro experimental platform that can reproduce the different clinical scenarios of AR (dilatation, perforation, and prolapse) and (ii) enrich our understanding of 4D flow MRI performance in quantifying different AR at these AR models. In addition, 3D hemodynamic visualization and quantification based on 4D flow MRI were conducted according to the various clinical scenarios. This protocol is not limited to AR and can be extended to other types of valvular disease studies that require a series of in vitro experiments and hemodynamic quantification.

<table><tbody><tr><td>3D modeling software(SolidWorks)</td><td>Dassault Syst&amp;egrave;mes SolidWorks Corporation</td><td></td><td>Waltham, MA, USA</td></tr><tr><td>3D printer</td><td>Zortrax S.A.</td><td></td><td>the construction of a three-dimensional object from a CAD model or a digital 3D model,(zortrax m200 plus, Zortrax S.A.,Olsztyn, Poland)</td></tr><tr><td>Dicom sort</td><td>Open source software</td><td></td><td>Jonathan Suever, Software Engineer</td></tr><tr><td>Ensight</td><td>Ansys</td><td></td><td>Flow visualization software (Canonsburg, PA, USA).</td></tr><tr><td>Expanded Polytetrafluoroethylene(ePTFE)</td><td>SANG-A-FRONTEC</td><td></td><td>Medical membrane (ePTFE,SANG-A-FRONTEC, Incheon, korea)</td></tr><tr><td>Itk snap software</td><td>Open source software</td><td></td><td>GNU General Public License,</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td>Natick, MA, USA</td></tr><tr><td>MRI</td><td>Siemens</td><td></td><td>3T, Erlangen, Germany</td></tr><tr><td>Scissors</td><td>Scanlan International Inc</td><td>n43 1765</td><td>7007-454, Scanlan International Inc., Saint Paul, USA</td></tr><tr><td>Suture</td><td>AILEE</td><td>NB530</td><td>Ailee, Polyamide suture, UPS 5-0</td></tr></tbody></table>

in vitro assessment of aortic regurgitation using four-dimensional flow magnetic resonance imaging

Aortic regurgitation (AR) refers to backward blood flow from the aorta into the left ventricle (LV) during ventricular diastole. The regurgitant jet arising from the complex shape is characterized by the three-dimensional flow and high-velocity gradient, sometimes limiting an accurate measurement of the regurgitant volume using 2D echocardiography. Recently developed four-dimensional flow magnetic resonance imaging (4D flow MRI) enables three-dimensional volumetric flow measurements, which can be used to accurately quantify the amount of the regurgitation. This study focuses on (i) magnetic resonance compatible AR model fabrication (dilatation, perforation, and prolapse) and (ii) systematic analysis of the performance of 4D flow MRI in AR quantification. The results indicated that the formation of the forward and backward jets over time was highly dependent on the types of AR origin. The amount of regurgitation volume bias for the model types were -7.04%, -33.21%, 6.75%, and 37.04% compared to the ground truth (48 mL) volume measured from the pump stroke volume. The largest error of the regurgitation fraction was around 12%. These results indicate that careful selection of imaging parameters is required when absolute regurgitation volume is important. The suggested in vitro flow phantom can easily be modified to simulate other valvular diseases such as aortic stenosis or bicuspid aortic valve (BAV) and can be used as a standard platform to test different MRI sequences in the future.

Three representative classes of aortic regurgitation models were fabricated, and one case without a valve was fabricated for comparison (Figure 3). The dilation model clearly showed incomplete closure of the valve leaflet due to the smaller-sized leaflets. A hole was punctured on one of the leaflets using scissors to mimic the perforation model. One leaflet of the prolapse model looked smaller than the other two leaflets because the two commissures were sutured at a position lower than the original height. There were no significant differences from the top view.
With the 3D velocity information acquired over time using 4D flow MRI, streamlines of normal and regurgitation jets were visualized during systole and diastole (Figure 6). The forward jet was straight in all models except for the perforation model. In the perforation model, a wall-biased jet occurred during the systole phase. The regurgitating jet showed a different velocity and shape according to the AR classification. In the case of without a valve, an overall forward and backward flow occurred. The regurgitant jet of the dilation model came out from the center and tended to change directions over time. The perforation and prolapse model regurgitant jet leaned toward the wall. The peak velocity of the forward and regurgitant jet was 0.28 m/s, -0.29 m/s in the model without valve, 2.03 m/s, -3.53 m/s in the dilatation model, 2.52 m/s, -3.13 m/s in the perforation model, and 2.76 m/s, -2.88 m/s in the prolapse model.
Figure 7&#160;shows the flow rate for each valve and the forward and regurgitant volumes in a 3D plane away from the valve base. The flow rates showed different waveforms and quantities for each model. The amount of regurgitation volume was 51.38 mL, 63.94 mL, 44.76 mL, and 30.22 mL for without valve, dilatation, perforation, and prolapse models, respectively. The bias for without valve, dilatation, perforation, and prolapse model were -7.04%, -33.21%, 6.75%, and 37.04%, respectively, compared to the ground truth (48 mL) measured from the pump stroke volume. The positive percentage values indicate underestimation while the negative percentage values represent over estimation. The regurgitation fraction error was -7.78%, -6.00%, 0.33% and -11.18% for without valve, dilatation, perforation and prolapse model, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63491/63491fig01.jpg" /> 
Figure 1: Workflow diagram of the protocol. This experimental protocol mainly consists of model fabrication, MRI scan, and data analysis. In the model fabrication step, the outer aortic root model and four different types of AR model (without valve, dilatation, prolapse, and perforation) are fabricated. During the MRI scan, scout imaging followed by multi-VENC scan and 4D flow MRI is performed. The data analysis part includes data sorting, image segmentation, velocity calculation, visualization, and quantification. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63491/63491fig02.jpg" /> 
Figure 2: Schematic and designed acrylic model of the aortic root (A) Geometrical characterization and parameters of the aortic root geometry. (B) Aortic root 3D model in multi-dimensional view. DA: diameter of sinotubular junction (STJ), DO: diameter of annulus, rmax: maximum sinus diameter, rmin: minimum sinus diameter, LA: height of sinus, LB: height of STJ. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63491/63491fig03.jpg" /> 
Figure 3: Aortic regurgitation frame and model (A) Geometrical information of the aortic valve frame which is used to hold the leaflet. Holes around the body of the frame is where the suture line passes. (B) Example of ePTFE membrane sutured valve. (C) En-face view of the in vitro models: without valve, dilation, perforation and prolapse fabricated in this study. The arrow indicates the damaged cusp. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63491/63491fig04.jpg" /> 
Figure 4: Material and fabrication step of ePTFE leaflet. (A) Using 3D printed leaflets as a guide, leaflets are made using ePTFE membrane. (B) Drawing, suturing, cutting and fixing steps of the ePTFE valve. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63491/63491fig05.jpg" /> 
Figure 5: Fabrication methods of different AR models. (A) Dilatation model, (B) perforation model, and (C) prolapse model. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63491/63491fig06.jpg" /> 
Figure 6: Streamline visualization according to aortic regurgitation type. A streamline visualization at systole (left of each panel) and diastole (right of each panel) according to aortic regurgitation type. (A) Model without valve (the diastole/systole image are the same due to lack of a valve), (B) dilatation, (C) perforation, and (D) prolapse. Systole and diastole data were taken where the inlet velocity is the highest and the lowest during the cardiac cycle. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63491/63491fig07.jpg" /> 
Figure 7: Flow rate and stroke volume. The flow rate and stroke volume for (A) model without valve, (B) dilatation, (C) perforation, and (D) prolapse. The flow rate and stroke volume are measured at the plane (solid line) three-diameter downstream to the valve annulus.&#160;The blue and red colors indicate the forward and regurgitating flows, respectively. <a href="https://www.jove.com/files/ftp_upload/63491/63491fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Ratio 
			(Do= 26 mm)</td>
			<td>DA/Do</td>
			<td>LA/Do</td>
			<td>LB/Do</td>
			<td>rmax/Do</td>
			<td>rmin/Do</td>
		</tr>
		<tr>
			<td>1.24</td>
			<td>1</td>
			<td>0.34</td>
			<td>0.82</td>
			<td>0.64</td>
		</tr>
	</tbody>
</table>
Table 1. Geometrical parameters of the aortic root geometry shown in Figure1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Temporal resolution</td>
			<td>0.025 ms/40 phases</td>
		</tr>
		<tr>
			<td>Spatial resolution</td>
			<td>2mm x 2mm/0.5 pixel per 1 mm</td>
		</tr>
		<tr>
			<td>Matrix</td>
			<td>96 x 160 x 26 pixel</td>
		</tr>
		<tr>
			<td>Slice thickness</td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Echo time</td>
			<td>2.54 ms</td>
		</tr>
		<tr>
			<td>Encoding velocity</td>
			<td>25-330 cm/s</td>
		</tr>
	</tbody>
</table>
Table 2. 4D Flow MRI sequence parameters in vitro.

Watch this Scientific Journal Video about In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging at JoVE.com

In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

Aortic regurgitation is an aortic valve heart disease. This manuscript demonstrates how four-dimensional flow magnetic resonance imaging can evaluate aortic regurgitation using in vitro heart valves mimicking aortic regurgitation.

In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

Aortic regurgitation (AR) refers to backward blood flow from the aorta into the left ventricle (LV) during ventricular diastole. The regurgitant jet ...

in-vitro-assessment-aortic-regurgitation-using-four-dimensional-flow

Research

JoVE Journal

Engineering

3.2K Views.  Kangwon National University. Aortic regurgitation is an aortic valve heart disease. This manuscript demonstrates how four-dimensional flow magnetic resonance imaging can evaluate aortic regurgitation using in vitro heart valves mimicking aortic regurgitation.

Video: In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

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 ...

Medicine

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 ...

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 ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Bioengineering

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

Quantitative MRI of Endothelial Permeability and Dysfunction in Atherosclerosis

We have developed an accurate, non-invasive, and easy-to-use method to quantify endothelial permeability and dysfunction in the arteries using Magnetic Resonance Imaging (MRI), named qMETRIC. This technique enables assessing vascular damage and cardiovascular risk associated with atherosclerosis in preclinical models and humans.

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

Four-Dimensional Computed Tomography-Guided Valve Sizing for Transcatheter Pulmonary Valve Replacement

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve replacement (TPVR), vary considerably. Three-dimensional (3D) computed tomography (CT) imaging for device size prediction is insufficient to assess the displacement of the right ventricular outflow tract (RVOT) and PA, which could increase the risk of stent misplacement and paravalvular leak. The aim of this study is to provide a dynamic model to visualize and quantify the anatomy of the RVOT to PA over the entire cardiac cycle by four-dimensional (4D) cardiac CT reconstruction to obtain an accurate quantitative evaluation of the required valve size. In this pilot study, cardiac CT from sheep J was chosen to illustrate the procedures. 3D cardiac CT was imported into 3D reconstruction software to build a 4D sequence which was divided into eleven frames over the cardiac cycle to visualize the deformation of the heart. Diameter, cross-sectional area, and circumference of five imaging planes at the main PA, sinotubular junction, sinus, basal plane of the pulmonary valve (BPV), and RVOT were measured at each frame in 4D straightened models prior to valve implantation to predict the valve size. Meanwhile, dynamic changes in the RV volume were also measured to evaluate right ventricular ejection fraction (RVEF). 3D measurements at the end of the diastole were obtained for comparison with the 4D measurements. In sheep J, 4D CT measurements from the straightened model resulted in the same choice of valve size for TPVR (30 mm) as 3D measurements. The RVEF of sheep J from pre-CT was 62.1 %. In contrast with 3D CT, the straightened 4D reconstruction model not only enabled accurate prediction for valve size selection for TPVR but also provided an ideal virtual reality, thus presenting a promising method for TPVR and the innovation of TPVR devices.

This study assessed a new methodology with a straightened model generated from the four-dimensional cardiac computed tomography sequence to obtain the desired measurements for valve sizing in the application of transcatheter pulmonary valve replacement.

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. Although mice are widely applied to shed light on the mechanisms of cardiovascular disease, mouse models of AR, especially those induced by surgery, are still paucity. Here, a mouse model of AR was described in detail which is surgically induced by disruption of the aortic valves under high-resolution echocardiography. In accordance with regurgitated blood flow, AR mouse hearts present a distinctive and clinically relevant volume overload phenotype, which is characterized by eccentric hypertrophy and cardiac dysfunction, as evidenced by echocardiographic and invasive hemodynamic evaluation. Our proposal, in a reliable and reproducible manner, provides a practical guide on the establishment and assessment of a mouse model of AR for future studies on molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

This protocol presents a practical guide on the surgery for creation of aortic regurgitation (AR) in the mouse. Assessment of the AR mouse by echocardiography and invasive hemodynamic measurement recapitulates its clinically relevant characteristics of volume overload-induced eccentric hypertrophy, suggesting its promising application in the study of cardiac hypertrophy.

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. ...

Biology

CFR Changes Post-Ischemia: Implications for Microvascular Dysfunction

Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice

After cardiac ischemia, there is often insufficient myocardial perfusion, even if flow has been successfully and completely restored in an upstream artery. This phenomenon, known as the "no-reflow phenomenon," is attributed to coronary microvascular dysfunction and has been associated with poor clinical outcomes. In clinical practice, a reduction in coronary flow reserve (CFR) is frequently used as an indicator of coronary artery disease. CFR is defined as the ratio of the peak flow velocity induced by pharmacologic or metabolic factors to the resting flow velocity.
This protocol focused on assessing the dynamic changes in CFR before and after ischemia-reperfusion (IR) using pulse wave Doppler measurements. In this study, normal mice exhibited the ability to increase the peak velocity of coronary blood flow up to two times higher than the resting values under isoflurane stimulation. However, after ischemia-reperfusion, the CFR at 1 h significantly decreased compared to the pre-operation baseline. Over time, the CFR showed gradual recovery, but it remained below the normal level. Despite the preservation of systolic function, early detection of microvascular dysfunction is crucial, and establishing a practical guide could aid doctors in this task, while also facilitating the study of cardiovascular disease progression over time.

Author Spotlight: Improved Localization and Monitoring of Coronary Flow Reserve Using Modified PLAX View in Mice

The present protocol describes a modified parasternal long-axis view for swift and precise localization of the left anterior descending artery. This approach is designed to be simpler and more user-friendly while facilitating the examination of dynamic changes in coronary flow reserve following myocardial ischemia-reperfusion in mice.

After cardiac ischemia, there is often insufficient myocardial perfusion, even if flow has been successfully and completely restored in an upstream ...

In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

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Chapters in this video

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

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

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

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Four-Dimensional Computed Tomography-Guided Valve Sizing for Transcatheter Pulmonary Valve Replacement

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice