This work was supported by NIH grant R01ES029967.

This study was conducted with approval of the Mount Sinai Institutional Review Board (HS-20-01011), and all subjects gave written informed consent.
1. Preparation before starting the protocol
<ol>
	<li>Appropriate folder structure is necessary for this program. Start by creating a main folder for the project anywhere on the computer by right-clicking and selecting the New Folder option in the files directory. All input DICOM files and results will be stored in this main folder. 
	NOTE: All DICOM headers required by this program are standard. Therefore, the program is translatable across scanner platforms.</li>
	<li>Within this main folder, create two new folders by right-clicking and selecting the New Folder option twice.</li>
	<li>Rename the first of these two folders to make it obvious that it stores raw data (e.g., Original_Data) by right-clicking on it and selecting the Rename option.
	<ol>
		<li>Create a new folder for a given patient within this Original_Data folder by right-clicking and selecting the New Folder option. Rename it as an anonymized patient identifier by right-clicking on the folder and selecting the Rename option. Import only the complete set of raw DICOM files for this patient into this folder.</li>
		<li>Repeat step 1.3.1 for all the patients to be analyzed in the program.</li>
	</ol>
	</li>
	<li>Rename the second of these two folders as Meta_Data for the storage of results by right-clicking on it and selecting the Rename option. This folder will be empty until the program is run and results are generated.</li>
	<li>Download the program file (Supplementary File 1) and the cover art image (Supplementary File 2) and move them from the downloads folder to the main project folder by dragging and dropping. The final setup of the project folder should look similar to Figure 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65458/65458fig01.jpg" /> 
Figure 1: Format of main project folder. This figure shows how the project&#39;s main folder should be structured and formatted for proper use of the program. <a href="https://www.jove.com/files/ftp_upload/65458/65458fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Launching the Program
<ol>
	<li>Open the program&#39;s code by double-clicking on its file in the project&#39;s main folder. This will open the software and display the program&#39;s code.</li>
	<li>Enter the editor window by single-clicking anywhere on it. Click the green Run button located in the top ribbon of the editor tab to launch the program. The initial program window, after opening, should look like the image in Figure 2. 
	NOTE: Written instructions on the expected actions from the user and the progress of result generation will be displayed in the bottom left area throughout the course of using the program.</li>
	<li>Click the Open DICOM button in the bottom left corner. This will open the files directory.</li>
	<li>Navigate to the project&#39;s main folder, the folder containing the original data, and to a patient to analyze. Single-click on the patient&#39;s folder so that it&#39;s highlighted and click Open.</li>
	<li>The images will now appear in three views: axial, sagittal, and coronal. Hovering and scrolling on a particular view will go through the slices in that view. The crosshairs on each view display the location of the pointer at that moment. Adjust the brightness and contrast in the bottom right area of the program by sliding the bars, as in Figure 3.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65458/65458fig02.jpg" /> 
Figure 2: Initial program window. The program, when initially launched, has the buttons laid out along with an art image. <a href="https://www.jove.com/files/ftp_upload/65458/65458fig02large.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/65458/65458fig03.jpg" /> 
Figure 3: Graphical user interface (GUI). Once images are loaded in, the program&#39;s GUI shows three anatomical views of the images along with crosshairs on each view, representing the cursor. <a href="https://www.jove.com/files/ftp_upload/65458/65458fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Analyzing coronary artery calcification
<ol>
	<li>When ready to start analyzing, click the 0 mg/mL Phantom button. Hover over the axial view and scroll up or down until the 0 mg/mL section of the phantom is visible.</li>
	<li>Move the cursor to the center of the 0 mg/mL phantom in the axial view. Now, without moving the cursor, observe the crosshairs in the sagittal and coronal views.</li>
	<li>Slowly scroll up and down a few slices until the crosshairs in all three views are in the center of the 0 mg/mL phantom. Single-click to place a 10-by-10 grid of points on the current slice and its neighboring two slices. Doing so will cause a red cluster of circles to appear in the axial view and a column of three points to appear in the sagittal and coronal views. These points will be used in the calculation of the weighting function.</li>
	<li>Repeat steps 3.1-3.3 for the 100 mg/mL phantom.</li>
	<li>If a phantom is not available or is unusable due to quality issues, click the No Phantom button for an aggregation of 10 sample phantoms and its corresponding weighting function to be used.</li>
	<li>When done selecting the phantoms, begin tracing each of the four coronary arteries by clicking on one of the artery buttons- LAD, LCA, LCX, or RCA.</li>
	<li>Hover over the axial view and scroll to navigate to either the proximal or distal end of the chosen artery. Observe the shape of the artery in this slice.</li>
	<li>If the shape of the artery is circular in the axial view of this slice and at most 5 mm in diameter, follow these sub-steps. If it is not circular, move on to step 3.9.
	<ol>
		<li>Place a point on the artery by single-clicking on the center of the artery in the axial view. The point placed will be displayed with a circle that is 5 mm in diameter. If the artery does not fit in the circle, delete it by double-clicking on it and move on to step 3.9 to draw an ROI. The point will appear in the sagittal and coronal views as well.</li>
		<li>If the artery is easier to visualize in the sagittal or coronal view, place a point there instead. Ensure it aligns with the center of the artery in the axial view.</li>
		<li>If a point placed is accidental or is not optimal, it can be deleted by rapidly double-clicking on it in any of the three views. The text in the bottom left area will give a notification that the point has been deleted.</li>
	</ol>
	</li>
	<li>If the shape of the artery is not circular in the axial plane, follow the steps below.
	<ol>
		<li>While ensuring the desired axial slice is in view, click on the Draw ROI button. In the pop-up window, scroll to zoom in/out and start single-clicking around the artery to trace it, as in Figure 4. 
		NOTE: ROIs, unlike points, can only be placed in the axial view.</li>
		<li>While the ROI is still open, the backspace key may be utilized to delete the previous point placed in the tracing of the artery. To close the ROI, either double-click where the last point is to be placed or double-click on the first point placed.</li>
		<li>Adjust the closed ROI further and refine by dragging the ROI&#39;s perimeter points or double-clicking on the perimeter of an ROI to add a point.</li>
		<li>Once the ROI is closed, two buttons will appear at the bottom of the pop-up window: Lock in and Redraw&#160;ROI. If the ROI needs to be redrawn, click the Redraw ROI button to clear the current one and draw a new one.</li>
		<li>When satisfied with the current ROI, click the Lock in button and close the pop-up window by clicking the red button in the top left corner (Mac) or the X button in the top right corner (PC) of the pop-up.</li>
		<li>A ROI can be deleted even once it is locked in by double-clicking on any of the perimeter points in the axial view of the main program window (not the pop-up).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65458/65458fig04.jpg" /> 
Figure 4: Draw ROI feature. When the Draw ROI option is chosen, a pop-up of the current axial slice appears. The yellow shows an ROI that was previously drawn on this slice. <a href="https://www.jove.com/files/ftp_upload/65458/65458fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="10">
	<li>Scroll up or down one slice in the axial view and repeat steps 3.8-3.9 until the end of the chosen artery is reached. When done, re-click on the finished artery&#39;s button to review the tracing of the artery and ensure no accidental points were placed. 
	NOTE: More than one point or ROI can be placed on any given axial slice. Extra lines connecting two ROIs on a slice can be ignored. If any slices for a given artery are not labeled, an error message will appear. Close the message and label the missed slice(s).</li>
	<li>Move on to the next of the four arteries and repeat steps 3.6-3.10.</li>
	<li>When all four arteries have been traced, click the SWCS button in the bottom right corner to generate the results. The bottom left area will show the progress and will display &#34;Done Processing&#34; when finished. Close the program window by clicking the red button in the top left corner (Mac) or the X button in the top right corner (PC).</li>
</ol>
4. Accessing the results
<ol>
	<li>To access the results for the case just analyzed, open the files directory and navigate to the project&#39;s main folder. Go into the Meta_Data folder and notice that a new folder with the same name as the original data folder for that subject has appeared.</li>
	<li>Within this folder, there will be three types of documents: CSVs, PNGs, and PDFs. Review the PDFs to get the final SWCS and Agatston score for the case, as well as the weighting function used. 
	NOTE: The CSVs store the coordinates of the various points/ROIs placed when analyzing this subject. Having these CSVs will allow this subject&#39;s images to be reopened in the program on a later day and have the previous points/ROIs appear automatically. Any changes made to the case, when reopened, will be automatically reflected in the CSVs.</li>
</ol>

Improving Coronary Artery Calcification Assessment with the SWCS Tool

<ol>
	<li>O'Malley, P. G., Taylor, A. J., Jackson, J. L., Doherty, T. M., Detrano, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+coronary+electron-bean+computed+tomography+for+coronary+heart+disease+events+in+asymptomatic+populations.">Prognostic value of coronary electron-bean computed tomography for coronary heart disease events in asymptomatic populations.</a> The American Journal of Cardiology. 85 (8), 945-948 (2000).</li><li>Budoff, M. 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=Assessment+of+coronary+artery+disease+by+cardiac+computed+tomography.">Assessment of coronary artery disease by cardiac computed tomography.</a> Circulation. 114 (16), 1761-1791 (2006).</li><li>Rumberger, J. A., Simons, D. B., Fitzpatrick, L. A., Sheedy, P. F., Schwartz, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+calcium+area+by+electron-beam+computed+tomography+and+coronary+atherosclerotic+plaque+area.">Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area.</a> Circulation. 92 (8), 2157-2162 (1995).</li><li>Mautner, G. 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=Coronary+artery+calcification:+assessment+with+electron+beam+CT+and+histomorphometric+correlation.">Coronary artery calcification: assessment with electron beam CT and histomorphometric correlation.</a> Radiology. 192 (3), 619-623 (1994).</li><li>Agatston, A. 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=Quantification+of+coronary+artery+calcium+using+ultrafast+computed+tomography.">Quantification of coronary artery calcium using ultrafast computed tomography.</a> Journal of the American College of Cardiology. 15 (4), 827-832 (1990).</li><li>Shea, 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=Spatially+weighted+coronary+artery+calcium+score+and+coronary+heart+disease+events+in+the+multi-ethnic+study+of+atherosclerosis.">Spatially weighted coronary artery calcium score and coronary heart disease events in the multi-ethnic study of atherosclerosis.</a> Circulation: Cardiovascular Imaging. 14 (1), e011981 (2021).</li><li>Liang, C. J., Budoff, M. J., Kaufman, J. D., Kronmal, R. A., Brown, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+quantifying+coronary+artery+calcification:+the+multi-ethnic+study+of+atherosclerosis+(MESA).">An alternative method for quantifying coronary artery calcification: the multi-ethnic study of atherosclerosis (MESA).</a> BMC Medical Imaging. 12, 14 (2012).</li><li>McCollough, C. H., 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=Coronary+artery+calcium:+a+multi-institutional,+multimanufacturer+international+standard+for+quantification+at+cardiac+CT.">Coronary artery calcium: a multi-institutional, multimanufacturer international standard for quantification at cardiac CT.</a> Radiology. 243 (2), 527-538 (2007).</li><li>Detrano, 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=Coronary+calcium+as+a+predictor+of+coronary+events+in+four+racial+or+ethnic+groups.">Coronary calcium as a predictor of coronary events in four racial or ethnic groups.</a> The New England Journal of Medicine. 358 (13), 1336-1345 (2008).</li><li>Budoff, M., 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=Cardiovascular+events+with+absent+or+minimal+coronary+calcification:+The+Multi-Ethnic+Study+of+Atherosclerosis+(MESA).">Cardiovascular events with absent or minimal coronary calcification: The Multi-Ethnic Study of Atherosclerosis (MESA).</a> American Heart Journal. 158 (4), 554-561 (2009).</li><li>Hecht, H. 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=2016+SCCT/STR+guidelines+for+coronary+artery+calcium+scoring+of+noncontrast+noncardiac+chest+CT+scans:+A+report+of+the+Society+of+Cardiovascular+Computed+Tomography+and+Society+of+Thoracic+Radiology.">2016 SCCT/STR guidelines for coronary artery calcium scoring of noncontrast noncardiac chest CT scans: A report of the Society of Cardiovascular Computed Tomography and Society of Thoracic Radiology.</a> Journal of Cardiovascular Computed Tomography. 11 (1), 74-84 (2017).</li><li>American College of Radiology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACR-NASCI-SPR+practice+parameter+for+the+performance+and+interpretation+of+cardiac+computed+tomography+(CT).">ACR-NASCI-SPR practice parameter for the performance and interpretation of cardiac computed tomography (CT).</a> American College of Radiology. , (2021).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

While the protocol for this program is relatively easy to follow, there are a few critical steps that are necessary for successful use and reliable results. Before starting, it is important to make sure the patient data that will be used in this program is anonymized to ensure patient confidentiality. The initial formatting and naming of the project's main folder must be correct for the program to recognize where to pull and place data. Incorrect naming and/or placement of folders, especially the Meta_Data folder, leads ...

Measuring the amount of calcium within arteries using computed tomography (CT) is an established way to assess the severity of coronary atherosclerosis. Knowing and quantifying the extent of atherosclerosis is key to determining the risk of future coronary heart disease1,2,3,4. The most common way of measuring calcium in the coronary arteries is using the Agatston score5. However, part of the Agatston score calculation relies on the intensity of the chosen pixels, measured in Hounsfield Units (HU). Any pixels less than 130 HU are not accounted for in the calculation. Similarly, calcifications with an area less than 1 mm2 are not considered. Due to these thresholds, the Agatston score is not sensitive to small, weakly attenuating foci of calcification, which may still be important in revealing the presence of subclinical disease6.
A previously described metric called the spatially weighted calcium score (SWCS) was proposed to assess the risk of atherosclerotic plaque in patients with low levels of calcification7. Unlike the Agatston score, the SWCS does not use signal thresholding to reduce the impact of image noise. Instead, it makes use of a phantom-an object with known concentrations of calcium hydroxyapatite (CHA) placed on the participant such that it is in the scan&#39;s field of view. Here, a phantom with 0 mg/mL, 50 mg/mL, 100 mg/mL, and 200 mg/mL CHA was used during development; however, in the current implementation of the graphical tool, only the 0 mg/mL and 100 mg/mL sections are required. The phantom is used to create a scan-specific weighting function, which is then used to weigh each of the user-selected pixels as well as its neighbors. Pixels with neighboring pixels that have a high attenuation level are given more weight than ones surrounded by pixels with lower attenuation levels. This process makes the SWCS tolerant to noise and comparable from scan to scan8. The SWCS is continuous and produces a score even when there are low levels of calcification, allowing for quantification of the extent of atherosclerosis when the Agatston score is zero. By allowing the evaluation of micro-calcification even when the Agatston score is zero, the SWCS may be important in revealing the presence of subclinical disease. This may allow a better understanding of the genetic, environmental, and other risk factors in atherosclerosis9,10. A previous study, which examined individuals with an Agatston score of zero at baseline and non-zero at a follow-up approximately 15 years later, observed that those with a higher SWCS at baseline had a higher coronary heart disease (CHD) event rate. The predictive power of the SWCS is especially important in younger populations, where the detection and monitoring of residual risk over a long term may be helpful6.
Presented here is a semi-automatic tool for calculating the SWCS along with the Agatston score. The tool utilizes a graphical user interface running on a compatible programming language. The user is able to interact with the images to generate a final series of reports, which include the two calcium scores. To start, the user selects a case, or a series of Digital Imaging and Communications in Medicine (DICOM) files, to input into the program. These images must be breath-held, electrocardiogram-gated CT scans, acquired only during diastole to avoid respiratory and cardiac motion. While the program is operational with any cardiac CT images, to produce meaningful results, the source images should meet the minimum clinical calcium scoring guidelines11,12. For reference, a slice thickness of 3 mm, peak tube voltage of 100 kVp, average CT dose index-vol of 1.19 mGy, and image resolution of 512 x 512 pixels are used in the study here. Any images that are not 512 x 512 pixels are resampled in the program automatically to ensure adequate and consistent resolution of small areas of calcification. Once the images are loaded, the user is able to see them in the axial, sagittal, and coronal views. One may then adjust the brightness and contrast of the images for better visualization before selecting the 0 mg/mL and 100 mg/mL sections of the phantom. Next, the user can trace each of the four coronary arteries-left anterior descending (LAD), left coronary artery (LCA), left circumflex (LCX), and right coronary artery (RCA)-by placing either a point, a region of interest (ROI), or a combination of both to allow for a thorough selection of an artery&#39;s pixels regardless of how the artery appears in the axial plane. The user may delete and replace or redraw points and ROIs as needed. Clicking the SWCS button generates the final reports. Cases are auto-saved so that images, along with the points and ROIs, can be reloaded at a later time. Written instructions are also available at every point while using the program, making the program easy to use.

<table><tbody><tr><td>Calcium Hydroxyapatite</td><td>Sigma-Aldrich</td><td>289396-100G</td><td>Suspended in EpoxAcast 690 resin for phantom creation</td></tr><tr><td>Clinical Cardiac CT Scanner</td><td>Siemens</td><td>SOMATOM Force Dual Source CT</td><td>Used for the source images; Any cardiac CT will be sufficient</td></tr><tr><td>EpoxAcast 690</td><td>Smooth-On</td><td>03641</td><td>Used for phantom creation</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2019a</td><td>Requires Image Processing Toolbox and Statistics and Machine Learning Toolbox; Any version compatible with and able to run version R2019a scripts is sufficient</td></tr><tr><td>Standard Computer</td><td>N/A</td><td>N/A</td><td>macOS or Windows operating system</td></tr><tr><td>syngo.via</td><td>Siemens</td><td>VB60A_HF04</td><td>Commercial software used for computing Agatston score for validation study</td></tr></tbody></table>

semi-automatic graphical tool for measuring coronary artery spatially weighted calcium score from gated cardiac computed tomography images

The current standard for measuring coronary artery calcification to determine the extent of atherosclerosis is by calculating the Agatston score from computed tomography (CT). However, the Agatston score disregards pixel values less than 130 Hounsfield Units (HU) and calcium regions less than 1 mm2. Due to this thresholding, the score is not sensitive to small, weakly attenuating regions of calcium deposition and may not detect nascent micro-calcification. A recently proposed metric called the spatially weighted calcium score (SWCS) also utilizes CT but does not include a threshold for HU and does not require elevated signals in contiguous pixels. Thus, the SWCS is sensitive to weakly attenuating, smaller calcium deposits and may improve the measurement of coronary heart disease risk. Currently, the SWCS is underutilized owing to the added computational complexity. To promote translation of the SWCS into clinical research and reliable, repeatable computation of the score, the aim of this study was to develop a semi-automatic graphical tool that calculates both the SWCS and the Agatston score. The program requires gated cardiac CT scans with a calcium hydroxyapatite phantom in the field of view. The phantom allows for deriving a weighting function, from which each pixel&#39;s weight is adjusted, allowing for the mitigation of signal variations and variability between scans. With all three anatomical views visible simultaneously, the user traces the course of the four main coronary arteries by placing points or regions of interest. Features such as scroll-to-zoom, double-click to delete, and brightness/contrast adjustment, along with written guidance at every step, make the program user-friendly and easy to use. Once tracing the arteries is complete, the program generates reports, which include the scores and snapshots of any visible calcium. The SWCS may reveal the presence of subclinical disease, which may be used for early intervention and lifestyle changes.

The representative results shown in this section display what successful use of the program entails. Here, a patient with an Agatston score greater than zero is used as an example. As discussed earlier, the results within a patient&#39;s metadata folder will have spreadsheets in the form of CSV files, images in the form of PNG files, and reports in the form of PDF files, as shown in Figure 5. The number of PNG files differs from case to case, since only snapshots of selected pixels with noti...

Watch this Scientific Journal Video about Advancing Cardiovascular Imaging - Introducing the Spatially Weighted Calcium Score for Early Disease Detection at JoVE.com

Author Spotlight: Advancing Cardiovascular Imaging - Introducing the Spatially Weighted Calcium Score for Early Disease Detection 

This video demonstrates the use of a novel graphical tool for measuring the spatially weighted calcium score (SWCS), an alternative to the Agatston score, for quantifying coronary artery calcification. The graphical tool computes SWCS based on image data from gated cardiac computed tomography and user-defined paths of the coronary arteries.

Semi-Automatic Graphical Tool for Measuring Coronary Artery Spatially Weighted Calcium Score from Gated Cardiac Computed Tomography Images

The current standard for measuring coronary artery calcification to determine the extent of atherosclerosis is by calculating the Agatston score from ...

semi-automatic-graphical-tool-for-measuring-coronary-artery-spatially

Research

JoVE Journal

Bioengineering

921 Views.  Icahn School of Medicine at Mount Sinai. De missie van het Biomedical Engineering and Imaging Institute is het creëren van innovatieve biomedische technologie en beeldvormingstechnologieën die kunnen worden gebruikt in de medische wetenschap en de klinische praktijk. Het doel is om technieken te identificeren en te ontwikkelen voor een vroege en nauwkeurige diagnose van ziekten, die vervolgens nuttig kunnen zijn bij het optimaliseren van effectievere behandelingen. Onze cardiovasculaire beeldvormingsgroep concentreert zich op beel...

Video: Auteur in de schijnwerpers: Advancing Cardiovascular Imaging - Introductie van de ruimtelijk gewogen calciumscore voor vroege opsporing van ziekten 

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

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 ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Medicine

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach warrants good early results, but adequate follow-up imaging after EVAR is imperative to maintain long-term positive outcomes. Potential graft-related complications are graft migration, infection, fraction, and endoleaks, with the last one being the most common. The most frequently used imaging after EVAR is computed tomography angiography (CTA) and duplex ultrasound. Dynamic, time-resolved computed tomography angiography (d-CTA) is a reasonably new technique to characterize the endoleaks. Multiple scans are done sequentially around the endograft during acquisition that grants good visualization of the contrast passage and graft-related complications. This high diagnostic accuracy of d-CTA can be implemented into therapy via image fusion and reduce additional radiation and contrast material exposure.
This protocol describes the technical aspects of this modality: patient selection, preliminary image review, d-CTA scan acquisition, image processing, qualitative and quantitative endoleak characterization. The steps of integrating dynamic CTA into intra-operative fluoroscopy using 2D-3D fusion-imaging to facilitate targeted embolization are also demonstrated. In conclusion, time-resolved, dynamic CTA is an ideal modality for endoleak characterization with additional quantitative analysis. It can reduce radiation and iodinated contrast material exposure during endoleak treatment by guiding interventions.

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

Longitudinal Quantification of Cardiovascular Calcification Using PET/CT Imaging

Novel Quantification Protocol for Cardiovascular Calcification Progression Using Longitudinal MicroPET/MicroCT Images

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of cardiovascular calcification. Due to their non-invasive nature, small research animals can be imaged at multiple time points. The challenge lies in the accurate quantification of cardiovascular calcification. Here, we provide a protocol, using images from the later disease stages as a template, to accurately quantify the progression of cardiovascular calcification in longitudinal studies. The protocol involves 1) the alignment of the chest area in multiple images from the same animal during a longitudinal study as the first step, 2) the definition of a region of interest (ROI) situated within the heart and the aorta at the site of larger calcium deposits that become apparent in later images, and 3) simultaneous segmentation and quantification of calcium deposits across all images acquired during the longitudinal study. This streamlined method enhances the accuracy of image analysis in following the progression of cardiovascular calcification by improving the precision of ROI definition and reducing the variability associated with earlier techniques that analyze individual scans independently.

Author Spotlight: Enhanced Quantification of Cardiovascular Calcification Progression for Longitudinal Micro PET/CT Studies in Small Research Animals

This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of ...

Retrospective Cardiac Gating with A Prototype Small-Animal X-ray Computed Tomograph

The CrumpCAT is a prototype small-animal X-ray computed tomography (CT) scanner developed at our research institution. The CMOS detector with a maximum frame rate of 29 Hz and similar Tungsten X-ray sources with energies ranging from 50 kVp to 80 kVp are widely used across commercially available preclinical X-ray CT instruments. This makes the described work highly relevant to other institutions, despite the generally perceived wisdom that these detectors are not suitable for gating the high heart rates of mice (~600 beats/min). The scanner features medium- (200 &#181;m) and high- (125 &#181;m) resolution imaging, fluoroscopy, retrospective respiratory gating, and retrospective cardiac gating, with iterative or filtered-back projection image reconstruction. Among these features, cardiac gating is the most useful feature for studying cardiac functions in vivo, as it effectively eliminates image blurring caused by respiratory and cardiac motion. 
Here, we describe our method for preclinical intrinsic retrospective cardiac-gated CT imaging, aimed at advancing research on in vivo cardiac function and structure analysis. The cardiac-gating method acquires a large number of projections at the shortest practical exposure time (~20 ms) and then retrospectively extracts respiratory and cardiac signals from temporal changes in raw projection sequences. These signals are used to reject projections belonging to the high motion rate inspiration phase of the respiratory cycle and to divide the remaining projections into 12 groups, each corresponding to one phase of the cardiac cycle. Each group is reconstructed independently using an iterative method to produce a volumetric image for each cardiac phase, resulting in a four-dimensional (4D) dataset. 
These phase images can be analyzed either collectively or individually, allowing for detailed assessment of cardiac function. We demonstrated the effectiveness of both approaches of the prototype scanner's cardiac-gating feature through representative in vivo imaging results.

We provide a comprehensive description of the intrinsic retrospective cardiac gating method of the CrumpCAT, a prototype small-animal X-ray computed tomography (CT) scanner designed and constructed at our research institution.

The CrumpCAT is a prototype small-animal X-ray computed tomography (CT) scanner developed at our research institution. The CMOS detector with a maximum ...

Semi-Automatic Graphical Tool for Measuring Coronary Artery Spatially Weighted Calcium Score from Gated Cardiac Computed Tomography Images

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video