We wish to acknowledge the Neonatal intensive care department of the John Hunter Hospital for allowing our ongoing work to be performed, along with the parents of our very small and precious participants.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants&#39; families included in the study. All images and video clips were de-identified following the acquisition.
1. Patient preparation
<ol>
	<li>Set up the ultrasound machine adjacent to the patient&#39;s cot and connect a three-lead electrocardiogram (see Table of Materials).</li>
	<li>Input the patient code and relevant details, such as body length and weight, and perform the echocardiogram according to the previously described standards12.</li>
</ol>
2. Image acquisition
<ol>
	<li>Specifically for BSI, obtain a shallow view of the LV in the apical four-chamber view with a narrow sector width, allowing an acquisition frame rate between 400-600 Hz.</li>
	<li>Open a color box over the left ventricular cavity, narrow maximally to include only the region from the mitral valve to the endocardial apex, and from the septal endocardial border to the lateral wall endocardial border.</li>
	<li>Increase the color gain to the point of speckling and reduce slightly. Set the color Doppler velocity scale limit to the appropriate diastolic velocity (20-30 cm/s in preterm infants) to maximally fill the color box with the slower-moving diastolic inflow.</li>
	<li>On the touchscreen control panel of the equipment (see Table of Materials), tap BSI mode to reveal the intracardiac flow directions and vortices in RAW color format. Adjust the BSI box position and size to include the flow region of interest and record at least two cardiac cycles.</li>
	<li>Repeat the procedure in the apical LV long-axis view or other views where intracardiac hemodynamic assessment is required (Figure 2 and Figure 3).</li>
</ol>
3. Image analyses
NOTE: The image analysis techniques for the LV vortex have been briefly described in previous work from our laboratory11. The protocol used for assessing intracardiac vortices is as follows (Figure 3 and Figure 4).
<ol>
	<li>Save two cardiac cycles from each respective patient to external media in their RAW DICOM format and transfer to a laboratory station with an image processing software (see Table of Materials) installed for detailed offline analyses.</li>
	<li>Once offline, identify the most prominent or main vortex. 
	NOTE: The main vortex is visualized as an elongated, oval-shaped, anti-clockwise rotating structure located in the upper left quadrant of the left ventricle near the septum, with the maximum vortex area found in late diastole (during the transmitral A-wave) in preterm infants (Video 1). The main vortex is usually found during the transmitral E-wave for older infants and children.</li>
	<li>Record the number of independent, complete oval-shaped vortices forming throughout the cardiac cycle for each clip.</li>
	<li>Measure the position of the main vortex relative to known landmarks within the LV. To determine the Vortex depth, using the &#34;distance measurement&#34; tool on the analysis software, measure the vertical distance from the vortex eye to the middle of the mitral valve annulus. For Vortex transverse position, measure the horizontal distance from the vortex eye to the endocardial border of the interventricular septum.</li>
	<li>Measure the vertical and horizontal edge-to-edge distances of the main vortex relative to the LV length and width to obtain the vortex shape. 
	NOTE: This also enables estimation of the vortex sphericity index as length divided by width.</li>
	<li>Using the &#34;tracing measurement&#34; tool on the analysis software, click on and trace the outermost vortex ring at the point where the main vortex is most prominent to determine the main vortex area.</li>
	<li>To assess Peak Vortex Formation Time (PVFT), record the cardiac frame when the vortex first appears (circular rings delineated) in the cardiac frame where the main vortex is most prominent and calculate the number of frames relative to the total number of frames in one cardiac cycle for the patient.</li>
	<li>To assess vortex duration, measure the frames from which the vortex first appears when the vortex loses its circular ring formation. Vortex duration is then calculated as the number of frames relative to that patient&#39;s total number of frames in one cardiac cycle (Figure 5).</li>
</ol>

Advancements in Echocardiography-Derived Blood Speckle Imaging Technology

<ol>
	<li>de Waal, K., Costley, N., Phad, N., Crendal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+diastolic+dysfunction+and+diastolic+heart+failure+in+preterm+infants.">Left ventricular diastolic dysfunction and diastolic heart failure in preterm infants.</a> Pediatric Cardiology. 40 (8), 1709-1715 (2019).</li><li>Lahmers, S., Wu, Y., Call, D. R., Labeit, S., Granzier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+titin+isoform+expression+and+passive+stiffness+in+fetal+and+neonatal+myocardium.">Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium.</a> Circulation Research. 94 (4), 505-513 (2004).</li><li>Chung, C. S., Hoopes, C. W., Campbell, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+relaxation+is+accelerated+by+fast+stretch,+not+reduced+afterload.">Myocardial relaxation is accelerated by fast stretch, not reduced afterload.</a> Journal of Molecular and Cellular Cardiology. 103, 65-73 (2017).</li><li>Pedrizzetti, G., La Canna, G., Alfieri, O., Tonti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vortex-an+early+predictor+of+cardiovascular+outcome.">The vortex-an early predictor of cardiovascular outcome.</a> Nature Reviews Cardiology. 11 (9), 545-553 (2014).</li><li>Rodriguez Munoz, D., 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=Left+ventricular+vortex+following+atrial+contraction+and+its+interaction+with+early+systolic+ejection.">Left ventricular vortex following atrial contraction and its interaction with early systolic ejection.</a> European Heart Journal. 34 (1), 1104 (2013).</li><li>Schmitz, L., Koch, H., Bein, G., Brockmeier, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+diastolic+function+in+infants,+children,+and+adolescents.+Reference+values+and+analysis+of+morphologic+and+physiologic+determinants+of+echocardiographic+Doppler+flow+signals+during+growth+and+maturation.">Left ventricular diastolic function in infants, children, and adolescents. Reference values and analysis of morphologic and physiologic determinants of echocardiographic Doppler flow signals during growth and maturation.</a> Journal of the American College of Cardiology. 32 (5), 1441-1448 (1998).</li><li>Marchese, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+vortex+analysis+by+high-frame+rate+blood+speckle+tracking+echocardiography+in+healthy+children+and+in+congenital+heart+disease.">Left ventricular vortex analysis by high-frame rate blood speckle tracking echocardiography in healthy children and in congenital heart disease.</a> International Journal of Cardiology. Heart &amp; Vasculature. 37, 100897 (2021).</li><li>Pierrakos, O., Vlachos, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+vortex+formation+on+left+ventricular+filling+and+mitral+valve+efficiency.">The effect of vortex formation on left ventricular filling and mitral valve efficiency.</a> Journal of Biomechanical Engineering. 128 (4), 527-539 (2006).</li><li>Mele, D., 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=Intracardiac+flow+analysis:+techniques+and+potential+clinical+applications.">Intracardiac flow analysis: techniques and potential clinical applications.</a> Journal of the American Society of Echocardiography. 32 (3), 319-332 (2019).</li><li>Nyrnes, S. A., Fadnes, S., Wigen, M. S., Mertens, L., Lovstakken, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+speckle-tracking+based+on+high-frame+rate+ultrasound+imaging+in+pediatric+cardiology.">Blood speckle-tracking based on high-frame rate ultrasound imaging in pediatric cardiology.</a> Journal of the American Society of Echocardiography. 33 (4), 493-503 (2020).</li><li>de Waal, K., Crendal, E., Boyle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+vortex+formation+in+preterm+infants+assessed+by+blood+speckle+imaging.">Left ventricular vortex formation in preterm infants assessed by blood speckle imaging.</a> Echocardiography. 36 (7), 1364-1371 (2019).</li><li>Nagueh, S. F., 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=Recommendations+for+the+evaluation+of+left+ventricular+diastolic+function+by+echocardiography:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for the evaluation of left ventricular diastolic function by echocardiography: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 29 (4), 277-314 (2016).</li><li>Takahashi, H., Hasegawa, H., Kanai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+averaging+of+two-dimensional+correlation+functions+for+velocity+vector+imaging+of+cardiac+blood+flow.">Temporal averaging of two-dimensional correlation functions for velocity vector imaging of cardiac blood flow.</a> Journal of Medical Ultrasonics. 42 (3), 323-330 (2015).</li><li>Kheradvar, A., 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=Echocardiographic+particle+image+velocimetry:+a+novel+technique+for+quantification+of+left+ventricular+blood+vorticity+pattern.">Echocardiographic particle image velocimetry: a novel technique for quantification of left ventricular blood vorticity pattern.</a> Journal of the American Society of Echocardiography. 23 (1), 86-94 (2010).</li><li>Phad, N. S., de Waal, K., Holder, C., Oldmeadow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+hypertrophy:+a+distinct+pattern+of+cardiac+remodeling+in+preterm+infants.">Dilated hypertrophy: a distinct pattern of cardiac remodeling in preterm infants.</a> Pediatric Research. 87 (1), 146-152 (2020).</li><li>Kheradvar, A., 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=Diagnostic+and+prognostic+significance+of+cardiovascular+vortex+formation.">Diagnostic and prognostic significance of cardiovascular vortex formation.</a> Journal of Cardiology. 74 (5), 403-411 (2019).</li><li>Cantinotti, 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=Intracardiac+flow+visualization+using+high-frame+rate+blood+speckle+tracking+echocardiography:+Illustrations+from+infants+with+congenital+heart+disease.">Intracardiac flow visualization using high-frame rate blood speckle tracking echocardiography: Illustrations from infants with congenital heart disease.</a> Echocardiography. 38 (4), 707-715 (2021).</li><li>Henry, 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=Bicuspid+aortic+valve+flow+dynamics+using+blood+speckle+tracking+in+children.">Bicuspid aortic valve flow dynamics using blood speckle tracking in children.</a> European Heart Journal-Cardiovascular Imaging. 22, 356 (2021).</li><li>Mawad, W., 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=Right+ventricular+flow+dynamics+in+dilated+right+ventricles:+energy+loss+estimation+based+on+blood+speckle+tracking+echocardiography-a+pilot+study+in+children.">Right ventricular flow dynamics in dilated right ventricles: energy loss estimation based on blood speckle tracking echocardiography-a pilot study in children.</a> Ultrasound in Medicine &amp; Biology. 47 (6), 1514-1527 (2021).</li><li>Kass, D. A., Bronzwaer, J. G. F., Paulus, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+mechanisms+underlie+diastolic+dysfunction+in+heart+failure.">What mechanisms underlie diastolic dysfunction in heart failure.</a> Circulation Research. 94 (12), 1533-1542 (2004).</li><li>Nagueh, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+diastolic+function:+understanding+pathophysiology,+diagnosis,+and+prognosis+with+echocardiography.">Left ventricular diastolic function: understanding pathophysiology, diagnosis, and prognosis with echocardiography.</a> JACC. Cardiovasc Imaging. 13, 228-244 (2020).</li><li>Carroll, J. D., Lang, R. M., Neumann, A. L., Borow, K. M., Rajfer, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+effects+of+positive+inotropic+and+vasodilator+therapy+on+diastolic+properties+in+patients+with+congestive+cardiomyopathy.">The differential effects of positive inotropic and vasodilator therapy on diastolic properties in patients with congestive cardiomyopathy.</a> Circulation. 74 (4), 815-825 (1986).</li></ol>

The authors have no disclosures or conflicts of interest to declare.

The importance of visualizing and understanding the intracardiac vortex 
There are many possible clinical applications of high-frame rate echocardiography-derived vortex imaging. Their ability to provide valuable insight into intracardiac flow dynamics has been the interest of recent studies16. Moreover, vortex imaging may allow the detection of pre-symptomatic changes in LV architecture and function in neonates, which may have a bearing on long-term cardiac remodeling into a...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65189">Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns</a>. The Authors section was updated. The affiliation for author Damien Vitiello has been updated to:&#160;Institute of Sport and Health Sciences of Paris (IS3P - URP 3625), Universit&#233; Paris Cit&#233;&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65189">Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns</a>. The Authors section was updated.

Erratum: Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns

Intracardiac blood flow patterns play a key role in cardiac development, starting in fetal morphogenesis and continuing throughout the lifespan1. Hemodynamic shear stress plays a pivotal role in the stimulation of cardiac chamber growth and architecture via the activation of specific genes2,3. This occurs at both the intrauterine stage and in the early stages of life, thus highlighting the importance of hemodynamic influence on early cardiac development and the carry-over into adulthood3.
The laws of fluid dynamics state that blood passing along a vessel wall move slower when closest to the wall and faster when in the center of a vessel, where resistance is lower. This phenomenon can be demonstrated in any large vessel with pulse wave Doppler as the typical Doppler velocity time integral envelope4. When blood enters a larger cavity such as the heart, the blood farthest from the endocardial surface continues to increase its velocity relative to the blood closest to that surface and create a rotational body of fluid, known as a vortex. Once created, vortices are self-propelling flow structures that typically draw in surrounding fluid via negative pressure gradients. Thus, a vortex can move a greater volume of blood than an equivalent straight jet of fluid, promoting greater cardiac efficiency4,5.
The literature suggests that the evolutionary purpose of vortices is to conserve kinetic energy, minimize shear stress, and maximize flow efficiency4,5,6. Specifically for the heart, this includes storing hemodynamic energy in a rotary motion, facilitating valve closure, and the propagation of blood flow toward the outflow tract, as seen in Figure&#160;1. Altered intracardiac blood flow patterns are expected in pathological situations such as volume-overloaded states and in cases with artificial valves7,8. Thus, herein lies the true diagnostic potential of vortices as early predictors of cardiovascular outcomes in adults.
Intracardiac hemodynamics have gained increasing interest in the literature in both adult and pediatric populations. Several modalities are available for the qualitative and quantitative assessment of intracardiac hemodynamics and were comprehensively summarised in a recent review, with a specific emphasis on the intracardiac vortex9. One modality with great promise is echocardiography-derived blood speckle imaging (BSI), which offers the ability to noninvasively measure a number of qualitative and quantitative vortex characteristics, described below, at a relatively low cost and with excellent reproducibility10. BSI is currently commercially available using a high-end cardiac ultrasound system with an S12 or S6 MHz probe. The speckle-tracking features are analogous to those used in tissue speckle tracking to study myocardial deformation11,12,13. Since red blood cells tend to move faster and with a higher Doppler frequency than the surrounding tissue, the two signals can be separated by applying a temporal filter. BSI uses a best-match algorithm to quantify the movement of blood speckles directly without using contrast agents. The blood velocity measurements can be visualized as arrows, streamlines, or path lines with or without underlying color Doppler images, and can highlight areas of complex flow10.
BSI has been shown to have good feasibility and accuracy for quantifying intracardiac blood flow patterns, with excellent validity compared to a reference phantom instrument and pulsed-Doppler7,10,11. Whilst still very novel, BSI is a promising clinical tool for the early diagnosis of various cardiac pathophysiologies. The clinical application of vortex imaging has shown promise in newborn infants. Specifically, the behavior of a vortex in the left ventricle (LV) may have long-term implications on cardiac remodeling and predisposition toward heart failure.
The mechanism linking vortices to left ventricular remodeling is still relatively unexplored, but has been recently investigated in our laboratory and is the subject of ongoing work11. This methodology article aims to describe the use of BSI in exploring intracardiac vortices and discuss the practical and clinical uses of vortices in assessing diastolic function in various populations. A secondary aim is to discuss the clinical relevance of BSI and present some of the work previously performed in neonates.

<table><tbody><tr><td>Tomtec Imaging Systems GmbH</td><td>Phillips</td><td>GmbH Corporation</td><td>Offline ultrasound image processing tool, used for calculating all vortex measurements</td></tr><tr><td>Vivid E95</td><td>General Electrics</td><td>NA</td><td>Cardiac Ultrasound device used to capture Echocardiography-derived Blood Speckle Imaging</td></tr></tbody></table>

assessing intracardiac vortices with high frame-rate echocardiography-derived blood speckle imaging in newborns

The left ventricle (LV) has a unique pattern of hemodynamic filling. During diastole, a rotational body or ring of fluid known as a vortex is formed due to the chiral geometry of the heart. A vortex is reported to have a role in conserving the kinetic energy of blood flow entering into the LV. Recent studies have shown that LV vortices may have prognostic value in describing diastolic function at rest in neonatal, pediatric, and adult populations, and may help with earlier subclinical intervention. However, the visualization and characterization of the vortex remain minimally explored. A number of imaging modalities have been utilized for visualizing and describing intracardiac blood flow patterns and vortex rings. In this article, a technique known as blood speckle imaging (BSI) is of particular interest. BSI is derived from high-frame rate color Doppler echocardiography and provides several advantages over other modalities. Namely, BSI is an inexpensive and noninvasive bedside tool that does not rely on contrast agents or extensive mathematical assumptions. This work presents a detailed step-by-step application of the BSI methodology used in our laboratory. The clinical utility of BSI is still in its early stages, but has shown promise within the pediatric and neonatal populations for describing diastolic function in volume-overloaded hearts. A secondary aim of this study is thus to discuss recent and future clinical work with this imaging technology.

The acquisition of vortex clips is comparable to the standard methodology universally employed in obtaining color Doppler clips. Pioneering studies in adults have described vortices using the apical two-, three-, and four-chamber views14. The LV vortex is a ring-like structure that moves from base to apex. BSI visualizes the internal diameter of the ring (Figure 2). A vortex ring is usually not symmetrical in shape, hence alternative imaging planes can show variable v...

Watch this Scientific Journal Video about Advancing Neonatal Cardiac Diagnostics with Echocardiography-Derived Blood Speckle Imaging at JoVE.com

Author Spotlight: Advancing Neonatal Cardiac Diagnostics with Echocardiography-Derived Blood Speckle Imaging 

The present protocol uses echocardiography-derived blood speckle imaging technology to visualize intracardiac hemodynamics in newborns. The clinical utility of this technology is explored, the rotational body of fluid within the left ventricle (known as a vortex) is accessed, and its significance in understanding diastology is determined.

Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns

The left ventricle (LV) has a unique pattern of hemodynamic filling. During diastole, a rotational body or ring of fluid known as a vortex is formed due ...

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1.3K Views.  University of Newcastle. We proberen te onderzoeken waarom te vroeg geboren baby's wanneer ze te vroeg geboren worden en opgroeien tot volwassenen een significant hoger risico op hartfalen hebben. We denken dat het te maken heeft met een abnormale hartontwikkeling omdat ze 10 of soms 15 weken te vroeg zijn geboren en waarbij de druk in het hart is veranderd. We kunnen dit soort veranderingen nu vastleggen door te kijken naar intracardiale bloedstroompatronen met bloedspikkelbeeldvorming.Hopelijk kunnen we baby...

Video: Auteur in de schijnwerpers: neonatale cardiale diagnostiek bevorderen met echocardiografie-afgeleide bloedspikkelbeeldvorming 

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 ...

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Developmental Biology

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, reliable, and real-time information on developmental hemodynamics in sick newborns. The comprehensive assessment is based on a multiparametric approach that overcomes the reliability issues of individual measurements, allows for earlier recognition of cardiovascular compromise and promotes enhanced diagnostic precision and timely management. TnECHO-driven research has led to an enhanced understanding of the mechanisms of illness and the development of predictive models to identify at-risk populations. This information may then be used to formulate a diagnostic impression and provide individualized guidance for the selection of cardiovascular therapies. TnECHO is based on the expert consultative model in which a neonatologist, with advanced training in neonatal hemodynamics, performs comprehensive and standardized TnECHO assessments. The distinction from point of care ultrasonography (POCUS), which provides limited and brief one-time assessments, is important. Neonatal hemodynamics training is a 1-year structured program designed to optimize image acquisition, measurement analysis, and hemodynamic knowledge (physiology, pharmacotherapy) to support cardiovascular decision-making. Neonatologists with hemodynamic expertise are trained to recognize deviations from normal anatomy and appropriately refer cases of possible structural abnormalities. We provide an outline of neonatal hemodynamics training, the standardized TnECHO imaging protocol, and an example of representative echo findings in a hemodynamically significant patent ductus arteriosus.

Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist.

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, ...

A Modified Sonographic Algorithm for Image Acquisition in Life-Threatening Emergencies in the Critically Ill Newborn

The use of routine point-of-care ultrasound (POCUS) is increasing in neonatal intensive care units (NICUs), with several centers advocating for 24 h equipment availability. In 2018, the sonographic algorithm for life-threatening emergencies (SAFE) protocol was published, which allows the assessment of neonates with sudden decompensation to identify abnormal contractility, tamponade, pneumothorax, and pleural effusion. In the study unit (with a consulting neonatal hemodynamics and POCUS service), the algorithm was adapted by including consolidated core steps to support at-risk newborns, aiding clinicians in managing cardiac arrest, and adding views to verify correct intubation. This paper presents a protocol that can be applied in the NICU and the delivery room (DR) in relation to three scenarios: cardiac arrest, hemodynamic deterioration, or respiratory decompensation. 
This protocol can be performed with a state-of-the-art ultrasound machine or an affordable handheld device; the image acquisition protocol is carefully detailed. This method was designed to be learned as a general competence to obtain the timely diagnosis of life-threatening scenarios; the method aims to save time but does not represent a substitute for comprehensive and standardized hemodynamic and radiological analyses by a multidisciplinary team, which might not universally be on call but needs to be involved in the process. From January 2019 to July 2022, in our center, 1,045 hemodynamic consultation/POCUS consults were performed with 25 patients requiring the modified SAFE protocol (2.3%), and a total of 19 procedures were performed. In five cases, trained fellows on call resolved life-threatening situations. Clinical examples are provided that show the importance of including this technique in the care of critical newborns.

Here, we present a protocol that can be applied in the neonatal intensive care unit and the delivery room in relation to three scenarios: cardiac arrest, hemodynamic deterioration, or respiratory decompensation. This protocol can be performed with a state-of-the-art ultrasound machine or an affordable handheld device; an image acquisition protocol is carefully detailed.

The use of routine point-of-care ultrasound (POCUS) is increasing in neonatal intensive care units (NICUs), with several centers advocating for 24 h ...

Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns

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