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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

assessing-intracardiac-vortices-with-high-frame-rate-echocardiography

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1.3K Views.  University of Newcastle. 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.

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

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 ...

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency Oscillations (HFOs) have emerged as potential presurgical biomarkers for the identification of the EZ in addition to Interictal Epileptiform Discharges (IEDs) and ictal activity. Although they are promising to localize the EZ, they are not yet suited for the diagnosis or monitoring of epilepsy in clinical practice. Primary barriers remain: the lack of a formal and global definition for HFOs; the consequent heterogeneity of methodological approaches used for their study; and the practical difficulties to detect and localize them noninvasively from scalp recordings. Here, we present a methodology for the recording, detection, and localization of interictal HFOs from pediatric patients with refractory epilepsy. We report representative data of HFOs detected noninvasively from interictal scalp EEG and MEG from two children undergoing surgery.
The underlying generators of HFOs were localized by solving the inverse problem and their localization was compared to the Seizure Onset Zone (SOZ) as this was defined by the epileptologists. For both patients, Interictal Epileptogenic Discharges (IEDs) and HFOs were localized with source imaging at concordant locations. For one patient, intracranial EEG (iEEG) data were also available. For this patient, we found that the HFOs localization was concordant between noninvasive and invasive methods. The comparison of iEEG with the results from scalp recordings served to validate these findings. To our best knowledge, this is the first study that presents the source localization of scalp HFOs from simultaneous EEG and MEG recordings comparing the results with invasive recordings. These findings suggest that HFOs can be reliably detected and localized noninvasively with scalp EEG and MEG. We conclude that the noninvasive localization of interictal HFOs could significantly improve the presurgical evaluation for pediatric patients with epilepsy.

High Frequency Oscillations (HFOs) have emerged as presurgical biomarkers for the identification of the epileptogenic zone in pediatric patients with medically refractory epilepsy. A methodology for the noninvasive recording, detection, and localization of HFOs with simultaneous scalp electroencephalography (EEG) and magnetoencephalography (MEG) is presented.

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency ...

A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic responses, the LSCI is used in the same way as a conventional Laser Doppler Imager (LDI). However, with a maximum skin depth of approximately 1 mm, the LSCI is designed to focus on mainly superficial blood flow. It is used to measure skin surface areas of up to 15 cm x 20 cm. The new technique introduced in this paper accounts for alternating speeds of microcirculations; i.e. both slow and fast flow flux measurement using the LSCI. The novel technique also overcomes LSCI&#39;s biggest shortcoming, which is high sensitivity to artifact movement. An adhesive opaque patch (AOP) is introduced for satisfactory recording of microcirculatory blood flow, by subtracting the LSCI signal from the AOP from the laser speckle skin signal. The optimal setting is also defined because the LSCI is most powerful when flux changes are measured relative to a reference baseline, with blood microcirculatory flux expressed as a percentage change from the baseline. These changes may be used for analyzing the status of the blood flow system.

This study introduces a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using a laser speckle contrast imager.

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic ...