Detection of retained microbubbles in carotid arteries with real-time low mechanical index imaging in the setting of endothelial dysfunction
Jeane M. Tsutsui, MD*,
Feng Xie, MD*,
Martin Cano, BS ,
James Chomas, PhD ,
Patrick Phillips, PhD,
Stanley J. Radio, MD,
John Lof, MS* and
Thomas R. Porter, MD, FACC*,*
* Section of Cardiology, University of Nebraska Medical Center, Omaha, Nebraska, USA
Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska, USA
Siemens Medical Solutions, Mountain View, California, USA
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Figure 1 (A) Example of the region of interest (ROI) used for the measurements of contrast acoustic intensity (AI) of the entire lumen (white line) and central lumen (yellow line). (B) Two-dimensional (2D) imaging of a normal carotid artery in the transverse view and with pulse sequence scheme (PSS) before intravenous microbubbles (MB) injection showing no background imaging, early after MB injection showing the complete luminal vessel opacification, and late after MB injection showing few MB in the central lumen. The time-intensity curve demonstrates the typical quantification of the mean AI in both the entire lumen ROI and the central lumen ROI. The late 50 frames after clearance of free-flowing MB were chosen for the calculation of endothelial AI, defined as the difference between the mean entire lumen AI minus the central lumen AI (see text for details).
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Figure 2 Example of flow cytometry showing histograms of red fluorescent intensity for perfluorocarbon-exposed sonicated dextrose albumin microbubbles incubated with R-phycoerythrin–labeled control monoclonal antibody (A), with R-phycoerythrin anti-mouse alone (B), and with serum and R-phycoerythrin–labeled monoclonal antibody to complement C3b, both at baseline (C) and after intralipid infusion (D). Note that incubation of microbubbles and anti-human C3b in the presence of serum drawn in the setting of hypertriglyceridemia resulted in a shift of the curve to the right, reflecting an increase in the mean fluorescent intensity.
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Figure 3 Carotid artery (CA) diameter response to intra-arterial infusion of acetylcholine, at baseline and during hypertriglyceridemia (A) and after CA balloon injury (B). Carotid response went from vasodilation at baseline to vasoconstriction after intralipid infusion and also after balloon injury, confirming the presence of endothelial dysfunction in both conditions.
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Figure 4 Transverse sectional images of carotid arteries obtained with low mechanical index real-time pulse sequence scheme at baseline (panels at left) and during hypertriglyceridemia (panels at right). Note that at baseline there was some adherence of microbubbles to the endothelium only in pig 4 (arrow) and no adherence in the other pigs. After intralipid infusion, we can see microbubbles adherence to the endothelium constituting an echogenic ring around the vessel lumen in all pigs.
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Figure 5 Pulse sequence scheme imaging showing microbubble retention to the endothelium in the carotid arteries (CAs) submitted to balloon-stretching (at left) and absence of retention in the contralateral control vessels (at right). In pig 5, the left CA was initially dissected, and the right CA was then submitted to the balloon dilation and considered for analysis. In this pig, the control demonstrated in the figure is the baseline imaging before dilation of the vessel. For an accompanying video corresponding to Figure 5 (Video 1), please see the September 1 issue of JACC at www.cardiosource.com/jacc.html.
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Figure 6 Example of time-intensity curve showing the difference between the mean acoustic intensity (AI) of the entire lumen (dark line) and the central lumen (gray line) regions (A), and transverse images of carotid artery after PESDA and Definity microbubbles (MB) injection in the setting of hypertriglyceridemia (B). Note the retention of PESDA MB around the vessel, not seen with Definity. Amplification of the curves in the late period after PESDA MB injection (arrow) demonstrates a resulting positive value of mean endothelial AI. On the other hand, after Definity MB injection the central lumen AI was slightly higher than the entire lumen AI with a resulting negative value of endothelial AI.
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Figure 7 The PESDA microbubbles fixed with formalin and examined under scanning electron microscopy (SEM) using a 0.22-µm Millipore filter, at (A) low magnification (bar = 10 µm; magnification 1,420x) and (B) high magnification (bar = 10 µm; magnification 7,400x). Under SEM the microbubbles were characterized as less electron-dense structures of different sizes, spherically shaped and not exhibiting any other surface features of hematopoietic cells.
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Figure 8 High magnification (bar = 10 µm; magnification 1,420x) scanning electron microscopy pictures (left panels) and their respective low mechanical index pulse sequence scheme (PSS) images (right panels). Panel A demonstrates the presence of retained microbubbles in the endothelium detected by PSS in the left injured carotid artery, and Panel B reveals the absence of microbubbles in the endothelium in the control right side. Scanning electron microscopy revealed sites of injury with endothelial denudation and attachment of microbubbles (black arrows) to the denuded endothelium only in the injured vessel (A) and normal-appearing endothelium in the control vessel (B).
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Figure 9 Scanning electron microscopy of carotid artery of a pig euthanized during hypertriglyceridemia at (A) low magnification (bar = 0.1 mm; magnification 462x) and (B) high magnification (bar = 10 µm; magnification 1,420x) showing the presence of microbubbles attached to the structurally normal endothelium (arrows).
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