A novel method for quantitative myocardial contrast echocardiography in mice.

The small size of the mouse heart frequently imparts technical challenges when applying conventional in vivo imaging methods for assessing heart function. Here, we describe the use of high-frequency ultrasound imaging in conjunction with a size-tuned blood pool contrast agent for quantitatively assessing myocardial perfusion in living mice. A perflurocarbon microbubble formulation exhibiting a narrow size distribution was developed, and echogenicity was assessed at 18 MHz in vitro. Adult mice were subjected to permanent ligation of the left anterior descending artery. Ultrasound imaging was performed on day 7, and a cohort of intact mice was used as a control. Parasternal long-axis cine clips were acquired at 18 MHz before and after contrast administration. Reduced ejection fraction and increased end-systolic volume were observed in infarcted compared with control mice. In control animals, washin of the contrast agent was visible in all myocardial segments. Reduced contrast enhancement was observed in apical-posterolateral regions of all infarcted mice. A novel method for reslicing of the imaging data through the time domain provided a two-dimensional presentation of regional contrast agent washin, enabling convenient identification of locations exhibiting altered perfusion. Myocardial segments exhibiting diminished contractility were observed to have correspondingly low relative myocardial perfusion. The contrast agent formulation and methods demonstrated here provide the basis for simplifying routine in vivo estimation of infarct size in mice and may be particularly useful in longitudinal evaluation of revascularization interventions and assessment of peri-infarct ischemia. NEW & NOTEWORTHY Murine myocardial contrast echocardiography frequently suffers from poor sensitivity to contrast. Here, we formulated a novel size-tuned microbubble contrast agent and validated it for use with ultra-high-frequency ultrasound. A novel data method for evaluating myocardial perfusion based on reslicing the imaging data through the time domain is presented.

Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow.

To improve ultrasound contrast agents targeted to the adhesion molecules P-selectin and VCAM-1 for the purpose of molecular imaging of atherosclerotic plaques, perfluorocarbon-filled phospholipid microbubble contrast agents were coupled by a polyethylene glycol-biotin-streptavidin bridge with mAb MVCAM.A(429), a sialyl Lewis(x) polymer (PAA-sLe(x)), or both (dual). Approximately three hundred thousand antibody molecules were coupled to the surface of each microbubble. Recombinant mouse P-selectin and/or VCAM-1 coated on flow chambers showed saturation of binding at approximately 15 ng/microl, resulting in 800 and 1200 molecules/microm(2) for P-selectin and VCAM-1, respectively. Dual substrates coated with equal concentrations of P-selectin and VCAM-1 had site densities between 50 and 60% of single substrates. When microbubbles were perfused through flow chambers at 5 x 10(6) microbubbles/ml (wall shear stress from 1.5 to 6 dyn/cm(2)) dual-targeted microbubbles adhered almost twice as efficiently as single-targeted microbubbles at 6 dyn/cm(2). The present study suggests that dual-targeted contrast agents may be useful for atherosclerotic plaque detection at physiologically relevant shear stresses.

Selectin ligands promote ultrasound contrast agent adhesion under shear flow.

Contrast-enhanced ultrasound imaging has shown promise in the field of molecular imaging. This technique relies upon the adhesion of ultrasound contrast agent (UCA) to targeted molecular markers of disease. This is accomplished by coating the surface of the contrast agent with a ligand that specifically binds to the intended molecular marker. Most UCA particles remain in the blood space, and their retention is influenced by the forces imposed by blood flow. For a UCA bound to a molecular target on the vascular endothelium, blood flow imposes a dislodging force that counteracts retention. Additionally, contrast agent adhesion to the molecular marker requires rapid binding kinetics, especially in rapid blood flow. The ability of a ligand:target bond complex to mediate fast adhesion and withstand dislodging force is necessary for efficient ultrasound-based molecular imaging. In the current study, we describe a flow-based adhesion assay which, combined with a novel automated tracking algorithm, enables quick determination of the ability of a targeting ligand to mediate effective contrast agent adhesion. This system was used to explore the adhesion of UCA targeted to the proinflammatory endothelial protein P-selectin via four targeting ligands, which revealed several interesting adhesive behaviors. Contrast agents targeted with glycoconjugate ligands modeled on P-selectin glycoprotein ligand 1 exhibited primarily unstable or transient adhesion, while UCA targeted with an anti-P-selectin monoclonal antibody exhibited primarily firm adhesion, although the efficiency with which these agents were recruited to the target surface was relatively low.

Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow.

Targeted ultrasound contrast materials (gas-filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm(2) wall shear stress) using a clustered polymeric form of the fast-binding selectin ligand sialyl Lewis(X). Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin-PEG-lipid. Biotinylated PSLe(x) (sialyl Lewis(X) polyacrylamide) or biotinylated anti-P-selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe(x) bubbles demonstrated specific adhesion, retention and slow rolling on P-selectin-coated plates. Efficiency of firm targeted adhesion to a P-selectin surface (140 molecules/microm(2)) was comparable for antibody-carrying bubbles and PSLe(x)-targeted bubbles at 0.68 dyne/cm(2) shear stress. At fast flow (4.45 dyne/cm(2)), PSLe(x)-targeted bubbles maintained their ability to bind, while antibody-mediated targeting dropped more than 20-fold. At lower surface density of P-selectin (7 molecules/microm(2)), targeting via PSLe(x) was more efficient than via antibody under all the flow conditions tested. Negative control casein-coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe(x)-bubbles were visualized on P-selectin-coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe(x)-microbubbles was comparable with targeting mediated by anti-P-selectin antibody, and significantly exceeded the accumulation of non-targeted control bubbles. In the inflamed femoral artery setting, PSLe(x)-mediated microbubble targeting was superior to antibody-mediated targeting.

Irreducible "pulled elbow" in a child. A case report.

"Pulled elbow" is a common pediatric injury that occurs when axial traction is applied to an arm that is extended while the forearm is pronated. These forces can create a transverse tear in the annular ligament, which permits subluxation of the radial head. In most cases, closed reduction can be performed by supinating the forearm and flexing the elbow. In the case of a five-year-old boy, surgical reduction of this injury was necessary to reduce the subluxated radial head.