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PURPOSE: The study describes the use of intravital microscopy (IVM) to assess the behavior of ultrasound contrast agents (UCAs), including targeted UCAs, in the microcirculation of rodents. MATERIALS AND METHODS: IVM was performed on various exteriorized organs: hamster cheek pouch, rat mesentery, liver, spinotrapezius muscle, and mouse cremaster muscle. A dorsal skin-fold chamber with MatBIII tumor cells was also implanted in rats. Nontargeted UCAs (SonoVue(®) and BR14) and targeted UCAs (BR55 and P-selectin targeted microbubbles) were tested. IVM was used to measure microbubble size, determine their persistence, and observe their behavior in the blood circulation. RESULTS: Intravenous and intra-arterial injections of high doses of UCAs did not modify the local microvascular hemodynamics. No microbubble coalescence and no increased size were observed. Adhesion of some microbubbles to leukocytes was observed in various microcirculation models. Microbubbles are captured by Kupffer cells in the liver. Targeted microbubbles were shown to adhere specifically to endothelial receptors without compromising local blood flow. CONCLUSION: These results support the safety of both targeted and nontargeted UCAs as no microvascular flow alteration or plugging of microvessels were observed. They confirm that binding observed with targeted microbubbles are due to the binding of these microbubbles to specific endothelial receptors.

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Myocardial infarction is frequently developed in canine and porcine models but exceptionally in non-human primates. The aim of this study was to develop a minimally invasive myocardial ischemic/reperfusion model in the monkey intended to be combined with imaging techniques, in particular myocardial contrast echocardiography (MCE). A balloon-tipped catheter was advanced via the femoral artery into the left anterior descending artery (LAD) under fluoroscopic guidance in ten anaesthetized male rhesus monkeys (Macaca mulatta). The balloon was inflated to completely occlude the vessel. Coronary angiography (CA) was performed to control the reality of the LAD occlusion/reperfusion. The ischemia period was followed by 3-6 h of reperfusion. Myocardial perfusion was evaluated during ischemia and at reperfusion by MCE using a novel ultrasound contrast agent (BR38). Occlusion was successfully induced during 18-50 min in nine out of the ten evaluated monkeys. ST segment elevation indicated myocardial ischemia. MCE showed complete transmural arrest of myocardial blood flow during the ischemia period and no persistent microvascular perfusion defects during reperfusion. A minimally invasive closed-chest model was successfully developed for creating myocardial ischemia in the rhesus monkey (Macaca mulatta). This technique could have an important role in mimicking acute coronary syndrome under physiologically and ethically-acceptable conditions. MCE provides non-invasively information on myocardial perfusion status, information not available from CA.

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OBJECTIVE: : To evaluate BR38, a new microbubble-based blood pool agent for contrast-enhanced ultrasound imaging. MATERIALS AND METHODS: : The size characteristics of BR38 microbubbles were measured by Coulter counting. The backscatter and attenuation coefficients were determined as a function of frequency. Additional measurements included the surface charge, osmolality, viscosity, and resistance to hydrostatic pressure. Extensive pharmacological and toxicological studies were conducted on the final formulation in rats and dogs. The blood levels and elimination of the gaseous component C4F10 were determined in the rabbit. Contrast-enhanced echographic examinations were performed in pigs focusing on the myocardium and the liver. Finally, safety testing and preliminary imaging experiments were performed in a Phase I clinical study in human volunteers. RESULTS: : BR38 suspensions are isotonic, nonviscous, and show a high resistance to hydrostatic pressure. Their backscatter coefficient is high at ≥ 2 MHz and attenuation shows a maximum at 4 MHz, slowly decreasing at higher frequencies. The no adverse effect levels of 1 µL/kg (rats) and 5 µL/kg (dogs) expressed as microbubble gas volume, observed in repeated toxicology studies, correspond to 50 and 250 times the expected imaging dose in human beings (0.02 µL/kg), respectively. No effects on cardiovascular and respiratory parameters were observed in rats and dogs. C4F10 is eliminated within minutes from blood and excreted in expired air. Imaging experiments showed strong and persistent enhancement of the myocardium and the liver. A late phase was observed in the liver, in animals and in human volunteers. No serious adverse events and no significant changes in vital signs, electrocardiographs, and laboratory tests were observed in Phase I human volunteers. CONCLUSIONS: : BR38 shows a very good safety profile. It is characterized by a long persistence and low shadowing. BR38 is a promising ultrasound blood pool agent for noncardiac and cardiac applications including myocardial perfusion imaging.

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OBJECTIVE: Sentinel lymph node (SLN) identification using intradermal micro-bubbles and contrast-enhanced ultrasound (CEUS) has been recently reported in swine models and patients with breast cancer. The objective of this study was to investigate the dynamics of intradermally administered microbubbles as they travel to draining SLNs in pigs. We also performed a detailed study of the passage of microbubbles through breast lymphatic channels in a small group of patients with breast cancer. METHODS: Nine anesthetized healthy pigs were used for the study, and 5 female patients with primary breast cancer were recruited. Pigs received intradermal injections of a microbubble contrast agent in several territories to access lymphatic drainage to regional lymph nodes. Patients had periareolar intradermal injection of the microbubble contrast agent. Ultrasound examination was performed in the real-time contrast pulse sequencing mode with a commercial scanner. RESULTS: Sentinel lymph nodes were identified rapidly (<1 minute) and consistently in pigs. Intradermal microbubble injection and CEUS were found to have perfect concordance with the Evans blue dye method in locating swine SLNs. In all 5 patients with breast cancer, the microbubble contrast agent entered breast lymphatic channels and traveled to draining ipsilateral axillary SLNs within 3 minutes. CONCLUSIONS: Intradermally injected microbubbles traverse readily though lymphatic channels in pigs and human breast tissue. The ability to rapidly identify SLNs in the diagnostic period would enable targeted biopsy and may facilitate preoperative axillary staging in patients with early breast cancer.

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The design of targeted ultrasound contrast agents for molecular imaging of myocardial ischemia-reperfusion (IR) requires the availability of an adequate in vivo model in a species in which cross reactivity with the target occurs. P-selectin (Psel) is an activation-dependent endothelial receptor that supports rapid and reversible cell adhesion in a flowing system. Together with E- and L-selectins it constitutes the selectin family of adhesion molecules. We investigated the myocardial expression of selectins in a closed chest minimally invasive monkey myocardial IR model. Catheter-based occlusion (30-50 min) followed by reperfusion (3 or 5 h) of left anterior descending artery (LAD) was performed in anesthetised monkeys. At the end of each procedure animals were killed, and their hearts were excised. The tissues were analyzed immunohistochemically using an anti-human Psel antibody (AK-6 clone) that cross reacts with rhesus monkey. Histopathological features confirm the presence of IR injuries in myocardial tissues. There was significant increase in the Psel expression in vessels from the IR areas. However, significantly higher Psel immunoreactivity was also seen in areas which are distant from IR injuries.

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The introduction of stable microbubble contrast agents and technological advances have recently made it feasible to perform quantitative measurements of microvascular damage by myocardial contrast echocardiography (MCE). Qontrast is a new software system far such measurements. It includes physiological filters, global rescale, regional rescale, automatic myocardial tracking, manual ECG trigger and parametric imaging. Qontrast was tested on 5 pigs given sulphur hexafluoride bubbles (I ml/min) and fluorescent microspheres (reference) after the induction of 50% and 100% stenosis of left anterior descending coronary artery. The image sequences were repeated four times using different ultrasound (US) equipment. A close correlation was found between the ratio risk area/control area by microspheres and the equivalent ratio risk area/control area (Sl x beta) by MCE, being approximately 0.9 far any contrast modality tested. Parametric MCE and SPELT were compared in 12 patients with recent myocardial infarction, including 119 segments. Agreement amounted to 83% (kappa: 0.53 far peak SI and 0.55 far SI x beta). The sensitivity and specificity of peak SI far detecting abnormal segmental tracer uptake were 67% and 88%; the values far Sl x beta were 70% and 87%. Parametric MCE is a promising imaging technique far the assessment of myocardial perfusion in patients with suspected or known coronary artery disease.

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