Targeson (United States)

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.

Obesity-related kidney disease occurs as a result of complex interactions between metabolic and hemodynamic effects. Changes in microvascular perfusion may play a major role in kidney disease; however, these changes are difficult to assess in vivo. Here, we used perfusion ultrasound imaging to evaluate cortical blood flow in a mouse model of high-fat diet-induced kidney disease. C57BL/6J mice were randomized to a standard diet (STD) or a high-fat diet (HFD) for 30 wk and then treated either with losartan or a placebo for an additional 6 wk. Noninvasive ultrasound perfusion imaging of the kidney was performed during infusion of a microbubble contrast agent. Blood flow within the microvasculature of the renal cortex and medulla was derived from imaging data. An increase in the time required to achieve full cortical perfusion was observed for HFD mice relative to STD. This was reversed following treatment with losartan. These data were concurrent with an increased glomerular filtration rate in HFD mice compared with STD- or HFD-losartan-treated mice. Losartan treatment also abrogated fibro-inflammatory disease, assessed by markers at the protein and messenger level. Finally, a reduction in capillary density was found in HFD mice, and this was reversed upon losartan treatment. This suggests that alterations in vascular density may be responsible for the elevated perfusion time observed by imaging. These data demonstrate that ultrasound contrast imaging is a robust and sensitive method for evaluating changes in renal microvascular perfusion and that cortical perfusion time may be a useful parameter for evaluating obesity-related renal disease.

Gammadelta (γδ) T lymphocytes respond quickly upon antigen encounter to produce a cytokine response. In this study, we sought to understand how functions of γδ T cells are differentially regulated compared to αβ T cells. We found that cholesterol, an integral component of the plasma membrane and a regulator of TCR signaling, is increased in γδ T cells compared to αβ T cells, and modulates their function. Higher levels of activation markers, and increased lipid raft content in γδ cells suggest that γδ T cells are more activated. Cholesterol depletion effectively decreased lipid raft formation and activation of γδ T cells, indicating that increased cholesterol content contributes to the hyper-activated phenotype of γδ T cells, possibly through enhanced clustering of TCR signals in lipid rafts. TCR stimulation assays and western blotting revealed that instead of a lower TCR threshold, enhanced TCR signaling through ERK1/2 activation is likely the cause for high cholesterol-induced rapid activation and proliferation in γδ T cells. Our data indicate that cholesterol metabolism is differentially regulated in γδ T cells. The high intracellular cholesterol content leads to enhanced TCR signaling and increases activation and proliferation of γδ T cells.

A large body of work has investigated the influence of the excitation pulse, agent size distribution, and the ambient pressure on the subharmonic response of microbubble contrast agents (MCA). The purpose of this study was to investigate the temporal evolution of the subharmonic emissions, i.e., whether the subharmonic response is influenced by the time elapsed since agent constitution. We measured subharmonic emissions from a commercial lipid-encapsulated contrast agent (Targestar-P®, Targeson Inc., San Diego) over the time span of 60 min. The excitation parameters were as follows: 10-MHz frequency, 30–290 kPa pressures, 60 cycles, and 1-kHz pulse repetition frequency. The subharmonic emissions were observed to increase by 11 dB over 60 min relative to those measured immediately after reconstitution. The most striking increase (>8 dB) was observed in the first 15 min. Although we did not observe a change in the agent size distribution, the pressure threshold for subharmonic emissions reduced by nearly two-fold within the time span of our measurements. This work demonstrates that time evolution of subharmonic emissions could bias quantitative estimates obtained from techniques such as subharmonic imaging and subharmonic-aided pressure estimation. Additionally, these findings suggest the possibility for improving subharmonic emission by careful agent design.

A large body of work has investigated the influence of the excitation pulse, agent size distribution and the ambient pressure on the subharmonic response of microbubble contrast agents (MCA). The purpose of this study was to investigate the temporal evolution of the subharmonic emissions, i.e., whether the subharmonic response is influenced by the time elapsed since agent constitution. We measured subharmonic emissions from a commercial lipid-encapsulated contrast agent (Targestar-P®, Targeson Inc, San Diego, CA,USA) over the time span of 60 minutes. The excitation parameters were: 10-MHz frequency, 30 - 290 kPa pressures, 60 cycles, and 1-kHz pulse repetition frequency. The subharmonic emissions were observed to increase by 11 dB over 60 minutes relative to those measured immediately after reconstitution. The most striking increase (> 8 dB) was observed in the first 15 minutes. Although we did not observe a change in the agent size distribution, the pressure threshold for subharmonic emissions reduced by nearly two-fold within the time span of our measurements. This work demonstrates that time evolution of subharmonic emissions could bias quantitative estimates obtained from techniques such as subharmonic imaging and subharmonic-aided pressure estimation. Additionally, these findings suggest the possibility for improving subharmonic emission by careful agent design.®

Just about a decade ago, targeted or molecular1 imaging with ultrasound contrast agents was deemed impossible. However, with the advances of ultrasound imaging equipment and improved design of ultrasound contrast agents, the old point of view could be reconsidered. Currently, ultrasound contrast agents are widely investigated as the imaging tools for molecular imaging of the specific markers, mostly for the targets located within the vascular bed.2 Most of the studies are performed in vivo in the animal model setting and clinical trials are expected to follow in the near future.

Focal drug delivery using ultrasound applied to microbubbles in the vascular system relies on the microbubbles being in close proximity to the vessel wall. In-vitro microbubble translation to a vessel wall under primary radiation force using an intravascular ultrasound (IVUS) catheter was tested using a wall-less flow phantom. Experiments were performed in 40% hematocrit blood to optimize the Pulse Repetition Frequency (PRF) for translating microbubbles to the edge of a channel under flow. An optimal PRF of 10 kHz in blood was selected for the IVUS catheter based on the results of the wall-less flow phantom experiments. Ex-vivo delivery of Dil, a hydrophobic fluorescent dye used as a model drug, was performed on excised porcine arteries under flow conditions of 30 ml/min with microbubbles incorporating Dil and both a 30 μs, 80 kPa PNP, 1.5 MHz Gaussian ramped sinusoid at 10kHz PRF for microbubble translation and 20% fractional bandwidth, 120 kPa PNP, Gaussian at 1kHz PRF for microbubble destruction. The results of Dil delivery were imaged with a fluorescent microscope and indicated a 665% greater intensity (in arbitrary units derived from fluorescence intensity) in the treated region versus the untreated region. This demonstrates that the IVUS catheter successfully provided localized delivery of the Dil marker to a porcine carotid artery.

A method for simultaneously pushing and imaging microbubbles is introduced. A programmable scanner with a broadband linear array was used to generate dual frequency pulses. For the range of flow velocities (2.65, 5.3, and 15.9 cm/s), microbubble concentrations (2.2 and 22 million/ml), and pushing frequencies (1.6, 3, 4, 6, 8 MHz), it was observed that the adherent microbubble signal in a 2 mm diameter vessel flow gelatin phantom reduced exponentially with increasing streaming velocities. Maximum adherent microbubble signal intensity was observed to scale with variations in the streaming microbubble concentration. The rate of increment in the adherent microbubble signal was found to be independent of the microbubble concentration. The adherent microbubble signal was maximized at the resonant frequency associated with the diameter corresponding to mean of the microbubble distribution. It was concluded that using the proposed dual frequency sequence, it may be feasible to use radiation force for optimizing the effect of targeted imaging and modulating drug delivery in large vessels with high shear rates.