Using an FDA-approved contrast agent (Definity®) and thrombolytic drug (rt-PA), we investigated ultrasound-enhanced thrombolysis in two whole-blood clot models. Porcine venous blood was collected from donor hogs and coagulated in two different materials. This method produced clots with differing compositional properties, as determined by routine scanning electron microscopy and histology. Clots were deployed in an ex vivo porcine thrombolysis model, while an intermittent ultrasound scheme previously developed to maximize stable cavitation was applied and acoustic emissions were detected. Exposure of clots to 3.15 μg/mL rt-PA promoted lysis in both clot models, compared to exposure to plasma alone. However, in the presence of rt-PA, Definity®, and ultrasound, only unretracted clots experienced significant enhancement of thrombolysis compared to treatment with rt-PA. In these clots, microscopy studies revealed loose erythrocyte aggregates, a significantly less extensive fibrin network, and a higher porosity, which may facilitate increase penetration of thrombolytics by cavitation.

Ultrasound contrast agents (UCAs) are used clinically to aid detection and diagnosis of abnormal blood flow or perfusion. Characterization of UCAs can aid in the optimization of ultrasound parameters for enhanced image contrast. In this study echogenic liposomes (ELIPs) were characterized acoustically by measuring the frequency-dependent attenuation and backscatter coefficients at frequencies between 3 and 30 MHz using a broadband pulse-echo technique. The experimental methods were initially validated by comparing the attenuation and backscatter coefficients measured from 50-μm and 100-μm polystyrene microspheres with theoretical values. The size distribution of the ELIPs was measured and found to be polydisperse, ranging in size from 40 nm to 6 μm in diameter, with the highest number observed at 65 nm. The ELIP attenuation coefficients ranged from 3.7  ±  1.0 to 8.0  ±  3.3 dB/cm between 3 and 25 MHz. The backscatter coefficients were 0.011  ±  0.006 (cm str)(-1) between 6 and 9 MHz and 0.023 ± 0.006 (cm str)(-1) between 13 and 30 MHz. The measured scattering-to-attenuation ratio ranged from 8% to 22% between 6 and 25 MHz. Thus ELIPs can provide enhanced contrast over a broad range of frequencies and the scattering properties are suitable for various ultrasound imaging applications including diagnostic and intravascular ultrasound.

Echogenic liposomes (ELIPs) are being developed for use as ultrasonic contrast agents and as drug carriers for ultrasound-targeted drug delivery. Physical and acoustical characterization of ELIPs is necessary in order to determine the optimum parameters for diagnostic and therapeutic applications. In this study, ELIP samples at concentrations of 10, 20, and 50 μg/ml were exposed to ultrasound in pulse-echo mode at center frequencies of 2.25, 3.5, 7.5, 10, 15, and 30 MHz. The received echoes were analyzed to determine the attenuation and backscatter coefficients and the results were compared to a theoretical computational model. The sample chamber contained two 50-μm tungsten wires as reference scatterers. The echoes from the wires were acquired before and after addition of ELIP to determine the attenuation coefficient. The backscatter coefficient was determined by averaging the square of the RF amplitude between the wires and accounting for transducer parameters. Each transducer was calibrated and characterized in deionized water using PVDF hydrophones. The peak attenuation coefficient occurred at 7.5 MHz while the backscatter coefficient increased with frequency. This was in agreement with the computational model. These results provide important information for determining the optimum acoustical parameters for ELIP exposure in diagnostic and therapeutic applications. [Work supported by NIH 2RO1 HL059586-04A2 and ASA Hunt Postdoctoral Research Fellowship.]

No abstract

Ultrasound acts synergistically with thrombolytic agents, such as recombinant tissue plasminogen activator (rt-PA), to accelerate thrombolysis. The aim of the study was to demonstrate the efficacy of 120-kHz ultrasound-enhanced rt-PA thrombolysis in a porcine hemorrhagic stroke model in vivo. Clots were formed by infusing 3 ml of autologous blood into the frontal white matter of 30 mixed-bred Yorkshire pigs (20.5–3.1 kg) and incubated for 3 h. For these nonsurvival studies, six pigs received rt-PA alone (0.3 cc of 0.107 mg/ml), six received ultrasound alone, six received rt-PA plus ultrasound, six were sham-exposed (saline only), and six were controls (no ultrasound or rt-PA treatment). The clots receiving ultrasound treatment were insonified with a peak-to-peak pressure of 0.48 MPa in situ (80% duty cycle, and PRF of 1.7 kHz) for 30 min. Clots treated with rt-PA alone exhibited a volume loss of 55.0% and clots treated with rt-PA and 120-kHz ultrasound had a significantly higher volume loss of 75.2% and a higher penetration of rt-PA. Thus, 120-kHz pulsed ultrasound enhancement of thrombolysis has been demonstrated both in vitro and in an in vivo porcine hemorrhagic stroke model.