Ultrasound-Enhanced rt-PA Thrombolysis in an ex vivo Porcine Carotid Artery Model

Abstract

Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model.

Cavitation thresholds of contrast agents in an in vitro human clot model exposed to 120-kHz ultrasound

“…A second PCD was aligned confocally with the blood clot sample in flow phantom. This secondary PCD, a focused 2.25-MHz transducer (595516C, Picker Roentgen GmbH, Espelkamp, Germany), has previously been utilized to detect cavitation at 120 kHz (Hitchcock et al, 2011). The PCD aperture is 1.9 cm and the focal length is 5.5 cm.…”

“…This finding is consistent with previous investigators using UCAs to nucleate cavitation at 120 kHz. Hitchcock et al (2011) applied continuous-wave 120 kHz ultrasound and Definity V R in an ex vivo porcine carotid artery model. They found that a maximal amount of UH energy was delivered over 30 min at an acoustic pressure of 0.44 MPa (peak to peak).…”

“…Ultrasound contrast agents (UCAs) are stabilized microbubbles that can be injected into the bloodstream and have previously been used to nucleate acoustic cavitation (Rota et al, 2006;Sboros, 2008). Initial studies have demonstrated the efficacy of UET in the presence of UCAs (Molina et al, 2006;Datta et al, 2008;Hitchcock et al, 2011). These studies demonstrated UET when acoustic emissions characteristic of stable cavitation, ultraharmonics (UHs) (Ilyichev et al, 1989;Datta et al, 2008) and the subharmonic (Neppiras, 1969), were present.…”

“…Two UCAs were used to nucleate cavitation, Definity V R and echogenic liposomes (ELIP). Definity V R , a commercially available UCA, has been shown previously to nucleate stable cavitation and enhance thrombolysis (Datta et al, 2008;Brown et al, 2011;Hitchcock et al, 2011). ELIP are potential theragnostic agents for the treatment of stroke Radhakrishnan et al, 2012;Kopechek et al, 2013) with the ability to encapsulate and release rt-PA upon exposure to ultrasound.…”

Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue

“…Each frequency spectrum was scaled to account for the energy lost due to windowing. A frequency domain comb filter was applied to the power spectrum ( Figure 3C) to extract the ultraharmonic (100 kHz bands at odd multiples of 500 kHz) and broadband 38,39 This process is depicted graphically in Figure 3.…”