Microbubbles Improve Sonothrombolysis In Vitro and Decrease Hemorrhage In Vivo in a Rabbit Stroke Model

Abstract

Introduction Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH. Materials and Methods Fresh blood clots (3–5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm2), with or without 1.12 × 108/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm2) for 60 minutes and intravenous tPA (0.1– 0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later. Results In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P < 0.0183 and P < 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19. Conclusion Combining tPAand MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

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

“…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.…”

Sonothrombolysis in acute stroke and myocardial infarction: A systematic review

“…A possible higher incidence of intracranial hemorrhage when using high frequency ultrasound as used in the study by Molina et al might be related to the enhanced effectiveness of TPA when administered in the presence of microbubbles. This supposition was supported by subsequent work of Culp et al, in a rabbit model of stroke, which showed that 1-Mhz ultrasound with TPA and microbubbles increases recanalization rates compared to ultrasound and TPA alone, without an increase in the incidence of intracranial hemorrhage [10]. They also showed effective sonolysis using microbubbles and ultrasound without TPA in a rabbit stroke model.…”

“…This can be achieved by the use of microbubbles [8,9], possibly even without the use of a fibrinolytic agent [10].…”

High-intensity focused ultrasound: past, present, and future in neurosurgery

“…20 Low-intensity ultrasound combined with systemic delivery of microbubble contrast agents has been shown to improve thrombolysis in the presence or absence of tissue plasminogen activator (tPA) in the past. 7,19,20,24 The Combined Lysis of Thrombus in Brain Ischemia Using Transcranial Ultrasound and Systemic tPA (CLOTBUST) trial and the Transcranial Low-Frequency Ultrasound-Mediated Thrombolysis in Brain Ischemia (TRUMBI) trial with unfocused, low-frequency (300 kHz) ultrasound have shown some promise, but with complications such as increased hemorrhage rates. 1,18 HIFU as a stand-alone method for thrombolysis seems to be more advantageous, as it reduces the risk of hemorrhage by eliminating the side effects of thrombolytic drugs as well as adequately causing thrombolysis without damage to the targeted vessels.…”