Accelerated sonothrombolysis with Definity in a xenographic porcine cerebral thromboembolism model

Abstract

Adjuvant ultrasound at 2 MHz with or without an ultrasound contrast agent improves the rate of thrombus resolution by recombinant tissue plasminogen activator (rt-PA) in laboratory and clinical studies. A sub-megahertz approach can further expand this therapy to a subset of patients with an insufficient temporal bone window, improving efficacy in unselected patient populations. The aim of this study was to determine if a clinical ultrasound contrast agent (UCA), Definity, and 220 kHz pulsed ultrasound accelerated rt-PA thrombolysis in a preclinical animal model of vascular occlusion. The effect of Definity and ultrasound on thrombus clearance was first investigated in vitro and subsequently tested in a xenographic porcine cerebral thromboembolism model in vivo. Two different microcatheter designs (end-hole, multi-side-hole) were used to infuse rt-PA and Definity at the proximal edge or directly into clots, respectively. Sonothrombolysis with Definity increased clot mass loss relative to saline or rt-PA alone in vitro, only when rt-PA was administered directly into clots via a multi-side-hole microcatheter. Combined treatment with rt-PA, Definity, and ultrasound in vivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline. In this porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity increased the probability of early successful reperfusion with rt-PA.

Figure 1

In vitro percent clot mass loss (A) and flow (B) for human whole blood clots with n = 10 for each treatment through each microcatheter. The end-hole microcatheter was positioned at the proximal face of the clot, and the multiple-side-hole microcatheter crossed the clot axially. Flow measurements spanned -0.2 mL/min for a fully occluded APA mimic (retrograde flow due to the treatment infusion) to 0.75 mL for a fully recanalized APA mimic (dotted lines). A one-way ANOVA was performed and significance was shown using compact letter display to identify statistically equivalent groups.

Figure 2

Representative digital subtraction angiographic images during in vivo procedure. Standard anatomy can be observed in (A). The common carotid artery (CCA) can be seen branching into the ascending pharyngeal artery (APA) and external carotid artery (ECA). The APA ends in the rete mirabile (Rete). The ECA is occluded using bare platinum and hydrogel coils (B), which diverts flow preferentially to the APA and reduces collateral circulation to the brain. The APA is successfully embolized with a human whole blood clot (mTICI < 2b) (C) and treated for 2 h with either sham, rt-PA only, or sonothrombolysis with Definity (2-h rt-PA only treatment arm shown with mTICI ≥ 2b) (D).

Figure 3

Reperfusion mTICI scores for (A) clots treated using the end-hole microcatheter or (B) the multiple-side-hole microcatheter. For plot (A), n = 12 for sham, n = 7 for rt-PA only, and n = 5 for sonothrombolysis with Definity. In plot (B), n = 5 for all three treatment arms. No recanalization up to or greater than mTICI = 2b was observed for any clots treated with the end-hole microcatheter (A). Reperfusion was accelerated for the rt-PA only treatment arm (75–105 min), and even more for sonothrombolysis with Definity (0–60 min) compared to sham (B). Percent APA lumen reduction, was used to quantify vasospasm based on DSA for the end-hole (C) and multiple side hole (D) microcatheters.

Figure 4

Representative longitudinal histology images of residual clot burden in APAs with Verhoeff-Van Giessen (VVG) stain. Residual clot volume can be seen in vessels in the sham treatment arm (A, D) rt-PA only treatment arm (B, E) or the sonothrombolysis with Definity treatment arm. Images A–C show vessels for which the perfusion was graded as ≥ 2b at 120 min on the mTICI scale and images D-F show vessels graded < 2b at 120 min.

Figure 5

Schematic of in vitro vascular occlusion model. A highly retracted human whole blood clot was deployed in a phantom of the ascending pharyngeal artery (APA) via an 8 F guide catheter and held in place by a rete phantom. A collateral tube simulated flow in the external carotid artery (ECA). A flow probe monitored flow through the occluded APA phantom. A passive cavitation detector (PCD) was aligned with the clot and field of the 220 kHz transducer for sonothrombolysis nucleated by Definity.