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In vitro and in vivo investigation of enzymatic clot lysis through non-thermal mechanisms using focused ultrasound waves as an adjunct to thrombolytic drug tenecteplase and in synergy with microbubbles

Papadopoulos, N. (2017). In vitro and in vivo investigation of enzymatic clot lysis through non-thermal mechanisms using focused ultrasound waves as an adjunct to thrombolytic drug tenecteplase and in synergy with microbubbles. (Unpublished Doctoral thesis, City, University of London)


In this thesis, the beneficial effect of ultrasound (US) energy is utilized for accelerating thrombolysis efficacy through non-thermal mechanisms. The outcome of the research, can be applied in the future for clinical use, providing alternative techniques (either noninvasive or invasive), for treating ischaemic stroke under MR guidance.

To study the effect of US on thrombolysis efficacy, three different in vitro circulating flow clot models were developed. Fully retracted porcine blood clots were treated with US waves as an adjunct to thrombolytic drug tenecteplase (TNK-tPA), in the presence or absence of microbubbles (MBs). Each one of the flow clot models used, was designed to reproduce a different physiologic situation of middle cerebral artery (MCA) occlusion, since it is the most common cause of stroke. In all the proposed treatment protocols, temperature elevation at beam focus never exceeded 1 0C, providing that the contribution of thermal mechanisms to clot lysis was negligible.

The first model, was reproducing a deep-seated MCA occlusion in a brain tissue. To provide a more realistic clinical environment, the study was conducted into a brain tissue mimicking phantom. Flow rate was set to 20 % of the maximum value occur in an open MCA and TNK-tPA dose was not exceeding the 30 % of the average maximum concentration in blood. Using 1.18 MHz focused US (FUS) waves, various experimental parameters that influence thrombolysis efficacy, were optimized. Maximum thrombolysis efficacy was observed when FUS pulses were used as an adjunct to thrombolytic drug in the presence of MBs. With this technique, 370 mg of clot mass was removed in 30 min, which may not be enough to achieve significant clinical benefits. However, further improvement was done (increase of flow rate and TNK-tPA concentration), to enhance thrombolysis efficacy.

The second model, was reproducing a MCA occlusion occurred superficially. Flow rate was increased four times and TNK-tPA concentration was doubled. The optimum operating mode parameters obtained before, were employed in this study in order to investigate the impact of frequency and acoustic intensity on thrombolysis efficacy. Study findings established that higher FUS frequencies (1.18 MHz), are associated with enhanced thrombolysis compared to lower frequencies (0.6 MHz). Also, a linear increasing dependence between acoustic intensity and thrombolysis efficacy was observed. After 30 min of treatment with 1.18 MHz FUS exposures in synergy with TNKtPA and MBs, 1050 mg of clot mass was removed, which should be sufficient for timely recanalization of an occluded cerebral artery.

The third model, was reproducing a MCA occlusion, treated invasively with a catheter-directed US device. The efficacy of two small planar ultrasonic transducers (operating at frequencies 3.7 and 5.2 MHz respectively), on clot lysis was evaluated. The values of flow rate and TNK-tPA concentration were kept the same with those used in the previous study. Using 3.7 MHz US waves in association with TNK-tPA and MBs for 30 min, 700 mg of clot mass was removed, showing that potentially, intravascular sonothrombolysis with such a transducer can be an effective method for treating stroke.

Finally, the knowledge gained in vitro for enhancing TNK-tPA induced thrombolysis, was translated in vivo using an animal model. A blood clot was artificially formed into the right carotid artery of a rabbit, mimicking a MCA occlusion in humans. The complete flow blockage as well as the recanalization procedure were monitored using a 3D time of flight MR angiography. This novel technique, clearly demonstrated that the combination of 1.18 MHz FUS pulses with MBs, strongly accelerated the action of TNKtPA, leading through non-thermal mechanisms to full artery recanalization within 19 min.

Publication Type: Thesis (Doctoral)
Publisher Keywords: ultrasound, stroke, clot, TNK-tPA, MBs, agar, intravascular, MRgFUS
Subjects: R Medicine
T Technology > TA Engineering (General). Civil engineering (General)
Departments: Doctoral Theses
School of Science & Technology > Engineering
School of Science & Technology > School of Science & Technology Doctoral Theses
Text - Accepted Version
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