Potentiating intra-arterial sonothrombolysis for acute ischemic stroke by the addition of the ultrasound contrast agents (Optison™ & SonoVue(®)).

Transcranial ultrasound in combination with intravenously administered ultrasound contrast agents (UCA) in the presence or absence of recombinant tissue plasminogen activator (rt-PA) has been widely evaluated as a new modality for treatment of ischemic stroke. Despite the successful demonstration of accelerated clot lysis there are inherent limitations associated with this modality such as inconsistency in temporal window thickness and/or potential serious cardiopulmonary reactions to intravenous administration of UCA that prevent broad application to ischemic stroke populations. As a complementary modality, we evaluated potential lysis enhancement by intra-arterial ultrasound with concurrent intra-clot delivery of UCA and rt-PA. To this end, clots were formed with average pore diameter similar to clinically retracted clots by adjusting the thrombin concentration. Physical characteristic and retention of UCA after delivery through the catheter as a function of clinically relevant flow rates of 6, 12, 18 ml/h were determined using a microscopic method. The ability of the UCA employed in this study, Optison and SonoVue, to penetrate into the clot was verified using ultrasound B-mode imaging. Clot lysis as a function of rt-PA concentration, 0.009 through 0.5 mg/ml, in the presence and absence of UCA diluted to 1:10, 1:100, and 1:200 v/v at two Peak rarefaction acoustic pressures of 1.3 and 2.1 MPa were evaluated using a weighing method. The study results suggest the addition of only 0.02 ml of 1:100 diluted UCA to rt-PA of 0.009, 0.05, 0.3, and 0.5 mg/ml can enhance the lysis rate by 3.9, 2.6, 1.9 and 1.8 fold in the presence of peak rarefaction acoustic pressure of 1.3 MPa and by 5.1, 3.4, 2.6, 3.1 in the presence of peak rarefaction acoustic pressure of 2.1 MPa, respectively. In addition, Optison and SonoVue demonstrated comparable effectiveness in enhancing the clot lysis rate. Addition of UCA to intra-arterial sonothrombolysis could be considered as a viable treatment option for ischemic stroke patients.

Effect of modulated ultrasound parameters on ultrasound-induced thrombolysis.

The potential of ultrasound to enhance enzyme-mediated thrombolysis by application of constant operating parameters (COP) has been widely demonstrated. In this study, the effect of ultrasound with modulated operating parameters (MOP) on enzyme-mediated thrombolysis was investigated. The MOP protocol was applied to an in vitro model of thrombolysis. The results were compared to a COP with the equivalent soft tissue thermal index (TIS) over the duration of ultrasound exposure of 30 min (p < 0.14). To explore potential differences in the mechanism responsible for ultrasound-induced thrombolysis, a perfusion model was used to measure changes in average fibrin pore size of clot before, after and during exposure to MOP and COP protocols and cavitational activity was monitored in real time for both protocols using a passive cavitation detection system. The relative lysis enhancement by each COP and MOP protocol compared to alteplase alone yielded values of 33.69 +/- 12.09% and 63.89 +/- 15.02% in a thrombolysis model, respectively (p < 0.007). Both COP and MOP protocols caused an equivalent significant increase in average clot pore size of 2.09 x 10(-2) +/- 0.01 microm and 1.99 x 10(-2) +/- 0.004 microm, respectively (p < 0.74). No signatures of inertial or stable cavitation were observed for either acoustic protocol. In conclusion, due to mechanisms other than cavitation, application of ultrasound with modulated operating parameters has the potential to significantly enhance the relative lysis enhancement compared to application of ultrasound with constant operating parameters.

Effect of ultrasound on enzymatic activity of selected plasminogen activators.

INTRODUCTION: Ultrasound has been shown to accelerate enzymatic fibrinolysis with adjunctive plasminogen activators. Additionally, ultrasound is known for interaction with biological substances on molecular level in sonodynamic therapy and sonochemistry. Therefore, we investigated the possibility of ultrasound affecting the biological activity of plasminogen activators used in thrombolysis treatment. MATERIALS AND METHODS: Four currently marketed plasminogen activators were evaluated: urokinase, streptokinase, alteplase, and reteplase. The tests were conducted in reconstituted, undiluted plasminogen activator. Each test contained a control and a test sample. The test sample was incubated in a water bath at temperatures of approximately 34 degrees C and exposed to ultrasound for 1h. The control was incubated in the same water bath as the test sample for the same duration but was not exposed to ultrasound. The ultrasound frequency and intensity used for this experiment were 1 MHz and 2.5-3.1W/cm2, respectively. For quantitative measurement of biological activity of the test and control samples of each plasminogen activator either specific chromogenic substrates or the fibrin clot liquefaction time was used. RESULTS: Student t-test was applied to compare treated vs. control group for each plasminogen activator. The p-value for urokinase, streptokinase, alteplase, and reteplase are 0.43, 0.76, 0.70, and 0.30, respectively. CONCLUSION: Ultrasound with a frequency of 1 MHz and intensities of 2.5-3.1W/cm2 had no statistically significant impact on biological activity of selected plasminogen activators.

Cavitational mechanisms in ultrasound-accelerated fibrinolysis.

The role of both inertial and stable cavitation was investigated during in vitro ultrasound-accelerated fibrinolysis by recombinant tissue plasminogen activator (rt-PA) in the presence and absence of Optison. A unique treatment configuration applied ultrasound, rt-PA and Optison to the interior of a plasma clot. Lysis efficacy was measured as clot weight reduction. Cavitational mechanisms were investigated by monitoring subharmonic and broadband noise. In the absence of Optison, 1.7 MHz pulsed ultrasound with 1.5 MPa peak-negative pressure applied for 30 min resulted in 45 +/- 19% lysis enhancement relative to rt-PA alone. Cavitation was not detected, indicating a role of noncavitational effects of ultrasound. The addition of Optison increased lysis enhancement to 88 +/- 25%. Inertial cavitation was present only at the start of the exposure, while low-amplitude subharmonic emissions persisted throughout. Additional protocols suggested a possible correlation between the increased lysis in the presence of Optison and the subharmonic emission, indicating a potentially important role of stable rather than inertial cavitation in microbubble-enhanced ultrasound-accelerated rt-PA-mediated thrombolysis.

Application of cavitation promoting surfaces in management of acute ischemic stroke.

High frequency, low intensity ultrasound has the potential to accelerate the clearance of thrombotic occlusion in the absence of cavitation. At high frequency ultrasound, high acoustic pressures, >5.2MPa, are required to generate cavitation in thrombus. The focus of this study was to reduce the cavitation threshold by applying materials with appropriate nucleation sites at the transducer-thrombus boundary to further augment sonothrombolysis. Heterogeneous and homogenous nucleation sites were generated on the outer surface of a polyimide tube (PI) using microfringed (MPI) and laser induced (LPI) microcavities. The cavitation threshold of these materials was determined using a passive cavitation detection system. Furthermore, the biological impact of both materials was investigated in vitro. The results revealed that both MPI and LPI have the potential to induce cavitation at acoustic pressure levels as low as 2.3MPa. In the presence of cavitation, thrombolysis rate could be enhanced by up to two times without any evidence of hemolysis that is generally associated with cavitation activities in blood. A prototype ultrasonic catheter operating at 1.7MHz frequency and acoustic pressure of 2.3MPa with either of MPI or LPI could be considered as a viable option for treatment of acute ischemic stroke.

Sonothrombolysis: an emerging modality for the treatment of acute ischemic and hemorrhagic stroke.

To date, it is believed that rapid removal of impedances hindering normal blood circulation in the brain would salvage ischemic tissue. Hence, most treatment modalities undergoing clinical evaluation for treatment of stroke are focused on faster recanalization in acute ischemic stroke or faster hematoma mass reduction in hemorrhagic stroke. Therapeutic ultrasound is among the promising emerging modalities being clinically evaluated to meet this purpose. This review provides an overview of existing clinical data in evaluating sonothrombolysis applications in treatment of acute ischemic and hemorrhagic stroke. Furthermore, the present status of clinical evaluation of microbubbles as a potential adjuvant to this modality is reviewed.

Stability of alteplase in presence of cavitation.

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.

Absence of biological damage from prolonged exposure to intravascular ultrasound: a swine model.

Ultrasound (US) has been used in IMS II (intravascular US) and CLOTBUST (transcranial US) clinical trials for thrombolysis. During the treatment, in addition to the targeted thrombus, other biological components, such as blood and vessel walls are subjected to long durations of US exposure. In this study we explored evidence of biological damage due to mechanical forces or thermal effects of US exposure at the frequency, intensity and duration employed for thrombolysis treatment. Biological effects were investigated by exposing swine ilio-femoral arteries bilaterally to an intravascular US generating catheter and a conventional catheter. A total of 12 animals each underwent 8h of exposure to intravascular pulsed US with a frequency of 2.2MHz and spatial peak time average intensity (I(SPTA)) of 6W/cm(2) per transducer (a total of six transducers per catheter) while the ultrasonic device surface temperature was maintained at 43 degrees C. The animals were euthanized either 24+/-3h or 28+/-3 days post treatment. A range of physiological and hematological parameters were evaluated pre-, post-, and during US exposure. The vascular diameter was determined pre- and post-US exposure using angiograms. Following euthanasia, each animal underwent a gross pathological examination, and the treated vessels and an unexposed vessel were excised for comparative histopathological evaluation. No evidence of biological damage was found at the end of 8h exposure to intravascular US.