Transcranial ultrasound-improved thrombolysis: diagnostic vs. therapeutic ultrasound

Ultrasound in Med. & Biol., Vol. 27, No. 12, pp. 1683–1689, 2001 Copyright © 2002 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/01/$–see front matter

PII: S0301-5629(01)00481-1

● Original Contribution TRANSCRANIAL ULTRASOUND-IMPROVED THROMBOLYSIS: DIAGNOSTIC VS. THERAPEUTIC ULTRASOUND STEPHAN BEHRENS,* KONSTANTINOS SPENGOS,* MICHAEL DAFFERTSHOFER,* HELMUT SCHROECK,‡ CARL E. DEMPFLE† and MICHAEL HENNERICI* *Department of Neurology, †First Department of Internal Medicine Universitaetsklinikum Mannheim, and ‡Institute of Physiology, University of Heidelberg, Mannheim, Germany (Received 25 April 2001; in final form 4 October 2001)

Abstract—Success of stroke treatment with rt-PA depends on rapid vessel recanalization. Enzymatic thrombolysis may be enhanced by additional transcranial application of ultrasound (US). We investigated this novel technique using a 185-kHz probe and compared it to standard diagnostic US. In vitro studies were performed in a continuous pressure tubing system. Clots were placed in a postmortem skull and treated with rt-PA together with or without transtemporal 185-kHz US insonation (2W/cm2) and in comparison to 1-MHz diagnostic US (0.5 W/cm2). Recanalization time was significantly (p < 0.01) shorter in the 185-kHz (14.1 min) and 1-MHz (17.1 min) US rt-PA treatment group compared to rt-PA treatment alone (29.3 min.). Flow rate was significantly higher (p < 0.025) and increased faster in the combined treatment group with rt-PA ⴙ 185-kHz US compared to rt-PA ⴙ 1-MHz US. We investigated the blood-brain barrier in rats after 90-min exposure time of the brain with 185-kHz US, but no damage was observed. Results suggest efficacy and safety of the 185-kHz transducer, which is superior to diagnostic US. Such a novel US probe may be able to optimize thrombolytic stroke treatment. (E-mail: [email protected]) © 2002 World Federation for Ultrasound in Medicine & Biology. Key Words: Cerebral revascularization, Ultrasonics, Thrombolysis, Blood-brain barrier.

group of stroke patients and the need to provide a 24-h interventional stroke team in limited centers to enhance the recanalization rate. More attractive is the complete fragmentation of the thromboembolic clot by means of US, either by pure US insonation at high intensities or at low US intensities in combination with thrombolytics. Pilot studies indicate that US frequencies (between 20 kHz and 1 MHz) lower than those used for diagnosis of intracranial vessel conditions (Aaslid et al. 1982) with different intensities (from 1 up to 35 W/cm2) may enhance rt-PA mediated recanalization time in patients treated for thrombotic coronary and peripheral artery occlusion by percutaneous (Siegel et al. 1989) or by endovascular application (Luo et al. 1993; Siegel et al. 1992). Experimental findings suggest that US application increases the transport of reactants into the clot and improves the penetration of the enzyme, in particular by reducing the fibrin polymerization of the clot (Francis et al. 1995; Siddiqi et al. 1995). Despite different experimentaI and limited clinical data in peripheral artery diseases, transcranial application of US has not been investigated systematically. Pilot studies suggested sufficient penetration and thrombolytic

INTRODUCTION Acute thromboembolic events with cerebral artery occlusion causes serious clinical and neurologic consequences, and vessel recanalization should be established as soon as possible to achieve a better functional outcome (Furlan et al. 1999). To date, the only established specific therapy for that purpose is the treatment with rt-PA applied within 3 to 6 h after symptom onset (Clark et al. 1999; NINDS and rt-PA Stroke Study Group 1995). Although thrombolytic therapy with IV application of rt-PA within a 3-h time window is approved in Canada and the USA, its efficacy is of limited success; this is mainly due to a poor recanalization rate of only 34% to 47% at 24 h (Del Zoppo et al. 1992; Von Kummer and Hacke 1992). Although local intraarterial application of thrombolytics increased the recanalization rate by 66% if applied within 6 h (Furlan et al. 1999), this gain of time is compensated by the disadvantage of a highly selected Address correspondence to: Michael Hennerici, M.D., Department of Neurology, Universitaetsklinikum Mannheim, University of Heidelberg, 68135 Mannheim, Germany. E-mail: [email protected]. uni-heidelberg.de 1683

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we submerged a connected-tubing perfusion system that included an obstructing fibrin thrombus. The pure fibrin thrombus was developed separately in a plastic tube and connected to the tubing system. Then, the frame with the plastic tube and the fibrin clot was mounted vertically in the low-pressure flowing system. The tubing system was filled with buffer (0.005 mol/L Tris, 0.1 mol/L NaCl, 0.005 mol/L EDTA), allowing a hydrostatic pressure of 400 mmH2O on top of the thrombus, at a distance of 45 mm from the surface of the skull at the position of the middle cerebral artery. The perfusate was clear without any visible microparticles and was collected per min in empty plastic micro test tubes (Safe lock 2.0 mL, Eppendorf-Netheler-Hinz-Gmbh, Hamburg, Germany), allowing the calculation of the flow per min through the thrombus. Total measurement-time was 60 min for each experiment, and we used 15 clots per treatment group. Flow rates were calculated at 1-min intervals. Tube recanalization was achieved when a continuous flow through the tubing system was present.

Fig. 1. Schematic drawing of the experimental setting. 1 ⫽ thrombus; 2 ⫽ human skull bone; 3 ⫽ 185-kHz transducer; 4 ⫽ amplifier and pulse-function generator; 5 ⫽ buffer reservoir; 6 ⫽ sound absorbing foam; 7 ⫽ perfusate collector.

effects of either low-frequency (Akiyama et al. 1998; Behrens et al. 1999) or diagnostic (Spengos et al. 2000) US through the skull in vitro and, hence, encouraged empiric assessments of US for thrombolysis even with standard US equipment (Alexandrov et al. 2000). To obtain basic data about the different US applicabilities and to identify the most efficient treatment, we compared the efficacy of focused US at FDA-approved intensities with nonfocused low-frequency US at higher intensities by measuring recanalization time and flow volume in a flow model of experimental intracranial vessel occlusion in vitro and tested, for safety reasons, the influence of non-FDA-approved therapeutic US on the integrity of the blood-brain barrier (BBB) in vivo. MATERIALS AND METHODS Flow apparatus An experimental continuous flow model (Spengos et al. 2000) to investigate acceleration of thrombolysis in vitro was designed (Fig. 1). Therefore, a temperaturecontrolled water tank (37°C ⫾ 0.5) was used, in which

Clot preparation and treatment Plasminogen-rich bovine fibrinogen (Fibrinogen Fraction I Typ IV, Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) was desalted using gel filtration chromatography with Sephadex G-25 M (PD10 columns Pharmacia Biotech). The column was equilibrated with 0.05 mol/L Tris, 0.1 mol/L NaCl, 0.005 mol/L EDTAbuffer. Fibrinogen-containing eluat fractions were diluted to a concentration of 6.5 mg fibrinogen/mL. Clots were prepared in 1-mL plastic tubes (Standartips 1000 ␮L, Eppendorf-Netheler-Hinz-Gmbh), which were closed with parafilm (Parafilm M; American National Can, Chicago, IL). In each tube, 300 ␮L fibrinogen solution was incubated with 100 ␮L bovine thrombin (Test-Thrombin 30 UI/mL; Dade-Behring Gmbh, Marburg, Germany) and stored for 3 h at room temperature. After that, the tubes containing the clots were connected to the tubing system as described before (Spengos et al. 2000). Human rt-PA (Alteplase, Boehringer Ingelheim, Ingelheim, Germany) was used and its concentration was optimized to achieve a recanalization time in the range of 30 min in the absence of US in our experimental setting; thus, representing a significant reduction of recanalization time compared to controls. These pilot studies indicated an efficient activation of bovine plasminogen present in the fibrinogen preparation by the recombinant human t-PA (Alteplase) used in this experiment. The present experiment was started by injecting 1 mL of 100 ␮g/mL rt-PA or placebo (0.9% NaCl) proximal close to the clot continuously over 2 min. Ultrasound exposure and application of rt-PA started simultaneously.

Ultrasound-accelerated thrombolysis ● S. BEHRENS et al.

Ultrasound apparatus US (185 kHz) was emitted by a piezoelectric crystal at the resonance frequency, and a prototype transducer (DWL-Co. Sipplingen, FRG/and Medizintechnik Basler AG, Zuerich, Switzerland; diameter ⫽ 60 mm) was used with a “spatial-peak, temporal-peak” intensity of 2 W/cm2 without passing through the skull and 1.27 W/cm2 after transmitting through the skull in a continuous sinus wave mode. The transducer was calibrated with a hydrophone before measuring. US (1 MHz) was emitted by a commercially available device (Multidop X4, DWL-Co.). The probe delivered pulsed-wave (PW) US with a spatial-peak, temporal-peak intensity of 524 mW/ cm2, an ultrasonic power of 226 mW and a pulse-repetition frequency of 16 kHz. Pulse duration was 26 ␮s and the focal length of the 1-MHz transducer was 15 mm. The ⫺6 dB pulse beam width was 0.67 cm on the x-axis and 0.664 on the y-axis for the 1-MHz transducer; the185-kHz transducer was nonfocused. The intensity of 524 mW/cm2 was measured without passing through the skull, but 347 mW/cm2 was measured in the focus of the US field after penetrating the skull. To optimize US coupling to the skull, all investigations were performed in a water tank (600 ⫻ 400 ⫻ 300 mm) with a temperature maintained at 37 ⫾ 0.5°C. Water was degassed by US at an increased temperature before starting the experiment. US wave reflections were reduced to a minimum by lining all sides, and the bottom, with soundabsorbing melamine foam (sound absorption coefficient ⫽ 1.15 at 1-MHz and 0.82 at 200-kHz; thickness 80 mm, Illsonic plano, Illbruck, Leverkusen, Germany). The test objects (a human formalin-fixed skull with a temporal bone thickness of 5 mm, and a plastic tube with fibrin clots) were placed in the water tank in front of the US transducer and the clots were positioned within the target focus of each US probe. The distance between the US probe (either 185 kHz or 1 MHz) and the clot was 45 mm. The US probe was positioned on the surface of the skull, allowing a transtemporal insonation of the clot. The indicated focus was confirmed with a cylindrical hydrophone before each measurement. The US signal of the 185-kHz transducer was generated by a frequency generator (Jupiter 500, Black & Star: 0.1 Hz–500 kHz) and amplified with a power amplifier (DWL/NeuroscanCo.; 500 Hz– 400 kHz. (⫺3 dB), output power 32 W, gain 120:1). Signals were picked up by a hydrophone (DWL Co.: piezoceramic cylinder hydrophone, calibrated by National Physical Laboratory Ltd., UK, average receive sensitivity level ⫺221.8 dB re 1V/␮Pa between 20 kHz and 300 kHz). The hydrophone signals were amplified by a simple operational amplifier (MAX410) circuit. All signals were measured with an oscilloscope (HM 203 to 7, Hameg, Frankfurt/M, Germany).

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Blood-brain barrier experiments Animal experiments were carried out according to the National Institute of Health - Guidelines for the care and use of laboratory animals, and approved by the local authorities (Regierungspraesidium Karlsruhe, Germany). We used male Sprague–Dawley rats (3 per group) between 300 and 350 g body weight. Animals were housed under diurnal lighting conditions and allowed access to food and water ad lib before the experiment. Rats were anesthetized with halothane (0.7% to 2%)-N2O-O2 gas mixture during the whole experiment. The animals had microcatheters placed under the stereomicroscope in the femoral artery, femoral vein and in the internal carotid artery after closing the pterygopalatine artery. Blood pressure (intraarterial measurement in the femoral artery) was monitored continuously, arterial pO2 and pCO2, pH and hematocrit were measured repeatedly during the experiment. Body temperature of the animals was maintained between 36.8 and 37.2°C with an automatic heating pad that was adjusted to the continuously monitored rectal temperature. After having placed the catheters, the animals were treated either with or without continuouswave US (185 kHz, 2 W/cm2) for a period of 90 min. The transducer was calibrated before in the water bath as described above and after coupling the transducer to the rat skull; the US field was controlled with a cylindrical hydrophone to exclude acoustic cavitation. To optimize the coupling of the US probe to the rat skull, we used a US coupling gel by covering the whole surface of the transducer with a layer of 1.5 cm gel. In total, 3 rats were exposed to the 185-kHz US transducer. The rats were positioned with the dorsal side downwards and the dorsal side of the skull positioned on top of the transducer. Temperature of the US coupling gel at the surface of the skull was measured with a thermometer and set at 40°C as maximum, but was not achieved after having excluded acoustic cavitation Afterward, 1.0 mL of 4% Evans blue solution was injected into the femoral vein and 10 min later the rats were decapitated. The brains were removed and frozen instantly at ⫺45°C. The rat brains were cut with the microtome at ⫺20°C into sections of 6-␮m thick. The sections were removed with the object holder and immediately transferred into cold acetone for dehydration to avoid diffusion of dye out of the capillaries. Finally, the brain sections were transferred to the fluorescence microscope and inspected in acetone. Brain regions-of-interest (ROI) were photographed and compared within the different treatment groups to obtain a documentation of capillaries with intact or damaged BBB. Statistical analysis The statistical significance of differences between mean values with SD were tested using a two-tailed

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Fig. 2. Comparison of the mean recanalization time (columns) in the different treatment groups. Each group n ⫽ 15. SEM are given as top lines. TC ⫽ transcranial insonation; no TC ⫽ direct insonation.

Student’s t-test for unpaired data. A p value ⬍ 0.05 was considered significant. ␣-correction was calculated in all cases of multiple group comparisons. Repeated measurement one-way analysis of variance (rm-ANOVA) was performed in the analysis of flow rate curves. RESULTS The longest recanalization time was observed in the control group without any treatment, with 38 min. (⫾ 6.2). A clearly shorter recanalization time of 29.3 min (⫾ 11.4) was observed in the rt-PA treatment group. Combined treatment of rt-PA and 185 kHz US further shortened the recanalization time significantly with transcranial (p ⬍ 0.01) (14.1 ⫾ 8.9 min) or direct (p ⬍ 0.001) insonation of the clot (8.4 ⫾ 3.04 min) in comparison to rt-PA treatment alone. Transcranial application of 1-MHz US in combination with rt-PA also significantly (p ⬍ 0.05) shortened the recanalization time (17.1 ⫾ 7.11 min.) compared to rt-PA treatment alone, but with a longer recanalization time than with transcranial application of 185 kHz US ⫹ rt-PA (Fig. 2). The mean flow rate was lowest in the control group without any treatment, with 33.67 ␮L/min after 15 min, 242.33 ␮L/min after 30 min, and 827.47 ␮L/min. after 60 min. After having added rt-PA, the mean flow rate increased to 668.93 ␮L/min vs. 2844.7 ␮L/min. vs. 3516.7 ␮L/min after 60 min. Combined treatment of rt-PA with transcranial 185 kHz US further increased the mean flow rate to 2846 ␮L/min after 15 min, 3394 ␮L/min after 30 min, and 3673 ␮L/min after 60 min. Treatment with 1 Mhz ⫹ rt-PA resulted in a lower mean flow rate of 1483 ␮L/min after 15 min and 2927 ␮L/min after 30 and 60 min. In comparison, the transcranial 185 kHz US ⫹ rt-PA treatment curve is statistically significantly (p ⫽ 0.012) different from the transcranial 1-MHz

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Fig. 3. Comparison of the mean flow rate (horizontal lines) in the different treatment groups. Each group n ⫽ 15 measurements at each time point. SEM are given as vertical lines. TC ⫽ transcranial insonation; no TC ⫽ direct insonation.

US ⫹ rt-PA treatment curve. Finally, direct insonation of the thrombus without transmitting through the skull showed the highest mean flow rate with 3996 ␮L/min after 15 min, vs. 4160 ␮L/min after 30 and 60 min (Fig. 3). Investigation of the BBB in rats exposed to CW-US for 90 min with an intensity of 2 W/cm2 at 185 kHz showed no leakage of the Evans blue dye out of the brain capillaries when analyzed under the fluorescence microscope (Fig. 4). On the other hand, we were able to detect a strong leakage of Evans blue out of brain capillaries in the hemisphere ipsilateral to the injection side after opening the BBB with mannitol (injected via internal carotid artery), in contrast to the noninfluenced BBB of the hemisphere contralateral to the injection side, as shown in Fig. 5.

Fig. 4. Fluorescence microscopy, magnification ⫻ 10⫺0: A rat brain section slice of the striatum after Evans blue injection and 90-min US exposure at 185 kHz with an intensity of 2 W/cm2. Integrity of the brain capillaries is obvious under the fluorescence microscope.

Ultrasound-accelerated thrombolysis ● S. BEHRENS et al.

Fig. 5. Fluorescence microscopy, magnification ⫻ 10⫺0: The left side shows a rat brain section slice of the striatum after Evans blue injection with a pretreatment of mannitol, but contralateral to the injection side and a little leakage of dye out of brain capillaries. The right side shows the same brain, but ipsilateral to the mannitol injection side, demonstrating a large leakage of dye out of the brain capillaries without US treatment.

DISCUSSION This study indicates efficacy in vitro and first safety data in vivo of transcranially applied, 185-kHz, continuous-wave US for therapeutic purposes in accelerating enzymatic-mediated thrombolysis. Even when externally supplied through the human skull, US transmission is high enough to shorten significantly not only the recanalization time, but also rt-PA mediated recanalization rate in a flow model in vitro. Our findings suggest that insonation with nonfocused US may be applicable to enhance enzymatic revascularization treatments, such as rt-PA thrombolysis, aimed at recanalizing occluded intracerebral vessels in acute ischemic stroke patients (Del Zoppo et al. 1992; Hacke et al. 1995, NINDS and rt-PA Stroke Study Group 1995). In contrast to diagnostic focused FDA-approved US at 2 MHz, which also showed efficacy in a pilot study with the disadvantage of a very narrow US target focus of approximately 0.5 cm3 (Alexandrov et al. 2000) or at 1 MHz used in this study, the present application at 185 kHz has a larger US field, according to the lower frequency that requires no exact localization of the thrombus position, which appears to be particularly difficult in occluded intracranial vessels without a flow signal. Our present results extend recently published data (Behrens et al. 1999; Spengos et al. 2000) and also suggest efficacy for potential therapeutic purpose on a low-frequency level. Other pilot investigations proved the therapeutical effect of high-intensity, low-frequency, continuous US in occluded peripheral vessels in vitro and in vivo (Rosenschein et al. 1991; Siegel et al. 1989; Steffen et al. 1994), resulting from the mechanical effects of US application. However, higher intensities such as 6.3 W/cm2 resulted

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in an initially fast reflow of the occluded femoral artery in rabbits, but to a higher rate of vessel reocclusion during the follow-up (Kornowski et al. 1994). This may be explained by platelet activation within the higher US intensity range (Williams et al. 1978). The results of further animal experiments (Kashyap et al. 1994; Luo et al. 1996; Nishioka et al. 1997) are consistent in demonstrating the potential of US for treatment of arterial or venous thrombosis. The effect is not activator-specific and is proven to enable increased transport of the thrombolytic agents into the clot. This enhanced permeation may be related to nonthermal mechanical microcavitation effects as a result of pressure changes in the medium exposed to the US field (Blinc et al. 1993; Francis et al. 1995). Although not significant, there is a trend to higher enhancement of rt-PA-mediated thrombolysis during direct US exposure without passing through the skull in the present study. This is in line with investigations indicating that transcranial insonation causes some loss of USenergy. A US frequency of 300 kHz allows an attenuation of only 0.2 N/cm when transmitting through the skull, but within 0.25- and 6.0-MHz, the increase of insertion loss through the skull is not only roughly proportional related to an increased frequency, but also directly linked to the thickness of the diploe¨ and the skull bone (Fry and Barger 1978; Heuter 1952). The use of a submerged continuous pressure-flow model, even although not pulsatile, adjusted to the position of the MCA, is a new aspect, demonstrating US efficacy in a setting that is, regarding the similar acoustical characteristics of brain and water density, sound velocity and characteristic sound impedance (Sorge and Hauptmann 1985), more adapted to the human physiology. It is conceivable that a pulsatile flow model with a higher pressure amplitude may further increase the penetration of the enzyme and, therefore, could be able to demonstrate stronger effects. Since we demonstrated efficacy of transcranially applied therapeutic US to enhance enzymatic thrombolysis (Behrens et al. 1999; Spengos et al. 2000), only a few have focused on the side effects after insonating brain tissue. CCw-US at 1 MHz with an intensity of 1.6 W/cm2 is known to have transient effects with potentiation of the inflammatory response by augmenting the adhesion of leucocytes to the endothelium (Maxwell et al. 1994). Also, nonthermal effects of therapeutic US exposure may lead to an increase of intracellular calcium by a perturbation of the membrane and, therefore, cause toxic in-cell activation (Mortimer and Dyson 1988). Toxicity from US heating is primarily related to the absolute increase of temperature, which may be minimized by optimal coupling of the US transducer to the skin and demonstrated negligible effects below 40°C (NCRP 1992). So far, our present investigation demonstrated no

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harm to the BBB when exposuring the rat brain for 90 min, with a stable integrity of the brain capillaries. The lack of increasing systemic body temperature in those rats exposed further suggests safety of therapeutic US application, in particular when transferring the setting to the clinical condition by coupling the transducer to the human skull with its larger surface. Other bioeffects of US are related to acoustic cavitation, which refers to the expansion and collapse of gas-bubbles resulting from sudden pressure changes in a liquid medium during the passage of the sound wave (Apfel 1982). This transient cavitation can produce additional local heating, liberation of free radicals and shock-waves that may cause peripheral and central cell damage (Dalecki et al. 1997, Daniels et al. 1995, Fahnestock et al. 1989), but is a more common phenomenon in high-intensity US fields and its significance and occurrence in low-intensity fields is inconsistent and still debatable. We finally conclude that transcranial application of “low”-frequency, continuous wave US may accelerate reperfusion and shorten the recanalization time of acute thromboembolically occluded intracerebral vessels by enhancing enzymatic thrombolysis with preliminary evidence of safety and, therefore, may help to improve clinical outcome in ischemic stroke, which needs to be confirmed in further animal studies. However, so far, diagnostic US showed efficacy at different levels, the application of FDA-approved diagnostic US, such as vessel monitoring, should be recommended in patients with rt-PA treated ischemic stroke, an adequate temporal bone window and a (lowered) flow signal of the targeted intracranial vessel. Acknowledgements—The authors thank the technical assistance of Mr. Georges Sam and the help of Mrs. Garcia-Knapp in the preparation of the manuscript. We also acknowledge Bertram Krumm for statistical support. This work has been supported by the BioRegio Foundation Rhein-Neckar.

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