In Vitro Sonothrombolysis Using Transcatheter Injection Of Microbubble And A New Therapeutic Ultrasound Device

Background:Thromboembolic disease, which leads to ischemic stroke, myocardial infarction, pulmonary artery or peripheral vessel occlusion, has become the leading cause of death and disability. Administration of thrombolytic drugs systemically through peripheral circulation or locally through an interventional catheter are both common and effective methods for thrombolysis in clinical. However, the limited time window of systemic thrombolysis hampers the use of intravenous thrombolytic drug, and the administration of high dose of thrombolytic drugs increases complications such as hemorrhage, so, this method is limited to a certain extent. Interventional thrombolysis treatment, which injects thrombolytic drug through the catheter directly to the thrombus, was not significant superior to standard intravenous treatment in the patient’s evaluation scale. Sonothrombolysis, is a promising approach based on ultrasound cavitation. Microbubbles(MBs), which served as cavitation nuclei to lower the acoustic pressure threshold required for inducing cavitation, are of vital importance in sonothrombolysis. The mechanism for this appears to be ultrasound(US) induced cavitation, which leads to axial shock wave, micro-jets, and high-velocity flow gradients that penetrate and destabilize the infrastructure of a thrombus, thus enhanced the thrombolysis effect significantly.One principal problem of sonothrombolysis is most thromboembolic are comply obstructive, cavitation only effect on the surface of the thrombus where MBs are present, thus reduced the effects of sonothrombolysis. If there were sufficient MBs within the thrombus, low intensity ultrasound would be able to produce persistent stable cavitation or low intensity initial cavitation, to achieve better thrombolysis effect and shorten the time for thrombolysis, which is very important to reduce infarct size of ischemic tissue. In order to solve the problem of insufficient microbubbles within the thrombus, this study proposes a new method of intraclot microbubbles mediated ultrasound thrombolysis(IMUT). By trapping the MBs in the thrombus through a micro catheter injection, the thrombus immediately becomes a sensitive target for ultrasound cavitation and thrombolysis. IMUT may have the following potential advantages: more efficient and rapid thrombolytic procedure, lower ultrasound intensity, less thrombolytic agent administration, fewer side effects to other tissues, and less financial cost.Objective:1. To compare the impacts of duty cycle and acoustic pressure on sonothrombolysis using a new therapeutic ultrasound device and a commercial physiotherapy US device.2. To investigate the in vitro effect of IMUT and compared to that of sonothrombolysis with microbubble injection through peripheral circulation. In addition, US destruction of Di O-labeled microbubble(MBDi O) was applied to trace the sonoporation effect of IMUT during thrombolysis.Materials and Methods1. Experimental apparatus:⑴ SL-10 therapeutic US device: manufactured by Shenzhen Welld Medical Electronics Co., Ltd., is a single-element, non-focused ultrasound generator. The transducer has a diameter of 2.5 cm and it is operated with the frequency of 1.0 MHz, the peak negative pressure ranges from 0.5 to 2.0 MPa and the duty cycle from 0.1% to 20%.⑵ Physiotherapy US device: a commercial physiotherapy Accusonic Plus-170 US device(AP-170, Metron Medical Australia Pty Ltd.) with a 2.5 cm circular transducer was used. The transducer is operated with the frequency of 1.0 MHz, the intensity ranges from 0.2 to 2.4 W/cm2 and the duty cycle from 20% to 100%.Peak negative pressure and acoustic intensity of these two instruments were calibrated with a HNC-0400 needle hydrophone(ONDA Company, Britain) at the Institute of Acoustics of Nanjing University.⑶ Ultrasound imaging system: a commercial high-resolution US micro-imaging system operating with a central frequency of 30 MHz(VeVo2100, Visual Sonics Company, Canada) were used to monitor the distribution of MBs within the clots.⑷ Laser scanning confocal microscope(LSCM): TCS SP5 laser confocal fluorescence microscopy(Leica Company, Germany) were used to trace the MBDiO during thrombolysis.2. Experiment reagents:⑴ Urokinase(UK): The UK is manufactured by Tianjin biochemistry pharmaceutical Co., LTD. The proposed UK dosage for endovascular thrombolysis ranges from 200,000 to 400,000 IU, hence the clots used in the experiment weighed about 0.25 to 0.35 g, which is approximately 1/10 the weight of clinical thromboses, so the UK dose was set at 20000 IU when administrated locally. The dose of UK was set at 1500 IU when administrated in the in vitro flowing system(25 ml in volume), which is comparable to the suggested 4400 IU/kg of body weight administrated systemically in a grown-up’s circulation(about 4.4 L).⑵ Fluorescent microbubbles(MBDi O): 10 mg of green fluorescent probe(3,3’-dioctadecyloxacarbocyanine perchlorate, Di O) was dissolved in 1 ml dimethy1 sulfoxide(DMSO) and then after mixed to put 0.1 ml in the 1.5 ml MBs to oscillation and mixed. lipid microbubbles with a concentration of(7-8)×109/ ml,⑶ Human blood clot: the blood from healthy adult volunteers was bathed for three hours to form clots in a 37 ℃ water-bath. The clot was washed three times with PBS, dried with filter papers and then the initial weight(W0) was taken.⑷ The circulation installation. The installation was consisted of a water tank, a homemade circulation tube and a peristaltic pump. The whole experiment process are conducted in a water tank at 37℃. The bottom of the tank was bedded with a 2 cm sound-absorbing sponge to reduce the reflected wave. Circulation tube flows with PBS with a, flow rate of 4.6 ml/s. Thrombus was inserted into the tube. The therapeutic transducer was fixed at 5 cm above the thrombus.3. Study protocolsFor comparing the thrombolytic effect of SL-10 and AP-170, sixty fresh human blood clots were equally divided into five experimental groups and one control group. Both US devices were operated at the frequency of 1.0 MHz. Three experimental groups were treated with SL-10 at three pressure levels(A: 1.02 MPa, B: 0.75 MPa, C: 0.51 MPa) with a duty cycle of 1.5%. The other two experimental groups were insonated with AP-170 at two pressure levels(D: 0.67 MPa, E: 0.57 MPa) with a duty cycle 50%. Sham US exposure was applied to the control group.For exploring the thrombolytic effects of IMUT, forty fresh human blood clots were divided into four groups. Intraclot administration of MBs and UK ultrasound thrombolysis(T1: IMUT +UK), Peripheral injection of MBs and UK ultrasound thrombolysis(T2: non-IMUT+UK) and two control groups with sham US(C1: Intraclot injection of MBs and UK, C2: Peripheral injection microbubble and UK into circulation). Therapeutic US was operated at the frequency of 1.0 MHz with the peak negative pressure of 512 kPa. The thrombolysis rate of clot was calculated after treatment. US release of MBDi O was observed by fluorescent distribution and intensity in thrombus after treatment.The thrombolytic rates were then calculated by weight lost of the clots:(W0-W1)/ W0×100%.W0 refers to the clot weight before treatment and W1 is the clot weight after treatment.Results:1. The thrombolytic rates of all experimental groups(A, B, C, D, E) were higher than that of the control(F)(P < 0.01). The thrombolytic rate of the thrombolysis US device(A, B, C) was higher than that of the physiotherapy US equipment(D, E)(P < 0.05). A higher thrombolytic rate was related to a higher acoustic pressure(r > 0.8, P < 0.01) when the frequency, pulse repetition frequency(PRF) and duty cycle were fixed.2. The thrombolysis rates of T1(37.70 ± 3.17%) were significantly higher than other three groups(P < 0.01). The thrombolysis rate of C1(14.37 ± 2.22%) were significantly higher than C2(7.90 ± 0.68%)(P < 0.05), but the T2(11.67 ± 1.13) thrombolysis rate contrast with C1, C2 were no statistical significance(P > 0.05). IMUT usually develop clusters of strong MBs echo in thrombus with acoustic shadow. After treatment, the echo was scattered and spread. LSCM found that US thrombolysis by IMUT left a large amount of bright fluorescent grains in thrombosis but rare fluorescent particles could be found in the thrombi by non-IMUT method.Conclusion:(1) The therapeutic ultrasound of thrombolytic rate was obviously higher than the control group. A higher thrombolytic rate was related to a higher acoustic pressure, and thrombolysis effect was more obvious in high acoustic pressure, low duty cycle model.(2) IMUT enhances the thrombolysis rate of US significantly at the same acoustic parameters and the same dose of UK.