Intraclot Microbubbles And Ultrasound Enhanced Catheter-directed Thrombolysis

Background:Thrombosis and consequent embolism is the fundamental pathological change of most cardiovascular diseases.Intravenous administration of tissue plasminogen activator(t-PA)or other thrombolystic agents is currently the predominant treatment of these diseases.However,there is a strict time limit(window period)for intravenous thrombolysis,and the safeness and effectiveness also need further improvement.Catheter-directed therapy(CDT)is an alternative choice for thrombosis diseases.Recently,with the use of more efficient thrombectomy device,evidence suggests that mechanical thrombectomy as an add-on to intravenous or intra-arterial administration of thrombolystic agent results in higher early recanalization rate,and is able to provide beneficial functional outcomes.Consequently,CDT is getting more and more recognition clinically.Ultrasound thrombolysis(UT)is a promising development of thrombolytic technique.The mechanism of UT,though not yet thoroughly clarified,is generally attributed to ultrasound induced cavitation mediated by endogenous or intravenous administered microbubbles(MB).Because of the lack of cavitation nuclei in vivo,MB,which serve as cavitation nuclei to lower the acoustic pressure threshold required to induce cavitation,are of great importance in UT.One most important problem in the study and application of external UT is the poor targeting of the thrombus,hence most clinical thrombosis diseases are obstructive with no blood flow for MB delivery.In the previous study,we have demonstrated that direct intraclot injection of MB combined with ultrasound was able to improve local urokinase throbolysis procedure.The difference between intraclot treatments(with and without ultrasound + MB)is also better than that of the systemic approaches.Another important problem in the study of transcutaneous UT is the lack of acoustic window in most thrombi that thrombolysis treatment was needed.Catheter directed intravascular ultrasound assisted thrombolysis is a new approach of UT.EkoSonic?Endovascular System was introduced in clinical practice in Europe and the United States,and has been proved able to accelerate clot resolution in acute pulmonary embolism,deep venous thrombosis,etc.However,due to the relatively high acoustic energy transmitted,real-time temperature monitoring and cooling system were indispensable to avoid possible vessel wall injury.These complex additional parts in consequence made the EkoSonic?catheter much more expensive than regular ones.This study is to firstly test the effectiveness and feasibility of intraclot MB enhanced external ultrasound thrombolysis in vivo,and to design a MB-mediated-cavitation based intravascular ultrasound catheter.This novel catheter would take advantage of the effect of MB-mediated ultrasound cavitation on thrombolysis,and the direct thrombus contact of intravascular ultrasound,and is expected to produce more effective and prompt thrombolytic effect.Objective:1.To investigate the possibility of external ultrasound combined with continuous intraclot infusion of MB to improve the effectiveness of catheter directed thromblysis in vivo.2.To design and manufacture a novel MB-mediated-cavitation based intravascular ultrasound catheter,and further test its capability to improve intraclot catheter directed t-PA thromblysis procedure in vitro,combining with continuous local infusion of MB.Materials and Methods:1.Experimental apparatus:⑴ Custom modified ultrasound system,a VINNO 70 diagnostic scanner(Vinno Technology Co.Ltd,Suzhou,China),equipped with a linear array probe(X4-12L).For sonothrombolysis capability,the scanner was specially modified.The modifications includes a therapeutic ultrasound(TUS)mode called Vflash with an adjustable treatment area super-imposed on the very low MI(0.04)Contrast Enhanced Ultrasonography(CEUS)mode.TUS impulses were emitting intermittently,for each TUS frame,an ensemble of one2.5 MHz,5 μsec pulse was delivered line by line at a line density of 32/cm in the treatment area.The beams were focused at the center of the treatment area.The treatment area was adjusted to cover the whole thrombus occluded vessel.The frame rate was set at 50 Hz,the TUS transmitting and intermittent time were set at 0.5 sec and 2.0 sec,respectively.The TUS mode was transmitted at an MI value of approximately 0.50.In a water tank test,TUSimpulses delivered in this setting was able to cavitate MB in the treatment area,leaving the MB outside the area basically unaffected.⑵ Syringe pump,Longerpumlp? TJP-3A,Baoding,China.⑶ Varioskan Flash Multiplate Reader 3001-1723,Thermo-Fisher Scientific Inc.,USA.⑷ Ultrasonic impedance analyzer,Agilent E4991 A,Agilent Technology Co.,Ltd.USA.⑸ Function generator,Hantek HDG 2022 B,Hantek Electronic Co.,Ltd.Qingdao,China.⑹ Calibrated needle hydrophone,ONDA HNC-0100,ONDA Company,USA.The diameter of the hydrophone sensor is 0.1 mm.⑺ Acoustic field scanning system(3D),Precision Acoustics Ltd.Britain.⑻ COMSOL Multiphysics 4.3a,COMSOL,Sweden.⑼ Confocal laser scanning microscopy,LEICA TCS SP5,Leica Microsystems Ltd.,Germany.2.Experimental reagents:⑴ Microbubble: Laboratory made lipid-coated perfluoropropane microbubble,Zhifuxian.The MB had a mean particle diameter of 1.95± 0.52 μm and a bubble concentration of 2-9×109/m L.⑵ Thrombolytic substance: recombinant tissue Plasminogen Activator(rt-PA),Boehringer Ingelheim pharmaceutical Co.,Ltd.Germany.⑶ Enzyme-Linked Immunosorbent Assay kit for rabbit plasma D-Dimer,Cloud-Clone Corp,Houston,USA.⑷ Anticoagulant bovine whole blood,Jiulong Biological Products Co.,Ltd.Zhengzhou,China.Stored at 4 ℃ before use.⑸ Anticoagulant bovine fresh frozen plasma,Jiulong Biological Products Co.,Ltd.Zhengzhou,China.Stored at-20 ℃,thawed within a 37 ℃ water-bath before use.⑹ Rabbit anti bovine fibrinogen antibody,BIOSS Biotechnology Co.,Ltd.Beijing,China.⑺ Goat anti rabbit Ig G,Zhongshan Golden Bridge Biotechnology Co.,Ltd.Beijing,China.Pre-labled with Fluorescein isothiocyanate(FITC)before use.3.Experimental animal:Forty healthy male New Zealand white rabbits,weighing between 2.5 and 3.0 kg,provided and quarantined by the Animal Laboratory of the Third Military Medical University,Chongqing,China.4.Study protocol:⑴ Modified diagnostic ultrasound and intraclot microbubbles enhanced catheter directed thrombolysis in vivo.(1)A rabbit model of acute obstructive inferior vena cava(IVC)thrombosis was established with a simulation of Virchow’s triad,including an endothelial injury(clamped with hemostatic forceps),flow stasis(blood flow blocked by clipping the vessel with a vascular clamp placed proximal to the previously clamped segment)and change of blood coagulation state(thrombin injection).(2)Randomized controlled study.The 40 animals were randomly divided into 4 groups as follows(n=10):CDT+UT.Rt-PA and MB administered into the intra-clot catheter,at the same time the Vflash mode of the scanner was switched on.UT alone.US and MB were applied in the same way,without any rt-PA administered.CDT alone.Rt-PA was given in the same way,with sham ultrasound exposure.Control.Only NS was administered into the intra-clot catheter,with sham ultrasound exposure.(3)Effectiveness evaluation:Thrombolysis efficacy(30 min and 60 min into treatment)was evaluated with a semi-quantitative score method based on the changes of B-mode ultrasound and CEUS.Quantitative measurement of plasma D-Dimer before & after treatment in groups.Pathological finding of the occluded IVC.(4)Safety observation: Gross examination of the harvested proximal segment of IVC,heart and pulmonary arteries to reveal any evidence of thrombo-embolism or local thrombus formation.⑵ Design and acoustic characteristics assessment of the novel intravascular ultrasound catheter probe.(1)Micro ultrasonic transducer selection and acoustic impedance test to decide the appropriate excitation frequency.(2)Acoustic field scanning and electro-acoustic conversion test on the single micro transducer probe.(3)Simulation of acoustic fields of single micro transducer probe,transducer group probe,and multiple transducer groups probe with COMSOL Multiphysics software,to optimize the combination scheme of micro transducers.(4)Microbubble cavitation capacity test of the novel intravascular ultrasound catheter probe with passive cavitation detection.Semi-quantitative analysis on the scattering signal was used to determine appropriate excitation voltages applied to the transducer to induce stable & initial cavitation.⑶ The novel MB-mediated-cavitation based intravascular ultrasound enhanced tPA thrombolysis in vitro.(1)Preparation of bovine whole blood clot: One ml of bovine whole blood mixed with33 μL 5% calcium chloride(containing 1.11 mg calcium chloride)in a clean 2.0 ml EP tube,then the tube was incubated in a 37 ℃ water-bath for 3 h to form bovine whole blood clot.(2)Established an in vitro thrombolytic test system for obstructive thrombi.(3)Randomized controlled study.Clots were randomly divided into 5 groups(n=10):US+MB+TPA.Intraclot ultrasound and MB enhanced t PA thrombolysis;US+NS+TPA.Intraclot ultrasound enhanced tPA thrombolysis;US+MB.Intraclot ultrasound and MB thrombolysis without tPA;TPA alone.Intraclot tPA thrombolysis,without ultrasound and MB;Control.Intraclot injection of same amount of saline.(4)Effectiveness evaluation: Mean clot weight loss of each group.(5)Mechanism analysis of thrombolysis: light microscopy observation of the hematoxylin and eosin stained sections of the residual thrombi;confocal laser scanning microscopy examination of the fibrin immunofluorescence stained sections of the residual thrombi.Results:1.Modified diagnostic ultrasound and intraclot microbubbles enhanced catheter directed thrombolysis in vivo.⑴ All animals were successfully thrombo-embolized in the IVC and were able to tolerated the whole procedure.Gross examination of the harvested proximal segment ofIVC,heart and pulmonary arteries of all animals did not reveal any evidence of thrombo-embolism or local thrombus formation.⑵ At 30 min into treatment,there was a significant higher thrombolysis efficacy score in CDT + UT group than that of the other groups(P < 0.05);at 60 min into treatment,both CDT + UT group and CDT alone group showed significant higher thrombolysis efficacy scores than that of UT alone group and control group(P < 0.05).⑶ After the modified ultrasound and intralot MB enhanced catheter directed thrombolysis,plasma D-Dimer concentration level significantly elevated(P < 0.05),while no significant differences before and after treatment were revealed in other groups.⑷ Pathological examination of the hematoxylin and eosin-stained occluded IVC revealed that most achieved complete or almost complete dissolution of the thrombi treated with CDT+UT,partial dissolution of the thrombi treated with UT or CDT alone,and only mild dissolution in the central of the thrombi treated with saline alone.2.Design and acoustic characteristics assessment of the novel intravascular ultrasound catheter probe.⑴ Acoustic impedance test determined that 1.5 MHz was the sub-optimal resonance frequency of the transducer.The acoustic field distribution of the miniature transducer could be characterized as a point sound source.The acoustic pressure of the miniature ultrasonic transducer decreased rapidly with the increase of distance between the transducer and the hydrophone.The peak acoustic pressure had a perfect linear correlation with the applied voltage within the measuring range(15-52.5 V).⑵ COMSOL Multiphysics simulation indicated that an uniform sound field can be formed with two back-to-back combining miniature transducers.When two or more transducer groups were parallel connected along the core,a super-imposed acoustic field can be formed along the long axis of the catheter.When the space between two transducer groups were not more than 5 mm,an ideal acoustic field can be formed along the catheter.⑶ Passive cavitation detection and semi-quantitative analysis on the scattering signal revealed only dominant moderate oscillations when the acoustic pressure of the transducer was 0.25 MPa.When the acoustic pressure of the transducer was 0.50 MPa,ultra-harmonic amplitude exceeded the noise amplitude by at least 3 dB,indicating dominant stable cavitation.The noise level was within 10 dB of the second harmonic level when theacoustic pressure of the transducer was 0.75 MPa or higher,indicating dominant initial cavitation.3.The novel MB-mediated-cavitation based intravascular ultrasound enhanced tPA thrombolysis in vitro.⑴ The mean clot weight loss of US+MB+TPA group was significantly higher than that of the other groups(P < 0.05).Mean clot weight loss of groups US+NS+TPA and TPA alone were both significantly higher than that of the intraclot saline injection control group(P < 0.05).There was no significant difference in mean clot weight loss between US+MB group and control group(P = 0.142).⑵ HE staining revealed loose change of the residual thrombi,varying in range and degree,except for those from the control group.Loose change was found more significant in the residual thrombi from US+MB+TPA group.The dense cross-linked fibrin network and cellular components trapped in the network of the residual thrombi from the control group were showed by confocal laser scanning microscopy examination of the fibrin immunofluorescence stained sections.The fibrin network appeared much denser and thicker near the transducer and microcatheter insertion site of the residual thrombi from the control group,probably due to clot retraction.For the residual thrombi from the 3 groups treated with tPA,release and lysis changes of the structure of fibrin network were noted.These changes were more significant near the transducer and microcatheter insertion site.Conclusion:1.Combining with intraclot infusion of MB,guided moderate MI longer pulses from a diagnostic ultrasound transducer was able to improve catheter directed thrombolysis procedure.This strategy has a possibility to achieve earlier clot removal,lower dosage of thrombolytic agent administered,and in consequence lower incidence of thrombolysis related side effects.2.Taking advantage of the effect of MB enhanced ultrasound cavitation on thrombolysis,and the direct thrombus contact of intravascular ultrasound,the novel MBmediated-cavitation based intravascular ultrasound can effectively enhance the thrombolytic effect of rt-PA.3.The pro-thrombolysis mechanism of ultrasound and intraclot MB relies on the effect of cavitation to increase the penetration of thrombolytics,to accelerate their fibrinolyticprocess,and to promote the diffusion of fibrin degradation products.