Intraclot Microbubble Mediated Ultrasound Thrombolysis In Vitro And A Clinical Study

BackgroundThrombotic disease is a common,frequently-occurring disease.Anticoagulant and thrombolytic therapy are its main treatment methods.Anticoagulation can prevent the progress of thrombus,while it can't dissolve it effectively.Thrombolytic therapy includes intravenous thrombolysis and catheter-directed thrombolysis(CDT).With the development of interventional therapy,CDT is widely used because it can inject thrombolytic drugs through the catheter directly into the thrombus.And due to higher intraclot thrombolytic drug concentration,the thrombolytic rate is higher.Sonothrombolysis is a promising approach based on thermal effect,mechanical effect and cavitation.And the cavitation intensity is closely related with microbubbles(MB)which serve as cavitation nuclei for inducing cavitation.While most clinical thrombi are obstructive that MB injected through venous can only contact with the surface of the thrombi,that the effect of sonothrombolysis is restricted.Therefore,this study is aimed to trapping the MB into the thrombus through a catheter.Then combining with the therapeutic ultrasound,the thrombus become favorable ultrasound cavitation targets.ObjectivesTo explore the thrombolytic efficiency of intraclot microbubbles mediated ultrasound thrombolysis(IMUT)combined with Urokinase(UK)in vitro.Further more,to explore the safety and effectiveness of treating lower limp deep vein thrombosis(DVT)by CDT combined with IMUT in a clinical study.Materials and MethodsPart ?1.Materials1.1 Circulation system: the system was consisted of a water tank,a peristaltic pump and circulation tubes with a collateral tube,which was used for simulating the opened collateral circulations in vivo.The circulating liquid was Phosphate buffer saline(PBS)with 10% freshfrozen bovine plasma,which could provide fibrinogen for activating the UK.The flow velocity of the circulating liquid was 5ml/min.And the system was kept at constant temperature(37?).And the bottom of the tank was bedded with a sound-absorbing sponge(2cm)to reduce the reflected wave.1.2 Therapeutic ultrasound device: the therapeutic ultrasound device(SL-10 Sonolyser,Welld Medical Electronics Co.,Ltd.,China)was used for IMUT.The transducer was operated at the frequency of 2MHz,the peak negative pressure of 708 kPa,and the duty factor of 0.05.The treatment time was 10 min.And the transducer was 5cm above the thrombus.1.3 Bovine clots: Fresh bovine blood(1ml)was put into a tube with inner diameter of 0.9cm.Then each tube was kept in a constant temperature oven(37?)for 3h to form a clot.In total 50 clots were needed.1.4 Urokinase: Each bottle of UK with 10~5 IU was dissolved in 0.5ml normal saline.1.5 Microbubbles: The MB named Zhifuxian were prepared by ourselves in Xinqiao Hospital.Before used,it had been vibrated for 90 s at high speed.And its concentration was(7-8)×10~9/ml.2.Methods2.1 Grouping:Fifty clots were equally divided into 5 groups randomly.Each group was treated by different combination of US,MB and UK: Clots in group 1 were treated by US,MB and UK,clots in group 2 were treated by US and MB,clots in group 3 were treated by US and UK,clots in group 4 were treated by UK only.Group 5 was the Control group,and the clots were not treated by US,MB,UK either.The dosage of MB was 0.02 ml and the dosage of Urokinase was 0.1ml.Each clot was treated in the circulation system for 10 min.2.2 Observations:2.2.1 Thrombolysis rate: Each clot was measured before and after treatment,then thrombolysis rate of each groups were calculated and compared.2.2.2 Hematoxylin-eosin(HE)staining: Histology changes of clots were detected through hematoxylin-eosin(HE)staining to evaluate the thrombolysis efficiency.2.2.3 Immunofluorescence: The fluorescence distribution and intensity of fibrous protein were observed via immunofluorescence(IF)to evaluate the fibrinolysis effeciency.Part?1.Grouping:The patients of lower limb deep vein thrombosis(DVT)was diagnosed from Ultrasound and D-dimer(more than 500?g/L).According to the inclusion criteria,exclusive criteria and elimination criteria,patients with the time since symptom onset time less than 14 d and the age from 25 to 70 years old,were included.The patients suffering from hemorrhage,coagulation disorder,pregnancy,pulmonary embolism and other unstable conditions such as severe renal impairment,liver dysfunction,were excluded.Experiment group: Patients who were up to the inclusion criteria,exclusive criteria and rejection criteria were treated by combining CDT and IMUT.Control group: According to the inclusion criteria,exclusive criteria and rejection criteria,patients with treating days fewer than 10 d from 2014 to 2015 were studied.They were treated by CDT alone.2.Materials2.1 Therapeutic ultrasound device: It was the same as used in vitro study.The transducer was operated at the frequency of 1MHz with a duty factor of 0.01.The pulse length was 100 cycles and the pulse reputation frequency was 100 Hz.The output of peak negative pressure was set at 0.75 MPa if the depth of thrombus was less than 3cm,or set at 1.0MPa if the depth of thrombus was more than 3cm.2.2 Catheter: The catheter named UniFuseTM made by Angio Dynamics company was used.And it provided a passage for injecting MB and UK into the thrombus.2.3 Contrast agent: SonoVue? was used in the clinical study.2.4 Urokinase: Each bottle of UK(2×10~5IU)was dissolved into 50 ml with normal saline.3.Methods3.1 Experiment group: An UniFuseTM catheter was inserted into the thrombus,then the thrombus surface projection was marked from common iliac vein to popliteal vein through trans-catheter contrast-enhanced ultrasonography.And the projection was the acoustic window during US therapeutic.UK and MB were injected into the thrombosis through the catheter simultaneously.The dilution ratio of SonoVue? was 1:4,and the injection speed was1 ml per 1.5min.Each point was exposed under US for 1.5min by incremental movement of2 cm until all the thrombus was exposed.The procedure was repeated once immediately.Part ?3.2 Control group: The patients in Control group were treated by CDT only,without IMUT.3.3 Evaluation index:3.3.1 Evaluation for safety: During the thrombolysis,blood routine,four blood coagulation indexes,and D-Dimer(D-D)were tested every other day.Complications such as thoracalgia,cough,and the blood pressure were detected every day.3.3.2 Evaluation for effectiveness: Treatment days and overall dosage of UK between the two groups(the experiment group and the historical Control group)were compared.Then we analyzed and evaluated the effectiveness of the two groups.3.3.3 Evaluation for side effects: Adverse reastions like hemorrhage,infection or pulmonary embolism were observed.ResultsPart ?1.The thrombolysis rate of clots in group 1(73.64 ± 14.16)% was much higher than that in group 2(47.97 ± 11.66)%,group 3(57.33 ± 8.65)%,group 4(50.85 ± 9.63)%,and group 5(29.76 ± 18.06)%,all P < 0.05.2.HE staining of the clots showed multiple thrombolysis foci with clots collapse in group 1,while the clots of the other groups were more compact than clots in group 1.3.The fibrin reticula in group 1 degraded massively,and only a little fluorescently-tagged fibrin could be captured.While the green fluorescence of fibrin in other groups,especially in group 5,was distinctly high.Part ?1.Fourteen patients were included in experiment group,and twenty three in historical control group.There was no difference of gender,age and time since symptom onset(P >0.05)between experimental group and control group.2.One patient in experiment group suffered light bleeding at the puncture site,but prognosis was favorable.No obvious abnormal blood routine or coagulation function were detected.No symptoms of pulmonary embolism like dyspnea or thoracalgia were observed.3.The average treatment duration in experiment group(5.21 ± 1.53)d was significantly less than that in control group(7.65 ± 2.27)d(P < 0.05).Also,the overall UK dosage used in experiment group(3.91 ± 1.66)× 106 IU was about 11.34% lower when compared withcontrol group(4.41 ± 1.93)×106 IU(P < 0.05).Conclusions1.The rate of thrombolysis could be enhanced by intraclot microbubbles mediated ultrasound thrombolysis combined with UK in vitro.2.This clinical study preliminarily indicated that the combination therapy of CDT and IMUT could be applied on DVT treatment with safety and effectiveness in fewer UK dosage and shorter treatment days.