| BackgroundVenous thromboembolism?VTE?,is a common and potentially fatal disease,and the incidence rate of VTE increases exponentially as the patient population increases in age.At present,the commonly used treatment methods are anticoagulation,thrombolysis,surgical thrombectomy,mechanical thrombectomy and so on.Anticoagulant therapy is the basis,which can effectively prevent the progress of thrombus,but cannot dissolve thrombus quickly and restore the patency of vein.Surgical thrombectomy and mechanical thrombectomy can quickly restore the recanalization of the blood vessel,but at the same time,it is easy to damage the vessel walls and the function of valve,and the lone-term effect is poor.According to Antithrombotic Therapy for VTE Disease CHEST Guide lines,patients are most likely to benefit from thrombolytic therapy.Thrombolytic therapy can be divided into systemic thrombolysis?ST?and catheter-directed thrombolysis?CDT?.Comparing with ST,CDT,which has been widely used in clinical,can improve the thrombolysis rate,reduce the dosage of thrombolytic drugs,short the treatment time and decrease the incidence rate of bleeding complications and PTS.Sonothrombolysis is a new thrombolytic therapy.The main mechanisms are cavitation effect and mechanical effect.The shock waves,microjets and radiation force released by microbubbles resonance and"implosion",can make thrombus become porous?sonoporation effect?,subsequently promote drugs penetration and thrombolysis.Microbubbles?MB?,as a key factor of inducing cavitation effect,can reduce the peak negative pressure threshold of cavitation and enhance the cavitation intensity.Recently,some scholars have conducted clinical studies by combining external therapeutic ultrasound?ETUS?with intravenous infusion of MB.and the results indicated the effect of thrombolysis assisted by ETUS combined with intravenous infusion of MB was not satisfactory.The reasons were because of the limited acoustic window for US and the MB injected via vein were not able to enter the obstructed thrombus,in turn,the ultrasonic cavitation effect was weakened.EKOS?catheter is a new thrombolytic therapy method for interventional vascular lumen,which combines catheter-directed thrombolysis and high-frequency ultrasound.Without MB,the EKOS?catheter has been used in the clinical treatment of PE,acute ischemic stroke and venous thrombosis,and has achieved positive results.Its advantage lies in the fact that the administration of the drugs through the multi-side holes of the sheath tube makes the thrombolytic drugs directly act on the inside of the thrombus while combining with intraluminal ultrasound irradiation,which can promote the drugs to penetrate into the thrombus,thus enhancing the efficacy of drug thrombolytic therapy.Therefore,we injected MB and urokinase?UK?into the thrombus through EKOS?catheter simultaneously to enhance the thrombolytic effect of UK.On this basis,an intravascular ultrasound thrombolytic catheter with adjustable ultrasound energy parameters is developed,which is conducive to microbubble cavitation,to maximize the advantages of microbubble-mediated ultrasound thrombolysis and obtain a more rapid and effective thrombolytic therapy.Objective1.To explore the therapeutic effect of EKOS?catheter combined with intrathrombotic injection of MB to enhance the thrombolysis of UK in vitro.2.To develop an intravascular ultrasound thrombolytic catheter with adjustable parameters,and to verify the efficacy of combining it with intrathrombotic injection of MB in enhancing the thrombolysis rate of UK in vitro.Materials and Methods1.Main experimental instruments and devices?1?Ekosonic Endovascular System:MACH44,manufactured by EKOS company of the United States,is equipped with multi-side holes drug delivery catheter,a matching US core wire containing 12 miniature ultrasound transducer elements?2.2 MHz,0.45 W?positioned along the treatment zone and a control system.?2?Self-made experimental prototype of ultrasonic thrombolytic catheter:Produced by Shenzhen Sonosray Technology Co.,Ltd.,the transmitting frequency is 2.8 MHz,the sound pressure gear is 0?11,the pulse repetition frequency is 50,100,200,300,500 and 1000 Hz,and the number of pulses is 15,30,50,80,100 and 300.?3?Micro peristaltic pump:BT-100E,Chongqing Jieheng peristaltic pump Co.,Ltd.?4?Dual-channel microinjection pump:LD-P2020?,Shanghai Lande medical machinery Co.,Ltd.?5?Constant temperature water bath box:HUBER company,Germany.?6?Needle hydrophone:HNC-0400,produced by ONDA company in the United States.?7?Digital oscilloscope:DSO9064A,produced by Agilent company of the United States.?8?Fluorescence microscope:BX3-CBH,produced by Olympus company,Japan.?9?Electronic balance:UTP-313,produced by Shanghai Huachao Electric Co.,Ltd.?10?Experimental apparatus for thrombolysis in vitro:It was composed of a micro peristaltic pump,Dual-channel microinjection pump,water bath box,Ekosonic Endovascular System and a self-made pipeline for simulating blood circulation.A tee piece was used at entrance of the pipeline to connect another pipe entrance for insertion of EKOS?catheter.A pipeline at both ends of the thrombus sample pool to simulate the open collateral circulation.The whole circulation pipeline was placed in a 37?constant temperature water bath.The circulating solution was phosphate buffer saline?PBS?containing 10%bovine plasma,and the peristaltic pump worked at the speed of 60 r/min.2.Main experimental reagents?1?Aseptic anticoagulant newborn bovine blood:500 ml,produced by Beijing Solarbio Technology Co.,Ltd.stored at 4?.?2?Bovine plasma:500 ml,produced by Zhengzhou Jiulong Biological Products Co.,Ltd.,stored at-20?.?3?Urokinase:105 IU/bottle,produced by Lizhu pharmaceutical factory of Lizhu group.?4?Sonazoid?:1.2×109/ml,produced by GE Medical Company of Norway.?5?SonoVue?:59 mg,produced by Bracco Suisse SA company.?6?Anhydrous calcium chloride:500 g,produced by Beijing Solarbio Technology Co.,Ltd.?7?PBS:1000 ml,produced by Wuhan Boster Bioengineering Co.,Ltd.?8?4%tissue cell fixative:500 ml,produced by Beijing Solarbio Technology Co.,Ltd.3.Experimental methodPart 1:EKOS?catheter combined with intrathrombotic injection of MB to enhance the thrombolysis of UK in vitro.?1?Measurement of the acoustic parameters of EKOS?catheter:The peak negative pressure?PNP?was measured and recorded at 1,2,3,4,and 5 mm below the transducer chip by hydrophone,respectively.?2?Preparation of thrombus:Each tube with 8.0 ml aseptic anticoagulant newborn bovine blood and 264?l?33?l/ml?calcium chloride solution?5%?were added,After gently mixing,put them into a constant temperature water bath at 37?for 3 hours.Before using,the unsolidified serum was removed and the thrombus was rinsed with PBS for three times.Then the initial mass of the thrombus?W0?was weighed by electronic balance after absorbing the thrombus surface liquid with filter papers.?3?Experimental groups:90 bovine thrombus samples in vitro were randomly divided into 6 groups:ultrasound?US?+MB+UK group?combined group?,US+UK group,US+MB group,US group,UK group and control group,each group had 15 samples.?4?Effectiveness evaluation:1.The thrombolysis rate of each group was calculated and compared.2.The distribution of erythrocyte and fibrin was observed under a light microscope.Part 2:Detection of acoustic parameters of self-made ultrasonic thrombolytic catheter and verification of the thrombolytic efficiency in vitro.?1?To detect acoustic parameters of self-made ultrasound thrombolytic catheter:Needle hydrophone was used to measure the PNP of different transducer chips,different sound pressure gears,different angles,without sheath and with sheath tube at 2 and 3 mm below the transducer chip by hydrophone,respectively.?2?Preparation of thrombus:The preparation method was the same as before.?3?Grouping:Clots were randomly divided into 3 groups:US+MB+UK group?combined group?,US+UK group and UK group.?4?Effectiveness evaluation:Thrombolysis efficacy of each group was calculated and compared.ResultsPart 1?1?The PNP of EKOS?catheter varied irregularly within the scope of 0.4?6.4 MPa,and the duty cycle is 24.86%.?2?The thrombolysis rate of combined group?41.9±4.5?%was close to that of US+UK group?42.0±3.3?%,which were significantly higher than that of the other groups?all P<0.05?.There was no significant difference between US+MB group?28.4±3.3?%and UK group?27.8±3.1?%in thrombolysis rate,which were significantly higher than that of US group?23.9±3.0?%and control group?17.5±3.7?%,and there were significant differences?both P<0.05?.?3?Histological examination of the thrombus in combined group and US+UK group showed that the thrombus contacting with EKOS?catheter were more disintegrated,and the distribution of red blood cells were sparser than the other groups.Part 2?1?The PNP of the self-made ultrasonic thrombolysis catheter transducer increases with the increase of the acoustic pressure gear and decreases with the increase of the distance from the hydrophone.The PNP measured with sheath is lower than that without sheath.The PNP measured from different angles fluctuated between 0.58 and 1.00 MPa.?2?The thrombolysis rate of combined group?38.9±1.8?%,was much higher than US+UK group?31.4±1.4?%and UK group?22.3±1.2?%,all P<0.05.Conclusions1.EKOS?catheter can obviously improve the thrombolysis rate of UK to bovine thrombus in vitro.However,MB has no significant effect on thrombolysis rate.2.The self-made ultrasonic thrombolysis catheter can enhance the thrombolysis effect of UK in vitro,and the addition of MB can further improve the thrombolysis rate. |
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