| Background: Sonothrombolysis(STL)as a new technology for treating thrombosis,has been studied for more than 20 years,but it is still not widely used in clinical practice.Microbubble mediated ultrasound thrombolysis can significantly improve the efficiency of thrombolysis,but its ability to enhance cavitation is limited due to its unable to enter the interior of the obstructed clot.In the previous study,we suggested an intraclot microbubbles mediated ultrasound thrombolysis(IMUT)by inject microbubble and urokinase directly into the thrombus.The ultrasonic stimulated microbubble cavitation,promoted the penetration of urokinase.In our previous study,we have confirmed that the thrombolysis effect of IMUT was significantly better than that of the injection of microbubbles in the peripheral circulation.The purpose of this study is to find a suitable combination of acoustic parameters for IMUT ultrasound thrombolysis,the pressure(Peak negative pressure)and duty cycle are two key parameters to determine the strength of cavitation,the present study was to explore the effect of sound pressure and duty cycle on the rate of thrombolysis in vitro.Objective: Explore different acoustic pressures and different duty cycles on the effect of in vitro IMUT.Materials and Methods: 1.The experimental apparatus:(1)DCT-700 therapeutic US device: Manufactured by Shenzhen Welld Medical Electronics co.,LTD,with a central frequency at 1 MHz,pulse repetition frequency 100 Hz,the range of sound pressure adjustable from 285 k Pa to 931 k Pa,and the duty cycle adjustable from 1% to 10%.(2)High frequency ultrasonic imaging system: The Vevo2100 ultra-high resolution ultrasonic imaging system,manufactured by Canada Visual Sonics.Contrast-enhancedultrasound imaging mode from a linar probe with a frequency of 20 MHz were used in this study.(3)Inverted microscope ADMIRB: inverted microscope manufactured by Germany LEICA company were used in this study.(4)In vitro experiment device:The over all experimental arrangement consisted of a 37? water tank bedded with a 2cm sound-absorbing sponge to reduce the reflected wave.Circulation tube were flowing with a rate of 4.6 ml/s with a precision microperistaltic pump.2.Laboratory Reagents:(1)microbubble,a lipid-coated microbubbles called Zhifuxian prepared by department ultrasound Xinqiao hospital.with a concentration of(7-8)×109 / ml,were used in this study.After a 15 times dilution,0.02 ml(about 0.9-1.7 ×107 MBs)were injected into each thrombus sample through a 21 G needle with multiple side holes produced by Hakko Co.,Ltd.Medical Device Division of JAPAN.(2)Urokinase produced by pharmaceutical factory of Guangdong Li Zhu group.A100000 iu/bottle were soluted in 0.5ml 0.9% sodium chloride.A 0.1 ml(about 20000 iu UK)were injected into each thrombus sample through the same needle.3.The experimental methods: Blood clots preparation: Fresh venous blood collected from healthy adult volunteers was quickly sub-divided into 1.0ml samples in EP tubes and incubated in a 37 ? water bath for three hours to form clots.Before each experiment,the clot was carefully removed from the tube,washed with a neutral phosphate buffer for three times,dried with filter papers and the initial weight(W0)was taken.Experiment 1: The effect of different acoustic pressure on Intraclot microbubbles mediated ultrasound thrombolysis 50 fresh blood clots were divided into four study groups and one control group(n = 10).After a bonus of microbubbles and UK was injected,the clots in the study groups were exposed to ultrasound at an acoustic pressure of 285 k Pa,512 k Pa,708 k Pa,or 931 k Pa,respectively.The ultrasound treatment time is for10 minutes,and the duty cycle was set at 10%.The clots in the control group received sham ultrasound exposure.Experiment 2: The effect of different duty cycle on Intraclot microbubblesmediated ultrasound thrombolysis 50 fresh blood clots were divided into four study groups and one control group(n = 10).After a bonus of microbubbles and UK was injected,The clots in the study groups were exposed to ultrasound at a duty cycle of 1%,2%,5% and 10% respectively.The ultrasound treatment time is for 10 minutes,and the acoustic pressure was set at 931 k Pa.The clots in the control group received sham ultrasound exposure.After each experiment,the residual clot was gently removed from the sample holder,washed with PBS,then the clot weight after treatment(W1)was taken.Observational Index:(1)Thrombolysis effect were the assessed by percentage weight loss of the clot:(W0-W1)/ W0×100%.(2)The microbubble distribution and changes before and after ultrasound exposure were observed using a Visual Sonics Vevo2100 ultra-high resolutionultrasound imaging system.(3)Histologic examination: Residual clots were fixed in 10% formaldehyde,then embedded in paraffin and were finally sectioned and stained with hematoxylin and eosin for observation using a LEICA DMIRB light microscope.Results: Experiment 1: Groups of thrombolysis rate comparison:Experiment 4 group(931 k Pa)thrombolysis rate(50.9±11.5%)is significantly higher than control group in thrombolysis rate(22.9±10.7%)and experiment 1 group(285 k Pa)thrombolysis rate(29.2±7.0%)(P<0.01);while the thrombolysis rate of experiment 3 group(708 k Pa)(46.2±15.6%)was higher than control group(P<0.05).No statistical difference of the thrombolysis rate was found between experiment 2 group(512 k Pa),3 group(708 k Pa),experiment 4 group(931 k Pa)(P > 0.05).HE dyeing imaging: After HE staining under observation group thrombus specimens,the control group visible destruction form of small defects,surrounding cells loose zone with thin and slightly uneven;As the pressure increases,red blood cell destruction form of defect increases gradually,the thrombus surrounding cells loose zone became uneventhe,then obvious thickening,even empty bubble is forming. Experiment 2: Groups of thrombolysis rate comparison : Experiment 4 group(DC=10%)of thrombolysis rate(52.6±14.6%),3 group(DC=5%)of thrombolysis rate(41.2±16.0%),experiment2 group(DC=2%)of thrombolysis rate(39.5±7.9%)were significantly higher than control group(22.9±10.7%,P<0.01);Experiment 4 group(DC=10%),Experiment 3 group(DC=5%)all higher than experiment 1 group(DC=1%)of thrombolysis rate(29.7±10.3%)(P<0.05);Experiment 4 group(DC=10%)of thrombolysis rate also higher than experiment 2group(DC=2%)(P<0.05);No statistical difference of the thrombolysis rate was found between Experiment 4 group(DC=10%)and experiment 3 group(DC=5%)(P > 0.05).After HE staining under ×100 to observe each group thrombus specimens,simple urokinase plus microbubble group its surrounding fibrin thrombus mesh belt thickness uniformity,good continuity,no significant difference;With increasing duty ratio,fibrin mesh belt surrounding the thrombus to fine,irregular,loss of free normal continuity,bubble formation and gradually increase.Simple urokinase plus microbubble group,visible urokinase fibrin degradation the formation of the small hole;with the increase of duty cycle,red cells destruction reduce more and more obvious,more fibrin degradation,formation of the hole is bigger and bigger.Conclusion: Intraclot microbubbles mediated ultrasound thrombolysis method enhanced ultrasound thrombolysis.The parameters of acoustic pressure as 708 k Pa and duty cycle more than 2% are favorable. |
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