| Backward and purposeDespite epicardial coronary artery reperfusion by percutaneous coronary intervention,distal hypo-perfusion or no-perfusion into the coronary microcirculation limits myocardial salvage during actue myocardial infraction,named No-reflow phenomenon.Thrombotic micro-embolization is a major component of No-reflow phenomenon,and always has a connection with additional myonecrosis,adverse left ventricular remodeling,and other worse prognosis after AMI,has become an heavy burden on individual families and society.Studies on microvascular preparations have reported the successful reperfusion of micro-embolization by delivering long-pulse-duration ultrasound(LUS)and lipped microbubbles(MBs)in vitro models,and this rapid and complete lysis occurred with the pulse length increased and the peak negative acoustic pressure increased.Later researches on rats hindlimb mirco-emboliazation model,using 1MHz,1.5MPa,5000cycle LUS also achieved the same thrombolysis effect,and considered these effects are most likely mediated by inertial cavitation-related increases in shear and activation of endothelial nitric oxide synthase.But researches also indicated that over-long pulse length would cause overheating in the areas of hypo-perfusion,resulting in necrosis of the cardiomyocytes and ultimately the opposite effect on thrombolysis.But due to the successfully thrombolysis effect from long pulse ultrasound has been achieved on most researches and the complexity of no-reflow phenomenon,it is necessary to further explore a better thrombolysis parameter on microcirculation.In this study,we continued to use the micro-embolization model on a rat hindlimb that has been published before to mimic no-flow phenomenon after percutaneous coronary intervention,and six different kinds of pulse length ultrasound,varying from 5cycle,100cyce,500cycle,1000cycle,5000cycle to 50000cycle,were delivered to treatment groups,while one control group received no therapeutic ultrasound but continuous microbubbles infusion and imagine ultrasound visualization.Contrast ultrasound was done to measure microvascular perfusion to explore the range of effective pulse length that cause micro-embolization thrombolysis.Hematoxylin and eosin(HE)staining on skeletal muscle was used to explore the influence of each LUS,finally to provide a better treatment to overcome the no-flow phenomenon.Materials and Methods1?Preparation of micro-thrombiMixing fresh rat blood with coagulation accelerator(1:10)made from 1000U Lyophilizing Thrombin Powder(Thrombin-1000U,Biosharp,GuangZhou,China)and 5%CaCL2 Solution(10ml)and in a glass vial and incubating at 37 ?temperature for 24h until the blood fully coagulated.Then grinded the blood clot and passed through a series of progressively narrower mesh plates(500?m,200?m)(TS-BXGSW,Filter Manufacture,ShangHai,China),then finally passed 200?m to achieve thrombi smaller than 200?m to induce micro-embolization on rats hindlimb.2?Microbubble preparationMicrobubbles filled with Perfluoropropane and coated with lipids were used to dissolve micro-thrombi and have contrast perfusion imagine at each experiment stages.The mean diameter was(2.46±0.05)?m,and the mean concentration was(2.99±0.19)×109/ml.3?Animal preparationForty-two Sprague Dawley(SD)male rats(n=42)weighting 250-300g were randomly divided into 7 groups and anesthetized with isoflurane(3%).Rats were placed on a thermostat plate to keep the rectal temperature at 37?.The right hindlimb was degloved for better visualization and delivering LUS.The right external jugular vein was cannulated with a Polyethylene tubing(PE-50,YongXin Polyethylene Manufacture,ShangHai,China)to continuous infusion of MBs for perfusion imaging.Another PE-50 tubing was advanced from the left common iliac artery into the abdominal aorta for administration of micro-thrombi into the right hindlimb.4?Ultrasound preparation(1)imaging ultrasound:Hindlimb muscle perfusion was measured by a clinical ultrasound imaging system(Sequoia 512,Acuson,Siemens,Germany)using contrast pulsed sequence mode from a liner array transducer(15L8,Siemens,Germany)at a mechanical index(MI)of 0.18 and a transmission frequency of 7.0MHz.Choosing the longitudinal section of hindlimb muscle(the midpoint between groin and knee joint)to visualize and have perfusion image at each experiment stage.MI 1.9 for burst-replenishment imaging and 0.18 for replenishment imaging during continuous infusion of MBs at 3ml/h.(2)Therapeutic Ultrasound:Ultrasound parameters(1MHz,MI 1.8,pulse interval 3S,5cycle,100cycle,500cycle,1000cycle,5000cycle,50000cycle)were set on an arbitrary function generator(33210A,Agilent),through a gated radio-frequency power amplifier(2200L,Electronics&Innovation,US)delivered to the treatment area,a flat single-element immersion transducer(Olympus NDT,US)was used to deliver the long pulse ultrasound.The whole treatment period last for 10 minutes.Then compare the treatment effects of different pulse length ultrasound after the experiment.5?Treatment protocolBy injecting micro-thrombi into the left common iliac artery to cause micro-emboolization on rats hindlimb.After the micro-embolization model had been successfully constructed,forty-two rats were randomly divided into one control group and six treatment groups:(1)one control group(n=6):continuous infusion of MBs and imaging ultrasound visualized,receiving no therapeutic ultrasound after embolization but perfusion imaging was performed at the same time points as the treatment groups.(2)six treatment groups(n=6):continuous infusion of MBs and imaging ultrasound visualized,each group receiving one pulse length ultrasound(from 5cycle?100cycle?500cycle?1000cycle?5000cycle to 50000cycle)after embolization and perfusion imaging was performed at each experimental stages.Then,each treatment group was given one kind of LUS during infusion of MBs(3ml/h),and control group received imagine ultrasound and MBs but no therapeutic ultrasound as described before.Perfusion imaging was performed at the first?fifth?tenth minutes when giving therapeutic ultrasound and control group had perfusion imaging at the same time points as the treatment groups.After the 10 minutes of treatment period,rats were sacrificed and HE staining were obtained from skeletal muscle to analyze the potential side effect from LUS.6?Imagine analysisThe images of hindlimb perfusion was analyzed by an offline image analysis software(MCE Version 2.7,University of Virginia,VA,USA).A is the peak plateau video intensity,which reflects vascular cross-sectional area.Histology of skeletal muscle was performed after treatment in each group and the fixed samples were stained by HE,and the sections were examined microscopically to detect whether LUS would have side effect in the treatment area.7?StatisticsData(peak plateau video intensity A)are expressed as the mean±SD.Using SPSS 19.0 to analysis all the datas.Differences within groups using repeated-measures ANOVA(P<0.01 was considered significant).LSD post hoc t-test was used to compare differences between each group and the difference at every experiment stages(P<0.01 was considered significant).Bonferroni post hoc t-test was used to compare differences at different experiment stages,and the significance was defined at P<0.002 to adjust for multiple comparisons.Results1?Changes of microvascular blood flow in ischemic hindlimb1.1?The comparison of peak plateau video intensity(A)in control group and six treatment groups:There was no significant difference between control group,5cycle,100cycle and 50000cycle groups,while 500cycle,1000cycle,5000cycle groups are much stronger than 5cycle,100cycle,50000cycle and control groups(P<0.01,compared to 500cycle,1000cycle and 5000cycle groups).1000cycle and 5000cycle groups were much stronger than 500cycle group(P<0.01),but there was no significant between 1000cycle group and 5000cycle group(P>0.01).1.2?The comparison of peak plateau video intensity(A)in each treatment groups:In all groups,the peak plateau video intensity(A)were significantly lower than Before-emboli after the micro-embolization(P<0.0001,compared with Before-emboli).In control group,5cycle,100cycle and 50000cycle treatment groups,A was persisted lower than Before-emboli and no longer different from emboli through the whole treatment period.In 500,1000 and 5000cycle groups,after the treatment for five minutes,A was significantly increased than emboli(P<0.001,compared with Emboli),and no longer different from Before-emboli throughout the experiment(P>0.01?compared with Before-emboli).The comparison at 10min between groups showed a higher thrombolytic effect in 1000cycle compared to 500cycle(P<0.01?1000cycle compared to 500cycle)and little difference were seen between 5000cycle and 500cycle(P>0.01,5000cycle compared to 500cycle),but there was no significant difference between 1000cycle and 5000cycle(P>0.01).2?HE-stained on skeletal muscleIn control group,5cycle and 100cycle groups,multiple occluded microvessels can be seen from hindlimb HE staining.But there was no obvious evidence of tissue bleeding or trauma.In 500cycle,1000cycle and 5000cycle groups,little thrombotic microvascular obstructions were visualized and little evidence of tissue bleeding or trauma,either.But when the pulse length increased up to 50000cycle,obvious bleeding of tissue can be seen in HE staining,indicating that the pulse length under 5000cycle has no side effect on hindlimb muscle,the pulse length over 50000cycle actually had side effect on hindlimb muscle.Conclusion(1)Long pulse ultrasound,with pulse length varied from 500 to 5000cycle,combined with microbubbles could restore microvascular blood flow after thrombotic microvascular obstruction.(2)There was no significant difference between 500cycle and 5000cycle,and no significant difference between 1000cycle and 5000cycle.But 1000cycle was much better than 500,so we recommended using 1000cycle.(3)There was no therapeutic effect but side effect when the pulse length increased to 50000cycle. |
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