| Object:1.To establish an in vitro simulated flow model,and to observe the effect of ultrasound irradiation combined with microbubbles on the potential enhanced thrombolysis.2.To define the effect difference of self-made lipid microbubbles and the dedicated SonoVue on thrombolysis in vitro.3.To compare the effects of different environment temperatures at 37℃and 41℃ on thrombolysis in vitro.4.To compare the effect of fresh thrombus and retracted blood clot on thrombolysis in vitro,and to understand the effect of thrombus age in thrombolysis in vitro.Method:1.Lipid microbubble preparation: Lipid microbubbles were prepared using dipalmitoyl-sn-glycerol-3-phosphocholine(DPPC),1,2-distearoyl-snglycero-3-phosphoeth-anolamine-nmaleimide(polyethyleneglycol)(DSPE-PE G),glycerol,perfluoropropane through mechanical shock method.2.Thrombus preparation:1 Fresh thrombus: Whole blood was draw from healthy volunteers using citric acid vacuum blood-collecting vessel,and blood coagulation was carried out with calcium chloride solution.After 3 hours of incubation at 37℃,the thrombus was stored in a refrigerator at 4℃ for 3 hours.2 Retracted blood clot: Whole blood was draw from healthy volunteersusing citric acid vacuum blood-collecting vessel,and blood coagulation was carried out with calcium chloride solution.After 3 hours of incubation at 37℃,the thrombus was stored in a refrigerator at 4℃ for 7 days.The test tube was taken out after 7 days,the translucent serum was separated from the blood clot to obtain a stable thrombus.3.Extracorporeal circulation device: the device consists of closed simulation blood vessels,thermostatic water bath,micro-peristaltic pump,drug injection channel.The closed pipe was placed in a constant temperature water bath during the entire experiment process,circulating pipe flow ratio was controlled at 4.5ml/s,the ultrasound irradiation probe was fixed 5 cm above the thrombus vertically.4.Experiment group: According to the different combinations of ultrasound irradiation dosages,microbubbles,urokinase,ambient temperature and thrombus age,the samples were randomly divided into one control groups and six experimental groups,each group consist of 10 samples;ultrasound irradiation parameters were set to frequency 1MHz,intensity 2w/cm2,duty ratio 50%.Group A: false ultrasound irradiation for 20 min,2 ml of normal saline,37℃ water bath,fresh thrombus.Group B: ultrasound irradiation for 20 min,1ml of self-made lipid microbubbles,1ml of saline,37℃ water bath,fresh thrombus.Group C: ultrasound irradiation for 20 min,1ml of urokinase,1ml of saline,37℃ water bath,fresh thrombus.Group D: ultrasound irradiation for 20 min,1ml of self-made lipid microbubbles,1ml of urokinase,37℃ water bath,fresh thrombus.Group E: ultrasound irradiation for 20 min,1ml of SonoVue,1ml of urokinase,37℃ water bath,fresh thrombus.Group F: ultrasound irradiation for 20 min,1ml of self-made lipid microbubbles,1ml of urokinase,41℃ water bath,fresh thrombus.Group G: ultrasound irradiation for 20 min,1ml of self-made lipid microbubbles,1ml of urokinase,37℃ water bath,retracted blood clot.5.SIEMENS ACUSON SC2000 and L9-1 linear ultrasonic probe were employed to measure the stenosis ratio and thrombus area.Thrombus weight loss ratio before and after thrombolysis,the tube stenosis ratio at thrombus placement site before and after thrombolysis,the maximal long axis area reduction ratio of thrombus before and after thrombolysis were measured and calculated.6.Statistical analysis: Seven sets of data accorded with normal distribution(P>0.05)and homogeneity of variance(P>0.05).Seven sets of data were expressed as mean ± standard deviation(),the overall comparison among multiple groups using one-way ANOVA,comparison between two groups using LSD-t.All with P<0.05 was statistically significant.All the data was analyzed using SPSS 21.0 statistical software.Results:1.There were significant differences of thrombus weight loss ratio before and after thrombolysis,the tube stenosis ratios at thrombus placement site before and after thrombolysis,the maximal long axis area reduction ratio of thrombus before and after thrombolysis in the seven groups(P<0.01).2.group A and B: There were significant differences of thrombus weight loss ratio and the maximal long axis area reduction ratio(P<0.01).thrombolysis weight loss ratio increased from 3% to 10%with an increase of233%,area reduction ratio from 2% to 7% with an increase of 250%,there was no significant difference of stenosis ratio(P>0.05).3.group C and D: There were significant differences of thrombus weight loss ratio and the maximal long axis area reduction ratio(P<0.01),thrombolysis weight loss ratio increased from 15% to 16% with an increase of7%,area reduction ratio from 10% to 14% with an increase of 40%,there was no significant difference in stenosis ratio(P>0.05).4.group D and E: There were significant differences of thrombus weight loss ratio,the maximal long axis area reduction ratio and the tube stenosis ratio at thrombus placement site(P<0.01),thrombolysis weight loss ratio increased from 16% to 21% with an increase of 31%,area reduction ratio from 14% to 25% with an increase of 79%,Stenosis ratio increased from 6%to 12% with an increase of 100%.5.group D and F: There were significant differences of thrombus weight loss ratio,the maximal long axis area reduction ratio and the tube stenosis ratio at thrombus placement site(P<0.01),thrombolysis weight loss ratio increased from 16% to 29% with an increase of 81%,area reduction ratio from 14% to 31% with an increase of 121%,Stenosis ratio increased from 6%to 13% with an increase of 117%.6.group G and A: There was no significant difference of thrombus weight loss ratio,the maximal long axis area reduction ratio and the tube stenosis ratio at thrombus placement site(P>0.05).7.group G and D: There were significant differences of thrombus weight loss ratio,the maximal long axis area reduction ratio and the tube stenosis ratio at thrombus placement site(P<0.01),thrombolysis weight loss ratio increased from 2% to 16% with an increase of 700%,area reduction ratiofrom 2% to 14% with an increase of 600%,Stenosis ratio increased from 3%to 6% with an increase of 200%.Conclusions:1.This experiment preliminary proved that the combination of ultrasound irradiation(f =1MHz,P =2.0w/cm2,duty ratio 50%)with microbubbles can enhance thrombolysis in the simulated model of vascular flow in vitro.2.Both SonoVue and self-made lipid microbubbles can be used to enhance thrombolysis,the effect of SonoVue is more significant.3.The temperature affects the dissolution of fresh thrombus in vitro,the and the higher temperature can enhance thrombolysis significantly.4.Microbubble-mediated ultrasound-enhanced urokinase thrombolysis is significantly affected by thrombus age without effect on the dissolution of retracted blood clots. |
PDF Full Text Request