| BackgroundDue to the special location of intracranial hematoma,if not treated in time,it is easy to lead to irreversible damage of brain tissue and deterioration of nerve function,and even cause death on account of cerebral hernia.Hematoma volume has been shown to be a key factor affecting the prognosis of patients.Therefore,the therapeutic strategy of hematoma removal and evacuation under surgical intervention is widely accepted in clinical.Traditional craniotomy is more likely to damage normal adjacent tissues and hit the prognosis of patients.Therefore,minimally invasive surgery(MIS)strategy,for example skull drilling and drainage,and fibrinolytic drug irrigation is favored in clinical application because of its advantages of simple and easy operation and less trauma,but the efficiency of hematoma evacuation through this strategy is low and the time required for catheter drainage is long,which increase the risks of infection and poor neurological recovery.In the past decades,diverse in-vivo and in-vitro experiments have proved that ultrasound can significantly enhance the thrombolytic efficiency of fibrinolytic drugs through its cavitation effect.Therefore,the strategy of MIS combined with ultrasound for the treatment of intracranial hematoma can theoretically improve the efficiency of hematoma evacuation and bring greater benefits to patients.Ultrasound catheter,a kind of ultrasound equipment used for intravascular thrombolysis,has been used to improve the hematoma lysis efficiency,through minimally invasive drilling operation and combined with fibrinolytic drugs,although the final results showed hematoma removal efficiency has elevated significantly,but the ultrasound catheter adjuvant treatment time is still too long,which limits the application and development of the strategy.Ultrasonic horn is a kind of power ultrasound equipment.Compared with ultrasound catheter,the rapid vibration of its tip can generate more intense cavitation effect in the liquid.Therefore,it is speculated that the therapeutic strategy of using the ultrasonic horn combined with fibrinolytic drugs to lyse and remove hematoma may achieve better results so as to meet clinical needs.Based on this theory,our research group designed and customized an ultrasonic horn equipment-Ultrasound Needle,which is multiparameter adjustable,and verified its mechanism of clot lysis when combined with fibrinolytic drugs,and discussed the effect of different working parameters and fibrinolytic drugs on this strategy in-vitro.Objectives1.To validate the feasibility and basic mechanism of Ultrasound Needle combined with fibrinolytic drugs lysing clot in-vitro.2.To explore the effects of different working parameters on the in-vitro lysis efficiency of Ultrasound Needle combined with urokinase.3.To discuss the disparity in in-vitro lysis efficiency of different types of fibrinolytic drugs(urokinase,reteplase)with the synergy of Ultrasound Needle.Materials and methods1、Experiment equipments1.1 Ultrasound Needle: it is customized from Jiangsu Baihang Ultrasonic Technology Co.,LTD.,the main body is a conical horn with a tip diameter of 1.8mm,and a needle sheath of 2.5mm is attached outside the rod for drug injection to the tip.The operating frequency is29.62 KHz,and there are three primary operating parameters in the host panel,which are Amplitude(%),(Duty cycle)DC(%)and(Plus repetition frequency)PRF(Hz).1.2 Microinfusion pump: LD-P2020 II,Shanghai Lande Medical Instrument Co.,LTD.1.3 Electronic balance: UTP-313,Shanghai Huachao Electric Appliance Co.,LTD.1.4 VINNO 70 integrated ultrasound theranostic system: Suzhou VINNO Technology Co.,LTD.2、Experiment reagents2.1 Aseptic anticoagulant newborn bovine whole blood: 500ml/bottle,Beijing Solarbio Science & Technology Co.,LTD.2.2 Urokinase: 100000IU/bottle,Wuhan Humanwell Pharmaceutical Co.,LTD.2.3 Membrane orange fluorescent probe Dil(1,1’-Dioctadecyl-filling level 3,3’-tetramethylindocarbocyanine perchlorate):10mg/bottle,Beijing Solarbio Science &Technology Co.,LTD.2.4 Recombinant human tissue type plasminogen activator derivative(Rteplase)for injection: 10mg/dose,China Resources Biotech Pharmaceutical Co.,LTD.3、Experiment methodsExperiment 1: Clot lysis efficiency enhanced by Ultrasound Needle combined with urokinase and its mechanism(1)In-vitro blood clot preparation: 200μL 5% calcium chloride solution and 10 m L sterile anticoagulant newborn bovine whole blood were mixed in a polypropylene centrifuge tube mold(50ml centrifuge tube was cut along a 25 ml scale line).The mixture was incubated in a 37℃ constant temperature water bath for 3h to form a blood clot,and then randomly allocated to each group.(2)The treatment plan exploring the influence of working parameters: The Ultrasound Needle was combined with urokinase to lyse blood clots,during which the Ultrasound Needle operating parameters PRF(100Hz)was fixed,and then Amplitude(%)and DC(%)were successively adjusted,and the highest temperature was recorded during the treatment.After that,the lysis weight of blood clot in each group was compared,and the optimal working parameters of Ultrasound Needle were selected.(3)Treatment plan under optimal working parameters: Ultrasound Needle was set the optimal working parameters,and the Ultrasound Needle group,urokinase group,Ultrasound Needle+normal saline group and Ultrasound Needle+urokinase group were respectively treated.(4)Mechanism study: Observe the internal permeability of fluorescent dye-Dil on blood clot under the action of Ultrasound Needle.Control group,only inject Dil.Ultrasound group,Dil was injected simultaneously with Ultrasound Needle treatment.(5)Evaluation indicators:① Blood clot lysis weight and lysis rate in each group.②Dil diffusion range under microscope.③Fibrinolysis performance under scanning electron microscopy.④The area of lysis region in gross specimen.Experiment 2: Comparison of the in-vitro lysis efficiency of urokinase and reteplase under with the synergy of Ultrasound Needle(1)In-vitro hematoma model: A sterile anticoagulant blood collection bag was used to collect blood from healthy adult volunteers through the cubital vein,and then 25 ml was taken and placed in a balloon mixed with 500μL 5% calcium chloride solution and 20 IU thrombin.The balloon was sealed and incubated in a 37℃ constant temperature water bath for1.5h/12h/24 h to form clot.Blood clots from the same volunteer were randomly assigned to different groups.(2)Treatment plan and grouping: Control group,Ultrasound Needle group,urokinase group,reteplase group,Ultrasound Needle+urokinase group and Ultrasound Needle+reteplase group.1.5h blood clots received all of the above treatment regiments,12/24 h blood clots only received the last two combined treatment regiments.The maximum temperature inside the clot was measured with a thermometer needle immediately after each treatment.(3)Evaluation indicators:① Evaluation blood clot lysis efficiency in each group: Relative end weight(post-treatment weight/initial weight).②The area changes of blood clot before and after treatment under the observation of two-dimensional ultrasound.③Fluorescence staining results of fibrin and HE staining results.④Scanning electron microscopy: Fibrin covering area on clot surface.ResultsExperiment 1: Clot lysis efficiency enhanced by Ultrasound Needle combined with urokinase and its mechanism(1)Working parameters discussion: The blood clot lysis weight increased with the increase of Amplitude(%)and DC(%),but reached the peak value(2.44±0.14g)when Amplitude(40%)and DC(20%),then decreased.The greater Amplitude(%)and DC(%),the higher the maximum temperature,and the influence of DC(%)increase on temperature rise is greater.(2)Lysis efficiency comparison under optimal parameters(PRF(100Hz),Amplitude(40%),DC(20%)): The lysis weight and lysis rate of the Ultrasound Needle+urokinase group were more than 4 times that of the urokinase group,and significantly higher than that of other groups(P<0.05).(3)Dil permeation results: After the action of Ultrasound Needle,the blood clot tissue was mechanically damaged,the gap was widened,and the Dil permeation was profound,and the fluorescence in full field could be seen under the microscope.(4)Scanning electron microscopy: Fibrin filaments lysed more thoroughly in blood clots in the Ultrasound Needle+urokinase group than in the Ultrasound Needle and urokinase group.(5)Gross specimen observation results: The area of clot lysis was the largest in the Ultrasound Needle combined with urokinase group,which was significantly higher than that in the urokinase group(P<0.05).Experiment 2: Comparison of the in-vitro lysis efficiency of urokinase and reteplase under with the synergy of Ultrasound Needle(1)The lysis efficiency under the synergy of ultrasound needle: Weighing results confirmed that the relative end weight of the combined treatment group(Ultrasound Needle+urokinase group,Ultrasound Needle+rteplase group)was significantly lower than that of the other treatment groups alone(P<0.05),the lysis efficiency was significantly improved.Two-dimensional ultrasound observation results,HE staining and fibrin fluorescence staining results also confirmed that the combined treatment group had stronger damage to the tissue of blood clots and better lysis effect compared with other groups.(2)The influence of clotting time on lysis efficiency: Under scanning electron microscope,the covering density of fibrin filament increased significantly with the increase of clotting time(1.5h vs 12 h vs 24 h,P<0.05),and the lysis efficiency of the corresponding combined group was significantly decreased successively(P<0.05).(3)Comparison of drug lysis efficiency: There was no difference in lysis efficiency between urokinase group and rteplase group(P<0.05),while the lysis efficiency of Ultrasound Needle+rteplase group was always significantly higher than that of Ultrasound Needle+urokinase group in 1.5h/12h/24 h clots(P<0.05).(4)Temperature change: The temperature rise in the combined group was more obvious than that in the urokinase group and the rteplase group,and the temperature rise increased slightly with the increase of clotting time,but the maximum temperature was still lower than that in the Ultrasound Needle group(43.86±1.12℃).Conclusions1.The Ultrasound Needle can destroy the blood clot tissue through its mechanical effect caused by the vibration of the tip,so as to promote the infiltration of fibrinolytic drugs and accelerate the lysis of clot.It has great potential to become a minimally invasive ultrasound therapeutic strategy for rapid evacuation of intracranial hematoma.2.Too high working parameters of Amplitude(%)and DC(%)will cause an obvious temperature rise and reduce the final lysis efficacy,among which DC(%)has a more significant influence.3.The increase of clotting time will reduce the in-vitro lysis efficiency of Ultrasound Needle combined with fibrinolytic drugs,but under the synergistic effect of Ultrasound Needle,the fibrinolytic efficiency of reteplase was always significantly higher than that of urokinase. |
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