| BackgroundThe incidence and mortality of acute myocardial infarction remains high depite great advances in modern therapeutic strategies.It is the major fatal and disabled disease in China,which has caused great medical burden to our country.The key to saving the dying myocardium,improving the cardiac function and the prognosis of the patients is the timely opening of the infarct related aretry(IRA)and the perfusion of the ischemic tissue.The main means of opening the IRA include emergency percutaneous coronary intervention(PCI)and drug thrombolysis.Whether PCI or thrombolytic therapy is used,although the blood flow in IRA was restored,the perfusion of microvasculature in myocardium remains low.This is known as coronary no reflow in clinical pratice.The incidence of coronary no reflow was high,up to 70%.More importantly,coronary no reflow is an important factor in short-term and long-term poor prognosis in patients with acute myocardial infarction.Therefore,the prevention and treatment of coronary no reflow is very important.The pathological mechanism of coronary no reflow mainly includes distal embolization and ischemia-reperfusion injury.Among them,microvascular thrombosis caused by distal microembolism and ischemia-reperfusion injury is the main cause of coronary no reflow.The distal protection device and thrombus aspiration catheter are the main instruments for the prevention of coronary no reflow.In recent years,clinical studies have proved that both thrombus aspiration catheter and distal protection device have limited effect on patients' clinical prognosis.More importantly,both of these two measures are the means of prevention,which are not effective for the thromboembolic blockage of the coronary microvessels.Thrombolytic therapy can effectively dissolve erythrocyte-rich microthrombus,but it may cause cerebral hemorrhage,which is limited in clinical use.Moreover,its thrombolytic effect on platelet-rich microthrombus is poor.Platelet glycoprotein ?b/?a receptor antagonist can effectively reduce microvascular in situ thrombosis on the basis of conventional dual antiplatelet therapy,but it is only a bailout measure because of high risk of bleeding.Therefore,it is urgent to find a new method for the prevention and treatment of coronary microvascular thrombosis.Ultrasound combined with microbubble treatment could have such potential.A large number of experiments have confirmed that ultrasound combined with microbubble treatment can effectively dissolve thrombus in vitro,in middle cerebral arteries,coronary arteries and peripheral arteries.The main mechanism responsible is cavitation effect.However,the previous studies of ultrasound combined microbubble treatment mainly focused on the field of large vessels.In recent years,researchers have begun to carry out related research in the field of microvasculature.Leeman and others confirmed that ultrasound combined with microbubble treatment could effectively dissolve the erythrocyte-rich microthrombi in vitro.Pacilla,Porter and others confirmed that ultrasound combined with microbubble treatment could improve the microvascular perfusion in hind limb models of microthrombi-induced no reflow in rats.More importantly,our team reported found that ultrasound combined with microbubble treatment could effectively dissolve mesenteric microthrombosis and cerebral microvascular thrombosis,and improve the prognosis of rat model of microthrombi-induced stroke.These studies provided an important theorectical basis for ultrasound combined with microbubble treatment to dissolve coronary microthrombi.However,up to now,no related study have been reported.ObjectiveTo explore whether ultrasound combined with microbubble treatment could improve myocardial perfusion and improve the prognosis of coronary no reflow by dissolution of platelet-rich microthrombi.This study is divided into four parts:1)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi in vitro;2)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi in mesenteric arterioles;3)the influence of thrombotic age on the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi;4)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi and improving outcomes of coronary no reflow in canine models.Main experimental instruments:The German company SIEMENS(Siemens)model for color ultrasonic diagnostic apparatus Sequoia 512;installation of MCE program(enhanced pulse sequence,Contrast Pulse Sequencing,CPS);equipped with 4V1C probe(frequency of 2?4.0MHz)and 15L8 probe(frequency of 7?14.0MHz),plane thickness is 5mm,the highest mechanical index(mechanical index,MI)1.9.Main experimental reagents:Perflutren lipid microbubbles:provided by the pharmacy department of Nanfang Hospital of Southern Medical University,microbubble envelope formed by the lipid materials(according to a certain proportion of DPPC,DPPA and DPPE-PEG 5000),inert perfluoropane wrapped by lipid envelope,milky white appearance,the average particle size of microbubbles was measured by the Coulter particle count and particle size analyzer:2.11 ± 1.05 um,the concentration is about 3.02?5.18 × 109/ml.Recombinant tissue plasminogen activator(rtPA):it is provided by the pharmacy department of Nanfang Hospital of Southern Medical University.It is stored in the refrigerator of 4 degrees Celsius,fresh prepared with normal saline.2.Method of experiment:1)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi in vitroPreparation of platelet-rich microthrombi:After anesthesia,saphenous vein of dogs was punctured and blood was drawn.Platelet-rich plasma was obtained after low speed centrifugation;bovine thrombin solution was added into the plasma and placed at room temperature for 30 minutes to get platelet-rich thrombus.Platelet-rich thrombus was placed in the refrigerator in 4 degrees Celsius for about 3 hours,letting thrombus fully contracting;then the thrombus was placed in liquid nitrogen for about 5 minutes,followed by mechanical grinding to get microthrombi.The microthrombi which pass through 100 micron filter but remain on the 70 micron filter were harvested.The histological examination of microthrombi:after preparation of platelet-rich microthrombi,HE staining and scanning electron microscopy were carried out for the histological examination of the microthrombi.Establishment of in vitro thrombolysis device:In vitro thrombolysis device simulating coronary no reflow mainly has the following components:(1)the rubber tube of about 4 cm;(2)filter of about 40 microns diameter;(3)the upper end of rubber tube is connected with the infusion pump for microbubble infusion;(4)the side pores are connected to the pressure transducer and microthrombus suspension infusion pipeline respectively;(5)thrombolytic device was placed in constant temperature water bath;(6)adjusting the baseline pressure of the system to 0 mmHg before infusion of microthrombi suspension;(7)microthrombi suspension was infused into the system via the side pores and adjust the pressure to about 40 mmHg.Experimental groups:The in vitro experiment was randomly assigned to 5 groups:(1)control(CON)group;(2)ultrasound(US)group;(3)recombinant tissue type plasminogen activator(rtPA)group;(4)ultrasound combined with microbubble(US ?MB)group;(5)recombinant tissue plasminogen activator + ultrasound combined with microbubbles(rt-PA + US + MB)group.The comparison of thrombolysis rate:the rate of thrombolysis is reflected by the percentage pressure change,Delta P%:Delta P%=(P 0-P 30)/P 0*100%,in this experiment P 30 represents the systemic pressure after treatment and P 0 indicates the pressure in the system before treatment.2)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi in mesenteric arteriolesPreparation of mesenteric platelet-rich microthrombi model:rats were anesthetized,the mesentery was gengtly pulled out of the abdomen cavity and placed on the self-made acrylic platform.The mesenteric arterioles of appropriate diameter(about 100 microns)were selected with the help of microscope.Tiny filter paper(about 0.5 mm × 5 mm),soaked by 5%FeCl3 solution,was gently placed on top of the selected target vessels.After about 5 minutes the filter paper was removed,and the platelet-rich microthrombi formed after about 5 to 10 minutes.Histological examination of mesenteric microthrombi:microvascular thrombus was examined by electron microscopy and HE staining to determine the histological characteristics of microthrombus in mesenteric arteriole.Experimental groups:the rat models of mesenteric platelet-rich microthrombus was randomly assigned to 5 groups(each group was 12)the same as the first part.Evaluation of thrombolytic effect:in each group,before and after modeling before and immediately after treatment three time points through microscopic video image acquisition system,and compared between the rate of recanalization,recanalization rate(%)= recanalization rats in each group the number/total number X 100%.3)the influence of thrombotic age on the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombiPreparation of rat models of hindlimb microthrombi:Rats were anesthetized,followed by layer-by-layer separation into the abdominal cavity.The left and right iliac arteries were isolated.The proximal end of the right iliac artery was clamped and the PE catheter was inserted.Microthrombi suspension(about 0.1ml)was injected slowly through indwelling PE catheter to establish the rat models of hindlimb microthrombi.Experimental group:the rat model of acute posterior limb thrombus embolism was randomly assigned to 5 groups(5 rats in each group),the sam as the first part.Evaluation of thrombolysis effect:each group was sampled by acoustic contrast imaging at 6 hours after modeling and two hours after treatment,and the microvascular blood flow in each group was compared.4)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi and improving outcomes of coronary no reflow in canine modelsPreparation of canine coronary no reflow model:Cut the skin layer,muscular layer and pleura of mongrel dogs after tracheal intubation and mechanical ventilation into the chest along the fourth intercostal chest space on the left side.The heart was exposed and the left anterior descending(LAD)was isolated gently by blunt dissection for about 1-1.5 cm.The 20F catheter was inserted into LAD,followed by immediately heparinization(bolus injection of 80 units/kg after continuous intravenous infusion to maintain volume)to prevent the formation of thrombus in the puncture site and fixation with suture to prevent slippage.Platelet-rich microthrombi suspension was injected into LAD under the guidance of the contrast echo to make sure that myocardial perfusion defect is roughly the same at the baseline.Experimental group:the coronary micro thrombus embolism model was randomly assigned to 5 groups,each group was 5,and the experimental group was the same as the first part.Evaluation of thrombolytic effect:myocardial contrast echocardiography was performed in all groups at after modeling and 6 hours after treatment,and the microvascular blood flow in each group was compared.After the images were collected,the animals were killed and the infarcted area was measured by TTC staining,and the ELISA method was used to determine the myocardial necrosis marker(CK-MB,TnI).Result1)the efficacy of ultrasound combined with microbubble treatment on dissolving microthrombi in vitroHistological examination of platelet-rich microthrombi:HE staining showed that the diameter of platelet-rich microthrombi is about 100 microns,mainly composed of platelet-fibrin mesh formed trabecular structure,among which a small amount of scattered red blood cells can be seen.Scanning electron microscope showed that platelet-rich microthrombi were in about 100 microns,which were mainly composed of platelets and fibrin formed reticular structure,intersected with a few biconcave red cells.The rate of thrombolysis(P%):the difference between the US group and CON group of thrombolysis rate was not statistically significant;RT PA group,US+MB group and rtPA+US+MB group.The thrombolysis rate were higher than CON group,the difference was statistically significant(n=8,p<0.001);rtPA group,US+MB group and rtPA+US+MB group with a rate higher than that of group US,there are statistically significant difference(n=8,p<0.001);US+MB group and rtPA+US+MB group.The thrombolysis rate were higher than rtPA group,the difference was statistically significant(n=8,p<0.001);the difference of US+MB group and RT PA+US+MB group was not statistically significant.2)ultrasound combined with microbubbles in the treatment of platelet rich thrombocytopeniaThe characteristics of mesenteric tissue:platelet rich microthrombi were observed under light microscope showed HE staining,mesenteric platelet rich microthrombi diameter of about 100 microns,mainly composed of trabecular structure of platelet fibrin network composed of the middle trabecular structure of red blood cells can be seen in a few scattered.It was observed under scanning electron microscope that the diameter of the mesenteric platelet rich microthrombus was about 100 microns,mainly composed of platelets and a small amount of red blood cells.Comparison of thrombolysis rate:US group,rtPA group and CON group had no significant difference in thrombolytic rate,US+MB group and RT PA+US+MB group had higher thrombolysis rate than CON group,the difference was statistically significant(n=12,p<0.001).3)study on the difference of efficiency of ultrasound combined with microbubbles in the treatment of thrombocytopenia with different age of coagulationPlatelet rich microthrombi histological examination results:WMT 3H group and WMT 24h group of micro thrombosis were dominated by platelet fibrin network structure,micro thrombus(WMT 3H and WMT 24h group)the shrinkage degree and the clotting time is closely related to micro thrombus age longer,thrombus contraction is more obvious,the specific performance with coagulation age increases,the fibrin network density.In vitro thrombolytic rate:the thrombolytic rate in the WMT 24h group was lower than that in the WMT 3H group(n=8,P<0.01).Skeletal muscle blood volume:the microvascular blood volume of skeletal muscle was measured by acoustic contrast before and after treatment.It was reflected by the AI value of sound intensity at platform stage.There was no significant difference in AI value between WMT 3H group and WMT 24h group before treatment(n=5,p>0.05).At 6 hours after treatment,the value of AI decreased(n=5,p<0.01)in the WMT 24h group compared with the WMT 3H group.4)ultrasound combined with microbubble therapy to improve the prognosis of coronary artery free reflow through dissolution microthrombusHistological examination results:H E and immunohistochemical staining were visible coronary micro artery thrombus in the light microscope,histological micro thrombus is mainly composed of platelets and fibrin,suggesting that in vitro platelet rich thrombus by intracoronary injection after successful entry into the left anterior descending control myocardium,and ultimately blockage in the coronary micro intravascular.Compared with CON group,US+MB group,microvascular obstruction decreased(n=5,P = 0.001).Myocardial microvascular perfusion:the value of VI US group and CON group after treatment(myocardial blood volume),beta(microvascular blood flow velocity)and MBF value(myocardial blood flow)of the difference was not statistically significant(n = 5,P = 1);RT PA group,US group and RT + MB-PA + US + MB group VI,beta and MBF values were higher than CON group,the difference was statistically significant(n = 5,p<0.05);US group and RT + MB-PA + US + MB group VI,beta and MBF values were significantly higher than that of RT-PA group,the difference was statistically significant(n = 5,p<0.05);the difference of US group and RT + MB-PA + US + MB group VI,beta and MBF values was not statistically significant(N5,p= 1).The area of myocardial infarction:US group and CON group difference after treatment of myocardial infarction area was not statistically significant(n=5,P = 1);RT PA group,US group and RT + MB-PA + US + MB group of myocardial infarction area were higher than CON group,the difference was statistically significant(n=5,p<0.001);US + MB group and RT-PA + US + MB group of myocardial infarction area were significantly higher than that of RT-PA group,the difference was statistically significant;the differences of US group and RT + MB-PA+ US + MB group the myocardial infarct size was not statistically significant(P =0.142).Myocardial necrosis markers:US group and CON group after treatment,the difference of CK-MB,TnI was not statistically significant(n=5,P = 1);RT PA group,US group and RT + MB-PA + US + MB group CK-MB and TnI were higher than CON group,the difference was statistically significant(n=5,p<0.05)US;MB group and RT +-PA + US + MB group CK-MB and TnI was higher than that of RT-PA group,the difference was statistically significant(n=5,p<0.05);the difference of US group and RT + MB-PA + US + MB group,CK-MB TnI was not statistically significant(P = 0.142).The left ventricular systolic function:US group and CON group after treatment EF(left ventricular ejection fraction(FS),left ventricular fractional shortening)the difference was not statistically significant(n=5,P = 1);RT PA group,US group and RT + MB-PA + US + MB group,EF FS were higher than CON group,the difference was statistically significant(n=5,p<0.05);US group and RT + MB-PA + US + MB group EF and FS was higher than that of RT-PA group,the difference was statistically significant(n=5,p<0.05);the difference of US group and RT + MB-PA +US + MB group EF and FS was not statistically significant(P = 0.142).Conclusion:1)ultrasound combined with microbubble treatment can effectively dissolve the platelet-rich microthrombi in vitro,and its thrombolytic effect is better than that of rt-PA.2)ultrasound combined with microbubble treatment can effectively dissolve the mesenteric microvascular thrombi,and its thrombolytic effect is better than that of rt-PA.3)the efficiency of ultrasound combined with microbubble treatment in lying platelet-rich microthrombi decreased with the increase of microthrombi age.4)ultrasound combined with microbubble treatment can effectively solve platelet rich thrombus in coronary microvessels,thereby improving myocardial perfusion and improving the prognosis of coronary no reflow,and there is no obvious adverse reaction. |
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