| BackgroundAcute ischemic stroke is usually caused by occlusion of major artery in the brain,and rapid reperfusion therapy is the fundamental treatment of acute ischemic stroke.At present,treatment options include noninvasive intravenous thrombolysis or invasive catheter-based endovascular treatments:intra-arterial thrombolysis,rmechanical thrombectomy,balloon angioplasty and stenting.Although the endovascular treatments are able to achieve 70?90%rate of recanalization,which is approximately three times higher than intravenous thrombolysis only,high rate of recanalization does not directly correlate with good clinical outcomes.One important reason for this mismatch may be microcirculation no re-flow,a phenomenon in which major vessel recanalization does not result in adequate downstream reperfusion.The pathophysiologic mechanisms of no-reflow phenomenon are various,mainly including ischemic injury,reperfusion injury,and microembolization by thrombi.The occluded micro thrombi originate from upstream clot fragmentation and in situ microvascular thrombosis,which causemicrovascular obstruction,leading to small areas of ischemia,infarction,and peri-inflammation,all of which might worsen the clinical symptoms,increase risk of intracranial hemorrhage as well as mortality.Studies found that platelets and fibrin,which is namely platelet-rich thrombi,are the main componentsof the micro thrombus in microcirculation.Thus,dissolve the micro platelet-rich thrombi will bring great benefits to the clinical outcomes after acute ischemic stroke.Unfortunately,platelet-rich thrombus shows a certain degree resistant to thrombolytic agents,as proved by evidences that the lytic effect of thrombolytic agents on platelet-rich thrombus is less than erythrocyte-rich thrombus.Currently,there is still a lack of effective strategy to prevent or dissolve the micro platelet-rich thrombi.Although coupled with the latest generation of mechanical thrombectomy devices and thrombus aspiration,micro platelet-rich thrombi still could be found in the microvascular bed downstream from the large occluded artery.Antiplatelet agents might also be feasible as an adjuvant treatment after endovascular treatments,however,data regarding the safety/benefit of antiplatelet agents in acute ischemic stroke are very limited.Against this background,there is a clear need for an effective therapy to dissolve the micro platelet-rich thrombi that improves the clinical outcomes after acute ischemic stroke.Recent years,with the development of ultrasonic,ultrasound and contrast agent has become more and more widely and importantin clinical application.They not only have important scientific significance in diagnosis but also show attractive prospects in the treatment field.The basic theory of the mechanism in the treatment is the ultrasonic cavitation effect,which is endogenous or exogenous microbubble cavitation nuclei process a series of dynamic process,such as oscillation,expansion,contraction,implosion,and so onit is peculiar to the ultrasonic propagation in the liquid.Multiple in vitro and in vivo studies have demonstrated that ultrasound and microbubbles can readily dissolve thrombi without thrombolytic agents.However,many in vitro studies were mainly focus on investigation of large thrombus.In addition,the thrombus used in such studies was erythrocyte-rich thrombus,and its preparation was homogeneous,using spontaneously clotting non-anticoagulated venous blood or recalcified citrated venous blood,which is far from physiological thrombus formation in artery.Differed from erythrocyte-rich thrombus,the platelet-rich thrombus is mainly formed in artery,which often is the main occluded thrombi in many acute arterial ischemia diseases.There are also evidences from a series of in vivo studies using acute arterial thrombosis model showed that ultrasound and microbubbles,along with thrombolytic agents,could effectively dissolved large platelet-rich thrombi.However,to date,it is still short of direct evidences supporting that ultrasound and microbubbles dissolve the micro platelet-rich thrombi in vivo.In conclusion,the purpose of this study was to determine whether ultrasound combined with microbubbles is efficient in dissolvingmicroplatelet-rich thrombi and whether microvascular recanalization actually resulted in better outcome of acute ischemic stroke.ObjectiveTo investigate whether sonothrombolysis by ultrasound and microbubbles would be potential to dissolve microplatelet-rich thrombi and whether microvascular recanalization actually result in better outcome of acute ischemic stroke.1.In vitro thrombolysis of platelet-rich thrombi by ultrasound and microbubbles.2.In vivo thrombolysis of abdominal aorta platelet-rich thrombi by ultrasound and microbubbles.3.In vivo thrombolysis of microvascular platelet-rich thrombi by ultrasound and microbubbles.4.Therapeutic effect of ultrasound and microbubbles on acute ischemic stroke induced by micro platelet-rich thrombi.Materials and Methods1.MaterialsPrincipal Experimental instruments:Doppler ultrasonic machine with ultrasonic contrasting mode(Sequoia,Siemens Medical Systems,Mountain View,Calif).A I5L8 transducer,probe frequency of 7?14MHz,with contrast pulse sequencing technology,which was used forimage acquisition.A treatment transducer 4V1C,probe frequency of 2-4 MHz,which was used for thrombolysis with parameters as below:frequency by 2 MHz and Mechanical Index by 1.5.Experimental reagents:"perflutren protein-type microbubbles developed by Department of Pharmacology,Nanfang Hospital,Southern Medical University,with perfluoropropane as core gas,at approximately 2.0-4.25 × 10~9 microbubbles/ml.A mean of 2.07±1.13 ?m in diameter.Urokinase was bought from Department of Pharmacology,Nanfang Hospital,Southern Medical University.Preservation at 4?,dilute it by saline when it will be used.2.Methods(1)In vitro thrombolysis of platelet-rich thrombi by ultrasound and microbubbles Preparation of platelet-rich thrombi:Arterial whole-blood samples weredrawn from the abdominal aorta.Platelet-rich plasma were acquired by centrifugation of arterial whole-blood.Addition of thrombin to PRP led to the formation of platelet-rich thrombi,then the thrombi were incubated at 37? for 30 min.All the thrombus were then stored at 4? for 3 hours until use,which ensured complete thrombus maturation and retraction.To histological examination,platelet-rich thrombus were prepared to haematoxylin-eosin stain and scanning electron microscope scanning.24 platelet-rich thrombi were respectively randomized to 4 groups for 30 min sonothrombolysis:US+MB,US only,UK only and sham groups.During the in vitroexperiment,0.2ml lipid-encapsulated microbubbles and 20,000 IU/L urokinase was added into the flow system at 0.08 ml/min by infusion pump via a stopcock.The 15L8 ultrasound transducer was used for measuring the changes of thrombi,lumen diameter;the 4V1C ultrasound transducer was used for thrombus dissolution,with the ultrasound parameters used for dissolution is 2MHz,MI=1.9.Lumen diameter at the site of thrombus was rmeasured by B-mode ultrasound.All the data and images were measured before and after thrombolysis.Percentage thrombus dissolution was calculated by weighing the thrombus before and after thrombolysis.After thrombolysis,the PBS within the flow system was collected to measure the diameter of thrombus debris by Coulter Counter.(2)In vivo thrombolysis of abdominal aorta platelet-rich thrombi by ultrasound and microbubblesThe abdominal aorta platelet-rich thrombosis model was made by external application of ferric chloride.To histological examination of thrombus formed in abdominal aorta,the abdominal aorta was taken out at the time of thrombosis,and prepared forimmunohistochemistry and HE stain.24 mice were randomly divided into 4 groups for 30mins sonothrombolysis:US+MB,US only,UK only and sham groups.During the in vivoexperiment,0.2ml lipid-encapsulated microbubbleand 20,000 IU/kg urokinase both were added into the flow system at 0.08 ml/min by infiusion pump via a stopcock.The application of thrombolysis was performed as above.18 mice were randomly divided into 3 groups for 30minsthrombolysis:MI=1.9,MI=1.5 and MI=0.9.0.2ml lipid-encapsulated microbubble was injected at 0.08 ml/min via a stopcock.All the mice were treated with ultrasound and microbubbles.The application of sonothrombolysis was performed as above.Appearance of thrombi in the abdominal aorta was measured by B-mode ultrasound,as well as the flow velocity was measured by pulse ultrasonic Doppler(3)In vivo thrombolysis of microvascular platelet-rich thrombi by ultrasound and microbubbles.The micro artery thrombosis model was made by external application of ferric chloride.To histological examination of thrombus formed in micro artery,themesentery was taken out at the time of thrombosis,and prepared forimmunohistochemistry and HE stain.24 mice were randomly divided into 4 groups for 30mins sonothrombolysis:US+MB,US only,UK only and sham groups.The process of experiment and parameters were the same as above.To evaluate the thrombolytic effect,microscopic images were acquired at pre-thrombosis,post-thrombosis and post-thrombolysis time points by intravital microscopy.The thrombolytic effect of each group were compared with rate of arterial stenosis.(4)Therapeutic effect of ultrasound and microbubbles on acute ischemic stroke induced by micro platelet-rich thrombi.The rat acute ischemic stroke model was induced by injection of micro platelet-rich thrombi suspension through internal carotid artery.30 min after the injection of micro platelet-rich thrombi,histologyexamination was made by immunohistochemistry and HE stain to confirm thrombus formed in cerebral micro artery.24 h after the injection,histology examination was performed by HE stain to confirm acute cerebral ischemic infarction.Neurological deficits scoreing was tested at 24h after surgery.24 mice were randomly divided into 4 groups for 30mins thrombolysis:US+MB,US only,UK only and sham groups.The process of experiment and parameters were the same as above.Both TTC stain and cerebral magnetic resonance images were made at 24h after surgery.The therapeutic effect of each group were compared with rate of cerebral infarction.Results(1)In vitro thrombolysis of platelet-rich thrombi by ultrasound and microbubblesHE stain showed that the main compositions of platelet-rich thrombus were platelet and fibrin,which shown obvious coral-like framework of agglutinated platelets and fibrin mesh,interspersed with few intact erythrocytes.At higher magnification of SEM images,the platelet-rich thrombus showed a clear appearance of fibrin pores,a few of erythrocytes trapped in the fibrin network.Compared with Control group,dissolution rate of UK and US-MB groups were both significantly higher(P<0.001).Compared with UK group,dissolution rate of US-MB group was significantly higher(P<0.001).B-mode ultrasound showed that Lumen areas was significantly increased after treatment in the UK and US+MB groups.Compared with Control group,the increased rate of cross areasof both UK and US-MB groups were significantly higher(both P<0.001).Compared with UK group,the increased rate of cross areasof US-MB group was significantly higher(P<0.001).The size of thrombus debris from US-MB group in the solution were smaller than 8 ?m,in diameter.The MBs and its fragments were 0-3?m in diameter,while the thrombi debris were 3?8?m in diameter.(2)In vivo thrombolysis of abdominal aorta platelet-rich thrombi by ultrasound and microbubblesHE stain showed acoral-like framework of agglutinated platelet and fibrin,which indicated the thrombus formed in abdominal aorta is platelet-rich thrombus,and immunohistochemistry indicated that GPIIb/IIIa antibody was highly expressed.B-mode ultrasound results:?Right after thrombosis,each group was showed non-occluded platelet-rich thrombi.Right after thrombolysis,no changes were showed in both Control group and US group,the size of thrombi was diminished in UK group,and the thrombi were totally dissolved in US-MB group.?Right after thrombosis,each group was showed non-occluded platelet-rich thrombi.Right after thrombolysis,no change was showed in MI=0.9 group,and the thrombi were totally dissolved in both MI=1.9 and MI=1.5 groups.Ultrasonic Doppler results:?Right after thrombosis,each group was showed non-occluded platelet-rich thrombi.Blood flow velocity were decreased in all groups.Right after thrombolysis,blood flow velocity of Control and US groups were still the same as pre-thrombolysis,velocity of UK group was slightly enhanced,but the velocity of US-MB group was almost the same as normal.?Right after thrombosis,each group was showed non-occluded platelet-rich thrombi.Blood flow velocity were decreased in all groups.Right after thrombolysis,blood flow velocity of MI=0.9 group was still the same as before,but the velocity of both MI=1.9 and MI=1.5 groups were almost the same as normal.Results of average flow velocity at different time points:? There is no difference of average flow velocity among all the groups at pre-thrombosis and post-thrombosis time points.After the treatment,only the velocity of US-MB group was significantly higher than other groups(P<0.05).?here is no difference of average flow velocity among all the MI groups at pre-thrombosis and post-thrombosis time points.After the treatment,velocity of MI=O.9 group was significantly lower than other groups,but there is no difference between the MI=1.9 group and MI=1.5 group.(3)In vivo thrombolysis of microvascular platelet-rich thrombi by ultrasound and microbubblesHE stain showed acoral-like framework of agglutinatedplatelet and fibrin,which indicated the thrombus formed in micro artery is platelet-rich thrombus,and immunohistochemistry indicated that GPIIb/IIIa antibody was highly expressed.The formation of micro platelet-rich thrombi:the velocity of the target vessel was normal before thrombosis.After thrombosis,it showed that swelling and contraction of endothelial cells,and the vessel wall became thicker,the leukocytes rolled and adhered to the vessel walls,then the micro platelet-rich was formed and getting bigger,resulting in slow blood velocity.Effect of thrombolysis:Microscopically,there is no difference about lumen and the blood flow among all the groups,no thrombus was showed in the lumen.After the treatment,thrombi in US-MB group was totally dissolved and the blood flow was recovered,while the thrombi in other groups gradually became completely occluded thrombus,the blood flow stagnated and stationary blood cells were full of the lumen.(4)Therapeutic effect of ultrasound and microbubbles on acute ischemic stroke induced by micro platelet-rich thrombiHistology examination:30 min after the injection of micro platelet-rich thrombi,HE stain showed acoral-like framework of agglutinated platelet and fibrin,which indicated the thrombus formed in the cerebral micro artery is platelet-rich thrombus,and immunohistochemistry indicated that GPIIb/IIIa antibody was highly expressed.24h after the injection,HE stain showed that there were multiple small infarctions in the brain.Karyopyknosis,cell shrinkage,cytolysisand separated neurocytes were showed in the small ischemic zone.Scoreing of neurological deficits:24h after the treatment,rats in Control group and US group showed obvious neurological deficits,while rats in UK group showed less obvious symptom and the rats in US-MB group showed almost no deficits.Compared with Control group,score of UK and US-MB groups were both significantly lower(both P<0.01);Compared with UK group,score of US-MB groups was significantly lower(both P<0.05).TTC stain and magnetic resonance scanning:Dispersive small infarctions were showed in both TTC stain and MRI images.Compared with Control group,the amount and area of infarction were obvious lower in US-MB group.The infarction rate of US-MB group was significantly lower than Control group and US group(P<0.001),as well as that of UK group was also significantly lower than Control group and US group(P<0.001),however,compared with UK group,the infarction rate of US-MB group was evidently lower.Conclusions1.Ultrasound and microbubble can effectively dissolve the platelet-rich thrombi in the setting of in vitro and large artery and micro artery in vivo.2.Ultrasound and microbubbles can play a therapeutic role in acute ischemic stroke induced by micro platelet-rich thrombi,decreasing the infarction and improving the neurological deficits. |
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