| OBJECTIVE:Ultrasound-mediated microbubble cavitation has been shown to improve local perfusion in irradiated areas of limbs or myocardial ischemic tissue in animal models,which is termed sonoperfusion.For the mechanism of sonoperfusion to increase local blood flow,relevant studies have confirmed that the cavitation effect of microbubbles can increase local perfusion by molecular pathways such as NO signaling pathway and ATP signaling pathway.However,most of the previous studies focus on the chronic ischemia model.In this study,acute rat hindlimb ischemia model was used to preliminarily study the effects of sonoperfusion on increasing local blood flow of acute ischemia tissue and its underlying signaling mechanism,in order to apply to early revascularization of peripheral arterial disease and acute myocardial infarction.METHODS:1.Animal models establishmentThe acute ischemia model of hindlimbs was established by randomly unilateral ligation of the external iliac artery for 10 minutes in male SPF SD rats(weighing 180-250g,aging 6-8 weeks).2.Quantitative evaluation of tissue perfusion by real-time contrast-enhanced ultrasound perfusion imagingThe jugular vein was indwelled,and the lipid microbubbles at a concentration of(2.98±0.15)× 10" 9/m L were slowly infused intravenously with a micro pump at a rate of 400 μL/min.Baseline imaging of low-mechanical index contrast ultrasound technique of both hindlimbs in rats used Sequoia 512 ultrasound system(SIEMENS,17L5 probe).Real-time contrast-enhanced ultrasound perfusion imaging was performed on both hindlimbs,after 10 minutes of continuous treatment(intravenous infusion of microbubble suspension at a rate of 600 μL/min during the treatment,the mechanical index was 1.9,each time high mechanical index destruction(flash)last for 3 seconds and triggered at 4 second intervals).3.Effect of ultrasound-mediated microbubble cavitation on blood perfusionRats were randomly divided into three groups(n=8 for each group):intermittent high mechanical index pulse combined with lipid microbubbles group(US+MB group),US alone group(US group)and MB alone group(MB group).Real-time ultrasound contrast perfusion imaging of both hind limbs was performed before modeling,after modeling,and after treatment.4.Duration of acute effects of sonoperfusionRats were randomly divided into 3 groups(n=9 for each group):US+MB group,US group and MB group.Real-time ultrasound contrast perfusion imaging for both hindlimbs were performed before and after modeling,and then after 10 minutes of treatment,contrast ultrasound perfusion imaging for both hindlimbs were performed immediately(0 minute)and at 5,10,15,20 and 25 minutes,respectively.5.Preliminary Study on molecular mechanism of sonoperfusionTwenty-seven SD rats were randomly divided into as ischemic cavitation group,ischemic cavitation+L-NAME group,and blank control group(n=9 for each group),among which Nω-nitro-L-arginine methyl ester hydrochloride(L-NAME)was an eNOS inhibitor and was pre-treated at a rate of 75 ug/kg IP 30 minutes before cavitation in the ischemic cavitation+L-NAME group.After modeling and pretreatment,the rats in the ischemic cavitation group,ischemic cavitation+L-NAME group were exposed to the treatment of ultrasound combined with microbubbles for 10 minutes.The blank control group were treated with no intermittent high mechanical index pulse and no microbubbles for 10 minutes.After treatment,contrast ultrasound perfusion imaging was performed immediately,and muscles of ischemic hindlimbs of rats at the same position of were taken.Then the tissue protein supernatant was extracted and isolated,and the concentration of p-eNOS was analyzed by an ELISA kit.RESULTS:1.Establishing an acute ischemia model of hindlimbs in ratsUltrasound contrast imaging showed that the blood flow perfusion of the hindlimb was reduced by 50%-70%(the microbubble filling area of the plateau was reduced by more than 50%-70%compared with the base map),indicating that the acute hindlimbs ischemia model was established successfully.2.Effect of ultrasound-mediated microbubble cavitation on blood perfusionAfter treatment,in the US+MB group,microvascular blood volume(A:41.824±7.836 vs 69.527 ± 6.570,P=0.000),and microvascular blood flow(MBF:6.451 ±1.021 vs 13.078 ± 1.967,P=0.000)were significantly increased in ischemic hindlimbs,while microvascular flux rate(β:0.156±0.0249 vs 0.193±0.0414,P=0.078)increased but it was not statistically significant.The US group showed an trend of improvement,but it was not statistically significant(P=0.311);and there was no change in the MB group(P=0.767).The improvement in tissue perfusion(A×β)is mainly based on the increase of A.3.Duration of acute effects of sonoperfusionThe acute effect of sonoperfusion worked and peaked immediately after treatment(P<0.05),then gradually weakened and disappeared after 25 minutes(P>0.05),while the US group peaked at 5 minutes after treatment(P<0.05)and almost disappeared after 15 minutes(P>0.05),but there was almost no change in the MB group after treatment(P>0.05).4.Preliminary Study on molecular mechanism of sonoperfusionInhibitory studies showed that the concentration of p-eNOS in ischemic hindlimbs was significantly increased after cavitation,but it was significantly inhibited by L-NAME with the inhibition of eNOS(P<0.05).CONCLUSION:Sonoperfusion can increase local perfusion in the irradiated area of acute ischemia tissue mainly by the increase of microcirculation perfusion,which mainly based on the growth of A rather than β.These effects work im mediately and can transiently reverse hindlimb acute ischemia tissue in rats for 25 minutes.After 25 minutes,the effect gradually weakened to disappear.The eNOS/NO signaling pathway is also a critical pathway for the acute effect of sonoperfusion.As a non-invasive treatment technique,sonoperfusion is expected to be applied clinically in the early revascularization in acute myocardial infarction and peripheral arterial disease. |
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