Ultrasound with Microbubble Contrast Agent and Urokinase for Thrombosis

This study was aimed at assessing the effects of urokinase (UK) in combination with ultrasound and microbubbles in in vitro and in vivo thrombolytic therapy for the treatment of deep vein thrombosis (DVT). Thrombi with formation times of 1, 3, 7, 14 and 21 d were used for thrombolysis. Forty-five adult mongrel dogs were used to evaluate thrombosis in vivo. Both in vitro and in vivo analyses revealed that UK?+?microbubbles had the best effect among the combinations. Thrombolysis <7 d was more effective at a thrombolysis rate of about 50%, but the thrombolytic effect of thrombi >7 d was poor at thrombolysis rates <30%. Ultrasound?+?UK significantly increased the thrombolysis rate of thrombi <7 d. These results suggest that the combination of ultrasound with microbubble contrast agents and UK may have a synergistic effect on thrombolysis.     

Effect of acoustic conditions on microbubble-mediated microvascular sonothrombolysis

Ultrasound (US) mediated microbubble (MB) destruction facilitates thrombolysis of the epicardial coronary artery in acute myocardial infarction (AMI) but its effect on microvascular thromboemboli remains largely unexplored. We sought to define the acoustic requirements for effective microvascular sonothrombolysis. To model microembolization, microthrombi were injected and entrapped in a 40 μm pore mesh, increasing upstream pressure, which was measured as an index of thrombus burden. MBs (2.0 × 10⁶ MBs/mL) were then infused while pulsed US (1 MHz) was delivered to induce MB destruction immediately adjacent to the thrombus. Upstream pressure decreased progressively during US delivery, indicating a reduction in thrombus burden. More rapid and complete lysis occurred with increasing peak negative acoustic pressure (1.5 MPa > 0.6 MPa) and increasing pulse length (5000 cycles > 100 cycles). Additionally, similar lytic efficacy was achieved at 1.5 MPa without tPA as was at 1.0 MPa with tPA. This model uniquely provides a means to systematically evaluate multiple acoustic and microbubble parameters for the optimization of microvascular sonothrombolysis. This treatment approach for thrombotic microvascular obstruction may obviate the need for adjunctive rt-PA and could have important clinical cost and safety benefits.     

超声介导下携尿激酶靶向微泡在兔动脉内溶栓后的电镜观察

目的 探讨携尿激酶靶向微泡溶解兔动脉内血栓后下的形态学改变。方法 在24只新西兰大白兔单侧股动脉制作富含血小板的混合性血栓,分成4组,每组6只:①单纯超声照射组(US);②单纯尿激酶静脉注射组(UK);③超声照射+微泡造影剂+尿激酶静脉注射组(US+ M+ UK);④超声照射+RGDS微泡造影剂+尿激酶组(US+ R+ UK)。将RGDS、微泡造影剂(SonoVue)和尿激酶通过直接联合法配制成携尿激酶的靶向微泡,超声照射30 min,多普勒血流仪监测血流量变化评价溶栓效果,然后对其行HE染色、电镜检查。结果 US+ R+ UK组血流量完全恢复,实现完全再通（P＜0.001）,HE染色显示血栓完全溶解,扫描电镜检查示血栓的纤维网状结构破坏,透射电镜显示血栓的大量降解碎片。结论血栓纤维蛋白网状结构的破坏、纤维蛋白的溶解是携尿激酶靶向微泡在兔动脉内溶解血栓的主要电镜表现。     

尿激酶RGDS微泡在体溶栓的血流频谱特点与可能机制的实验研究

目的 用直接连接法制备的携尿激酶RGDS超声微泡溶解在体血栓,分析其血流频谱的特征性改变及可能的溶栓机制.方法 42只新西兰大白兔单侧股动脉制成富含血小板的混合性血栓模型,分成7组,每组6只:①单纯超声照射组(US);②超声照射+造影剂注射组(US+M);③单纯尿激酶静脉注射组(UK);④超声照射+微泡造影剂+尿激酶静脉注射组(US+M+UK);⑤超声照射+RGDS微泡造影剂组(US+R);⑥RGDS微泡造影剂+尿激酶静脉注射组(R+UK);⑦超声照射+RGDS微泡造影剂+尿激酶组(US+R+UK),进行在体溶栓治疗.将RGDS、微泡造影剂(SonoVue)和尿激酶通过直接联合法配制成6 ml的混悬液后经耳缘静脉注射,超声照射30 min,应用脉冲多普勒血流仪持续监测血流流速,对血流量和频谱变化特点进行分析.结果 120 min后US、UK、US+M、US+R及US+M+UK组血流量均未实现再通,R+UK组血流量实现部分再通,US+R+UK组血流量完全再通(P＜0.001).血流频谱特点为:未再通的各组在整个溶栓过程中血流频谱为持续小、低幅杂波;再通的R+UK组和US+R+UK组溶栓时血流频谱出现持续高幅、杂乱的波,US+R+UK组溶栓时超声波与血流频谱产生共振变化.结论 在体溶栓时通与不通的血流频谱各具特点,超声共振效应是促进溶栓的可能机制.     

Inertial Cavitation Ultrasound with Microbubbles Improves Reperfusion Efficacy When Combined with Tissue Plasminogen Activator in an In Vitro Model of Microvascular Obstruction

We have previously reported that long-tone-burst, high-mechanical-index ultrasound (US) and microbubble (MB) therapy can restore perfusion in both in vitro and in vivo models of microvascular obstruction (MVO). Addition of MBs to US has been found to potentiate the efficacy of thrombolytics on large venous thrombi; however, the optimal US parameters for achieving microvascular reperfusion of MVO caused by microthrombi, when combined with tissue plasminogen activator (tPA), are unknown. We sought to elucidate the specific effects of US, with and without tPA, for effective reperfusion of MVO in an in vitro model using both venous and arterial microthrombi. Venous- and arterial-type microthrombi were infused onto a mesh with 40-μm pores to simulate MVO. Pulsed US (1 MHz) was delivered with inertial cavitation (IC) (1.0 MPa, 1000 cycles, 0.33 Hz) and stable cavitation (SC) US (0.23 MPa, 20% duty cycle, 0.33 Hz) regimes while MB suspension (2 × 10⁶ MBs/mL) was infused. The efficacy of sonoreperfusion with these parameters was tested with and without tPA. Sonoreperfusion efficacy was significantly greater for IC + tPA compared with tPA alone, IC, SC and SC + tPA, suggesting lytic synergism between tPA and US for both venous- and arterial-type microthrombi. In contrast to our previous in vitro studies using 1.5 MPa at 5000 US cycles without tPA, the IC regime employed herein used 90% less US energy. These findings suggest an IC regime can be used with tPA synergistically to achieve a high degree of fibrinolysis for both thrombus types.     

超声联合全氟戊烷相变纳米粒治疗冠状动脉血栓栓塞的溶栓效率及安全性研究

目的 探索超声联合相变纳米粒靶向助溶人工血管内血栓的溶栓效率,评价其对于急性心肌梗死(AMI)冠状动脉血栓栓塞治疗的有效性及生物安全性。方法 取家兔颈总动脉血制备成统一规格约0.2ml条形血栓块;采用声振乳化法制备全氟戊烷(DDFP)脂质相变型纳米粒;实验分为三组：PBS对照组(A组),声诺维微泡组(B组),DDFP相变纳米粒组(C组);将各组血栓条块置入体外溶栓模拟循环装置的人工血管中,同时联合低强度聚焦超声仪(LIFU)靶向辐照。优化LIFU各项参数(时间、功率等),综合LIFU和纳米粒的安全性探索最佳溶栓条件。采用Malvern激光测量仪等检测相变纳米粒粒径等一般性能;称量溶栓前后血栓重量评价溶栓效率并观察比较血栓HE染色内部结构;CCK-8评价不同浓度纳米粒和不同强度LIFU对脐静脉内皮细胞(HUVECs)存活率的影响。结果 相变纳米粒粒径大小为(542.6± 28.4)nm;荧光显微镜示纳米粒液体相时结构稳定,大小均一;当LIFU的参数调整为辐照时间：20min、功率为6W时,纳米粒的相变转化率、溶栓效率及HUVECs细胞的存活率均较高;溶栓后C组血栓HE染色显示红细胞及血小板梁数量显著减少,质地松软,结构疏松。结论 LIFU联合DDFP相变纳米粒可有效助溶冠状动脉内血栓,这种治疗方式不仅高效,且具有较好的生物安全性,为AMI提供了一种新的治疗方式。     

靶向和非靶向微泡联合尿激酶超声溶栓的电镜表现

目的 比较靶向和非靶向微泡联合尿激酶超声溶栓的电镜表现。方法 将精氨酸-甘氨酸-天冬氨酸-丝氨酸片段（RGDS）与尿激酶（UK）以及超声微泡（SonoVue）通过机械振荡法,制备成靶向微泡。于30只新西兰大白兔单侧股动脉制备在体混合性血栓,并分为单纯超声辐照组（US组）、超声辐照+非靶向微泡造影剂+UK组（US+M+UK组）、超声辐照+靶向微泡造影剂+UK组（US+RGDS+UK组）。通过超声及多普勒血流仪观察溶栓效果,然后对股动脉离体标本行HE染色,并观察电镜表现。结果 溶栓20 min后,与US组和US+M+UK组比较,US+RGDS+UK组血流量明显恢复（P均<0.05）,US组与US+M+UK组比较差异无统计学意义（P均>0.05）。US+M+UK组HE染色显示管腔内充满血栓,血小板梁呈颗粒状、不致密,扫描电镜示粗大束状的胶原纤维上疏松附着少量细小纤维蛋白丝,大部分断裂;透射电镜示血栓大部分溶解为空泡状,可见白细胞或血小板降解的碎片。US+RGDS+UK组HE染色显示血栓完全溶解;扫描电镜示血栓的纤维网状结构被破坏,纤维蛋白完全的溶解;透射电镜示血栓降解为高电子密度的颗粒。结论 血栓结构的空泡化、纤维蛋白网架结构完全崩解和纤维蛋白的完全溶解是靶向微泡和UK联合溶栓的主要电镜改变。     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

New Insights into Mechanisms of Sonothrombolysis Using Ultra-High-Speed Imaging

Thrombotic arterial occlusion is the principal etiology for acute cardiovascular syndromes such as stroke, myocardial infarction and unstable angina. Exposing the thrombus to ultrasound and microbubbles facilitates thrombus disruption, making “sonothrombolysis” a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of such a strategy, and hence clinical translation, is constrained by an incomplete understanding of mechanisms by which ultrasound-induced microbubble vibrations disrupt blood clots. We posit that previously reported sonothrombolytic efficacy using inertial cavitation regimes was due, at least in part, to mechanical clot disruption by oscillating microbubbles. To test this hypothesis, we optically characterized lipid microbubble interactions with thrombus in the presence of ultrasound using a recently developed ultra-high-speed microscopy imaging system to visualize microbubble acoustic behaviors at megahertz frame rates. A microscope/acoustic stage designed for the system allowed an experimentally created thrombus and microbubbles to be insonified at a co-localized acoustic and optical focus during synchronized high-speed imaging. Under inertial cavitation conditions, large-amplitude microbubble oscillations caused thrombus deformation and pitting. Acoustic radiation forces (Bjerknes forces) further augmented microbubble-thrombus interaction. These observations suggest that a direct mechanical effect of oscillating lipid microbubbles on an adjacent thrombus may play a role in mediating clot disruption in the presence of specific ultrasound conditions.     

Improved Sonothrombolysis from a Modified Diagnostic Transducer Delivering Impulses Containing a Longer Pulse Duration

Although guided high-mechanical-index (MI) impulses from a diagnostic ultrasound transducer have been used in preclinical studies to dissolve coronary arterial and microvascular thrombi in the presence of intravenously infused microbubbles, it is possible that pulse durations (PDs) longer than that used for diagnostic imaging may further improve the effectiveness of this approach. By use of an established in vitro model flow system, a total of 90 occlusive porcine arterial thrombi (thrombus age: 3–4 h) within a vascular mimicking system were randomized to 10-min treatments with two different PDs (5 and 20 μs) using a Philips S5-1 transducer (1.6-MHz center frequency) at a range of MIs (from 0.2 to 1.4). All impulses were delivered in an intermittent fashion to permit microbubble replenishment within the thrombosed vessel. Diluted lipid-encapsulated microbubbles (0.5% Definity) were infused during the entire treatment period. A tissue-mimicking phantom 5 cm thick was placed between the transducer and thrombosed vessel to mimic transthoracic attenuation. Two 20-MHz passive cavitation detection systems were placed confocal to the insonified vessel to assess for inertial cavitational activity. Percentage thrombus dissolution was calculated by weighing the thrombi before and after each treatment. Percentage thrombus dissolution was significantly higher with a 20-μs PD already at the 0.2 and 0.4 MI therapeutic impulses (54 ± 12% vs. 33 ± 17% and 54 ± 22% vs. 34 ± 17%, p < 0.05 compared with the 5-μs PD group, respectively), and where passive cavitation detection systems detected only low intensities of inertial cavitation. At higher MI settings and 20-μs PDs, percentage thrombus dissolution decreased most likely from high-intensity cavitation shielding of the thrombus. Slightly prolonging the PD on a diagnostic transducer improves the degree of sonothrombolysis that can be achieved without fibrinolytic agents at a lower mechanical index.     

3-D measurement of body tissues based on ultrasound images with 3-D spatial information

In this study, we developed a new method to perform 3-D measurements between the recorded B-scans using the corresponding spatial location and orientation of each B-scan, without the need to create a 3-D volume. A portable ultrasound (US) scanner and an electromagnetic spatial locator attached to the US probe were used. During data collection, the US probe was moved over the region-of-interest. A small number of B-scans containing interesting anatomical information were captured from different body parts and displayed in a 3-D space with their corresponding locations recorded by the spatial locator. In the B-scan planes, the distance between any two points, as well as the angle between any two lines, could be calculated. In validation experiments, three distances and three angles of a custom-designed phantom were measured using this method. In comparison with the results measured by a micrometer, the mean error of distance measurement was −0.8 ± 1.7 mm (−2.3 ± 3.6%) and that of angle measurement was −0.3 ± 2.9° (−0.1 ± 4.1%). The lengths of the first metatarsals and the angles between the first metatarsals and the middle part of the tibias of three subjects were measured in vivo using magnetic resonance imaging (MRI) and the US method by two operators before and after MRI scanning. The overall percentage differences of the length and angle measurements were 0.8 ± 2.2% and 2.5 ± 3.6%, respectively. The results showed that this US method had good repeatability and reproducibility (interclass correlation coefficient values > 0.75). We expect that this new method could potentially provide a quick and effective approach for the 3-D measurement of soft tissues and bones in the musculoskeletal system.     

Enhancement of fibrinolysis by EF6265 [(S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic acid], a specific inhibitor of plasma carboxypeptidase B

Plasma procarboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is converted by thrombin into the active enzyme, carboxypeptidase B (CPB)/activated TAFI. Plasma CPB down-regulates fibrinolysis by removing carboxy-terminal lysines, the ligands for plasminogen and tissue-type plasminogen activator (tPA), from partially degraded fibrin. To target thrombosis in a new way, we have identified and optimized a phosphinic acid-containing inhibitor of CPB, EF6265 [(S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino) propyl]hydroxyphosphinoyl]methyl]heptanoic acid] and determined both the pharmacological profile and pathophysiological role of CPB in rat thrombolysis. EF6265 specifically inhibited plasma CPB activity with an IC(50) (50% inhibitory concentration) of 8.3 nM and enhanced tPA-mediated clot lysis in a concentration-dependent manner. EF6265 decreased detectable thrombi (percentage of glomerular fibrin deposition; control, 98 +/- 1.1; EF6265, 0.1 mg/kg, 27 +/- 9.1) that had been generated by tissue factor in a rat microthrombosis model with concomitant increases in plasma D-dimer concentration (control, <0.5 microg/ml; EF6265, 0.1 mg/kg, 15 +/- 3.5 microg/ml). EF6265 reduced plasma alpha2-antiplasmin activity to a lesser extent than tPA. In an arteriovenous shunt model, EF6265 (1 mg/kg) enhanced exogenous tPA-mediated thrombolysis under the same conditions that neither EF6265 nor tPA (600 kIU/kg) alone reduced thrombi. EF6265 (1 and 30 mg/kg) did not affect the bleeding time in rats. Moreover, it did not prolong the bleeding time evoked by tPA (600 kIU/kg). These results confirm that circulating procarboxypeptidase B functions as a fibrinolysis inhibitor's zymogen and validates the use of CPB inhibitors as both an enhancer of physiological fibrinolysis in microcirculation and as a novel adjunctive agent to tPA for thromboembolic diseases while maintaining a small effect on primary hemostasis.     

诊断超声联合微泡空化溶栓治疗中心静脉导管堵塞的体外实验观察

目的 探讨体外模型评价诊断超声联合微泡空化溶栓治疗中心静脉导管堵塞的效果.方法 建立中心静脉置管堵塞的体外模型后,在模拟人体血管系统中输入微泡混悬液,按诊断超声分为低机械指数组,中机械指数组,高机械指数组(每组各重复20次)进行治疗.观察比较各组导管再通率及血栓溶解率.结果 高机械指数组导管再通率及血栓溶解率较其余两组明显增加(均P＜0.05).,结论 体外实验结果表明,高机械指数超声联合微泡空化效应能有效解决中心静脉导管置管堵塞问题.     

载组织型纤溶酶原激活物及精-甘-天-丝氨酸四肽超声微泡造影剂的实验研究

目的 制备一种同时携带组织型纤溶酶原激活物(Tissue plasminogen activator,tPA)与精-甘-天-丝氨酸四肽(RGDS)靶向配体的新型脂质超声微泡造影剂.方法 以冷冻干燥法制备包载tPA的脂质超声微泡造影剂并应用共价键桥连剂法结合RGDS配体.光镜及荧光显微镜观察其形态;测量其粒径、表面电位、pH值;以ELISA法测定tPA包封率;流式细胞仪检测RGDS携带率;琼脂糖纤维蛋白平板法检测其纤溶活性;以该造影剂对正常家兔肝脏进行静脉声学造影,绘制时间-强度曲线,检测其成像功能.结果 微泡膜上同时携带tPA和RGDS,浓度(7～9)×109/ml,粒径(2.08±0.93) μm,表面电位-51.3,pH值5.58,tPA包封率(81.12±2.44)%,RGDS携带率(94.49±6.19)%.微泡在超声辐照条件下显示溶栓活性.声学造影后,该造影剂能明显增强实验兔肝实质回声.结论 以冷冻干燥法和共价键桥连剂结合法能成功制备同时包载tPA并携带RGDS的脂质超声微泡造影剂,将为血栓的靶向释药促溶治疗提供一条新的可行之路.     

制备RGDS/rt-PA双负载靶向微泡并初步评价其靶向性溶栓效果

目的 制备RGDS/rt-PA双负载靶向微泡,分析其对富血小板血栓（PRT）的靶向性和溶栓效果。方法 制备不同剂量梯度的RGDS及rt-PA单负载靶向微泡,以其最佳结合剂量、按照不同加入顺序（先加入RGDS,再加入rt-PA;先加入rt-PA,再加入RGDS;将RGDS及rt-PA混匀后加入）分别制备RGDS/rt-PA双负载靶向微泡,检测其结合率。比较裸微泡、单负载及双负载微泡的物理特性（直径、浓度及pH值）,不同剂量梯度的同一配体与微泡的单负载结合率,同一剂量梯度的不同配体与微泡的单负载结合率,加入顺序不同的配体与微泡的双负载结合率。制备体外PRT模型,检测RGDS/rt-PA双负载靶向微泡的声学显像特征、靶向溶栓能力。结果 不同微泡间的物理特性差异均无统计学意义（P均>0.05）。不同剂量梯度的rt-PA、RGDS与微泡单负载结合率的差异均有统计学意义,同一剂量梯度的rt-PA与微泡单负载的结合率均低于RGDS （P均<0.05）。加入顺序不同的配体与微泡的双负载结合率的差异有统计学意义（F=16.090,P=0.004）。将RGDS/rt-PA双负载靶向微泡注入血栓模型管腔后,超声可见均匀分布的点状高回声,血栓边界回声明显增强;扫描电镜下可见纤维蛋白网状结构明显破坏、纤维束断裂成细沙状,并可见变形融合的血细胞。结论 RGDS/rt-PA双负载靶向微泡性质稳定,与配体的结合率高,声学显像特征好,具备一定的PRT靶向性及溶栓能力。     

超声破坏微泡联合粒细胞集落刺激因子促进大鼠心肌血管新生的实验研究

目的 探讨超声破坏微泡联合粒细胞集落刺激因子(G-CSF)促进大鼠心肌血管新生的有效性.方法 40只健康成年Wistar大鼠随机分为4组.A组在超声微泡治疗前一天行G-CSF皮下注射预处理;B组仅行超声微泡治疗;C组仅行G-CSF皮下注射;D组为对照.于治疗后2周取材,HE染色光镜观察心肌结构变化,免疫组化法检测心肌组织中VEGF及CD34表达,评定心肌血管新生疗效.结果 A组心肌中有大量VEGF和CD34表达,新生血管较多;B组心肌中有部分VEGF和CD34表达,新生血管相对较少;C组及D组仅有极少量VEGF和CD34表达,未见明显新生血管.结论 超声微泡可刺激心肌内源性VEGF分泌,促进心肌血管新生,G-CSF能明显增强其血管新生作用.     

血管内超声导管联合微泡增强尿激酶溶栓的实验研究

目的探讨EKOS■超声导管联合血栓内注射微泡增强尿激酶体外溶栓治疗效果。方法采用水听器法测量EKOS■超声导管峰值负压。将90份体外牛血血栓样本根据其实验方法随机分为6组,分别为:超声+微泡+尿激酶联合组(联合组)、超声+尿激酶组(US+UK组)、超声+微泡组(US+MB组)、超声组(US组)、尿激酶组(UK组)和对照组,每组15份,称取各组溶栓前后的血栓质量,计算并比较其溶栓率。对溶栓处理后各组血栓行HE染色并于光镜下观察红细胞和纤维蛋白的分布情况。结果EKOS■超声导管的峰值负压在0.4~6.4 MPa,呈无规律动态变化。联合组溶栓率为(41.9±4.5)%,与US+UK组(42.0±3.3)%接近,均显著高于其余各组,差异均有统计学意义(均P<0.05);US+MB组溶栓率为(28.4±3.3)%,UK组为(27.8±3.1)%,两组比较差异无统计学意义,但均高于US组(23.9±3.0)%和对照组(17.5±3.7)%,差异均有统计学意义(均P<0.05)。光镜下见联合组和US+UK组与EKOS■超声导管接触处的血栓较多崩解,红细胞分布较其余各组更稀疏。结论EKOS■超声导管可明显提高尿激酶对体外牛血血栓的溶栓率,在此基础上增加微泡对溶栓率无明显提高。     

Correlation between inertial cavitation dose and endothelial cell damage in vivo

Previous in vivo studies have demonstrated that vascular endothelial damage can result when vessels containing gas-based microbubble ultrasound contrast agent (UCA) are exposed to MHz-frequency pulsed ultrasound (US) of sufficient pressure amplitudes, presumably as a result of inertial cavitation (IC). The hypothesis guiding this research was that IC is the primary mechanism by which the vascular endothelium (VE) is damaged when a vessel is exposed to pulsed 1-MHz frequency US in the presence of circulating UCA. The expectation was that a correlation should exist between the magnitude and duration of IC activity and the degree of VE damage. Rabbit auricular vessels were exposed in vivo to 1.17-MHz focused US of variable peak rarefaction pressure amplitude (1, 3, 6.5 or 9 MPa), using low duty factors (0.04% or 0.4%), pulse lengths of 500 or 5000 cycles, with varying treatment durations and with or without infusion of a shelled microbubble contrast agent. A broadband passive cavitation detection system was used to measure IC activity in vivo within the targeted segment of the blood vessel. The magnitude of the detected IC activity was quantified using a previously reported measure of IC dose. Endothelial damage was assessed via scanning electron microscopy image analysis. The results supported the hypothesis and demonstrate that the magnitude of the measured IC dose correlates with the degree of VE damage when UCA is present. These results have implications for therapeutic US-induced vascular occlusion.     

体外超声波溶解血栓实验及血栓凝龄对溶栓效应的影响的研究

以超声波及／或尿激酶对健康人静脉血形成的不同凝龄体外血栓行溶栓实验。结果显示，超声波具有优于尿激酶的溶栓效应（Ｐ＜０．０５）并可强化尿激酶的溶栓效应。超声波加用尿激酶较单纯超声波溶栓效果更佳（Ｐ＜０．０５）。超声波的溶栓效应与血栓凝龄有关，血栓愈陈旧，则超声波溶栓效应愈差（单纯超声波组ｒ＝－０．７４１，ｎ＝９６；超声波加尿激酶组ｒ＝－０．８０２，ｎ＝９６）。结论为，超声波具有肯定的溶栓效应和强化尿激酶溶栓效应，血栓形成时间愈短，超声波溶栓效果愈佳     

Microbubble Stability is a Major Determinant of the Efficiency of Ultrasound and Microbubble Mediated in vivo Gene Transfer

In the search for an efficient nonviral gene therapy approach for the treatment of genetic disorders of cardiac and skeletal muscle such as Duchenne muscular dystrophy, ultrasound in combination with contrast enhancing microbubbles has emerged as a promising tool for safe and site-specific enhancement of gene delivery. Indeed, microbubble-enhanced gene transfer (MBGT) has been investigated for a wide variety of target sites using both reporter and therapeutic genes. Although a range of different microbubbles have been used for MBGT studies, comparison of their efficiencies is difficult because microbubble concentration and the ultrasound settings used for the application vary considerably. Only two studies to date have attempted a direct comparison of commercially available microbubbles, and both concluded that not all microbubbles show the same efficiencies with MBGT. Thus far, the reason for this is unclear. Here, the efficiency of three commercially available microbubbles—Optison, SonoVue and Sonazoid—was analyzed to understand the microbubble properties that are important for their function as an effective enhancer for gene transfer in vivo. In this study, plasmid DNA or antisense oligonucleotides were delivered by systemic injection with MBGT, focused on the heart. Gene delivery to the heart with equalized concentrations of the three microbubbles showed that Optison and Sonazoid are more efficient in MBGT compared with SonoVue, which showed the weakest gene transfer to the myocardium. Investigations into the properties of these microbubbles showed that size and shell composition did not directly influence MBGT, whereas the microbubbles with increased stability in an ultrasound field showed better MBGT results than those degrading faster. Moreover, the microbubble concentration used for MBGT was also found to be an important factor influencing the efficiency of MBGT. In conclusion, the stability of a microbubble was shown to be a major influential factor for its performance in MBGT, as is the concentration of the microbubbles used. These findings emphasize the importance of detailed investigations into the properties of microbubbles to allow the production of a microbubble specifically designed for optimum performance with MBGT. (E-mail: d.wells@imperial.ac.uk)