Ultrasound exposure can increase the membrane permeability of human neutrophil granulocytes containing microbubbles without causing complete cell destruction

Activated polymorphonuclear neutrophil (PMN) granulocytes can bind and subsequently phagocytose microbubbles used as ultrasound (US) contrast agents. The purpose of the present study was to assess insonation effects on cell membrane integrity and metabolic activity of activated PMN. Furthermore, we investigated whether or not there is an acoustic threshold at which insonation of PMN results in increase of membrane permeability without causing complete cell destruction. PMN isolated from healthy volunteers were activated with phorbol myristate acetate (PMA) for 15 min to allow phagocytosis of albumin and lipid microbubbles and were subsequently exposed to US with a mechanical index between 0.15 and 1.8. Apoptosis, loss of membrane integrity and formation of cell fragments were evaluated by measurement of lactate dehydrogenase leakage and by double staining with annexin V and propidium iodide, using flow cytometry. Neutrophil superoxide anion generation was measured photometrically. Insonation of activated PMN in the presence of microbubbles amplified apoptosis and lactate dehydrogenase leakage and induced loss of membrane integrity and complete cell destruction with increasing acoustic pressures. The bioeffects observed by insonation with high mechanical indices (1.0 to 1.8), and particularly the formation of cell fragments, were significantly more pronounced in the presence of albumin microbubbles. Insonation in the presence of lipid microbubbles increased cell membrane permeability, but caused significantly less cell destruction and left the metabolic activity of activated PMN uninfluenced. Thus, both albumin and lipid microbubbles induce apoptosis and membrane injury during insonation of activated PMN. However, insonation in the presence of lipid microbubbles seems to influence cell viability to a smaller extent. This could be of advantage in the setting of US-guided local drug delivery. In this setting, increase of membrane permeability may allow bioactive substances to enter into cells, which survive the US treatment, and specifically modify their function.     

动脉血栓靶向超声显像的体内实验研究

目的探讨白蛋白血栓靶向超声造影剂体内增强动脉血栓超声显像的效果.方法采用FeCl3溶液诱发30只健康新西兰大白兔腹主动脉非梗阻性新鲜血栓形成;经耳缘静脉团注0.04 ml/kg的白蛋白超声造影剂进行超声造影,分为靶向组(20只)和非靶向组(10只);采用视觉观察评价血栓显像增强效果.结果 29只(29/30)兔模型建立成功;造影后视觉观察,靶向组18例达到2级增强效果,1例为1级增强效果,1例死亡;非靶向组仅2例达到1级增强效果,两组比较差异显著(P<0.01).结论白蛋白血栓靶向超声造影剂可显著增强兔腹主动脉内新鲜血栓超声显像效果.     

诊断超声联合微泡对兔VX2肿瘤的血流增强效应

目的探讨不同机械指数(MI)的诊断超声联合微泡对兔VX_2肿瘤的血流增强效应。方法选取健康雄性新西兰白兔40只,采用单侧大腿内侧瘤组织块接种法接种VX_2肿瘤,造模成功后将其随机分为实验1组(MI=0.3)、实验2组(MI=0.7)、实验3组(MI=1.4)及对照组,每组各10只。抽取0.2 ml"脂氟显"用5.0 ml生理盐水稀释后经耳缘静脉通道匀速推入,同时分别经不同机械指数辐照肿瘤,对照组予以超声假照,时间均为5 min。储存治疗前后动态造影图像,并用造影分析软件分析,记录峰值强度(PI)和曲线下面积(AUC)。结果实验1组、实验3组治疗前后PI比较差异均有统计学意义(P=0.028、0.018),实验2组、对照组治疗前后PI比较差异无统计学意义(P=0.994、0.978);实验3组治疗前后AUC值比较差异有统计学意义(P=0.009),实验1、2组及对照组治疗前后AUC值比较差异均无统计学意义(P=0.099、0.497、0.898)。结论低能量诊断超声(MI=0.3)联合微泡可丰富兔VX_2肿瘤血供,高能量诊断超声(MI=1.4)可减少血流灌注。     

诊断超声介导微泡提高肝纤维化通透性及促进基因传递

目的 探讨诊断超声联合微泡对肝纤维化组织通透性的影响及其介导基因转染肝纤维化大鼠的有效性。方法 采用二甲基亚硝胺(DMN)法建立大鼠肝纤维化模型,80只大鼠在建模第4周末随机分为:模型对照组、单纯微泡组、单纯超声组和诊断超声联合微泡组。分别进行肝纤维化微血管通透性实验和基因转染实验,采用激光共聚焦显微镜观察伊文思蓝(EB)在肝纤维化组织内分布情况,同时定量检测肝纤维化组织内EB的含量,评估不同分组微血管通透性。荧光显微镜下观察含增强型绿色荧光蛋白报告基因的质粒转染大鼠肝纤维化模型的基因表达情况。结果 激光共聚焦显微镜显示诊断超声联合微泡组纤维化肝实质内可见明显的EB红色荧光。诊断超声联合微泡组纤维化肝组织中EB含量明显高于其他3组(P<0.05)。荧光显微镜下观察,相比其余3组,诊断超声联合微泡组增强型绿色荧光蛋白最多,基因转染效率最高。结论 诊断超声联合微泡在提高纤维化肝脏微血管通透性的同时可促进基因传递。     

微泡激励的超声空化阻断正常肝血流灌注的初步研究

目的探讨采用脉冲式超声激励微泡空化阻断兔正常肝脏血流的可行性及阻断的病理改变。方法健康新西兰大白兔24只随机分成3组,分别是超声微泡组、单纯超声组、假照组。经兔耳缘静脉注射脂质微泡,剂量0.1 ml/kg;同时超声治疗头垂直辐照兔肝脏300 s,超声能量以脉冲式发射,频率1.2 MHz,平均声强0.89 W/cm~2。靶区治疗前后进行超声造影检查和定量分析。最后,获取该区域肝组织标本,行病理学检查。结果超声微泡组治疗后肝组织血流灌注基本消失,而单纯超声组、假照组治疗前后无明显变化。超声微泡组治疗前后肝实质平均灰阶值(GSV)分别为115.27±3.8和1.16±0.7,治疗后肝实质GSV显著低于治疗前(P0.01),单纯超声及假照组治疗前后GSV无显著差别。病理检查,超声微泡治疗组肝组织出现大片充血、出血、血栓形成等。结论微泡增强的脉冲式超声空化可以造成正常肝的血流灌注暂时性阻断或者显著下降,阻断机制可能是肝实质出血、水肿和血栓形成。     

Quantification of microbubble destruction of three fluorocarbon-filled ultrasonic contrast agents

The assessment of myocardial blood velocity using ultrasonic contrast agents is based on the premise that the vast majority of contrast microbubbles within a myocardial region can be destroyed by an acoustic pulse of sufficient magnitude. Determination of the period of time after destruction that a region of myocardium needs to reperfuse may be used to assess myocardial blood velocity. In this study, we investigated the acoustic pressure sensitivity of three solutions of intravenous fluorocarbon-filled contrast agents and the magnitude of acoustic pulse required to destroy the contrast agent microbubbles. A novel tissue-mimicking phantom was designed and manufactured to investigate the relationships between mean integrated backscatter, incident acoustic pressure and number of frames of insonation for three fluorocarbon-filled contrast agents (Definity(R), Optison(R), and Sonazoid(R), formerly NC100100). Using a routine clinical ultrasound (US) scanner (Acuson XP-10), modified to allow access to the unprocessed US data, the contrast agents were scanned at the four acoustic output powers. All three agents initially demonstrated a linear relationship between mean integrated backscatter and number of frames of insonation. For all three agents, mean integrated backscatter decreased more rapidly at higher acoustic pressures, suggesting a more rapid destruction of the microbubbles. In spite of the fact that there was no movement of microbubbles into or out of the beam, only the results from Definity(R) suggested that a complete destruction of the contrast agent microbubbles had occurred within the total duration of insonation in this study.     

伊文思蓝定量评估超声造影对鼠胎盘屏障通透性影响的实验研究

目的探讨不同机械指数诊断性超声造影对胎盘屏障通透性的影响。方法怀孕14～16d（孕中期）清洁级SD鼠60只,经尾静脉注射剂量为1ml/kg的“声诺维”超声造影剂,在不同机械指数下（MI：0.13,1.0,1.4）超声辐照孕鼠子宫,辐照时间（连续5min,间歇10s）,分光光度法测定胎盘及胎鼠组织内伊文思蓝（EB）含量。结果不同机械指数下的超声造影,胎鼠组织内EB含量与对照组相比差异无统计学意义。结论高机械指数及低机械指数诊断超声造影均不会导致孕鼠胎盘屏障通透性增加。     

In vitro platelet activation by an echo contrast agent.

OBJECTIVE: We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. METHODS: Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5-75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 microL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. RESULTS: The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). CONCLUSIONS: In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.     

Effects of transcranial ultrasound and intravenous microbubbles on blood brain barrier permeability in a large animal model

We sought to determine whether transtemporal-applied 1-MHz ultrasound-induced microbubble destruction may be a safe method of transiently altering blood brain barrier (BBB) permeability for drug delivery in a large animal model. Endothelial cells are an integral component of the BBB but also prevent passage of potentially therapeutic drugs. Ultrasound-mediated destruction (UMD) of microbubbles has been shown to disrupt this barrier in small animals when ultrasound is delivered through bone windows. However, the effects of temporal bone attenuation and scattering in a large animal may limit the clinical application of such a technique. Twenty-four pigs were studied. One-MHz pulsed-wave ultrasound at 2.0 W/cm(2) (20% duty cycle) across the temporal bone was applied for 30 min after intravenous injections of either albumin-coated perfluorocarbon microbubble (PESDA, 8 pigs), lipid-encapsulated perfluorocarbon microbubbles (LEMB, 8 pigs) or ultrasound alone (8 pigs). BBB leak was quantified at 30 and 120 min after insonation using Evans blue. Serial magnetic resonance imaging (MRI) was performed in nine of the pigs (3 for each group) to quantify Gadolinium leak within the parenchyma. Peak negative pressures decreased ten-fold when ultrasound was transmitted across the pig temporal bone. Despite this, spectrophotometric analysis showed that both IV LEMB and PESDA combined with transtemporal ultrasound resulted in a significant increase in Evans blue extravasation across BBB of the treated side at 30 min after insonation (p < 0.001; compared with ultrasound alone) but not at 120 min. There was significant retention of Gadolinium within the insonified parenchyma at 60 and 90 min after insonation, but not at 120 min. Oxygen saturation and arterial pressures were not changed after any microbubble injection. Intravenous microbubbles, combined with transtemporal ultrasound, can transiently increase BBB permeability in a large animal. This induced opening of BBB is reversible and may be a safe noninvasive method of achieving drug or gene delivery across the BBB.     

Dynamic Visualization of Lymphatic Channels and Sentinel Lymph Nodes Using Intradermal Microbubbles and Contrast‐Enhanced Ultrasound in a Swine Model and Patients With Breast Cancer

Objective. Sentinel lymph node (SLN) identification using intradermal micro‐bubbles and contrast‐enhanced ultrasound (CEUS) has been recently reported in swine models and patients with breast cancer. The objective of this study was to investigate the dynamics of intradermally administered microbubbles as they travel to draining SLNs in pigs. We also performed a detailed study of the passage of microbubbles through breast lymphatic channels in a small group of patients with breast cancer. Methods. Nine anesthetized healthy pigs were used for the study, and 5 female patients with primary breast cancer were recruited. Pigs received intradermal injections of a microbubble contrast agent in several territories to access lymphatic drainage to regional lymph nodes. Patients had periareolar intradermal injection of the microbubble contrast agent. Ultrasound examination was performed in the real‐time contrast pulse sequencing mode with a commercial scanner. Results. Sentinel lymph nodes were identified rapidly (<1 minute) and consistently in pigs. Intradermal microbubble injection and CEUS were found to have perfect concordance with the Evans blue dye method in locating swine SLNs. In all 5 patients with breast cancer, the microbubble contrast agent entered breast lymphatic channels and traveled to draining ipsilateral axillary SLNs within 3 minutes. Conclusions. Intradermally injected microbubbles traverse readily though lymphatic channels in pigs and human breast tissue. The ability to rapidly identify SLNs in the diagnostic period would enable targeted biopsy and may facilitate preoperative axillary staging in patients with early breast cancer.     

Acoustic techniques for assessing the Optison destruction threshold.

OBJECTIVE: The purpose of this study was to identify the pressure threshold for the destruction of Optison (octafluoropropane contrast agent; Amersham Health, Princeton, NJ) using a laboratory-assembled 3.5-MHz pulsed ultrasound system and a clinical diagnostic ultrasound scanner. METHODS: A 3.5-MHz focused transducer and a linear array with a center frequency of 6.9 MHz were positioned confocally and at 90 degrees to each other in a tank of deionized water. Suspensions of Optison (5-8x10(4) microbubbles/mL) were insonated with 2-cycle pulses from the 3.5-MHz transducer (peak rarefactional pressure, or Pr, from 0.0, or inactive, to 0.6 MPa) while being interrogated with fundamental B-mode imaging pulses (mechanical index, or MI,=0.04). Scattering received by the 3.5-MHz transducer or the linear array was quantified as mean backscattered intensity or mean digital intensity, respectively, and fit with exponential decay functions (Ae-kt+N, where A+N was the amplitude at time 0; N, background echogenicity; and k, decay constant). By analyzing the decay constants statistically, a pressure threshold for Optison destruction due to acoustically driven diffusion was identified. RESULTS: The decay constants determined from quantified 3.5-MHz radio frequency data and B-mode images were in good agreement. The peak rarefactional pressure threshold for Optison destruction due to acoustically driven diffusion at 3.5 MHz was 0.15 MPa (MI=0.08). Furthermore, the rate of Optison destruction increased with increasing 3.5-MHz exposure pressure output. CONCLUSIONS: Optison destruction was quantified with a laboratory-assembled 3.5-MHz ultrasound system and a clinical diagnostic ultrasound scanner. The pressure threshold for acoustically driven diffusion was identified, and 3 distinct mechanisms of ultrasound contrast agent destruction were observed with acoustic techniques.     

Observation on the integrity of the blood-brain barrier after microbubble destruction by diagnostic transcranial color-coded sonography.

OBJECTIVE: To investigate alteration of the blood-brain barrier from ultrasonic contrast agent destruction by diagnostic transcranial color-coded sonography using gadolinium-enhanced magnetic resonance imaging. METHODS: Healthy male volunteers received 10 mL (400 mg/dL) of Levovist (SH U 508A; Schering AG, Berlin, Germany; n = 6) or 3 mL of Optison (FS069; Mallinckrodt Inc, St Louis, MO; n = 4) followed by 0.3 mmol/kg magnetic resonance imaging contrast agent (Magnevist; Schering) intravenously. Then transcranial color-coded sonography was performed with a conventional color duplex sonographic system, which insonated the brain in a slightly angulated axial plane with temporal average intensity of less than 700 mW/cm2 or acoustic pressure amplitude of less than 2.69 MPa, attenuated by the temporal bone. Before, immediately after, and 2 hours after insonation, T1-weighted axial magnetic resonance imaging was performed. All magnetic resonance images were individually assessed, and T1 signal intensities were measured in 2 regions of interest in both hemispheres at the 3 time points. RESULTS: No focal contrast enhancement or damage to the brain and no significant difference between T1 signal intensities in the right and left brain regions could be detected during early or late phases when either ultrasonic contrast agent was used. CONCLUSIONS: This bioeffects study gives further evidence of the safety of ultrasonic destruction of Levovist and Optison microbubbles by diagnostic transcranial color-coded sonography. However, more subtle local effects may have been missed by gadolinium-enhanced magnetic resonance imaging. Studies on diagnostic contrast-enhanced transcranial color-coded sonography as well as microbubble-based drug delivery strategies should consider ultrasonic contrast agent microbubble characteristics and concentration as well as ultrasound transmission power levels.     

Nanodroplet-Mediated Low-Energy Mechanical Ultrasound Surgery

Mechanical ultrasound surgery methods use short, high-intensity pulses to fractionate tissues. This study reports the development of a two-step technology for low-energy mechanical ultrasound surgery of tissues using nanodroplets to reduce the pressure threshold. Step 1 consists of vaporizing the nanodroplets into gaseous microbubbles via megahertz ultrasound excitation. Then, low-frequency ultrasound is applied to the microbubbles, which turns them into therapeutic warheads that trigger potent mechanical effects in the surrounding tissue. The use of nanoscale nanodroplets coupled with low-frequency ultrasound reduces the pressure threshold required for mechanical ultrasound surgery by an order of magnitude. In addition, their average diameter of 300 nm can overcome challenges associated with the size of microbubbles. Optimization experiments were performed to determine the ultrasound parameters for nanodroplet vaporization and the subsequent microbubble implosion processes. Optimal vaporization was obtained when transmitting a 2-cycle excitation pulse at a center frequency of 5 MHz and a peak negative pressure of 4.1 MPa (mechanical index = 1.8). Low-frequency insonation of the generated microbubbles at a center frequency of 850, 250 or 80 kHz caused enhanced contrast reduction at a center frequency of 80 kHz, compared with the other frequencies, while operating at the same mechanical index of 0.9. Nanodroplet-mediated insonation of ex vivo chicken liver samples generated mechanical damage. Low-frequency treatment at a mechanical index of 0.9 and a center frequency of 80 kHz induced the largest lesion area (average of 0.59 mm²) compared with 250- and 850-kHz treatments with the same mechanical index (average lesions areas of 0.29 and 0.19 mm², respectively, p < 0.001). The two-step approach makes it possible to conduct both the vaporization and implosion stages at mechanical indices below 1.9, thus avoiding undesired mechanical damage. The findings indicate that coupled with low-frequency ultrasound, nanodroplets can be used for low-energy mechanical ultrasound surgery.     

超声介导微泡破裂法对VEGF体外转染成纤维细胞蛋白表达影响的实验研究

目的利用ELISA检测法观察诊断剂量的超声介导微泡破裂法对VEGF体外转染成纤维细胞蛋白表达的影响并探讨该种转染方法的最佳超声条件。方法实验分组:单纯VEGF质粒转染组;超声 造影剂微泡 VEGF质粒转染组,该组并根据MI(机械指数)分为三组1.2;1.4;1.6。转染后利用ELISA法对各组成纤维细胞的VEGF蛋白表达进行定量检测;利用锥虫蓝染色法比较不同超声强度对细胞活性的影响。结果VEGF质粒转染成纤维细胞后,24h开始即有较高水平的VEGF蛋白表达,表达量于48 h7、2 h呈下降趋势;超声 造影剂微泡 VEGF质粒组(MI1.4、1.6)细胞VEGF蛋白表达量最高,但2组间差异无显著性(P>0.05);以上2组与单纯质粒转染组及MI为1.2组比较有显著性差异(P<0.01);超声 造影剂(MI1.2)组与单纯质粒转染组亦有显著性差异(P<0.05)。锥虫蓝染色法显示:随着超声强度的增加,细胞死亡率呈上升趋势。结论诊断剂量的超声介导微泡破裂转染法能够提高VEGF体外转染成纤维细胞蛋白的表达;使用的超声剂量大,转染率高,但细胞损害大;反之,细胞损伤小,转染率低。     

Activation of microbubbles by short-pulsed ultrasound enhances the cytotoxic effect of cis-diamminedichloroplatinum (II) in a canine thyroid adenocarcinoma cell line in vitro

Ultrasound targeted microbubble destruction has succeeded in delivering drugs and genes. This study was designed to explore characteristics of ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. Canine thyroid adenocarcinoma cells were exposed to short-pulsed diagnostic ultrasound in the presence of cis-diamminedichloroplatinum (II) (cisplatin) and ultrasound contrast agent Sonazoid^® microbubbles. The cytotoxic effect of cisplatin was enhanced by short-pulsed diagnostic ultrasound and microbubbles. Incubation time with microbubbles influenced the cytotoxic effect of cisplatin. However, exposure duration did not affect the cytotoxic effect of cisplatin. Therefore, short-pulsed diagnostic ultrasound may activate microbubbles near cells and deliver cisplatin into cells. In addition, activation of microbubbles may be concluded in a short time. Our results suggest that short exposure duration could be potentially sufficient to induce efficient drug delivery by ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound.     

In vivo quantification of ultrasound targeted microbubbles to enhance cancer assessment

Contrast‐enhanced ultrasound with targeted microbubble contrast agents is an emerging technique for imaging biological processes at the molecular level. The accumulation of targeted microbubbles at tissue sites overexpressing specific molecular markers increases the backscattered signal for noninvasive evaluations of diseases. The aim of this preliminary study was to combine molecular imaging with an in vivo contrast agent quantification to support the early diagnosis of the pathology and to enhance the assessment of neoplastic tissues. Tumor growth was induced by subcutaneous injection of prostate cancer cells in four rats. Microbubbles targeted to tissue factor (TF) were administered. A vascularized region located in proximity to the tumor and centered around the focus depth was analyzed in each animal. The backscattered signals (i.e. the radio‐frequency data) were acquired during two different perfusion conditions to evaluate the contribution of attached microbubbles. After image generation by means of a multi‐pulse contrast‐enhanced technique, a nonlinear regression method based on the support vector machine was employed to estimate the contrast agent concentrations in cubic voxels (1‐mm side length). The number of attached microbubbles per mm³ was estimated based on a multi‐dimensional vector of features extracted from the processed radio‐frequency signals. A significant correlation (p < 0.05) between the size of the tumors and the estimated microbubble concentration was found, thus opening the possibility for combining molecular imaging and contrast agent concentration mapping to refine pathology evaluation. Copyright © 2016 John Wiley & Sons, Ltd.     

The sonographic evaluation of tubal patency with stimulated acoustic emission imaging.

OBJECTIVES: Experimental and clinical data suggest that insonation of echo-enhancing contrast agents with high acoustic power produces disintegration of microbubbles, resulting in a phenomenon called stimulated acoustic emission (SAE). The purpose of this study was to investigate whether SAE might be detected by transvaginal sonography and whether this technique may be useful in the assessment of tubal patency by hysterosalpingo-contrast sonography (SAE-HyCoSy). METHODS: Patients booked for X-ray hysterosalpingography (HSG) for infertility evaluation also received SAE-HyCoSy. The order of the two procedures was established in each patient by randomization after placement of a transcervical balloon catheter. For SAE-HyCoSy, the ultrasound contrast medium Levovist was injected, with the acoustic power set at the maximum level permitted on ultrasound machines employing dedicated algorithms. Conventional HSG was performed for comparison. RESULTS: Seventy-seven Fallopian tubes were examined in 41 patients. In all cases it was possible to obtain the SAE phenomenon. In 10 tubes (13%) proximal filling was not observed by both SAE-HyCoSy and HSG. In the remaining 67 tubes, free spill from the distal end of the lumen was demonstrated in 96% of cases (64/67) with SAE-HyCoSy and in 97% of cases (65/67) with HSG. Disagreement between the two techniques was observed in five tubes only, with a Cohen's kappa coefficient of 0.76 (95% confidence interval, 0.56-0.96). CONCLUSION: SAE techniques were successfully applied to HyCoSy and allowed the visualization of the free spill of contrast agent into the peritoneal cavity in the majority of cases. SAE-HyCoSy showed good agreement with HSG in this preliminary study.     

Treatment of deeply located acute intravascular thrombi with therapeutic ultrasound guided by diagnostic ultrasound and intravenous microbubbles.

OBJECTIVE: We sought to determine the added value of simultaneous imaging of intravenously infused microbubbles that are being used to dissolve an intravascular thrombus with therapeutic ultrasound (TUS). METHODS: In a chronic canine arteriovenous graft occluded by a thrombus, TUS (1 MHz) was applied through a 6-cm-thick tissue-mimicking phantom (measured mean +/- SD peak negative pressure through the phantom, 958 +/- 104 kPa) during an intravenous infusion of either saline (n = 6 occlusions) or lipid-encapsulated microbubbles (ImaRx Therapeutics, Inc, Tucson, AZ). Therapeutic ultrasound was intermittently applied during the microbubble infusion either at set time intervals (n = 6 occlusions) or when simultaneous diagnostic ultrasound (DUS) indicated a sustained presence of microbubbles (n = 12 occlusions). Success was defined as return of rapid flow within the graft (grade 3 flow). RESULTS: Diagnostic ultrasound showed microbubbles moving through small channels within the thrombus before angiographic evidence of flow in the graft. This guided the timing of TUS application better than using set time intervals. Angiographic clearance of the thrombus and restoration of grade 3 flow at 45 minutes of treatment were seen in 33% of deeply located thrombosed grafts treated with TUS at set time intervals and 92% of grafts treated with TUS guided by DUS (P < .001 compared with set time intervals). CONCLUSIONS: The use of TUS with intravenous microbubbles has a high success rate in recanalizing deeply located thrombosed arteriovenous grafts when performed with DUS guidance.     

High-speed optical observations of contrast agent destruction

Ultrasound contrast agents are now available since a few years and used for diagnostic purposes. Improved diagnostic decisions have been made possible with new imaging methods that are mainly based on the nonlinear properties of gas microbubbles. Since it is well known that contrast agents are destroyed by ultrasound when the acoustic pressure exceeds a threshold, extremely low acoustic pressures were applied to achieve enhanced contrast image quality. However, destruction of contrast microbubbles is not necessarily undesirable, since it is beneficial in, for example, destruction/reperfusion imaging and recently in drug delivery. We investigate in this experimental study the destruction dynamics of a contrast agent consisting of nitrogen bubbles encapsulated in a double polymer/albumin wall shell. This is accomplished using an ultrafast camera Brandaris that operates at a frame rate of 25 MHz and records 128 frames. The measurements were performed with an ultrasound sine burst of 10 cycles at 1.7 MHz. Different acoustic pressures were applied and various microsphere sizes were examined. The results show three different zones depending on the applied pressure and bubble size: these are nondestruction zone, transient zone and destruction zone. The nondestruction zone is reached for either very small microspheres or low mechanical indices (MI) (<0.3). In the destruction zone lie either large microspheres (5 microm or higher) even when irradiated at low MIs or small microspheres (<5 microm) when the MI is above 0.6. The optical observations revealed that the destruction of the microspheres is characterized by shell rupture and gas release. The release of the gas gives rise to new free microbubble that lasts for a few milliseconds and then disappears due to dissolution. In the transient zone, the microspheres are mainly compressed in the first few cycles but no expansion is induced. After intense compressions, the shell fissures and gas escapes in the last cycles of the burst or during a second burst depending on the initial size and MI. These optical recordings are important to investigate contrast bubble destruction and can help in amplifying or minimizing this process. Indeed, bubble disruption remains the basis of most current sensitive methods for detecting perfusion with contrast agents and is an essential component of perfusion quantification with microbubbles, in addition to drug delivery applications and pressure measurements.     

Low-Amplitude Non-linear Volume Vibrations of Single Microbubbles Measured with an “Acoustical Camera”

Contrast-enhanced ultrasound imaging is based on the detection of non-linear vibrational responses of a contrast agent after its intravenous administration. Improving contrast-enhanced images requires an accurate understanding of the vibrational response to ultrasound of the lipid-coated gas microbubbles that constitute most ultrasound contrast agents. Variations in the volume of microbubbles provide the most efficient radiation of ultrasound and, therefore, are the most important bubble vibrations for medical diagnostic ultrasound imaging. We developed an “acoustical camera” that measures the dynamic volume change of individual microbubbles when excited by a pressure wave. In the work described here, the technique was applied to the characterization of low-amplitude non-linear behaviors of BR14 microbubbles (Bracco Research, Geneva, Switzerland). The amplitude dependence of the resonance frequency and the damping, the prevalence of efficient subharmonic and ultraharmonic vibrations and the amplitude dependence of the response at the fundamental frequency and at the second harmonic frequency were investigated. Because of the large number of measurements, we provide a statistical characterization of the low-amplitude non-linear properties of the contrast agent.