微泡声学造影剂气体构成对二次谐波显像的作用

目的探讨二次谐波成像条件下改变微泡造影剂的气体构成是否能够增强经静脉注射后的心肌显像效果。方法对１０条开胸犬经静脉注射含有不同气体的声振右旋糖酐白蛋白微泡造影剂ＲＡ（空气,ｒｏｏｍａｉｒ）、ＳＦ６（六氟化硫,ｓｕｌｆｕｒｈｅｘａｆｌｕｏｒｉｄｅ）、ＦＸ系列（含有氟碳气体Ｃ３Ｆ８、Ｃ４Ｆ１０、Ｃ５Ｆ１２）。二次谐波显像条件下取左心室短轴切面观,观察心肌造影效果。结果含有氟碳气体的微泡造影剂经静脉注射后二次谐波显像下产生强烈的心肌显影,心肌声学密度分别为（１４．７２±２．７）ｄＢ（ＦＸ３３０）、（１４．３±２．４ｄＢ）（ＦＸ４３０）、（１３．７８±２．２４）ｄＢ（ＦＸ５３０）;氟硫气体仅存在轻微的心肌显像［（６．２４±１．５８）ｄＢ］,但心腔显影强烈［（２９．４８±６．０２）ｄＢ］,空气造影剂经静脉注射后几乎不产生心肌显影［（２．５３±０．７８）ｄＢ］,心腔内造影剂亦较弱［（１１．３±４．９６）ｄＢ］。结论不同气体构成的微泡声学造影剂二次谐波显像效果差异显著。氟碳气体声振右旋糖酐白蛋白是较好的经静脉声学造影剂。     

Image enhancement by acoustic conditioning of ultrasound contrast agents

A novel contrast imaging technique has been developed for use with microbubble contrast agents. It employs two acoustic fields: there is an excitation field for conditioning microbubbles and an imaging field for detecting microbubbles. The maximum increases (due to microbubble conditioning) in scattered first and second harmonic signals were 14.5 and 16 dB, respectively. This technique is unique for effectively enhancing the blood-to-tissue image contrast.     

Vascular flow and perfusion imaging with ultrasound contrast agents

Current techniques for imaging ultrasound (US) contrast agents (UCA) make no distinction between low-velocity microbubbles in the microcirculation and higher-velocity microbubbles in the larger vasculature. A combination of radiofrequency (RF) and Doppler filtering on a low mechanical index (MI) pulse inversion acquisition is presented that differentiates low-velocity microbubbles (on the order of mm/s) associated with perfusion, from the higher-velocity microbubbles (on the order of cm/s) in larger vessels. In vitro experiments demonstrate the ability to separate vascular flow using both harmonic and fundamental Doppler signals. Fundamental and harmonic Doppler signals from microbubbles using a low-MI pulse-inversion acquisition are compared with conventional color Doppler signals in vivo. Due to the lower transmit amplitude and enhanced backscatter from microbubbles, the in vivo signal to clutter ratios for both the fundamental (-11 dB) and harmonic (-4 dB) vascular flow signals were greater than with conventional power Doppler (-51 dB) without contrast agent. The processing investigated here, in parallel with conventional pulse-inversion processing, enables the simultaneous display of both perfusion and vascular flow. In vivo results demonstrating the feasibility and potential utility of the real-time display of both perfusion and vascular flow using US contrast agents are presented and discussed.     

靶向超声造影评价裸鼠肾癌新生血管的实验研究

目的 探讨以血管内皮生长因子受体2 (VEGFR2)为靶点的靶向超声造影评价裸鼠肾癌新生血管的应用价值.方法 建立人肾透明细胞癌皮下移植瘤裸鼠模型,制备携VEGFR2抗体的靶向造影剂(MBV),每只裸鼠分别随机注射普通造影剂(MBC)和MBV并行超声造影检查,比较两种造影剂的视频强度,并对肿瘤组织行VEGFR2免疫组化检测.结果 超声造影示MBV组视频强度(6.50±1.43)Db,MBC组视频强度(2.59±0.99)Db,两者差异有统计学意义(P<0.01),且MBV组持续强化时间较MBC组长,免疫组化结果证实肿瘤血管内皮VEGFR2高表达.结论 以VEGFR2为靶点的靶向造影剂可特异性增强肾癌显影,对评估肿瘤新生血管有重要价值.

Abstract：

Objective To investigate the value of contrast enhanced ultrasound(CEUS) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in murine tumor models.Methods Established human renal cell carcinomas(RCC) subcutaneous xenograft tumor model in nude mice,microbubbles targeted to VEGFR2(MBV) was prepared,control microbubbles(MBC) and MBV were injected respectively in each mouse,the intensity of each microbubble was compared,the expressions of VEGFR2 in tumors were tested by immunohistochemistry.Results CEUS imaging showed the intensity of MBV and MBC was (6.50±1.43)dB,(2.59±0.99)dB,respectively.There was significantly higher intensity when using MBV compared with MBC (P<0.01).The time to wash out was longer in MBV contrast group compared with MBC group.Immunohistochemistry showed VEGFR2 was highly expressed in novel vessel walls.Conclusions Contrast microbubbles targeted to VEGFR2 can specially enhance the images of RCC and has tremendous significance in the assessment of angiogenesis.     

Effect of Microbubble Size on Fundamental Mode High Frequency Ultrasound Imaging in Mice

High-frequency ultrasound imaging using microbubble (MB) contrast agents is becoming increasingly popular in pre-clinical and small animal studies of anatomy, flow and vascular expression of molecular epitopes. Currently, in vivo imaging studies rely on highly polydisperse microbubble suspensions, which may provide a complex and varied acoustic response. To study the effect of individual microbubble size populations, microbubbles of 1–2 μm, 4–5 μm and 6–8 μm diameter were isolated using the technique of differential centrifugation. Size-selected microbubbles were imaged in the mouse kidney over a range of concentrations using a Visualsonics Vevo 770 ultrasound imaging system (Visualsonics, Toronto, Ontario, Canada) with a 40-MHz probe in fundamental mode. Results demonstrate that contrast enhancement and circulation persistence are strongly dependent on microbubble size and concentration. Large microbubbles (4–5 and 6–8 μm) strongly enhanced the ultrasound image with positive contrast, while 1–2 μm microbubbles showed little enhancement. For example, the total integrated contrast enhancement, measured by the area under the time-intensity curve (AUC), increased 16-fold for 6–8 μm diameter microbubbles at 5 × 10⁷ MB/bolus compared with 4–5 μm microbubbles at the same concentration. Interestingly, 1–2 μm diameter microbubbles, at any concentration, did not measurably enhance the integrated ultrasound signal at tissue depth, but did noticeably attenuate the signal, indicating that they had a low scattering-to-attenuation ratio. When concentration matched, larger microbubbles were more persistent in circulation. However, when volume matched, all microbubble sizes had a similar circulation half-life. These results indicated that dissolution of the gas core plays a larger role in contrast elimination than filtering by the lungs and spleen. The results of this study show that microbubbles can be tailored for optimal contrast enhancement in fundamental mode imaging. (E-mail: mb2910@columbia.edu)     

自制脂膜氟烷声学造影剂增强肾脏灰阶显像的实验研究

目的　探讨自制脂膜氟烷声学造影剂“脂氟显”对正常肾灰阶显像的增强效果。方法　 10只健康家兔经耳缘静脉团注 0 .0 1ml/kg的脂氟显 ,采用视觉评分和视频分析 ,观察肾皮质、髓质及肾窦的二维灰阶增强效果。结果　脂氟显不仅能明显增强肾皮质、髓质及肾窦的灰阶显像 ,而且持续时间长 ,肾皮质明显增强时间长达 5 2min ,肾髓质为 2min 2 0s ,肾窦为 1min 40s。结论　脂氟显能长时间显著增强肾脏的灰阶显像     

Measurement of radial artery contrast intensity to assess cardiac microbubble behavior

OBJECTIVES: We sought to determine whether analysis of the contrast signal from the radial artery is better able to reflect changes in left ventricular (LV) microbubble dynamics than the signal from the LV itself. BACKGROUND: Assessment of microbubble behavior from images of the LV may be affected by attenuation from overlying microbubbles and nonuniform background signal intensities. The signal intensity from contrast in a peripheral artery is not affected by these artifacts and may, thus, be more accurate. METHODS: After injection of a contrast bolus into a peripheral vein, signal intensity was followed simultaneously in the LV and radial artery. The measurements were repeated using continuous, triggered, low and high mechanical index harmonic imaging of the LV. RESULTS: Peak and integrated signal intensities ranged from 25 dB and 1550 dB/s, respectively, with radial artery imaging to 5.6 dB and 471 dB/s with ventricular imaging. Although differences in microbubble behavior during the different imaging protocols could be determined from both the LV and radial artery curves, analysis of the radial artery curves yielded more consistent and robust differences. CONCLUSIONS: The signal from microbubbles in the radial artery is not affected by shadowing and is, thus, a more accurate reflection of microbubble behavior in the LV than the signal from the LV itself. This may have important implications for the measurement of myocardial perfusion by contrast echocardiography.     

新型声学造影方法评价兔肾脏VX2肿瘤血流灌注的实验研究

目的　探索新型声学造影技术在评价肾脏ＶＸ２ 肿瘤血流灌注方面的诊断价值。方法　５ 只患有ＶＸ２肾肿瘤的新西兰白兔经耳缘静脉注射ＦＸ５３０ 声学造影剂,用二次谐波和基波显像观察兔肾造影效果,分析使用视觉评分和视频密度定量。结果　新型声学造影方法显著地增强了肾肿瘤的超声影像、实时显示了肿瘤异常血流灌注状态,即呈“充盈缺损”状负性显影。ＶＸ２ 肿瘤与肾皮质的视频密度灰阶差值从造影前的１６６ 提高到４５８３（ 基波）和８０１７（ 二次谐波） 。结论　新型声学造影技术可以反映肾脏ＶＸ２ 肿瘤的异常血流灌注,显著提高肿瘤影像清晰度。     

自然组织谐波成像与基波成像在心脏声学造影中的对比观察

组织谐波成像是新近开展的一种超声成像技术，其探头接受声波的频率是发射频率的两倍（分别为１．７５ＭＨｚ和３．５ＭＨｚ），我们将该技术应用于心脏声学造影检查，观察其对造影剂微气泡的显像效果，并与传统的基波成像技术相比较，发现前者右心显影更强，密度更高，散在造影剂微气泡颗粒更粗，更亮，并能清晰显示左心少量的穿肺的造影剂，这是传统方法难于探及的，但要达到心肌显影，还需改进造影剂的制备，开发出新的心肌声学造影剂。     

CPS及CCI灰阶超声造影成像技术诊断肝脏局灶性病变应用价值

目的探讨灰阶超声造影成像(CE-GUS)诊断肝占位性病变的应用价值.方法应用CPS及AD-CCI成像技术检查39个肝脏局灶性病变,32个病灶同期检查基波彩色多普勒增强超声(CE-CDFI). 结果 CE-GUS病灶内部血流信号显示率97.44%,高于CE-CDFI(P<0.05).CE-GUS诊断符合率为92.3%(36/39),明显高于常规超声检查(23.1%)及CE-CDFI(50%),P<0.05.结论 CPS及CCI灰阶超声造影成像技术能清楚显示肝脏及肝肿瘤血液灌注,对肝脏局灶性病变的鉴别诊断有实用价值.     

Second Harmonic and Subharmonic for Non-Linear Wideband Contrast Imaging Using a Capacitive Micromachined Ultrasonic Transducer Array

When insonified with suitable ultrasound excitation, contrast microbubbles generate various non-linear scattered components, such as the second harmonic (2H) and the subharmonic (SH). In this study, we exploit the wide frequency bandwidth of capacitive micromachined ultrasonic transducers (CMUTs) to enhance the response from ultrasound contrast agents by selective imaging of both the 2H and SH components simultaneously. To this end, contrast images using the pulse inversion method were recorded with a 64-element CMUT linear array connected to an open scanner. In comparison to imaging at 2H alone, the wideband imaging including both the 2H and SH contributions provided up to 130% and 180% increases in the signal-to-noise and contrast-to-tissue ratios, respectively. The wide-frequency band of CMUTs offers new opportunities for improved ultrasound contrast agent imaging.     

Treatment of Microvascular Micro-embolization Using Microbubbles and Long-Tone-Burst Ultrasound: An in Vivo Study

Despite epicardial coronary artery reperfusion by percutaneous coronary intervention, distal micro-embolization into the coronary microcirculation limits myocardial salvage during acute myocardial infarction. Thrombolysis using ultrasound and microbubbles (sonothrombolysis) is an approach that induces microbubble oscillations to cause clot disruption and restore perfusion. We sought to determine whether this technique could restore impaired tissue perfusion caused by thrombotic microvascular obstruction. In 16 rats, an imaging transducer was placed on the biceps femoris muscle, perpendicular to a single-element 1-MHz treatment transducer. Ultrasound contrast perfusion imaging was performed at baseline and after micro-embolization. Therapeutic ultrasound (5000 cycles, pulse repetition frequency = 0.33 Hz, 1.5 MPa) was delivered to nine rats for two 10-min sessions during intra-arterial infusion of lipid-encapsulated microbubbles; seven control rats received no ultrasound–microbubble therapy. Ultrasound contrast perfusion imaging was repeated after each treatment or control period, and microvascular volume was measured as peak video intensity. There was a 90% decrease in video intensity after micro-embolization (from 8.6 ± 4.8 to 0.7 ± 0.8 dB, p < 0.01). The first and second ultrasound–microbubble sessions were respectively followed by video intensity increases of 5.8 ± 5.1 and 8.7 ± 5.7 dB (p < 0.01, compared with micro-embolization). The first and second control sessions, respectively, resulted in no significant increase in video intensity (2.4 ± 2.3 and 3.6 ± 4.9) compared with micro-embolization (0.6 ± 0.7 dB). We have developed an in vivo model that simulates the distal thrombotic microvascular obstruction that occurs after primary percutaneous coronary intervention. Long-pulse-length ultrasound with microbubbles has a therapeutic effect on microvascular perfusion and may be a valuable adjunct to reperfusion therapy for acute myocardial infarction.     

载10-羟基喜树碱液态氟碳纳米粒制备及其表征、体外增强超声显像效果观察

目的 研制一种包裹液态氟碳（PFP）的新型载药纳米级超声造影剂,考察其基本特性并观察其体外增强超声显像效果。方法 采用旋转蒸发和探头超声法制备载10-羟基喜树碱（10-HCPT）的液态氟碳（PFP）纳米粒,在光学显微镜和透射电镜下观察纳米粒形态,采用马尔文仪测量纳米粒粒径和电荷。采用紫外分光光度计测定纳米粒包封率,并应用低强度聚焦超声（LIFU）辐照载药液态氟碳纳米粒溶液,观察纳米粒相变情况及其增强超声显像效果。结果 制备的载药脂质纳米粒外观为乳白色混悬液,在油镜及透射电镜下观察,载药纳米粒形态规则,分布均一,平均粒径约（500.82±25.97）nm,表面平均电位为（-47.77±3.09）mV;在体外经LIFU辐照后,可见纳米粒发生液气相变形成微泡,在基波模式和谐波模式下,均可显著增强超声显影。结论 成功研制了包裹液态氟碳的载药脂质纳米粒,可望成为一种新型的多功能超声造影剂。     

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.     

Theoretical and experimental characterisation of magnetic microbubbles

In addition to improving image contrast, microbubbles have shown great potential in molecular imaging and drug/gene delivery. Previous work by the authors showed that considerable improvements in gene transfection efficiency were obtained using microbubbles loaded with magnetic nanoparticles under simultaneous exposure to ultrasound and magnetic fields. The aim of this study was to characterise the effect of nanoparticles on the dynamic and acoustic response of the microbubbles. High-speed video microscopy indicated that the amplitude of oscillation was very similar for magnetic and nonmagnetic microbubbles of the same size for the same ultrasound exposure (0.5 MHz, 100 kPa, 12-cycle pulse) and that this was minimally affected by an imposed magnetic field. The linear scattering to attenuation ratio (STAR) was also similar for suspensions of both bubble types although the nonlinear STAR was ~50% lower for the magnetic microbubbles. Both the video and acoustic data were supported by the results from theoretical modelling.     

纳米级超声微泡造影剂的制备及其性状的初步研究

目的制备适用于超声诊断的纳米级超声微泡造影剂,并对其性状进行初步研究。方法通过低速离心分级分离法制备纳米级的脂膜超声微泡,检测微泡大小形态、表面电位、粒径、分布,测试其与抗体结合情况,体外初步测试其增强显像效果。结果微泡呈圆形,分布均匀,粒径均值为623.4nm;微泡表面电位均值1.3mV,与抗体结合情况良好,体外初步测试能明显增强显像效果。结论通过低速离心分级分离法可以制备纯度较高的纳米级超声微泡,为超声造影剂的小型化和靶向性发展提供重要的工作平台。     

Spectral normalization for ultrasonic contrast microbubble detection

Ultrasonic contrast agents consisting of microbubbles are used to assess tissue perfusion. The microbubbles are highly reflective and nonlinear and thus produce harmonics that are stronger than those from tissues. However, the magnitude of harmonic signals resulting from a region with microbubbles also depends on the acoustic pressure of incident ultrasound and the attenuation of intervening tissues in the ultrasound path. Therefore, the harmonic magnitude, as used in traditional harmonic imaging, may not be a reliable indicator of the presence or absence of microbubbles, and hence, tissue perfusion. To compensate for these effects, we present two parameters defined as the ratio of the harmonic to the fundamental component (HFR) and the ratio of the harmonic to squared fundamental (HSFR). A simplified model is used to illustrate the usefulness of these two parameters. Experiments show that both parameters improve detection of microbubbles and that HSFR performs better than HFR.     

纳米级超声造影剂制备及成像的实验研究

目的 探讨纯度较高的纳米级超声微泡造影剂的理化性质及体内成像效果.方法 机械振荡法与低速离心法结合制备纳米级微泡,观察微泡形态,检测其平均粒径,并观察其增强正常兔肝显影的情况,并与微米级微泡造影剂进行比较.结果 光镜以及透射电镜观察,微泡呈圆形,分布均匀,无聚集现象.Zeta SIZIER 3000测定微泡粒径均值为623.4 nm,微泡表面电位均值1.3 mV.注射纳米级造影剂于新西兰大白兔体内能持续增强肝的显像效果,与微米级造影剂对比无明显差异.结论 纳米级的微泡造影剂大小合理,分布均匀,显像效果强,为下一步研制小型化靶向造影剂奠定了基础.     

Effect of ultrasound-activated microbubbles on the cell electrophysiological properties

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs (n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles' oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels.     

Ultrasound contrast harmonic imaging of abdominal organs

Ultrasound contrast agents consist of microbubbles, which are the most effective acoustic backscatters. The interaction between the insonating ultrasound beam and the microbubbles is very complex and basic understanding of their behavior under various sound fields has been fundamental to the development of improved methods of visualizing and displaying the contrast agents. Although echo enhancers have been under development for a long time, their clinical applications have been limited to enhancing the Doppler signals in difficult cases. However, recent advances in harmonic imaging and the development of new tissue-specific contrast agents stand to broaden the scope of ultrasound diagnostic potential beyond simply rescuing failed Doppler examinations. This article reviews the current and potential applications of ultrasound contrast harmonic imaging in the abdomen.