诊断性超声造影对胎盘屏障通透性影响的实验研究

目的探讨不同机械指数的诊断性超声造影对胎盘屏障通透性的影响，以了解超声造影检查中超声强度的安全性。 方法14～16d（孕中期）清洁级SD孕鼠，经尾静脉注射剂量为1ml／kg的“声诺维”超声造影剂，不同机械指数（MI：0．13、1．0、1．4）的超声对孕鼠子宫进行连续及间歇辐照，时间5min，在荧光显微镜下观察胎盘及胎鼠组织伊文思蓝（Evans blue，EB）自发红色荧光情况。 结果诊断剂量超声造影，大体观察胎盘呈深蓝色，胎鼠呈半透明浅粉色。在荧光显微镜下，胎盘内可见明显的EB自发的红色荧光斑块，在胎鼠体内未见EB荧光显像。 结论高机械指数及低机械指数诊断性超声造影均不会导致孕鼠胎盘屏障通透性增加。     

脑超声造影中超声强度对血脑屏障通透性的影响

目的探讨不同机械指数的诊断性超声在超声造影中对血脑屏障通透性的影响,以了解脑超声造影检查中超声强度的安全应用范围。方法50只清洁级SD大鼠,经尾静脉注射剂量为1ml/kg的“脂氟显”超声造影剂,辅以不同机械指数的超声进行辐照,观察超声照射后血脑屏障通透性的变化。结果在MI等于0.4时,血脑屏障通透性与对照组相比无统计学差异,当MI≥0.8时,血脑屏障通透性增加,且随着超声能量的进一步提高血脑屏障的通透性增加。结论高机械指数的体表超声在超声造影中可导致血脑屏障通透性增加,但应用适当强度的超声在进行大脑超声造影时仍是安全的。     

Effects of Focused Ultrasound and Microbubbles on the Vascular Permeability of Nanoparticles Delivered into Mouse Tumors

Ultrasound sonication with microbubbles (MBs) was evaluated for enhancement of the release of nanoparticles from vasculature to tumor tissues. In this study, tumor-bearing Balb/c mice were insonicated with focused ultrasound (FUS) in the tumors after the injection of MBs (SonoVue^®) and then lipid-coated quantum dot (LQD) nanoparticles (130 ± 25 nm) were injected through the tail vein. We studied the effects of the injected MB dose (0–300 μL/kg), sonication duration (0–300 s) and treatment-procedure sequence on the accumulation of nanoparticles in the tumors 24 h after the treatment and the time response of the accumulation (0.5–24 h). After the treatment, the mice were sacrificed and perfused and then the tumor tissues were harvested for quantifying the amount of nanoparticles using graphite furnace atomic absorption spectrometry (GF-AAS). The results showed that pulsed-FUS sonication with MBs can effectively enhance the vascular permeability for LQD nanoparticle delivery into the sonicated tumors. It indicates that this technique is promising for a better nanodrug delivery for tumor chemotherapy. (E-mail: winli@ntu.edu.tw)     

低频诊断超声联合脂氟显微泡开放小鼠血脑屏障的可逆性研究

目的探讨低频诊断超声联合脂氟显微泡开放小鼠血脑屏障(blood-brain barrier,BBB)的可逆性及伊文思蓝(Evans blue,EB)透过率。方法健康C57BL/6小鼠48只,随机分为对照组6只和观察组42只。观察组再随机分为0、0.5、1、2、3、8、10 h亚组各6只,低频诊断超声辐照同时经尾静脉注射脂氟显微泡1 mL/kg,并分别于超声辐照0、0.5、1、2、3、8、10 h经尾静脉注射质量分数2%EB溶液50 mg/kg;对照组不进行低频诊断超声辐照,经尾静脉注射1 mL/kg生理盐水后注射质量分数2%EB溶液50 mg/kg。注射1 h后处死小鼠取脑组织,观察脑组织蓝染深度及面积,采用紫外分光光度法定量分析脑组织EB含量。结果对照组及观察组8、10 h亚组未见脑实质蓝染,观察组0、0.5、1、2、3 h亚组辐照区脑实质均有不同程度蓝染,且1 h时蓝染深度最深、面积最大;观察组0、0.5、1、2、3 h亚组脑组织EB含量[(45.43±2.89)、(50.94±1.25)、(79.79±1.12)、(51.55±1.20)、(31.60±1.77)μg/g]均高于对照组[(8.32±0.12)μg/g](P<0.05),8、10 h亚组脑组织EB含量[(8.34±0.09)、(8.31±0.07)μg/g]与对照组比较差异无统计学意义(P>0.05);观察组0、0.5、1 h亚组脑组织EB含量依次升高(P<0.05),1、2、3 h亚组脑组织EB含量依次降低(P<0.05)。结论低频诊断超声联合脂氟显微泡开放小鼠BBB呈动态可逆的过程,BBB在超声辐照后1 h开放程度最大,于超声辐射8 h恢复正常。     

Pulmonary Capillary Hemorrhage Induced by Different Imaging Modes of Diagnostic Ultrasound

The induction of pulmonary capillary hemorrhage (PCH) is a well-established non-thermal biological effect of pulsed ultrasound in animal models. Typically, research has been done using laboratory pulsed ultrasound systems with a fixed beam and, recently, by B-mode diagnostic ultrasound. In this study, a GE Vivid 7 Dimension ultrasound machine with 10?L linear array probe was used at 6.6?MHz to explore the relative PCH efficacy of B-mode imaging, M-mode (fixed beam), color angio mode Doppler imaging and pulsed Doppler mode (fixed beam). Anesthetized rats were scanned in a warmed water bath, and thresholds were determined by scanning at different power steps, 2?dB apart, in different groups of six rats. Exposures were performed for 5?min, except for a 15-s M-mode group. Peak rarefactional pressure amplitude thresholds were 1.5?MPa for B-mode and 1.1?MPa for angio Doppler mode. For the non-scanned modes, thresholds were 1.1?MPa for M-mode and 0.6?MPa for pulsed Doppler mode with its relatively high duty cycle (7.7?×?10^?3 vs. 0.27?×?10^?3 for M-mode). Reducing the duration of M-mode to 15?s (from 300?s) did not significantly reduce PCH (area, volume or depth) for some power settings, but the threshold was increased to 1.4?MPa. Pulmonary sonographers should be aware of this unique adverse bio-effect of diagnostic ultrasound and should consider reduced on-screen mechanical index settings for potentially vulnerable patients.     

脑超声造影中超声造影剂剂量对血脑屏障通透性的影响

目的探讨不同剂量超声造影剂在超声造影中对血脑屏障通透性的影响。方法60只清洁级SD大鼠,给予相同机械指数的超声进行辐照,并经尾静脉注射不同剂量的“脂氟显”超声造影剂,观察超声照射后血脑屏障通透性的变化。结果在造影剂剂量为50μl/kg时,血脑屏障通透性即与对照组相比产生显著性统计学差异,且随着超声造影剂剂量的增加其引起血脑屏障通透性也随之增加。结论在相同能量强度超声场中,超声造影剂微泡数目是引起不同生物学效应的重要因素。     

Spatial Distribution of Ultrasound Targeted Microbubble Destruction Increases Cardiac Transgene Expression But Not Capillary Permeability

Ultrasound targeted microbubble destruction (UTMD) has evolved as a promising tool for organ specific gene and drug delivery. Using DNA-loaded microbubbles, cardiac transfection has been shown to be feasible. However, two-dimensional properties of the ultrasound beam limit cardiac transgene expression to the focal zone, thus, reducing its potential therapeutic effect. The aim of this study was to test if spatial distribution of ultrasound targeted microbubble destruction in the heart could lead to augmented transgene expression or increased capillary permeability. Lipid microbubbles containing plasmids with a luciferase transgene were used to target rat hearts. The diagnostic ultrasound probe was fixed in a mid-short axis view with a gel stand-off between the chest and probe. Ultrasound (1.3 MHz) with a mechanical index of 1.6 was intermittently applied to rats during microbubble infusion. Rats were randomized to either stay in that position or move horizontally in a cranio-caudal direction (3 mm sweep) relative to the ultrasound probe during UTMD. After 4 days, organs were harvested and analyzed for reporter gene expression. Another group of rats received Evans Blue, followed by UTMD with unloaded microbubbles. Again, rats were randomized into a static or moving group. Hearts were harvested to evaluate extravasation of Evans Blue. Moving rats in a cranio-caudal direction significantly increased transgene expression by 19-fold in the anterior heart, by sixfold in the posterior heart and by 32-fold in the apex. Interestingly, Evans Blue extravasation was not augmented in the moving group. Spatial distribution of UTMD may increase transgene expression due to sonication of larger areas in the heart. In contrast, capillary permeability does not increase, indicating less capillary damage. (E-mail: raffi.bekeredjian@med.uni-heidelberg.de)     

诊断超声对不同年龄大鼠睾丸组织Fas/FasL蛋白表达的研究

目的 :探讨诊断超声对不同年龄组大鼠睾丸组织生精细胞 Fas/ Fas L 蛋白表达的影响。方法 :36只 SD雄性大鼠随机分为 6组 : 组 (出生后 30天组 )、 组 (出生后 30天对照组 )、 组 (出生后 45天组 )、 组 (出生后 45天对照组 )、 组 (出生后 6 0天组 ,即性成熟组 )、 组 (出生后 6 0天对照组 )。应用 HP 85 0 0彩色多普勒超声诊断仪对大鼠睾丸组织统一进行 30分钟的持续照射 ,2 4小时后处死动物取睾丸进行免疫组化分析 ,观察标本中Fas/ Fas L表达情况。结果 :不同年龄组大鼠睾丸组织生精细胞同时表达 Fas和 Fas L,其表达率随年龄增长有所增加 ,性成熟组两组基因的表达率明显高于其它两个年龄段 (P<0 .0 0 1)。超声照射后各组 Fas和 Fas L的表达率均明显增高 (P<0 .0 0 1) ;但 组和 组之间无差异 (P>0 .0 5 ) ,而 组两种基因的表达率均明显高于其它实验组 (P<0 .0 0 1) ,Fas表达率达 (81.5 6± 16 .2 4) % ,Fas L表达率达 (87.91± 17.36 ) % ,尤其是强阳性细胞增加更为明显。结论 :诊断超声照射不同年龄组大鼠睾丸组织 30分钟可引起 Fas和 Fas L 高表达 ,从而导致细胞凋亡     

产科诊断超声对宫内绒毛细胞DNA的影响

目的：研究产科诊断超声的安全性。方法：用诊断超声照射早孕妇子宫内的胚囊组织,照射时间长短不同,24小时后采取绒毛组织,测定绒毛细胞DNA单元链断裂和双链断裂。结果：产科诊断超声照射10分钟以内,DNA单链断裂及双链断裂无增加,照射20分钟以上,DNA单链断裂和双链断裂增加,结论：诊断超声在产科应用于早孕时,应有明显的适应症,且使用最小剂量照射早孕胚囊。     

Creating Brain Lesions with Low-Intensity Focused Ultrasound with Microbubbles: A Rat Study at Half a Megahertz

Low-intensity focused ultrasound was applied with microbubbles (Definity, Lantheus Medical Imaging, North Billerica, MA, USA; 0.02 mL/kg) to produce brain lesions in 50 rats at 558 kHz. Burst sonications (burst length: 10 ms; pulse repetition frequency: 1 Hz; total exposure: 5 min; acoustic power: 0.47–1.3 W) generated ischemic or hemorrhagic lesions at the focal volume revealed by both magnetic resonance imaging and histology. Shorter burst time (2 ms) or shorter sonication time (1 min) reduced the probability of lesion production. Longer pulses (200 ms, 500 ms and continuous wave) caused significant near-field damage. Using microbubbles with focused ultrasound significantly reduced acoustic power levels and, therefore, avoided skull heating issues and potentially can extend the treatable volume of transcranial focused ultrasound to brain tissues close to the skull.     

诊断用超声对人体宫内绒毛组织的影响

本文报道诊断用超声辐射人体宫内胚胎绒毛引超的脂质过氧化改变。将55名早期孕妇随机分成正常对照组(Ⅰ组)、辐射10分钟(Ⅱ组)、20分钟(Ⅲ组)、30分钟(Ⅳ组)以及超声辐射20分钟于7～10天后观察组(Ⅴ组),共五组。结果表明,常规诊断用超声连续辐射胎囊20分钟可致人体宫内绒毛细胞脂质过氧化改变(P_(Ⅰ·Ⅲ)<0.05)。丙二醛(MDA)值随超声辐射剂量增加而升高,而谷胱甘肽过氧化物酶(GSH-px)活性随超声辐射剂量增加而下降(P_(Ⅲ·Ⅳ)<0.05)。上述改变在作用7～10天后基本恢复正常(P_(Ⅲ·Ⅴ)<0.05、P_(Ⅰ·Ⅴ)>0.05),此结果提示这种损伤作用是可复性的。     

Influence of Microbubble Size and Pulse Amplitude on Hepatocyte Injury Induced by Contrast-Enhanced Diagnostic Ultrasound

Recent research has found that contrast-enhanced diagnostic ultrasound (CEDUS) has the potential to induce localized injury in the liver, with clearly observable effects for contrast agent doses higher than the recommended dose and maximal mechanical index values. This study was undertaken to assess effects with intermittent exposure at lower contrast doses of infusion and at reduced output to determine thresholds. In addition, microbubble (MB) suspensions with enhanced content of larger MBs were tested. Exposure from a phased array probe (GE Vivid 7 Dimension, GE Vingmed Ultrasound, Horten, Norway) was applied at 1.6 MHz and 1-s intermittent frame trigger for 10 min with infusion of MB suspension with normal (1.8 µm), medium (3.1 µm) and large (5.3 µm) mean MB diameters. The bio-effect endpoint was the count of hepatocytes stained with Evans blue dye in frozen sections. For the normal MBs, the count increased for clinically relevant infusion dosages, but leveled off above 20 µL/kg/min. The evidence of injury declined with time from 30 min to 4 h and was lacking at 24 h. The exposure thresholds in terms of peak rarefactional pressure amplitude, divided by the square root of frequency (in situ mechanical index) were 1.7, 1.3 and 1.2 for the normal-, medium- and large-sized MB suspensions. The enhanced efficacy for larger MBs lends support to the two-criterion model for cavitational microvascular injury during CEDUS. Overall, CEDUS in liver appears to have markedly less potential for induction of tissue injury than has been reported in other tissues, which indicates a satisfactory safety profile for CEDUS using recommended parameters in normal liver.     

Generation of Reactive Oxygen Species in Heterogeneously Sonoporated Cells by Microbubbles with Single-Pulse Ultrasound

To develop and realize sonoporation-based macromolecule delivery, it is important to understand the underlying cellular bioeffects involved. It is known that an appropriate level of reactive oxygen species (ROS) is necessary to maintain normal physiologic function, but excessive ROS triggers adverse downstream bioeffects. However, it is still unclear whether a relationship exists between intracellular ROS levels and sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33 µs duration and 0.70?MPa peak negative pressure) and imaging the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and the concentration of intracellular ROS indicator to evaluate the extent of sonoporation and ROS change, respectively. Our results revealed that the intracellular ROS level was correlated with the degree of the sonoporation. (i) Within ~120?s of the onset of ultrasound, during which membrane perforation and complete membrane resealing occurred, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane and positively correlated with the degree of the sonoporation. (ii) In the following 270?s (120–390?s post-exposure), ROS generation in reversibly sonoporated cells gradually increased and was positively correlated with the degree of the sonoporation. (iii) The ROS level in irreversibly sonoporated cells reduced to depletion during this time interval. It is possible that ROS generation in reversibly sonoporated cells can impact their long-term fate. These results thus provide new insight into the biological response to sonoporation.     

超声联合微泡对卡铂化疗A549细胞凋亡影响的实验研究

目的 探讨超声联合微泡是否协同卡铂诱导人肺癌A549细胞凋亡。方法 设置对照组、US组、USMB组、CBP组、CBP+US组、CBP+USMB组,其中卡铂采用最适宜剂量50μg/ml,超声辐照参数采用最适宜声强0.6W/cm_²,辐照时间60s。处理后培养24小时,每组分别消化、离心、收集3个独立样本,加入FITC标记的Annexin-V室温避光30 min,再加入PI避光反应5 min,应用流式细胞仪检测各组A549细胞凋亡率。应用SPSS 26.0分析各组凋亡率。结果 A549细胞总凋亡率由高到低依次为CBP+USMB组、CBP+US组、CBP组、USMB组、US组。CBP+US组与CBP组比较,早期凋亡率差异无统计学意义(P=0.964)。CBP+USMB组与CBP组、CBP+US组比较,早期凋亡率差异均有统计学意义(均P<0.01)。CBP+US组、CBP+USMB组与CBP组两两比较,晚期、总凋亡率差异均有统计学意义(均P<0.01)。 结论 US、USMB联合卡铂化疗可以协同诱导A549细胞凋亡。     

低频超声联合微泡经颅开放血脑屏障初步研究

目的探讨低频超声联合微泡经颅靶向开放血脑屏障及在临床应用价值。方法①经静脉注射微泡后,用频率为43kHz、强度为2W/cm2,持续超声波辐射大鼠头部5min;②采用荧光显微镜伊文氏蓝(EB)、电镜镧示踪法以及透射电镜观察脑组织的超微结构变化。结果经颅超声波与微泡联合能短暂地促进伊文氏蓝和镧离子通过多种形式跨越血脑屏障。结论低频超声联合微泡能可逆的、靶向的、局部的开放血脑屏障,并为进一步研究药物进入脑实质内提供新的策略。     

585例婴儿出生身高,体重与母亲孕期超声辐照次数关系分析

为探讨产科超声诊断安全性，我们随机抽取５８５例婴儿调查其出生身高、体重与母亲孕期Ｂ超检查史及其它影响因素，并进行简单相关和逐步多元回归统计分析。简单相关分析结果显示：婴儿身高与母亲孕期Ｂ超检查史无显著性差异，婴儿体重与母亲Ｂ超检查史呈正相关关系，有显著性（Ｐ＜０．０５）。婴儿身高与体重呈正相关关系，有显著性（Ｐ＜０．０５）。逐步多元回归分析婴儿身高、体重与母亲孕期Ｂ超检查史均无显著性差异。     

The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots

In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3–9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.     

声学造影剂增强超声辐照对血管内皮细胞膜通透性作用的研究

目的　探讨诊断级超声辐照对血管内皮细胞膜通透性的影响及声学造影剂的增强作用。方法　用连续多普勒超声辐照人脐血管内皮培养细胞 (HUVEC) ,照射条件为频率 1.9MHz,TIS 0 .8,增益 80 % ,ISATA为 80 .0 m W/ cm2 ,时间持续 6 0 s,添加或不添加声学造影剂全氟显。在荧光显微镜下观察培养液中大分子荧光素物质 FD 5 0 0进入细胞内的情况 ,用流式细胞仪计测荧光染色阳性的细胞。结果　荧光染色阳性细胞的百分率 :无超声辐照组或单纯添加全氟显组分别为 4 .2 %± 1.6 %和 3.0 %± 0 .9%。经超声辐照后为 2 4 .0 %± 5 .5 % ,添加全氟显辐照后显著增加至 6 6 .6 %± 4 .1% (P<0 .0 0 1)。结论　诊断级超声辐照具有增加 HUVEC膜通透性的作用 ,声学造影剂可使这种作用显著增强