Rapporteur report: Mechanisms and interactions

The first two talks of this session described the basic characteristics of the interaction of ultrasound with matter, the first (by V.F. Humphrey) concentrating on the physical interactions, with the second (by W.D. O’Brien, Jr.) focusing on the biophysical effects and mechanisms. The regimes covered by the first and second talks were of primarily high-frequency (O(MHz)) ultrasound in liquids and tissue, respectively. In contrast, the third and final talk of the session (by M. Alves-Pereira) discussed observations made by her group of the vibroacoustic effects of low-frequency noise and infrasound, propagating through air, on humans and animals.     

Effects of shear stress on endothelial cells: possible relevance for ultrasound applications

This review forms part of a series of papers resulting from a workshop on safety of ultrasound applications. The physical effects of ultrasound include generation of steady streaming in large fluid volumes, and micro-streaming around contrast bubbles. Such streaming induces shear stress acting on the vascular endothelium. This review provides a discussion on the levels of endothelial shear stress associated with diagnostic ultrasound applications, and on the biological effects of shear stress acting on the endothelial cells. Depending on vessel size and ultrasound characteristics, shear stresses associated with streaming and micro-streaming may exceed the physiological levels associated with the flow of blood by many orders of magnitude. The resulting biological effects could range anywhere from activation of normal shear stress sensors such as ion channels, damage of the endothelial surface layer, reversible perforation of the membrane, to cell detachment and lysis. The possible presence of such biological effects does not necessarily mean that the effects are harmful for the individual. However, considering the ever-increasing use of ultrasound, a further investigation into these shear stress-related effects, using both experiments and modelling, is desired. Apart from safety concerns, such effects may provide a base for strategies aimed at targeted delivery of drugs.     

Ultrasound and matter--physical interactions

The basic physical characteristics of ultrasound waves are reviewed in terms of the typical displacements, velocities, accelerations and pressures generated in various fluid media as a function of frequency. The effects on wave propagation of interfaces are considered, and the way in which waves are reflected, transmitted and mode converted at interfaces introduced. Then the nonlinear propagation of high amplitude ultrasound is explained, and its consequences, including the generation of harmonic frequencies and enhanced attenuation, considered. The absorption of ultrasonic waves and the resulting heat deposition in absorbing media are described together with factors determining the resulting temperature rises obtained. In the case of tissue these include conduction and perfusion. The characteristics of cavitation in fluid media are also briefly covered. Finally, secondary nonlinear physical effects are described. These include radiation forces on interfaces and streaming in fluids.     

超声波均质化对仙台病毒ELISA测试中抗原稳定性的影响

目的研究超声波均质化（homogenization）处理对于仙台病毒在ELISA测试中抗原稳定性的影响。方法对BHK-21细胞内扩增培养的仙台病毒通过差速离心,富集后使用超声波做均质化处理,和未处理的病毒分别包被酶标板,使用标准免疫小鼠血清和SPF小鼠血清对包被平板进行测试,比较样品测试孔间测量数据的变异率。结果对免疫血清做梯度稀释,各梯度样品在未经超声处理仙台病毒抗原包被ELISA检测中测量值的变异率在1.97%～6.02%之间;在经超声处理仙台病毒抗原包被ELISA检测中测量值的变异率在0.53%～2.26%之间;SPF血清测量值的变异率均高于免疫血清;所有样品在经超声处理的病毒抗原包被ELISA检测中测量值的变异率均较小。结论超声波处理有效的提高了仙台病毒抗原的均质性,在ELISA测试中提高了抗原的稳定性。     

Induction of adhesion-dependent signals using low-intensity ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with α(5)β(1)-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes. The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal, causes integrin redistribution, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions, and thereby induces directional migration. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement. In this protocol we describe the method by which 30 mW/cm(2), 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair. The method uses recombinant fibronectin fragments to engage α(5)β(1)-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts. The positive effect of ultrasound on repair mechanisms is well documented, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.     

超声引导与传统PICC 置管术在老年患者中应用的对比性研究

目的:比较超声引导下PICC置管术与传统PICC置管术在老年患者中的应用情况,评价其效果。方法:回顾性分析2009年1月至2010年12月期间解放军总医院南楼老年患者PICC置管情况,记录超声引导及传统PICC置管两种方法的成功率与并发症情况。结果:超声引导组共512例次,传统PICC组共384例次,超声引导组均选择肘上静脉置管,传统PICC置管组均选择肘下静脉;超声引导组一次置管成功率及总成功率分别为96.7%,99.6%,传统PICC组分别为78.6%,86.2(P<0.01);超声引导组相关并发症发生率5.9%,对照组为12.0%(P<0.01)。结论:采用超声引导下PICC穿刺置管术显著提高了置管成功率,降低了PICC相关并发症发生率,与传统PICC组比较优势明显,值得临床推广。     

肿瘤的超声波治疗

文中就超声波热疗,高强度聚焦超声以及低频超声波在肿瘤治疗中的应用进行了描述。早期主要是利用超声波的热效应来治疗肿瘤,近年来兴起的高强度聚焦超声是热疗法的另一发展。它瞬间使肿瘤组织温度升至65℃以上,导致靶区组织凝固和坏死来达到对肿瘤的“热切除”,是一种安全、有效的肿瘤治疗手段,具有无限潜力。尽管低频超声治疗肿瘤的机制尚不明了,但因其可以诱导细胞凋亡,为肿瘤的治疗提供了新的途径,其治疗作用已受到重视。     

Free Radical Formation by Ultrasound in Aqueous Solutions. A Spin Trapping Study

Our recent spin trapping studies of free radical generation by ultrasound in aqueous solutions are reviewed. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions exposed to ultrasound lead to the thermal dissociation of water vapor into H atoms and OH radicals. Their formation has been confirmed by spin trapping. Sonochemical reactions occur in the gas phase (pyrolysis reactions), in the gas-liquid interfacial region, and in the bulk of the solution (radiation-chemistry reactions). The high temperature gradients in the interfacial regions lead to pyrolysis products from non-volatile solutes present at sufficiently high concentrations. The sonochemically generated radicals from carboxylic acids, amino acids, dipeptides. sugars, pyrimidine bases. nucleosides and nucleo-tides were identified by spin trapping with the non-volatile spin trap 3.5-dibromo-2.6-dideuterio-4-nitrosobenzenesulfonate. At low concentrations of the non-volatile solutes. the spin-trapped radicals produced by sonolysis are due to H atom and OH radical reactions. At higher concentrations of these non-volatile solutes, sonolysis leads to the formation of additional radicals due to pyrolysis processes (typically methyl radicals). A preferred localization of non-volatile surfactants (compared to analogous non-surfactant solutes) was demonstrated by the detection of pyrolysis radicals at 500-fold lower concentrations. Pyrolysis radicals were also found in the sonolysis of aqueous solutions containing only certain nitrone spin traps. The more hydrophobic the spin trap, the lower the concentration at which the pyrolysis radicals can be observed. The effect of varying the temperature of collapsing transient cavities in aqueous solutions of different rare gases and of N₂O on radical yields and on cell lysis of mammalian cells was investigated.     

Comparison of ultrasound and skinfold measurements in assessment of subcutaneous and total fatness

Ultrasound (A-scan mode) and skinfold methods were evaluated in the measurement of subcutaneous fat thickness and prediction of total fat weight (by whole body potassium counting). Based on intraobserver correlations on 39 men at 15 body sites, skinfold caliper measurements were more reproducible than ones obtained by ultrasound. Measurements made with the two techniques at the same site typically produced different mean estimates of fat thickness. However, scores were often highly correlated with each other, indicating similar relative rankings of subjects by each technique. Skinfolds were more highly correlated with total fat weight than were ultrasound measurements, but body weight and anthropometric measures had even higher correlations with total fat weight. Anthropometric measurements were highly correlated with fatness because of their association with body weight, and when this relationship was statistically controlled for, they typically lost their predictive effectiveness. Multiple regression analyses revealed that the best predictors of fat weight were body weight along with skinfold and ultrasound measurements. These results suggest that skinfolds are a more effective means of assessing subcutaneous fat than ultrasound, especially when the large difference in cost of equipment is considered.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.