Presence Of Microbubbles Research Articles

Influence of fluid shear and microbubbles on bacterial detachment from a surface.

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s(-1)) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.

A basic study on sonoporation with microbubbles exposed to pulsed ultrasound.

Sonoporation is an ultrasound technique that enables large molecules that normally do not penetrate the cell membrane to pass through it. Recent studies show that pulsed ultrasound in the presence of microbubbles increases the permeability of the cell membrane. However, the mechanism and basic properties of this sonoporation remain unclear. We thus investigated the mechanism of generation and frequency of occurrence of sonoporation, as well as the repair of a cell membrane damaged by microbubbles. The spatial relationship between microbubbles and cells was observed microscopically when cells were sonicated with pulsed ultrasound. Effects of microbubbles on the cells were observed with a high-speed camera, and the ratio of cell membrane damage and repair was examined using fluorescent microscopy. Damage to the cell membrane, caused mainly by mechanical effects of the expansion and contraction of microbubbles, significantly increased the permeability of the cell membrane. The frequency of cell membrane damage was closely associated with the presence of microbubbles and increased with increase in acoustic pressure. The ratio of repair of damaged cells was about 70% during 3 min after a single shot of pulsed ultrasound, indicating that repair of damaged cell membranes requires little time. We examined the frequency of occurrence of cell membrane damage and repair in sonoporation using pulsed ultrasound and microbubbles. Our results should prove useful for improving pulsed-ultrasound sonoporation.

Continuous Monitoring of Middle Cerebral Artery Recanalization with Transcranial Color-Coded Sonography and Levovist

Treatment of acute ischemic stroke with intravenous recombinant tissue plasminogen activator (rtPA) is relatively ineffective in patients with large vessel occlusion. Numerous experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and acceleration of lysis by US is enhanced in the presence of microbubbles used as echo-contrast agents. The purpose of this study was to evaluate the feasibility of continuous monitoring of middle cerebral artery (MCA) recanalization using transcranial color-coded sonography (TCCS) and intravenously administered microbubbles. Recanalization of middle cerebral artery (MCA) mainstem occlusion was assessed using continuous monitoring with TCCS and intravenously administered galactose-based microbubbles (Levovist) in 8 consecutive patients with acute ischemic stroke treated with intravenous rt-PA within 3 hours of symptom onset. Recanalization at one hour occurred in 4 of 8 patients. The median NIHSS score was 21 (range 10 to 28) at baseline, 15 (range 0 to 24) at 1 h, and 11 (range 0 to 22) at 24 h. Asymptomatic hemorrhagic transformation (HT) was demonstrated on brain imaging in 6 patients. This study demonstrates the feasibility of continuous monitoring of MCA recanalization using TCCS and Levovist, in acute stroke patients. The findings suggest a high rate of asymptomatic HT in monitored patients. Although all HTs were asymptomatic and did not preclude early clinical improvement, particular attention should be given to the incidence and clinical significance of HT in future studies using these methods.

Membrane disruption by optically controlled microbubble cavitation.

In fluids, pressure-driven cavitation bubbles have a nonlinear response that can lead to extremely high core-energy densities during the collapse phase—a process underpinning phenomena such as sonoluminescence1 and plasma formation2. If cavitation occurs near a rigid surface, the bubbles tend to collapse asymmetrically, often forming fast-moving liquid jets that may create localized surface damage3. As encapsulated microbubbles are commonly used to improve echo generation in diagnostic ultrasound imaging, it is possible that such cavitation could also lead to jet-induced tissue damage. Certainly ultrasonic irradiation (insonation) of cells in the presence of microbubbles can lead to enhanced membrane permeabilization and molecular uptake (sonoporation)4,5,6,7, but, although the mechanism during low-intensity insonation is clear8, experimental corroboration for higher pressure regimes has remained elusive. Here we show direct observational evidence that illuminates the energetic micrometre-scale interactions between individual cells and violently cavitating shelled microbubbles. Our data suggest that sonoporation at higher intensities may arise through a synergistic interplay involving several distinct processes.

Impact of bubbles on ultrasound safety

Microbubbles in a medium function to concentrate the energy of the ultrasound to which it is exposed, and the focused energy has thermal, chemical and mechanical effects on surrounding biological tissue. Thermal effects: When a microbubble vibrates, it extracts energy from the ultrasound to which it is exposed. Part of this power is used for re-radiation of acoustic waves, but most of the extracted power is converted into heat due to adiabatic compression of encapsulated gas, viscosity of surrounding water and friction of the shell. Generated heat can cause an additional temperature rise in biological tissue. Chemical effects: The presence of microbubbles reduces the pressure threshold to produce inertial cavitation bubbles. Symmetrical contraction of an inertial cavitation bubble will result in very high temperature and pressure inside the bubble and generate highly reactive sonochemicals. Mechanical effects: Behavior of microbubbles exposed to ultrasound can cause mechanical stress to the surrounding tissue. Asymmetric contraction of an inertial cavitation bubble generates a microjet-like small stream that has a localized but unfavorable mechanical effect, such as membrane damage of a cell.

Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles

Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings—fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma. (E-mail: nsheikov@bwh.harvard.edu)

A study on premature ventricular contractions caused byultrasound exposure with microbubbles using cultured ventricular muscle cells

It has been shown that diagnostic ultrasound examination using a contrast agent cancause premature ventricular contractions (PVCs). In this study, we investigated a usefulness ofa new technique using cultured cardiac myocytes to study mechanisms of PVC production.Cardiac myocytes were isolated from neonatal rats and cultured on a cover glass. The coverglass was attached to an observation chamber in which it was possible to observe changes inmyocytes during ultrasound exposure. In the experiments, cardiac myocytes were exposed topulsed ultrasound in the presence and absence of microbubbles. The pressure amplitudes(peak-negative pressures) were set at 5 steps, −0.28, −0.55, −0.73, −0.92 and −1.1 MPa,and threshold pressure to produce a PVC was recorded. The results showed that the presence ofmicrobubbles attached to a cell reduces threshold pressure for producing PVCs, and it wasconcluded that our method is useful for studying the mechanisms of PVC production.

Contrast-enhanced ultrasound: beyond the liver.

Most of the work and interest in CEUS has been focused on the liver, and therefore this technique has been established as a reliable tool for detection and characterization of liver lesions. However, microbubbles are distributed within the blood and appear wherever there is blood supply. The properties of Sono Vue and the high sensitivity of recent ultrasound equipment to the presence of microbubbles have shown that CEUS is potentially very useful in many organs and vascular structures. During the study of new possible applications of this technique, we found areas where CEUS, in selected cases, contributes significantly towards the diagnosis. Of these, the field of parenchymal trauma may at present be one of the most promising applications deserving more extensive study with the prospect of giving CEUS a central part in standard trauma procedures.

Harmonic US in hepatobiliary disease

Harmonic ultrasound (US) is a new sonographic imaging method that exploits the effect of nonlinear propagation on the acoustic signal as it travels through the human body or tissue. It produces sonographic images by using second harmonic signals generated by tissue itself during this nonlinear propagation of insonated acoustic energy. The resultant advantages are improved lateral resolution, reduced side lobe artifact, and increased signal-to-noise ratio. Our recent clinical studies using tissue harmonic US showed that tissue harmonic US provided significantly better image quality in both solid and cystic hepatic structures than conventional imaging, and enabled significantly improved image quality and improved diagnostic accuracy in evaluation of gallbladder disease. Harmonic US enhances the signals from contrast over those from tissue, since the contrast agent microbubbles generate far more harmonic energy than tissue does. New promising contrast-enhanced US techniques are pulse inversion harmonic, coded harmonic angio (CHA), and agent detection imaging (ADI). CHA uses codes to suppress the fundamental signal, and uses decoding techniques optimized for contrast agents, to suppress tissue signal, thus, improving sensitivity and uniquely optimizing for contrast signal visualization in dynamic flow states. ADI is a microbubble-specific mode that is designed for optimal detection of stimulated acoustic emission from a fragile agent such as Levovist, and is characterized as its high mechanical index that provides extreme sensitivity to the presence of microbubble. Our recent clinical experience of contrast-enhanced harmonic US with Levovist will be presented in various diseases of hepatobiliary system.

Microbubble-augmented Ultrasound Declotting of Thrombosed Arteriovenous Dialysis Grafts in Dogs

Transcutaneous low-frequency ultrasound (LFUS) can effectively lyse clots in the presence of microbubbles. This study was designed to test the commercially available human albumin microspheres injectable suspension octafluoropropane formulation, Optison, to establish efficacy and assess US parameters of intensity and wave modes in a canine model of a thrombosed arteriovenous (dialysis) graft. Arteriovenous grafts in five dogs were cannulated, temporarily ligated, and thrombosed. Different declotting techniques were randomized to treat nine groups. Control groups involved direct saline (4.5 mL) clot injection in 0.5-1.0-mL increments. One group underwent peripheral intravenous microbubble injection (13.5 mL). Six groups underwent direct incremental clot injection of 4.5 mL of microspheres with LFUS for 30 minutes in 3-5-minute increments with use of various intensity settings in continuous-wave and pulsed-wave (PW) modes. At each increment, angiography was used to grade flow, declotting, and overall success. One hundred four procedures showed success in all 24 high-intensity PW modes (1.2-2.0 W/cm(2)); only one of 20 control experiments was successful (P <.0001). Medium-intensity modes yielded intermediate success rates. Lowest-intensity direct-injection groups and intravenous and control groups ranked lower. Results at 30 minutes were better than at 15 minutes (P <.0001). LFUS with direct injection of microbubbles is effective in lysing moderate-sized clots and recanalizing thrombosed arteriovenous grafts. It best succeeds at the higher range of intensity settings tested in PW mode. Further development is justified.

Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats

The generation of ultrasound (US) bioeffects using a clinical imaging system is controversial. We tested the hypothesis that the presence of microbubbles in the US field of a medical imager induces biologic effects. Both kidneys of anesthetized rats were insonified for 5 min using a medical imaging system after the administration of microbubbles. One kidney was insonified using a continuous mode (30 Hz) and the opposite kidney was insonified using an intermittent (1 Hz) technique. The microbubbles were exposed to three different transducer frequencies and four transducer output powers. After insonification, the animals were euthanized, the kidneys were removed and their gross appearance scored under “blinded” conditions using a defined scale. After the administration of microbubbles, US imaging of the kidney caused hemorrhage in the renal tissue. The severity and area of hemorrhage increased with an increase in the transducer power and a decrease in the transducer frequency. Intermittent insonification in the presence of microbubbles produced a greater degree of renal hemorrhage than continuous imaging techniques. (E-mail: james.wible@tycohealthcare.com)

Temperature Effects on the Gas Hold-up in Agitated Vessels

Previous work found the gas hold-up in a tall, 0.15 m3 vessel, agitated by three impellers, to decrease considerably with temperature. This was attributed to a decrease in liquid viscosity, a decrease in surface tension or a decrease in gas density. The aim of this work was to investigate the influence of gas density on the gas hold-up. First of all, it was attempted to reproduce the data found in literature. It was shown that contaminants had a significant impact on the gas hold-up. Experiments with increasing and decreasing amounts of tap water (as the source of the contamination) found the gas hold-up to be affected by a strongly absorbing non-ionic surface active agent. Experiments in a system exhibiting low conductivity and low gas hold-up showed that the gas hold-up decreased by 15% for temperatures between 295 and 350K. Experiments with helium as sparging gas found that gas density does have a lowering effect on the gas hold-up. The gas hold-up did not change for temperatures above 350K, which may be due to the increased presence of micro-bubbles in a near-boiling system.

Effectiveness of transcranial and transthoracic ultrasound and microbubbles in dissolving intravascular thrombi.

To examine the effectiveness of 1 -MHz and 40-kHz ultrasound with and without microbubbles in fragmenting thrombi in attenuated conditions. First, an vitro transcranial model was used to examine the ability of these frequencies to fragment thrombi in the presence or absence of perfluorocarbon-exposed sonicated dextrose albumin microbubbles. Second, an in vivo transthoracic model was used to test the effectiveness of these same frequencies with intravenous perfluorocarbon-exposed sonicated dextrose albumin in fragmenting left circumflex coronary thrombotic occlusions. In the in vitro model, both transcranial 1-MHz and 40-kHz ultrasonic frequencies were effective at fragmenting thrombi only in the presence of microbubbles. In the in vivo model, 1-MHz ultrasound with intravenous perfluorocarbon-exposed sonicated dextrose albumin angiographically recanalized only 4 of 14 occlusions but was consistently effective at improving myocardial blood flow to the risk area even in the absence of angiographic recanalization. Both 40-kHz and 1-MHz ultrasound with perfluorocarbon-exposed sonicated dextrose albumin improved regional wall-thickening and electrocardiographic abnormalities (P < .05 compared with control or ultrasound alone). Transcranial and transthoracic ultrasound in the presence of intravenous microbubbles can improve flow to ischemic regions and should be considered as a supplement to current pharmacologic therapy.

Bubbles and microcirculatory disorders.

Doppler methods are in daily clinical use for the assessment of blood flow in various vessels. With the adjunct of an ultrasound (US) contrast agent, weaker signals, for instance in smaller vessels, can be detected. However, even in the presence of an US contrast agent, colour, spectral or even power Doppler can only detect blood flow in vessels exceeding 200‐300 µm. The microvasculature is not detectable because the flow velocity in capillaries, at approximately 1 mm/s, is actually lower than the tissue motion velocity and is therefore filtered out by the equipment. It was recently demonstrated that using US contrast agents, together with novel bubble-specific imaging modes, allows the blood flow detection threshold to be lowered and the presence of microbubbles even in very small vessels to be detected. Detection of flow in such smaller vessels increases considerably the clinical utility of US, in particular for the detection of reductions in vascularity, for instance in ischaemic or infarcted tissues, or increases in vascularity, for instance in tumor angiogenesis. These bubble-specific methods, which have emerged from extensive research carried out on US contrast agents during the last decade, make use of the nonlinear properties of bubbles. Harmonic B-mode, harmonic power Doppler, phase or pulse inversion, power pulse inversion, coherent contrast imaging, and power modulation are some of the most popular of these novel imaging methods. By detecting specifically bubble signals and reducing the background signals from the surrounding structures, these methods dramatically enhance the image contrast-to-tissue ratio. Studies making use of contrast agents in combination with harmonic power Doppler have shown that vessels of approximately 40 µm in diameter can be detected in the kidney [1]. Pulse inversion imaging provides an even better resolution than harmonic imaging. Moreover, it allows the use of low-power pulses, thus preventing bubble destruction. In the following article, the new opportunities offered by the combination of new US contrast agents and novel imaging modes will be illustrated by a number of studies carried out with SonoVue, a novel ultrasound contrast medium constituted by phospholipid-stabilized microbubbles of sulfur hexafluoride (SF6) [2].

Sonar performance in turbid and bubbly environments

The presence of both solid particles and gas bubbles in coastal waters may have a significant effect on sound propagation, particularly at high frequencies, and may therefore be partially responsible for the observed variability in high-frequency sonar performance in shallow waters. Suspended particles increase volume attenuation through the processes of thermo-viscous absorption and scattering. Microbubbles also attenuate sound through viscous and thermal dissipation and scattering. The presence of microbubbles in the water column may also modify the sound speed and result in a dispersive medium. The effects of dilute suspensions of solid particles on the sound speed may generally be neglected for practical sonar applications. Algorithms for estimating the acoustic absorption coefficient and the speed of sound in water containing suspensions of fine mineral particles and populations of microbubbles have been developed. These have been incorporated into a sonar performance prediction model based on the ray method. Results are presented from this model which show that, for populations of suspended mineral particles and microbubbles which are commonly encountered in shallow-water environments, the predicted sonar performance is significantly different from that predicted when the additional effects are not taken into account. []

Microbubbles associated with mitral valve prostheses in pediatric patients

Objectives: To assess whether microbubbles are associated with mitral valve prostheses in children and to investigate their relationship to exercise. Background: Bright, highly mobile echoes are seen in left heart chambers of patients with mechanical mitral valve prostheses. The clinical importance of those microbubbles is not yet known but they survive long enough to reach the cerebral circulation. No such studies have been reported in children. Methods: There were 20 male and 10 female patients with a mean age of 10.4 years (range 2–16 years). Transthoracic echocardiography with calculation of cardiac output and ejection fraction was performed at baseline and after exercise following mechanical mitral valve replacement. Studies were evaluated for the presence of microbubbles. The mean time from operation to time of study was 18 months (range 0.2–89 months) Results: Of the 30 children evaluated, 28 (93%) had evidence of microbubbles. Exercise increased the number of microbubbles in 15 of the 26 children. This was positively associated with an increase in cardiac output. Conclusions: (1) Microbubbles are common observed in children with mechanical mitral prostheses and are augmented by exercise. (2) This phenomenon is of a special concern in children due to their expected longevity with the prosthesis.

Bacterial stress responses to 1-megahertz pulsed ultrasound in the presence of microbubbles.

Members of a panel of stress-responsive biosensors have been used to study the effect of megahertz frequency ultrasound on Escherichia coli. Insonification causes acoustic cavitation, the collapse of oscillating microbubbles in solution, which can damage bacterial cells. A focused 1-MHz ultrasound transducer, capable of generating a spatial peak pulse average intensity of 500 W/cm2, was used to treat liquid bacterial cultures. Stress-responsive promoters fused to luxCDABE allowed the continuous measurement of light produced as a result of protein damage, DNA damage, oxidative stress, and membrane perturbation. A promoter responsive to ammonia limitation was not transcriptionally activated under test conditions. In contrast to bacteria in exponentially growing cultures, those in stationary-phase cultures were more resistant to the effects of ultrasound treatment. Quantification of the degree of acoustic cavitation due to symmetric bubble collapse was measured by a 20-MHz passive transducer, the output of which appears to be only partially correlated with cellular damage and survival. The methods and results summarized here provide the basis for further investigation into applications, including the purification of water samples.

Acoustical nonlinearity parameter of liquids with microbubbles

The gas bubbles in liquid are the strong scatters of the sound propagation in such liquid. A liquid containing microbubbles is used as an effective ultrasound contrast agent in medical diagnosis to improve the contrast of the ultrasonic image. However, the existence of bubbles in liquid may enhance its nonlinearity parameter. In our previous paper, the preliminary results of some ultrasound contrast agents were reported and their large values of the nonlinearity parameter were obtained. This paper is devoted to an experimental demonstration of the influence of microbubbles on the nonlinearity parameter B/A values. These include measuring the dependence of B/A values of Echovist 300 on time. Results show that B/A values decrease with time (from 2678 to 16 over 45 min). B/A values of human blood with a different portion of Echovist were also studied. Larger B/A values are obtained with more Echovist. B/A values of a kind aqua, such as 76% Injectio Meglumini Diatrizoatis Composita (MDC), were studied before and after sonication with different bubble contents. Results indicate that the values of nonlinearity parameter depend on the presence of microbubbles obviously. Some explanation of this effect is discussed. [Work supported by NSF of China.]

Tissue harmonic imaging in cardiology and radiology applications

Harmonic imaging, the formation of ultrasound images from the harmonic components of the sonification signal, has been used in diagnostic ultrasound applications that utilize microbubble contrast agents. In the presence of microbubbles the harmonic signals are mainly due to nonlinear bubble oscillations. Recently, harmonic imaging has been applied without contrast agents, the harmonic signals arising from nonlinear propagation. Near-field artifacts and aberrations are reduced, and tissue borders are enhanced. The harmonic signals are at a lower amplitude and suffer more attenuation than the fundamental. However, the wide dynamic range, digital architecture, and signal processing capabilities of modern diagnostic ultrasound systems make it possible to utilize this tissue-generated harmonic energy for image formation. The nonlinear parabolic wave equation is used to model tissue propagation. A time domain numerical solution for a nonaxisymmetric source function is presented. Theory and experiments demonstrate harmonic generation in tissues. In vitro animal tissue experiments and in vivo collected radio frequency data are shown. A novel broadband pulsing scheme that uses two pulses with a phase difference of 180° is introduced, which results in cancellation of the propagated fundamental component and doubling of the second harmonic. Cardiac and abdominal harmonic images are shown.

New prospects for ultrasound contrast agents

Considering the several suggestions regarding the future developments of echocontrast agents, there is a striking difference between the few compounds actually available on the market and used in clinical practice and those undergoing experimental clinical trials. It is therefore difficult to predict what will be the actual impact of these agents in the next future. Future developments will probably go beyond color enhancement which was the end-point till a very short time ago. They can be schematically summarized as follows: (1) development of new substances which enhance both color and gray scales; (2) use of new-generation substances, such as BR1™ (Bracco, Milan, Italy) and EchoGen™ (Sonus, Bothell, WA), which use a gas other than air, such as perfluorate compounds which are more stable and gurantee longer and stronger effects; (3) use of more complex compounds acting at different levels. For example, SHU 536A (Sonovist) produces resonance phenomena with the second and third harmonics, and also stimulated acoustic emission which permits the morphological study of liver parenchyma. Other promising compounds are liposomes and aerosomes. Among the new possibilities in recording and observing phenomena, we can distinguish two main application fields: one is based on the physics of ultrasound and related to the presence of microbubbles in an acoustic field. These phenomena are generally obtained increasing the emission acoustic pressure, which eventually results in microbubble destruction and they are called nonlinear because there is no direct relationship between emission and return frequencies. These phenomena, which are detectable only with dedicated equipment, include: the resonance phenomenon with harmonic emission; intermittent harmonic emission and stimulated acoustic emission. The other application field is not strictly related to ultrasound physics and includes all the systems which can detect the presence of microbubles qualitatively or quantitatively. Other possible applications are related to the possibility of acquiring not only morphological but also functional data, especially in cardiology and neurology. Finally, targeted agents are potentially capable of demonstrating receptor sites or specific molecules, which may open very interesting therapeutic routes.