Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects.

The purpose of this study was to develop a unified model capable of explaining the mechanisms of interaction of ultrasound and biological tissue at both the diagnostic nonthermal, noncavitational (<100 mW · cm(-2)) and therapeutic, potentially cavitational (>100 mW · cm(-2)) spatial peak temporal average intensity levels. The cellular-level model (termed "bilayer sonophore") combines the physics of bubble dynamics with cell biomechanics to determine the dynamic behavior of the two lipid bilayer membrane leaflets. The existence of such a unified model could potentially pave the way to a number of controlled ultrasound-assisted applications, including CNS modulation and blood-brain barrier permeabilization. The model predicts that the cellular membrane is intrinsically capable of absorbing mechanical energy from the ultrasound field and transforming it into expansions and contractions of the intramembrane space. It further predicts that the maximum area strain is proportional to the acoustic pressure amplitude and inversely proportional to the square root of the frequency (ε A,max ∝ P(A)(0.8f - 0.5) and is intensified by proximity to free surfaces, the presence of nearby microbubbles in free medium, and the flexibility of the surrounding tissue. Model predictions were experimentally supported using transmission electron microscopy (TEM) of multilayered live-cell goldfish epidermis exposed in vivo to continuous wave (CW) ultrasound at cavitational (1 MHz) and noncavitational (3 MHz) conditions. Our results support the hypothesis that ultrasonically induced bilayer membrane motion, which does not require preexistence of air voids in the tissue, may account for a variety of bioeffects and could elucidate mechanisms of ultrasound interaction with biological tissue that are currently not fully understood.

Low intensity ultrasound perturbs cytoskeleton dynamics.

Therapeutic ultrasound is widely employed in clinical applications but its mechanism of action remains unclear. Here we report prompt fluidization of a cell and dramatic acceleration of its remodeling dynamics when exposed to low intensity ultrasound. These physical changes are caused by very small strains (10-5) at ultrasonic frequencies (106 Hz), but are closely analogous to those caused by relatively large strains (10-1) at physiological frequencies (100 Hz). Moreover, these changes are reminiscent of rejuvenation and aging phenomena that are well-established in certain soft inert materials. As such, we suggest cytoskeletal fluidization together with resulting acceleration of cytoskeletal remodeling events as a mechanism contributing to the salutary effects of low intensity therapeutic ultrasound.

A novel ischemia reperfusion injury hereditary tissue model for pressure ulcers progression.

Ischemia reperfusion injury (IRI) involvement in pressure ulcers (PU) progression via a surge of oxidative stress and inflammatory responses is well documented. IRI strongly depends on the mechanical loading history. We present a generalized IRI model considering external loading, dynamic tissue healing capacity, accumulating mechanical and reperfusion-mediated damages and competing repair processes of saturating nature. Reperfusion depends on strain and strain rate to enhance loading history sensitivity. Tissue-specific ulceration susceptibility is assumed dependent on variable accumulated damage. We study damage evolution under cyclic loading having several strain expulsion profiles and demonstrate load relief history has critical impact on PU progression. Abrupt load removal generally follows existing models representing extreme repair/damage. We show (first time in silico) that under certain conditions (previously experimentally identified), IRI becomes repairing rather than damaging. In particular, we recapitulate the preconditioning and postconditioning IRI hallmarks. Finally, it is customary among physicians and nurses to promptly alleviate mechanical load applied to patients lying in bed for extended periods and in risk of developing PUs. We demonstrate this practice can be harmful. If load removal is performed early while reperfusion is still beneficial, then this conduct is suitable. However, if critical tissue damage has been crossed, then abrupt expulsion can constitute the worst-case scenario for patient outcome. If no preliminary patient documentation is available, we recommend gradual load removal since risks of accelerated damage eventually leading to ulceration supersede the improved repair potential benefit.

The beginning of a new era: treatment of erectile dysfunction by use of physical energies as an alternative to pharmaceuticals.

This introductory manuscript aims to familiarize the concept of the ability of certain forces or energies applied on the penis. This concept is described and discussed in more detail for three optional applicative energies; shock wave energy via mechano-transduction, ultrasound energy via its theoretical unique effect on the cellular membrane, specifically cyclic separation of the two phospholipid layers, creating biochemical, functional and structural tissue changes. Radio frequency energy via its heating effect is proven to induce immediate changes on collagen strucures and on realignment of collagen fibers, as well as induction of local vasodilation. Applying any of these energies on the erectile tissue may potentially affect biochemical processes, which through different mechanisms lead to a beneficial clinical effect on erectile function.

Bubble growth within the skin by rectified diffusion might play a significant role in sonophoresis.

Low frequency ultrasound has successfully been used for enhancing transdermal transport of a variety of different molecules. This phenomenon is referred to as sonophoresis. Several attempts have been made to investigate the enhancing mechanism in order to modulate the overall process. In this study we assess whether rectified diffusion is a process that occurs within the skin, which could eventually lead to channeling and thereby to transdermal sonophoresis. The model presented in this paper is based on the following postulate: gas bubbles are randomly distributed within the lipid bilayers of the stratum corneum (SC). As the skin is subjected to ultrasound, gas bubbles grow by rectified diffusion. During this period, bubbles may merge with the outer or inner boundaries of the SC, or merge with neighboring bubbles. Eventually, channels are created, allowing drugs to easily penetrate through the most significant barrier to transdermal delivery, the SC. As a result, transdermal transport rate is enhanced. In this work, a mathematical model has been formulated, in which permeability enhancement of the SC is linked to channels, possibly created by means of rectified diffusion. Sonophoresis may result from various mechanisms that act in synergy. The present model predicts that rectified diffusion might be one of the factors that lead to sonophoresis during ultrasound treatment.

Ultrasound-induced angiogenic response in endothelial cells.

Mechanical forces are known to affect endothelial cell (EC) function and can promote the formation of mature, muscular arterioles and arteries (arteriogenesis). The present study explores the possible angiogenic role of ultrasonic irradiation on EC phenotype using an in-vitro approach. Therapeutic ultrasound (TUS) stimulation at 1-MHz frequency was applied to bovine aortic endothelial cells (BAECs) in 2-D monolayer cultures and 3-D spheroid cultures. An angiogenic EC phenotype was characterized by the proliferation rate, migration, sprouting and Flk-1 expression in response to ultrasound stimulation. Irradiation lasting as long as 30 min caused a down-regulation and redistribution of Flk-1, increased EC proliferation rates and enhanced migration and sprouting in the 3-D spheroid cultures. The ultrasound-mediated EC stimulation in monolayers may be attributed to stable cavitation and micro-streaming, which are induced by pulsating microbubbles near the EC surface. Three-dimensional EC spheroid cultures surrounded by a highly viscous gel phase also exhibited ultrasound-induced angiogenic characteristics, although microbubbles may not participate in this response because of the impeding medium. The described in-vitro influence of low-intensity ultrasound on angiogenic EC phenotype has implications for TUS as a safe and controlled noninvasive stimulus for vascular regeneration.

Subharmonic response of encapsulated microbubbles: conditions for existence and amplification.

The response of encapsulated microbubbles at half the ultrasound insonation frequency, termed subharmonic response, may have potential applications in diagnosis and therapy. The subharmonic signal, emitted by Definity microbubble cloud sonicated by ultrasound was studied theoretically and experimentally. The size distribution of the microbubbles was optically analyzed and resonance frequency of 2.7 MHz was determined. An asymptotic model has been developed that generates subharmonic response of a single and of a cloud of bubbles of various sizes. Threshold conditions for existence and the intensity of the subharmonic signal are predicted to depend on microbubbles size distribution and shell properties, as well as on the driving field frequency and pressure. Thin tubes filled with Definity solution were insonated at acoustic pressures from 100 to 630 kPa. The intensities of the emitted fundamental harmonics and subharmonics were measured. At frequency 5.5MHz, twice the resonance frequency, the subharmonic signals were observed only at pressures greater than 190 kPa. The subharmonic to fundamental harmonics intensity ratio was within -12 to -1 dB. The experimental results showed good correlation with the theoretical results in the range of validity of the asymptotic solution, thus supporting the model assumptions.

Cavitation bioeffects.

Acoustic cavitation takes place when tiny gas bubbles oscillate, grow, and collapse in liquid under the influence of ultrasonic field. This study reviews cavitation bioeffects that are found both in vivo and in vitro when exposed to either low- or high-power acoustics. Proposed mechanisms are discussed here as well based on theoretical studies, simulations and test bench experiments. Bioeffects are induced in living tissue once the gas bubble is, for instance, within a blood vessel in close vicinity to the endothelium or to the red blood cells. Conditions for inducing various bioeffects are discussed - from severe damage, such as cell necrosis, to delicate alterations, such as increased permeability of cell membrane. Present and potential applications for therapeutic purpose from stone pulverization and tissue ablation to gene transfection and transdermal delivery are reviewed including the growing use of artificial microbubbles.

Stability of an encapsulated bubble shell.

The stability of an encapsulated bubble filled with gas is studied where gas is allowed to diffuse out of the bubble. A mechanistic model that takes into account shell stiffness and surface tension is considered. A critical shell radius for loss of mechanical stability is derived based on a technique adapted for small radius, where surface tension effects become substantial. A new parameter is defined that determines the relative importance of surface tension forces and shell stiffness for shell stability. The developed technique allows to predict, for a given bubble population and gas saturation level of the surrounding liquid, a range of bubble sizes which may collapse in time. Surface tension effects are dominant in determining the critical radius but have a negligible effect on the minimal radius for collapse. The influence of the surface tension on the stability of the shell is illustrated for Optison, a typical ultrasound contrast agent.

Cell-Type-Selective Effects of Intramembrane Cavitation as a Unifying Theoretical Framework for Ultrasonic Neuromodulation.

Diverse translational and research applications could benefit from the noninvasive ability to reversibly modulate (excite or suppress) CNS activity using ultrasound pulses, however, without clarifying the underlying mechanism, advanced design-based ultrasonic neuromodulation remains elusive. Recently, intramembrane cavitation within the bilayer membrane was proposed to underlie both the biomechanics and the biophysics of acoustic bio-effects, potentially explaining cortical stimulation results through a neuronal intramembrane cavitation excitation (NICE) model. Here, NICE theory is shown to provide a detailed predictive explanation for the ability of ultrasonic (US) pulses to also suppress neural circuits through cell-type-selective mechanisms: according to the predicted mechanism T-type calcium channels boost charge accumulation between short US pulses selectively in low threshold spiking interneurons, promoting net cortical network inhibition. The theoretical results fit and clarify a wide array of earlier empirical observations in both the cortex and thalamus regarding the dependence of ultrasonic neuromodulation outcomes (excitation-suppression) on stimulation and network parameters. These results further support a unifying hypothesis for ultrasonic neuromodulation, highlighting the potential of advanced waveform design for obtaining cell-type-selective network control.

Time and pressure dependence of acoustic signals radiated from microbubbles.

Encapsulated microbubbles are considered to be microsensors for in vivo blood pressure measurements in the cardiovascular system. To study the potential of this method, we developed a simulation and an experimental set-up that relate various characteristics of radiated acoustic signals from the microbubbles to the varying ambient pressure. Both the simulation and the experiment show that the radiated pressure from microbubbles generates a significant subharmonic component, which is modulated by changes in the ambient pressure. A time-dependent decrease of the steady-state radii within a population of microbubbles causes a phase reversal phenomenon, which explains the observed time delay in the build-up of the subharmonic modulation response. Additionally, we identify a frequency-capturing effect that indicates the termination of the nonlinear behavior of the microbubbles. Our research suggests that these subharmonic signals can be used for in vivo blood pressure measurements and highlights some of the considerations that need to be addressed in developing such techniques.

Ultrasound attenuation by encapsulated microbubbles: time and pressure effects.

Ultrasound (US) contrast agents (UCA) consist of artificial encapsulated microbubbles filled with low-diffusivity gas. This study evaluated, both experimentally and theoretically, the behavior of a cloud of encapsulated microbubbles while the surrounding pressure was modified within the physiological range. The theoretical analysis included calculation of US attenuation caused by a bubble cloud. The radius and gas content of each bubble were determined from a solution of a diffusion problem. Shell permeability and rigidity were taken into account. Both experiments and theory demonstrated that, for fixed ambient pressures, higher pressures result in increased rate of attenuation decay. Pulsatile ambient pressure induces pulsations of attenuation of the same frequency. In general, theoretical predictions are in good agreement with experimental data.

Shear stress induced by a gas bubble pulsating in an ultrasonic field near a wall.

Some of the effects that therapeutic ultrasound has in medicine and biology may be associated with steady oscillations of gas bubbles in liquid, very close to tissue surface. The bubble oscillations induce on the surface steady shear stress attributed to microstreaming. A mathematical simulation of the problem for both free and capsulated bubbles, known as contrast agents, is presented here. The simulation is based on a solution of Laplace's equation for potential flow and existing models for microstreaming. The solution for potential flow was obtained numerically using a boundary integral method. The solution provides the evolution of the bubble shape, the distribution of the velocity potential on the surface, and the shear stress along the surface. The simulation shows that significant shear stresses develop on the surface when the bubble bounces near the tissue surface. In this case, pressure amplitude of 20 kPa generates maximal steady shear stress of several kilo Pascal. Substantial shear stress on the tissue surface takes place inside a circular zone with a radius about half of the bubble radius. The predicted shear stress is greater than stress that causes hemolysis in blood and several orders of magnitude greater than the physiological stress induced on the vessel wall by the flowing blood.

Extracorporeal acute cardiac pacing by high intensity focused ultrasound.

Ultrasound has been shown to produce Premature Ventricular Contractions (PVC's). Two clinical applications in which acute cardiac pacing by ultrasound may be valuable are: (1) preoperative patient screening in cardiac resynchronization therapy surgery; (2) Emergency life support, following an event of sudden death, caused by cardiac arrest. Yet, previously the demonstrated mean success rate of extra-systole induction by High Intensity Focused Ultrasound (HIFU) in rats is below 4.5% (Miller et al., 2011). This stands in contrast to previous work in rats using ultrasound (US) and ultrasound contrast agents (UCAs), where success rates of close to 100% were reported (Rota et al., 2006). Herein, bi-stage temporal sequences of accentuated negative pressure (rarefaction) and positive pressure HIFU transmission (insonation) patterns were applied to anaesthetized rats under real-time vital-signs monitoring and US imaging. This pattern of insonation first produces a gradual growth of dissolved gas cavities in tissue (cavitation) and then an ultrasonic impact. Results demonstrate sequences of successive successful HIFU pacing. Triggering insonation at different delays from the preceding ECG R-wave demonstrated successful HIFU pacing induction from mid ECG T-wave till the next ECG complex's PR interval. Spatially focusing the beam at different locations allows cumulative coverage of the whole left ventricle. Analysis of the acoustic wave patterns and temporal characteristics of paced PVCs is suggested to provide new insight into the mechanisms of HIFU cardiac pacing. Specifically, the observed HIFU pacing temporal success rate distribution suggests against sarcomere length modulation current being the dominant cellular level mechanism of HIFU cardiac pacing and may allow postulating that membrane deformation currents are dominant at the applied insonation conditions.

Towards multifocal ultrasonic neural stimulation II: design considerations for an acoustic retinal prosthesis.

Ultrasound waves, widely used as a non-invasive diagnostic modality, were recently shown to stimulate neuronal activity. Functionally meaningful stimulation, as is required in order to form a unified percept, requires the dynamic generation of simultaneous stimulation patterns. In this paper, we examine the general feasibility and properties of an acoustic retinal prosthesis, a new vision restoration strategy that will combine ultrasonic neuro-stimulation and ultrasonic field sculpting technology towards non-invasive artificial stimulation of surviving neurons in a degenerating retina. We explain the conceptual framework for such a device, study its feasibility in an in vivo ultrasonic retinal stimulation study and discuss the associated design considerations and tradeoffs. Finally, we simulate and experimentally validate a new holographic method--the angular spectrum-GSW--for efficient generation of uniform and accurate continuous ultrasound patterns. This method provides a powerful, flexible solution to the problem of projecting complex acoustic images onto structures like the retina.

Mechanical parameters determining pharyngeal collapsibility in patients with sleep apnea.

The relative impact of mechanical factors on pharyngeal patency in patients with obstructive sleep apnea is poorly understood. The present study was designed to evaluate parameters of the "tube law" on pharyngeal pressure-flow relationships and collapsibility in patients with obstructive sleep apnea. We developed a mathematical model that considered the collapsible segment of the pharynx to represent an orifice of varying diameter. The model enabled us to assess the effects of pharyngeal compliance (C), neutral cross-sectional area (A(o)), external peripharyngeal pressure (P(ex)), and the resistance proximal to the site of collapse on flow mechanics and pharyngeal collapsibility [critical pressure (P(crit))]. All parameters were measured in 15 patients with obstructive sleep apnea under propofol anesthesia, both at rest and during mandibular advancement and electrical stimulation of the genioglossus. The data was used both to confirm the validity of the model and to compare expected and actual relationships between the tube-law parameters and the pharyngeal pressure-flow relationship and collapsibility. We found a close correlation between predicted and measured P(crit) (R = 0.98), including changes observed during pharyngeal manipulations. C and A(o) were closely and directly interrelated (R = 0.93) and did not correlate with P(crit). A significant correlation was found between P(ex) and P(crit) (R = 0.77; P < 0.01). We conclude that the pharynx of patients with obstructive sleep apnea can be modeled as an orifice with varying diameter. Pharyngeal compliance and A(o) are closely interrelated. Pharyngeal collapsibility depends primarily on the surrounding pressure.

Modeling linear vibration of cell nucleus in low intensity ultrasound field.

Therapeutic ultrasound of low to medium intensity is known to induce alterations in structure and functioning of cells and tissues, both in vivo and in vitro. Such effects, including excitation or inhibition of action potentials, enhanced angiogenesis rate, increased membrane permeability and changes in molecular expression, cannot be attributed in many cases to rising temperatures or the presence of gas bubbles. This study attempts to find a possible alternative explanation for the cases where neither thermal effects nor cavitation mechanisms count. We focus our attention on the complex and dense structure of cell cytoplasm, looking for periodic separating forces and relative motion between intracellular elements, such as the nucleus, and the structure in which they are embedded. It is hypothesized that relative oscillatory displacements between intracellular elements of different densities might appear in cells in response to low intensity therapeutic ultrasound (LITUS). Those displacements might induce alterations in cell structure and functioning. A linear model is constructed and solved for a spherical object, representing a typical organelle such as the nucleus, within a homogenous viscoelastic medium that vibrates uniformly. The structure in which the object is embedded is described by four different rheologic models, including viscous fluid, elastic solid, and Voigt and Maxwell viscoelastic constructs. It is found that cyclic intracellular displacements comparable with and even larger than the mean thermal fluctuations may be obtained due to LITUS irradiation in conditions where the relative motion of organelles is dominated by elastic response, or where the effective viscosity of the cytoplasm is low. Resonance frequency at which intracellular vibration of maximal amplitude is obtained is found to lie within the low LITUS frequency range, i.e., tens to hundreds of kHz. Local intracellular strain on the order of 0.5% is found for 1 microm organelle in 10 microm cell under typical LITUS settings. It is suggested that fatigue-like, cumulative effect underlie the transfer of the intracellular strain into biologic alterations.

Investigations into pulsed high-intensity focused ultrasound-enhanced delivery: preliminary evidence for a novel mechanism.

Pulsed high-intensity focused ultrasound (HIFU) exposures without ultrasound contrast agents have been used for noninvasively enhancing the delivery of various agents to improve their therapeutic efficacy in a variety of tissue models in a nondestructive manner. Despite the versatility of these exposures, little is known about the mechanisms by which their effects are produced. In this study, pulsed-HIFU exposures were given in the calf muscle of mice, followed by the administration of a variety of fluorophores, both soluble and particulate, by local or systemic injection. In vivo imaging (whole animal and microscopic) was used to quantify observations of increased extravasation and interstitial transport of the fluorophores as a result of the exposures. Histological analysis indicated that the exposures caused some structural alterations such as enlarged gaps between muscle fiber bundles. These effects were consistent with increasing the permeability of the tissues; however, they were found to be transient and reversed themselves gradually within 72 h. Simulations of radiation force-induced displacements and the resulting local shear strain they produced were carried out to potentially explain the manner by which these effects occurred. A better understanding of the mechanisms involved with pulsed HIFU exposures for noninvasively enhancing delivery will facilitate the process for optimizing their use.

Modeling photothermal and acoustical induced microbubble generation and growth.

Previous experimental studies showed that powerful heating of nanoparticles by a laser pulse using energy density greater than 100 mJ/cm(2), could induce vaporization and generate microbubbles. When ultrasound is introduced at the same time as the laser pulse, much less laser power is required. For therapeutic applications, generation of microbubbles on demand at target locations, e.g. cells or bacteria can be used to induce hyperthermia or to facilitate drug delivery. The objective of this work is to develop a method capable of predicting photothermal and acoustic parameters in terms of laser power and acoustic pressure amplitude that are needed to produce stable microbubbles; and investigate the influence of bubble coalescence on the thresholds when the microbubbles are generated around nanoparticles that appear in clusters. We develop and solve here a combined problem of momentum, heat and mass transfer which is associated with generation and growth of a microbubble, filled with a mixture of non-vaporized gas (air) and water vapor. The microbubble's size and gas content vary as a result of three mechanisms: gas expansion or compression, evaporation or condensation on the bubble boundary, and diffusion of dissolved air in the surrounding water. The simulations predict that when ultrasound is applied relatively low threshold values of laser and ultrasound power are required to obtain a stable microbubble from a single nanoparticle. Even lower power is required when microbubbles are formed by coalescence around a cluster of 10 nanoparticles. Laser pulse energy density of 21 mJ/cm(2) is predicted for instance together with acoustic pressure of 0.1 MPa for a cluster of 10 or 62 mJ/cm(2) for a single nanoparticle. Those values are well within the safety limits, and as such are most appealing for targeted therapeutic purposes.