Spatial Peak Pressure Research Articles

Transcranial focused ultrasound induces sustained synaptic plasticity in rat hippocampus

Transcranial focused ultrasound (tFUS) neuromodulation provides a promising emerging non-invasive therapy for the treatment of neurological disorders. Many studies have demonstrated the ability of tFUS to elicit transient changes in neural responses. However, the ability of tFUS to induce sustained changes need to be carefully examined. In this study, we use the long-term potentiation/long term depression (LTP/LTD) model in the rat hippocampus, the medial perforant path (mPP) to dentate gyrus (DG) pathway, to explore whether tFUS is capable of encoding frequency specific information to induce plasticity. Single-element focused transducers were used for tFUS stimulation with ultrasound fundamental frequency of 0.5 MHz and nominal focal distance of 38 mm tFUS stimulation is directed to mPP. Measurement of synaptic connectivity is achieved through the slope of field excitatory post synaptic potentials (fEPSPs), which are elicited using bipolar electrical stimulation electrodes and recorded at DG using extracellular electrodes to quantify degree of plasticity. We applied pulsed tFUS stimulation with total duration of 5 min, with 5 levels of pulse repetition frequencies each administered at 50 Hz sonication frequency at the mPP. Baseline fEPSP is recorded 10 min prior, and 30+ minutes after tFUS administration. In N = 16 adult wildtype rats, we observed sustained depression of fEPSP slope after 5 min of tFUS focused at the presynaptic field mPP. Across all PRFs, no significant difference in maximum fEPSP slope change was observed, average tFUS induced depression level was observed at 19.6%. When compared to low frequency electrical stimulation (LFS) of 1 Hz delivered to the mPP, the sustained changes induced by tFUS stimulation show no statistical difference to LFS for up to 24 min after tFUS stimulation. When both the maximum depression effects and the duration of sustained effects are both taken into account, PRF 3 kHz can induce significantly larger effects than other PRFs tested. tFUS stimulation is measured with a spatial-peak pressure amplitude of 99 kPa, translating to an estimation of 0.43 °C temperature increase when assuming no loss of heat. The results suggest the ability of tFUS to encode sustained changes in synaptic connectivity through mechanism which are unlikely to involve thermal changes.

Experimental Validation of k-Wave: Nonlinear Wave Propagation in Layered, Absorbing Fluid Media.

Models of ultrasound propagation in biologically relevant media have applications in planning and verification of ultrasound therapies and computational dosimetry. To be effective, the models must be able to accurately predict both the spatial distribution and amplitude of the acoustic pressure. This requires that the models are validated in absolute terms, which for arbitrarily heterogeneous media should be performed by comparison with measurements of the acoustic field. In this article, simulations performed using the open-source k-Wave acoustics toolbox, with a measurement-based source definition, were quantitatively validated against measurements of acoustic pressure in water and layered absorbing fluid media. In water, the measured and simulated spatial-peak pressures agreed to within 3% under linear conditions and 7% under nonlinear conditions. After propagation through a planar or wedge-shaped glycerol-filled phantom, the difference in spatial-peak pressure was 8.5% and 10.7%, respectively. These differences are within or close to the expected uncertainty of the acoustic pressure measurement. The -6 dB width and length of the focus agreed to within 4% in all cases, and the focal positions were within 0.7 mm for the planar phantom and 1.2 mm for the wedge-shaped phantom. These results demonstrate that when the acoustic medium properties and geometry are well known, accurate quantitative predictions of the acoustic field can be made using k-Wave.

Ultrasound-induced cell lysis and sonoporation enhanced by contrast agents

The enhancement of ultrasound-induced cell destruction, lysis, and sonoporation in low cell concentration suspensions (2×105/mL) by the presence of contrast agents (gas bubble to cell ratio =3D 230) was demonstrated using cervical cancer cells (HeLa S3) suspensions containing micron-size denatured albumin microspheres filled with air (Albunex=AE) or octafluoropropane (OptisonTM). The suspensions were insonificated by 2-MHz continuous or toneburst ultrasound in the near field. The spatial peak pressure amplitude was 0.2 MPa. The enhancement of cell destruction due to Optison was shown to be much higher than that due to Albunex for similar bubble concentration and ultrasound conditions. For toneburst exposures, significant lysis and sonoporation only occurred in the presence of a contrast agent. The majority of the bioeffects observed occurred in the first 5 min of exposure. The relationship between the enhancement of bioeffects and duty cycle of toneburst ultrasound appears to indicate that both stable gas spheres of contrast agents and cavitation nuclei created by the disruption of the gas spheres play a significant role in causing the bioeffects.

Observation of acoustic streaming near Albunex® spheres

Using Albunex® spheres (a contrast agent for ultrasonic imaging), a specially designed microscopic device, a video peak storage technique and micron-size Latex spheres as tracers, acoustic streaming patterns around a single and a pair of Albunex® spheres of a few tens micrometer radius in a 160-kHz ultrasonic standing wave field have been recorded. The spatial peak pressure amplitude of the standing wave was estimated to be of the order of 0.5 MPa with optical interferometry. The acoustic streaming velocity was in the range of 50–100 μm/s.

Gas-body-based contrast agent enhances vascular bioeffects of 1.09 MHz ultrasound on mouse intestine

Anesthetized hairless mice were exposed to continuous or pulsed 1.09-MHz ultrasound with or without prior injection of a gas-body-based ultrasound contrast agent. Albunex® at a dose of 10 mL/kg increased the production of intestinal hyperemia, petechia and hemorrhages by continuous ultrasound. For pulsed ultrasound, with 10 μs pulses and 0.01 duty cycle, petechiae were produced for exposures as low as 1 MPa spatial peak pressure amplitude with added gas bodies. The enhancement of petechiae production was robust for pulsed exposure; for example, at 2.8 MPa, an average of 227 petechiae was obtained with added gas bodies, which was 30 times more than without the agent. The production of petechia was roughly proportional to the dosage of Albunex ® for pulsed exposure. Results did not appear to be strongly dependent on pulsing parameters, but long bursts (0.1 s) were somewhat more effective than pulses (10 μs). The observed vascular bioeffects appeared to involve both thermal and nonthermal mechanisms for continuous exposure, but to result primarily from gas-body activation for pulsed exposure.

Defects of bilayers generated by ultrasound

Transdermal drug delivery is handicapped by the low skin permeability caused by highly ordered structure of lipid bilayers in the outer human skin layer. It has been reported that ultrasound can increase the permeability of human skin [S. Mitragotri, D. Blankschtein, and R. Langer, Science 269, 850–853 (1995)]. The enhancement was attributed to acoustic cavitation but the underlying physical mechanism is not fully understood. As a model, dipalmitoylphosphatidylcholine (DPPC) lipid bilayers are insonicated by ultrasound of two submegahertz frequencies (168 and 707 kHz). The free-field spatial peak pressure amplitudes of both are measured to be 6×105 Pa. Bilayer defects, which have average diameters of tens to hundreds of nanometers and can be detected by an atomic force microscope, are generated within less than 0.5 min. The number of the defects grows with time. The defect growth rate at the 168-kHz frequency is about 3.5 times that at the 707-kHz frequency.

Ultrasound contrast agent enhances vascular damage in mouse intestines

Either Albunex®, a gas-body based contrast agent, or 5% albumin in saline without gas bodies was administered to anesthetized hairless mice at 10 ml/kg by retro-orbital injection. The mice then were exposed at the midsection to 1.09 MHz of ultrasound for 100 s, either continuous (cw) or pulsed (pw, 10 μs pulses repeated at 1 kHz), in a 37 °C water bath. After exposure, the intestines were examined to count petechiae and hemorrhages into the intestinal lumen. cw thresholds for statistically significant petechiae production were 0.7 MPa spatial peak pressure amplitude without added gas bodies, and 0.5 MPa with added gas bodies. At 0.7 MPa the mean counts (five mice) were 22 without and 120 with added gas bodies. A statistically significant number of hemorrhages occurred at 0.7 MPa (three per mouse) with the contrast agent. The pw thresholds with added agent were 1.4 MPa for petechiae and 2.8 MPa for hemorrhage. At 2.8 MPa, average counts were 227 petechiae and 2.4 hemorrhages. No statistically significant vascular damage occurred for pw exposure up to 2.8 MPa without contrast agent. In terms of mechanisms, the effects appeared to be partly thermal for cw, but primarily associated with ultrasonic cavitation for pw exposure. [Work supported by NIH CA42947.]

Acoustic streaming near Albunex spheres

Acoustic streaming near bubbles in an external standing-wave sound field has been an interesting research topic for decades. It was relatively difficult to visualize the acoustic streaming pattern experimentally, especially for small bubbles due to the instability of the bubbles and difficulties associated with the optical technique. Using Albunex spheres (a contrast agent for ultrasonic imaging), a specially designed microscope device, and a video peak store technique, several acoustic streaming patterns around a single and a pair of Albunex spheres of tens of micrometers radius in a 160-kHz ultrasonic standing wave field have been recorded. Spatial peak pressure amplitude of the standing wave was estimated to be in the order of 0.5 MPa by using optical interferometry. The acoustic streaming velocity was in the range of 50–100 μ/s.

Generation and growth of bilayer defects induced by ultrasound.

Transdermal drug delivery is handicapped by the low skin permeability caused by highly ordered structure of lipid bilayers in the outer human skin layer. It has been reported that ultrasound can increase the permeability of human skin. The enhancement was attributed to acoustic cavitation but the underlying physical mechanism is not fully understood. As a model, dipalmitoylphosphatidylcholine (DPPC) lipid bilayers are insonicated by ultrasound of two submegahertz frequencies (168 kHz and 707 kHz). The free-field spatial peak pressure amplitudes of both are measured to be 6×105 Pa. Bilayer defects, which have average diameters of tens to hundreds of nanometers and can be detected by an atomic force microscope, are generated within less than 0.5 min. The number of the defects grows with time. The defect growth rate at the 168-kHz frequency is about 3.5 times that at the 707-kHz frequency.

Transfection of a reporter plasmid into cultured cells by sonoporation in vitro

Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex ®, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H 2O 2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 μg/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10 6 cells at 0.8-MPa exposure. The luciferase production was greater for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex ® was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube exposure system yields plasmid transfection with subsequent transient gene expression.

Ultrasound contrast agents nucleate inertial cavitation in vitro

Some ultrasound contrast agents contain stable bodies of gas, and this study was undertaken to determine if these agents could provide nuclei for inertial cavitation. Inertial cavitation was detected and assessed by the measurement of the sonochemical hydrogen peroxide after exposure to 2.17-, 2.95- or 3.8-MHz ultrasound. A noncavitating system was obtained by removing cavitation nuclei from the rotating tube exposure chambers by vacuum degassing, and from the phosphate-buffered saline medium by filtering. Albunex ® added at 10 −2, 10 −3 or 10 −4 dilutions, or Levovist ® added at 2 mg mL −1, 0.2 mg mL −1 or 0.02 mg mL −1 all initiated significant H 2O 2 production for 2.17-MHz ultrasound at 0.41 MPa or higher spatial peak pressure amplitude for 5 min exposure gated at 0.25 s on and off with 60-rpm rotation. Not rotating the tube virtually eliminated H 2O 2 production. For 2.5-min continuous exposure, both agents initiated significant H 2O 2 production for 2.95-MHz exposure at 0.58 MPa or higher, but not for 3.8-MHz exposure up to 1.16 MPa. Bubble-based ultrasound contrast agents therefore appear to be able to provide nuclei for inertial cavitation in the rotating tube exposure system.

Heating as a mechanism for ultrasonically-induced petechial hemorrhages in mouse intestine

Anesthetized hairless mice were exposed to 1.035 MHz unfocused ultrasound in a temperaturecontrolled water bath. Visible petechial hemorrhages and hyperemia were produced in the intestines. Histologically, the petechiae appeared to be localized mostly to the lamina propria of the mucosa. For 2 min exposure, the number of petechiae and length of hyperemic tissue both increased with increasing spatial peak pressure amplitude above a threshold of about 0.4–0.57 MPa. The magnitude of these effects increased, and the threshold decreased, with increasing exposure duration from 1 min to 4 min. The effects also increased for increasing bath temperature from 32°C to 42°C. The effects decreased markedly for 1 ms burst-mode exposure with 2 ms and 4 ms repetition periods with constant pressure amplitude, but were essentially constant for constant temporal-average intensity. Both effects were also produced by heating from radio-frequency diathermy at 10.35 MHz. The effects were associated with abdominal temperatures above about 42–43°C, which were estimated with a thermocouple at the end of the exposure. The hyperemia is a clearly thermal effect, while the petechiae have been previously associated with the cavitation, mechanism. However, the ultrasonically-induced petechiae observed in this study do not represent an unequivocal marker for cavitation, but rather appear to be attributable to heating. These findings help to reduce the expectation of ultrasonic cavitation during medical therapy treatments.

Transport and harvesting of suspended particles using modulated ultrasound

Polystyrene particles of 9 μm diameter were acoustically concentrated along the axis of a water-filled cylindrical waveguide containing a 3 MHz standing wave field. Modulation of the acoustic field enabled transport of the concentrated particles in the axial direction. Four modulations were investigated; 1, a fixed frequency difference introduced between two transducers; 2, ramping the transducer frequency; 3, tone burst, i.e. sound that is pulsed on and off, allowing intermittent sedimentation under gravity; and 4, switching the sound off to allow continuous sedimentation. The most efficient transport (leaving the fewest particles in suspension) of clumps to one end of the container was achieved with method 1 above. In this system the maximum speed of transport of the axial clumps was 24 mm s -1. A theory developed here for the transport of particles in a pseudo (i.e. slowly moving) standing wave field predicts an upper limit, which increases with particle size, for the speed of an entrained body. For a single 9 μm diameter particle in a field with a spatial peak pressure amplitude of 0.4 MPa this speed would be 0.5 mm s -1. The higher experimental speeds observed here emphasize the value of acoustically concentrating particles into relatively large clumps prior to initiating transport.

A survey of the acoustic output of ultrasonic Doppler equipment.

Measurements of the acoustic output generated by a variety of clinical ultrasonic Doppler instruments have been carried out. The instruments surveyed include continuous wave Doppler units for cardiovascular investigations, fetal monitors, stand-alone pulsed Doppler equipment and 'duplex' scanners working in Doppler mode. Acoustic measurements have been made using a calibrated PVDF membrane hydrophone, and a milliwatt radiation force balance. Almost all the pulsed Doppler and duplex systems investigated could generate spatial-peak, temporal-average intensities in water which exceeded 100 mW cm-2, with a maximum of 825 mW cm-2 measured. These intensities were also reached by some continuous-wave Doppler systems, particularly those incorporated into duplex scanning systems. Fetal monitoring equipment was found to operate typically at lower intensities. Doppler units have been found to show a very wide variation in pulse length, repetition frequency and spatial peak pressure, and a wider range of pulse average intensities than previously reported. Units were found to vary considerably in their control of total acoustic power whilst operating parameters such as gate width and range were altered.