Junru Wu

Ultrasound, cavitation bubbles and their interaction with cells ☆

This article reviews the basic physics of ultrasound generation, acoustic field, and both inertial and non-inertial acoustic cavitation in the context of localized gene and drug delivery as well as non-linear oscillation of an encapsulated microbubble and its associated microstreaming and radiation force generated by ultrasound. The ultrasound thermal and mechanical bioeffects and relevant safety issues for in vivo applications are also discussed.

Evidence of connective tissue involvement in acupuncture

Acupuncture needle manipulation gives rise to “needle grasp,” a biomechanical phenomenon characterized by an increase in the force necessary to pull the needle out of the tissue (pullout force). This study investigates the hypothesis that winding of connective tissue, rather than muscle contraction, is the mechanism responsible for needle grasp. We performed 1) measurements of pullout force in humans with and without needle penetration of muscle; 2) measurements of pullout force in anesthetized rats, with and without needle rotation, followed by measurements of connective tissue volume surrounding the needle; 3) imaging of rat abdominal wall explants, with and without needle rotation, using ultrasound scanning acoustic microscopy. We found 1) no evidence that increased penetration of muscle results in greater pullout force than increased penetration of subcutaneous tissue; 2) that both pullout force and subcutaneous tissue volume were increased by needle rotation; 3) that increased periodic architectural order was present in subcutaneous tissue with rotation, compared with no rotation. These data support connective tissue winding as the mechanism responsible for the increase in pullout force induced by needle rotation. Winding may allow needle movements to deliver a mechanical signal into the tissue and may be key to acupunctures therapeutic mechanism.

A model for longitudinal and shear wave propagation in viscoelastic media

Relaxation models fail to predict and explain loss characteristics of many viscoelastic materials which follow a frequency power law. A model based on a time-domain statement of causality is presented that describes observed power-law behavior of many viscoelastic materials. A Hookes law is derived from power-law loss characteristics; it reduces to the Hookes law for the Voigt model for the specific case of quadratic frequency loss. Broadband loss and velocity data for both longitudinal and shear elastic types of waves agree well with predictions. These acoustic loss models are compared to theories for loss mechanisms in dielectrics based on isolated polar molecules and cooperative interactions.

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 x 10(5)/mL) by the presence of contrast agents (gas bubble to cell ratio = 230) was demonstrated using cervical cancer cells (HeLa S3) suspensions containing micron-size denatured albumin microspheres filled with air (Albunex) or octafluoropropane (Optison). The suspensions were insonificated by 2-MHz continuous or tone burst ultrasound in 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 tone burst 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 tone burst 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.

Experimental study of the effects of Optison concentration on sonoporation in vitro.

Lethal sonoporation and reparable sonoporation were observed in Jurkat lymphocytes in suspension with the addition of varying amounts of Optison, a commercially available bubble-based contrast agent. For given ultrasound (US) exposure conditions (spatial peak-pressure amplitude of 0.2 MPa, duty cycle 10% and 2-MHz frequency), sonoporation was directly related to the bubble-to-cell ratio (in a range from 0 to 230). It was found that the nearest bubble-cell spacing was also related to the occurrence frequency of bioeffects. A constant bubble-to-cell ratio often provided very different results for two different initial cell concentrations (200,000 cells/mL and 600,000 cells/mL), with the higher cell concentration generally exhibiting higher levels of sonoporation. In contrast, a constant bubble-to-cell spacing provided similar results between the two initial cell concentrations. The frequency of reparable and lethal sonoporation was seen to decay as the inverse-cube power of the nearest bubble-cell spacing. Significant reparable sonoporation was observed at a bubble-cell spacing that was 10 microm larger than the minimum spacing at which significant lethal sonoporation was observed. Preliminary analysis also suggests the possibility of a step-wise increase in lethal sonoporation as spacing decreases; further experiment is needed.

Theoretical study on shear stress generated by microstreaming surrounding contrast agents attached to living cells

Numerical calculations have shown that shear stress associated with microstreaming surrounding encapsulated stable bubbles of contrast agents, near living cells driven by 0.12-MPa acoustic pressure amplitude ultrasound (US) at 1 MHz or 2 MHz, may be large enough to generate reparable sonoporation of the cells. Some encapsulated bubbles that have mechanically weak shells may break into free bubbles under the above-mentioned sound field. When that happens, the shear stress caused by microstreaming surrounding the free bubble increases dramatically and may play an important role in lethal sonoporation and fragmentation of cells during the early stage of US exposure.

Reparable sonoporation generated by microstreaming

Reparable sonoporation was observed in Jurkat lymphocytes in suspension exposed to a vibrating Mason horn tuned to 21.4 KHz. The diameter of the horn tip was 400 microm and its transverse displacement amplitude was 7.8 microm. It was found that the shear stress associated with microstreaming surrounding the Mason-horn tip was the primary reason for the cell reparable sonoporation. The threshold shear stress was determined to be 12 +/- 4 Pa for exposure time up to 7 min. It was also found that the shorter the exposure time, the greater the threshold.

The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro

Previous studies have demonstrated that the efficiency of gene/drug delivery can be enhanced under ultrasound (US) exposure with the presence of US contrast agent microbubbles, due to the acoustic cavitation-induced sonoporation. However, obstacles still remain to achieve controllable sonoporation outcome. The general hypotheses guiding present studies were that inertial cavitation (IC) activities accumulated during US exposure could be quantified as IC dose (ICD) based on passive cavitation detection (PCD), and the assessment of sonoporation outcome should be correlated with ICD measurements. In current work, MCF-7 cells mixed with PEI:DNA complex and UCD microbubbles were exposed to 1-MHz US pulses with 20-cycle pulse and varied acoustic peak negative pressure (P(-); 0 (sham), 0.3, 0.75, 1.4, 2.2 or 3.0MPa), total treatment time (0, 5, 10, 20, 40 or 60s), and pulse-repetition-frequency (PRF; 0, 20, 100, 250, 500, or 1000Hz). Then, four series experiments were conducted: (1) the IC activities were detected using a PCD system and quantified as ICD; (2) the DNA transfection efficiency was evaluated with flow cytometry; (3) the cell viability was examined by PI dying then measured using flow cytometry; and (4) scan electron microscopy was used to investigate the sonoporation effects on the cell membrane. The results showed that: (1) the ICD generated during US exposure could be affected by US parameters (e.g., P(-), total treatment time, and PRF); (2) the pooled data analyses demonstrated that DNA transfection efficiency initially increased linearly with the increasing ICD, then it tended to saturate instead of trying to achieve a maximum value while the ICD kept going up; and (3) the measured ICD, sonoporation pore size, and cell viability exhibited high correlation among each other. All the results indicated that IC activity should play an important role in the US-mediated DNA transfection through sonoporation, and ICD could be used as an effective tool to monitor and control the US-mediated gene/drug delivery effect.

Electrical impedance along connective tissue planes associated with acupuncture meridians

BackgroundAcupuncture points and meridians are commonly believed to possess unique electrical properties. The experimental support for this claim is limited given the technical and methodological shortcomings of prior studies. Recent studies indicate a correspondence between acupuncture meridians and connective tissue planes. We hypothesized that segments of acupuncture meridians that are associated with loose connective tissue planes (between muscles or between muscle and bone) visible by ultrasound have greater electrical conductance (less electrical impedance) than non-meridian, parallel control segments.MethodsWe used a four-electrode method to measure the electrical impedance along segments of the Pericardium and Spleen meridians and corresponding parallel control segments in 23 human subjects. Meridian segments were determined by palpation and proportional measurements. Connective tissue planes underlying those segments were imaged with an ultrasound scanner. Along each meridian segment, four gold-plated needles were inserted along a straight line and used as electrodes. A parallel series of four control needles were placed 0.8 cm medial to the meridian needles. For each set of four needles, a 3.3 kHz alternating (AC) constant amplitude current was introduced at three different amplitudes (20, 40, and 80 μAmps) to the outer two needles, while the voltage was measured between the inner two needles. Tissue impedance between the two inner needles was calculated based on Ohms law (ratio of voltage to current intensity).ResultsAt the Pericardium location, mean tissue impedance was significantly lower at meridian segments (70.4 ± 5.7 Ω) compared with control segments (75.0 ± 5.9 Ω) (p = 0.0003). At the Spleen location, mean impedance for meridian (67.8 ± 6.8 Ω) and control segments (68.5 ± 7.5 Ω) were not significantly different (p = 0.70).ConclusionTissue impedance was on average lower along the Pericardium meridian, but not along the Spleen meridian, compared with their respective controls. Ultrasound imaging of meridian and control segments suggested that contact of the needle with connective tissue may explain the decrease in electrical impedance noted at the Pericardium meridian. Further studies are needed to determine whether tissue impedance is lower in (1) connective tissue in general compared with muscle and (2) meridian-associated vs. non meridian-associated connective tissue.

Ultrasound evidence of altered lumbar connective tissue structure in human subjects with chronic low back pain.

BackgroundAlthough the connective tissues forming the fascial planes of the back have been hypothesized to play a role in the pathogenesis of chronic low back pain (LBP), there have been no previous studies quantitatively evaluating connective tissue structure in this condition. The goal of this study was to perform an ultrasound-based comparison of perimuscular connective tissue structure in the lumbar region in a group of human subjects with chronic or recurrent LBP for more than 12 months, compared with a group of subjects without LBP.MethodsIn each of 107 human subjects (60 with LBP and 47 without LBP), parasagittal ultrasound images were acquired bilaterally centered on a point 2 cm lateral to the midpoint of the L2-3 interspinous ligament. The outcome measures based on these images were subcutaneous and perimuscular connective tissue thickness and echogenicity measured by ultrasound.ResultsThere were no significant differences in age, sex, body mass index (BMI) or activity levels between LBP and No-LBP groups. Perimuscular thickness and echogenicity were not correlated with age but were positively correlated with BMI. The LBP group had ~25% greater perimuscular thickness and echogenicity compared with the No-LBP group (ANCOVA adjusted for BMI, p < 0.01 and p < 0.001 respectively).ConclusionThis is the first report of abnormal connective tissue structure in the lumbar region in a group of subjects with chronic or recurrent LBP. This finding was not attributable to differences in age, sex, BMI or activity level between groups. Possible causes include genetic factors, abnormal movement patterns and chronic inflammation.