Cheri X. Deng

ULTRASONIC SPECTRUM ANALYSIS FOR TISSUE ASSAYS AND THERAPY EVALUATION

Ultrasonic spectrum analysis procedures have been developed to measure tissue morphologic features that are not well depicted with conventional ultrasonography. This article reviews some of the applications of spectral techniques and provides an expanded theoretical framework showing how measured spectral features are related to the spatial autocorrelation function descriptive of tissue microstructure. Explicit relationships are obtained that describe how linear‐regression spectral parameters are related to the effective mean sizes, concentrations, and relative mechanical properties of scattering centers in tissue. In vitro, in vivo, and clinical results are presented illustrating how these techniques can be used to evaluate tissue alterations induced by ultrasonic hyperthermia and ablative treatments of tumors. These results show that ultrasonic spectrum analysis can provide quantitative information regarding changes in microstructure attributes. Spectral parameter images in two and three dimensions demonstrate how such procedures can map the spatial extent and severity of these changes, thereby providing a quantitative basis for assessing the results of tumor therapy.

Radiation-force technique to monitor lesions during ultrasonic therapy.

This report describes a monitoring technique for high-intensity focused ultrasound (US), or HIFU, lesions, including protein-denaturing lesions (PDLs) and those made for noninvasive cardiac therapy and tumor treatment in the eye, liver and other organs. Designed to sense the increased stiffness of a HIFU lesion, this technique uniquely utilizes the radiation force of the therapeutic US beam as an elastographic push to detect relative stiffness changes. Feasibility was demonstrated with computer simulations (treating acoustically induced displacements, concomitant heating, and US displacement-estimation algorithms) and pilot in vitro experimental studies, which agree qualitatively in differentiating HIFU lesions from normal tissue. Detectable motion can be induced by a single 5 ms push with temperatures well below those needed to form a lesion. Conversely, because the characteristic heat diffusion time is much longer than the characteristic relaxation time following a push, properly timed multiple therapy pulses will form lesions while providing precise control during therapy.

Direct evidence of cavitation in vivo from diagnostic ultrasound

Recent increases in the pressure output of diagnostic ultrasound scanners have led to an interest in establishing thresholds for bioeffects in many organs including the lungs of mammals. Damage may be mediated by inertial cavitation, yet there have been no such direct observations in vivo. To explore the hypothesis of cavitation-based bioeffects from diagnostic ultrasound, research has been performed on the thresholds of damage in rat lungs exposed to 4.0-MHz pulsed Doppler and color Doppler ultrasound. A 30-MHz active cavitation detection scheme complementing these studies provides the first direct evidence of cavitation in vivo from diagnostic ultrasound pulses.

Ultrasonic spectrum analysis for tissue evaluation

Spectrum analysis procedures have been developed to improve upon the diagnostic capabilities afforded by conventional ultrasonic images. These procedures analyze the frequency content of broadband, coherent echo signals returned from the body. They include calibration procedures to remove system artifacts and thereby provide quantitative measurements of tissue backscatter. Several independent spectral parameters have been used to establish databases for various organs; several investigations have shown that these parameters can be used with statistical classifiers to identify tissue type. Locally computed spectra have been used to generate sets of images displaying independent spectral parameters. Stained images have been derived by analyzing these parameter images with statistical classifiers and using color to denote tissue type (e.g., cancer). This report describes spectrum analysis procedures, discusses how measured parameters are related to physical tissue properties, and summarizes results describing estimator precision. It also presents illustrative clinical results showing how such procedures are being adapted to address specific clinical problems for a number of organs. This report indicates where further developments are needed and suggests how these techniques may improve image segmentation for three-dimensional displays and volumetric assays.

Spatiotemporally controlled single cell sonoporation

This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.

Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

Controlled permeation of cell membrane by single bubble acoustic cavitation

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5MHz) ultrasound pulse (duration 13.3 or 40μs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d=0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106±0.032μm (n=70) for acoustic pressure of 1.5MPa (duration 13.3μs), and increased to 0.171±0.030μm (n=125) for acoustic pressure of 1.7MPa and to 0.182±0.052μm (n=112) for a pulse duration of 40μs (1.5MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles.

Ultrasound application in the presence of microbubbles is a promising strategy for intracellular drug and gene delivery, but it may also trigger other cellular responses. This study investigates the relationship between the change of cell membrane permeability generated by ultrasound-driven microbubbles and the changes in intracellular calcium concentration ([Ca(2+)](i)). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1MHz, 10-15cycles, 0.27MPa) in the presence of a Definity(TM) microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca(2+)](i) and dye loss from the cells were measured with preloaded fura-2. The ultrasound exposure generated fragmentation or shrinking of the microbubble. Only cells adjacent to the ultrasound-driven microbubble exhibited propidium iodide uptake with simultaneous [Ca(2+)](i) increase and fura-2 dye loss. The amount of PI uptake was correlated with the amount of fura-2 dye loss. Cells with delayed [Ca(2+)](i) transients from the time of ultrasound application had no uptake of PI. These results indicate the formation of non-specific pores in the cell membrane by ultrasound-stimulated microbubbles and the generation of calcium waves in surrounding cells without pores.

Nitro-Oleic Acid Inhibits Angiotensin II–Induced Hypertension

Rationale Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. Objective The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced hypertension. Methods and Results We show that systemic administration of OA-NO2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)&ggr; activation. Rather, OA-NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. Conclusions These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension.

The size of sonoporation pores on the cell membrane

Sonoporation generates transient pores on the cell membrane and has been exploited as a promising intracellular drug and gene delivery strategy. The size of the pores in the membrane resulting from sonoporation determines the size of molecules or agents that can be delivered using the technique. However, size information has not been readily available due to the challenges in measuring dynamic, submicron-sized pores. Post ultrasound assays such as AFM or SEM, often time consuming and labor intensive, have been used to gauge pore size but are intrinsically limited to static measurements that may not accurately represent the relevant size. We have previously demonstrated the utility of voltage clamp techniques for monitoring sonoporation in real time via the trans-membrane current (TMC) change of a single cell under voltage clamp. Using Xenopus oocytes as the model system in this study, the TMC change during sonoporation (0.2 s, 0.3 MPa, 1 MHz) of single cells in solution with Definity microbubbles was recorded in a whole cell voltage clamp configuration. As the changes of the TMC are related to the diffusion of ions through the pores on the membrane, they can potentially provide relevant information of the pore size generated in sonoporation. By controlling the microbubble concentration, experiments were designed to allow measurement of the TMC corresponding to a single pore on the membrane. An electro-diffusion model was developed to relate the TMC with pore size from the ion flow through the pores on the membrane.