S. Lori Bridal

Parametric (integrated backscatter and attenuation) images constructed using backscattered radio frequency signals (25–56 MHz) from human aortae in vitro

Quantitative ultrasonic tissue characterization using backscattered high-frequency intravascular ultrasound could provide a basis for the objective identification of lesions in vivo. Representation of local measurements of quantitative ultrasonic parameters in a conventional image format should facilitate their interpretation and thus increase their clinical utility. Toward this goal, the apparent integrated backscatter, the slope of attenuation (25-56 MHz) and the value of the attenuation on the linear fit at 37.5 MHz were measured using the backscattered radio frequency signals from in vitro human aortae. Local estimations of these ultrasonic parameters from both normal and atherosclerotic aortic segments were displayed in a B-scan format. The morphological features of these parametric images corresponded well to features of histological images of the same regions. The attenuation from 25-56 MHz of seven segments of the medial layer (both with and without overlying atheroma) were measured using the multinarrow-band backscatter method. The average attenuation in the media at 24 degrees C +/- 3 degrees C was 45 +/- 16 dB/cm at 25 MHz and 102 +/- 13 dB/cm at 50 MHz. This work represents progress toward the development of quantitative imaging methods for intravascular applications.

Correlation of ultrasonic attenuation (30 to 50 MHz) and constituents of atherosclerotic plaque

The ultrasonic integrated attenuation and the slope of attenuation (30-50 MHz) were measured in vitro at 20 degrees +/- 2 degrees C using radio frequency signals backscattered from human aortae. Ultrasonic measurements and histologic classifications were made in a total of 124 local regions from 58 independent segments of aortae. Values of the integrated attenuation were significantly higher in collagen-lipidic (142 +/- 51 dB cm-1, n = 18), and lipidic regions (139 +/- 53 dB cm-1, n = 11) compared to regions of normal media (97 +/- 20 dB cm-1, n = 44) and dense collagen (107 +/- 33 dB cm-1, n = 43). The most elevated integrated attenuation values were observed in calcified regions (245 +/- 93 dB cm-1, n = 8). The slope of attenuation was significantly higher in lipidic than in normal media (p = 0.002), dense collagen (p = 0.0007) or collagenlipidic (p = 0.04) regions. The correlation between attenuation and local tissue composition was used to establish ranges of values of integrated attenuation that are most likely to indicate specific tissue types. Images of the local tissue type were constructed. Comparison of these quantitative images with the corresponding histologic sections demonstrates that attenuation measurements offer promise for the in vivo characterization of plaque structure and composition.

Clinical relevance of contrast-enhanced ultrasound in monitoring anti-angiogenic therapy of cancer: Current status and perspectives

Angiogenesis regulation is one of the newest fronts in the fight against cancer. Anti-angiogenic therapy is based on inhibiting factors required to solicit vessel formation thus cutting-off the tumors supply of nutriments and oxygen. Initial vascular response is followed by formation of necrosis. Volumetric regression occurs more tardively. Effective monitoring of this new therapeutic approach thus requires imaging techniques that can detect early microvascular changes. A number of clinical studies provide evidence that contrast-enhanced ultrasound (CEUS) can provide early indication of tumor response to anti-angiogenic therapy. More sophisticated imaging and analysis techniques for CEUS and contrast agents targeted for adhesion to anti-angiogenic markers have also demonstrated promise in animal model studies. This review underlines the relevance of CEUS for anti-angiogenic therapy monitoring by summarizing the current clinical results, emerging CEUS techniques and preclinical data.

Noninvasive Contrast-enhanced US Quantitative Assessment of Tumor Microcirculation in a Murine Model: Effect of Discontinuing Anti-VEGF Therapy

PURPOSEnTo determine, by using contrast material-enhanced ultrasonography (US), how quickly renal tumors grafted in mice begin to revascularize after stopping bevacizumab treatment.nnnMATERIALS AND METHODSnAll experiments were approved by the regional ethics committee. A human tumor cell line SK-NEP-1 was grafted at day 0 in the left kidney of 50 nude mice. Forty-two mice developed tumors and longitudinal follow-up was performed on 32 surviving mice. From day 13, 14 controls received biweekly saline; 11 mice received biweekly bevacizumab until day 35 (continuous); and seven received biweekly bevacizumab until day 22, then biweekly placebo until day 35 (discontinued). Contrast-enhanced US was performed on days 13, 14, 22, 27, and 35. Once the injected contrast material distribution reached an equilibrium phase, high-acoustic pressure pulses were applied to destroy microbubbles in the capillary bed in the imaged plane. Reperfusion was monitored, and time-signal intensity (SI) curves were obtained from the linear average of SIs in intratumoral and matched-depth renal cortex regions of interest. A kinetic parameter calculated from reperfusion curves reflects local perfusion, normalized with respect to adjacent renal cortex perfusion. Normalized perfusion obtained from each group was compared with that from the other groups and with necrosis percentages and microvascular density assessed histologically at day 35. Comparisons were made by using analyses of variance and Tukey-Kramer tests.nnnRESULTSnThe lowest excised mean tumor weights (+/- standard deviation) corresponded to the longest bevacizumab-treatment duration: 1.4 g +/- 1.1 (continuous-treatment) compared with 2.3 g +/- 2.1 (discontinued) and 3.7 g +/- 1.9 (control) (P = .01). On day 35, the respective control and continuously treated groups had comparable and significantly larger necrotic areas: 37% +/- 14 and 32% +/- 17 larger than the discontinued-treatment group (15% +/- 9; P < .05). Normalized perfusion increased significantly with time (P = .02) in the discontinued-treatment group after therapy ceased (day 22).nnnCONCLUSIONnNoninvasively measured contrast-enhanced US parameters demonstrated tumor revascularization after stopping antiangiogenic therapy in this murine tumor model.

RANKL Induces Organized Lymph Node Growth by Stromal Cell Proliferation

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.

Ultrasonic Backscatter and Attenuation (11–27 MHz) Variation with Collagen Fiber Distribution in Ex Vivo Human Dermis

This ex vivo study explores the relationship of ultrasonic attenuation and backscatter to dermal microarchitecture by comparing ultrasonic measurements of these parameters (11–27 MHz) to a microscopic analysis of three parameters describing the collagen distribution (mean thickness and spacing of collagen bundles along the insonification direction and the percent area occupied by collagen). Skin samples (N = 31) were obtained from patients undergoing breast or abdominal reduction surgery. Radio-frequency (rf) signals were acquired in a B-scan format using an ultrasound system developed for skin imaging (Ultrasons Technologies, Tours, France). Ultrasonic data were analyzed to calculate average integrated backscatter (IBS in dB) and frequency dependence of backscatter (n, dimensionless) of each specimen at depths centered approximately 370, 620 and 880 μm beneath the skin surface. Average integrated attenuation coefficient (IA in dB.cm−1) and frequency dependence of attenuation coefficient (β in dB.cm−1.MHz−1) were estimated across the depth between 240 and 1,000 μm. The three collagen distribution parameters were estimated using digitized microcopic fields from matched regions of histological sections stained with hematoxylin-eosin-saffron. No significant correlation was identified between collagen distribution parameters and IA or β. For the most superficial depth studied in abdominal skin, n was inversely correlated to collagen bundle thickness (r = −0.67, p = 0.002) and percent area (r = −0.65, p = 0.003). At the same depth, IBS was inversely correlated to percent area of collagen (r = −0.51, p = 0.03). The rather high collagen packing (48 to 82% area) measured in histological sections and the inverse relationship observed between IBS and percent area of collagen suggest that a packing factor should be included in models relating skin collagen distribution to ultrasound spectral parameters. A better understanding of the relationship between ultrasound parameters and the microarchitecture of the dermis should help to interpret changes in ultrasonic parameters observed during in vivo ultrasonic skin examinations.

Optimization of attenuation estimation in reflection for in vivo human dermis characterization at 20 MHz

In vivo skin attenuation estimators must be applicable to backscattered radio frequency signals obtained in a pulse-echo configuration. This work compares three such estimators: short-time Fourier multinarrowband (MNB), short-time Fourier centroid shift (FC), and autoregressive centroid shift (ARC). All provide estimations of the attenuation slope (/spl beta/, dB.cm/sup -1/.MHz/sup -1/); MNB also provides an independent estimation of the mean attenuation level (IA, dB.cm/sup -1/). Practical approaches are proposed for data windowing, spectral variance characterization, and bandwidth selection. Then, based on simulated data, FC and ARC were selected as the best (compromise between bias and variance) attenuation slope estimators. The FC, ARC, and MNB were applied to in vivo human skin data acquired at 20 MHz to estimate /spl beta//sub FC/, /spl beta//sub ARC/, and IA/sub MNB/, respectively (without diffraction correction, between 11 and 27 MHz). Lateral heterogeneity had less effect and day-today reproducibility was smaller for IA than for /spl beta/. The IA and /spl beta//sub ARC/ were dependent on pressure applied to skin during acquisition and IA on room and skin-surface temperatures. Negative values of IA imply that IA and /spl beta/ may be influenced not only by skins attenuation but also by structural heterogeneity across dermal depth. Even so, IA was correlated to subject age and IA, /spl beta//sub FC/, and /spl beta//sub ARC/ were dependent on subject gender. Thus, in vivo attenuation measurements reveal interesting variations with subject age and gender and thus appeared promising to detect skin structure modifications.

Dual-mode registration of dynamic contrast-enhanced ultrasound combining tissue and contrast sequences

This study proposes a new method for automatic, iterative image registration in the context of dynamic contrast-enhanced ultrasound (DCE-US) imaging. By constructing a cost function of image registration using a combination of the tissue and contrast-microbubble responses, this new method, referred to as dual-mode registration, performs alignment based on both tissue and vascular structures. Data from five focal liver lesions (FLLs) were used for the evaluation. Automatic registration based on the dual-mode registration technique and tissue-mode registration obtained using the linear response image sequence alone were compared to manual alignment of the sequence by an expert. Comparison of the maximum distance between the transformations applied by the automatic registration techniques and those from expert manual registration reference showed that the dual-mode registration provided better precision than the tissue-mode registration for all cases. The reduction of maximum distance ranged from 0.25 to 9.3mm. Dual-mode registration is also significantly better than tissue-mode registration for the five sequences with p-values lower than 0.03. The improved sequence alignment is also demonstrated visually by comparison of images from the sequences and the video playbacks of the motion-corrected sequences. This new registration technique better maintains a selected region of interest (ROI) within a fixed position of the image plane throughout the DCE-US sequence. This should reduce motion-related variability of the echo-power estimations and, thus, contribute to more robust perfusion quantification with DCE-US.

Relationship Between Ultrasonic Attenuation, Apparent Integrated Backscatter (30 To 50 Mhz) and the Composition of Atherosclerotic Plaque

Because of the important impact of atherosclerosis on public health, the evaluation of the potential evolution of atherosclerotic lesions and their response to interventional therapy is a subject of much current concern. Studies in vitro have demonstrated that the ultimate biological behavior of an atherosclerotic plaque depends, not only on its extent, but also on its biochemical composition and structure.1 An in vivo means for identifying the composition and structure of atherosclerotic plaques would be useful in guiding diagnosis and treatment. Using appropriate signal analysis methods, the acoustic attenuation and the backscatter coefficient may be estimated from echographic signals.2 These parameters are related to tissue structure and composition and may therefore, offer additional quantitative information useful for the characterization of atherosclerotic plaques.3−12 We have recently described a method for the construction of quantitative images using local estimations of the attenuation and the apparent integrated backscatter.11 The current work examines the sensitivity of measurements of the ultrasonic attenuation and the apparent integrated backscatter (30 to 50 MHz) to differences in the local tissue composition in the arterial wall (collagen, lipids, calcifications, and media).

High-frequency (20 to 40 MHz) acoustic response of liquid-filled nanocapsules

Liquid-core nanoparticles are promising candidates for targeted ultrasound-controlled therapy, but their acoustic detection remains challenging. High-frequency (20 to 40 MHz) tone burst sequences were implemented with a programmable ultrasound biomicroscope to characterize acoustic response from perfluorooctyl bromide-core nanoparticles with thick poly(lactide-coglycolide) (PLGA) shells. Radio-frequency signals were acquired from flowing solutions of nanoparticles with two different shell-thickness-to-particle-radius ratios, solid PLGA nanoparticles, and latex nanobeads (linear controls). Normalized fundamental (20 MHz) and second-harmonic power spectral density (PSD) increased with particle concentration and was highest for the thinnest shelled particles. The second-harmonic PSD was detectable from the nanoparticles for peak rarefactional pressures (PRP) from 0.97 to 2.01 MPa at 23 cycles and for tone bursts from 11 to 23 cycles at 2.01 MPa. Their second-harmonic-to-fundamental ratio increased as a function of PRP and number of cycles. Within the same PRP and cycle ranges, the second-harmonic-to-fundamental ratios from matched concentration solutions of latex nanobeads and solid PLGA nanoparticles was more weakly detectable but also increased with PRP and number of cycles. Nanoparticles were detectable under flow conditions in vitro using the contrast agent mode of a high-frequency commercial scanner. These results characterize linear acoustic response from the nanoparticles (20 to 40 MHz) and demonstrate potential for their high-frequency detection.