Volumetric and Simultaneous Photoacoustic and Ultrasound Imaging With a Conventional Linear Array in a Multiview Scanning Scheme.

Volumetric, multimodal imaging with precise spatial and temporal coregistration can provide valuable and complementary information for diagnosis and monitoring. Considerable research has sought to combine 3-D photoacoustic (PA) and ultrasound (US) imaging in clinically translatable configurations; however, technical compromises currently result in poor image quality either for PA or ultrasonic modes. This work aims to provide translatable, high-quality, simultaneously coregistered dual-mode PA/US 3-D tomography. Volumetric imaging based on a synthetic aperture approach was implemented by interlacing PA and US acquisitions during a rotate-translate scan with a 5-MHz linear array (12 angles and 30-mm translation to image a 21-mm diameter, 19 mm long cylindrical volume within 21 s). For coregistration, an original calibration method using a specifically designed thread phantom was developed to estimate six geometrical parameters and one temporal offset through global optimization of the reconstructed sharpness and superposition of calibration phantom structures. Phantom design and cost function metrics were selected based on analysis of a numerical phantom and resulted in a high estimation accuracy for the seven parameters. Experimental estimations validated the calibration repeatability. The estimated parameters were used for the bimodal reconstruction of additional phantoms with either identical or distinct spatial distributions of US and PA contrasts. The superposition distance of the two modes was within < 10% of the acoustic wavelength, and a wavelength-order uniform spatial resolution was obtained. This dual-mode PA/US tomography should contribute to more sensitive and robust detection and follow-up of biological changes or the monitoring of slower-kinetic phenomena in living systems such as the accumulation of nanoagents.

Non-Invasive Ultrasonic Description of Tumor Evolution.

PURPOSE: There is a clinical need to better non-invasively characterize the tumor microenvironment in order to reveal evidence of early tumor response to therapy and to better understand therapeutic response. The goals of this work are first to compare the sensitivity to modifications occurring during tumor growth for measurements of tumor volume, immunohistochemistry parameters, and emerging ultrasound parameters (Shear Wave Elastography (SWE) and dynamic Contrast-Enhanced Ultrasound (CEUS)), and secondly, to study the link between the different parameters. METHODS: Five different groups of 9 to 10 BALB/c female mice with subcutaneous CT26 tumors were imaged using B-mode morphological imaging, SWE, and CEUS at different dates. Whole-slice immunohistological data stained for the nuclei, T lymphocytes, apoptosis, and vascular endothelium from these tumors were analyzed. RESULTS: Tumor volume and three CEUS parameters (Time to Peak, Wash-In Rate, and Wash-Out Rate) significantly changed over time. The immunohistological parameters, CEUS parameters, and SWE parameters showed intracorrelation. Four immunohistological parameters (the number of T lymphocytes per mm2 and its standard deviation, the percentage area of apoptosis, and the colocalization of apoptosis and vascular endothelium) were correlated with the CEUS parameters (Time to Peak, Wash-In Rate, Wash-Out Rate, and Mean Transit Time). The SWE parameters were not correlated with the CEUS parameters nor with the immunohistological parameters. CONCLUSIONS: US imaging can provide additional information on tumoral changes. This could help to better explore the effect of therapies on tumor evolution, by studying the evolution of the parameters over time and by studying their correlations.

Monitoring Dual VEGF Inhibition in Human Pancreatic Tumor Xenografts With Dynamic Contrast-Enhanced Ultrasound.

BACKGROUND: Association of drugs acting against different antiangiogenic mechanisms may increase therapeutic effect and reduce resistance. Noninvasive monitoring of changes in the antiangiogenic response of individual tumors could guide selection and administration of drug combinations. Noninvasive detection of early therapeutic response during dual, vertical targeting of the vascular endothelial growth factor pathway was investigated in an ectopic subcutaneous xenograft model for human pancreatic tumor. METHODS: Dynamic contrast-enhanced ultrasound 12 MHz was used to monitor tumor-bearing Naval Medical Research Institute mice beginning 15 days after tumor implantation. Mice received therapy from 15 to 29 days with sorafenib (N = 9), ziv-aflibercept (N = 11), combined antiangiogenic agents (N = 11), and placebo control (N = 14). Sorafenib (BAY 43-9006; Nexavar), a multikinase inhibitor acting on Raf kinase and receptor tyrosine kinases-including vascular endothelial growth factor receptors 2 and 3-was administered daily (60 mg/kg, per os). Ziv-aflibercept (ZALTRAP), a high-affinity ligand trap blocking the activity of vascular endothelial growth factor A, vascular endothelial growth factor B, and placental growth factor was administered twice per week (40 mg/kg, intraperitoneally). RESULTS: Functional evaluation with dynamic contrast-enhanced ultrasound indicated stable tumor vascularization for the control group while revealing significant and sustained reduction after 1 day of therapy in the combined group (P = .007). There was no survival benefit or penalty due to drug combination. The functional progression-free survival assessed with dynamic contrast-enhanced ultrasound was significantly higher for the 3 treated groups; whereas, the progression-free survival based on tumor size did not discriminate therapeutic effect. CONCLUSIONS: Dynamic contrast-enhanced ultrasound, therefore, presents strong potential to monitor microvascular modifications during antiangiogenic therapy, a key role to monitoring antiangiogenic combining therapy to adapt dose range drug.

In Vivo Multiparametric Ultrasound Imaging of Structural and Functional Tumor Modifications during Therapy.

Longitudinal imaging techniques are needed that can meaningfully probe the tumor microenvironment and its spatial heterogeneity. Contrast-enhanced ultrasound, shear wave elastography and quantitative ultrasound are ultrasound-based techniques that provide information on the vascular function and micro-/macroscopic tissue structure. Modifications of the tumor microenvironment induced by cytotoxic and anti-angiogenic molecules in ectopic murine Lewis lung carcinoma tumors were monitored. The most heterogenous structures were found in tumors treated with anti-angiogenic drug that simultaneously accumulated the highest levels of necrosis and fibrosis. The anti-angiogenic group presented the highest number of correlations between parameters related to vascular function and those related to the micro-/macrostructure of the tumor microenvironment. Results suggest how patterns of multiparametric ultrasound modifications can be related to provide a more insightful marker of changes occurring within tumors during therapy.

Local Transverse-Slice-Based Level-Set Method for Segmentation of 3-D High-Frequency Ultrasonic Backscatter From Dissected Human Lymph Nodes.

OBJECTIVE: To detect metastases in freshly excised human lymph nodes (LNs) using three-dimensional (3-D), high-frequency, quantitative ultrasound (QUS) methods, the LN parenchyma (LNP) must be segmented to preclude QUS analysis of data in regions outside the LNP and to compensate ultrasound attenuation effects due to overlying layers of LNP and residual perinodal fat (PNF). METHODS: After restoring the saturated radio-frequency signals from PNF using an approach based on smoothing cubic splines, the three regions, i.e., LNP, PNF, and normal saline (NS), in the LN envelope data are segmented using a new, automatic, 3-D, three-phase, statistical transverseslice-based level-set (STS-LS) method that amends Lankton's method. Due to ultrasound attenuation and focusing effects, the speckle statistics of the envelope data vary with imaged depth. Thus, to mitigate depth-related inhomogeneity effects, the STS-LS method employs gamma probabilitydensity functions to locally model the speckle statistics within consecutive transverse slices. RESULTS: Accurate results were obtained on simulated data. On a representative dataset of 54 LNs acquired from colorectal-cancer patients, the Dice similarity coefficient for LNP, PNF, and NS were 0.938 ± 0.025, 0.832 ± 0.086, and 0.968 ± 0.008, respectively, when compared to expert manual segmentation. CONCLUSION: The STS-LS outperforms the established methods based on global and local statistics in our datasets and is capable of accurately handling the depth-dependent effects due to attenuation and focusing. SIGNIFICANCE: This advance permits the automatic QUS-based cancer detection in the LNs. Furthermore, the STS-LS method could potentially be used in a wide range of ultrasound-imaging applications suffering from depth-dependent effects.

Quantification of tumor perfusion using dynamic contrast-enhanced ultrasound: impact of mathematical modeling.

Dynamic contrast-enhanced ultrasound has been proposed to monitor tumor therapy, as a complement to volume measurements. To assess the variability of perfusion parameters in ideal conditions, four consecutive test-retest studies were acquired in a mouse tumor model, using controlled injections. The impact of mathematical modeling on parameter variability was then investigated. Coefficients of variation (CV) of tissue blood volume (BV) and tissue blood flow (BF) based-parameters were estimated inside 32 sub-regions of the tumors, comparing the log-normal (LN) model with a one-compartment model fed by an arterial input function (AIF) and improved by the introduction of a time delay parameter. Relative perfusion parameters were also estimated by normalization of the LN parameters and normalization of the one-compartment parameters estimated with the AIF, using a reference tissue (RT) region. A direct estimation (rRTd) of relative parameters, based on the one-compartment model without using the AIF, was also obtained by using the kinetics inside the RT region. Results of test-retest studies show that absolute regional parameters have high CV, whatever the approach, with median values of about 30% for BV, and 40% for BF. The positive impact of normalization was established, showing a coherent estimation of relative parameters, with reduced CV (about 20% for BV and 30% for BF using the rRTd approach). These values were significantly lower (p < 0.05) than the CV of absolute parameters. The rRTd approach provided the smallest CV and should be preferred for estimating relative perfusion parameters.

Reproducibility of Contrast-Enhanced Ultrasound in Mice with Controlled Injection.

PURPOSE: Sensitivity of contrast-enhanced ultrasound (CEUS) to microvascular flow modifications can be limited by intra-injection variability (injected dose, rate, volume). PROCEDURES: To evaluate the effect of injection variability on microvascular flow evaluation, CEUS was compared between controlled and manual injections where enhancement was assessed in vitro within a flow phantom, in normal murine kidney (N = 12) and in murine ectopic tumors (N = 10). RESULTS: For both in vitro and in vivo measurements in the renal cortex, controlled injections significantly improved reproducibility of functional parameter estimation. Their coefficient of variation (CV) in the renal cortex ranged from 4 to 19 % for controlled injection vs. 5 to 43 % for manual injections. For measurements in tumors, controlled injection only decreased the CV significantly for the mean transit time. In tumors, multiple injections of contrast agent with a 15-min delay between each were shown to strongly modify contrast uptake by facilitating penetration of microbubbles. CONCLUSION: Improved reproducibility of CEUS assessments in murine models should provide more robust quantification of flow parameters and more sensitive evaluation of tumor modifications in therapeutic models.

VEGFR2-Targeted Contrast-Enhanced Ultrasound to Distinguish between Two Anti-Angiogenic Treatments.

The aim of this study was to evaluate the capacity of BR55, an ultrasound contrast agent specifically targeting vascular endothelial growth factor receptor 2 (VEGFR2), to distinguish the specific anti-VEGFR2 therapy effect of sunitinib from other anti-angiogenic effects of a therapy (imatinib) that does not directly inhibit VEGFR2. Sunitinib, imatinib and placebo were administered daily for 11 d (264 h) to 45 BalbC mice bearing ectopic CT26 murine colorectal carcinomas. During the course of therapy, B-mode ultrasound, contrast-enhanced ultrasound and VEGFR2-targeted contrast-enhanced ultrasound were performed to assess tumor morphology, vascularization and VEGFR2 expression, respectively. The angiogenic effects on these three aspects were characterized using tumor volume, contrast-enhanced area and differential targeted enhancement. Necrosis, microvasculature and expression of VEGFR2 were also determined by histology and immunostaining. B-Mode imaging revealed that tumor growth was significantly decreased in sunitinib-treated mice at day 11 (p < 0.05), whereas imatinib did not affect growth. Functional evaluation revealed that the contrast-enhanced area decreased significantly (p < 0.02) and by similar amounts under both anti-angiogenic treatments by day 8 (192 h): -23% for imatinib and -21% for sunitinib. No significant decrease was observed in the placebo group. Targeted contrast-enhanced imaging revealed lower differential targeted enhancement, that is, lower levels of VEGFR2 expression, in sunitinib-treated mice relative to placebo-treated mice from 24 h (p < 0.05) and relative to both placebo- and imatinib-treated mice from 48 h (p < 0.05). Histologic assessment of tumors after the final imaging indicated that necrotic area was significantly higher for the sunitinib group (21%) than for the placebo (8%, p < 0.001) and imatinib (11%, p < 0.05) groups. VEGFR2-targeted ultrasound was able to sensitively differentiate the anti-VEGFR2 effect from the reduced area of tumor with functional flow produced by both anti-angiogenic agents. BR55 molecular imaging was, thus, able both to detect early therapeutic response to sunitinib in CT26 tumors as soon as 24 h after the beginning of the treatment and to provide early discrimination (48 h) between tumor response during anti-angiogenic therapy targeting VEGFR2 expression and response during anti-angiogenic therapy not directly acting on this receptor.

Automatic motion estimation using flow parameters for dynamic contrast-enhanced ultrasound.

Dynamic contrast-enhanced ultrasound (DCE-US) sequences are subject to motion which can disturb functional flow quantification. This can make estimated parameters more variable or unreliable. Methods that compensate for motion are therefore desirable. The most commonly used motion correction techniques in DCE-US register the images in the sequence with respect to a user-selected reference image. However, this image may not include all features that are representative of the whole sequence. Moreover, image-based registration neglects pertinent, functional-flow information contained in the DCE-US sequence. An operator-free method is proposed that combines the motion estimation and flow-parameter quantification (M/Q method) in a single mathematical framework. This method is based on a realistic multiplicative model of the DCE-US noise. By computing likelihood in this model, motion and flow parameters are both estimated iteratively. First, the maximization is accomplished by estimating functional and motion parameters. Then, a final registration based on a non-parametric temporal smoothing of the sequence is performed. This method is compared to a conventional (mutual information) registration method where all the images of the sequence are registered with respect to a reference image chosen by an expert. The two methods are evaluated on simulated sequences and DCE-US sequences acquired in patients (N = 15). The M/Q method demonstrates significantly (p < 0.05) lower Dice coefficients and Hausdorff distance than the conventional method on the simulated data sets. On the in vivo sequences analysed, the M/Q methods outperformed the conventional method in terms of mean Dice and Hausdorff distance on 80% of the sequences, and in terms of standard deviation of Dice and Hausdorff distance on 87% of the sequences.

Modeling the envelope statistics of three-dimensional high-frequency ultrasound echo signals from dissected human lymph nodes.

This work investigates the statistics of the envelope of three-dimensional (3D) high-frequency ultrasound (HFU) data acquired from dissected human lymph nodes (LNs). Nine distributions were employed, and their parameters were estimated using the method of moments. The Kolmogorov Smirnov (KS) metric was used to quantitatively compare the fit of each candidate distribution to the experimental envelope distribution. The study indicates that the generalized gamma distribution best models the statistics of the envelope data of the three media encountered: LN parenchyma, fat and phosphate-buffered saline (PBS). Furthermore, the envelope statistics of the LN parenchyma satisfy the pre-Rayleigh condition. In terms of high fitting accuracy and computationally efficient parameter estimation, the gamma distribution is the best choice to model the envelope statistics of LN parenchyma, while, the Weibull distribution is the best choice to model the envelope statistics of fat and PBS. These results will contribute to the development of more-accurate and automatic 3D segmentation of LNs for ultrasonic detection of clinically significant LN metastases.

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.

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 highfrequency detection.

Echo-power estimation from log-compressed video data in dynamic contrast-enhanced ultrasound imaging.

Ultrasound (US) scanners typically apply lossy, non-linear modifications to the US data for visualization purposes. The resulting images are then stored as compressed video data. Some system manufacturers provide dedicated software for quantification purposes to eliminate such processing distortions, at least partially. This is currently the recommended approach for quantitatively assessing changes in contrast-agent concentration from clinical data. However, the machine-specific access to US data and the limited set of analysis functionalities offered by each dedicated-software package make it difficult to perform comparable analyses with different US systems. The objective of this work was to establish if linearization of compressed video images obtained with an arbitrary US system can provide an alternative to dedicated-software analysis of machine-specific files for the estimation of echo-power. For this purpose, an Aplio 50 system (Toshiba Medical Systems, Tochigi, Japan), coupled with dedicated CHI-Q (Contrast Harmonic Imaging Quantification) software by Toshiba Medical Systems, was used. Results were compared with two approaches that apply algorithms to estimate relative echo-power from compressed video images: commercially available VueBox software by Bracco Suisse SA (Geneva, Switzerland) and in-laboratory software called PixPower. The echo-power estimated by CHI-Q analysis indicated a strong linear relationship versus agent concentration in vitro (R(2) ≥ 0.9996) for dynamic range (DR) settings of DR60 and DR80, with slopes between 9.22 and 9.57 dB/decade (p = 0.05). These values approach the theoretically predicted dependence of 10.0 dB/decade (equivalent to 3 dB for each concentration doubling). Echo-power estimations obtained from compressed video images with VueBox and PixPower also exhibited strong linear proportionality with concentration (R(2) ≥ 0.9996), with slopes between 9.30 and 9.68 dB/decade (p = 0.05). On an independent in vivo data set (N = 24), the difference in echo-power estimation between CHI-Q and each of the other two approaches was calculated after excluding regions that contain pixels affected by saturated or thresholded pixel values. The mean difference in estimates (expressed in decibels) was -0.25 dB between VueBox and CHI-Q (95% confidence interval: -0.75 to 0.26 dB) and -0.17 dB between PixPower and CHI-Q (95% confidence interval: -0.67 to 0.13 dB). To achieve linearization of data, one of the approaches (VueBox) requires calibration files provided by the software manufacturer for each machine type and setting. The other (PixPower) requires empirical correction of the imaging dynamic range based on ground truth data. These requirements could potentially be removed if US system manufacturers were willing to make relevant information on the applied processing publically available. Reliable echo-power estimation from linearized data would facilitate inclusion of different US systems in multicentric studies and more widespread implementation of emerging techniques for quantitative analysis of contrast ultrasound.

Fast in vivo imaging of amyloid plaques using µ-MRI Gd-staining combined with ultrasound-induced blood-brain barrier opening.

Amyloid plaques are one of the major microscopic lesions that characterize Alzheimer's disease. Current approaches to detect amyloid plaques by using magnetic resonance imaging (MRI) contrast agents require invasive procedures to penetrate the blood-brain barrier (BBB) and to deliver the contrast agent into the vicinity of amyloid plaques. Here we have developed a new protocol (US-Gd-staining) that enables the detection of amyloid plaques in the brain of an APP/PS1 transgenic mouse model of amyloidosis after intra-venous injection of a non-targeted, clinically approved MRI contrast agent (Gd-DOTA, Dotarem®) by transiently opening the BBB with unfocused ultrasound (1 MHz) and clinically approved microbubbles (Sonovue®, Bracco). This US-Gd-staining protocol can detect amyloid plaques with a short imaging time (32 min) and high in-plane resolution (29 µm). The sensitivity and resolution obtained is at least equal to that provided by MRI protocols using intra-cerebro-ventricular injection of contrast agents, a reference method used to penetrate the BBB. To our knowledge this is the first study to demonstrate the ability of MR imaging to detect amyloid plaques by using a peripheral intra-venous injection of a clinically approved NMR contrast agent.

Correlation and agreement between contrast-enhanced ultrasonography and perfusion computed tomography for assessment of liver metastases from endocrine tumors: normalization enhances correlation.

We studied correlation and agreement between perfusion parameters derived from contrast-enhanced ultrasonography (CEUS) and computed tomography (CT). Both techniques were performed in 16 patients with proven liver metastases from endocrine tumor. Replenishment study after ultrasound-induced destruction of microbubbles was used for CEUS quantification. CEUS-derived relative values of blood flow, blood volume and mean transit time were compared with perfusion CT-derived parameters measured in the same tumors. Significant correlation was observed between CEUS normalized values and CT absolute tumor values for blood flow (r = 0.58; p = 0.018), blood volume (r = 0.61; p = 0.012) and mean transit time (r = 0.52; p = 0.037). Correlation was not significant for non-normalized values. Agreement between CEUS normalized values and perfusion CT relative values was significant (p < 0.04). Estimated bias between CEUS and CT for relative perfusion values was -1.38 (-5.02; 2.27) for blood flow, +0.26 (-0.79; 1.31) for blood volume and +0.21 (-0.46; 0.87) for mean transit time. We conclude that normalization markedly increased correlation between CEUS- and CT-derived perfusion values and allowed agreement assessment.

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.

Noninvasive contrast-enhanced US quantitative assessment of tumor microcirculation in a murine model: effect of discontinuing anti-VEGF therapy.

PURPOSE: To determine, by using contrast material-enhanced ultrasonography (US), how quickly renal tumors grafted in mice begin to revascularize after stopping bevacizumab treatment. MATERIALS AND METHODS: All 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. RESULTS: The 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). CONCLUSION: Noninvasively measured contrast-enhanced US parameters demonstrated tumor revascularization after stopping antiangiogenic therapy in this murine tumor model.

Real-time chirp-coded imaging with a programmable ultrasound biomicroscope.

Ultrasound biomicroscopy (UBM) of mice can provide a testing ground for new imaging strategies. The UBM system presented in this paper facilitates the development of imaging and measurement methods with programmable design, arbitrary waveform coding, broad bandwidth (2-80 MHz), digital filtering, programmable processing, RF data acquisition, multithread/multicore real-time display, and rapid mechanical scanning (

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 tumor's 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.

Ultrasound biomicroscopy: a powerful tool probing murine lymph node size in vivo.

Invasive cell-counting in lymph node (LN) is the current reference to assess LN changes due to inflammation, immunodeficiency and cancer in murine models. This work evaluates whether ultrasound biomicroscopy (UBM) can measure LN size alterations noninvasively for a large range of sizes (0.1 mm3 to 22 mm3). Correlation was assessed (rho = 0.91, p < 0.0001) between invasive cell count and LN volume estimated with UBM (24, 2 to 28-week-old, C57BL/6 mice; 13 same-strain, transgenic mice presenting LN hyperplasia). UBM LN modification screening was applied in a skin-graft rejection model and compared with cell-counting (15 mice). UBM LN-size follow-up with fine temporal sampling was demonstrated from 9 d of age (minimum area 0.13 mm2). Reliability (intraclass correlation coefficient [ICC] > 0.84) and variability of UBM evaluations compared favourably with invasive cell count. UBM provides a noninvasive alternative to cell-counting in mice for early detection and longitudinal screening of LN modifications. This can enable significant reduction in the number of mice and exploration of LNs that would be too small to dissect for cell count.