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Plane-wave imaging (PWI) with angular compounding has gained in popularity over recent years, because it provides high frame rates and good image properties. However, most linear arrays used in clinical practice have a pitch that is equal to than the wavelength of ultrasound. Hence, the presence of grating lobes is a concern for PWI using multiple transmit angles. The presence of grating lobes produces clutter in images and reduces the ability to observe tissue contrast. Techniques to reduce or eliminate the presence of grating lobes for PWI using multiple angles will result in improved image quality. Null subtraction imaging (NSI) is a nonlinear beamforming technique that has been explored for improving the lateral resolution of ultrasonic imaging. However, the apodization scheme used in NSI also eliminates or greatly reduces the presence of grating lobes. Imaging tasks using NSI were evaluated in simulations and physical experiments involving tissue-mimicking phantoms and rat tumors in vivo. Images created with NSI were compared with images created using traditional delay and sum (DAS) with Hann apodization and images created using a generalized coherence factor (GCF). NSI was observed to greatly reduce the presence of grating lobes in ultrasonic images, compared to DAS with Hann and GCF, while maintaining spatial resolution and contrast in the images. Therefore, NSI can provide a novel means of creating images using PWI with multiple steering angles on clinically available linear arrays while reducing the adverse effects associated with grating lobes.

Ensuring the consistency of spectral-based quantitative ultrasound estimates in vivo necessitates accounting for diffraction, system effects, and propagation losses encountered in the tissue. Accounting for diffraction and system effects is typically achieved through planar reflector or reference phantom methods; however, neither of these is able to account for the tissue losses present in vivo between the ultrasound probe and the region of interest. In previous work, the feasibility of small titanium beads as in situ calibration targets (0.5–2 mm in diameter) was investigated. In this study, the importance of bead size for the calibration signal, the role of multiple echoes coming from the calibration bead, and sampling of the bead signal laterally through beam translation were examined. This work demonstrates that although the titanium beads naturally produce multiple reverberant echoes, time-windowing of the first echo provides the smoothest calibration spectrum for backscatter coefficient calculation. When translating the beam across the bead, the amplitude of the echo decreases rapidly as the beam moves across and past the bead. Therefore, to obtain consistent calibration signals from the bead, lateral interpolation is needed to approximate signals coming from the center of the bead with respect to the beam.

BACKGROUND Tomatoes contain carotenoids that have the potential to alter the effects of external beam radiation therapy (EBRT). OBJECTIVES We hypothesized that dietary lyophilized tomato paste (TP) would reduce apoptosis within carotenoid-containing nonneoplastic tissues in EBRT-treated TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. METHODS Male TRAMP mice (n = 73) were provided an AIN-93G diet or a modified AIN-93G diet containing 10% TP (wt:wt) at 4 wk of age. Prostate tumor growth was monitored by ultrasound. The caudal half of the mouse was irradiated with 7.5 Gy (Rad) or 0 Gy (sham) at 24 wk of age or after the tumor volume exceeded 1000 mm3 with a Cobalt-60 source. Mice were euthanized 24 h postradiation. Carotenoids and α-tocopherol were measured by HPLC and compared by a t test. Tissues were assessed for radiation-induced changes (hematoxylin and eosin) and apoptosis [cleaved caspase-3 (CC3)] and compared by Kruskal-Wallis test or Freedman-Lane's permutation test. RESULTS Serum concentrations of lycopene (52% lower), phytoene (26% lower), and α-tocopherol (22% lower) were decreased in TP-fed irradiated mice (TP-Rad) compared with TP-fed sham mice (P

Ultrasound localization microscopy can image microvasculature in vivo without sacrificing imaging penetration depth. However, the reliance on super-resolution inference limits the applicability of the technique as subpixel tissue motion can corrupt microvascular reconstruction. Consequently, the majority of previous pre-clinical research on this super-resolution procedure has been restricted to low motion experimental models with ample motion correction and/or data rejection, which precludes the imaging of organ sites with that exhibit a high degree of respiratory and other motion. In this paper, we present a novel anesthesia protocol in rabbits that induces safe controllable periods of apnea to enable the long imaging acquisition times required for ultrasound localization microscopy. We apply this protocol to a VX2 liver tumor model undergoing sorafenib therapy and compare the results to super-resolution images from a conventional high-dose isoflurane anesthesia. We found that the apneic protocol was necessary to correctly identify the poorly vascularized tumor cores, as verified by immunohistochemistry, and to reveal the tumoral microvascular architecture.

Quantitative ultrasound (QUS) was used to classify rabbits that were induced to have liver disease by placing them on a fatty diet for a defined duration and/or periodically injecting them with CCl(4). The ground truth of the liver state was based on lipid liver percents estimated via the Folch assay and hydroxyproline concentration to quantify fibrosis. Rabbits were scanned ultrasonically in vivo using a SonixOne scanner and an L9–4/38 linear array. Liver fat percentage was classified based on the ultrasonic backscattered radio-frequency (RF) signals from the livers using either QUS or a 1D convolutional neural network (CNN). Use of QUS parameters with linear regression and canonical correlation analysis (CCA) demonstrated that the QUS parameters could differentiate between livers with lipid levels above or below 5%. However, the QUS parameters were not sensitive to fibrosis. The CNN was implemented by analyzing raw RF ultrasound signals without using separate reference data. The CNN output the classification of liver as either above or below a threshold of 5% fat level in the liver. The CNN outperformed the classification utilizing the QUS parameters combine with a support vector machine (SVM) in differentiating between low and high lipid liver levels, i.e., accuracies of 74% versus 59% on the testing data. Therefore, while the CNN did not provide a physical interpretation of the tissue properties, e.g., attenuation of the medium or scatterer properties, the CNN had much higher accuracy in predicting fatty liver state and did not require an external reference scan.

The use of wireless implanted medical devices (IMDs) is growing because they facilitate monitoring of patients at home and during normal activities, reduce the discomfort of patients, and reduce the likelihood of infection associated with trailing wires. Currently, radio frequency (RF) electromagnetic waves are the most commonly used method for communicating wirelessly with IMDs. However, due to the restrictions on the available bandwidth and the employable power, data rates of RF-based IMDs are limited to 267 kb/s. Considering standard definition video streaming requires data rates of 1.2 Mb/s and high definition requires 3 Mb/s, it is not possible to use the RF electromagnetic communications for high data rate communication applications such as video streaming. In this work, an alternative method that utilizes ultrasonic waves to relay information at high data rates is introduced. An advanced quadrature amplitude modulation (QAM) modem with phase-compensating, sparse decision feedback equalizer (DFE) is tailored to realize the full potential of the ultrasonic channel through biological tissues. The proposed system is tested in a variety of scenarios, including both simulations with finite impulse response (FIR) channel models, and real physical transmission experiments with ex vivo beef liver and pork chop samples as well as in situ rabbit abdomen. Consequently, the simulations demonstrated that video-capable data rates can be achieved with millimeter-sized transducers. Real physical experiments confirmed data rates of 6.7, 4.4, 4, and 3.2 Mb/s through water, ex vivo beef liver, ex vivo pork chop, and in situ rabbit abdomen, respectively.

BACKGROUND: Dietary tomato products or lycopene protect against prostate carcinogenesis, but their impact on the emergence of castration-resistant prostate cancer (CRPC) is unknown. OBJECTIVE: We hypothesized that tomato or lycopene products would reduce the emergence of CRPC. METHODS: Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were castrated at 12–13 wk and the emergence of CRPC was monitored by ultrasound in each study. In Study 1, TRAMP mice (n = 80) were weaned onto an AIN-93G-based control diet (Con-L, n = 28), a 10% tomato powder diet (TP-L, 10% lyophilized w/w, n = 26), or a control diet followed by a tomato powder diet after castration (TP-Int1, n = 26). In Study 2, TRAMP mice (n = 85) were randomized onto a control diet with placebo beadlets (Con-Int, n = 29), a tomato diet with placebo beadlets (TP-Int2, n = 29), or a control diet with lycopene beadlets (Lyc-Int, n = 27) following castration (aged 12 wk). Tumor incidence and growth were monitored by ultrasound beginning at an age of 10 wk. Mice were euthanized 4 wk after tumor detection or aged 30 wk if no tumor was detected. Tissue weights were compared by ANOVA followed by Dunnett's test. Tumor volumes were compared using generalized linear mixed model regression. RESULTS: Ultrasound estimates for the in vivo tumor volume were strongly correlated with tumor weight at necropsy (R(2) = 0.75 and 0.94, P

The emergence of in-body medical devices has provided a means of capturing physiological or diagnostic information and streaming this information outside of the body. Currently, electromagnetic-based communications make up the bulk of in-body medical device communication protocols. Traditional electromagnetic-based solutions are limited in their data rates and available power. Recently, ultrasound was investigated as a communication channel for through-tissue data transmission. To achieve real-time video streaming through tissue, data rates of ultrasound need to exceed 1 Mbps. In a previous study, we demonstrated ultrasound communications with data rates greater than 30 Mbps with two focused ultrasound transducers using a large footprint laboratory system through slabs of lossy tissues. While the form factor of the transmitter is also crucial, it is obvious that a large, focused transducer cannot fit within the size of a small in-body medical device. Several other challenges for achieving high-speed ultrasonic communication through tissue include strong reflections leading to multipath effects and attenuation. In this work, we demonstrate ultrasonic video communications using a mm-scale microcrystal transmitter with video streaming supplied by a camera connected to a Field Programmable Gate Array (FPGA). The signals were transmitted through a tissue-mimicking phantom and through the abdomen of a rabbit in vivo . The ultrasound signal was recorded by an array probe connected to a Verasonics Vantage system and decoded back to video. To improve the received signal quality, we combined the signal from multiple channels of the array probe. Orthogonal frequency division multiplexing (OFDM) modulation was used to reduce the receiver complexity under a strong multipath environment.

Untreated nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) lead to irreversible liver damage. We hypothesized that a low-fat diet (LFD) or a high-fat diet (HFD) with soy protein isolate (SPI) would be an effective intervention to halt or reverse NAFLD progression. To test these hypotheses, we conducted 2 studies. In the first study, we fed an HFD to 7-week-old C57BL/6J mice to induce NAFLD compared to an LFD (control). Hepatic steatosis was monitored by quantitative ultrasound (QUS) scans (in vivo and ex vivo). Animals were euthanized after 0, 2, 4, and 6 weeks of feeding. In the second study, 7-week-old mice were randomized onto an LFD or HFD with SPI intervention after 4 weeks of feeding HFD. Animals from each group were scanned with QUS and euthanized after 4, 9, and 12 weeks of feeding. Animals fed the HFD developed NAFLD (100%) and NASH (80%) characterized by increased liver weight, lipid accumulation, and histological scores for inflammation by 4 weeks in the first study. In the second study, the LFD ameliorated this NAFLD phenotype after 5 weeks of feeding; however, the SPI intervention failed to significantly attenuate NAFLD. QUS parameters were significantly increased with the HFDs (P < .05) and steatosis grade (P < .05) and were positively correlated with hepatic lipid concentrations. In conclusion, dietary modification may be effective at reversing NAFLD and NASH at early stages. Furthermore, QUS may become a valuable tool to track hepatic steatosis. Additional studies are needed to further evaluate the effectiveness of these interventions.

OBJECTIVES To show that quantitative ultrasound biomarkers attenuation (AC) and backscatter (BSC) coefficients are effective tools to detect early changes in acute pancreatitis, using a cerulein-induced pancreatitis rat model. METHODS Sprague-Dawley rats (n = 68) were divided into 8 groups: uninjected cage controls, saline-injected controls, and cerulein-injected rats euthanized at 2, 4, 15, 24, 48, and 60 hours after injection. Pancreatic AC and BSC (25-55 MHz) were estimated in vivo (Vevo 2100, VisualSonics, Toronto, CA) and ex vivo (40-MHz transducer). The pancreas of each rat was evaluated histopathologically. RESULTS Changes in both in vivo and ex vivo AC and BSC relative to controls reflected temporal histomorphologic changes. Overall, there were decreased AC and BSC at early time points and then rebound toward control values over time. Maximal in vivo AC and BSC decreases occurred at 2 hours after cerulein injection. Attenuation coefficient changes corresponded well with early pancreatic edema and acinar cell vacuolation, with rebound as edema decreased, autophagy/cellular death occurred, and histiocytic infiltrates and fibrosis manifested. Backscatter coefficient decreased early but rebounded as autophagy and apoptosis increased, only to fall as acinar atrophy peaked, and fibrosis and histiocytic infiltration increased. CONCLUSIONS Cerulein-induced pancreatitis is an excellent model for studying ultrasonic AC and BSC biomarkers during the early stages of acute pancreatitits, reflecting microscopic structural changes. Edema followed by cell shrinkage and apoptosis, then histiocytic infiltration and fibrosis, has certain similarities with the morphologies of some forms of pancreatic carcinoma. This suggests that quantitative ultrasound may be very useful for early detection of disease onset or response to therapy for not only acute pancreatitis but also pancreatic cancer.

OBJECTIVES: The primary objective was to determine the effect of dietary tomato on prostate cancer (PCa) development and progression in overweight/obese, transgenic mice prone to PCa (TRAMP mice). Secondary objectives included assessing histological, inflammatory, angiogenic, and metabolic changes in prostate tumors at different time points of cancer progression. METHODS: Four-week-old TRAMP mice were randomly assigned to consume one of four diets (n = 45/diet): control (CON) or obesogenic (OB), both with and without 10% freeze-dried tomato powder (TP). Prostate tumor incidence and growth were monitored via ultrasound imaging. Mice were terminated one or four weeks following tumor development to assess early and later molecular changes in the tumors. RESULTS: OB diets led to greater body weight over time (45.2 ± 1.0 g at 24 weeks of age) when compared with CON diets (33.2 ± 0.8 g; p

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and can often lead to fibrosis, cirrhosis, cancer and complete liver failure. Liver biopsy is the current standard of care to quantify hepatic steatosis, but it comes with increased patient risk and only samples a small portion of the liver. Imaging approaches to assess NAFLD include proton density fat fraction estimated via magnetic resonance imaging (MRI) and shear wave elastography. However, MRI is expensive and shear wave elastography is not proven to be sensitive to fat content of the liver (Kramer et al. 2016). On the other hand, ultrasonic attenuation and the backscatter coefficient (BSC) have been observed to be sensitive to levels of fat in the liver (Lin et al. 2015; Paige et al. 2017). In this study, we assessed the use of attenuation and the BSC to quantify hepatic steatosis in vivo in a rabbit model of fatty liver. Rabbits were maintained on a high-fat diet for 0, 1, 2, 3 or 6 wk, with 3 rabbits per diet group (total N = 15). An array transducer (L9-4) with a center frequency of 4.5 MHz connected to a SonixOne scanner was used to gather radio frequency (RF) backscattered data in vivo from rabbits. The RF signals were used to estimate an average attenuation and BSC for each rabbit. Two approaches were used to parameterize the BSC (i.e., the effective scatterer diameter and effective acoustic concentration using a spherical Gaussian model and a model-free approach using a principal component analysis [PCA]). The 2 major components of the PCA from the BSCs, which captured 96% of the variance of the transformed data, were used to generate input features to a support vector machine for classification. Rabbits were separated into two liver fat-level classes, such that approximately half of the rabbits were in the low-lipid class (≤9% lipid liver level) and half of the rabbits in the high-lipid class (>9% lipid liver level). The slope and the midband fit of the attenuation coefficient provided statistically significant differences (p value = 0.00014 and p value = 0.007, using a two-sample t test) between low and high-lipid fat classes. The proposed model-free and model-based parameterization of the BSC and attenuation coefficient parameters yielded classification accuracies of 84.11 %, 82.93 % and 78.91 % for differentiating low-lipid versus high-lipid classes, respectively. The results suggest that attenuation and BSC analysis can differentiate low-fat versus high-fat livers in a rabbit model of fatty liver disease.

The success of any minimally invasive treatment procedure can be enhanced significantly if combined with a robust noninvasive imaging modality that can monitor therapy in real time. Quantitative ultrasound (QUS) imaging has been widely investigated for monitoring various treatment responses such as chemotherapy, radiation, and thermal therapy. Previously, we demonstrated the feasibility of using spectral-based QUS parameters to monitor high-intensity focused ultrasound (HIFU) treatment of in situ tumors in euthanized rats [ Ultrasonic Imaging 36(4), 239–255, 2014]. In the present study, we examined the use of spectral-based QUS parameters to monitor HIFU treatment of in vivo rat mammary adenocarcinoma tumors (MAT) where significant tissue motion was present. HIFU was applied to tumors in rats using a single-element transducer. During the off part of the HIFU duty cycle, ultrasound backscatter was recorded from the tumors using a linear array co-aligned with the HIFU focus. A total of 10 rats were treated with HIFU in this study with an additional sham-treated rat. Spectral parameters from the backscatter coefficient, i.e., effective scatterer diameter ( ESD ) and effective acoustic concentration ( EAC ), were estimated. The changes of each parameter during treatment were compared with a temperature profile recorded by a fine-needle thermocouple inserted into the tumor a few millimeters behind the focus of the HIFU transducer. The mean ESD changed from $121 \pm {6}$ to $81 \pm {8}\, \upmu {\rm{m}}\, (p{\text{-value}}=0.0002)$ , and the EAC changed from $33 \pm {2}$ to $46 \pm {3}\, \hbox{dB/cm}^{3}\, (p{\text{-value}}=0.0002)$ during HIFU exposure as the temperature increased on average from $38.7 \pm 1.0 ^\circ{\rm C}$ to $64.2 \pm 2.7 ^\circ{\rm C}$ . The changes in ESD and EAC were linearly correlated with the changes in tissue temperature during the treatment. When HIFU was turned off, the $ESD$ increased from $81 \pm {8}$ to $121 \pm {7}\,\upmu{\rm{m}}$ and the EAC dropped from $46 \pm {3} $ to $36\pm {2}\, \hbox{dB/cm}^{3}$ as the temperature decreased from $64.2 \pm 2.7 ^\circ{\rm C}$ to $45 \pm 2.7 ^\circ{\rm C}$ . QUS was demonstrated in vivo to track temperature elevations caused by HIFU exposure.

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

OBJECTIVES: Tomatoes contain carotenoids and other potent antioxidants that may protect the surrounding tissue from the detrimental effects of external beam radiation therapy, while reducing rates of prostate carcinogenesis. The objective of this study was to determine whether dietary lyophilized tomato paste (TP) alters early inflammatory and oxidative events following a single dose of radiation and leads to a more successful therapeutic outcome. METHODS: Male Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice (n = 76) were provided a powdered AIN-93 G diet (Control) or a modified AIN-93 G diet containing 10% TP (w/w) at 4 weeks of age. Mice were monitored by ultrasound for in vivo tumor detection and 3-D volumetric measurement biweekly. Once tumors reached a volume of 1000 mm(3), the caudal half of the mouse was irradiated with 7.5 gy (Rad, n = 18–19 per diet) or 0 gy (sham, n = 16–20 per diet) with a Cobalt-60 source. Mice were euthanized 24 hours after radiation or sham treatment. Antioxidants (carotenoids and α-tocopherol) were measured by high performance liquid chromatography (HPLC) in the serum, tumor, prostate, and liver. Sections of tumor, liver, kidneys, bladder, lymph, bladder and intestines were stained by hematoxylin and eosin (H&E) and cleaved-caspase 3 were assessed for radiation-induced changes and apoptosis. Inflammatory markers (C-reactive protein, IL-6, IL-17A, TNFα, IFNγ, and IL-10) were measured in serum, liver, prostate, tumor, and epididymal adipose tissues by ELISA. RESULTS: This study is the first to explore the effects of TP on the tumor microenvironment following irradiation. Initial results suggest that TP consumption does not alter circulating or tissue (liver and prostate) concentrations of inflammatory markers (C-reactive protein, TNFα, IFNγ, IL-6, IL-17, or IL-10). We hypothesize that TP-Rad will maintain similar levels of circulating concentrations of antioxidants (carotenoids and α-tocopherol) compared to sham-treated mice. Additionally, we hypothesize that TP will reduce markers of cell damage in surrounding tissues. CONCLUSIONS: This study will provide important preclinical data to inform future clinical trials evaluating approaches to lessen extra-prostatic damage from radiation therapy and thus improve therapeutic outcomes. FUNDING SOURCES: This work was supported by USDA NIFA ILLU-971–334.

Quantitative ultrasound estimates based on the backscatter coefficient (BSC) have demonstrated the ability to detect and classify disease beyond the capabilities of traditional B-mode imaging. Quantitative ultrasound has been able to differentiate live from apoptotic cells or oncotic cells in vitro with high-frequency ultrasound,1,2 monitor changes in tissue microstructure due to high-intensity focused ultrasound at high frequencies in a preclinical model,3 and monitor changes due to chemotherapy clinically at 6 MHz.4 Tumors have been characterized and differentiated using quantitative ultrasound at high frequencies in preclinical models of thyroid cancer,5 at high frequencies in clinical applications for ocular tumors,6,7 and at clinical frequencies in human patients for breast tumors8 and prostate cancers.9,10 Also, the BSC has been proven valuable for diagnosing fatty liver disease clinically11 and for monitoring physiologic and pharmacologic responses in renal function at clinical frequencies in an animal model.12,13 One of the BSC features that is often cited as an advantage is its system independence; ie, the same BSC value for a given tissue can be estimated from any imaging platform. Previous studies have demonstrated system independence of BSC estimates in well-characterized phantoms.14–17 These investigations applied single-element imaging systems measuring backscatter levels for strong glass bead scatterer phantoms (1–12 MHz),14,15 single-element imaging systems for probing weakly scattering agar-in-agar phantoms (1–13 MHz),16 and clinical array imaging systems for estimating BSC for phantoms with glass bead scatterers (1–15 MHz).17 Parametric estimates based on the BSC have demonstrated the ability to differentiate different types of orthotopic mouse tumors ex vivo (16–24 MHz),18 to identify regions of micrometastases within resected lymph nodes clinically (26.5 MHz center),19 and to identify dominant sources of backscattering in the renal cortex (frequencies covering 2.5–15 MHz).12,13,20,21 The frequency ranges in these studies cover the range being investigated in this study: namely, from the clinical frequencies to the lower end of the small-animal pre-clinical imaging frequencies, covering, for example, 1 to 21 MHz. This study addresses the need for demonstrating that in vivo interlaboratory measurements can be used to distinguish different types of tumors, while being able to obtain consistent BSC results from all systems. Previous studies22,23 examined BSC agreement across single-element and clinical imaging systems for an in vivo rodent fibroadenoma model over a frequency range of 1 to 14 MHz. The heterogeneity of the benign fibroadenomas provided a near worst-case scenario to evaluate quantitative ultrasound performance. The study presented herein seeks to expand on this work using clinical and preclinical array imaging systems to test their capability to detect known tissue differences while maintaining good agreement in BSC values across all systems. Mouse (4T1) and rat (MAT) mammary tumor models were used in a comparative experiment with 3 array-based ultrasound imaging systems (Siemens, Ultrasonix, and VisualSonics) and 5 different transducers covering a range of frequency bandwidths. Backscatter coefficient agreement between systems was assessed, as well as the ability to differentiate the two tumor types.

Dietary patterns with cardiovascular benefits have been recommended, but the relative contributions of individual foods and food components, alone or in combination, remain undefined. Male ApoE(-/-) mice were fed either a purified AIN-93G control diet, a Western diet (WD), or a WD with 10% tomato powder (TP), 2% soy germ (SG), or the combination, for 4 wk (n = 10 per group). Plasma total cholesterol and triglycerides were measured with enzymatic colorimetric kits, and serum amyloid A (SAA) was measured by ELISA. Liver lipids were extracted with chloroform:methanol, and triglycerides, free and esterified cholesterol measured with enzymatic colorimetric kits. Expression of Cyp27a1, Cyp7a1, Abcg5, and Abcg8 in the liver was determined by quantitative polymerase chain reaction. Sections of the aortic root and aorta were cut and stained with hematoxylin and eosin (H&E) to assess extent of atherosclerotic lesions. WD-fed animals had greater liver and adipose weights, plasma cholesterol and SAA, hepatic lipids, and atherosclerosis than AIN-93G animals. TP and SG did not decrease atherosclerosis as measured by H&E-stained sections of the aortic root, aortic arch, and descending aorta. The TP diets further increased plasma cholesterol, but also led to increased expression of the Abcg5/8 transporters involved in cholesterol efflux. Addition of SG alone to the WD attenuated WD-induced increases in plasma cholesterol, liver lipids, and gonadal adipose weight. The results of this study do not support the use of either TP or SG for reduction of atherosclerosis, but suggest some beneficial effects of SG on lipid metabolism in this model of cardiovascular disease.

Ultrasound contrast agents (UCAs) are used to enhance ultrasound imaging of the cardiovascular system. Ultrasound contrast agents are administered intravenously as a solution of micrometer-sized bubbles, consisting of an inert gas encased in a shell of phospholipid or albumin. When ultrasound is applied to the heart or blood vessels, the ultrasonic waves interact with UCAs in the circulation, causing the bubbles to oscillate or collapse. The interaction of ultrasound with UCAs opacifies the blood, allowing for improved imaging of cardiac structure and function,1 arterial stenosis,2 vascularization of atherosclerotic plaques,3 and other aspects of the cardiovascular system. When used for cardiac left ventricular opacification, UCAs can enhance the visual quality of ultrasound scans in patients who are difficult to image and improve diagnostic accuracy.4,5 While UCAs have clear clinical utility in detection and diagnosis of cardiovascular disease, concerns have been raised over their safety. There have been reports of adverse cardiovascular events after ultrasound with UCA administration in animal models and humans. In experimental studies from our research group and others, UCAs have been shown to induce cardiac arrhythmias6 and hemorrhage,7–9 impair endothelium-dependent vasodilatation,10 and alter atheroma thickness and the circulating biomarker von Willebrand Factor (vWF).11 Reports of cardiopulmonary complications and deaths in human clinical patients associated with UCA administration added to these concerns, and patients with preexisting cardiovascular disease appeared to be at increased risk.12 As a result, the US Food and Drug Administration (FDA) mandated warning labels for 2 commercially available UCAs, Definity (Lantheus Medical Imaging, North Billerica, MA) and Optison (GE Healthcare, Princeton, NJ) in 2007. The reported adverse events highlight the importance of further research in this area. The biological effects of contrast ultrasound may be a function of the ultrasound pressure level or UCA concentration, so it is necessary to test for adverse events using a variety of scenarios and experimental models. In this study, we used the ApoE−/− mouse model of atherosclerosis to determine thresholds for arterial damage at several different ultrasound pressure levels, with concomitant UCA administration. We fed groups of mice either a standard chow diet or a high-fat, high-sugar, cholesterol-containing Western diet to modulate the progression of atherosclerosis after a single contrast ultrasound exposure. We hypothesized that contrast ultrasound would induce vascular injury and accelerate the progression of atherosclerosis, that this effect would be pressure dependent, and that the threshold for effects of contrast ultrasound on biomarkers would be lower than FDA limits established for clinical ultrasound imaging.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and can often lead to fibrosis, cirrhosis, cancer and complete liver failure. Liver biopsy is the current standard of care to quantify hepatic steatosis, but it comes with increased patient risk and only samples a small portion of the liver. Imaging approaches to assess NAFLD include proton density fat fraction (PDFF) estimated via MRI and shear wave elastography. However, MRI is often prohibitively expensive and shear wave elastography is not sensitive to fat content of the liver [1]. On the other hand, ultrasonic attenuation and the backscatter coefficient (BSC) have been observed to be sensitive to levels of fat in the liver. In this study, we explored the use of attenuation and a principal component analysis (PCA) of the BSC to detect and quantify hepatic steatosis in vivo in a rabbit model of fatty liver. Rabbits were maintained on a high fat diet for 0, 1, 2, 3 or 6 weeks with three rabbits per diet group (total N = 15). For analysis, rabbits were separated into three classes based on the total lipid content of the livers estimated using the Folch method: low fat ( 10%). An array transducer L9-4 with center frequency of 4 MHz connected to a SonixOne scanner was used to gather RF backscattered data in vivo from rabbits. The RF signals were used to estimate an average attenuation and BSC for each rabbit. The first five principal components of the PCA from the BSCs were used as input features to a support vector machine (SVM) for classification and comparison to total liver lipid levels. The slope of the attenuation coefficient provided statistically significant differences (p < 0.00058, using two-sample t-test) between low and high lipid fat groups. The proposed PCA-SVM based classification system yielded a classification accuracy of 63.7%, 32.6% and 71.2% for the low, medium and high fat groups, respectively. The results suggest that attenuation and BSC analysis can differentiate low versus high fat livers in a rabbit model. This work was supported by a grant from NIH (R21EB020766).

Identifying real-time changes in tissues via quantitative ultrasound (QUS) approaches are clinically significant, particularly if QUS changes correspond to early detection of disease or provide early assessment of treatment success. Thus, understanding sequential steps in disease progression is key for success. Cerulein-induced inflammation of the pancreas (pancreatitis) in rodent models causes a significant release of pancreatic enzymes into blood, and it induces interstitial edema and inflammatory cell infiltration into the pancreas. This degree of pancreatitis is relatively mild: all animals survive the induction of pancreatitis that resolves itself in ∼7 days. This makes this model an excellent one for studying ultrasonic attenuation coefficient and backscatter coefficient over time. The edematous stroma, cell shrinkage and death, followed by repopulation by dedifferentiated acinar cells, has certain similarities with the morphologies of some forms of pancreatic carcinoma (PCa). The study has several unique contributions: 1) The cerulein-induced pancreatitis model can be used as a surrogate for the PCa model, particularly for detecting early responses to PCa onset/treatment. 2) Understanding the progression of acute pancreatitis per se is imperative for successful intervention and treatment, especially in the early phases of the disease, before other clinical changes are manifest. Pancreatic AC and BSC (25–55 MHz) were estimated in vivo and ex vivo at baseline (no cerulein injections) and from 2 to 168 hr after cerulein injections. Following euthanasia, the pancreas was evaluated histopathologically and biochemically. There are significant measureable early effects on both in vivo AC and BSC (and their respective ex vivo estimates) relative to baseline controls that reflect the temporal biochemical and morphological effects of cerulein. The general trend is decreased AC and BSC at early time points and then rebound increases relative to controls at later time points. The greatest AC and BSC decreases occurred at 2 hr post-cerulein injections [29% AC (in dB/cm-MHz) decrease and 34% BSC (in dB) decrease, at 25 MHz)], suggesting high likelihood for early detection of either disease onset or response to therapy using quantitative ultrasound measures.

All viruses strategically alter the antiviral immune response to their benefit. The vaccinia virus (VACV) K1 protein has multiple immunomodulatory effects in tissue culture models of infection, including NF-κB antagonism. However, the effect of K1 during animal infection is poorly understood. We determined that a K1L -less vaccinia virus (vΔK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity was correlated with diminished virus replication in intranasally infected mice. However, in intradermally inoculated ears, vΔK1L replicated to levels nearly identical to those of VACV, implying that the decreased immune response to vΔK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vΔK1L induced slightly less edema than vK1L, as revealed by histopathology and noninvasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vΔK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vΔK1L-infected ears. While these results identified the biological basis for smaller lesions, they remained puzzling; because K1 antagonizes NF-κB in vitro , antiviral gene expression was expected to be higher during vΔK1L infection. Despite these diminished innate immune responses, vΔK1L vaccination induced a protective VACV-specific CD8 + T cell response and protected against a lethal VACV challenge. Thus, vΔK1L is the first vaccinia virus construct reported that caused a muted innate immune gene expression profile and decreased immune cell infiltration in an intradermal model of infection yet still elicited protective immunity. IMPORTANCE The vaccinia virus (VACV) K1 protein inhibits NF-κB activation among its other antagonistic functions. A virus lacking K1 (vΔK1L) was predicted to be less pathogenic because it would trigger a more robust antiviral immune response than VACV. Indeed, vΔK1L was less pathogenic in intradermally infected mouse ear pinnae. However, vΔK1L infection unexpectedly elicited dramatically reduced infiltration of innate immune cells into ears. This was likely due to decreased expression of cytokine and chemokine genes in vΔK1L-infected ears. As such, our finding contradicted observations from cell culture systems. Interestingly, vΔK1L conferred protective immunity against lethal VACV challenge. This suggests that the muted immune response triggered during vΔK1L infection remained sufficient to mount an effective protective response. Our results highlight the complexity and unpredictable nature of virus-host interactions, a relationship that must be understood to better comprehend virus pathogenesis or to manipulate viruses for use as vaccines.

OBJECTIVES: The objective was to determine whether dietary tomato or lycopene supplementation impacted the expression of biomarkers associated with aggressive tumors and poorer prognostic outcomes in castration-resistant prostate cancer (CRPC). METHODS: Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were castrated at 12 weeks of age to mimic the effects of androgen deprivation therapy (ADT) leading to CRPC. Mice were separated into three dietary groups (n = 30/group) and fed powdered, modified AIN-93 G diets containing 10% tomato powder (TP), lycopene beadlets matched for lycopene content of TP (LYCO), or placebo beadlets (PLAC). Tumors were detected and monitored for growth by ultrasound scan. After 5 weeks of CRPC growth, mice were euthanized, and prostate and tumor tissues were collected. Histological analysis was used to identify presence of tumor neuroendocrine (NE) phenotype (by synaptophysin [syp] expression) and androgen receptor (AR) expression. ELISA and Western blot were used to quantify interleukin-6 (IL-6) and STAT3, and RT-PCR was used to measure downstream targets of both AR and STAT3. RESULTS: No differences were observed between AR expression or presence of the NE phenotype between dietary groups. However, lower tumor weight was associated with both AR and syp expression in mice fed TP. A positive correlation (P = 0.03) was observed between reduced tumor IL-6 (P = 0.012) and reduced phosphorylation (activation) of STAT3 (P = 0.017) in tumors of mice fed TP when compared to PLAC or LYCO. RNA analysis showed reduced expression of genes related to invasion (mmp-2; P = 0.006), cell proliferation (cdk1; P = 0.01), NE phenotype (ngfr; P = 0.001), and androgen metabolism (srd5a2; P = 0.03) in tumors of TP-fed mice. CONCLUSIONS: Physiological levels of dietary tomato, but not lycopene supplementation, reduced the expression of molecular biomarkers typically upregulated in CRPC tissues that are associated with tumor growth and metastasis. FUNDING SOURCES: This research is supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health.

This contribution demonstrates that quantitative ultrasound (QUS) capabilities are platform independent, using an in vivo model. Frequency-dependent attenuation estimates, backscatter coefficient, and effective scatterer diameter estimates are shown to be comparable across four different ultrasound imaging systems with varied processing techniques. The backscatter coefficient (BSC) is a fundamental material property from which several QUS parameters are estimated; therefore, consistent BSC estimates among different systems must be demonstrated. This study is an intercomparison of BSC estimates acquired by three research groups (UIUC, UW, ISU) from four in vivo spontaneous rat mammary fibroadenomas using three clinical array systems and a single-element laboratory scanner system. Because of their highly variable backscatter properties, fibroadenomas provided an extreme test case for BSC analysis, and the comparison is across systems for each tumor, not across the highly heterogeneous tumors. RF echo data spanning the 1 to 12 MHz frequency range were acquired in three dimensions from all animals using each system. Each research group processed their RF data independently, and the resulting attenuation, BSC, and effective scatterer diameter (ESD) estimates were compared. The attenuation estimates across all systems showed the same trends and consistently fit the power-law dependence on frequency. BSCs varied among the multiple slices of data acquired by each transducer, with variations between transducers being of a similar magnitude as those from slice to slice. Variation between BSC estimates was assessed via functional signal-to-noise ratios derived from back-scatter data. These functional signal-to-noise ratios indicated that BSC versus frequency variations between systems ranged from negligible compared with the noise level to roughly twice the noise level. The corresponding functional analysis of variance (fANOVA) indicated statistically significant differences between BSC curves from different systems. However, root mean squared difference errors of the BSC values (in decibels) between different transducers and imaging platforms were less than half of the BSC magnitudes in most cases. Statistical comparison of the effective scatterer diameter (ESD) estimates resulted in no significant differences in estimates from three of the four transducers used for those estimates, demonstrating agreement among estimates based on the BSC. This technical advance demonstrates that these in vivo measurements can be made in a system-independent manner; the necessary step toward clinical implementation of the technology.

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The success of any minimally invasive treatment procedure can be enhanced significantly if combined with a robust noninvasive imaging modality that can monitor therapy in real time. Quantitative ultrasound (QUS) imaging has been widely investigated for monitoring various treatment responses such as chemotherapy, radiation, and thermal therapy. Previously, we demonstrated the feasibility of using spectral-based QUS parameters to monitor high-intensity focused ultrasound (HIFU) treatment of in situ tumors in euthanized rats [Ultrasonic Imaging 36(4), 239–255, 2014]. In the present study, we examined the use of spectral-based QUS parameters to monitor HIFU treatment of in vivo rat mammary adenocarcinoma tumors (MAT) where significant tissue motion was present. HIFU was applied to tumors in rats using a single-element transducer. During the off part of the HIFU duty cycle, ultrasound backscatter was recorded from the tumors using a linear array co-aligned with the HIFU focus. A total of 10 rats were treated with HIFU in this study with an additional sham-treated rat. Spectral parameters from the backscatter coefficient, i.e., effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were estimated. The changes of each parameter during treatment were compared with a temperature profile recorded by a fine-needle thermocouple inserted into the tumor a few millimeters behind the focus of the HIFU transducer. The mean ESD changed from 121 ± 6 to 81 ± 8 μm(p-value = 0.0002), and the EAC changed from 33 ± 2 to 46 ± 3 dB/cm3 (p-value = 0.0002) during HIFU exposure as the temperature increased on average from 38.7 ± 1.0° C to 64.2 ± 2.7° C. The changes in ESD and EAC were linearly correlated with the changes in tissue temperature during the treatment. When HIFU was turned off, the ESD increased from 81 ± 8 to 121 ± 7 μm and the EAC dropped from 46 ± 3 to 36 ± 2 dB/cm3 as the temperature decreased from 64.2 ± 2.7° C to 45 ± 2.7° C. QUS was demonstrated in vivo to track temperature elevations caused by HIFU exposure.

In this pilot study, focused ultrasound (FUS) was used to produce hyperthermia in cervical lymph nodes of rats having experimental autoimmune encephalomyelitis (EAE) to alleviate symptoms associated with EAE. EAE was induced in dark agouti rats, and EAE scores were recorded over 21 d. At the onset of EAE symptoms, rats were treated with FUS to induce temperatures of 43–44°C for 20 min in the superficial cervical lymph nodes. An EAE remittance score was tallied for all rats, defined as the maximum EAE score observed minus the minimum EAE score observed after the maximum EAE was reached. On average, the peak remittance score for FUS-treated rats was 1.14 ± 0.48 versus 0.33 ± 0.27 for sham-treated rats. These differences were statistically significant ( p = 0.037). Therefore, FUS treatment of cervical lymph nodes in rats with EAE resulted in a significant reduction in EAE score.

Cardiovascular diseases affect one-quarter of men and women between the ages of 20 and 39 years; this number more than doubles after the age of 40 years.1 These statistics include ischemia affecting the heart, brain, kidney, and limbs, due to atherosclerosis or diabetes. Current treatments are contraindicated for certain populations due to their invasiveness. For that reason, ultrasound (US) has been explored as a new strategy for myocardial and limb ischemia over the past decade. Ultrasound has been cited to cause upregulation of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibro - blast growth factor,2,3 decreased wound healing time,4 in addition to the induction of angiogenesis in hind limb ischemia.5 Recently, microbubbles have been explored as a means of drug delivery in an effort to treat ischemia.6–8 Studies have shown that US and ultrasound contrast agents (UCAs) induce neovascularization9,10 and arteriogenesis11,12 and improve cardiac function8 in ischemic models. However, an equal amount of literature details the ability of US and UCAs to create damage to various tissues. Ultrasound and UCAs have been documented to create undesired bioeffects, including premature ventricular contractions,13 hemorrhage,14,15 capillary disruptions,16 and lesions17 in normal animal models. Bioeffects studies focus on 0-day effects in normal models, while methods to induce angiogenesis focus on 3 to 28 days after US exposure, typically with ischemic models. Acute (0-day) and effect days are rarely addressed in the same study. As such, little is known about the mechanism of US-UCA–induced angiogenesis. One of the reasons for the disconnect between US’s biological effects and therapy is that there is a lack of understanding of mechanisms initiating the angiogenic response. Literature shows a wide range of US peak rare - factional pressures (Prs) has been used5,8,11,18,19 with no dose-effect examination of the therapy. A relatively low Pr (0.25 MPa) was demonstrated to induce an angiogenic response,7 while Fuji et al19 found a much higher Pr (4.5 MPa) to be beneficial. When UCAs are used, Pr is the exposure quantity of particular importance because it affects the behavior of the UCA. As incident Pr increases, the UCA becomes less stable and eventually collapses.20,21 The UCAs behavior affects the vasculature and initiation of a biological response.22,23 Therefore, in an effort to connect the 0-day (day of exposure) bioeffects with subsequent therapeutic responses, this study seeks to examine the role of the UCAs, and relate the physical mechanism by which US-UCA interaction induces angiogenesis, via a dose-effect study on Pr.

Ischemia-related heart disease affects more than 80 million people in the United States.1 Current interventions include ablation, angioplasty, revascularization, and bypass surgery. The invasive nature of these techniques excludes patients with myocardial ischemia who are not amenable to surgical intervention. For this reason, alternative methods of revascularization in ischemic cardiac muscle have been explored. Over the past 2 decades, cellular, molecular, and genetic therapy attempts have been made to find a clinically relevant treatment.2–6 Ultimately, it is the invasiveness or lack of site specificity that provides the largest obstacle for therapeutic effectiveness of angiogenic treatments. Current research has suggested that ultrasound (US)-ultrasound contrast agent (UCA) treatment can be therapeutically beneficial, providing a noninvasive way to spatially and temporally target ischemic tissues.7–10 This type of angiogenic therapy can be used as an alternative to high-risk percutaneous intervention or bypass graft surgery. Several studies report a reparative response to US-UCA exposure and state that inertial cavitation, or microbubble collapse, is possibly required for angiogenesis to occur.8,11–14 Numerous small-scale studies have shown promising results; however, when large-scale double-blind studies were conducted, they showed limited effects.15 A major impediment for progress to clinical applicability is, perhaps, the lack of understanding of the biophysical mechanisms that connect US-UCAs to neovascularization. It is important to establish a basic knowledge of capillary changes that occur with US-UCA exposure so that US-UCA-induced angiogenesis can be assessed for therapeutic benefit and so that existing therapies can be improved. Current studies in the literature involving US-UCA-induced angiogenesis use peak rarefactional US pressure (Pr) varying between 0.1 and 1.0 MPa.8,12,16,17 These Pr values have been shown to collapse approximately 20% to 70% of UCAs at 1 MHz.18 Therefore, the question remains as to what causes the angiogenic response. Studies involving US-UCA-induced angiogenesis show increases in the collateral blood supply ranging from 5% to 35% with respect to their controls.8–12 However, the effectiveness of the therapy used in each study cannot be correlated to Pr levels or any other parameter used. Also, these studies do not directly correlate a UCA mechanism to the angiogenic response.8–12 In addition, most studies involving US-UCA therapy use ischemic models without exploring the underlying mechanism. Furthermore, the time points for angiogenic assessment (ie, days postexposure [DPE]) were quite varied, ranging from 3 to 28 DPE.8,16,17,19,20 The goals of this study were to explore the influence of UCA collapse cavitation, the angiogenic progression in a normal animal model, and determine whether and if so when the capillary density (as a measure of angiogenesis) increases subsequent to US-UCA exposure in an effort to connect the acute (0 day) bioeffect to the end goal of angiogenesis. This study used a Pr of 3.8 MPa to collapse about 100% of the UCAs18 in an effort to correlate a mechanism with the angiogenic response. Previous work has shown effectiveness at lower Pr values; however, the underlying premise is that angiogenesis is damage induced and involves a reparative response.9,12,13 This study inspects that underlying premise. Examination of UCA collapse cavitation and subsequent angiogenic effects is integral to understanding how to increase angiogenesis. In this study, rats were randomly assigned to 0, 3, 6, 13, 20, and 27 DPE groups to investigate the progression of capillary density and inflammation in response to a Pr sufficient to cause collapse cavitation of UCAs in a normal animal model. Capillary density was quantified in capillaries per square millimeter via immunohistochemical (IHC) staining (CD31 and CD34).

To translate quantitative ultrasound (QUS) from the laboratory into the clinic, it is necessary to demonstrate that the measurements are platform independent. Because the backscatter coefficient (BSC) is the fundamental estimate from which additional QUS estimates are calculated, agreement between BSC results using different systems must be demonstrated. This study was an intercomparison of BSCs from in vivo spontaneous rat mammary tumors acquired by different groups using 3 clinical array systems and a single-element laboratory scanner system.Radio frequency data spanning the 1- to 14-MHz frequency range were acquired in 3 dimensions from all animals using each system. Each group processed their radio frequency data independently, and the resulting BSCs were compared. The rat tumors were diagnosed as either carcinoma or fibroadenoma.Carcinoma BSC results exhibited small variations between the multiple slices acquired with each transducer, with similar slopes of BSC versus frequency for all systems. Somewhat larger variations were observed in fibroadenomas, although BSC variations between slices of the same tumor were of comparable magnitude to variations between transducers and systems. The root mean squared (RMS) errors between different transducers and imaging platforms were highly variable. The lowest RMS errors were observed for the fibroadenomas between 4 and 5 MHz, with an average RMS error of 4 x 10(-5) cm(-1)Sr(-1) and an average BSC value of 7.1 x 10(-4) cm(-1)Sr(-1), or approximately 5% error. The highest errors were observed for the carcinoma between 7 and 8 MHz, with an RMS error of 1.1 x 10(-1) cm(-1)Sr(-1) and an average BSC value of 3.5 x 10(-2) cm(-1)Sr(-1), or approximately 300% error.This technical advance shows the potential for QUS technology to function with different imaging platforms.

Epidemiological studies have shown dietary magnesium (Mg) intake and serum Mg levels to be inversely correlated with the development of atherosclerosis. We hypothesized that low levels of Mg would promote atherosclerotic plaque development in rabbits. New Zealand white rabbits (4 months old, n = 22) were fed an atherogenic diet containing 0.12% (−Mg), 0.27% (control), or 0.43% (+Mg) Mg for 8 weeks. Blood samples were obtained at baseline, 2, 4, 6, and 8 weeks and were assayed for total cholesterol, high-density lipoprotein (HDL), non-HDL, triglycerides (TG), C-reactive protein, serum Mg, and erythrocyte Mg. Aortas from −Mg had significantly more plaque, with an intima thickness 42% greater than control and 36% greater than +Mg. Serum cholesterol levels rose over time, and at 8 weeks, −Mg had the highest and +Mg the lowest total and non-HDL cholesterol and TG levels, although these results did not reach significance. Over time, serum Mg levels increased, and erythrocyte Mg levels decreased. C-reactive protein significantly increased in all groups at 4 and 6 weeks but returned to baseline levels by 8 weeks. This study supports the hypothesis that inadequate intake of Mg results in an increase in atherosclerotic plaque development in rabbits.

In a previous study, it was hypothesized that ultrasound-induced lung damage was related to the transfer of ultrasonic energy into the lungs (W. D. O'Brien et al. 2002, "Ultrasound-induced lung hemorrhage: Role of acoustic boundary conditions at the pleural surface," J. Acoust. Soc. Am. 111, 1102-1109). From this study a technique was developed to: 1) estimate the impedance (Mrayl) of fresh, excised, ex vivo rat lung versus its level of inflation (cm H(2)O) and 2) predict the fraction of ultrasonic energy transmitted into the lung (M. Oelze et al. 2003, "Impedance measurements of ex vivo rat lung at different volumes of inflation." J. Acoust. Soc. Am. 114, 3384-3393). In the current study, the same technique was used to estimate the frequency-dependent impedance of lungs from rats, rabbits, and pigs of various ages. Impedance values were estimated from lungs under deflation (atmospheric pressure, 0 cm H(2)O) and three volumes of inflation pressure [7 cm H(2)O (5 cm H(2)O for pigs), 10 cm H(2)O, and 15 cm H(2)O]. Lungs were scanned in a tank of degassed 37 degrees C water. The frequency-dependent acoustic pressure reflection coefficient was determined over a frequency range of 3.5-10 MHz. From the reflection coefficient, the frequency-dependent lung impedance was calculated with values ranging from an average of 1.4 Mrayl in deflated lungs (atmospheric pressure) to 0.1 Mrayl for fully inflated lungs (15 cm H(2)O). Across all species, deflated lung (i.e., approximately 7% of the total lung capacity) had impedance values closer to tissue values, suggesting that more acoustic energy was transmitted into the lung under deflated conditions. Finally, the impedance values of deflated lungs from different species at different ages were compared with the thresholds for ultrasound-induced lung damage. The comparison revealed that increases in ultrasonic energy transmission corresponded to lower injury threshold values.

Diagnostic ultrasound imaging is enhanced by the use of circulating microbubble contrast agents (UCAs), but the interactions between ultrasound, UCAs, and vascular tissue are not fully understood. We hypothesized that ultrasound with a UCA would stress the vascular tissue and increase levels of heat shock protein 70 (Hsp70), a cellular stress protein.Male New Zealand White rabbits (n = 32) were fed a standard chow diet (n = 4) or a 1% cholesterol, 10% fat, and 0.11% magnesium diet (n = 28). At 21 days, 24 rabbits on the cholesterol diet were either exposed to ultrasound (3.2-MHz f/3 transducer; 2.1 MPa; mechanical index, 1.17; 10 Hz pulse repetition frequency; 1.6 microseconds pulse duration; 2 minutes exposure duration at 4 sites along the aorta) with the UCA Definity (1× concentration, 1 mL/min; Lantheus Medical Imaging, North Billerica, MA) or sham exposed with a saline vehicle injection (n = 12 per group). Four rabbits on the cholesterol diet and 4 on the chow diet served as cage controls and were not exposed to ultrasound or restrained for blood sample collection. Animals were euthanized 24 hours after exposure, and aortas were quickly isolated and frozen in liquid nitrogen. Aorta lysates from the area of ultrasound exposure were analyzed for Hsp70 level by Western blot. Blood plasma was analyzed for cholesterol, Hsp70, and von Willebrand factor, a marker of endothelial function.Plasma total cholesterol levels increased to an average of 705 mg/dL. Ultrasound did not affect plasma von Willebrand factor, plasma Hsp70, or aorta Hsp70. Restraint increased Hsp70 (P.001, analysis of variance).Restraint, but not ultrasound with the UCA or cholesterol feeding, significantly increased Hsp70.

Dietary patterns with cardiovascular benefits have been recommended, but the relative contributions of individual foods and food components, alone or in combination, remain undefined. Male ApoE−/− mice were fed either a purified AIN-93G control diet, a Western diet, or a Western diet with 10% tomato powder (TP), 2% soy germ (SG), or the combination, for four weeks (n=10 per group). Plasma total cholesterol and triglycerides were measured with enzymatic colorimetric kits, and serum amyloid A (SAA) was measured by ELISA. Liver lipids were extracted with chloroform:methanol, and triglycerides, free and esterified cholesterol measured with enzymatic colorimetric kits. Expression of Cyp27a1, Cyp7a1, Abcg5, and Abcg8 in the liver was determined by quantitative polymerase chain reaction. Sections of the aortic root and aorta were cut and stained with H&E to assess extent of atherosclerotic lesions. Western diet-fed animals had greater liver and adipose weights, plasma cholesterol and SAA, hepatic lipids, and atherosclerosis than AIN-93G animals. TP and SG did not decrease atherosclerosis as measured by H&E-stained sections of the aortic root, aortic arch and descending aorta. The TP diets further increased plasma cholesterol, but also led to increased expression of the Abcg5/8 transporters involved in cholesterol efflux. Addition of SG alone to the Western diet attenuated Western-diet-induced increases in plasma cholesterol, liver lipids and gonadal adipose weight. The results of this study do not support the use of either TP or SG for reduction of atherosclerosis, but suggest some beneficial effects of SG on lipid metabolism in this model of cardiovascular disease.

Arterial injury resulting from the interaction of contrast agent (CA) with ultrasound (US) was studied in rabbit auricular arteries and assessed by histopathologic evaluation and s-thrombomodulin concentrations. Three sites on each artery were exposed (2.8 MHz, 5-min exposure duration, 10-Hz pulse repetition frequency, 1.4-mus pulse duration) using one of three in situ peak rarefactional pressures (0.85, 3.9 or 9.5 MPa). Saline, saline/CA, and saline/US infusion groups (n = 28) did not have histopathologic damage. The saline/CA/US infusion group (n = 10) at exposure conditions below the FDA mechanical index limit of 1.9 did not have histopathologic damage, whereas the saline/CA/US infusion group (n = 9) at exposure conditions above the FDA limit did have damage (5 of 9 arteries). Lesions were characteristic of acute coagulative necrosis. Mean s-thrombomodulin concentrations, a marker for endothelial cell injury, were highest in rabbits exposed to US at 0.85 and 3.9 MPa, suggesting that vascular injury may be physiological and not accompanied by irreversible cellular injury.

Attenuation coefficient and propagation speed of intercostal tissues were estimated as functions of temperature (22, 30, and 37/spl deg/C) from fresh chest walls from eight 10- to 11-week-old female Sprague-Dawley (SD) rats, eight 21to 24-week-old female Long-Evans (LE) rats, and ten 6- to 10-week-old mixed sex Yorkshire (York) pigs. The primary purpose of the study was to estimate the temperature dependence of the intercostal tissue's attenuation coefficient so that accurate estimates of the in situ (at the pleural surface) acoustic pressure levels could be made for our ultrasound-induced lung hemorrhage studies. The attenuation coefficient of intercostal tissue for both species was independent of the temperature at the discrete frequencies of 3.1 MHz (-0.0076, 0.0065, and 0.016 dB/cm//spl deg/C for SD rats, LE rats, and York pigs, respectively) and 6.2 MHz (-0.015, 0.014, and 0.014 dB/cm//spl deg/C for SD rats, LE rats, and York pigs, respectively). However, the temperature-dependent regressions yielded a significant temperature dependency of the intercostal tissue attenuation coefficients in SD and LE rats (over the 3.1 to 9.6 MHz frequency range); there was no temperature dependency in York pigs (over the 3.1 to 8.6 MHz frequency range). There was no significant temperature dependency of the intercostal tissue propagation speed in SD rats; there was a temperature dependency in LE rats and York pigs (-0.59, -1.6, and -2.9 m/s//spl deg/C for SD rats, LE rats, and York pigs, respectively). Even though the attenuation coefficient's temperature dependency was significant from the linear regression functions, the differences were not very great (-0.040 to -0.13, 0.011 to 0.18, and 0.055 to 0.10 dB/cm//spl deg/C for SD rats, LE rats, and York pigs, respectively, over the data frequency range). These findings suggest that it is not necessary to determine the attenuation coefficient of intercostal tissue at body temperature (37/spl deg/C), but rather it is sufficient to determine the attenuation coefficient at room temperature (22/spl deg/C), a much easier experimental procedure.

Attention coefficient and propagation speed of intercostal tissues were estimated from chest walls removed postmortem (pm) from 15 5.3 /spl plusmn/ 2.3-day-old, 19 31 /spl plusmn/ 6-day-old, and 15 61 /spl plusmn/ 3-day-old crossbred pigs. These ultrasonic propagation properties were determined from measurements through the intercostal tissues, from the surface of the skin to the parietal pleura. The chest walls were placed in a 0.9% sodium chloride solution, sealed in freezer bags, and stored at -15/spl deg/C prior to measurements. When evaluated, chest-wall storage time ranged between 1 and 477 days pm. All chest walls were allowed to equilibrate to 22/spl deg/C in a water bath prior to evaluation. There was an age dependency of the intercostal tissue propagation speed, with the speed increasing with increasing age. The attenuation coefficient of intercostal tissue was shown to be independent of the age of the pig at the discrete frequencies of 3.1 and 6.2 MHz. For pig intercostal tissues, the estimated attenuation coefficient over the 3.1-9.2 MHz frequency range was A = 1.94f/sup 0.90/ where A is in decibels per centimeter (dB/cm) and f is the ultrasonic frequency in megahertz. In order to determine if there was an effect of storage time pm on estimates of attenuation coefficient, a second experiment was conducted. Five of the youngest pig chest walls measured on day 1 pm in the first experiment were stored at 4/spl deg/C prior to the first evaluation then stored at -15/spl deg/C before being measured again at 108 days pm. There was no difference in the estimated intercostal tissue attenuation coefficient as a function of storage time pm.

In a previous study [J. Acoust. Soc. Am. 108, 1290 (2000)] the acoustic impedance difference between intercostal tissue and lung was evaluated as a possible explanation for the enhanced lung damage with increased hydrostatic pressure, but the hydrostatic-pressure-dependent impedance difference alone could not explain the enhanced occurrence of hemorrhage. In that study, it was hypothesized that the animal's breathing pattern might be altered as a function of hydrostatic pressure, which in turn might affect the volume of air inspired and expired. The acoustic impedance difference between intercostal tissue and lung would be affected with altered lung inflation, thus altering the acoustic boundary conditions. In this study, 12 rats were exposed to 3 volumes of lung inflation (inflated: approximately tidal volume; half-deflated: half-tidal volume; deflated: lung volume at functional residual capacity), 6 rats at 8.6-MPa in situ peak rarefactional pressure (MI of 3.1) and 6 rats at 16-MPa in situ peak rarefactional pressure (MI of 5.8). Respiration was chemically inhibited and a ventilator was used to control lung volume and respiratory frequency. Superthreshold ultrasound exposures of the lungs were used (3.1-MHz, 1000-Hz PRF, 1.3-micros pulse duration, 10-s exposure duration) to produce lesions. Deflated lungs were more easily damaged than half-deflated lungs, and half-deflated lungs were more easily damaged than inflated lungs. In fact, there were no lesions observed in inflated lungs in any of the rats. The acoustic impedance difference between intercostal tissue and lung is much less for the deflated lung condition, suggesting that the extent of lung damage is related to the amount of acoustic energy that is propagated across the pleural surface boundary.

Quantitative ultrasound (QUS) imaging is hypothesized to map temperature elevations induced in tissue with high spatial and temporal resolution. To test this hypothesis, QUS techniques were examined to monitor high-intensity focused ultrasound (HIFU) exposure of tissue. In situ experiments were conducted on mammary adenocarcinoma tumors grown in rats and lesions were formed using a HIFU system. A thermocouple was inserted into the tumor to provide estimates of temperature at one location. Backscattered time-domain waveforms from the tissue during exposure were recorded using a clinical ultrasonic imaging system. Backscatter coefficients were estimated using a reference phantom technique. Two parameters were estimated from the backscatter coefficient (effective scatterer diameter (ESD) and effective acoustic concentration (EAC). The changes in the average parameters in the regions corresponding to the HIFU focus over time were correlated to the temperature readings from the thermocouple. The changes in the EAC parameter were consistently correlated to temperature during both heating and cooling of the tumors. The changes in the ESD did not have a consistent trend with temperature. The mean ESD and EAC before exposure were 120 ± 16 μm and 32 ± 3 dB/cm3, respectively, and changed to 144 ± 9 μm and 51 ± 7 dB/cm3, respectively, just before the last HIFU pulse was delivered to the tissue. After the tissue cooled down to 37°C, the mean ESD and EAC were 126 ± 8 μm and 35 ± 4 dB/cm3, respectively. Peak temperature in the range of 50-60°C was recorded by a thermocouple placed just behind the tumor. These results suggest that QUS techniques have the potential to be used for non-invasive monitoring of HIFU exposure.

Communication between physicians and their financial advisors is critical. Often, physicians are reluctant to discuss financial matters, but in today's environment, communication is important. Practice management, revenue generation, and personal taxes are areas that require year-long interaction between the parties. Practice management is an area where the CPA can assist with suggestions of best practices. Revenue generation is maximized by a physician who knows and understands his or her office. Personal taxes are important, not only on April 15! How can a physician work with a CPA in terms they both understand? A few guidelines will enable a smooth communication process.

Diagnostic ultrasound imaging (US) is enhanced by the use of circulating microbubble contrast agents (UCAs), but the interactions between US, UCAs, and vascular tissue are not well understood. We h...

The cholesterol-fed rabbit is useful for atherosclerosis research. We describe development of a low-magnesium (Mg) cholesterol-containing diet to accelerate atherosclerosis in this model. Male New Zealand White rabbits were fed either chow or one of four atherogenic diets: 1% cholesterol 10% fat 0.11% Mg, 1% cholesterol 10% fat 0.40% Mg, 2% cholesterol 20% fat 0.11% Mg, or 2% cholesterol 20% fat 0.40% Mg. While feed intake decreased in cholesterol-fed rabbits, they were able to maintain their body weights. Rabbits consuming cholesterol experienced profound hypercholesterolemia and tissue lipid accumulation, with plasma cholesterol levels above 1500 mg/dl for all groups at the completion of the study. Liver and spleen lipid content and liver cholesterol content also increased. Aortic arch atheroma thickness was greatest in 1% cholesterol 10% fat 0.11% Mg animals. Tissue Mg levels decreased in cholesterol-fed animals compared to chow-fed controls, despite equal or greater serum Mg levels. Our results indicate that the 1% cholesterol 10% fat 0.11% Mg diet was optimal at promoting hypercholesterolemia and atherosclerosis while minimizing health complications for the animals. The low Mg cholesterol diet will be useful to other biomedical researchers interested in utilizing the rabbit for cardiovascular disease research.

Attenuation coefficients of intercostal tissues were estimated from chest walls removed postmortem (pm) from 41 6-to-7-week-old female ICR mice and 27 10-to-11-week-old female Sprague-Dawley rats. These values were determined from measurements through the intercostal tissues, from the surface of the skin to the parietal pleura. Mouse chest walls were sealed in plastic wrap and stored at 4 degrees C until evaluated, and rat chest walls were sealed in Glad-Lock Zipper sandwich bags, and stored at -15 degrees C. When evaluated, chest wall storage time ranged between 1 and 2 days pm for mice and between 41 and 110 days pm for rats. All chest walls were allowed to equilibrate to 22 degrees C in a water bath prior to evaluation. For both mouse and rat intercostal tissues, the estimated frequency normalized attenuation coefficient was 1.1 dB/cm-MHz. In order to determine if there was an effect of storage time on estimates of attenuation coefficient, an independent experiment was conducted. The intercostal tissues from six mouse chest walls were evaluated at three time points (1, 22, and 144 days pm), and from six rat chest walls were evaluated at four time points (1, 22, 50, and 125 days pm). There was no difference in the estimated intercostal tissue attenuation coefficient as a function of time postmortem.

Multiple sclerosis (MS) is a prevalent neurological disease among adults worldwide. Disease modifying therapies are only modestly effective for slowing long-term progression of pathological and disability outcomes. One approach to treating MS is to reduce the burden of lymphocytes entering the central nervous system. We hypothesized that using focused ultrasound (FUS) to heat the cervical lymph nodes in rats with EAE, an animal model of MS, would alleviate symptoms of EAE by reducing lymphocyte burden. EAE was induced in rats through injection of myelin oligodendrocyte glycoprotein (MOG) and EAE disability scores were recorded over 21 days post injection. The cervical lymph nodes of rats with EAE were treated at day 9 and day 12 post MOG injection using FUS to elevate the temperature to 43-44 °C for 20 minutes. On average the EAE remittance score for FUS treated rats was 1.14 ± 0.48 versus 0.33 ± 0.27 for sham treated rats. These differences were statistically significant (p = 0.037). Remittance of the EA...

Pancreatitis is inflammation of the pancreas. Cerulein-induced pancreatitis in animal models can cause a significant increase in pancreatic enzymes in blood, as well as interstitial edema and inflammatory cell infiltration in the pancreas. This degree of pancreatitis is mild; all animals survive the induction of pancreatitis and resolves within ~6 days after induction, making this an excellent model to study the attenuation coefficient (AC: dB/cm-MHz) and backscatter coefficient (BSC: 1/cm-sr) of the pancreas temporally. The edematous stroma and shrinkage and dedifferentiation of acinar cells, has certain similarities with the morphology in some forms of pancreatic carcinoma. The pancreas’ AC and BSC (20-50 MHz) were estimated in vivo and ex vivo at baseline (before cerulein injections) and at 24 h, 48 h and 72 h after cerulein injections in Sprague-Dawley rats (N = 24). AC and BSC showed the same trends whereby (relative to baseline values) the 24-h AC decreased ~0.3 dB/cm-MHz and BSC decreased ~1-2 dB w...