Contrast ultrasound imaging of the aorta alters vascular morphology and circulating von Willebrand factor in hypercholesterolemic rabbits.

OBJECTIVES: Ultrasound contrast agents (UCAs) are intravenously infused microbubbles that add definition to ultrasonic images. Ultrasound contrast agents continue to show clinical promise in cardiovascular imaging, but their biological effects are not known with confidence. We used a cholesterol-fed rabbit model to evaluate these effects when used in conjunction with ultrasound (US) to image the descending aorta. METHODS: Male New Zealand White rabbits (n = 41) were weaned onto an atherogenic diet containing 1% cholesterol, 10% fat, and 0.11% magnesium. At 21 days, rabbits were exposed to contrast US at 1 of 4 pressure levels using either the UCA Definity (Lantheus Medical Imaging, Inc, North Billerica, MA) or a saline control (n = 5 per group). Blood samples were collected and analyzed for lipids and von Willebrand factor (vWF), a marker of endothelial function. Animals were euthanized at 42 days, and tissues were collected for histologic analysis. RESULTS: After adjustment for pre-exposure vWF, high-level US (in situ [at the aorta] peak rarefactional pressure of 1.4 or 2.1 MPa) resulted in significantly lower vWF 1 hour post exposure (P = .0127; P(adj) < .0762). This difference disappeared within 24 hours. Atheroma thickness in the descending aorta was lower in animals receiving the UCA compared to animals receiving saline. CONCLUSIONS: Contrast US affected the descending aorta, as evidenced by two separate outcome measures. These results may be a first step in elucidating a previously unknown biological effect of UCAs. Further research is warranted to characterize the effects of this procedure.

Temperature dependent ultrasonic characterization of biological media.

Quantitative ultrasound (QUS) is an imaging technique that can be used to quantify tissue microstructure giving rise to scattered ultrasound. Other ultrasonic properties, e.g., sound speed and attenuation, of tissues have been estimated versus temperature elevation and found to have a dependence with temperature. Therefore, it is hypothesized that QUS parameters may be sensitive to changes in tissue microstructure due to temperature elevation. Ultrasonic backscatter experiments were performed on tissue-mimicking phantoms and freshly excised rabbit and beef liver samples. The phantoms were made of agar and contained either mouse mammary carcinoma cells (4T1) or chinese hamster ovary cells (CHO) as scatterers. All scatterers were uniformly distributed spatially at random throughout the phantoms. All the samples were scanned using a 20-MHz single-element f/3 transducer. Quantitative ultrasound parameters were estimated from the samples versus increases in temperature from 37 °C to 50 °C in 1 °C increments. Two QUS parameters were estimated from the backscatter coefficient [effective scatterer diameter (ESD) and effective acoustic concentration (EAC)] using a spherical Gaussian scattering model. Significant increases in ESD and decreases in EAC of 20%-40% were observed in the samples over the range of temperatures examined. The results of this study indicate that QUS parameters are sensitive to changes in temperature.

Ultrasonic backscatter coefficient quantitative estimates from high-concentration Chinese Hamster Ovary cell pellet biophantoms.

Previous work estimated the ultrasonic backscatter coefficient (BSC) from low-concentration (volume density <3%) Chinese Hamster Ovary (CHO, 6.7-µm cell radius) cell pellets. This study extends the work to higher cell concentrations (volume densities: 9.6% to 63%). At low concentration, BSC magnitude is proportional to the cell concentration and BSC frequency dependency is independent of cell concentration. At high cell concentration, BSC magnitude is not proportional to cell concentration and BSC frequency dependency is dependent on cell concentration. This transition occurs when the volume density reaches between 10% and 30%. Under high cell concentration conditions, the BSC magnitude increases slower than proportionally with the number density at low frequencies (ka<1), as observed by others. However, what is new is that the BSC magnitude can increase either slower or faster than proportionally with number density at high frequencies (ka>1). The concentric sphere model least squares estimates show a decrease in estimated cell radius with number density, suggesting that the concentric spheres model is becoming less applicable as concentration increases because the estimated cell radius becomes smaller than that measured. The critical volume density, starting from when the model becomes less applicable, is estimated to be between 10% and 30% cell volume density.

Ultrasonic backscatter coefficient quantitative estimates from Chinese hamster ovary cell pellet biophantoms.

A cell pellet biophantom technique is introduced, and applied to the ultrasonic backscatter coefficient (BSC) estimate using Chinese hamster ovary (CHO) cells. Also introduced is a concentric sphere scattering model because of its geometrical similarities to cells with a nucleus. BSC comparisons were made between the concentric sphere model and other well-understood models for mathematical verification purposes. BSC estimates from CHO cell pellet biophantoms of known number density were performed with 40 and 80 MHz focused transducers (overall bandwidth: 26-105 MHz). These biophantoms were histologically processed and then evaluated for cell viability. Cell pellet BSC estimates were in agreement with the concentric sphere model. Fitting the model to the BSC data yielded quantitative values for the outer sphere and inner sphere. The radius of the cell model was 6.8 ± 0.7 µm; the impedance of the cytoplasm model was 1.63 ± 0.03 Mrayl and the impedance of the nuclear model was 1.55 ± 0.09 Mrayl. The concentric sphere model appears as a new tool for providing quantitative information on cell structures and will tend to have a fundamental role in the classification of biological tissues.

Estimation of the acoustic impedance of lung versus level of inflation for different species and ages of animals.

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.

Hemorrhage near fetal rat bone exposed to pulsed ultrasound.

Ultrasound-induced hemorrhage near the fetal rat skull was investigated to determine if the damage could be correlated with temporal-average intensity. A 0.92-MHz f/1 spherically focused transducer (5.1-cm focal length) was used to expose the skull of 18- to 19-day gestation exteriorized Sprague-Dawley rat fetuses (n = 197). There were four ultrasound-exposed groups (n = 36 each), one sham exposed group (n = 36) and one cage control group (n = 17). Three of the ultrasound-exposed groups had the same peak compressional (10 MPa)/peak rarefactional (6.7 MPa) pressure but different spatial-peak temporal-average intensities (I(TA)) of 1.9, 4.7 and 9.4 W/cm(2); the pulse repetition frequency (PRF) was varied (100, 250 and 500 Hz, respectively). The fourth ultrasound-exposed group had a peak compressional (6.7 MPa)/peak rarefactional (5.0 MPa) pressure and corresponding I(TA) of 4.6 W/cm(2); PRF was 500 Hz. Hemorrhage occurrence increased slightly with increasing I(TA), as well as peak rarefactional pressure and PRF, but the hemorrhage area did not correlate with any of the exposure parameters.

Vascular lesions and s-thrombomodulin concentrations from auricular arteries of rabbits infused with microbubble contrast agent and exposed to pulsed ultrasound.

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.

Lesions of ultrasound-induced lung hemorrhage are not consistent with thermal injury.

Thermal injury, a potential mechanism of ultrasound-induced lung hemorrhage, was studied by comparing lesions induced by an infrared laser (a tissue-heating source) with those induced by pulsed ultrasound. A 600-mW continuous-wave CO2 laser (wavelength approximately 10.6 microm) was focused (680-microm beamwidth) on the surface of the lungs of rats for a duration between 10 to 40 s; ultrasound beamwidths were between 310 and 930 microm. After exposure, lungs were examined grossly and then processed for microscopic evaluation. Grossly, lesions induced by laser were somewhat similar to those induced by ultrasound; however, microscopically, they were dissimilar. Grossly, lesions were oval, red to dark red and extended into subjacent tissue to form a cone. The surface was elevated, but the center of the laser-induced lesions was often depressed. Microscopically, the laser-induced injury consisted of coagulation of tissue, cells and fluids, whereas injury induced by ultrasound consisted solely of alveolar hemorrhage. These results suggest that ultrasound-induced lung injury is most likely not caused by a thermal mechanism.

Threshold estimation of ultrasound-induced lung hemorrhage in adult rabbits and comparison of thresholds in mice, rats, rabbits and pigs.

The objective of this study was to assess the threshold and superthreshold behavior of ultrasound (US)-induced lung hemorrhage in adult rabbits to gain greater understanding about species dependency. A total of 99 76 +/- 7.6-d-old 2.4 +/- 0.14-kg New Zealand White rabbits were used. Exposure conditions were 5.6-MHz, 10-s exposure duration, 1-kHz PRF and 1.1-micros pulse duration. The in situ (at the pleural surface) peak rarefactional pressure, p(r(in situ)), ranged between 1.5 and 8.4 MPa, with nine acoustic US exposure groups plus a sham exposure group. Rabbits were assigned randomly to the 10 groups, each with 10 rabbits, except for one group that had nine rabbits. Rabbits were exposed bilaterally with the order of exposure (left then right lung, or right then left lung) and acoustic pressure both randomized. Individuals involved in animal handling, exposure and lesion scoring were blinded to the exposure condition. Probit regression analysis was used to examine the dependence of the lesion occurrence on in situ peak rarefactional pressure and order of exposure (first vs. second). Likewise, lesion depth and lesion root surface area were analyzed using Gaussian tobit regression analysis. Neither probability of a lesion nor lesion size measurements was found to be statistically dependent on the order of exposure after the effect of p(r(in situ)) was considered. Also, a significant correlation was not detected between the two exposed lung sides on the same rabbit in either lesion occurrence or size measures. The p(r(in situ)) threshold estimates (in MPa) were similar to each other across occurrence (3.54 +/- 0.78), depth (3.36 +/- 0.73) and surface area (3.43 +/- 0.77) of lesions. Using the same experimental techniques and statistical approach, great consistency of thresholds was demonstrated across three species (mouse, rat and rabbit). Further, there were no differences in the biologic mechanism of injury induced by US and US-induced lesions were similar in morphology in all species and age groups studied. The extent of US-induced lung damage and the ability of the lung to heal led to the conclusion that, although US can produce lung damage at clinical levels, the degree of damage does not appear to be a significant medical problem.

Differentiation and characterization of rat mammary fibroadenomas and 4T1 mouse carcinomas using quantitative ultrasound imaging.

Scatterer properties like the average effective scatterer diameter and acoustic concentration were determined in vivo using a quantitative ultrasound (QUS) technique from two tumor phenotypes grown in animal models. These tumor models included spontaneously occurring mammary fibroadenomas in rats and transplanted 4T1 mammary carcinomas in mice. The scatterer properties of average scatterer diameter and acoustic concentration were estimated using a Gaussian form factor from the backscattered ultrasound measured from both types of tumors. QUS images of the tumors were constructed utilizing estimated scatterer properties from regions in the tumors. The QUS images showed a clear distinction between the two types of tumors and a statistically significant difference existed between their estimated scatterer properties. The average scatterer diameter and acoustic concentration for the mammary fibroadenomas were estimated to be 105 +/- 25 microm and -15.6 +/- 5 dB(mm(-3)), respectively. The average scatterer diameter and acoustic concentration for the carcinomas was estimated to be 28 +/- 4.6 microm and 10.6 +/- 6.9 dB(mm(-3)), respectively. The distinctions in the scattering properties are clearly seen in the QUS images of the tumors and indicate that QUS imaging can be useful in differentiating between different types of mammary tumors.

Optimization of a Low Magnesium, Cholesterol-Containing Diet for the Development of Atherosclerosis in Rabbits.

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.

Inadequate dietary magnesium intake increases atherosclerotic plaque development in rabbits.

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.

Impedance measurements of ex vivo rat lung at different volumes of inflation.

A previous study [J. Acoust. Soc. Am. 111, 1102-1109 (2002)] showed that the occurrence of ultrasonically induced lung hemorrhage in rats was directly correlated to the level of lung inflation. In that study, it was hypothesized that the lung could be modeled as two components consisting of air and parenchyma (contiguous tissue [pleura and septa]). The speed of sound and lung impedance would then depend on the fractional volume of air in the lung. According to that model, an inflated lung should act like a pressure-release surface for sound incident from tissue onto a tissue-lung boundary. A deflated lung containing less air should allow more acoustic energy into the lung tissue because the impedance was more closely matched to the contiguous tissues. In the study reported herein, a measurement technique was devised to calculate the impedance of seven rat lungs, ex vivo, under deflation (atmospheric pressure) and three volumes of inflation pressure (7-cm H2O, 10-cm H2O, and 15-cm H2O). Lungs were dissected from rats and immediately scanned in a tank of degassed 37 degrees C water. The frequency-dependent acoustic pressure reflection coefficient was measured over a frequency range of 3.5 to 10 MHz. From the reflection coefficient, the frequency-dependent lung impedance was calculated with values ranging from an average of 1 Mrayls in deflated lungs to 0.2 Mrayls for fully inflated lungs. Lung impedance calculations showed that deflated lungs had an impedance closer to water (1.52 Mrayls) than inflated lungs. At all volumes of inflation, the lungs acted as pressure-release surfaces relative to the water. The average of the four lung impedance values (deflated, 7-cm H2O, 10-cm H2O, and 15-cm H2O) at each level of inflation was statistically different (p<0.0001).

Ultrasound-induced lung hemorrhage: role of acoustic boundary conditions at the pleural surface.

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.