In this paper, we detail blood flow studies in a rat mammary adenocarcinoma model using contrast-enhanced ultrasound. Imaging was implemented on a Siemens Elegra, and utilized the subharmonic phase-inversion detection scheme introduced last year by Chomas, et al. Parameters used in these studies include a transmission frequency of 6 MHz, receive center frequency of 3 MHz, and transmitted pressures ranging from 800 kPa -2.2 MPa. A base study was completed to observe normal tumor growth over a five-week period. Measures of mean replenishment time and integrated enhancement were evaluated and compared to the percentage of viable tumor observed with histology. Results obtained for replenishment measures in tumors were also compared with those obtained in the normal kidney cortex. Mean replenishment time intervals of 6-14 seconds were observed in the tumors. The histogram of replenishment times within a given tumor plane has a wide distribution. We hypothesize this may be due to variably and poorly perfused tissue, abnormal vascular architecture, and tortuosity. The zeroth and first moments of the histograms of integrated enhancement provided an additional method of analysis.
Ultrasound contrast agents can be used to receive signals from vessels significantly smaller than the effective resolution of an ultrasound system. The signal from these microbubbles is coherent across pulses as long as the microbubble is intact, looking like a moving reflector that is more echogenic than blood cells and shifted in spectral mean. When the microbubble is destroyed, the received signal decorrelates between pulses. In-vitro optical and acoustical experiments observing single bubble echoes were conducted. Three mechanisms of destruction were observed optically: static diffusion, pressure-driven diffusion, and fragmentation. Fragmentation was often observed at pressures above 1.4 MPa, while static diffusion and acoustical diffusion were most frequently responsible for bubble destruction at low transmitted pressure. Correlation analysis between echoes received from the same bubble provided analytical evidence of bubble destruction; moreover, a detection scheme based on decorrelation was designed and tested.
Ultrasound contrast agents provide new opportunities to image vascular volume and flow rate directly. To accomplish this goal, new pulse sequences can be developed to detect specifically the presence of a microbubble or group of microbubbles. We consider a new scheme to detect the presence of contrast agents in the body by examining the effect of transmitted phase on the received echoes from single bubbles. In this study, three tools are uniquely combined to aid in the understanding of the effects of transmission parameters and bubble radius on the received echo. These tools allow for optical measurement of radial oscillations of single bubbles during insonation, acoustical study of echoes from single contrast agent bubbles, and the comparison of these experimental observations with theoretical predictions. A modified Herring equation with shell terms is solved for the time-dependent bubble radius and wall velocity, and these outputs are used to formulate the predicted echo from a single encapsulated bubble. The model is validated by direct comparison of the predicted radial oscillations with those measured optically. The transient bubble response is evaluated with a transducer excitation consisting of one-cycle pulses with a center frequency of 2.4-MHz. The experimental and theoretical results are in good agreement and predict that the transmission of two pulses with opposite polarity will yield similar time domain echoes with the first significant portion of the echo generated when the rarefactional half-cycle reaches the bubble.
To determine the ability of contrast material-enhanced ultrasonography (US) to assess replenishment time in a rat kidney and adenocarcinoma tumor model.Mammary adenocarcinoma cells were implanted into the subcutaneous tissues of the flank of 11 rats. Resultant tumors were imaged serially with contrast-enhanced US and compared with images of the rat kidney, a highly perfused normal organ. The US acquisition and processing methods yield images of perfused tumor regions and the times required to achieve 80% replenishment. Findings at contrast-enhanced computed tomography (CT) and light microscopy of hematoxylin-eosin-stained tumor tissue were compared. Paired Student t test was performed to compare the accuracy of US with that of histologic examination and CT in the detection of viable tumor regions.Replenishment of the kidney cortex microvasculature requires 1-5 seconds compared with a replenishment time of 6-14 seconds in tumors. Over the time course of tumor growth, the mean perfusion time becomes progressively longer, and a wider range of perfusion times is detected. Comparison of findings at US, CT, and histologic examination suggested that all three methods yield correlated estimates of the percentage of viable perfused tumor cells. Results of the t test suggested that the viable tumor percentages observed at US are not significantly different from those observed at CT and histologic examination (US vs CT, P =.92; US vs histologic examination, P =.94).Repeated measurements of microvascular flow rate can be accomplished in a rat animal model with a minimally invasive technique.
Various applications of contrast-assisted ultrasound, including blood vessel detection, perfusion estimation, and drug delivery, require controlled destruction of contrast agent microbubbles. The lifetime of a bubble depends on properties of the bubble shell, the gas core, and the acoustic waveform impinging on the bubble. Three mechanisms of microbubble destruction are considered: fragmentation, acoustically driven diffusion, and static diffusion. Fragmentation is responsible for rapid destruction of contrast agents on a time scale of microseconds. The primary characteristics of fragmentation are a very large expansion and subsequent contraction, resulting in instability of the bubble. Optical studies using a novel pulsed-laser optical system show the expansion and contraction of ultrasound contrast agent microbubbles with the ratio of maximum diameter to minimum diameter greater than 10. Fragmentation is dependent on the transmission pressure, occurring in over 55% of bubbles insonified with a peak negative transmission pressure of 2.4 MPa and in less than 10% of bubbles insonified with a peak negative transmission pressure of 0.8 MPa. The echo received from a bubble decorrelates significantly within two pulses when the bubble is fragmented, creating an opportunity for rapid detection of bubbles via a decorrelation-based analysis. Preliminary findings with a mouse tumor model verify the occurrence of fragmentation in vivo. A much slower mechanism of bubble destruction is diffusion, which is driven by both a concentration gradient between the concentration of gas in the bubble compared with the concentration of gas in the liquid, as well as convective effects of motion of the gas-liquid interface. The rate of diffusion increases during insonation, because of acoustically driven diffusion, producing changes in diameter on the time scale of the acoustic pulse length, thus, on the order of microseconds. Gas bubbles diffuse while they are not being insonified, termed static diffusion. An air bubble with initial diameter of 2 microns in water at 37 degrees C is predicted to fully dissolve within 25 ms. Clinical ultrasound contrast agents are often designed with a high molecular weight core in an attempt to decrease the diffusion rate. C3F8 and C4F10 gas bubbles of the same size are predicted to fully dissolve within 400 ms and 4000 ms, respectively. Optical experiments involving gas diffusion of a contrast agent support the theoretical predictions; however, shelled agents diffuse at a much slower rate without insonation, on the order of minutes to hours. Shell properties play a significant role in the rate of static diffusion by blocking the gas-liquid interface and decreasing the transport of gas into the surrounding liquid. Static diffusion decreases the diameter of albumin-shelled agents to a greater extent than lipid-shelled agents after insonation.
New techniques for the assessment of tissue perfusion and the local delivery of drugs have been developed using ultrasound contrast agents. A perfusion estimator, based on the destruction and wash-in of ultrasound contrast agents, has been developed and applied to tumor models. This technique requires the transmission of a train of pulses with varying amplitude, frequency, and phase within the train. High-amplitude and low-frequency pulses are applied to destroy the agent, and low-amplitude higher-frequency pulses are used to monitor the refresh of contrast agents into the sample volume. The signal processing techniques applied following data acquisition utilize the subharmonic component of the received signal. Mechanistic studies of contrast agent destruction, a Rayleigh–Plesset based signal model, and experimental data acquired from a rat tumor model will be included in our presentation. In addition, studies of the local delivery of drugs using oil-based drug delivery vehicles have been conducted. Results of these studies demonstrate that the probability of agent fragmentation and the size of the resulting fragment can be controlled using ultrasonic parameters. [We acknowledge the support of NIH CA 76062.]
Tumor biologists indicate that angiogenesis is required for tumor growth beyond about 1 mm/sup 3/. Furthermore, regions of increased and decreased vascular density are each on the order of 500 /spl mu/m. Therefore, it is for tumors whose size is on the order of 1 mm/sup 3/ that the authors would like to detect this increased vascular density, and a resolution of at least 500 /spl mu/m is critical. In a small clinical study the authors were able to detect localized vasculature in all malignant masses of diameter 1 cm/sup 3/ or greater, with associated vessels of a size 300 /spl mu/m or greater. In order to detect very low velocity blood flow in vessels of diameter less than 300 /spl mu/m, the authors evaluate the feasibility of both nominal 50 MHz color flow mapping without contrast, and 7 MHz color flow mapping with contrast agent. The authors tested these strategies with LNCaP athymic mice, a human prostate tumor model. In a very high frequency study, they detected low velocity flow in vessels as small as 50 /spl mu/m within and surrounding the tumor. In a 7 MHz contrast study, vessels less than 200 /spl mu/m were not detected with color flow without a contrast agent. With a contrast agent, the dense vasculature within and surrounding the tumors was detected, however, individual vessels can not be resolved. These tumor models are a new tool to evaluate imaging systems, with both development and morphology being similar to human tumors. The authors have detected both high speed flow in A-V shunts and low velocity flow in these tumor models. Here, they establish the feasibility of 7 MHz contrast assisted imaging for detection of smaller vessels, with the goal of detecting angiogenesis in its earliest stages (i.e. for a 1 mm/sup 3/ tumor).
