In this project we (1) demonstrated the ability of the DFU to detect stationary microbubbles in tissue, (2) performed a comprehensive calibration of the sizing capabilities of the device using bubbles of known size, and (3) performed a mix of human and animal experiments to explore the usefulness of tissue bubble detection.

First, we demonstrated that DFU could detect both ultrasonic contrast agent and decompression bubbles in tissue. This demonstrated the ability of DFU to detect small bubbles in tissue. The next step was to assess whether bubbles could be detected after exercise in normal humans. Exercise has been postulated to create small bubbles in tissue, and these bubbles are thought to increase bubble formation and decompression sickness risk during subsequent decompression stress after exercise. But, these small bubbles had never been directly detected in tissue.

To do this, in the past year we surveyed for bubbles in the legs of human subjects before and after cycle ergometer exercise using DFU. Six normal human subjects aged (28-52) were studied. Eleven marked sites on the left thigh and calf were imaged on each subject using standard imaging ultrasound. Subjects then rested in a reclining chair for 2 hours prior to exercise. For the hour before exercise a series of baseline measurements were taken at each site using DFU. A minimum of 6 baseline measurements was taken at each site. The subjects then exercised at 80% of their age-adjusted maximal heart rate for 30 minutes on an upright bicycle ergometer. After exercise, the subjects returned to the chair and multiple post-exercise measurements were taken at the marked sites with the CDFI. Measurements continued until no further signals consistent with bubbles were returned or one hour had elapsed. All the subjects had signals consistent with bubbles at a least one site after exercise.

The most likely explanation for these results is that exercise does produce gas-filled micronuclei (bubbles). This is the first demonstration that these micronuclei can be detected after exercise, and these results have important implications for decompression sickness diagnosis and treatment.

Another application for this technology is bubble monitoring during coronary artery bypass surgery or valve replacement surgery. Patients who have coronary artery bypass surgery are at risk for having solid and gaseous emboli reach the brain when they are on the "pump" (the cardiopulmonary bypass circuit). The Creare dual-frequency ultrasound unit could be used to monitor for bubbles in the bypass circuit and could distinguish between solid and gaseous emboli.

Creare is also applying the knowledge gained on the bubble acoustics knowledge and expertise gained in this effort to a Department of Energy project to mitigate cavitation damage in the Spallation Neutron Source (SNS) being developed at Oak Ridge National Laboratory. In this facility, a large acoustic wave is produced in the mercury spallation target when proton pulses very rapidly and repeatedly enter the mercury. The acoustic wave reflects off the vessel walls and causes the mercury to cavitate which results in severe damage to the vessel when the SNS is operated at the desired full power level. Creare is characterizing the ability of various stabilized bubbles to dampen the large acoustic wave and, thereby, mitigate the resulting cavitation damage.

* Micropipette-generated bubbles of optically-verifiable sizes were used as a standard with which to calibrate and validate the intravascular bubble sizing capability of the instrument. This work has been completed and presented in abstract form. It is being prepared for publication.

* Definity® stabilized bubble-based ultrasound contrast agent were used as a known source of nonlinear bubble mixing signal for tissue bubble detection validation. Solid polymer microspheres were used as comparative standard since they reflect ultrasound but do not produce nonlinear mixing signals characteristic of bubbles. Solutions of known concentrations of ultrasound contrast agent and solid polymer microspheres were injected into the thigh of an anesthetized swine and imaged using the dual-frequency ultrasound bubble detection and sizing device. These results have been published in the journal Undersea and Hyperbaric Medicine.

* A swine model of decompression sickness was used to produce nitrogen bubbles in tissue and blood. Anesthetized pigs weighing 20 kg were exposed to 4.5 ATA for 120 minutes and then brought to 1 ATA. This work has been completed and presented in abstract form. It is being prepared for publication.

* Exercise was used as a mechanism to increase levels of pre-existing extravascular bubbles in human subjects. This work has been accepted for publication in the Journal of Applied Physiology.

Detecting and sizing bubbles intravascularly (a new and unique capability) allows for bubble size histograms to be constructed during the development and treatment of DCS. The change of bubble size distribution during decompression stress may indicate DCS severity. Before using the device either for quantitative research or operations, the sizing ability needs to be quantified in optimal in-vitro conditions. The calibration system developed for this project can create monodisperse distributions of microbubbles at the sizes likely to occur during decompression stress. Work in the past year has focused on resolving technical problems in the calibration setup and the calibration is moving forward.

Tissue bubble detection is also a unique capability. The CDFI potentially can detect very small bubbles (the possible precursors of larger bubbles in tissue or blood) and identify larger bubbles in areas with symptoms of pain or discomfort consistent with DCS. Last year, a significant accomplishment was the demonstration of the ability to detect ultrasound contrast bubbles (Definity®) injected into tissue (in anesthetized swine). This work has been extended significantly. In a series of studies, the power levels needed to detect bubbles in tissue were established and the concentration of injected bubbles that could be detected was determined. Also, we validated that the CDFI was specific for bubbles and did not detect ultrasonic reflectors the same size as injected bubbles. Microspheres of the same size as the bubbles were also injected into tissue and were not detected by the CDFI.

For the coming year, the plan is to: (a) complete the in-vitro calibration, (b) track bubble sizes during decompression stress in anesthetized swine, (c) determine if signals consistent with bubbles can be detected in swine muscle before or after decompression stress and (d) assess if these signals change after exercise or immobilization.

Another application for this technology is bubble monitoring during coronary artery bypass surgery or valve replacement surgery. Patients who have coronary artery bypass surgery are at risk for having solid and gaseous emboli reach the brain when they are on the "pump" (the cardiopulmonary bypass circuit). The Creare dual-frequency ultrasound unit could be used to monitor for bubbles in the bypass circuit and could distinguish between solid and gaseous emboli. A collaboration with Dr. Donny Likosky is underway to advance this work.

The cavitation approach can be used to determine to assess the gas saturation in fluids such as hydraulic fluid, which is important for industrial and aviation applications. Creare is currently developing an instrument based on this concept for the U.S. Air Force.

Creare is also applying the knowledge gained on the bubble acoustics knowledge and expertise gained in this effort to a Department of Energy project to mitigate cavitation damage in the Spallation Neutron Source (SNS) being developed at Oak Ridge National Laboratory. In this facility, a large acoustic wave is produced in the mercury spallation target when proton pulses very rapidly and repeatedly enter the mercury. The acoustic wave reflects off the vessel walls and causes the mercury to cavitate which results in severe damage to the vessel when the SNS is operated at the desired full power level. Creare is characterizing the ability of various stabilized bubbles to dampen the large acoustic wave and, thereby, mitigate the resulting cavitation damage.

Efforts continued to calibrate the sizing capability of our dual-frequency ultrasound bubble-sizing device. Previous work has shown that bubbles can be detected and sized intravascularly in-vivo, but a careful in-vitro calibration is essential for future work with the device. Performing this calibration is technically challenging. We continue to make enhancements to the device and the experimental setup to aid in the completion of this calibration. The main challenges are producing and measuring monodisperse streams of bubbles in various sizes, and removing or understanding sources of non-linear signals that can mask bubble signals. We currently believe that the main source of non-bubble, non-linear signal is due to non-linear ultrasound propagation, which is the basis for harmonic imaging. Predicting the magnitude of this masking signal is confounded by the presence of standing waves, which are very difficult to eliminate from a calibration setup. Efforts were made to understand ultrasonic standing waves in both the tissue phantom and water tank experimental setups, and the impact that they may have on the production of sum and difference signals that indicate the presence of bubbles. In addition we have made adjustments to the calibration setup to minimize the interaction between the transmitted pump and image signals. Calibration of the sizing capability of the device is ongoing and should be completed by the end of the year.

Stationary microbubble detection in-vivo

This was the main effort of the year. Several dilutions of ultrasonic contrast agent were injected into the thigh of an anesthetized swine. The ability of the device to detect stationary microbubbles at different ultrasonic pressures and concentrations was demonstrated and characterized. The results of this effort have been submitted for publication.

Decompression Studies

Decompression studies using a swine model have been performed to detect decompression-induced bubble formation in tissue. Swine were pressurized at 4.5ATA for 2hrs and decompressed over 5 minutes. Each swine was monitored with clinical and dual-frequency ultrasound for 1-2 hours post-dive (depending on conditions). Results to date are as of yet inconclusive. It is widely speculated that bubbles form in tissue following decompression, but detecting native tissue bubbles is difficult since the concentration, locations and sizes of normally-occurring microbubbles in tissue is unknown. Current efforts to detect decompression-induced stationary bubbles in tissue are focused on determining the correct frequencies and pressures to use.

Detecting and sizing bubbles intravascularly (a new and unique capability) allows for bubble size histograms to be constructed during the development and treatment of DCS. The change of bubble size distribution during decompression stress may indicate DCS severity. Before using the device either for quantitative research or operations, the sizing ability needs to be quantified in optimal in-vitro conditions. The calibration system developed for this project can create monodisperse distributions of microbubbles at the sizes likely to occur during decompression stress. Work in the past year has focused on improving the optical sizing capability (used for comparison to the ultrasonically determined size), developing tissue equivalent phantoms, and reducing standing waves in the in-vitro test setup. A full calibration is currently underway.

Tissue bubble detection is also a unique capability. The CDFI potentially can detect very small bubbles (the possible precursors of larger bubbles in tissue or blood) and identify larger bubbles in areas with symptoms of pain or discomfort consistent with DCS. Interpreting tissue bubble signals, however, requires knowledge of other potential sources of false bubble signals in tissue. Work in this past year has identified the mechanisms for false positive signals and methods to minimize them have been developed. A significant accomplishment was the demonstration of the ability to detect contrast bubbles (Definity®) injected into tissue (in anesthetized swine). Bubble signals were returned only from the area where Definity® had been injected and not from other areas, including tissues like bone, which strongly reflects ultrasound. False bubble signals were not detected. This demonstrated the ability of the system to detect stationary bubbles in tissue.

In the past year the first human tissue bubble studies were performed. Additionally, a new technique to measure gas saturation in tissue is being explored. In-vitro work has shown that the point at which bubbles are created by cavitation varies with gas saturation. This offers a potential new way to determine gas saturation in tissue, which could potentially lead to a new way to assess DCS risk.

For the coming year, the plan is to: (a) complete the in-vitro calibration, (b) track bubble sizes during decompression stress in anesthetized swine, (c) determine if signals consistent with bubbles can be detected in human muscle and assess if these signals change after exercise or immobilization and (d) develop the cavitation threshold as a method to measure gas saturation in tissue or blood.

Another application for this technology is bubble monitoring during coronary artery bypass surgery or valve replacement surgery. Patients who have coronary artery bypass surgery are at risk for having solid and gaseous emboli reach the brain when they are on the "pump" (the cardiopulmonary bypass circuit). The Creare dual-frequency ultrasound unit could be used to monitor for bubbles in the bypass circuit and could distinguish between solid and gaseous emboli. A collaboration with Dr. Donny Likosky is underway to advance this work.

The cavitation approach can be used to determine to assess the gas saturation in fluids such as hydraulic fluid, which is important for industrial and aviation applications. Creare is currently developing an instrument based on this concept for the U.S. Air Force.

The bubble generator that has been developed for this project is currently being supplied to a large industrial manufacturer in the pharmaceuticals industry to calibrate a Doppler-based detector, which monitors a manufacturing process.

Creare is also applying the knowledge gained on the bubble acoustics knowledge and expertise gained in this effort to a Department of Energy project to mitigate cavitation damage in the Spallation Neutron Source (SNS) being developed at Oak Ridge National Laboratory. In this facility, a large acoustic wave is produced in the mercury spallation target when proton pulses very rapidly and repeatedly enter the mercury. The acoustic wave reflects off the vessel walls and causes the mercury to cavitate which results in severe damage to the vessel when the SNS is operated at the desired full power level. Creare is characterizing the ability of various stabilized bubbles to dampen the large acoustic wave and, thereby, mitigate the resulting cavitation damage.

The detection of frequent, unexplained false bubble signals had been complicating bubble detection. Work in the past year successfully identified the mechanism underlying a major source of false bubble signals. These signals originated from previously undetected electrical interference. After solving this problem an in-vivo test of tissue bubble detection was performed using Definity® ultrasonic contrast agent. Experiments in a water tank showed that a small amount of Definity® produced bubble signals, while water did not. Subsequently, Definity® was injected into thigh tissue in an anesthetized swine. Measurements were taken at several points on the thigh and bubble signals were only returned from the area where Definity® had been injected. False bubble signals were not detected, even from strong ultrasound reflectors like bone.

Calibration

Previous in-vivo studies have shown that bubbles can be detected and sized transthoracically during decompression stress. Before the information from in-vivo studies can be used scientifically, however, a calibration of the device against bubbles of known size is essential. This calibration, however, is technically challenging since it requires (1) streams of small bubbles of known size, (2) phantoms that match tissue attenuation characteristics and (3) the elimination of standing ultrasonic waves from the test setup, which can confound the results.

This past year, tissue-like phantoms were built using special polyurethane formulations that mimic the attenuation properties of tissue. Measurements were taken to determine the presence and magnitude of standing ultrasonic waves created in the phantom and test setup during calibration. Once this was characterized, countermeasures were employed to minimize standing waves. Currently, an initial set of calibration data is under review to ascertain whether all the technical factors involved in calibration have been solved.

Cavitation

The amount of dissolved inert gas within a tissue strongly influences whether bubbles will form in the tissue during decompression stress. At present, however, no method exists to assess gas saturation within tissue.

As the amount of ultrasonic energy sent into the tissue increases, it reaches a point where bubbles form spontaneously. This bubble formation process is called cavitation. When the bubbles cavitate, ultrasonic signals at multiple harmonics can be detected indicating that cavitation has occurred. Work in the past year has explored whether the point at which bubbles cavitate within a fluid could be used to assess gas saturation. We have shown that the bubble cavitation threshold (as measured by the returned ultrasonic signals) decreases with increasing gas saturation in the liquid. Further work is underway to determine if this method could be useful to measure gas saturation in tissue and hence as a measure for DCS risk.

In addition to its NASA application, this technology could be used to improve the safety and efficiency of diving operations.

a. establish the appropriate transducer configurations, electronic settings and instrument enhancements to detect and size bubbles reliably in-vivo--experiments demonstrated that bubbles could be detected as they move through the right ventricle and right atrium. These experiments established the technical knowledge (transducer position relative to anatomical features, equipment settings, etc.) needed to monitor bubbles during subsequent decompression experiments.

b. compare the new bubble monitoring technique to Doppler, and use it to investigate decompression sickness -- aim has been advanced by comparing the signals obtained with the dual frequency device to a standard clinical ultrasound instrument.

c. develop the capability to size small bubbles in tissue-- aim has been advanced through a variety of in vitro and in vivo studies.

The newly developed ability to construct bubble size histograms has a major impact on our research. The histograms provide a novel way to study the time course and treatment of decompression sickness as it is manifested in the bubbles that form in the vasculature. Currently, no other technique exists that allows for bubble size histograms to be constructed during decompression stress.