Diagnostic Ultrasound High Mechanical Index Impulses Restore Microvascular Flow in Peripheral Arterial Thromboembolism.

We sought to explore mechanistically how intermittent high-mechanical-index (MI) diagnostic ultrasound impulses restore microvascular flow. Thrombotic microvascular obstruction was created in the rat hindlimb muscle of 36 rats. A diagnostic transducer confirmed occlusion with low-MI imaging during an intravenous microbubble infusion. This same transducer was used to intermittently apply ultrasound with an MI that produced stable or inertial cavitation (IC) for 10 min through a tissue-mimicking phantom. A nitric oxide inhibitor, L-Nω-nitroarginine methyl ester (L-NAME), was pre-administered to six rats. Plateau microvascular contrast intensity quantified skeletal microvascular blood volume, and postmortem staining was used to detect perivascular hemorrhage. Intermittent IC impulses produced the greatest recovery of microvascular blood volume (p < 0.0001, analysis of variance). Nitric oxide inhibition did not affect the skeletal microvascular blood volume improvement, but did result in more perivascular hemorrhage. IC inducing pulses from a diagnostic transducer can reverse microvascular obstruction after acute arterial thromboembolism. Nitric oxide may prevent unwanted bio-effects of these IC pulses.

Improvements in cerebral blood flow and recanalization rates with transcranial diagnostic ultrasound and intravenous microbubbles after acute cerebral emboli.

OBJECTIVES: Intravenous microbubbles (MBs) and transcutaneous ultrasound have been used to recanalize intra-arterial thrombi without the use of tissue plasminogen activator. In the setting of acute ischemic stroke, it was our objective to determine whether skull attenuation would limit the ability of ultrasound alone to induce the type and level of cavitation required to dissolve thrombi and improve cerebral blood flow (CBF) in acute ischemic stroke. MATERIALS AND METHODS: In 40 pigs, bilateral internal carotid artery occlusions were created with 4-hour-old thrombi. Pigs were then randomized to high-mechanical index (MI = 2.4) short-pulse (5 microseconds) transcranial ultrasound (TUS) alone or a systemic MB infusion (3% Definity) with customized cavitation detection and imaging system transmitting either high-MI (2.4) short pulses (5 microseconds) or intermediate-MI (1.7) long pulses (20 microseconds). Angiographic recanalization rates of both internal carotids were compared in 24 of the pigs (8 per group), and quantitative analysis of CBF with perfusion magnetic resonance imaging was measured before, immediately after, and at 24 hours using T2* intensity versus time curves in 16 pigs. RESULTS: Complete angiographic recanalization was achieved in 100% (8/8) of pigs treated with image-guided high-MI TUS and MBs, but in only 4 of 8 treated with high-MI TUS alone or 3 of 8 pigs treated with image-guided intermediate-MI TUS and MBs (both P < 0.05). Ipsilateral and contralateral CBF improved at 24 hours only after 2.4-MI 5-microsecond pulse treatments in the presence of MB (P < 0.005). There was no evidence of microvascular or macrovascular hemorrhage with any treatment. CONCLUSIONS: Guided high-MI impulses from an ultrasound imaging system produce sustained improvements in ipsilateral and contralateral CBF after acute cerebral emboli.

Microbubble cavitation imaging.

Ultrasound cavitation of microbubble contrast agents has a potential for therapeutic applications such as sonothrombolysis (STL) in acute ischemic stroke. For safety, efficacy, and reproducibility of treatment, it is critical to evaluate the cavitation state (moderate oscillations, stable cavitation, and inertial cavitation) and activity level in and around a treatment area. Acoustic passive cavitation detectors (PCDs) have been used to this end but do not provide spatial information. This paper presents a prototype of a 2-D cavitation imager capable of producing images of the dominant cavitation state and activity level in a region of interest. Similar to PCDs, the cavitation imaging described here is based on the spectral analysis of the acoustic signal radiated by the cavitating microbubbles: ultraharmonics of the excitation frequency indicate stable cavitation, whereas elevated noise bands indicate inertial cavitation; the absence of both indicates moderate oscillations. The prototype system is a modified commercially available ultrasound scanner with a sector imaging probe. The lateral resolution of the system is 1.5 mm at a focal depth of 3 cm, and the axial resolution is 3 cm for a therapy pulse length of 20 µs. The maximum frame rate of the prototype is 2 Hz. The system has been used for assessing and mapping the relative importance of the different cavitation states of a microbubble contrast agent. In vitro (tissue-mimicking flow phantom) and in vivo (heart, liver, and brain of two swine) results for cavitation states and their changes as a function of acoustic amplitude are presented.

Effects of attenuation and thrombus age on the success of ultrasound and microbubble-mediated thrombus dissolution.

The purpose of this study was to examine the effects of applied mechanical index, incident angle, attenuation and thrombus age on the ability of 2-D vs. 3-D diagnostic ultrasound and microbubbles to dissolve thrombi. A total of 180 occlusive porcine arterial thrombi of varying age (3 or 6 h) were examined in a flow system. A tissue-mimicking phantom of varying thickness (5 to 10 cm) was placed over the thrombosed vessel and the 2-D or 3-D diagnostic transducer aligned with the thrombosed vessel using a positioning system. Diluted lipid-encapsulated microbubbles were infused during ultrasound application. Percent thrombus dissolution (%TD) was calculated by comparison of clot mass before and after treatment. Both 2-D and 3-D-guided ultrasound increased %TD compared with microbubbles alone, but %TD achieved with 6-h-old thrombi was significantly less than 3-h-old thrombi. Thrombus dissolution was achieved at 10 cm tissue-mimicking depths, even without inertial cavitation. In conclusion, diagnostic 2-D or 3-D ultrasound can dissolve thrombi with intravenous nontargeted microbubbles, even at tissue attenuation distances of up to 10 cm. This treatment modality is less effective, however, for older aged thrombi.

The stripe artifact in transcranial ultrasound imaging.

OBJECTIVE: Transcranial images are affected by a "stripe artifact" (also known as a "streak artifact"): two dark stripes stem radially from the apex to the base of the scan. The stripes limit the effective field of view even on patients with good temporal windows. This study investigated the angle dependency of ultrasound transmission through the skull to elucidate this artifact. METHODS: In vivo transcranial images were obtained to illustrate the artifact. In vitro hydrophone measurements were performed in water to evaluate transcranial wavefronts at different incidence angles of the ultrasound beam. Both a thin acrylic plate, as a simple bone model, and a human temporal bone sample were used. RESULTS: The imaging wavefront splits into two after crossing the solid layer (acrylic model or skull sample) at an oblique angle. An early-arrival wavefront originates from the direct longitudinal wave transmission through water-bone interfaces, while a late-arrival wavefront results from longitudinal-to-transverse mode conversion at the water-bone interface, propagation of the transverse wave through the skull, and transverse-to-longitudinal conversion at the bone-water interface. At normal incidence, only the direct wavefront (without mode conversion) is observed. As the incidence angle increases, the additional "mode conversion" wavefront appears. The amplitude of the transcranial wavefront decreases and reaches a minimum at an incidence angle of about 27°. Beyond that critical angle, only the mode conversion wavefront is transmitted. CONCLUSIONS: The stripes are a consequence of the angle-dependent ultrasound transmission and mode conversion at fluid-solid interfaces such as between the skull and the surrounding fluidlike soft tissues.

Material characterization of the encapsulation of an ultrasound contrast microbubble and its subharmonic response: strain-softening interfacial elasticity model.

Two nonlinear interfacial elasticity models--interfacial elasticity decreasing linearly and exponentially with area fraction--are developed for the encapsulation of contrast microbubbles. The strain softening (decreasing elasticity) results from the decreasing association between the constitutive molecules of the encapsulation. The models are used to find the characteristic properties (surface tension, interfacial elasticity, interfacial viscosity and nonlinear elasticity parameters) for a commercial contrast agent. Properties are found using the ultrasound attenuation measured through a suspension of contrast agent. Dynamics of the resulting models are simulated, compared with other existing models and discussed. Imposing non-negativity on the effective surface tension (the encapsulation experiences no net compressive stress) shows "compression-only" behavior. The exponential and the quadratic (linearly varying elasticity) models result in similar behaviors. The validity of the models is investigated by comparing their predictions of the scattered nonlinear response for the contrast agent at higher excitations against experimental measurement. All models predict well the scattered fundamental response. The nonlinear strain softening included in the proposed elastic models of the encapsulation improves their ability to predict subharmonic response. They predict the threshold excitation for the initiation of subharmonic response and its subsequent saturation.

Effect of molecular weight, crystallinity, and hydrophobicity on the acoustic activation of polymer-shelled ultrasound contrast agents.

Polymer-shelled microbubbles are applied as ultrasound contrast agents. To investigate the effect of the polymer on microbubble preparation and acoustic properties, polylactides with systematic variations in molecular weight, crystallinity, and end-group hydrophobicity were used. Polymer-shelled cyclodecane filled capsules were prepared by emulsification, and the cyclodecane was removed by lyophilization to obtain hollow capsules. Complete removal of cyclodecane from the microcapsules was only achieved for short chain (about M(w) 6000) crystalline polymers. The pressure threshold for acoustic destruction of the microbubbles was found to increase with molecular weight. Noncrystalline polymers showed a higher threshold for destruction than crystalline polymers. Hydrophobically modified short chain crystalline polymers showed the steepest increase in acoustic destruction after the threshold as a function of the applied pressure, which is a favorable characteristic for ultrasound mediated drug delivery. Microcapsules made with such polymers had an inhomogeneous surface including pores through which cyclodecane was lyophilized efficiently.

Oil-filled polymer microcapsules for ultrasound-mediated delivery of lipophilic drugs.

The use of ultrasound contrast agents as local drug delivery systems continues to grow. Current limitations are the amount of drug that can be incorporated as well as the efficiency of drug release upon insonification. This study focuses on the synthesis and characterisation of novel polymeric microcapsules for ultrasound-triggered delivery of lipophilic drugs. Microcapsules with a shell of fluorinated end-capped poly(L-lactic acid) were made through pre-mix membrane emulsification and contained, apart from a gaseous phase, different amounts of hexadecane oil as a drug-carrier reservoir. Mean number weighted diameters were between 1.22 microm and 1.31 microm. High-speed imaging at approximately 10 million fames per second showed that for low acoustic pressures (1 MHz, 0.24 MPa) microcapsules compressed but remained intact. At higher diagnostic pressures of 0.51 MPa, microcapsules cracked, thereby releasing the encapsulated gas and model lipophilic drug. Using conventional ultrasound B-mode imaging at a frequency of 2.5 MHz, a marked enhancement of scatter intensity over a tissue-mimicking phantom was observed for all differently loaded microcapsules. The partially oil-filled microcapsules with high drug loads and well-defined acoustic activation thresholds have great potential for ultrasound-triggered local delivery of lipophilic drugs under ultrasound image-guidance.

The influence of acoustic transmit parameters on the destruction of contrast microbubbles in vitro.

In this study, the destruction of the contrast agent Sonazoid (GE Healthcare, Oslo, Norway) was measured in vitro as a function of centre frequency (2-3 MHz), acoustic amplitude (0.66-1.6 MPa), pulse length (2-16 cycles) and PRF (0.5-8.0 kHz). Up to 82% of microbubbles were destroyed after exposure to a single 1.6 MPa acoustic pulse (16 cycles, 2.5 MHz and PRF of 1.0 kHz), while at a low amplitude of 0.66 MPa, fractional destruction increased gradually from 0 to 40% after exposure to 9 (identical) pulses. Fractional destruction increased from approximately 8 to 66% as pulse length was changed from 2 to 16 cycles following exposure to a single 2.5 MHz, 1.3 MPa pulse. As the PRF was increased from 0.5 to 8.0 kHz, shorter exposure time intervals (from 4.8 to 1.2 ms) were needed to achieve the same fractional destruction of 80%. Conversely, as the transmit frequency was increased from 2 to 3 MHz the fractional destruction decreased (by more than half within the first 3 pulses). The influence of changes in acoustic pressure and duty cycle on the destruction of Sonazoid microbubbles was highly statistically significant (p < or = 0.01) with a threshold around 0.67 MPa for a duty cycle of 0.0064. In conclusion, the fractional destruction increases with the duty cycle and the acoustic pressure amplitude and decreases with ultrasonic transmit frequency. Better understanding of the influence of the ultrasound transmit parameters on the destruction of contrast microbubbles should help improve existing contrast-assisted imaging modalities and may help develop new techniques for better use of contrast agents.

In vivo perfusion estimation using subharmonic contrast microbubble signals.

OBJECTIVE: The purpose of this study was to quantify perfusion in vivo using contrast-enhanced subharmonic imaging (SHI). METHODS: A modified LOGIQ 9 scanner (GE Healthcare, Milwaukee, WI) operating in gray scale SHI mode was used to measure SHI time-intensity curves in vivo. Four dogs received intravenous contrast bolus injections (dose, 0.1 mL/kg), and renal SHI was performed. After 3 contrast agent injections, a microvascular staining technique based on stable (nonradioactive) isotope-labeled microspheres (BioPhysics Assay Laboratory Inc, Worcester, MA) was used to quantify the degree of perfusion in 8 sections of each kidney. Low perfusion states were induced by ligating surgically exposed segmental renal arteries followed by contrast agent injections and microvascular staining. Digital clips were transferred to a personal computer, and SHI time-intensity curves were acquired in each section using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Subharmonic fractional blood volumes were calculated, and the perfusion was estimated from the initial slope of the fractional blood volume uptake averaged over 3 injections. Subharmonic perfusion data were compared with the gold standard (ie, the microspheres) using linear regression analysis. RESULTS: In vivo gray scale SHI clearly showed flow and, thus, perfusion in the kidneys with almost complete suppression of tissue signals. In total, 270 SHI time-intensity curves were acquired, which reduced to 94 perfusion estimates after averaging. Subharmonic perfusion estimates correlated significantly with microsphere results (r = 0.57; P < .0001). The best SHI perfusion estimates occurred for high perfusion states in the anterior of the kidneys (r = 0.73; P = .0001). The corresponding root mean square error was 2.4%. CONCLUSIONS: Subharmonic perfusion estimates have been obtained in vivo. The perfusion estimates were in reasonable to good agreement with a microvascular staining technique.

Real-time excitation-enhanced ultrasound contrast imaging.

A new nonlinear contrast specific imaging modality, excitation-enhanced imaging (EEI) has been implemented on commercially-available scanners for real-time imaging. This novel technique employs two acoustic fields: a low-frequency, high-intensity ultrasound field (the excitation field) to actively condition contrast microbubbles, and a second lower-intensity regular imaging field applied shortly afterwards to detect enhanced contrast scattering. A Logiq 9 scanner (GE Healthcare, Milwaukee, WI) with a 3.5C curved linear array and an AN2300 digital ultrasound engine (Analogic Corporation, Peabody, MA) with a P4-2 phased array transducer (Philips Medical Systems, Bothell, WA) were modified to perform EEI on a vector-by-vector basis in fundamental and pulse inversion harmonic grayscale modes. Ultrasound contrast microbubbles within an 8 mm vessel embedded in a tissue-mimicking flow phantom (ATS Laboratories, Bridgeport, CT) were imaged in vitro. While video intensities of scattered signals from the surrounding tissue were unchanged, video intensities of echoes from contrast bubbles within the vessel were markedly enhanced. The maximum enhancement achieved was 10.4 dB in harmonic mode (mean enhancement: 6.3 dB; p = 0.0007). In conclusion, EEI may improve the sensitivity of ultrasound contrast imaging, but further work is required to assess the in vivo potential of this new technique.

Characterization of ultrasound contrast microbubbles using in vitro experiments and viscous and viscoelastic interface models for encapsulation.

Zero-thickness interface models are developed to describe the encapsulation of microbubble contrast agents. Two different rheological models of the interface, Newtonian (viscous) and viscoelastic, with rheological parameters such as surface tension, surface dilatational viscosity, and surface dilatational elasticity are presented to characterize the encapsulation. The models are applied to characterize a widely used microbubble based ultrasound contrast agent. Attenuation of ultrasound passing through a solution of contrast agent is measured. The model parameters for the contrast agent are determined by matching the linearized model dynamics with measured attenuation data. The models are investigated for its ability to match with other experiments. Specifically, model predictions are compared with scattered fundamental and subharmonic responses. Experiments and model prediction results are discussed along with those obtained using an existing model [Church, J. Acoust. Soc. Am. 97, 1510 (1995) and Hoff et al., J. Acoust. Soc. Am. 107, 2272 (2000)] of contrast agents.

In vivo pressure estimation using subharmonic contrast microbubble signals: proof of concept.

Changes in ambient pressure affects the reflectivity of ultrasound contrast microbubbles leading to an excellent correlation between subharmonic signals and hydrostatic pressure. The aortas of two dogs were scanned with an experimental pulse-echo system to validate in vivo pressure estimation based on subharmonic microbubble signals. Results matched well with instantaneous pressure measurements (from 20-60 mmHg) obtained simultaneously with a pressure catheter (root mean square errors <27%).

On the usefulness of the mechanical index displayed on clinical ultrasound scanners for predicting contrast microbubble destruction.

OBJECTIVE: The purpose of this study was to evaluate the mechanical index (MI) displayed on clinical ultrasound scanners as a predictor of exposure conditions related to the destruction of sonographic microbubble contrast agents. METHODS: Sonazoid (GE Healthcare, Oslo, Norway) and Optison (GE Healthcare, Princeton, NJ) microbubbles were injected into a tissue-mimicking flow phantom. Gray scale imaging was performed with 4 different scanners and 3 different transducers (3.5 MHz curved linear, 2.5 MHz convex, and 7.5 MHz linear array), and the MI displayed by the scanner was varied from 0.2 to 1.5 by changing the system output power. All other scanning parameters were kept constant. Downstream changes in echogenicity were monitored with a PowerVision 7000 scanner (Toshiba America Medical Systems, Tustin, CA) as an indirect measure of bubble destruction. Video intensity changes within the flow tube were determined as a function of MI for the different scanner/transducer combinations, and the best linear fit was determined. RESULTS: At a displayed MI of 0.7, different scanner/transducer combinations exhibited a range in video intensity from +16% to -3% of baseline for Sonazoid and from +8% to -71% for Optison. At an MI of 0.3, reductions in video intensity of up to 32% were produced. These results indicate a wide range in bubble destruction at identical MI values. Likewise, regression analysis found no linear fits for all scanner/transducer combinations (r2 < 0.046). CONCLUSIONS: The MI displayed on clinical ultrasound scanners does not predict the degree of microbubble destruction and should not be used by itself to define exposure conditions for destruction of microbubble contrast agents.

Multi-frequency harmonic arrays: initial experience with a novel transducer concept for nonlinear contrast imaging.

Nonlinear contrast imaging modes such as second harmonic imaging (HI) and subharmonic imaging (SHI) are increasingly important for clinical applications. However, the performance of currently available transducers for HI and SHI is significantly constrained by their limited bandwidth. To bypass this constraint, a novel transducer concept termed multi-frequency harmonic transducer arrays (MFHA's) has been designed and a preliminary evaluation has been conducted. The MFHA may ultimately be used for broadband contrast enhanced HI and SHI with high dynamic range and consists of three multi-element piezo-composite sub-arrays (A-C) constructed so the center frequencies are 4f(A) = 2f(B) = f(C) (specifically 2.5/5.0/10.0 MHz and 1.75/3.5/7.0 MHz). In principle this enables SHI by transmitting on sub-array C receiving on B and, similarly, from B to A as well as HI by transmitting on A receiving on B and, likewise, from B to C. Initially transmit and receive pressure levels of the arrays were measured with the elements of each sub-array wired in parallel. Following contrast administration, preliminary in vitro HI and SHI signal-to-noise ratios of up to 40 dB were obtained. In conclusion, initial design and in vitro characterization of two MFHA's have been performed. They have an overall broad frequency bandwidth of at least two octaves. Due to the special design of the array assembly, the SNR for HI and SHI was comparable to that of regular B-mode and better than commercially available HI systems. However, further research on multi-element MFHA's is required before their potential for in vivo nonlinear contrast imaging can be assessed.

Image enhancement by acoustic conditioning of ultrasound contrast agents.

A novel contrast imaging technique has been developed for use with microbubble contrast agents. It employs two acoustic fields: there is an excitation field for conditioning microbubbles and an imaging field for detecting microbubbles. The maximum increases (due to microbubble conditioning) in scattered first and second harmonic signals were 14.5 and 16 dB, respectively. This technique is unique for effectively enhancing the blood-to-tissue image contrast.

Subharmonic signal generation from contrast agents in simulated neovessels.

Detection and measurement of blood flow in neovessels around a tumor can yield prognostic information about the tumor. Early detection and classification may help differentiate benign and malignant tumors; thus, improving patient management. This can be accomplished by injecting ultrasonic contrast agents and measuring the backscattered signals from them. Use of the subharmonic backscattered signals from these agents may be better than fundamental or second harmonic components because of the negligible subharmonics generated by the surrounding tissue. Preliminary results on the detection and measurement of subharmonic signal components up to 12 dB (at increasing pressures) from very small tubes (200 to 300 microm diameter) are reported, demonstrating the possibility and potential application of subharmonic imaging in detecting tumor angiogenesis.

Comparing contrast-enhanced ultrasound to immunohistochemical markers of angiogenesis in a human melanoma xenograft model: preliminary results.

This study compared contrast-enhanced ultrasound (US) measures of tumor neovascularity with molecular markers of angiogenesis in a human melanoma xenograft model. A total of 14 mice were implanted with a human melanoma cell line (WM-9) in the thigh. After 2 to 3 weeks, a tumor, approximately 12 mm in diameter, developed. The US contrast agent Optison (Mallinckrodt, St. Louis, MO) was injected in a tail vein (dose: 0.4 to 0.6 mL/kg). Power Doppler and pulse-inversion harmonic imaging (HI) were performed with an Elegra scanner (Siemens Medical Systems, Issaquah, WA) and a 7.5 MHz linear array. Frame-rates of 30 Hz and 0.5 Hz (intermittent imaging) were used for pulse-inversion HI. After surgical removal, specimens were sectioned in the same planes as the US images. Immunohistochemical stains for endothelial cells (CD31), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and cyclooxygenase-2 (COX-2) were performed. Two observers graded the stains (for intensity and percent area), and two other observers graded the US imaging modes (for fractional tumor neovascularity) on the same scale from 0 to 3. Of the 14 mice, 4 failed for technical reasons (i.e., n = 10). Linear regressions indicated statistically significant correlations between percent area stained with COX-2 and power Doppler (r = -0.789; p < 0.01), as well as intermittent pulse-inversion HI (r = -0.795; p < 0.05). There was a trend toward significance between percent area stained with VEGF and intermittent pulse-inversion HI (r = -0.720; 0.05 < p < 0.10). No other comparisons were significant. In conclusion, contrast-enhanced US measures of tumor neovascularity in a human melanoma xenograft model appear to provide a noninvasive marker of angiogenesis corresponding to expression of COX-2. However, the sample size of this study is small and, until further studies have been conducted, these conclusions are preliminary.

Contrast-enhanced two- and three-dimensional sonography for evaluation of intra-abdominal hemorrhage.

OBJECTIVE: To determine whether a contrast agent enhances sonographic detection of bleeding sites in the abdomen and whether contrast-enhanced three-dimensional sonography provides additional information compared with contrast-enhanced two-dimensional sonography. METHODS: Bleeding sites were created within the livers (n = 3), spleens (n = 5), and kidneys (n = 3) of 3 dogs. A sonographic contrast agent with vascular and parenchymal enhancement capabilities was administered intravenously at a dose of 0.02 mL/kg. Before and after each contrast agent injection, the bleeding sites were imaged with two- and three-dimensional sonography in gray scale harmonic imaging and color flow modes. Sonographic findings were compared with gross pathologic findings. RESULTS: Noncontrast-enhanced sonography was not able to show the specific location of the active bleeding in any of the organs evaluated. The contrast agent enhanced the sonographic detection of blood flow in normal vessels and extravasated blood from damaged vessels or organs in all cases. Intrasplenic and intrahepatic hematomas were better identified on delayed imaging sequences because there was marked enhancement of the normal parenchyma, whereas the hematomas remained unenhanced. Reconstructed three-dimensional sonography showed spatial relationships of the bleeding sites and surrounding structures. Gross pathologic findings were consistent with the contrast-enhanced sonographic results. CONCLUSIONS: Contrast-enhanced sonography improves the detection and evaluation of abdominal bleeding sites. Contrast-enhanced three-dimensional sonography appears to provide additional information when compared with two-dimensional sonography.