The objective of this preliminary study was to examine the spatial correlation between microbubble (MB)-induced vessel wall displacements and resultant microvascular bioeffects. MBs were injected into venules in ex vivo rat mesenteries and insonated by a single short ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 1.5%u20135.6 MPa. MB and vessel dynamics were observed under ultra-high speed photomicrography. The tissue was examined by histology or transmission electron microscopy for vascular bioeffects. Image registration allowed for spatial correlation of MB-induced vessel wall motion to corresponding vascular bioeffects, if any. In cases in which damage was observed, the vessel wall had been pulled inward by more than 50% of the its initial radius. The observed damage was characterized by the separation of the endothelium from the vessel wall. Although the study is limited to a small number of observations, analytic statistical results suggest that vessel invagination comprises a principal mechanism for bioeffects in venules by microbubbles.

Ultrasound-activated microbubbles were used as actuators to deform microvessels for quantifying microvessel relaxation timescales at megahertz frequencies. Venules containing ultrasound contrast microbubbles were insonified by short 1 MHz ultrasound pulses. Vessel wall forced-deformations were on the same microsecond timescale as microbubble oscillations. The subsequent relaxation of the vessel was recorded by high-speed photomicrography. The tissue was modeled as a simple Voigt solid. Relaxation time constants were measured to be on the order of ~10 µs. The correlation coefficients between the model and 38 data sets were never lower than 0.85, suggesting this model is sufficient for modeling tissue relaxation at these frequencies. The results place a bound on potential numerical values for viscosity and elasticity of venules.

High-speed photomicrography was used to study the translational dynamics of single microbubbles in microvessels of ex vivo rat mesenteries. The microbubbles were insonated by a single 2 microsecond ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 0.8-4 MPa. The microvessel diameters ranged from 10-80 micrometers. The high-speed image sequences show evidence of ultrasound-activated microbubble translation away from the nearest vessel wall; no microbubble showed a net translation toward the nearest vessel wall. Microbubble maximum translation displacements exceeded 20 micrometers. Microjets with the direction of the jets identifiable were also observed; all microjets appear to have been directed away from the nearest vessel wall. These observations appear to be characteristic of a strong coupling between ultrasound-driven microbubbles and compliant microvessels. Although limited to mesenteric tissues, these observations provide an important step in understanding the physical interactions between microbubbles and microvessels.

To provide insight into the mechanisms of microvascular damage induced by ultrasound-activated microbubbles, experimental studies were performed to correlate microvascular damage to the dynamics of bubble-vessel interactions. High-speed photomicrography was used to record single microbubbles interacting with microvessels in ex vivo tissue, under the exposure of short ultrasound pulses with a center frequency of 1 MHz and peak negative pressures (PNP) ranging from 0.8%u20134 MPa. Vascular damage associated with observed bubble-vessel interactions was either indicated directly by microbubble extravasation or examined by transmission electron microscopy (TEM) analyses. As observed previously, the high-speed images revealed that ultrasound-activated microbubbles could cause distention and invagination of adjacent vessel walls, and could form liquid jets in microvessels. Vessel distention, invagination, and liquid jets were associated with the damage of microvessels whose diameters were smaller than those of maximally expanded microbubbles. However, vessel invagination appeared to be the dominant mechanism for the damage of relative large microvessels.

Transient interactions among ultrasound, microbubbles, and microvessels were studied using high-speed photomicrography. We observed liquid jets, vessel distention (motion outward against the surrounding tissue), and vessel invagination (motion inward toward the lumen). Contrary to current paradigms, liquid jets were directed away from the nearest vessel wall and invagination exceeded distention. These observations provide insight into the mechanics of bubble-vessel interactions, which appear to depend qualitatively upon the mechanical properties of biological tissues.

Vascular bioeffects produced by ultrasound contrast agent microbubbles are primarily manifested as damage to microvessels. The objective of this work is to directly observe the transient dynamics of bubble-vessel interactions and correlate the observed interactions with associated vascular damage. Microbubbles were perfused into microvessels in ex vivo rat mesenteries and then excited by a single 2 us long ultrasound pulse at 1 MHz. Meanwhile, 14 high-speed photomicrographic images were acquired using 50 ns shutter speeds. The targeted region was then examined by histology and transmission electron microscopy (TEM). Image registration was used to identify the specific vessels that the corresponding high-speed images were captured. The recorded high-speed images revealed that bubble-vessel interactions caused vessel wall distention (motion outward against the surrounding tissue) and invagination (motion inward toward the lumen). Invagination exceeding distention was observed in 60 out of 70 cases. Significant vessel invagination was correlated with vascular damage that was characterized by a separation of the endothelium from the surrounding tissue as revealed by both the histology and TEM analyses. The separation of the endothelium from the surrounding tissue is consistent with damage caused by tensile stresses at the vessel walls that lead to vessel invagination. This suggests that invagination may be an important mechanism by which microbubbles cause vascular damage.

The application of microbubbles for both diagnostic macro- and molecular imaging and therapeudic ultrasound requires an understanding of the coupled interactions involving microbubble dynamics with the surrounding compliant microvessel. In this study, ultra-high speed microphotography was used to directly observe transient behaviors of microbubbles in microvessels of ex vivo rat mesenteries. Definity^® microbubbles were perfused through the vasculature, and then excited by a 2-µs long ultrasound pulse with a center frequency of 1 MHz with peak negative pressures between 0.8-7.2 MPa. These amplitudes span the diagnostic to therapeutic pressure levels. During insonation, ultrahigh speed images were captured with 50-ns exposure time and 150-ns or 300-ns interframe time. The recorded images show a wonderful assortment of microbubble dynamics, including oscillation, translation, jetting, coalescence and fragmentation. These microbubble behaviors were coupled with the dynamic responses of the vessel wall, which showed distention, invagination, and even rupture.

Cavitation is thought to be one mechanism for vessel rupture during shock wave lithotripsy treatment. However, just how cavitation induces vessel rupture remains unknown. In this work, a high-speed photomicrography system was set up to directly observe the dynamics of bubbles inside blood vessels in ex vivo rat mesenteries. Vascular rupture correlating to observed bubble dynamics were examined by imaging bubble extravasation and dye leakage. The high-speed images show that bubble expansion can cause vessel distention, and bubble collapse can lead to vessel invagination. Liquid jets were also observed to form. Our results suggest that all three mechanisms, vessel distention, invagination and liquid jets, can contribute to vessel rupture.

Interactions between bubbles and nearby boundaries have been studied for some time; however, the direct interactions between bubbles and tissue boundaries, especially blood vessel walls, have not been studied to a large extent. In this work highspeed microscopy was used to study the dynamical interaction between microbubbles and microvessels of ex vivo rat mesentery subjected to a single pulse of ultrasound. Ultrasound contrast agent microbubbles were injected into the blood vessels of rat mesentery subsequent to having the blood flushed out. India ink was used to increase the contrast between microvessels and surrounding tissues. Tissue samples were aligned at the focus of both an ultrasound transducer with a center frequency of 1 MHz and an inverted microscope coupled to a high speed camera. Fourteen high-speed microphotographic images were acquired for each experiment using 50 ns shutter speeds. Observations of the coupled dynamics between bubbles and vessels ranging from 10 micrometer to 100 micrometer diameters under the exposure of ultrasound of peak negative pressure within the range of 1 MPa to 7.8 MPa suggest that the vessel wall dilates during bubble expansion, and invaginates during bubble contraction. A significant finding is that the ratio of invagination to distension is usually >1 and large circumferential strains can be imposed on the vessel wall during vessel invagination. In addition, the surrounding tissue response was also quantified. Based on these studies, we hypothesize that vessel invagination is the dominant mechanism for the initial induction of vascular damage via cavitation.

Understanding the interaction of acoustically activated microbubbles with small blood vessels is important for designing better imaging schemes, and for targeting and drug delivery applications. To understand the fundamental mechanisms of this interaction, high-speed microscopy was used to observe microbubble dynamics in microvessels of ex vivo rat mesenteries exposed to a single pulse of ultrasound with a center frequency of 1 MHz and peak negative pressure (PNP) of 1.2 MPa or 11 MPa. It was found that microbubble oscillation caused adjacent microvessel dilation, invagination and even rupture on a microsecond time scale. In small microvessels, microbubble contacted with the vessel wall during expansion under both low and high pressure levels, and microvessel dilation generated by microbubble expansion was larger than invagination induced by bubble collapse. Specifically, under 11 MPa PNP insonation, a small microvessel (17 mum) dilated to 2.7times, and then invaginated to 0.4times of its original diameter, followed by extravasation of re-expanding daughter microbubbles indicating that the microvessel had been ruptured. For large microvessels, microbubbles did not contact with the vessel wall during expansion, and generated much less dilation than invagination at both pressure levels. In one case, a large microbubble caused the wall of a 100 mum microvessel to form a jet-like structure during invagination.