Blood vessel rupture by cavitation
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.
KeywordsCavitation Vessel rupture High-speed imaging
Acute injury to the kidney occurs in the clinical application of shock wave lithotripsy (SWL). The primary feature of SWL-induced tissue injury is the rupture of blood vessels, with small vessels particularly susceptible to injury . Cavitation is thought to play a prominent role in SWL-induced vascular rupture .
The study of how cavitation bubbles damage surrounding materials dates back almost a century, when the collapse of vapor bubbles near propeller blades was found to be the prime mechanism associated with damage to the blades . Extensive studies have shown that cavitation bubbles cause erosion to ship propeller blades by the formation of liquid jets. It has been suggested that liquid jet impact is also one mechanism by which cavitation damages vessels . In addition, vessel distention associated with cavitation bubble expansion has also been proposed as a mechanism contributing to vessel rupture .
To understand precisely how cavitation induces vascular injury, the most direct method is to observe the dynamics of bubbles in blood vessels in real time. In this way, bubble dynamics and their interactions with blood vessels can be investigated. Zhong et al.  used high-speed imaging to observe the interactions of bubbles with vessel phantoms (cellulose hollow fibers and silicon tubing). The vessel phantoms ruptured in response to large-scale cavitation; however, such in vitro tubing does not match the viscoelastic properties of real tissue. Observations of bubble dynamics in actual vessels embedded within surrounding tissue has been reported only once . In that pioneering work, streak and strobe imaging were used; however, the transient dynamics of bubbles and vessels were not fully captured.
In this work, a high-speed photomicrography system was set up to directly visualize transient interactions between ultrasound-activated bubbles and blood vessels, especially microvessels whose diameters are comparable to bubble sizes. Bubble-induced vessel distention (outward push into the tissue), invagination (inward pull into the lumen) and liquid jets were all observed to be associated with vessel rupture, indicated by extravasation of bubble fragments as well as a dye injected into the vessels.
Animal tissue preparation
The animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Washington. Rat mesenteries were used as our animal model because they have good optical transparency and are easy to access. In our experience, not all strains of rats have rich mesenteric vascular networks. We examined the mesenteries of Sprague–Dawley and F344 rats (both ordered from Charles River Labs) and found that F344 rat mesenteries usually have much higher vessel densities than Sprague–Dawley rats. Therefore, F344 rats were used in our study.
Blood vessels (such as those illustrated in Fig. 1b) with diameters ranging from 10 to 100 μm were identified for targeting. Ultrasound contrast microbubbles [7, 8] were perfused into the blood vessels to serve as cavitation nuclei for visualization (that is, selected nuclei were used in focusing the microscopic system described below). Green India ink at a volume concentration less than 3% was added to the perfusate to increase the contrast of the vessels compared to the surrounding tissue, and also to indicate blood vessel leakage.
The microscope was aligned confocally with a high-intensity focused ultrasound (HIFU) transducer (H102; Sonic Concepts, Bothell, WA, USA) with a 1 MHz center frequency, a 63 mm geometrical focal length and a 0.9 f-number (see Fig. 2a). The transducer was positioned directly opposite to the microscope objective. It was driven by one cycle sinusoidal signal produced by a function generator (33120A; Hewlett Packard, Palo Alto, CA, USA) and amplified by a power amplifier (ENI A150; ENI, Rochester, NY, USA). Due to ringing of the transducer, the acoustic output pulse was about 2 cycles long as determined using a fiber-optic hydrophone (FOPH 2000; RP Acoustics, Leutenbach, Germany). An example of the pressure waveform measured at the location of the tissue sample (in the absence of tissue) is shown in Fig. 2b. In this figure, we can see both the incident wave from the transducer and the reflected wave from the microscope objective. Ultrasound pulses with peak negative pressures (PNP) of 4–7 MPa were used in this study. We note that a shock wave generator was not used as a source, but instead a HIFU transducer was used to generate signals with PNP close to those used in SWL. It should also be noted that to get rid of the reflected ultrasound wave, the transducer could be located at a 45° angle with respect to the top surface of the objective, instead of being directly above the objective as shown in Fig. 2a. In this way, the reflected wave from the objective would not impact the targeted region on the tissue sample. We did not do so because the alignment of the optical and acoustic system is much easier in the present setup. Moreover, as shown in Fig. 2b, the amplitude of the reflected wave is only about 1/3 of the incident wave’s amplitude. The impact of the reflected pulse is thus reduced compared to the incident pulse.
A high-speed camera (Imacon 200; DRS Hadland, Cupertino, CA, USA) was connected to the microscope and synchronized with the ultrasound transducer. When contrast agent microbubbles were observed flowing through the field of view of the microscope, the ultrasound pulse was generated and the camera was triggered to capture a 14-frame sequence of images at a shutter speed of 50 ns. The delay of the first frame was set to the ultrasound traveling time from the transducer to the tissue. The interframe time was set to 150, 300 or 600 ns to examine bubble dynamics over different time periods.
Results and discussion
Vessel rupture associated with vessel distention and invagination
Figure 5b and c are color images captured before and 1 min after the high-speed image sequence was taken. This delay was the time needed to take the high-speed images and switch imaging modalities from using the high-speed camera to a color CCD camera. The dye that was initially inside the blood vessel in Fig. 5b extravasated into the interstitial space (Fig. 5c), leading to a reduction of contrast and thus vessel clarity. The lower bigger vessel appears to have remained intact.
Vessel distention caused by bubble expansion pushing against the vessel wall was previously observed by Zhong et al.  in a vessel phantom and was considered to be one of the mechanisms leading to vessel rupture. Our study showed that vessel distention from acoustically activated bubbles can also occur in real blood vessels. And as we noted above, vessel invagination induced by collapsing bubbles may lead to high strains on the vessel wall which may also contribute to vessel rupture and may be more important in some conditions.
Small vessel rupture involving liquid jets
Unlike the examples shown above, in this particular example we hypothesize liquid jetting as a mechanism for vascular rupture. The formation of liquid jets is controversial, as bubble expansion is somewhat constrained by the vessel walls, which is thought to limit the strength of collapse that leads to jet formation. However, our observations clearly show that liquid jets can form in constrained environments. Even though bubble expansion is constrained by the vessel wall and surrounding tissue, the rebounding of the vessel wall during bubble collapse can ‘inject’ momentum back into the liquid  that may contribute to the formation of liquid jets. We observed that in larger vessels, at least twice the size of the maximum bubble diameter, liquid jets are always directed away from the nearest vessel wall, not towards it. In those cases, damage due to liquid jets impinging on the vessel wall is probably not occurring. But for small vessels like that shown here, a liquid jet can impact the opposite vessel wall leading to damage.
High-speed imaging was used to record cavitation within blood vessels in an ex vivo rat mesentery preparation exposed to an ultrasound source generating negative pressures in the range produced by shock wave lithotripters. The data provide visual evidence that cavitation can cause obvious rupture of microvessels smaller than 20 μm in diameter as indicated by bubble fragments growing outside the vessel and dye extravasation. The precise mechanisms that were observed included vessel distention, invagination and liquid jetting. All experiments involved a single ultrasound pulse lasting no more than a few microseconds. Future work will involve the use of shock waves that mimic actual SWL devices.
Work supported by NIH grants EB000350, AR053652, DK043881 and DK070618.
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