Original contribution
Optical monitoring of ultrasound-induced bioeffects in glass catfish

https://doi.org/10.1016/j.ultrasmedbio.2003.08.005Get rights and content

Abstract

This study is an investigation of the therapeutic ultrasound (US) effects on the blood vessels of optically transparent fish in vivo. Although many investigators have characterized cavitation in vivo using remote-sensing methods (i.e., measuring the acoustic emissions caused by oscillating bubbles) very few have made direct observations of cavitation-induced damage. Anesthetized glass catfish, which are optically transparent, was injected with the contrast agent, Optison®, and then insonified at pressures that ranged from 0.5–10 MPa (peak negative pressures). Two focused transducers were used in these experiments to cover a frequency range of 0.7–3.3 MHz. Sonications were pulsed with pulse durations of 100, 10, 1, 0.1 and 0.01 ms and a pulse repetition frequency (PRF) of 1 Hz. The entire length of one sonication at a specific pressure level was 20 s. An inverted microscope combined with a digital camera and video monitor were used optically to monitor and record US interaction with the blood vessels in the tail of the anesthetized fish at 200× magnification. The effects of the burst sonication were analyzed visually at each pressure level. For the 1.091-MHz sonications, the first type of damage that occurred due to the US interaction was structural damage to the cartilage rods that comprise the tail of the fish, and was characterized by a disintegration of the lining of the rod. Damage to the rods occurred, starting at 3.5 MPa, 3.1 MPa, 4.1 MPa and 5.5 MPa for the 100-ms, 10-ms, 1-ms and 100-μs sonications, respectively. The formation of large gas bubbles was observed in the blood vessels of the fish at threshold values of 3.8 MPa, 3.8 MPa and 5.3 MPa, for the 100-ms, 10-ms and 1-ms sonications, respectively. Neither gas bubble formation nor hemorrhaging was observed during 100-μs sonications. Bubble formation was always accompanied by an increase of damage to the rods at the area surrounding the bubble. At 1.091 MHz, petechial hemorrhage thresholds were observed at 4.1 MPa, 4.1 MPa and 6.1 MPa, respectively, for the three pulse durations. The thresholds for damage were the lowest for the 0.747-MHz sonications: they were 2.6 MPa for damage to the rods, 3.7 MPa for gas bubble formation and 2.4 MPa for hemorrhaging. ([email protected])

Introduction

Much research is currently being done to understand ultrasound (US) induced bioeffects both in vitro and in vivo. Specifically, the use of contrast agents containing microbubbles for diagnostic US has increased the possibility of damage caused by US. Examples of observed damage include hemorrhaging and petechiae formation Dalecki et al., 1995, Miller and Gies, 1998, Miller and Gies, 2000, Carstensen et al., 1990, hemolysis Dalecki et al., 1997, Carstensen et al., 1993, Poliachik et al., 1999, Everbach et al., 1997 and structural damage to tissue and vasculature Hynynen et al., 1996, Brayman et al., 1999, Miller and Quddus, 2000, Frenkel et al., 1999. Mechanical bioeffects have been attributed to the occurrence of acoustic cavitation, most probably inertial cavitation.

Many investigators have used the mouse intestine model system to assess the possible association of vascular damage with ultrasonic activation of US contrast agent gas bodies circulating in the blood. Miller and Gies (1998) exposed anesthetized hairless mice to continuous and pulsed 1.09-MHz US with and without prior injection of Albunex® (a gas-body-based contrast agent). The addition of contrast agent during in vivo US exposure enhanced the production of vascular damage in intestinal tissue. The threshold for hemorrhaging at their pulsing conditions (100-s duration, 10-μs pulse, 1-ms pulse repetition frequency PRF, 0.01 duty factor) was found to be 2.4 MPa.

In later work, Miller and Gies (2000) extended previous work to include 0.4- and 2.4-MHz US. Also, several different contrast agents (Albunex®, Levovist®, PESDA and Optison®) were tested in this model system at 2.3 MHz to determine if vascular bioeffects can be expected from the different gas-body compositions used in different contrast agents. The induction by US of petechiae and hemorrhages in the mouse intestine was examined with injection of contrast agents. Exposures were performed at 0.4, 1.09 and 2.4 MHz, using both continuous and pulsed (10-μs and 1-ms PRF) exposures for 100 s. They found that contrast agents greatly increase the numbers of petechiae, especially for pulsed-mode exposure. Higher-pressure amplitudes were required for petechiae production at the higher frequencies. The thresholds for petechiae in terms of the peak negative pressure for the 2.3-MHz exposure were between 1.8 and 2.6 MPa.

Work was done by Dalecki et al. (1995) to determine if diagnostic levels of pulsed US produce intestinal hemorrhage in the mouse. They determined thresholds for intestinal hemorrhage as a function of frequency. Exposure was performed for 5 min using pulsed US with a pulse length of 10 μs and duty cycle of 0.001. Intestinal damage ranged from small petechiae to hemorrhagic regions of extravasations extending 4 mm or more along the intestine. For all frequencies, the extent of intestinal damage increased with increasing exposure level. Yet, for a given exposure level, lower frequencies were more effective in producing hemorrhage than were higher frequencies. Not much damage was seen at 2.4 and 3.6 MHz. They found threshold exposures for the production of intestinal hemorrhage with US (peak negative pressure) to be 0.9, 1.5, 3.9 and 2.8 MPa, respectively, for 0.7-, 1.1-, 2.4- and 3.6-MHz frequencies.

Miller and Quddus (2000) investigated a hypothesis that clinical diagnostic US exposure could induce bioeffects with Optison® gas bodies at 2.5 MHz. They found that hundreds of petechiae hemorrhages and extravasations of the Evans blue dye resulted from the exposure with injected contrast agent bodies, whereas no effects were seen without the injected gas bodies. These effects occurred at pressure amplitudes above about 0.64 MPa. They suggested that diffuse microscale effects might occur over large volumes of tissue, which might not be readily apparent clinically.

In later work, Dalecki et al. (1997) studied ultrasonically-induced hemolysis with and without Albunex®. They exposed murine hearts at either 1.15 or 2.35 MHz for 5 min with a pulse length of 10 μs and PRF of 100 Hz. They found the threshold for hemolysis to be about 1.9 MPa (peak negative pressure) at 1.15 MHz. Their failure to obtain hemolysis in the absence of added bubbles suggests that the number of appropriate cavitation nuclei in the blood of normal animals is very small.

Clearly, the current picture of the bioeffects induced and incurred is not complete and more data are needed essentially to establish the relationship between the bioeffects and the exposure parameters. This requires an experimental set-up that allows a large number of exposures to be performed for each study. Our aim for this study was to extend the previous studies by directly observing the tissue damage in vivo during pulsed US. This is possible in optically transparent animal systems. This approach has been used in the past for investigating the effects of acoustic streaming and standing waves in fish Martin et al., 1982, Martin et al., 1983, Dyson et al., 1974.

Section snippets

Animal specimens

The muscles and skin of the glass catfish (Kryptopterus bicirrhus Minor, Siluridae family) are optically transparent. The fish were approximately 5 cm in length and weighed between 0.4 g and 0.6 g. The area of the fish that was sonicated was the tail of the fish, which is comprised of cartilage rods, minor blood vessels and capillary vessels flowing along and through the rods. The cartilage rods have a lining approximately 10-μm thick. Rod thickness is, on the average, 50 μm. Prior to

Us-induced bioeffects on the blood vessels and cartilage rod structure in the tail of the fish

The optical field of view of the tail contained two or three cartilage rods, depending on the size of the fish. All sonications were observed at 200x magnification, to have as wide a field of view as possible, while still being able to observe the microscopic structure of the fish. The optical field of view at 200× magnification was 1.5 mm × 1 mm. This provided a resolution of 2 μm/pixel. The US-induced bioeffects that occurred in the fish were damage to the structure of the cartilage rods in

Discussion

The results from this study demonstrate bioeffects due to US after injection with a contrast agent containing microbubbles. The experimental set-up described in this paper allows the visualization of US-induced damage in biological tissue in real-time. For the 1.091-MHz frequency sonications, the first apparent damage was to the cartilage rods in the tail of the fish. These effects could be cavitation, radiation force or acoustic streaming. To determine the magnitude of the streaming effects in

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