Effect of microbubble contrast agent during high intensity focused ultrasound ablation on rabbit liver in vivo

Materials and methods HIFU ablations (intensity of 400 W/cm 2 for 4 s, six times, with a 5 s interval between exposures) were performed upon 16 in vivo rabbit livers before and after intravenous injection of a microbubble contrast agent (0.8 ml). A Wilcoxon signed rank test was used to compare mean ablation volume and time required to tissue ablation on real-time US. Shape of ablation and pattern of coagulative necrosis were analyzed by Fisher's exact test. Results The volume of coagulative necrosis was significantly larger in the combination microbubble and HIFU group than in the HIFU alone group ( P < 0.05). Also, time to reach ablation was shorter in the combination microbubble and HIFU group than in the HIFU alone group ( P < 0.05). When analyzing the shape of tissue ablation, a pyramidal shape was more prevalently in the HIFU alone group compared to the combination microbubble and HIFU group ( P < 0.05). Following an analysis of the pattern of coagulative necrosis, non-cavitary necrosis was found in ten and cavitary necrosis in six of the samples in the combination microbubble and HIFU group. Conversely, non-cavitary necrosis occurred in all 16 samples in the HIFU alone group ( P < 0.05). Conclusion HIFU of in vivo rabbit livers with a microbubble contrast agent produced larger zones of ablation and more cavitary tissue necrosis than without the use of a microbubble contrast agent. Microbubble contrast agents may be useful in tissue ablation by enhancing the treatment effect of HIFU. Keywords High-intensity focused ultrasound Contrast agent Microbubble Rabbit High-intensity focused ultrasound (HIFU) causes tissue death mainly via heat and cavitation mechanisms. It is mainly used to treat malignant cancers in the liver, pancreas and prostate, but it is also used to treat benign diseases such as uterine fibroids [1–5] . A microbubble contrast agent is a gas bubble of 2–5 μm in diameter, which has been prepared for the purpose of improving the detection and characterization of focal liver lesions [6,7] . Microbubble contrast agents have been used for diagnosis of tumors, and as a tool in the follow-up process after radiofrequency ablation and TACE [8] . Another field in which the use of microbubbles has been applied regards therapeutic application; furthermore, many studies in various areas have been conducted investigating changes of microbubbles in the acoustic field, but they are still in the research stage [9–17] . We have found many undesirable adverse effects following HIFU treatment in liver cancer patients, many of them were associated with high acoustic intensity or prolonged sonication duration. If the therapeutic efficiency of HIFU can be improved by changing the acoustic properties of target tissues, it could lead to shorter exposure time and lower acoustic power, which would reduce the incidence of adverse effects. Notably, in ex vivo studies, several authors have reported that a microbubble contrast agent can enhance tissue ablation within an acoustic field [18–22] . Up to the present, however, in vivo studies of the effect of microbubbles on the therapeutic efficiency of HIFU have rarely been conducted [23–25] ; out of which only two in vivo studies on the liver have been performed [24,25] . Accordingly, because the effects of a microbubble contrast agent cannot be predicted during HIFU treatment, these types of contrast media are not yet used in a clinical setting. In this study, we investigated the enhancing effects of the microbubble contrast agent SonoVue (Bracco, Milan, Italy) on HIFU ablation. 1 Materials and methods 1.1 Animals All experiments were approved by our animal ethics committee. Twenty male rabbits (Xamtako, Gyeonggi-do, Korea) between 2 and 3 months-old, with a mean weight of 2.5–3 kg, were used in all experiments. All the experiments and procedures were performed under complete anesthesia. Anesthesia was induced by intramuscular injection of Zolazepam ([15 mg/kg of body weight] Zoletil 50; Varbac lab, France) and xylazine ([5 mg/kg] Ronpum; Bayer Pharma, Puteau, France). A booster anesthetic injection at the same dose was administered every 30 min. With the use of a booster anesthetic injection immediately before the initiation of HIFU, rabbit pain-related movement was prevented. Before HIFU ablation, the skin was shaved and degassed with vacuum pump. 1.2 Sonographic contrast agent The microbubble agent (SonoVue; Bracco, Milan, Italy) was reconstituted according to the manufacturer's instructions, and diluted in 5 ml saline. SonoVue is a lipid-shelled US contrast agent composed of microbubbles containing sulfur hexafluoride gas. Its standard dose for an adult patient consists of 2 ml of suspension of bubbles in saline solution; accordingly, in rabbits weighing 3 kg, the optimal dose was approximately 0.2 ml. Mean size of the microbubbles were approximately 2.5 μm. The final concentration of sulfur hexafluoride was 45 μg/ml. Before each injection, microbubbles were resuspended by shaking the vial. An intravenous bolus of the contrast agent was injected via the rabbit's ear vein. An AU3 US imaging device (Esaote, Genoa, Italy) was used for real-time imaging of the HIFU system, which was used to confirm that the contrast agent had reached the rabbit liver; furthermore, the hyperechoic change was visible in real-time. Twenty seconds following the administration of the contrast agent, HIFU ablation was performed. For the Grayscale image, the conventional harmonic image was obtained using a 3.5–5.0 MHz probe. 1.3 HIFU ablation An ultrasound therapy system (model JC; Chongqing Haifu Technology, Chongqing, China) was used in this study. The focused ultrasound transducer used in this study had a focal length of 135 mm, a diameter 150 mm, and an operating frequency of 0.8 MHz. An AU3 US imaging device (Esaote, Genoa, Italy) was used as the real-time imaging unit of the system. This 3.5–5.0 MHz imaging probe was located in the center of the HIFU transducer. The water tank was filled with degassed deionized water. Preliminary experiments, to determine the dose that resulted in a hyperechoic area suggesting an optimal degree of tissue ablation during the HIFU treatment in rabbit liver, were performed using four rabbits. Preliminary experiments determined that, the total acoustic power during HIFU treatment in a single exposure dot mode at an intensity of 400 W/cm 2 , an exposure duration of 4 s, a 5 s interval between exposures and a total of six HIFU exposures was sufficient to occur hyperechoic change in rabbit liver. Single exposure dot mode means ablation of one spot without moving the HIFU transducer. Ablation was repeated six times on the same spot both before and after microbubble injection. In the current study, the main experiment was performed using these parameters, using a total of 16 rabbits. All parameter settings were predetermined on the HIFU machine and ablation was performed automatically. Each anesthetized rabbit was placed into a degassed water bath in a prone position. Ablations were performed twice per rabbit, once before and once after microbubble injection, in different lobes. All ablations were performed by the first author (D, J, C). To prevent movement, a specially designed holder was used ( Fig. 1 ). According to pre-determined parameters, tissue ablation was performed on one lobe prior to the infusion of the microbubble contrast agent; the lobes were randomly selected for HIFU ablation. Anatomically, a rabbit's liver is composed of a right lobe, a middle one, and several small-sized caudate lobes. The right and middle lobes are large, and located anteriorly. Accordingly, tissue ablation was performed on right and middle lobe. Two regions were separated by more than 3 cm to ensure that they were not reached by heat from a nearby exposure. The therapeutic depth on the liver surface was consistently set at approximately 1.5 cm beneath the skin. A rabbit's liver is surrounded by the sternum and rib; therefore, when possible, the subcostal region, which was not surrounded by these structures, was chosen. During the HIFU treatment, the hyperechoic change of tissue was confirmed via real-time imaging. The hyperechoic area on the ultrasound image was presumed to relate to the cavitation, microbubbles produced from water boiling, rising temperature of the target tissue, as well as the formation of coagulated necrosis. In order to evaluate hyperechoic change objectively, pre- and post-ablation echoes of the target area were automatically compared with using GrayVal 1.0 software (Chongqing Haifu Technology, Chongqing, China; JC HIFU system). This method was helpful as it was difficult to determine echoic changes visually. Accordingly, the time elapsed until tissue ablation was defined as the time required for the target to show a hyperechoic change on the real-time US. Approximately 3 min after ablation, until the effects of the HIFU treatment abated, animals were given intravenous injections of 0.2 ml of SonoVue, followed by injections of 2 ml of physiologic saline to flush before secondary HIFU exposure. On the real-time imaging system, the hyperechoic change was found to occur in the liver parenchyma and within the vessels. Microbubbles appeared in the rabbit liver about 15 s after injection, and sustained a stable concentration for 1 min, and then decreased, as observed via sonography. Therefore, HIFU ablation was performed 20 s after the injection in the other lobe ( Fig. 2 ). To make sure that a sufficient amount of microbubbles reached the tissue between the sessions of HIFU ablation, a resting time of 5 s was provided. A total of six exposures were performed for 24 s, and total HIFU ablation time, including resting time, was 54 s. Respiratory movement control was not conducted. 1.4 Examination of tissue After all HIFU ablations were performed, the animal was euthanized with intravenous pentobarbital (Fatal Plus; Vortech Pharmaceuticals, Dearborn, Mich) and the animals were moved to the animal care facility. The rabbit liver was removed en bloc immediately after the animal was euthanized. Ablated tissue was sectioned, and the resultant coagulation was evaluated. All sections were independently examined by two of the authors (S.E.J and J.M.L), who were blinded to the results. A total of 32 specimens, 16 each, were analyzed in the combination microbubble and HIFU group, and HIFU alone group. The two short-axis ( X , Y, perpendicular to sound source), long-axis ( Z , along sound source) diameters of the coagulated region in the liver of each animal were measured by means of gross examination with calipers. The volume of ablation could be accurately calculated according to the definition of V = ∏ × L × W × D /6 ( L : length, W : width, D : depth). In the cases where ablation zones induced by HIFU were broadly abutted to the hepatic capsule, the precise portion of the ablation zone beyond the border of the hepatic capsule could be disregarded. However, this error was not thought to influence the total ablation volume considerably on the supposition that the depth of focal ablation was same. The shape of ablation was classified as spherical or pyramidal. Pattern of coagulative ablation was classified into two types: The occurrence of tissue ablation accompanying the internal presence of a cavity was defined as cavitary necrosis; whereas, the occurrence of homogeneous tissue ablation without the interior presence of a cavity was defined as non-cavitary necrosis. 1.5 Statistical analysis Mean ablation volume and onset time of tissue ablation on the real-time US system were compared by Wilcoxon signed rank test using SPSS 12.0 software (SPSS, Chicago, IL). Shape of the focal region and pattern of coagulative necrosis were analyzed by Fisher's exact test using SPSS 12.0 software (SPSS, Chicago, IL). A P value of less than 0.05 was considered to indicate a significant difference. 2 Results 2.1 Volume and time to reach tissue ablation The volume of coagulative necrosis was significantly larger in the combination microbubble and HIFU group than in the HIFU alone group (1.75 ± 0.21 ml vs. 0.83 ± 0.13 ml, P < 0.05, Wilcoxon signed rank test). Time until tissue ablation was significantly shorter in the combination microbubble and HIFU group compared with the HIFU alone group (8.6 ± 1.3 s vs. 12.1 ± 1.8 s, P < 0.05, Wilcoxon signed rank test) ( Table 1 ). 2.2 Shape and pattern of coagulative necrosis In regard to the shape of ablation, a pyramidal shape was evident in 13 cases (81.2%) and a spherical shape in three cases (18.8%) of the HIFU alone group ( Fig. 3 ). In the combination microbubble and HIFU group, there was a pyramidal shape in three cases (18.7%) and a spherical shape in 13 cases (81.2%) ( P < 0.05, Fisher's exact test). In the analysis of the pattern of coagulative necrosis, non-cavitary necrosis occurred in ten cases (62.5%) and cavitary necrosis in six cases (37.5%) of the combination microbubble and HIFU group ( Fig. 4 ). Non-cavitary necrosis occurred in all 16 cases (100%) of the HIFU alone group ( P < 0.05, Fisher's exact test) ( Table 2 ). 3 Discussion The major effects of HIFU on tissue ablation are a result of thermal changes to the tissue [26] . The thermal effect is generated by the absorption of acoustic energy, which induces coagulative necrosis by transiently raising the tissue temperature to >60 °C. There are also some non-thermal effects of HIFU, such as cavitation, acoustic streaming and shear stresses. Because coated microbubbles are filled with gas, they are highly compressible, and when exposed to an ultrasound field, the varying pressure causes them to expand and contract. Cavitation is one of the effects by which the HIFU induces mechanical tissue injury, but it does not occur at a lower intensity acoustic field. Cavitation is defined as the generation of a gas cavity and its movement within an acoustic field; i.e., the therapeutic action of HIFU [14–17] . The exposure of the bubble to a high acoustic field results in violent oscillations and rapid growth of the bubble during the rarefaction phase, which eventually leads to its violent collapse and destruction. Microbubbles burst explosively, leading to the occurrence of tissue necrosis, this would cause a strong mechanical injury to the tissue and then form a cavity within the ablated tissue. While the cavitation effect caused by HIFU observed in tissues plays a minor role in tissue ablation, the effect resulting from microbubbles infused from outside is expected to take a major role in tissue ablation. Another effect of the microbubble on the tissue ablation regards heating. The heating effect of the microbubbles has been experimentally investigated both in vitro and in vivo , both types of study suggest microbubble contrast agents enhance the heating effect in HIFU treatment [27] . Cavitary coagulative necrosis was present in significantly more samples in the combination microbubble and HIFU group. To our knowledge, there have been two peer-reviewed in vivo studies demonstrating the effect of microbubbles on HIFU during liver tissue ablation [24,25] . They found that HIFU-induced lesions were larger in animals in the presence of microbubbles than in those given saline. Although there are many variables when comparing our study to that of Kaneko et al. and Luo et al., such as type and dose of microbubble contrast agent, depth, time, power, etc., these results are similar to those of our study. It is questionable, however, whether our results are actually applicable to HIFU treatment of cancer in the human body. This is because the microbubbles had dual effects upon coagulative tissue necrosis, i.e., the facilitation of the cavitation effect and the suppression of sonic attenuation, following the HIFU ablation. Reflection, refraction, scatter and diffraction of ultrasound in the propagation path may interfere with the focusing of an ultrasound beam in tissue. These phenomena were further strengthened with the passage of the HIFU beam through the microbubble, which may have reduced the volume of tissue ablation. Although a decrease in volume was not found in our study, the human liver presents a longer path for the HIFU beam, increasing the likelihood that the size of tissue ablation could decline because of the attenuation effects mentioned above. Further studies are warranted to examine the dual effects of microbubbles. Based on our results, possible clinical applications are as follows. First, when a hyperechoic area is not formed inside a tumor during HIFU treatment, it can be made by using a microbubble agent, and HIFU ablation can be conducted with the area as a center to treat the tumor effectively. Second, in cases of hypervascular tumor, such as hepatocellular carcinoma, the concentration of microbubbles within the tumor is relatively higher than hypovascular tumor. It is therefore probable that the treatment effects could be enhanced in these tumors. Our study had some limitations, mainly related to the need for more replicates. First, no studies have been conducted to examine whether microbubble agents are effective in a clinical setting. So, further studies are required to examine whether the effects of microbubbles on tissue ablation during the HIFU treatment could be harnessed for the treatment of patients with cancer. Second, we performed this experiment only under specific HIFU sonication settings (400 W/cm 2 , duration 4 s, etc.) and microbubble agent administration (0.8 ml IV injection), and the results may not apply to other methodological settings. It is possible that adjustment of intensity and duty cycle could induce different results. 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