Bioeffects Of Microbubble-enhanced Ultrasound In Liver And Its Impact On Liver Ethanol Ablation

BackgroundsPercutaneous ethanol ablation （PEA） is a safe and effective method for treating smallliver cancer. PEA was first introduced by two research teams in the1980s. Theydemonstrated that absolute ethanol was able to induce chemical ablation of small, nodularhepatocellular carcinoma （HCC） when administered as ultrasound-guided injections. Theefficacy of ethanol ablation was later confirmed by numerous reports and it was widelyaccepted for clinical use.The limitations of PEA are also obvious. The anti-tumor effect of PEA is based on itschemical ablation by ethanol. Due to the quick washout by the tumor or liver circulation, it maybe difficult to accumulate a high concentration of the injected ethanol at the injection site.Generally, the PEA indication for liver tumors has been restricted to small tumors thatare smaller than3cm in diameter. HCCs usually develop minute, local intrahepaticmetastasis with an incidence that increases with tumor size.However, the quick washout of ethanol by blood perfusion prevents its accumulationwithin tumors. We previously found that liver blood perfusion can be temporally arrestedfor15-30minutes with microbubble-enhanced, pulsed therapeutic ultrasound, which couldbe used to delay the quick ethanol washout by liver circulation during a EA procedure.Objective1. To explore the bioeffects of arresting hepatic blood perfusion using microbubbleenhanced acoustic cavitation in rabbits.2. To investigate the impact of microbubble-enhanced ultrasound （MEUS） on canethanol ablation of normal liver.Materials and methods1. MaterialsTherapeutic ultrasound device:A therapeutic ultrasound device （CZ-960, Mianyang Sonic Electronic Ltd. Mianyang, China） was applied to generate pulsed, high-pressure amplitude US. used in the study. Thetransducer was driven by a specially designed power amplifier. The transducer was operatedat831KHz with a400cycle pulse length and a pulse repetition frequency of9Hz. Theacoustic pressure （peak negative pressure） output was4.26MPa. The transducer wasworking in an intermittent mode of6seconds on and6seconds off. The actual workingduty cycle came out to be approximately0.22%.Microbubbles:A lipid-coated microbubbles, Zhifuxian, were used for both the nucleation of the UScavitation and the enhancement of contrast enhanced ultrasound （CEUS）. Zhifuxian wasprepared by lyophilization of two lipids in suspension,（DPPG and DSPE）, and thenagitated with perfluoropropane gas using a high-speed mechanical amalgamator. Themicrobubbles had a mean particle diameter of2μm and a bubble concentration of9×10¹⁰/mL. For the CEUS study, a bolus injection of0.02mL/kg microbubble suspensionwas injected. For the nucleation for MEUS treatment, the microbubble was constantlyadministered at a rate of approximately0.6mL/min with a total dose of0.1mL/kg.2. Methods2.1Twenty-one healthy New Zealand rabbits were randomized to the ultrasound onlygroup, one MEUS session group and two MEUS sessions group. The livers were surgicallyexposed and insonated by pulsed therapeutic ultrasound for5min with a peak negativepressure of4.26MPa. The lipid microbubble was intravenously injected at0.1ml/kg. Theone session group was treated by MEUS once and the two sessions group doubled thetreatment within1h interval. The microbubble was replaced by3ml saline in theultrasound only group. CEUS was performed before and immediately,30min,60min and48h after treatment with peak intensity （PI） analysis. Liver specimens were harvested48hafter treatment.2.2We performed the study on twenty-four healthy New Zealand rabbits weighing2.0-2.5kg. Nine animals were assigned for the purpose of studying the joint ablative abilityof MEUS and EA. Another nine animals were assigned for treatment with MEUS but notEA. EA alone was performed in the remaining six animals. The latter two groups served ascontrols. We treated surgically exposed rabbit liver with a combination of MEUS and EA.The controls were treated with only MEUS or EA at a level of0.05mL. MEUS treatment was performed with a high-pressure amplitude, pulsed therapeutic ultrasound device andintravenous injection of microbubbles. The therapeutic ultrasound was operated with anacoustic pressure of4.26MPa and a duty cycle of0.22%. CEUS was performed to estimatethe liver blood perfusion. The livers were harvested for necrotic volume measurements48hours after treatment.All procedures were performed in accordance with the approval of the InstitutionalAnimal Care and Use Committee of the Hospital.ResultsIn the one session group, the treated region showed significant non-enhanced perfusiondefect, and the PI dropped from （-51.9±4.3）dB to （-62.5±4.8）dB after insonation. However,blood perfusion recovered and the PI rose to（-52.0±4.6）dB after48h. In the two sessionsgroup, the perfusion defect seemed larger than that of the one session group, and the PIdisplayed a similar variation. The liver blood perfusion of the ultrasound only groupshowed no differences before and after treatment. Pathological examination found thatminute to small sized necrotic nodules scattered the livers in two MEUS groups.CEUS demonstrated that the liver perfusion was temporally arrested, with a significantpeak intensity decline from-46.9±3.8dB to-64.0±3.3dB, after MEUS treatment. Themean necrotic volume of the MEUS+EA treated livers （3.3±2.3cm3） was over10timeslarger than that of the EA only （0.3±0.2cm3）. The liver necrosis treated by MEUS onlywas minor, scattered and immeasurable. The results indicate that MEUS disruption of livercirculation can greatly promote the liver EA.ConclusionHepatic blood perfusion could recover within48h after MEUS treatment with minorliver necrosis.MEUS disruption of liver circulation can greatly promote the EA ablation of liver.