| Background:Tumor angiogenesis is the critical factor in tumor growth and metastasis. Fast developed neovascularization transfers requisite oxygen and nutrition to tumor cells to maintain cell activities. Disruption of tumor neovasculature may effectively inhibit or even decimate tumor cells by cutting off the supply of nutrient. Anti-angiogenesisi has been studied for decades and by now several drugs focusing on specific targets of endothelial cells have been promoted in clinical. However, the limited effects and patients dependency restricted their application. Tumor neovasculature, which is much more fragile and incomplete than that of normal tissues, is more likely to be damaged by physical effects caused by microbubble-enhanced ultrasound (MEUS). Previous studies proved that MEUS can cause serious pathological damages (hemorrhage, thrombosis, edema et al.) to tumor neovasculature and may block the blood perfusion for as long as24hours. Repeated reinforcement of this effect could inhibit tumor growth and reduce metastasis, which indicates that MEUS do have the effects of anti-angiogenesis.Percutaneous ethanol ablation (PEA) is a safe, effective and minimally invasive treatment for tumor in clinical. It is the most widely used chemical ablation therapy due to the simple operating, less cost, less complication, and less damages. However, the quick washout of ethanol by tumor circulation limits the application of this therapy to small tumor which is less than3cm in diameter. Assumption came up that combining PEA with MEUS may increase ablation volume of ethanol by blocking blood perfusion and hence preventing washout of ethanol. So rabbits are designed to accept combined therapy of MEUS and PEA to obtain indicators to evaluate the therapeutic effect. Objective:To evaluate the synergistic effect of microbubble-enhance ultrasound on percutaneous ethanol ablation of VX2tumor by observing necrotic ratio, growth inhibition ratio and life cycle of different groups, and thus evaluate the clinical application value of this combined therapy. This new method might offer an effective way in improving the ablation volume of percutaneous ethanol injection therapy.Materials and Methods:1. Materials:(1) Therapeutic ultrasound device:two different customized machines were used in this experiment.①CZ-960, a non-focused high pressure device manufactured by Mianyang Sonic Electronics Co., Ltd. The transducer of the device emit pulsed ultrasound at a frequency of831KHz, pulse length, emission/pause time, pulse recurrence frequency, and peak acoustic pressure are adjustable. Settings used in this experiment were as follows: peak negative pressure for4.26MPa, duty cycle for0.22%.②DCT-700, a newly designed digital, integrated ultrasound device manufactured by Shenzhen Wilder Medical Electronics Co., Ltd. Similar as CZ-960, pulse length, emission/pause time, pulse recurrence frequency, and peak acoustic pressure are adjustable. Settings were also the same.(2) Ultrasound imaging system:S2000diasonography manufactured by German Siemens Ltd. A high frequency linear-array probe with an emission frequency of4-9MHz was used in this experiment to perform contrast enhanced ultrasound(CEUS). The time-intensity curve (TIC) was analyzed using the built-in software.(3) Microbubbles:Lipid-coated microbubbles with perfluoropropane as gas core in it were applied both as the cavitaion nuclei and the contrast agent for enhanced ultrasonography. The concentration of MBs is4-9×109/ml and ninety-eight percent of the microbubbles are less than8μm in diameter. The dose for enhanced ultrasonography was0.02ml/kg and that for ultrasound therapy was0.1ml/kg.(4) Animals:Sixty healthy New Zealand rabbits weighing from2.0kg to2.5kg were bought and used in this experiment. Shutos bearing VX2tumor were offered by Chongqing Institution of Medical Ultrasound Engineering. 2. Methods:(1) Thirty rabbit models bearing subcutaneous VX2tumor were randomly assigned into four groups:microbubble-enhanced ultrasound therapy group, percutaneous ethanol ablation group, combined therapy group and control group. In the microbubble-enhanced ultrasound therapy group, tumors were exposed to pulsed therapeutic ultrasound for5minutes while0.2ml MBs (diluted by3ml saline) was injected intravenously. In the percutaneous ethanol ablation group,0.1ml ethanol was injected into tumor under ultrasound guidance. In the combined group, both microbubble-enhanced ultrasound therapy and percutaneous ethanol injection were applied. While in control group, no treatment was applied. CEUS was performed before, right after and24hours after treatment to image the tumor blood perfusion. The peak intensity value (PI) and area under the curve (AUC) were analyzed. Animals were executed after24hours and tumors were then removed, fixed, sectioned and stained to get the necrotic ratio.(2) Thirty rabbit models bearing subcutaneous VX2tumor were established, grouping and treatments were the same as the first part. All the tumors were conducted CEUS before and right after treatment. The long and short diameters of each tumor were measured on the4th,8th,12th,16th,20th,24th, and28th days respectively by ultrasonic examination. Survival span was calculated by recording the natural death time of each rabbit.Results:1. In the control group, the contrast perfusion of VX2tumors showed no significant change after treatment, while in the three experimental groups the perfusion of tumors decreased greatly after treatment. Compared to that before treatment, the PI and AUC value in24hours later decreased25.2%and28.1%respectively in microbubble-enhanced ultrasound group(P<0.05). In percutaneous ethanol ablation group the reduction was28.1%and34.7%(P<0.05), and in combined group it was65.8%and70.3%(P<0.05). The tumor necrotic ratios of microbubble-enhanced ultrasound group, percutaneous ethanol ablation group, control group and combined group weren (32.3±8.8)%,(37.5±9.9)%,(21.9±8.3)%, and (56.6±16.8)%, respectively. Tumor necrotic ratio in combined group was significantly higher than other groups(P<0.05). Pathological injuries of all experimental groups are coagulation necrosis. 2. Within the observation period of twenty-eight days, the average tumor volume of microbubble-enhanced ultrasound group, percutaneous ethanol ablation group, control group and combined therapy group increased from (0.55±0.35) cm,(0.41±0.12) cm,(0.59±0.23) cm3, and (0.70±0.37) cm3to (39.34±17.35) cm3,(41.94±14.77) cm3,(13.49±8.49) cm3, and (43.60±19.13) cm3, respectively. On the28th day, the average tumor volume of combined therapy group was significantly smaller than other three groups(P<0.05). The survival span of each group were:(84±19) days for combination group,(58±4) days for control group,(68±8) days for microbubble-enhanced ultrasound group, and (70±9) days for percutaneous ethanol ablation group. The survival span of treated rabbits in combined therapy group was much longer than other groups(P<0.05).Conclusions:Microbubble-enhanced ultrasound combined with percutaneous ethanol ablation can remarkably increase tumor necrosis. For long-term effects, the combined therapy could inhibit the growth rate of tumors and thus prolong the survival span of rabbits bearing tumors. This experiment might offer a new effective method for promoting PEA ablation. |
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