The Study On The Impact Of Focused Ultrasound On The Ethanol Ablation Of Liver Tumors In Rabbits

Background:As a local interventional therapy,percutaneous ethanol ablation(PEA)has been practiced in the clinic since 1983 and has been widely used to treat small hepatocellular carcinoma(HCC).PEA is a safe and effective method with negligible complications and was considered the primary percutaneous treatment for HCC in the early1990s.HCC is highly vascularized with abundant nourishing blood vessels that flush ethanol out,leading to decreased local concentration and shorter residence time of ethanol.Moreover,liver cirrhosis is frequently present as a comorbidity with its multiple fiber septa constraining ethanol diffusion and limiting the ablation area and necrosis rate for HCC.With emerging technologies,PEA is now inferior to radiofrequency ablation(RFA)and microwave ablation(MWA)in treating HCC.Even so,PEA is not irrelevant;this method still plays a role in the treatment of some HCCs with difficult locations,such as those adjacent to the hepatic hilum,diaphragm,or bile ducts,which is a relative contraindication to thermal ablation.Therefore,improving local ethanol concentration and obtaining a larger ablation volume is a problem to be solved.There are various studies showing that there is no difference between surgery and ablative therapies in small HCC with diameter fewer than 2cm in terms of long-term survival.More researches have combined ethanol ablation with transhepatic arterial chemotherapy and embolization(TACE),radiofrequency ablation or microwave ablation to achieve an improvement in both ablative effect and patient survival.The basic principle for these studies is to reduce the wash-out effect on ethanol brought by other therapies and thus reinforce ablative efficacy.However,these combined treatment can increase trauma,treatment duration and financial burden,without changing the indications to ethanol ablation.High-intensity focused ultrasound(HIFU)is a relatively non-invasive therapeutic method,and HIFU exposure does not increase the metastasis of tumors.Several studies have found thatwhen HIFU causes damage to tissues,both vaporization and cavitation bring about the formation of microbubble clouds.There are also studies that have found by injecting ethanol the local cavitation threshold could be decreased due to the relatively low boiling point of ethanol.Acoustic cavitation and thermal effects are the two major physical effects of HIFU,and both of these effects can effectively vaporize the ethanol injected into the HIFU focus because ethanol has a low boiling point of 78.3�C and a relatively low cavitation threshold.Previous studies have found by injecting ethanol into liver tissue,the cavitation threshold in the HIFU-targeted area could be obviously decreased,thus reinforcing the cavitating activities during the HIFU exposure,increasing the temperature of the focus and shortening the treatment duration.In light of this,we assumed that once ethanol is vaporized by HIFU,a bubble cloud may form and is likely to be trapped in local tissues and thus,the ethanol bubbles cannot be easily washed away by the tumor or liver bloodstream.In that case,ethanol could probably accumulate locally with a higher concentration,and a greater ablation zone might be created.In addition,ethanol,as cavitation nuclei,could increase ultrasonic cavitation which causes improved local tissue permeably and ethanol diffusion,resulting in a larger ablation area.In this study,we attempted to improve EA using focused US(FUS),which is non-invasive and coupling cone focused,with a lower intensity than HIFU.Objective:1.The objective of this study is to investigate the efficacy and safety of the combined treatment by simultaneously injecting ethanol and applying FUS in rabbit liver;to testify the enlarged ablation volume caused by ethanol vaporization in the combined treatment.2.Based on the experiment above,to verify the ablating efficacy of the combined therapy in VX2 liver tumors in rabbits.Materials and Methods:1.Experimental apparatus:(1)Commercial therapeutic ultrasound equipment(CZ180A,Sonic Electronic Ltd,Mianyang,China).The air-backed transducer was built with a plane circular ring disk with a25 mm outer diameter and 5 mm inner diameter and was operated with a frequency of 1.0MHz and a duty cycle of 50%.A plastic coupling cone to guide the self-focusing wave was set on the front end of the transducer,and a pumped circulating water device was placed inside the machine,providing degassed water for acoustic coupling and heat dissipation.A needle hydrophone(HNA-0400,Onda Corporation)adjusted by a precision 3-D motion stage was set up in water to measure the acoustic output of the transducer at a range of 10 mm outside the tip.The results showed that the peak negative pressure was 1.4 MPa�15%,and the corresponding actual acoustic intensity(I_SPTA)was 33.0 W/cm²�20%.(2)VINNO 70 Color Doppler Ultrasound system(manufactured by Vinno Technology Co.Ltd,Suzhou,China)with a high frequency linear array probe(X4-12L)was operated at a frequency range of 4?12 MHz.CEUS was performed using a low mechanical index setting.(3)AZ8856 Dual channel digital thermometer(Heng Xin Technologies Co.Ltd,Taiwan,China),with a temperature monitoring range of-200?1760?.(4)WRT-MI Miniature needle type temperature sensor(Shenggao Measuring&Controlling Technology Co.Ltd,Guangzhou,China),with a temperature monitoring range of-50?125?.The needle has a length of 100 mm and a diameter 0.6 mm.2.Experimental reagentsAbsolute ethanol(Chongqing Chuandong Chemical Co.Ltd.),concentration(CH₃CH₂OH)?99.7%.Sonazoid?,a phospholipid-coated perfluorobutane microbubbles(GE Healthcare,Oslo,Norway)were used both as an ultrasound contrast agent and as cavitation nuclei.Microbubble(MB)suspension was made by diluting 16?l of microbubbles drawn from vial to 4 ml saline solution.The MB had a mean diameter of 2.1?m and concentration of 6�108/ml.3.Experimental animals:Fifty-nine healthy New Zealand white rabbits of either sex,aged 3�6 months and weighing 2.0�2.5 kg,were provided by the Laboratory Animal Center of the Second Affiliated Hospital of Army Medical University.Thirty-nine rabbits were involved in the experiment on efficacy and safety of HIFU combining ethanol ablation in liver;VX2 liver tumor model was established in other 20 rabbits.4.Experimental methods:Experiment 1:The safety and efficacy of focused ultrasound on the ethanol ablation of rabbit liver.For the treatment of normal liver,39 rabbits were divided randomly into three groups to receive either FUS alone(n=12),ethanol ablation(EA)alone(n=12),or EA+FUS(n=12)combination treatment,and a control group(n=3)underwent upper abdominal open surgery only.In the FUS group,the central region of the middle and right lobes was only exposed to FUS for 20 s.In the EA group,0.2 m L absolute ethanol was slowly injected(approximately20 s)into the same target site at a depth of approximately 10 mm with a fine needle(21 G�180 mm,PEIT needle,Hakko Co.Ltd.)under 2-D US guidance.For EA+FUS combination therapy,the ethanol injection was given while the injection point was simultaneously and exactly exposed to FUS for 20 s(equal to the injection time).Blood samples of three rabbits randomly selected from each of the FUS,EA,and EA+FUS groups(n=3)and the blank control group were collected to test liver function for 7consecutive days.Forty-eight hours after treatment in the remaining rabbits in the FUS,one rabbit was selected for histological examination,and the remaining 8 rabbits again received FUS treatment with temperature measurements for 60 s.All of the treated liver lobes in the EA group and EA+FUS group were collected,one was selected from each group for histological examination,and then all of the liver lobes were harvested to calculate the ablation volume.One liver sample of each group was fixed in 10%formaldehyde,embedded in paraffin,sectioned,and stained with haematoxylin and eosin(H&E)for light microscopyExperiment 2:Impact of focused ultrasound on the ethanol ablation of VX2 liver tumors in rabbits.Implantation of a solid VX2 tumor in the liver was carried out in 20 rabbits.Vital VX2tumor samples harvested from a carrier rabbit were cut into tissue cubes approximately 1 mm³in size.Then,the cubic tumor samples were implanted into the central region of the liver via an 18-gauge core needle under ultrasound guidance.Finally,the abdomen was carefully closed.Penicillin was administered at 800,000 IU per day for the following 3 days.When the resulting tumors reached a size of approximately 10�15 mm in diameter,the rabbits were randomly distributed to different groups.20 tumor-bearing animals were divided randomly into two groups to receive either EA alone(n=10)or EA+FUS(n=10)combination treatment.Before treatment,all rabbits received CEUS to evaluate the baseline tumor perfusion and determine the tumor volume.Then,during the Kupffer phase of Sonazoid-CEUS,the maximum section of the perfusion defect was found,and the length(L),height(H),and width(W)were measured;these three diameters are perpendicular to each other.To approximately calculate the tumor volume(V),the following ellipsoidal formula was used:V=?LWH/6.The tumor was scanned,and a maximal section of the perfusion defect was found.Then,the needle was inserted into the central region of the tumor.For the tumors in the EA group,0.3 m L ethanol was slowly injected,and in the EA+FUS group,tumors received a 0.3 m L ethanol injection and were synchronously exposed to FUS for 20 s.Forty-eight hours after treatment,all the treated liver lobes were harvested.One liver lobe in each group was randomly selected for gross observation.Then,the tumors were separated from the remaining liver lobes for histological examination.All histological examination samples were fixed in 10%formaldehyde for 24 h,embedded in paraffin,sectioned,and stained with haematoxylin and eosin(H&E)for light microscopy.The entire tumor area and residual tumor area of each section were manually traced and calculated by Image-Pro Plus software(Media Cybernetics,Inc.)for tumor necrosis rate.Results:Experiment 1:The safety and efficacy of focused ultrasound on the ethanol ablation of rabbit liver.(1)In the EA+FUS group,a dense hyperechoic bubble cloud of alcohol vaporisation could be seen in the target region after treatment,and48 h later,the bubble cloud was obviously reduced,while only a mild and small bright area around the needle tip was seen after EA injection.CEUS showed an irregular perfusion defect with scattered peripheral lesions in the EA group,and a larger and more spherical perfusion defect formed in the EA+FUS group.For FUS-treated livers,no obvious changes were observed on2-D or CEUS.(2)The ALT levels peaked at 24 h and then gradually dropped to baseline levels at 7 days after treatment in every group.The AST levels peaked immediately after treatment and then gradually dropped to the baseline level 72 h after treatment in every group.There were no significant differences in the ALT and AST levels between the FUS group and the control group at any time point(p>0.05),and no significant differences were found between the EA group and the EA+FUS group(p>0.05).(3)The average temperature of the target area at the beginning was 35.96�0.96�C.The temperature rose to a mean peak value of 44.93�1.67�C after FUS treatment(thermal dose<240 CEM43�C)and then gradually decreased.(4)The mean ablation liver volume induced by EA+FUS combination therapy(1.46�0.30 cm³)was approximately 3 times the volume induced by EA alone(0.51�0.17 cm³,p<0.001).(5)The necrotic area was small,irregular,and scattered when treated with EA alone,while in the EA+FUS-treated liver,the area was relatively larger and had an elliptical shape.There were no visible necrotic areas in livers treated by FUS alone.Experiment 2:Impact of focused ultrasound on the ethanol ablation of VX2 liver tumors in rabbits.(1)A dense hyperechoic bubble cloud with a similar shape to the tumor was observed immediately after EA+FUS treatment.Forty-eight hours later,the CEUS imaging showed a round-shaped perfusion defect in EA+FUS group,but peripheral blood perfusion could be frequently observed in EA group.(2)Before the ablation of VX2 liver tumors,the average tumor volumes measured by CEUS were not significantly different between the EA group(0.67�0.19 cm3)and the EA+FUS group(0.71�0.18 cm3).The average necrosis rate of the tumors was 90.27�4.59%in the EA+FUS group,which was significantly higher than that of the EA group(63.55�8.06%,p<0.001).Conclusion:(1)Ethanol ablation combined with FUS could significantly increase ablation effect by achieving a larger ablation volume and a more regular ablation area in rabbit liver without additional liver damage.This combining therapy had the potential to reach complete response.The efficacy and safety of the combined therapy was verified.(2)Ethanol ablation combined with FUS could significantly increase necrosis rate in VX2 liver tumor in rabbit.The present research proved the effect of the combined therapy in enhancing ethanol ablation in liver tumors,which provided a novel,simple and non-invasive method for optimizing the traditional ethanol ablation.