Enhancing Effects Of Microbubble Contrast Agent During Radiofrequency Ablation In Vivo

BackgroundRadiofrequency ablation (RFA) was introduced in the early19th century, but in recent years RFA has been continuously growing to be in popularity for the treatment of localized tumors in the liver, kidney, thyroid. As a new minimally invasive treatment for localized tumors, RFA has shown the greatest potential in experimental and clinical research owing to its low invasiveness, its simplicity, its favorable cost-effectiveness, and the potential to perform the procedure on an out-patient basis. Percutaneous thermal tissue ablation is performed with radiofrequency current. A needle is placed into the tumor with imaging guidance, in a fashion similar to a routine percutaneous biopsy. Alternating radiofrequency current agitates ions in the tissue surrounding the needle, creating frictional heat, which denatures and destroys tissue at predictable temperatures, in a relatively predictable volume.Ultrasonography guidance plays an important role in ablation therapy, being used for different purposes such as targeting the tumor, monitoring the ablative procedure and assessing treatment response. These functions are essential for a successful ablation therapy. The treatment area is monitored ultrasonographically for increasing echogenicity during the procedure. This increase in echogenicity corresponds to the formation of tissue and water vapor microbubbles from the heated tissue and is used to roughly estimate the boundaries of the treatment sphere. However, discrepancies between the spread of microbubbles and the ablated area were reported. This may be due to artefacts from gas bubbles formation during the ablation process, the gas bubble formation during the ablation process was irregular, varied in shape and the margins of hyperechogenic area were usually equivocal. The main objective of thermal ablation is to induce in situ coagulation necrosis resulting in a complete loss of microcirculation in the targeted tumor with a restrained damage to the surrounding normal tissues. To avoid damage to important organs (stomach intestine, gall bladder, bile duct, diaphragm, recurrent laryngeal nerve, carotid artery) in patients is major significance. Damage to the adjacent organs is not very rare in the clinical application that may put patients under risk. This complication can be avoided if the real-time observation of the ablated area is possible during the procedure.Microbubble contrast agents have been used for diagnosis of tumors, and as a tool in the follow-up process after radiofrequency ablation. Recent studies have shown that microbubble contrast agents, in addition to improving diagnostic sonographic images because of their acoustic properties, which can enhance backscattered signals, play an important role in enhancing the therapeutic efficiency of high in tensity focused ultrasound (HIFU). Microbubble contrast agents are able to improve the thermal and cavitation effects of HIFU by enlarging coagulated volumes and accelerating temperature increase. More studies showed that HIFU therapy efficiency raised with the increased of dosage of microbubble contrast agents. HIFU can destroy liver tissue by heating which is similar to RFA. Inspired by the applications of enhancement by microbubble contrast agents in HIFU, we speculated that the introduction of microbubble contrast agents during RFA may help the ablation in some way, perhaps as it facilitates HIFU. With a combination of microbubble contrast agents during RFA, by observing the area of hyperechogenicity, the damage to the adjacent vessels and organs could be reduced, and prediction of boundaries of ablated areas by the spread of microbubbles would be more reliable. However, few reports concentrated on RFA combined with microbubble contrast agents immediately after ablation have been published.ObjectiveIn this study, we investigated the effects of the sonographic microbubble contrast agent SonoVue (Bracco, Milan, Italy) on RFA via measurements of the coagulated volumes, hyperechogenicity formation during ablation, light microscopy with hematoxylin-eosin (H&E) staining and ablation time in animal experiments. The effects of dosage of microbubble contrast agent SonoVue on RFA were further studied. This method was also applied in the treatment of benign thyroid nodules to evaluate the safety and efficacy.Materials and methods1. Research object①Animals:Fifty-two New Zealand white rabbits weighing2.0to3.0kg were purchased from the Laboratory Animal Center of Nanfang Hospital. The experimental procedures in the study were approved by the Animal Care and Use Committee of the university.②Patients:A total of twenty-four benign thyroid nodules in24euthyroid patients were treated with US-guided RFA between April2012and December2012. The inclusion criteria of this study were as follows:fine-needle aspiration cytology and US findings were compatible with a benign nodule; there was not any sonographic criteria for a malignancy including ill-defined margins, microcalcifications and enlarged lymph nodes; the nodule was more than1.0cm in size; contrast-enhanced ultrasonography showed infusion of contrast agents in thyroid nodules. This study population consisted of8males and16females, aged29-65(mean age,(50.67±11.76) years). This study was approved by the institutional review board of the Nanfang Hospital, and written informed consent was obtained from all patients prior to US-guided fine-needle aspiration biopsy and RF ablation.2. Microbubble contrast agentThe microbubble contrast agent SonoVue (Bracco, Milan, Italy), including the active substance sulfur hexafluoride, was reconstituted according to the manufacturer’s instructions. The bubble concentration was in the range of1to5×108microbubbles/mL, with90%of the microbbules smaller than8μm in diameter.3. Devices for radiofrequency ablation and ultrasoundRadiofrequency ablation was applied percutaneously under real-time ultrasonic guidance (IU-22, Philips, Bothell, Washington, USA) using probe (L9-3). The RFA system (CelonENT, Celon AG medical instruments, Berlin, Germany) consisted of a generator supplying energy to an electrode in conjunction with a straight internally electrode needle antenna (18G, with a1-cm active tip). The output power was set to5W. The treatment continued until power "roll-off’occurred, indicating power output into the tissue reaching the pre-defined impedance threshold. When the ablation was completed, the noninsulated electrode would create theoretically elliptical lesion, the largest and a perpendicular diameters from the longitudinal view were lcm,0.6cm respectively according to the manufacturer’s instructions.4. Radiofrequency ablation protocol(1) Animal experiment:①Groups:RFA was performed with or without SonoVue on two areas per rabbit liver in20rabbits under ultrasound guidance. They were divided as group1and group2based on RFA with or without SonoVue. Another32rabbits were randomly assigned into four groups to investigate the enhancing effect of SonoVue of different dosage. RFA was performed once per rabbit liver. Group1,2and3received RFA combined with different doses of SonoVue, group4only received RFA. Group1,2, and3was O.lml/kg,0.2ml/lkg and0.3ml/kg respectively.㏑FA procedure:Each rabbit was anesthetized by injection of a3%pentobarbital solution (1mL/kg) by ear vein injections. After anesthesia, animals were placed with the supine position, and the abdomen was shaved. For each injection, SonoVue was injected intravenously through the auricular vein in a bolus, followed by a1-mL physiologic saline flush to ensure that no residual contrast agent remained in the intravenous catheter. The needle was inserted into the liver tissue under regular B-mode imaging, moreover, the needle was not adjacent to the extra-hepatic organs or large vessels. The RFA needle was kept at the same place throughout an ablation. In each rabbit, RFA was firstly performed without combination of SonoVue as a control to create a lesion in group2. In group1, before RFA, one injection of0.05ml/kg SonoVue was firstly given intravenously and RFA was conducted when microbubbles emerged in the rabbit liver to create a lesion in group1. Another3groups in different doses of SonoVue received RFA when microbubbles appeared in the rabbit liver, the control group received RFA without combination of SonoVue. The ablation time was recorded after completing the ablation. During the procedure of ablation, the spread of microbubbles was observed by conventional gray-scale US. Contrast-enhanced ultrasound was performed to evaluate the ablated area. After ablations, the whole liver was dissected and the tissue volume of coagulation necrosis was determined. Liver tissues were examined under light microscopy with H&E staining.(2) Clinical research:24patients were randomly divided into two groups, only one solid benign nodule was selected in every patient. Each group had12nodules. The first shot was put into experiment. Group I only received RFA, group II was performed RFA in combination with microbubble contrast agent SonoVue. The patients were treated with2%lidocaine for the local anesthesia of the puncture site and around the thyroid gland. The skin was incised by lmm. RFA was conducted when there was infusion of microbubble contrast agents in the nodule. The ablation time was recorded after completing the ablation. During the procedure of ablation, the spread of microbubbles was observed by conventional gray-scale US.5. Statistical analysisResults are shown as mean values±SD. The qualitative variables were tested for significance with chi-square test or Kruskal-Wallis H-test and where necessary with Fisher’s exact test. The statistical significance of quantitative data was assessed by one-way ANOVA. This was followed by multiple comparisons using LSD to identify where the differences lay. The correlation between hyperechogenic area and the size of ablation was analyzed by linear regression test. P<0.05was considered statistically significant. Data analysis was performed with statistical software (SPSS for Windows version13.0SPSS Inc. Chicago, IL, USA).Results1. Effects of microbubbles on RFAIn group1, after RFA assisted with SonoVue, an evenly increased echogenicity with clear boundaries from surrounding normal liver parenchyma was detected. The shape of hyperechogenicity was more easily observed in group1. In addition, the shapes were well matched between the spread of microbubbles and the defects in CEUS as observed on ultrasonography in group1. After ablation, the irregular ablated regions in group2were found with diffuse coarse hyperechogenicity and differentiated from unablated tissues, the margin of microbubbles was equivocal which was difficult to estimate the boundaries of the ablated area. The total number of ablations for clear visualized hyperechogenicity was15vs.7ablations for partial or not clearly visualized hyperechogenicity (P<0.05). The volumes of ablation areas closely correlated between the size of the area of hyperechogenicity and that of coagulation tissue in group1(r2=0.803), while in group2, the correlation was less strong. Measured by conventional gray-scale US, CE-US and pathology, there was no significant difference in volume of ablated regions between group1and group2(P>0.05). It was remarkable that the ablating time in group1was greatly reduced compared with that in group2(P<0.001). Pathological examiniation showed the irreversible necrosis of liver tissues in the ablation area in both groups.2. Effects of different doses of microbubble contrast agents on RFAMeasured by conventional gray-scale US and pathology, there was no significant difference in volume of ablated regions among4groups (P>0.05). The volumes of hyperechogenicity and coagulation area were not obviously changed with the increase of microbubble contrast agent (P>0.05). The ability of improving the visualization of hyperechogenicity was the same among different SonoVue groups (P>0.05). Under the light microscope, the morphologic characteristics of the coagulated tissue were not significantly different among4groups. RFA with different doses of SonoVue produced irreversible necrosis in all groups. The ablating time in different SonoVue groups was greatly reduced compared with that in control group, but the ablating time did not changed with the increase of SonoVue (P>0.05).3. RFA combined with microbubbles in treatment of benign thyroid noduleIn group Ⅱ, after RFA assisted with SonoVue, an evenly increased echogenicity with clear boundaries from surrounding normal liver parenchyma was detected. The shape of hyperechogenicity was more easily observed in group Ⅱ. In addition, the shapes were well matched between the spread of microbubbles and the defects in CEUS as observed on ultrasonography in group Ⅱ. After ablation, the irregular ablated regions in group Ⅰ were found with diffuse coarse hyperechogenicity and differentiated from unablated tissues, the margin of microbubbles was equivocal which was difficult to estimate the boundaries of the ablated area. The visualization of hyperechogenicity was better when RFA was conducted in combination with SonoVue (P=0.041). It was remarkable that the ablating time in group Ⅱ was greatly reduced compared with that in group2in achieving the same effects (P=0.020).Conclusions1. After RFA assisted with SonoVue, an evenly increased echogenicity with clear boundaries from surrounding normal liver parenchyma was detected. By observing the area of hyperechogenicity carefully, the damage to the adjacent vessels and organs could be reduced or even prevented if RFA was performed with microbubble agents.2. RFA assisted with microbubbles and RFA alone both produced irreversible necrosis in liver tissues. Moreover, there was no significant difference in the volume of ablated tissues.3. Hyperechoic area had good correlation with the ablated area in the presence of microbubble contrast agents. RFA combined with microbubble contrast agents is potentially a viable technique for delineating thermal lesions.4. The infusion of microbubble contrast agents resulted in a shorter ablating time for achieving the same effects in RFA, which may reduce the incidence of adverse effects.5. The improvement of visualization of the ablated area and shortened ablation time has close relation with the introduction of microbubble contrast agents during RFA, however, there was no confirmation that these effects were related to doses of microbubble contrast agents.