| BackgroundAs one of thermal ablation techniques, microwave ablation (MV) has beenwidely accepted and used for treatment of many kinds of tumors involving liver,lung, kidney and bone. Compared with other ablation techniques, the microwaveablation has several advantages such as larger ablation volumes, absence ofimpedance problem, simultaneously multiple applicators, and optimal heating ofcystic masses. Microwave ablation used clinically is usually based on not thetissue temperatures but the parameters of power and duration. Thus, not only thetemperature fluctuations occur and are difficult to control, but also the parametersare not uniform for the different tissue properties. Recently, with theimprovement of temperature measurement technology, quantitative real-timemonitoring of the ablation temperature has become true, which will make the application of the thermal ablation will be more safe, effective and precise. Forthe real-time adjustment of its power, temperature-controlled microwave canmaintain an ablative temperature, and will further promote the microwaveablation. Moreover, some reports considered microwave cell death (MCD)induced by microwave ablation in tissues is unique in that microscopicmorphology is well preserved, while enzymatic activity is lost. However, there isnot a definitively standerd and further study for the temperature-controlledmicrowave ablation.ObjectiveWe have designed three experiments to find the optimized ablation parametersof temperature-controlled microwave, and evaluate the detection method. We alsopreliminary revealed the devitalization mechanism.Methods1．We detected the heating efficacy of the MAS-II microwave workstation, anddecided the power up limit and the samples volume. We applied MV on humancervical carcinoma cell line HELA and human osteosarcoma cell line MG-63with differnt temperatures and durations ranged from40℃to60℃and5min to30min. After24hours incubation, we observed the cell survival changed bytemperature-controlled microwave through cell adherent ability and morphology,and using CCK-8cytoactive detection.2．We applied MV on human cervical carcinoma cell line HELA and humanosteosarcoma cell line MG-63with differnt temperatures and durations rangedfrom40℃to60℃and5min to30min. Immediately after MV, we observed thecell viability changed by temperature-controlled microwave using flow cytometerassay and Calcein-AM staining detection.3．We observed the HELA and MG-63cells devitalized by temperature- controlled microwave using NADH-diaphorase staining assay, TUNEL andelectron microscope detection. We eveluated the applyment of the NADH-diaphorase staining (detection method commonly uesed) in temperature-controlled microwave, and combined with the entire experimental resultspreliminary revealed the devitalization mechanism.Results1． The MAS-II microwave workstation has fast samples heating rate,repeatability, temperatures stability, and short time to response to the temperaturechanges. We set the microwave power parameters at0-300W automatic poweradjustment, and each liquid sample volume was30ml. After24hours incubation,two kinds of cells suffered from MV at45℃30min and45℃20min respectivelylose the adherent ability，and when the MV was at50℃25min and50℃20minrespectively the metabolism stopped detected by CCK-8cytoactive detection.2．HELA and MG-63cells suffered from temperature-controlled microwavewere devitalized at55℃15min and55℃5min respectively，immediately assayedafter microwave treatment by flow cytometer and calcein-AM staining detection.3．HELA and MG-63cells suffered from temperature-controlled microwave at55℃15min and55℃5min respectively，manifested the NADH-diaphoraseinactivation detected by NADH-diaphoras staining, and normal cells’morphologyand negative nuclear staining by TUNEL. The morphologic features werevacuolization，cytoplasm lost, electron density decreased, rounding-up of nuclear,nuclear heterochromatin and margination, few of mitochondria and endoplasmicreticulum, secondary lysosomes increased, and increased disorder microvillidetected by electron microscop.ConclusionsThe temperature-controlled microwave can devitalize HELA and MG-63 tumor cells, and the immediate threshold is55℃15min and55℃5min. TheNADH-diaphorase staining can be used to detect the efficacy of thetemperature-controlled microwave and the cells devitalized can manifestmorphology and enzymes changes in accordance with microwave cell death(MCD). However, the ultrastructures can change apparently. The detailedmechanism needs further research. |
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