Treatment Of VX2 Hepatic Tumor In Rabbits With Radiofrequency Ablation: A Study Of Ghost Cells In Ablation Zones

1. BackgroundThe surgical resection is considered to be the most promising first choice therapeutic option for many malignant tumors in human, but, only about 20 percent of the patients with hepatic carcinoma are eligible for surgical resection because of various reasons, such as severe impairment of hepatic functional reserve, extrahepatic metastasis, involvement of the portal vein, severe extrahepatic disease and so on[1-4]. Many patients have to accept other non-surgical treatments. Therefore, some local ablative therapies have been hot spot,for instance, percutaneous ethanol injection (PEI), percutaneous acetic acid injection (PAI), radiofrequency ablation(RFA), cryoablation therapy(CST), microwave coagulation therapy(MCT), interstitial laser photocoagulation (ILP). Among them, RFA has gained the widest acceptance because of its minimal invasion, safety, effectiveness, repeatability in local therapy for primary and secondary liver malignancies [4-9].Radiofrequency ablation technique has been used to treat liver tumor since the early 1990s, at RFA, high-frequency alternating current (350–500 kHz) is used to generate frictional heat energy with ionic vibration within the tumor cells which results in the final destruction of the tumor tissue. At temperatures of greater than 60°C, there is denaturation of intracellular protein and lipid bilayers, which leads to irreversible cell death. Even at lower temperatures (50°–55°C), irreversible cell damage can be achieved with the process of ablation for 4–6 minutes[10].If temperature is not high enough to cause necrosis,another pattern of cell necrosis,i.e.,apoptosis may occur[11].Moreover,tumor tissue inactivated under high temperature may act as tumor vaccine as a result of altered cell immunophenotype,thus further triggering the antitumor immunity and exerting special antitumor effect.In addition,blood vessels in tumor are circuitous and irregular,with slow blood flow within,and are lack of innervation;therefore after heating of liver tumor,heat loses slowly,and temperature rises more quickly in the tumor than in normal tissues.Meanwhile,intracellular acidic metabolites exhibit stronger lethal effect on tumor cells after heating than before heating,which further enhances the effect of thermotherapy on tumor[12,13].Although RFA has been widely used to treat various hepatic tumors and has got good effects, Unfortunately, incomplete ablation often are caused, the remnant tumors are produced , due to the size, number and anatomic distribution of tumor nodules and other factors . In the past years,pathology examination had been considered the most scientific method on evaluate the therapeutic efficacy,but further research found that hematoxylin-eosin-stain could not scientific evaluate the necrosis range in ablation area. In a study by Miao[14] et al, a"wet"electrode with interstitial infusion of hypertonic saline was tested for the RFA of liver tumor in rabbits. They studied the ablated lesions from hours to days after RFA therapy, the ablated tissues appeared almost unchanged microscopically,they called"ghost"phenomenon. Although being not yet widely known, this ghost phenomenon occurs following most of the thermotherapies as a consequence of sudden tissue coagulation or fixation. The ghost phenomenon is normally found in the large area of the lesion peripheral to the central electrode track. At the hematoxylin-eosin staining, the ablated nonviable tissues were stained only slightly lighter than viable tissues.But the suddenly ablated tissues appeared almost intact and constitute, which should be more carefully differentiated from unablated viable tissues. Failure in this respect may cause misevaluation of the therapy[15,16].In the past years,foreign researchers only reported this ghost cell phenomenon at the hematoxylin-eosin staining[14,17,18],they did not made deeper research.After all, how long the ghost cell existence? What time ghost cell vanish in the ablation zones? What significance of ghost cell existence? At present no system research has been reported. Foreign researchers usually evaluate ablation zones after RFA by histochemical stain,they confirmed that this method is characteristic and effective to evaluate the necrosis range[15,18-23]. Resort to this method can discriminate the ghost cells from remnant tumor , it also provide possibility for the study of ghost cells. 2. ObjectivesBased on the animal test, this study is designed to observe the changes and existence of ghost cells in rabbit hepatic VX2 tumor model after treatment with radiofrequency ablation .3. Methods and Results(1)Establish the liver model of VX2 tumor lines in rabbits The VX2 tumor tissue was minced into 1mm×1mm×1mm cubes[24], and transplanted into the livers of 48 New Zealand rabbits.After 2 weeks the size and growth of the tumor were observed. Results:①All of the animals were survivaled after operation,the occurrence rate of tumor was 100%,the tumors were located in the left liver lobes,two animals were observed tumor metastasize in the liver.②The VX2 tumors seemed to assume similar spherical,no obviously peplos, protrude the surface of liver, the diameter is about 1.5cm,there was obvious gray necrosis in the center in part of the tumor.③Observed with light microscope,the tumor cells were arranged irregularly with invasive growth potential.④Many tumors metastasized could be observed in the liver,lung and enterocoelia lymphonodes on the postoperative of 3 weeks.(2) Radiofrequency ablation in vivo rabbit livers to explore radiofrequency control conditionsMany studies indicated that at temperatures of greater than 60°C could lead to irreversible cell death, which can ensure the ablation thoroughly[18,19].The treatment were established in vivo liver of 4 New Zealand rabbits, to observe ablation zones at temperatures of greater than 60°C with identical treatment power and different electrode expanse diameters. Results:①The treatment power was 20W, 12 ablation zones were generated after the treatment.②At 6mm,8mm and 10mm of electrode expanse diameters,average diameter of ablation zones was 9.75mm,13.5mm,16.5mm,the area at temperatures of greater than 60°C was 14.75mm,18.75mm,21.5mm。③At the same electrode expanse diameter,the diameter of ablation zones are smaller than the diameter of temperatures greater than 60°C areas.(3)Treatment of VX2 hepatic tumor in rabbits with radiofrequency ablation :a study of ghost cells in ablation zonesRFA was applied by controlling the ablation condition with the rabbit hepatic tumor models which were established with VX2 tumor at early time. The tissue samples were cut at 0,1,2,4,8,12 weeks after treatment, the hematoxylin-eosin-stained specimens and NADH vital stained specimens were obtained from the close section,to observe the changes and existence of ghost cells. Results:①The NADH staining showed nonviable tissue in ablation areas,the ghost cells can be observed in the ablation zones between hyperraemia zone of ablation perimeter area and the center charring area of elecrtode track,the number of ghost cells are parum and distributed with focus shape.②The morphous of ghost cells in ablation zones was to maintain at least four weeks after RFA.After 12 weeks, the appearance of ghost cells would vanished.4. Main Conclusions(1)The metastasis model of VX2 tumor lines established in rabbits has such characteristics as easy reproduction, short period of tumor occurrence, high success rate, high stability. It is a perfect metastasis model in vivo established in big animal with normal immune system.(2) Radiofrequency ablation in vivo rabbit livers to explore radiofrequency control conditions is considerable significance for the post treatment of VX2 tumor in rabbit livers, it provid scientific evidence for ensure the ablation thoroughly.(3) The ghost cells can be observed in the ablation zones between hyperraemia zone of ablation perimeter area and the center charring area of elecrtode track,the number of ghost cells are parum and distributed with focus shape.(4) The morphous of ghost cells in ablation zones was to maintain at least four weeks after RFA, the whole structure of cells become blured 8 weeks later, after 12 weeks, the appearance of ghost cells would vanished.(5) Hematoxylin-eosin-stain is not reliable for the judgement of therapeutic efficacy in ablation area after RFA, in order to avoid erroneous judgement it must add NADH stain.