| Preface When the liver tumors are inoperable due to various reasons, tumor thermal ablation that destroys the tumor in situ and spares the hepatic tissue is considered to be the most suitable treatment option. It is used most extensively in liver tumors and the therapeutic effect is positive. Thermal-physical treatment of tumor has many advantages, but the freezing or heating treatment alone has some limitations. The disadvantage of cryotherapy is the high local recurrence rate at the cryo-site after cryoablation. In hepatic cryosurgery, the potential complication of large-volume tissue freezing is systemic response in the nature of multiorgan failure, which has been known as “cryoshock”. On the other hand, there are several defects with regard to thermotherapy, such as unexpected residual viable tissue because of the heat-sinking effect and the risk of stimulating metastasis from the primary site induced by heating. Taking both of the advantages and disadvantages of cryosurgery and thermotherapy into consideration, researchers propose a new thermal technique which combines the cryosurgery and radiofrequency ablation(RFA) to overcome the disadvantages of each treatment alone and improve the curative effects while minimizing damage to the surrounding healthy tissue. When the alternating cooling and heating treatment is applied to the tumor, it experiences greater thermal stress within a short time than cooling or heating treatment alone. On the other side, vascular injury and blood stagnation induced by cryotherapy might prevent tumor cells from metastasis. But there is no study in the treatment of liver tumors. In this paper, we evaluate the anticancer effects of the alternating freezing and heating therapy in established transplanted rabbit VX2 hepatic tumor model by using contrast enhanced ultrasound(CEUS) and contrast enhanced computed tomography(CECT).Part One Experimental Study of the Effectiveness of Alternate Cooling and Heating Ablation in Normal Rabbit LiverObjective To investigate the safe and effective protocol and reliable estimated methods of alternate cooling and heating ablation in normal rabbit liver, and provide the foundation for the alternate cooling and heating treatment of liver tumors in vivo.Materials and Methods Control group and ablation group were established. According to the various critical temperature of the edge, the rabbits in the ablation group were divided into heating alone group, cooling alone group and alternative cooling and heating group. The effectiveness of the different ablation modalities were evaluated through HE staining immediately after ablation and arrive at the effective ablation condition of liver tissue to maximize the protection of the surrounding liver parenchyma. Under this condition, twenty normal rabbits underwent alternated cooling and heating ablation in livers and rabbits were monitored with US and CT at 1, 3, 7 and 14 days after ablation to observe the dynamic changes of the ablated lesions. Five rabbits were sacrificed randomly to observe the pathological changes of the ablation lesions after performing examination at corresponding time points. The maximum lesion size measured on CEUS and CECT was then compared with the histopathologically determined lesion size.Results Separate cooling, separate heating and alternative cooling and heating ablation could cause liver cell degeneration, hyperemia and hemorrhage of different degrees. Compared with separate hyperthermia or cryosurgery, alternating freezing and heating ablation could lead to more serious injury. The ablation effectiveness varied from the critical temperature of the edge. Lower the freezing temperature or higher the heating temperature, the greater the scope of the ablation zone and the ablation effect of tissue was more obvious. A mild cooling and heating ablation mode(0℃/43℃) was safe and effective. Within one week after the ablation, congestion and edema of tissue and the acute inflammatory cells infiltration were seen surrounding the central coagulation necrosis. The plain CT scan showed hypodense or isodense,while routine ultrasound showed mixed echo area mainly hypoechoic. CECT and CEUS showed filling defect of contrast media in the ablated areas which were surrounded by ring enhancement on arterial phase. At 7 to 14 days after ablation, congestion and edema around the necrotic tissue improved, and the ablation lesions also showed extensive coagulative necrosis and were surrounded by chronic inflammatory cells infiltration, fibroplasia and calcification. Both enhanced performance showed ring enhancement gradually reduced until it disappeared. Follow-up imaging displayed that ablation areas shrunk correlating with time and fibrous repair. CEUS and CECT may reveal the shape and extent of ablated lesion. The lesion sizes measured with CEUS, CECT and liver specimens were well correlated(P>0.05).Conclusions The alternate cooling and heating may cause much more serious damage in rabbit liver. Mild alternated cooling and heating ablation(0℃/43℃) in normal rabbit liver could produce coagulation necrosis. CECT and CEUS imaging could well reflect the pathological changes of the liver lesions in the different periods and accurately demonstrate the range of the ablated lesions, which could be used to evaluate the therapeutic effect of alternated cooling and heating treatment in liver tumors.Part Two Therapeutic Effect and Imaging Evaluation of the Alternate Cooling and Heating Treatment in the VX2 Rabbit Liver Tumor ModelObjective To estimate survival after alternate cooling and heating treatment in the VX2 rabbit liver tumor model, and to explore the role of CEUS and CECT in the evaluation of the therapeutic effect.Materials and Methods In this study, the VX2 liver tumor model was established by implanting the VX2 carcinoma into the liver of the rabbit. Conventional ultrasonography was used to monitor the growth of the tumor, which was important for determining the experiment time. On sixteenth day after implantation, ten tumor-bearing rabbits were treated with alternated cooling and heating treatment after we reviewed their imaging features on US and CT. Ten untreated tumor-bearing animals served as the control group. All rabbits were monitored for 6 months after treatment and observed the progression of ablated lesions with US and CT at 1, 7, 14 and 30 days posttreatment to assess the treatment effectiveness. Necroscopy was executed at the end of the experiment or at the time of death. Survival analyses were performed using the Kaplan-Meier method.Results Ten animals came from the alternating treatment group, with a mean survival period of 156.5 days. Alternating freezing and heating treatment resulted in a good recovery of VX2 rabbits, and the weight of rabbits successfully cured was significantly increased(P<0.05). Ten tumor-bearing animals of the control group were not treated with alternate cooling and heating. Their mean survival period was 59.7 days and all ten rabbits died of tumor progression. Compared with the controls, rabbits in the treatment group lived significantly longer(P<0.05). Seven of ten(70%) animals in the treatment group showed complete elimination and survived to the defined end point of six months without metastasis and recurrence, but rabbits in the control group did not reach the scheduled end point. One day after treatment, the volume of ablation lesions were larger than those of the pre-treatment tumors(P<0.05). A gradual reduction of the lesion’s size was found at 14 and 30 days in treated rabbits(P<0.05). All rabbits in the control group developed metastasis, while only three of ten treated rabbits(30%) did so(P<0.05). CEUS and CECT may reveal the shape and extent of ablated lesion. Complete ablation of the tumor demonstrated no contrast-enhancement. Residual tumor was performance as nodular or irregular thick ring enhancement on CEUS and CECT.Conclusions Alternated cooling and heating treatment can inhibit tumor growth and metastasis and prolong the survival time of the rabbit with VX2 liver tumor. It is an effective method for tumor therapy. Both CEUS and CECT are suitable for the accurate evaluation of the ablated lesions. CEUS has the potential superiority in assessment of treatment effectiveness because it can reveal the variation of flow perfusion in real time.Part Three Experimental Research of the Anti-tumor Effects Induced by Alternate Cooling and Heating Treatmenttreatment in the VX2 rabbit liver tumor. Objective To investigate the biological effects of alternate cooling and heatingMaterials and Methods Tumor-bearing rabbits were randomly divided into treatment group and control group. Alternate cooling and heating treatment was performed in VX2 liver tumor model. We observed the therapeutic results using CEUS. Three rabbits were sacrificed and the livers specimens were harvested for pathologic analysis before treatment, immediately after treatment and at 1, 3, 7 and 14 days posttreatment respectively. The tumors and ablated lesions were observed with HE staining and TUNEL staining at different time. HSP70 expression was detected by immunohistochemistry staining. Pathological changes such as tumor cell necrosis, apoptosis and HSP70 expression were observed with an optical microscope.Results HE staining showed tumor cell necrosis and hyperemia and hemorrhage of the ablation lesions immediately after alternate cooling and heating treatment. But in the ablation region some tumor cells were still alive. On the first day and third day after treatment, necrosis range was gradually increased and the tumor cells became smaller, fewer and tend to death. The acute inflammatory cells infiltration was seen surrounding the central coagulation necrosis. At 7 and 14 days after ablation, ablation lesions showed extensive coagulative necrosis and were surrounded by chronic inflammatory cells infiltration such as multinucleated giant cells and lymphocyte. It also showed formation of fibroplasia and calcification. TUNEL staining showed that apoptosis of tumor cells was mainly located in the edge of the ablation lesions. There was no obvious apoptosis immediately after treatment. One day after treatment, tumor cell apoptosis was apparente and apoptotic index reached its peak. Three days after treatment, there were still apoptotic cells in the ablation lesions and apoptotic cells gradually decreased to 14 days after treatment. Immediately after treatment, HSP70 expression in the nuclei of tumor cells was obvious. One day later, the expression in the nuclear drastically reduced, and gradually increased in the cytoplasm. HSP70 expression in the cytoplasm was significantly increased on day 7 and dropped on day 14 in the ablation region. Alternate cooling and heating treatment induced greater HSP70 expression than control group.Conclusions Alternate cooling and heating treatment of VX2 rabbit liver tumor resulted in tumor necrosis. Direct tumor-killing effect of alternated cooling and heating was not the only treatment factor. It could trigger inflammatory response of the ablated region, which maybe play an important role in inducing immune response of the body. It could also induce tumor cell apoptosis and increase expression of HSP70 in the ablation zone. |
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