Effect Of High Intensity Focused Ultrasound Ablation On Metastasis Of Human Neuroblastoma In Animal Model

PART ONE:ESTABLISHMENT OF HUMAN NEUROBLASTOMA MODEL IN INFANCY SD RATObjective:To establish model in infant SD rats’adrenal area by injecting human neuroblastoma cells (human SK-N-SH cell lines) under euthyphoria, which are orthotopic cell transplantation model that can reflect the clinical appearance of human neuroblastoma, thus to provide the experimental animal model for the following study.Methods:A humen neuroblastoma cell (human SK-N-SH cell line) was cultured’in vitro.(1～3)×10(7) cells undergoing exponential growth were collected in0.2ml of suspension and injected into the left adrenal area in infant SD rat under euthyphoria to establish a orthotopic transplantation tumor model. Since5days after transplantation, SD rats were given B ultrasound and Doppler color flow imagines examination at different time points. The ultrasonographic images were observed, whereas mean tumor volumes were calculated and the growth curve was drawn synchronously. The SD rats were randomly selected at7day after transplantation, sacrificed after anesthesia for gross anatomy and pathological examination. The morphology of the tumor tissue was observed, the expressions of NSE in the tumor were analyzed by immunohistochemisty. The rate of successful model establishment was calculated. The experimental procedure was accorded wieh Animal Ethical Standards. All observed date were calculated by using Kaplan-Meier method, Student’s t test.etc respectively. Differences were considered statistically significant when the P value was less than0.05.Results:The successful rate of model was100%(35/35), there were two rats with widespread metastasis. Tumor had been showed and located accurately by B ultrasound imaging since5days after transplantation. The blood supply surrounding tumor focus was showed abundant by Doppler color flow imaging. According to the B ultrasound examination, at9-12days after transplantation, tumor growed quickly; after17th day, tumor become reduced in volum. Natural extinction was found in two rats after transplantation from30th day, and others regressed after3months. Histopathological observation suggested tumor cell showed disorder and nest, neoplastic cells with round, oval or irregular shape, and with many karyokinesis. The positive expression rate of NSE in the tumor tissues was100%. Human neuroblastoma orthotopic model within the infant SD rats’ adrenal area is established successfully, the tumor modeling time is short and tumor grows with abundant blood supply. This model has the advantages of simplicity, stability and high success rate.It lays a good foundation for the study on which the biological characteristics of tumor in body with nomal immune system. It is also an economic and ideal experimental model which will contribute to the physical therapeutic research for adrenal area tumor, such as neuroblastoma. PART TWO:EFFECT OF HIGH INTENSITY FOCUSED ULTRASOUND ABLATION ON METASTASIS IN INFANCY SD RAT BEARING HUMAN NEUROBLASTOMA IN THE ADRENAL AREAObjective:To investigate the effect and associsted mechanism of high-intensity focused ultrasound ablation on metastasis in infant SD rat bearing human neuroblastoma.Methods:Infant SD rats baring human neuroblastoma were divided into two groups at random:HIFU-treat group and surgey group, meanwhile establish normal control group. Treated tumor and the status of metastasis were observed by B ultrasound and Doppler color flow imaging examination at3,7,14,21and28days aftertreatment. At3,14,28days after treatment, cenral tissues (tumor tissues by HIFU ablation), marginal tissues (skin and muscle tissues), and other adjacent organs (kidney, adrenal, spleen, liver, bowel, lung, etc) were examined by gross anatomy and light microscope with HE stain. At3,7,14,21and28days after treatment, immune changes and the expression of VEGF in blood serum after HIFU ablation were tested in infant SD rat model.Results:Significance increase of recovery rate in HIFU-treat group was fond compared with surgery group (88.9%vs.48.9%, P<0.01).Compared with the surgery group, there was a significance decrease of metastatic rate in HIFU-treat group according to observation through gross anatomy and light microscope with HE stain. The metastatic rate was6.7%(1/15) vs.46.7%(7/15),20%(3/15) vs.60%(9/15) and0(0/15) vs.46.7(7/15) respectively at3d,2w,4w after treatment. In HIFU-treat group, CDFI did not find blood signal in all treated target regions. And B-ultrasound showed echo free zone with irregular shape and size or clustered high-level echo calcification was observed in tumor on the3-7days after HIFU treatment. The metastasis was found in4rats of HIFU-treat group and20rats of the surgery group, the difference between these two groups was significant (P<0.01). The results showed a significance increase in the peripheral blood of CD3+、CD4+lymphocytes (P<0.05) and the ratios of CD4+/CD8+(P<0.05) in infancy SD rats bearing human neuroblastoma after HIFU treatment, but no statistical differences were found pre-and postoperatively (P>0.05). Compared to the values in either surgery group, there were a significant increase of CD3+、 CD4+level(P<0.05) and CD4+/CD8+ratios(P<0.05), and a significant decrease of CD8+level(P<0.05) in HIFU-treat group and normal control group, but compared with the values in either HIFU-treat group or normal control group, there was no significant difference of CD3+、CD4+、CD8+level and CD4+/CD8+ratios(P>0.05). There was a significance decrease of serum-VEGF concentrations in infancy SD rats bearing human neuroblastoma after HIFU treatment compared with that before treatment (P<0.01), and the levels in the HIFU group were all lower than those in the surgery group at day3,14, and28after treatment respectively, and there was statistical difference at day14and day28(P<0.01). There was a decreasing tendency of serum-VEGF concentration in SD rats after surgery compared with those before treatment, and there was a significant decrease at-day14(P<0.05). Compared with the values in normal control group, there was a significant increase of serum-VEGF concentration in HIFU group and surgery group both before treatment (P<0.01), and after operation there were also increased serum-VEGF concentration in HIFU group (at day3, P<0.01) and surgery group (P<0.01).Conclusion:The expression of VEGF is suppressed in the peripheral blood and the cell-mediated immune function is improved after high-intensity focused ultrasound ablation in the infancy SD rats bearing human neuroblastoma, these may inhibit the tumor growth and metastasis, and offer the basis for HIFU ablition children neuroblastoma in clinical practice.