The Improvement Of Catheterization Technique Of VX2 Rabbit Hepatoma Model And Its Experimental Study After Interventional Therapy

BackgroundHepatocellular carcinoma(HCC) is one of the common malignant tumors in China, especially in the southeast coast area. At least 130 000 people died of this disease every year. It is ranked in the second among the incidence of all kinds of the malignant. Although transcatheter arterial chemoembolization(TACE) has increasingly become the leading non-surgical method for treating HCC and great progress has been achieved in recent 20 years, tumor recurrence and metastasis are still the important impeding factors in the prognosis of disease. Currently, extracellular matrix(ECM) is presumed to be very crucial for tumor reccurence and metastasis. Matrix metallonroteinases(MMPs) can promote the procession of hepatocarcinoma cells invading the surrounding tissues by degrading the ECM. Among the family of matrix metallonproteinases, MMP-2 and MMP-9 have more close correlation with the tumor invasion by destroying the type IV collagen in the basement membrane. In this investigation we try to study the improvement of the hepatic arterial catheterization technique of VX2 rabbit hepatoma model, the DSA and CT manifestations and the pathomorphology of hepatoma after interventional treatment, and the influence of interventional treatment on the MMP-2 and MMP-9 expression in VX2 rabbit hepatoma model.Objective1. To prepare New Zealand rabbit hepatoma models with VX2 cell line, and evaluate the improvement of the hepatic arterial catheterization technique;2. To observe DSA and CT manifestations and pathomorphology of VX2 rabbit hepatoma model after interventional therapy;3. To observe the expression of MMP-2 and MMP-9 in VX2 rabbit hepatoma model after interventional therapy.Materials and Methods1. Preparation of the rabbit hepatoma model and the catheterization technique of hepatic arteryVX2 cell line was put into left liver lobe of 40 New Zealand rabbits respectively with the open implantation method, and 36 models were made successfully. After 2 weeks, 26 rabbits in the experiment group underwent improved catheterization of hepatic artery by means of microsurgery technique, 10 rabbits in the contrast group were catheterized with 3F micro-catheter by traditional technique.2. Animal grouping23 rabbits were proved catheterizated successfully by DSA in the experiment group which underwent improved catheterization of hepatic artery by means of microsurgery technique, they were randomly divided into three groups, rabbits in the experimental group A (8 models) were treated by embolism with lipiodol before the hepatic artery were deligated; rabbits in the experimental group B (8 models) were treated by lipiodol embolization and chemoembolization before the hepatic artery were deligated; and the remained 7 models were treated without any therapy, as the control group C.3. Manifestations in imaging and pathological morphological examination and the MMP-2 and MMP-9 expression analysis in immunohistochemistry examination1 week after the catheterization, the tumor growth rate (TGR) of three groups was compared by CT scan. After that 23 rabbits were all killed, and the patho-morphological changes were observed under light microscope with HE staining, and the expression of MMP-2 and MMP-9 in VX2 rabbit hepatoma were observed with immunohistochemistry examination and compared. The results were reviewed double-blindly by two pathological experts.4. Statistical analysisSPSS package version 10. 0 was used in the analysis of the success rate of catheterization, the tumor growth rate of the models after interventional therapy, and the expression analysis of MMP-2 and MMP-9 in VX2 rabbit hepatoma. P<0. 05 was considered as significant differences.Results1. Success rate of inoculationIn 40 rabbits with VX2 tumor inoculation, 36 rabbits were proved implanted successfully by CT scan, and the inoculation success rate was 90%. 2 rabbits died of anesthesia, and the other 2 rabbits were considered with unsuccessful implantation because of extensive metastasis in abdominal cavity caused by VX2 tumor. CT scan showed soft-tissue mass with low-density in left lobe of liver. The gross samples of the left lobe were found distributed with white nodules in various sizes and with clear demarcation. 2. Success rate of catheterizationThe success rate of catheterization was 88% (23/26) in the experimental group, which underwent improved catheterization of hepatic artery by means of microsurgery technique, but 40% (4/10) in the control group, which were catheterized with 3F micro-catheter by traditonal technique. There was significant difference between the two groups (P=0. 006).3. Observation of blood supply of VX2 tumors in DSATotal 27 rabbits were catheterized successfully in experimental and control groups. DSA could display secondary branch of hepatic artery and tumor stain. 89% of VX2 hepatomas (24/27) showed obvious tumor stain in hepatic artery angiograms. The VX2 hepatomas in deep part of liver had homogeneous or nodous tumor stain, around which hypervascularity with disordered neovasculariy and enlargement feeding artery were observed. Three VX2 hepatomas in superficial partof liver had hypervascularity with disordered neovasculariy only. The blood supply could be observed in all 27 VX2 hepatomas by DSA.4. Comparison of tumor growth rate (TGR) of VX2 rabbit hepatoma after interventional therapyCT scans showed that the tumor size of all VX2 hepatomas in three groups got larger as time went on, with tumor growth rate (TGR) of (111. 98±57. 40) %, (87. 87±64. 70)%and (194. 07±109. 87)%respectively. The statistical analysis showed the difference among the three groups was significant (F= 3. 645, P = 0. 045 ), and the TGR of group B was significantly lower than that of group C (P=0. 017)5. Pathological morphologic observations(1) Tissue pathological observation of control group(group C) The hepatic structure of rabbits in control group was approximately normal. The hepatocytes were cord-shaped arrangement and a few were found fatty degeneration. Blood sinus expanded slightly. Portal area was infiltrated by a small quantity of lymphocytes. The inoculated VX2 cell mass was seen in left hepatic lobe and the cells appeared as obvious heteromorphism, dislaying round or oval carcinoma cell nests, mitotic figures or pathological mitotic figures, and even tumor giant cells. Fasciolar necrosis was observed in some tumor tissue. In the area surrounding VX2 cell mass, apomorphosis, apoptosis, necrosis of tumor cells and sporadic lymphocyte infiltration were found.(2) Tissue pathological observation of experimental groups(group A and B)Massive necrosis of tumor cells and karyopyknosis and karyorrhexis were observed in tumor tissues of left hepatic lobe, with a few residual tumor cells in peripheral area. Mesenchymal blood vessels displayed dilated and congested, and the focal hemorrhagic regions were seen. Fibrous tissues swelled. Many inflammatory cells, especially neutrophils, were infiltrated around the necrotic area. In the two experimental groups, obvious necrosis ofhepatocytes was seen in the region arround the tumor tissues, especially in group B. Most of hepatocytes around the tumor tissues were degenerated. VX2 cell mass, apomorphosis and parenchymatous degeneration were found in the two groups, which indicated that the embolic materials such as lipiodol could damage the normal tissue.6. Expression of MMP-2 protein in three groupsMMP-2 protein was located in the cytoplasm of VX2 hepatoma cells. There was significant difference among expression of MMP-2 protein in three groups ( x=12. 259, P=0. 002), but there was no difference between groupAand group B ( x~2 = 0. 000, P= 1. 000). The positive rate of MMP-2 protein in experimental group after interventional therapy (group A and group B) was significiantly lower than that in control group (group C) (x~2=6. 674, P=0. 010). 7. Expression of MMP-9 protein in three groupsMMP-9 protein was located in the cytoplasm of VX2 hepatoma cells. There was significant difference among expression of MMP-9 protein in three groups (x = 10. 022, P--0. 007), but there was no difference between group A and group B (x~2=0. 000, P= 1. 000). The positive rate of MMP-2 protein in experimental group after interventional therapy (group A and group B) was significiantly lower than that in control group (group C) (x~2=6. 572, P=0. 010).Conclusions1. The preparation of VX2 rabbit hepatoma model was simple and convenient. It was a ideal animal model for researching hepatocarcinoma;2. The improved catheterization of hepatic artery using microsurgery technique had higher success rate compared with catheterization with 3F micro-catheter by traditional technique, and could decrease X-ray exposure greatly for the experiment workers. It is a useful and promising experiment method;3. Interventional therapy could delay the growth of VX2 rabbit hepatoma and degrade the positive expression of MMP-2 and MMP-9 protein;4. The embolic materials as lipiodol could damage the normal tissue. These embolic materials should be avoid or reduce to enter the normal hepatic tissue in the treatment of the hepatocarcinoma.