| Development of a rat model for orthotopic liver transplantation for hepatocellular carcinoma:BACKGROUNDHepatocellular carcinoma (HCC) is one of the world's most common malignant diseases. Surgical resection is of limited benefit in HCC accompanied by severe liver cirrhosis or multicentric hepatic cancer.Liver transplantation (LT) has been advocated as a salvage treatment for unresectable hepatocellular carcinoma. LT is the only simultaneous treatment of cirrhosis as well as of HCC. Long-term results after liver transplantation for HCC have been disappointing, largely because of the high recurrence rate. Factors to high recurrence rate were as follows: 1) Undetected micrometastases; 2) tumor that embolizes at the time ofliver transplant from manipulation of the liver; 3) the tumor growth-promoting effects of immunosuppressive agents. To evaluate tumor recurrence patterns after OLT in patients with HCC, it is necessary to have a reproducible animal model with a tumor growth pattern allowing accurate assessment of results. There have been several groups that have used rat models of hepatoma to examine the effect of liver transplantation on the survival of rats.1. Rat model that had tumors induced with diethylnitrosamine1) Advantage: these animals typically had multiple tumors at the time of transplantation. Liver transplantation alone resulted in prolonged survival.2) Disadvantage: In animals with multiple tumors, transplantation for cure may not be possible because of early metastatic disease and a different biology of these tumors.2. Animal model of intrahepatic tumor implantation (IHTI) using human hepatocellular carcinoma in nude mice. Tumor fragments from surgical specimens of human hepatoma were implanted in livers of nude mice. Animals were examined for evidence of metastases, and if pulmonary nodules were discovered, these tumors were passaged subcutaneously and eventually reimplanted into livers of nude mice. These tumors would then readily re-metastasize and were considered suitable to study patterns of metastases and potential treatment.3. Techniques of IHTI for intrahepatic tumor model.1) Advantage: reproducible animal model with a tumor growth pattern allowing accurate assessment of results.2) Disadvantage: high rates of artificial tumor dissemination during tumor implantation, which interferes with the evaluation of therapyMATERIALS AND METHODS1. Maintenance of tumor line Walker-256: Walker-256 line from Shanghai Medical Industrial Institution2. Experimental animals: Inbred male SD rats, 12-18 weeks old andweighing220-250 g.3. Intrahepatic tumor implantation model. A 1-mm cube of suspension containing 10~7 Walker-256 carcinosarcoma cells was implanted subcutaneously under light anesthesia. One week after inoculation, a palpable tumor generated at the injected site. Viable tumor tissue was excised under sterile conditions. Tissue was diced into approximately 1 mm×1mm×1 mm fragments. Experimental rats were anesthetized with ether in tube. A small midline incision beneath the metasternum was made and the left lateral lobe of the liver was extracted. A small, superficial incision into the liver was made, 1X1X1 mm cube of Walker-256 tumor was implanted into the liver incision . Incision in the liver was closed with a 7-0 suture. The technique for intrahepatic tumor implantation is also performed as same as described by Yang et al.4. Orthotopic liver transplantation (OLT) the day 14: Syngeneic OLT was performed with the cuff technique without rearterilization, as described by Kamada, under ether anesthesia.5. These animals were separated into 2 groups:Group A (n=25): receiving saline i.p for 14 days after OLT Group B. receiving paclitaxel for 14 days after OLT6. Five recipients from each group were sacrificed at days 21, 28, 32, 42, respectively. Parts of the liver tumors were excised and frozen in liquid nitrogen. Ten percent neutral buffered formalin was injected into the lungs through the trachea, liver tissue and lung. Step sections of livers were processed routinely for hematoxylin and eosin staining for identification of liver lesions. Quantitative analysis Lung metastatic nodules: Lung metastatic lesions were counted under a light microscope and the total areas of lung tissues per animal were measured with the assistance of an image analyzer, thus degree of metastasis according to the number of metastastic lesion per lung was analyzed.7. Statistical analysis: U test was utilized for the comparison of the number of lung and liver metastases. Statistical calculations were performed using SPSS 11.0.RESULTS1. Survival: Recipients who died within 48 hours after OLT were excluded from the study. In the current study, four recipient rats failed to survive 48h in the group A, and two in group B. One more died during day 2-21 in group A, another one in group B, respectively, which were also excluded from the study. Suvival rate at days 21 was 82%.2. the rate of lung metastases was 0 (0/5), 20% (1/5), 60% (3/5), 60% (3/5) at week 3, 4, 5,6 in group A, and 0 (0/5), 0 (0/5), 40%(2/5), 33% (2/6) in group B. A significant difference was found between two groups in term of No. (No. /cm2) Area (mm2/cm2) of lung metastasis.CONCLUSION1. A reproducile model for orthotopic liver transplantation for hepatocellular carcinoma was established.2. OLT could improve the survival of the rat bearing the walker-256 and deter the lung metastases.3. Paclitaxel could decrease the lung metastases in term of No. (No. /cm2) Area (mm2/cm2) of lung nodules.Immunosuppressive effect of paclitaxel on liver transplant ratsObjective To study effect of paclitaxel on liver transplant rats. Methods Orthotopic liver transplantation (OLT) was performed in Lewis-to-BN rats (n=45) who were randomly divided into three groups, and paclitaxel and saline was injected i.p daily (days 1-14), respectively: (1) Control group (n=15) treated by saline at dosage 0.5 ml/kg per day: (2) low dosage paclitaxel group (n=15): treated by paclitaxel at dose 0.5 mg/kg per day; (3) high dosage paclitaxel group (n=15) : treated by paclitaxel at dose 1.0 mg/kg per day. Survival, liverfunction, IFN-γ/ IL-4 (Th1/Th2) and histopathological findings were compared among groups, and T helper precursor frequencies by limiting dilution analysis and CD4~+ cells apoptosis by two-color staining of annexin V were determined. Results Paclitaxel could protect allografts against immunologic rejection (rejection activity index according to Banff criteria: control 5.2 ± 0.8 vs. low dosage 3.8 ± 0.4 or high dosage 3.6 ± 0.5, U=23. 0 and 23. 5, respectively, both P<0.05), and prolonged the recipients survival (control: 10.6±1.9d vs. low dosage: 16.1 ± 3.4d, log rank=9.06, F<0.05; low dosage: 16.1±3.4d vs. high dosage: 25.9±4.1d, Jog rank= 7.81, P <0.05), meanwhile treatment of paclitaxel could contribute to a significant decrease in Th-p frequencies (logarithmic control: 3.42±0.48 vs. low dosage: 1.55±0.58 and high dosage: 1.14±0.52, t=8.9 and 11.8, respectively, both P <0.05), and a shift of the Thl/Th2 balance toward Th2 direction (control: 448. 7%±149.1% vs. low dosage: 107.4%±41.4% and high dosage: 51.4%±15.9%, t=4.93 and 5.92, respectively, both P <0.05) through promoting apoptosis of CD4~+ cells (control: 10.7% vs. low dosage :43.7% and high dosage: 55.6%, x2=27.49 and 93.4, respectively, both P<0.05). Conclusion: Paclitaxel could protect rat liver graft from acute rejection by decreasing Th-p frequencies, and inducing a shift of the Th1/Th2 balance toward Th2 direction through promoting apoptosis of CD4~+ cells.
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