Establishment of VX2 rabbit liver tumor model
All experiments were approved by the Animal Care Committee in China. The animals (purchased from Wuhan Wanqianjiaxing Biotechnology Co.,LTD.SCXK 2016-0011) were treated according to National Institutes of Health guide for the care and use of Laboratory animals and all efforts were made to minimize the number of rabbits used and their suffering. A total of 100 adult Japanese white rabbits weighing 3.0-3.5 kg were initially used in the study. The methods of VX2 carcinoma strain transplantation was followed as described by Virmani et al . Two weeks after inoculation, the harvested VX2 tumor fragment implanted into the medial left liver lobe to generate liver tumors in carrier rabbits. On the 15 day after the implantation, each rabbit was undergoing magnetic resonance imaging (MRI, 1.5-T, Magnetom Avanto; Siemens Medical Solutions, Erlangen, Germany) and 96 of them detected the VX2 tumor. The rabbits’ position and the T1WI and T2WI imaging parameters were described as previously . The epigastria and backs of the rabbits were shaved before procedure.
The 96 rabbits with VX2 liver tumor were randomly divided into 4 groups: 1) TACE group (consisting of 0.4 mg doxorubicin that was mixed into 0.4 ml iodized oil); 2) RFA group; 3) TACE+RFA group; 4) TAE+RFA group. RFA was performed in TACE+RFA and TAE+RFA groups 15 min after TACE or TAE [polyvinyl alcohol particles (PVA) 150-250 μm in size (Cook, Bloomington, IN, USA)] treatment . Each above groups were further randomly divided into subgroup A (15 rabbits) and subgroup B (9 rabbits). Five rabbits from each subgroup A were sacrificed by sodium pentobarbital (100 mg/kg IV) on days 1, 3, 7 after procedures, respectively. The subgroup B was followed up until animal's death (Fig. 1). All rabbits were treated humanely during the experiment as reported as previously .
The TACE and TAE procedures were performed according to digital subtraction angiography (DSA; Multistar, Siemens, Erlangen, Germany) . The rabbits that suffered TACE, TAE or without treatment were placed on to a circuit pad in the supine position and RFA procedure was performed following an adequate anesthesia .
Hepatocellular necrosis score
The severity of injury in live tissue surrounding the zone of necrosis or coagulation was blindly graded according to modified Suzuki criteria . The sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded on a scale of 0 to 4. Absence of necrosis, congestion, or centrilobular ballooning was scored as 0, whereas the presence of severe congestion/ballooning and 60% or greater lobular necrosis was scored as 4. Six fields of each sample were evaluated under light microscope with 100 magnification.
Serum and liver samples collection
Serum samples were collected 1 day before operation and on days 1, 3 and 7 after operation. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by standard enzymatic procedures. For TACE group, livers were incised along the long axis of tumor, its largest and the smallest diameters were measured on the largest slice. For RFA, TACE+RFA and TAE+RFA groups, the affected area of liver was incised along the long axis of the RFA electrode tract and the samples were sectioned at 3-5 mm intervals. All samples were preserved in 4% paraformaldehyde for pathologic analysis.
Proinflammatory Cytokine Response
Serum TNF-α, the most important acute-phase response cytokine produced by hepatic macrophages, was assessed with ELISA by using polyclonal TNF-α goat anti-rabbit antibody (USCN Life Science, Wuhan, China) according to the manufacturer’s instructions.
Hematoxylin - Eosin and heat shock protein 70 immunohistochemistry staining
The slides embedded in paraffin were sectioned at 4 μm. Hematoxylin - Eosin (HE) staining was performed in the standard fashion. The heat shock protein 70 (HSP70) levels of the samples were determined by immunohistochemistry staining . All slides were evaluated with 200 magnification by two pathologists in a blinded manner. In TACE group, 5 photographs of HSP70 staining were taken from the liver tissue surrounding the zone adjacent to the necrosis. In RFA, TAEC+RFA and TAE+RFA groups, 5 photographs were taken from liver tissue surrounding the coagulation zone, respectively. The HSP70 expression was automatically calculated by integrated optical density (IOD) using Image-Pro Plus software (version 6.0, Media Cybernetics, Bethesda, MD, USA).
Enzyme-linked immunosorbent assay of HSP70 in peripheral blood
One milliliter of peripheral blood taken from each rabbit’s ear vein before treatment and on days 1, 3 and 7 after the treatment was placed into tubes containing no anticoagulants to separate serum (kept at -20°C). The serum HSP70 and tumor necrosis factor (TNF)-α were quantitated by Enzyme-linked immunosorbent assay (ELISA, Abcam PLC., Cambridge, UK).
Ninety-six well microtiter plates were coated with mouse anti-rabbit HSP70 capture antibodies in carbonate buffer overnight. 100 μL of the samples were added into the plates, incubated for 2 h. Plates were subsequently washed and PBS-gelatin buffer containing diluted biotin anti-HSP70 was added and incubated at room temperature for 1.5 h; the plates were washed again and incubated with streptavidin–HRP diluted in PBS gelatin for 20 min. Then the plate was washed to remove excess HRP conjugate and added Tetramethyl benzidine (TMB) substrate solution into each well. After the reaction stopped, the colorimetric reaction produced was read at a wavelength of 450 nm. The amount of signal was directly proportional to the HSP70 level in the sample and it was expressed as ng/ml.
The apoptosis index (AI) of hepatocyte apoptosis in liver and tumor cells were assessed with the method of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). The results were semi quantitatively scored by averaging the number of apoptotic cells per field at 200 × magnification. Five microscopic fields of each sample were evaluated.
Liver tissues surrounding the ablation or embolized zone were immunostained with anti-Ki-67 antibodies (monoclonal mouse anti-rat Ki-67 antigen; MIB-5; Dako, Carpinteria, California), which allowed to detect all active parts of the cell cycle and was a strong positive correlation with proliferating antigen expression and bromode-oxyuridine / thymidine incorporation . The immunostained sections were counterstained with HE. The proliferation index was defined as the percentage of Ki-67 positive hepatocytes per total hepatocyte by counting 6 fields for each section at 200× magnification.
Data analysis was carried out with SPSS software (Chicago, IL, U.S.A.). Quantitative data were expressed as means ± SD and analyzed by ANOVA. The survival curves were constructed by the Kaplan-Meier method and compared by log-rank test and χ2 tests for survival analysis. Differences in counts between each groups and different time points were analyzed by the Kruskal-Wallis H test and Mann-Whitney U test. P value <0.05 was considered to indicate statistical significance.