Radiofrequency ablation combined with transcatheter therapy in rabbit VX2 liver tumors: effects and histopathological characteristics

Abstract

Background

Transarterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) treatment (TACE-RFA) has been confirmed superior to TACE or RFA alone in animal liver tumors. TACE before RFA was shown to increase hepatocellular damage. Further optimization of the combination strategy for transcatheter arterial embolization (TAE) or TACE combined with RFA is warranted.

Purpose

To determine the optimal strategy for radiofrequency ablation combined with transcatheter therapies in VX2 liver tumors in a rabbit model.

Material and Methods

Twenty-four Japanese White rabbits with VX2 liver tumors were randomly divided into four groups: TACE-RFA (TACE-RFA group), transcatheter arterial embolization (TAE) combined with RFA treatment (TAE-RFA group), RFA only group, and TACE only group. Blood samples were collected 1 day before the operation and at 3 and 7 days postoperatively. Seven days after the operation, maximal diameters of coagulation or infarcted zones in the gross specimens, CT images, histopathological characteristics, tumor necrotic rate, and growth rate were compared.

Results

Significantly larger mean long-axis (P < 0.05) and short-axis (P < 0.05) diameters of coagulation and infarction were observed in the TACE-RFA group compared with the TAE-RFA, RFA, and TACE groups on day 7; and the TAE-RFA group showed a significant (P < 0.05) increase versus the RFA and TACE groups on day 7. There were no significant differences in tumor growth rate (109.3 ± 37.5 vs. 119.0 ± 43.1%, P = 0.45) and necrotic rate (89.5 ± 12.0 vs. 83.5 ± 9.3%, P = 0.73) between the TACE-RFA and TAE-RFA groups. TACE-RFA was more effective for achieving tumor destruction than the other treatment strategies, but led to increased rabbits discomfort and more severe liver dysfunction compared with TAE-RFA.

Conclusion

TAE-RFA appears to be a beneficial therapeutic modality for treating VX2 liver tumors in a rabbit model.

Keywords

Transcatheter arterial chemoembolization radiofrequency ablation VX2 liver tumor transcatheter arterial embolization

Introduction

Transcatheter arterial chemoembolization (TACE) is widely used as an alternative treatment for unresectable liver tumors (1). During the past decade, because of its ease of use and minimal invasiveness, radiofrequency ablation (RFA) appeared to be an effective and safe treatment method for unresectable primary liver tumors (2,3). However, local tumor recurrence has been observed following RFA, suggesting the persistence of viable tumor cells at the margin of the ablation zone (4). Indeed, the efficacy of RFA is inherently affected by the tumor volume and by the so-called “heat sink” effect (5 –7). To overcome this problem, certain groups studied combination therapies to increase the diameter of coagulation necrosis, including TACE (8) and vascular occlusion (6,9,10).

Clinical studies have demonstrated that TACE combined with RFA treatment (TACE-RFA) are superior to TACE or RFA alone (11 –13). However, non-superselective TACE was showed to increase hepatocellular damage, stimulating systemic proinﬂammatory cytokines release and inhibiting hepatocytes proliferation (14). Upper gastrointestinal hemorrhage, hepatic failure, atrophy or cirrhosis, pulmonary embolism, and cholecystitis are the most common side-effects of TACE (15).

Hepatic primary malignant lesions receive most (90–100%) of their blood supply from the hepatic artery (16,17). Thus, embolization of this tumor-feeding vessel can induce ischemia and hypoxia within the tumor and, subsequently, tumor necrosis. Recently, clinical studies demonstrated that single-session combined transcatheter arterial embolization (TAE) combined with RFA (TAE-RFA) was an effective and safe treatment in patients with unresectable liver malignancies (18,19).

To the best of our knowledge, there are few studies about the comparison of TACE-RFA and TAE-RFA for the treatment of liver tumors. The rabbit VX2 tumor model is currently an optimal animal liver tumor model for the development of catheter-directed therapies (20). In this study, a rabbit VX2 liver tumor model and a standardized embolization protocol were used to compare the tumor necrotic rate, growth rate and liver dysfunction in TACE-RFA, TAE-RFA, RFA alone, and TACE alone to determine the optimal combination strategy for treating rabbit VX2 liver tumor.

Material and Methods

Animals and rabbit VX2 liver tumor models

Our experiments were approved by the Animal Care Committee of Hubei Province. Adult Japanese White rabbits (3.0–3.5 kg, 4–5 month old) were used in the study. The VX2 carcinoma strain was maintained by successive transplantation into the hind limb of carrier rabbits by deep intramuscular injection. Two weeks after inoculation, a palpable nodule was detected in a carrier rabbit. The tumor was harvested under strict sterile conditions. After the recipient rabbit was anesthetized with intravenous pentobarbital sodium (30–35 mg/kg), tumor implantation was performed as outlined by Virmani et al. (21). Tumor evaluation was performed using 1.5-T magnetic resonance imaging (MRI) (Magnetom Avanto; Siemens Medical Solutions, Erlangen, Germany) to detect the localization and size of the VX2 tumor 15 days after implantation. All animals underwent T2-weighted (T2W) MRI with the following parameters: TR/TE, 3700/87 ms; slice thickness, 4 mm; intersection gap, 15%; 168 Hz/pixel BW; field of view, 200 × 200 mm²; matrix, 320 × 320; turbo factor, 11; averages, 2.

Three days before the procedure, the epigastria and backs of the rabbits were shaved. Twenty-four rabbits were confirmed by MRI as successful for the establishment of VX2 liver tumor, and were randomly divided into four treatment groups (n = 6 in each group): TACE-RFA, TAE-RFA, RFA alone, and TACE alone. All 24 rabbits were treated humanely during the experiments. If necessary, one-quarter of the initial dose of 30–35 mg/kg of pentobarbital sodium was administered intravenously every 30 min. Following the procedures, the animals were monitored until they recovered from anesthesia. They were then placed in cages.

TACE and TAE procedures

The TACE and TAE procedures were performed under digital subtraction angiography (DSA) guidance (Angiostar Plus, Siemens Medical Solutions, Munich, Germany) as described by Wang et al. (16). Celiac arteriography was performed by hand bolus injection of contrast medium to delineate the blood supply to the liver and to confirm the location of the tumor. A 1.9 F (EcheconTM-14, EV3, Plymouth, MN, USA) coaxial catheter system was then advanced into the left hepatic artery. TACE was infused using a mixture of doxorubicin (Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou, China) (2 mg/kg body weight) with iodized oil (Lipiodol Ultra-Fluid, Guerbet, Paris, France) (0.1 mL/kg body weight). TAE was performed using polyvinyl alcohol particles (PVA), 150–250 µm in size (Cook, Bloomington, IN, USA), mixed with contrast media. After confirmation of the tumor-supplying artery by angiography, TACE or TAE was carefully performed in all experimental animals under DSA to avoid the retrograde reflux of the embolic materials out of the tumor-supplying arteries. The embolization endpoint of TACE or TAE was the complete stasis of antegrade blood flow. For the TACE-RFA or TAE-RFA group rabbits, RFA was implemented 15 min later according to Mostafa et al. (22).

Radiofrequency ablation

RFA was performed using the RITA 1500 system (RITA Medical Systems, Mountain View, CA, USA) and a 14-gauge Starburst multiarray electrode (RITA Medical Systems). A circuit pad was placed on the back of the rabbit, and the liver was exposed through a subxiphoid abdominal incision. Then, the electrode was placed in the center of the tumor through part of normal liver tissue and expanded to 1 cm in diameter. Radiofrequency energy was applied for 5 min for each rabbit and the power output was set at 20 W. The electrode had a designated tip temperature of 90 ± 5℃. At the end of ablation, thermocoagulation was performed along the needle track to avoid tumor seeding.

CT scanning and sample collection

Seven days after the procedure, contrast-enhanced computed tomography (CT) (Somatom Definition AS 128 Siemens, Erlangen, Germany) was performed with a slice thickness of 2 mm, and included the entire liver field with contrast injection at a rate of 1 mL/s through the auricular vein in the supine position (Fig. 1a). Blood samples were collected from all rabbits 1 day before the operation, and 3 and 7 days after the operation. Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using standard enzymatic procedures.

Fig. 1.

Images from rabbits in the TACE-RFA group 1 week after the procedure. (a) Contrast-enhanced CT scans showing high-density iodized oil in the center of the low-density area, which was an oval-shaped non-enhancement zone of ablation with gas bubbles (long arrow). A contrast enhancement zone (arrowheads) was observed around the ablation area. (b) Gross specimens of the liver showing a core of ablated tissue with a surrounding narrow rim of hemorrhagic necrosis on the surface of the liver. (c) The two largest slices in the middle of the ablated area showing a white-brown completely ablated zone with an irregular margin (short arrows), surrounded by yellow fibrous tissue. The damaged hepatic vessels (arrows) and tumor (arrowheads) were within the coagulation area. (d) The contrast between the two largest slices, one of which was directly preserved in paraformaldehyde and the other stained with TTC and then preserved in paraformaldehyde. TTC staining of the largest slice revealed a 1.6 × 3.3 cm ablated zone.

Animals were sacrificed following CT scanning, and the livers were removed and dissected for gross and pathological analysis (Fig. 1b). Each affected area was sectioned at 3–5 mm intervals in a sectional manner along the RFA electrode tract (Fig. 1c). One of the largest slices in the middle was soaked in 2% 2,3,5-triphenyl tetrazolium chloride (TTC) at room temperature for 15 min to detect the extent of the white coagulated zones and the surrounding infarcted zones (22). Following the replacement of TTC with phosphate-buffered saline (PBS), an image was taken of the largest slice. The remaining slices were directly preserved in 4% paraformaldehyde.

The maximum long- and short-axis diameters of the affected area were measured in the largest slices with TTC staining by two observers who were blinded to treatment. The key endpoints of the measurement were white coagulation without TTC staining in the zone of coagulation and reddish tissue change with patchy areas of TTC staining in the region of the infarction zone (Fig. 1d).

Pathological evaluation

Sections preserved in 4% paraformaldehyde were stained with hematoxylin and eosin for histopathological examination. The evaluation of the histopathological slides in each treatment group was performed by a pathologist with 5 years of experience in the examination of ablated tissue.

Evaluation of tumor growth

The tumor was longitudinally cut for the evaluation of tumor necrosis. The tumor volume was calculated according to the following formula: a × b²/2, where “a” and ‘ “b” represented the maximum and minimum diameters of the tumor, respectively. The tumor necrosis rate was calculated as the necrosis area divided by the entire area of the tumor. The tumor growth rate was evaluated by the ratio of tumor volume before and after therapy.

Statistical analysis

Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data are shown as mean ± SD. One-way ANOVA was used to compare maximum diameter of coagulation necrosis, tumor growth, and necrosis rates in the different groups on day 7. The Mann-Whitney U test was used to compare ALT and AST between two groups at different time points. A P value <0.05 was considered statistically significant.

Results

Comparison of maximum diameter of coagulation necrosis

Prior to any procedure, VX2 liver tumors showed a low signal area on T1-weighted images, and a high signal area on T2W images on MRI. The maximum diameter of the tumors in the 24 rabbits was 13.8 ± 2.5 mm. The volume of iodized oil used in the TACE-RFA and TACE groups was 0.35 ± 0.04 mL. During operation, a rabbit in the TAE-RFA group died from intraperitoneal bleeding, and a rabbit in the RFA group died from pneumothorax. Two days after operation, a rabbit in the TACE-RFA group died from liver function failure. Operations were successfully performed in all remaining animals. Three days after the procedure, the remaining animals had recovered to normal diet and physical activity. No more animals died.

The mean maximum long- and short-axis diameters were measured in the gross specimens of the four groups (Table 1). The differences in the mean long- and short-axis diameters between the TACE-RFA group and the other groups were statistically significant (P < 0.05). In addition, the mean maximum long- and short-axis diameters of the ablation zones in the TAE-RFA group showed a significant (P < 0.05) increase compared with the RFA and TACE groups.

Table 1.

Comparison of maximal diameter of coagulation necrosis in each treatment group.

Groups (n)	Long diameter (mm)	Short diameter (mm)
TACE-RFA (5)	37.9 ± 2.2	14.7 ± 1.9
TAE-RFA (5)	30.1 ± 0.4*	12.7 ± 1.4*
RFA (5)	22.7 ± 2.9*^†	10.1 ± 0.8 *^†
TACE^‡ (6)	19.8 ± 1.4*^†	7.7 ± 1.5*^†

P < 0.05: TACE-RFA group compared with the other groups.

†

P < 0.05 TAE-RFA group compared with the RFA and TACE groups.

‡

Diameters of infarcted zones by TACE group.

Effect of different treatments on tumor growth and necrosis

Seven days after treatment, there were no significant differences in tumor growth rate (109.3 ± 37.5 vs. 119.0 ± 43.1%, P = 0.45) and necrotic ratio (89.5 ± 12.0 vs. 83.5 ± 9.3%, P = 0.73) between the TACE-RFA and TAE-RFA groups. The tumor growth rate was significantly decreased while the necrosis ratios were increased in the TACE-RFA and TAE-RFA groups compared with the RFA and TACE groups (Table 2).

Table 2.

Tumor growth and tumor necrotic rate in different groups.

Groups (n)	Tumor growth rate (%)	Tumor necrotic ratio (%)
TACE-RFA (5)	109.34 ± 37.46	89.50 ± 12.03
TAE-RFA (5)	118.97 ± 43.11	83.48 ± 9.28
RFA (5)	269.15 ± 39.94*^†	66.53 ± 14.29^‡ §
TACE (6)	325.48 ± 51.31*^†	58.50 ± 13.57^‡ §

P < 0.001, <0.001: TACE-RFA group compared with the RFA and TACE groups.

†

P < 0.001, <0.001: TAE-RFA group compared with the RFA and TACE groups.

‡

P = 0.009, <0.001: TACE-RFA group compared with the RFA and TACE groups.

P = 0.045, <0.001: TAE-RFA group compared with the RFA and TACE groups.

Changes in liver function

ALT and AST levels in the TACE-RFA group were not significantly different from the other groups 1 day before the operation (P > 0.05). ALT and AST levels in the four groups peaked on day 3 and decreased on day 7. The TACE-RFA group showed the highest ALT and AST levels on days 3 and 7 (all P < 0.01). On days 3 and 7, the TACE group showed marked increases in ALT and AST levels, significantly greater than in the TAE-RFA and RFA groups (all P < 0.01). On day 3, the TAE-RFA group showed a marked increase in ALT and AST levels, significantly greater than in the RFA group (P < 0.05, respectively). ALT and AST levels in the TAE-RFA group were not significantly different from the RFA group on day 7 (P > 0.05) (Fig. 2).

Fig. 2.

Changes in serum ALT and AST levels before and after operation (Mean ± SD). ^&P < 0.01: ALT and AST levels in the TACE-RFA group were significantly different compared with the other groups on days 3 and 7 after operation. ^$P < 0.01: ALT and AST levels in the TACE group were significantly different compared with the TAE-RFA and RFA groups on days 3 and 7. ^#P < 0.05: ALT and AST levels in the TAE-RFA group were significantly different compared with the RFA group on day 3, but ^#P < 0.05: on day 7.

Image changes and histopathological characteristics

TACE-RFA group

Unenhanced CT scans showed high-density deposition of iodized oil in an oval-shaped defect, which corresponded to coagulation necrosis with gas bubbles. Contrast-enhanced CT scans (Fig. 1a) showed a low-density area surrounded by enhancement areas, but no contrast enhancement in the regions of low-density coagulation necrosis. This contrast enhancement zone was inflammatory edema surrounding the ablation area.

Gross specimen showed typical coagulation necrosis with an irregular margin surrounded by yellow fibrous tissue. The damaged hepatic vessels and tumor were located within the coagulation area (Fig. 1c). In TTC-stained slices, the white zone without TTC staining was the irregular edge of the ablated zone, and the red zone with TTC staining was normal liver parenchyma (Fig. 1d).

Histological examination of the ablation zones revealed a typical coagulation necrosis, pyknotic nuclei, and streamlined cytoplasm in the region of the tumor (Fig. 3a and b) and a number of ghost-like cells with eosinophilic cytoplasm in the areas of normal liver tissue (Fig. 3c). Visible cells survived in the perivascular regions at the edge of the ablation zone (Fig. 3b and c). Fibroid tissues surrounded the ablation zone with inflammatory cell infiltration (Fig. 3c and d).

Fig. 3.

Histopathological changes in the TACE-RFA group. (a) Section of tumor tissue (200 × magnification) showed typical coagulative necrosis (zone a) and tumor cells undergoing necrosis (zone b). (b) Viable tumor cells (200× magnification) were observed around three tumor vessels (arrows). Tumor cells located at a distance from the perivascular regions showed undergoing coagulation necrosis (arrowheads). (c) Zone A (100× magnification) had typical coagulation necrosis and an irregular burned edge with periportal cell survival (arrows) towards the edge of the ablation. The fibroid tissue with infiltration of inflammatory cells (zone F) and hepatocytes (zone H) surrounding zone A. (d) The enlarged inset of zone H (200× magnification) seen in C demonstrates hepatocytes undergoing ballooning degeneration.

TAE-RFA group

Unenhanced CT scans showed an oval-shaped defect, which corresponded to the coagulation necrosis area. Contrast-enhanced CT scans showed the low-density area surrounded by enhancement areas (Fig. 4a). Gross specimen showed more completely coagulation necrosis than the TACE-RFA group. Some liquefied tissue had been lost in the central zone of the ablation area, and left a cavity in some cases. Ablated zone was surrounding by regular yellow fibrous tissue (Fig. 4b). Liver tissue around the ablation zone showed three distinct histopathologic zones (Fig. 4c). Typical necrosis coagulation and tumor cells undergoing necrosis with some survival tumor cells in it were present in the region of the tumor (Fig. 4d).

Fig. 4.

Images from rabbits in the TAE-RFA group. (a) On contrast-enhanced CT, contrast enhancement zones (short arrows) surrounded oval-shaped ablation area. (b) Gross specimen showing more completely coagulation necrosis. Central zone of ablation showed complete destruction of the parenchyma and tumor, including a cavity where some ablated tumor tissue had been lost (short arrows). Ablated zone was surrounding by regular yellow fibrous tissue. (c) Section (200× magnification) showing three distinct histopathologic zones: zone 1 revealed a typical coagulation necrosis; zone 2 was regular fibroid tissue with infiltration of inflammatory cells; and zone 3 was normal hepatocytes. (d) Section of tumor tissue (200× magnification) showing two histopathologic zones: coagulative necrosis (zone 1), and tumor cells undergoing necrosis (zone 2) with some survival tumor cells.

RFA group

The RFA group displayed almost the same images as in the TAE-RFA group on unenhanced and contrast-enhanced CT scans. Air bubbles could be seen within the ablation area (Fig. 5a). The RFA group showed an oval-shaped, light brown ablated zone surrounded by a regular yellow fibrous capsule (Fig. 5b). Liver slices around the ablation zone showed three distinct histopathologic zones, similar to the TAE-RFA group (Fig. 5c). Histological examination of the ablation zones was similar to Fig. 4d in the TAE-RFA group in the region of the tumor (Fig. 5d). However, more islands of viable tumor cells or hepatocyte nests were found in perivascular regions at the edge of the ablation zone compared with the TACE-RFA and TAE-RFA groups.

Fig. 5.

Images from rabbits in the RFA group. (a) On contrast-enhanced CT, contrast enhancement zones (short arrows) surrounded the ablation area within an air bubble (long arrow). (b) Gross specimen showing coagulation necrosis. Regular margin of the ablated zone was surrounded by yellow fibrous tissue. (c) Image (100× magnification) showing three distinct histopathologic zones: zone 1 revealed a typical coagulation necrosis; zone 2 was regular fibroid tissue with infiltration of inflammatory cells; and zone 3 was normal hepatocytes. (d) Section of tumor tissue (200× magnification) showing two histopathologic zones: coagulative necrosis (zone 1) and some tumor cells undergoing necrosis in survival tumor cells (zone 2).

TACE group

Unenhanced CT scans showed a large high-density deposition of iodized oil with fewer low-density coagulation necrosis areas than in the other groups (Fig. 6a). Contrast-enhanced CT scans showed that the inflammatory edema surrounding the necrosis area was not very important. Infarcted necrosis in the TACE group also had an irregular edge, but small areas in the form of interspersed islands with TTC staining were observed in the infarction zone without TTC staining. In addition, small areas in the form of interspersed islands with TTC staining were small pieces of normal liver or residual tumors in complete infarcted necrosis due to incomplete embolization. Normal hepatocytes with infiltration of inflammatory cells and fibroid tissue with significant infiltration of inflammatory cells in the perivascular regions, which surrounded the irregular edges of the infarction zone, was apparent on histological examination (Fig. 6c). The TACE group had typical infarcted tumor necrosis and many survival tumor cells with exudation of red blood cells, which were more significant than in the TACE-RFA group (Fig. 6d).

Fig. 6.

Images from rabbits in the TACE group. (a) Contrast-enhanced CT scans showing high-density iodized oil surrounding low-density area which was necrosis in central tumor (zone1). (b) Gross specimen showed central necrosis in tumor (zone 1), red infarcted necrosis (zone 2), and fish-like tumor tissue (zone 3). Irregular margin of infarcted necrosis was surrounded by yellow fibrous tissue. (c) Image (100× magnification) showing two distinct histopathologic zones surrounding the infarcted necrosis: 1 was normal hepatocytes with significant infiltration of inflammatory cells (white arrow); and 2 was fibroid tissue with infiltration of inflammatory cells in the perivascular regions (black arrow). (d) Section of tumor tissue (200× magnification) showing tumor infarcted necrosis with many survival tumor cells and exudation of red blood cells.

Discussion

Embolization of the portal vein, hepatic artery, or both during RFA is reportedly able to increase coagulation area in porcine model of normal liver tissue (8,23), which was also reported by Mostafa et al. (22). However, they reported that there were no significant differences in coagulation volumes in rabbits that underwent TAE with 300–500 µm embospheres prior to RFA and in those that underwent RFA alone. Tanaka et al. (24) determined that TAE with the use of 40 µm microspheres before RFA enhanced the efficacy of RFA more than the use of larger particles. We observed significant differences in the coagulation areas between the RFA and the TAE-RFA groups. We used a superselective catheterization technique to embolize tumor-feeding arteries with 150–250 µm PVA by using a 1.9 F microcatheter. These results suggest that TAE with smaller size PVA adequately reduced rabbit VX2 liver tumors perfusion and sufficiently increased heat retention.

In liver slices around the ablation zone obtained from the TACE-RFA group, microscopic images revealed six distinct histopathological zones from the center of the ablation zone to the periphery, including the typical coagulation necrosis zone, the pronounced inflammatory reaction zone, viable cells surrounding the portal veins in the form of islands, undergoing coagulation necrosis zone, fibroid tissue with irregular burned edges and large numbers of hepatocytes undergoing ballooning degeneration. By contrast, the images in the TAE-RFA and RFA groups only showed three histopathological zones. The regular burned edges in the TAE-RFA and RFA groups may be due to the single effect of hyperthermia.

Moreover, the large numbers of hepatocytes undergoing ballooning degeneration surrounding the ablation zone in the TACE-RFA group may be the local chemotherapy’s adverse reaction to normal hepatocytes (25). However, the local chemotherapy’s adverse reaction to normal hepatocytes may have an additional effect at the periphery of the ablation zones in tumors, which has the potential to increase the volume of tumor destruction and RFA efficacy, while RFA heating was lowest and the eradication of tumor cells was difficult in the TACE-RFA group. In TACE-RFA, local hyperthermia with RFA was used to increase the synergistic effect of chemotherapy as an apoptotic enhancer by increasing local drug uptake and drug metabolism by solid tumors (26). Dudar et al. (27) revealed that hyperthermia increased the vascular endothelial pore size and thus allowed a greater deposition of doxorubicin. Lencioni et al. (28) proposed that RFA redistributed the chemoembolic material to the periphery of the ablation zone, with an associated increase in tumor destruction.

Mostafa et al. (22) found that tumors treated with TACE showed numerous islands of viable tumor cells, particularly adjacent to vessels, indicating incomplete tumor treatment. In the present study, gross specimen showed evidence of inadequate treatment from TACE alone. There were a number of islands of visible tumor cells adjacent to blood vessels in the necrotic areas. This phenomenon was observed in all groups, but least in the TACE-RFA and TAE-RFA groups. Thus, TACE prior to RFA is able to increase tumor heating and coagulation by decreasing tumor blood flow and vascular-mediated tissue cooling to reduce the “heat sink” effect (7,22). The multifactorial TACE-RFA strategy showed the best treatment effectiveness, and its effects may be summarized as: reduction of tumor blood flow, effects of hyperthermia, local chemotherapy and their synergistic effect.

It is important to underline that there were no significant differences in the rates of tumor necrotic rate and growth rate between the TACE-RFA and TAE-RFA groups in our study. Chemoembolization in TACE-RFA and TACE groups led to moderate to severe hepatocellular necrosis in adjacent liver tissues, which was confirmed by hepatocytes undergoing ballooning degeneration surrounding the irregular edges of the infarction zone and fibroid tissue. However, this phenomenon was rare in the TAE-RFA and RFA groups, which may be due to the absence of local chemotherapy’s adverse reaction in normal hepatocytes (14,25).

The chemotherapy drugs used in TACE were determined to aggravate hepatic apoptosis and liver fibrosis in non-tumor areas (29). Wang et al. (14) found that TACE led to moderate to severe hepatocellular necrosis in the adjacent liver tissues, which activates systemic proinflammatory cytokine release, as revealed by significantly increased plasma TNF-α level. In the present study, liver function was assessed by plasma AST and ALT. Changes in plasma AST and ALT levels in the TACE-RFA and TACE groups were in accordance with Wang et al. (14). At the same time, we observed hepatocytes undergoing ballooning degeneration surrounding the ablation zone in the TACE-RFA group and normal hepatocytes with significant infiltration of inflammatory cells near the infarcted necrosis in the TACE group. These phenomenons contributed to the local chemotherapy’s adverse reactions in normal hepatocytes.

In our study, three rabbits died from complications (3/24, 12.5%), including massive hepatic necrosis, pneumothorax, and intraperitoneal bleeding. Due to the small diameter of rabbit liver arteries, it was difficult to undergo superselective chemoembolization in the TACE-RFA and TACE groups. Therefore, some of the iodized oil mixed with doxorubicin refluxed to the normal liver lobes. This event may have led to the loss of appetite in four rabbits in the TACE group, and in five rabbits in the TACE-RFA group during the first 3 days after the procedure, and the death of a rabbit due to massive hepatic necrosis in the TACE-RFA group 2 days after the procedure.

The present study had several limitations. In the present study, we only wanted to evaluate the synergistic effects of TACE or TAE combined with RFA, and to compare tumor necrotic ratio, tumor growth rate, and liver dysfunction in all groups. The effects of TACE and TAE were difficult to evaluate in complete ablation with a considerable security margin in combined treatments. Hence, we did not focus the evaluation on the effect of TACE or TAE in combined treatments. The rabbit liver lobes are thin, and we did not place the electrode directly in the center of the tumor but obliquely punctured through part of the normal liver. Nevertheless, certain rabbits were not able to tolerate treatment following booster injections of pentobarbital sodium during RFA and thermal damage was observed in the nearby liver lobes. According to the gross specimen of the dead rabbit in the TAE-RFA group, we speculated that the reason for intraperitoneal bleeding was hilar vascular injury due to the rabbit’s restlessness when it awoke from incomplete anesthesia during the procedure. The pneumothorax that occurred in the RFA group may have been due to the same reason.

In conclusion, TACE-RFA was more effective for achieving tumor destruction than TAE-RFA, RFA alone or TACE alone. TACE-RFA showed no difference in achieving a lower tumor growth rate and a higher tumor necrotic ratio compared with TAE-RFA. However, TACE-RFA led to more severe liver dysfunction compared with TAE-RFA. Hence, TAE-RFA appears to be a beneficial therapeutic modality for treating rabbit VX2 liver tumor model. Further studies need to be conducted to determine the extent of the clinical benefits that may be achieved by TAE-RFA. Thus, TAE-RFA may be used as a standard treatment for the treatment of larger liver tumors.

Footnotes

Funding

Hubei Province Natural Science Foundation project (2010CDB07607).

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