© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60098-3

 

 

Contributors: vGTM proposed the study. vLKP, HLT and vTJD performed the experiments and collected and analysed the data. vLKP and HLT wrote the first draft. RJJ evaluated the pathological slides and wrote the histological results. All authors contributed to the design and interpretation of the study and to further drafts. vLKP is the guarantor.

Funding: None.

Ethical approval: This study was approved by the Institutional Animal Ethics Committee of the Academic Medical Center of the University of Amsterdam (BEX 11-0986).

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

 

 

 

 

(Hepatobiliary Pancreat Dis Int 2013;12:622-629)

 

KEY WORDS: hypertrophy; liver regeneration; animal model; transarterial embolization; hepatic artery

For patients with primary or secondary liver tumors, the treatment deemed most effective is liver resection. For this intervention, however, a minimum volume of remnant liver is required for sufficient liver function and volume. If the future remnant liver (FRL) is too small, patients are not indicated for tumor resection. Unfortunately, in patients with malignant liver tumors, less than 15% to 20% are eligible for surgical resection.

 

Portal vein embolization (PVE), in which either the right or left branch of the portal vein is occluded, is an option to increase the volume of the FRL by inducing atrophy of the embolized liver lobe, and consequently, hypertrophy of the contralateral, non-occluded liver lobe.^[1] However, there is evidence that after PVE the regenerating liver releases growth factors and cytokines, which also enhance tumor growth.^[1-8] In addition, PVE results in a compensatory hyperperfusion of the ipsilateral hepatic artery, which will boost the growth of tumors mainly fed by the hepatic artery, such as hepatocellular carcinoma and colorectal liver metastases.

 

Embolizing the hepatic arteries supplying the tumor-bearing liver segments is a conceivable treatment option, alone or in combination with PVE. Transcatheter arterial embolization (TAE), with or without additional chemotherapy (TACE), may be offered to patients with primary liver tumors or liver metastases who are unsuitable for resection. Embolizing the tumor-supplying artery will decrease arterial perfusion of the tumor, resulting in selective ischemia of the tumor and possible reduction of tumor size. There are many articles published on the technique of embolization using polyvinyl alcohol particles, gelatin sponge, or lipiodol with or without additional chemotherapeutic agents as doxorubicine, cisplatin, mitomycin C or their combinations, as described in the review of Marelli et al.^[9] However, as hepatocellular carcinoma is known to be relatively insensitive for chemotherapeutic agents, the role of embolization is probably more important than chemotherapy.^[9] Another additional effect of arterial liver embolization as described already in 1998 by Vogl et al is the unexplained hypertrophy response on the non-embolized liver segments.^[10]

 

We explored the effects of an arterial embolization strategy as described above in a standardized animal model in which tumor growth rate can be assessed in relation with TAE. The VX2 liver tumor model in rabbits is used, enabling evaluation of liver regeneration response and tumor changes after TAE. The present study therefore aimed to determine the effects of TAE on tumor volume, but more importantly on the hypertrophy response of the non-tumor-bearing, non-embolized liver lobe in a VX2 liver tumor model in rabbits.

 

 

Twenty-three female New Zealand white rabbits (Harlan, Charles River, France) were acclimatized for 2 weeks under standardized laboratory conditions, including a temperature-controlled room with a 12-hour light/dark cycle and access to standard chow and water ad libitum. The rabbits had a mean weight of 3253±288 g. The Institutional Animal Ethics Committee of the Academic Medical Center of the University of Amsterdam approved this study protocol.

 

After anesthetization of the rabbit, four tumor fragments of 0.5×0.5 mm each were injected superficially in the subcapsular area of the left-medial liver lobe using a 16-gauge (16G) angiocatheter. After the angiocatheter was removed, the liver capsule was manually compressed, and the abdomen was closed in two layers. Five rabbits served as a control group, and 18 rabbits were used for embolization experiments. Two weeks after implantation, the injected VX2 carcinoma had an acquired sufficient mass in the rabbit model to be used for the TAE experiments. Two weeks after TAE, and thus four weeks after tumor implantation, the rabbits were sacrificed.

 

The animals were anesthetized by intramuscular injection of 25.0 mg/kg ketamine (Nimatek, Eurovet, Bladel, the Netherlands) and 0.2 mg/kg dexmedetomidine (Dexdomitor, Orion Corporation, Espoo, Finland). The eyes were protected from drying out using an eye cream (Oculentum simplex, Pharmachemie, Haarlem, the Netherlands). After subcutaneous injection of 0.03 mg/kg buprenorphine (Temgesic, Reckitt Benckiser Healthcare Limited, Hull, GreatBritain) and 0.2 mg/kg enrofloxacin (Baytril, Bayer Healthcare, Berlin, Germany) the rabbit was placed in a supine position. Heart rate and arterial oxygen saturation were measured by pulse oximetry (Hewlett Packard M1165A model 56S, Andover, MA, USA) on the hind leg throughout the procedure.

 

Hepatic artery embolizations were performed by an interventional radiologist (vLKP) with over 10 years of experience. The left central auricular artery, located in the center of the left ear, was punctured and canulated with an 18-gauge (18G) catheter (Hospira Venisystems, Lake Forest, IL, USA). A 3Fr Renegade microcatheter (Boston Scientific, Place Natick, MA, USA) with a 0.014 inch Transend-ex wire (Boston Scientific, Place Natick, MA, USA) was subsequently introduced into the 18G catheter, and under fluoroscopic guidance using a mobile C-arm (Oldelft Benelux, Veenendaal, the Netherlands) advanced toward a retrograde direction through the external carotid artery and the common carotid artery into the aortic arch and the descending aorta. An aortogram was performed, and the origin of the celiac trunk was located. After canulating the celiac trunk and passing the gastric and gastro-duodenal branches, the common hepatic artery was catheterized and the feeding branches of the tumor were visualized. Previous experiments already showed that the small caliber of the tumor feeding vessels made it impossible to embolize with small particles without having spill to the surrounding arterial branches. Therefore a super-selective embolization was performed using one or two 2-mm straight platinum coil (Boston Scientific, Place Natick, MA, USA) as close to the tumor as possible. Finally, control angiography was performed to assure complete embolization of the target vessels, and to verify flow in the spared right cranial and the caudal hepatic arterial branches. After removing the catheter, the puncture site was manually compressed to avoid bleeding.

 

Using a mobile C-arm Exposcop 8000 (Ziehm Imaging, Nürnberg, Germany), we performed angiography at different time points during the experiments. During TAE, the first angiography was done to observe hepatic arterial anatomy and tumor vascularization. Immediately after TAE, control angiography was performed to check the result of complete tumor embolization. Finally, on day 14 after TAE the third angiography was executed to confirm complete occlusion of the hepatic artery.

 

Tumor growth was determined by CT volumetry. With a 64-slice CT scan (Brilliance 64-channel, Philips, Eindhoven, the Netherlands), contrast-enhanced multiphasic CT scans were made for the arterial (15 seconds), portal (30 seconds), and venous phase (45 seconds) after injection of contrast solution (3 mL Visipaque, GE Healthcare, Waukesha, WI, USA) in an ear vein, which was subsequently flushed with 4 mL of sterile physiological saline. On each section of the CT scan, total liver, caudal liver lobe and tumor(s) were outlined manually after which total liver volume (TLV), caudal liver lobe volume (CLV) and tumor volume (TV) were calculated, respectively. CT scans were made 14 days before TAE (on the day of tumor implantation), directly after TAE, and on days 3, 7, 10 and 14 after TAE. During CT scans, the rabbits were anesthetized and positioned in a supine position. After the last CT volumetry, the rabbits were sacrificed.

 

Tumor growth rate (TGR) after TAE was calculated by dividing the calculated TV on x days after TAE (dx) by the calculated TV two weeks after tumor implantation (d0), i.e. the following formula: TGR=TVdx/TVd0. The volume ratio of the caudal, non-embolized liver lobe to the TLV (CLV, %) was calculated by dividing the CLV by the TLV minus the TV, using the following formula: CLV(%)=CLV/(TLV-TV)×100%. In a similar way as CLV, atrophy of the cranial liver lobe volume (CRLV) was calculated with the formula: CRLV (%)=(TLV-CLV-TV)/TLV×100%.

 

The body weight of each rabbit was obtained before sacrifice. After the animal was killed, the entire liver and the caudal liver lobes were weighed using a precision scale (Sartorius, Göttingen, Germany).

 

Liver function and liver damage parameters were assessed in blood samples by routine clinical chemistry. These samples were obtained before tumor implantation, after two weeks of tumor growth, and before TAE. Following TAE, blood samples were taken 3 hours after the procedure, and on post-TAE days 1, 3, 7, 10 and 14. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH) and bilirubin were determined to serve as liver damage parameters. Liver synthesis function was determined by measuring plasma prothrombin time and albumin.

 

Biopsies from the embolized liver lobes and the non-embolized liver lobes were taken at sacrifice. Tissue samples were routinely fixed in 4% formalin (48 hours) and then processed to paraffin tissue blocks. Four µm sections of these blocks were cut and stained with hematoxylin and eosin. The hematoxylin and eosin slides were blindly evaluated by an experienced liver pathologist.

 

Portal inflammation was indiscriminately graded as follows: 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). Sinusoidal dilation was graded as: 0 (absent), 1 (mild; involving ≤1/3 of the (centro-) lobular area), 2 (moderate; involvement ≤2/3 of the liver parenchyma), or 3 (severe; involving >2/3 of the liver parenchyma). Portal edema was scored by determining the percentage of portal tracts involved: 0 (not present), 1 (<25%), 2 (25%-50%), 3 (51%-75%), and 4 (>75%). The presence of areas with the merging necrosis of liver parenchyma was scored as: 0 (absent), 1 (involving <25% of the parenchyma), 2 (involving 25%-50% of the parenchyma), 3 (involving 51%-75% of the parenchyma), and 4 (involving >75% of the parenchyma).

 

For the evaluation of hepatocyte proliferation in normal, non-tumorous liver parenchyma of the embolized cranial liver lobes and the non-embolized caudal liver lobes, staining with the proliferation marker Ki-67 was performed (monoclonal mouse anti-rat Ki-67 antigen, clone MIB-5, Dako Cytomation, Glostrup, Denmark). The immunostained sections were counterstained with hematoxylin. The immunostained sections were quantified in 10 fields of view per section (original magnification ×40) using a Leica CMLB microscope (Leica Microsystems, Wetzlar, GmbH, Rijswijk, the Netherlands) and expressed as percentage of the total amount of pixels in the field of view.

 

Statistical analysis was performed with SPSS 18.0. and GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data were tested for normal distribution, and equal variances. Values were expressed as mean±SEM, unless otherwise stated. Continuous, non-parametric data were compared (TAE versus sham) by the Mann-Whitney U test. The Wilcoxon's signed-rank test was used for non-parametric continuous data for different time points within the groups. Correlation between variables (total/caudal liver volume as measured by CT volumetry and actual liver weight determined at sacrifice) was tested using the Pearson's product-moment correlation coefficient. Histological specimens were evaluated using Fisher's exact test and the Chi-square test where appropriate. Since most of the histology scores are ordinal (there is a ranking in the categories), the linear by linear association test was also used, which is identical to the Fisher's exact test. The median of differences between cranial and caudal hepatocyte proliferation, as determined by Ki-67 staining, was evaluated using Wilcoxon's signed-rank test. Statistical significance was accepted when P<0.05.

 

 

Selective angiography and subsequent embolization of the hepatic artery using a minimally invasive route succeeded in all procedures. All initial angiographies, performed before the embolization procedure, showed a normal blood supply by the hepatic artery and an arterial hypervascularization of the tumor in the cranial liver lobes. The control angiography, following super selective embolization, showed no flow in the embolized hepatic artery, and normal flow in the caudal artery and the gastro-duodenal artery. Angiography performed after 14 days and before sacrifice again showed normal blood flow in the hepatic artery but no flow in the arterial branches feeding the tumor. The tumor was clearly visible on angiography, as a hypo-vascular mass within the normal enhancing parenchyma of the cranial liver lobe.

 

CT volumetry was used to calculate absolute total volume measurements. The absolute data and CLV rates are presented in Tables 1 and 2, respectively. The CLV before TAE, calculated 14 days after tumor implantation, was 22.7±0.7%. The CLV increased to 24.4±1.1% (P=0.172), 25.3±1.3% (P=0.081), 25.0±0.8% (P=0.032), and 26.6±1.0% (P=0.004) on days 3, 7, 10 and 14, respectively. In the sham group, there were no significant differences over time. When the time points were analyzed one-by-one comparing both groups, differences were observed from day 7 till 14, post-TAE (Table 2 and Fig. 1A). The volume percentage of the cranial liver lobe significantly decreased from 76.6±0.7% on day 0, to 70.8±1.7% on day 3, 69.9±2.1% on day 7, 68.4±2.1% on day 10, and 64.9±2.5% on day 14 (Fig. 1B). The TLVs as measured by means of CT volumetry correlated well with liver weight at sacrifice (r=0.965, P<0.001) (Fig. 2A). This was also the case for the caudal liver lobe calculated by CT volumetry, which correlated strongly with the actual weight of the caudal liver lobe at sacrifice (r=0.905, P<0.001) (Fig. 2B).

 

TV increased over time in both groups (Fig. 3A). Considering the separate time points post-embolization, TVs remained stable between days 3 and 7, after which re-growth was seen. The mean volume of the tumor mass after TAE was smaller than that of the sham group, but there was no statistical difference between the groups after 14 days. TGR increased over time in both groups, with a lower trend seen in the TAE-group, although no significant differences were observed between the two groups (Fig. 3B).

 

There was a transient elevation of plasma ALT and AST levels after TAE (Fig. 4). Also, GGT, LDH and albumin were shortly significantly increased, with peak concentrations on days 1 and 3, returning to normal within 7 days (data not shown).

 

Histopathological evaluation of sections of the tumor demonstrated no signs of inflammation. The tumor showed confluencing areas of necrosis and apoptosis. A capsule of fibrotic tissue was seen around the tumor (Fig. 5A). Although 60%-70% of the tumor was necrotic, approximately 30%-40% of the tumor still contained viable tumor cells; whereas in the sham group no tumor necrosis was seen markedly. The sections of parenchyma of the embolized cranial liver lobe demonstrated normal liver structure without tissue necrosis nor apoptosis, but with minimal portal inflammation. Also, mild sinusoidal dilatation was observed in the cranial liver lobe. In accordance with the volumetric results as mentioned above, the caudal liver lobe contained a significantly higher number of proliferating hepatocytes in the Ki-67 stained slides, in contrast to the cranial liver lobe. The median number of proliferating hepatocytes per field of view in the cranial liver lobe (Fig. 5B) was 18 (range 9-106), in contrast to 24 (range 11-77) in the caudal liver lobe (P=0.004) (Fig. 5C).

 

 

For patients with primary or metastatic liver tumor which is unresectable due to insufficient FRL-volume, preoperative PVE is a successful technique to induce hypertrophy of the FRL, and to minimize the risk of extensive liver resection.^[11-13] In recent years, however, an increasing number of articles have been published showing induction of tumor growth as side effect of PVE.^{[6, 14, 15]} Therefore, TAE alone or sequentially in combination with PVE has been suggested to induce hypertrophy of the FRL and in the meantime to prevent tumor growth.^{[10, 16, 17]} A similar hypertrophy response has also been described after hepatic arterial radioembolization using yttrium-90.^[18] Besides destructing the tumor, the radiation treatment causes intrahepatic parenchymal damage, fibrosis and portal hypertension, triggering a regeneration response of the untreated, contralateral lobe.

 

Our study was limited to assessment of the effects of bland hepatic artery embolization on tumor growth and liver regeneration. However, as hepatocellular carcinoma is known to be relatively insensitive for chemotherapeutic agents, the role of embolization is probably more important than chemotherapy.^[9] Causing ischemia to the tumor by the embolization is the most important factor in causing necrosis and reducing the TV. The technique of TAE, using a transauricular approach, appears to be feasible. In our study, only minor spasm caused by the microcatheter and guidewire was seen in the small target vessels, but this did not impede the embolization process. In previous experiments on rabbits, spill of polyvinyl alcohol particles was seen in non-targeted arterial branches, causing ischemia of the stomach and duodenum (data not shown). Therefore, the present experiments were conducted using a single 2 mm microcoil, resulting in complete occlusion of the tumor feeding arterial branches without damaging the surrounding liver tissue. Although arterial embolization did cause atrophy of the tumor-bearing cranial liver lobes, normal liver parenchymal structure with minimal portal inflammation and mild sinusoidal dilatation in the cranial lobes was seen by histological examination.

 

Despite arterial embolization, increased TV was seen on CT volumetry after an initial steady state. TV was smaller than that in the sham group but this difference was not significant. On histopathological examination, however, massive necrosis and apoptosis of the tumor was seen, whereas tumors in the sham group did not show any necrosis or apoptosis. The percentage of necrosis was probably underestimated, as during sacrifice of the rabbits, necrotic parts of the tumor were not included in the histological sections.

 

Although histologically undamaged parenchyma was seen in the cranial lobe after embolization of the tumor, a significant hypertrophy response was apparent in the caudal, non-embolized lobe. These observations indicate that arterial embolization of only the hepatic arterial branches supplying the tumor causes significant tumor necrosis and in addition a hypertrophy response of the non-embolized caudal lobe, in the absence of histological damage of the embolized lobe. The findings at CT volumetry were confirmed by the increased regeneration response observed using Ki-67 staining of histological sections.

 

This is in line with the publication of Vogl et al^[10] who already in 1998 described a 10% volume decrease of the embolized lobe after embolization of the right hepatic artery in patients with an initially unresectable cholangiocarcinoma, and a hypertrophy response of the unaffected left lobe, with a mean increase of 37%. In 2011 Denecke et al^[19] compared TAE with PVE in patients with cholangiocarcinoma and also described a hypertrophy response after arterial embolization. The increase of the FRL-volume, however, was less than that after PVE.

 

It is believed that the regeneration response after PVE is not only caused by the hemodynamic changes after occlusion of part of the portal system, but also by the upregulation of several humoral mediators (HGF, TNF-α, TGF-α, IL-6 and insulin) released after hepatocyte damage, causing a regeneration response that leads to hypertrophy of the non-embolized lobe.^[19] Although the exact mechanism is not specifically investigated, it is assumed that the hypertrophy response after arterial embolization is mediated by the same pathways.^[20] The fact that PVE has a superior hypertrophy response compared with arterial embolization can be explained by its greater effect on overall hepatic blood perfusion. The question remains, however, whether the regeneration response of the caudal lobe is caused only by the damage incurred in the tumor after embolization. On basis of the results of this study, this seems highly suggestive.

 

In conclusion, the transauricular approach of hepatic artery embolization is a feasible technique in a rabbit VX2 tumor model. Super-selective, bland arterial coil embolization causes massive necrosis of the tumor, despite increase of volume on CT volumetry. Atrophy of the tumor-bearing liver lobe is seen after arterial embolization of the tumor with a concomitant hypertrophy response of the non-embolized lobe, despite absence of histological damage of the tumor-surrounding liver parenchyma.

 

 

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