Introduction

Surgical resection has been considered as the primary choice for a radical cure of liver cancer. But the resectability rate of hepatocellular carcinoma (HCC) is only about 20%-30% and its recurrence and metastasis are common even after radical resection.[1-4]And the resectability rate of metastatic liver cancer is only about 5%-25%.[5]Thus non-surgical treatments have been the focus of research in this field. In these treatments such as transcatheter arterial embolization(TAE), percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), and microwave coagulation,[6-8]PEI has been widely used clinically because of its easy manipulation and high safety.[9-11]In recent years, new agents including acetic acid, hot saline and hot distilled-water have broaden the indications for the treatment of this disease; but these treatments have the same shortcomings like local diffusion, unequal permeation and effect of tumor mesenchyma, which result in tumor recurrence at injection position.[12,13]If an agent is available for control or killing of the tumor, the completeness of the treatment and the survival rate of patients can be improved.

Recently, Tamai et al[14] have reported research of percutaneous alkali injection in animals. In our research, the controllability of necrotic lesion volume and the completeness of necrosis were observed after injection of low concentration sodium hydroxide solution in normal liver parenchyma, and the relationship between concentration, dose and lesion volume was also studied in order to assess its possibility in injection treatment of liver cancer instead of ethanol.

Methods

Materials and experimental design

 Experiments were performed in 27 New Zealand rabbits weighing 2.5 kg to 3.0 kg, 20 male and 7 female rabbits, provided by the Experimental Animal Center at Zhejiang University, Hangzhou, China. Sodium hydroxide solution prepared at the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China at concentrations of 1%, 2.5%, 5% was injected respectively into the liver at three different volumes in the range between 0.2 and 1.0 ml, using 22G PTC needles (Hakko Medical Co. Ltd., Japan).

The animals were divided into 9 groups (Aa, Ab, Ac, Ba, Bb, Bc, Ca, Cb and Cc) by a 3×3(three-by-three) factorial design, each consisting of 3 rabbits. Group A was given sodium hydroxide solution at a concentration of 5%, while B 2.5% and C 1%. Each group received 3 different doses of a (0.2 ml), b(0.5 ml) and c(1.0 ml). Each rabbit was injected for one time. Then another 3 rabbits, which were taken as side-effect group were dropped with 0.5 ml sodium hydroxide solution at concentrations of 1%, 2.5% and 5% respectively in their liver lobe space.

Experimental procedure

Anesthesia

Before injection, the rabbits were injected 1% pentothal sodium via the vein of ear at a dose of 3 ml/kg. After anesthesia the rabbits were fixed on the operation table in supine position and their upper abdomen were sheared for ultrasonography.

Injection

All solutions were injected percutaneously into the largest liver lobe rapidly using a 22G needle, guided by ultrasound. Each time the needle tip was placed at a depth of 1.5 cm from the liver theca. In the side-effect group, 0.5 ml 1%, 2.5% and 5% sodium hydroxide solutions were dropped respectively in the space between two liver lobes.

Monitoring and measurements

Sonographic examinations were performed with a Sequoia-512 ultrasound scanner (Siemens, Co.Ltd., Germany) with a small-part transducer(15L8W). B-mode was set at a frequency of 8-13 MHz. The lesion and echo change during the injection procedure and 15 minutes after the injection were observed by ultrasound continuously. The lesion volume was measured by ultrasound with the following equation: V=4/3π×a×b×c; a: occipitofrontal diameter, b: transverse diameter, and c: longitude diameter. Seven days later, each rabbit was examined again ultrasonographically for the image change of local lesion.

Blood and pathological examination

After injection the rabbits were fed with regular food for 7 days, after that they were sacrificed. The liver was then removed, fixed in 10% neutral buffered formalin for histopathological examination. The liver specimens were stained with hematoxylin and eosin. Liver and kidney function of the rabbits was examined before injection and compared with that 7 days later.

Side-effect observation

All the rabbits were sacrificed 7 days after injection. Then their abdominal cavity was examined for enterocoelia synechia and ascites.

Statistical analysis

The data were expressed as mean±SD and analyzed by a computer software SAS 8.02 for Windows. The necrotic lesion volumes were analyzed by  factorial design ANOVA, and the liver and kidney function indexes were analyzed by Student’s t test. A P value less than 0.05 was considered statistically significant.

Results

Ultrasonographic changes during and 7 days after the injection

When injected with sodium hydroxide solution at a concentration of 5%, a transient hyper-echo area was found from the needle tip (Fig.1). The hyper-echo area extended rapidly and stopped in some certain area forming an oval local necrotic lesion with the border showing clearly hyper-echo. This course continued only from 1 to 5 seconds. Subsequently, from the needle tip the hyper-echo area demonstrated low-echo or non-echo, slightly enlarged, and stopped extending in a very short time (Fig.2). This low-echo or non-echo area had a clear boundary to the surrounding normal hepatic parenchyma. The diameter of the lesion increased a little but kept static after 10 minutes. Seven days later ultrasonography showed that the area was of homogeneous hyper-echo (Fig.3). The ultrasound changes after injection of 2.5% sodium hydroxide were just similar to those after injection of 5% of sodium hydroxide solution. But injection of 1% sodium hydroxide produced no obvious ultrasound image changes momentarily and after that the focal lesion only demonstrated a slightly high echo. Hence it was difficult to measure the size of the lesion because of blurred boundary. And another 7 days later, the same lesion still showed a slightly high echo. This image suggested the lesion be incomplete necrosis, which was histopathologically confirmed. Since 1% sodium hydroxide solution did not cause complete necrosis, this solution was not an ideal injection agent. All ultrasound and histopathological changes of the different injection groups after injection of sodium hydroxide are shown in Table 1.

Necrotic lesion volume in different groups

Since 1% sodium hydroxide solution did not produce complete necrosis, which was monitored ultrasonographically, this concentration was rejected and the experiment was modified to a 2×3 factorial design. Lesion volume between the different groups was compared (Table 2).

ANOVA showed no significant difference in lesion

volume between groups A (5%) and B (2.5%)(F=0.11, P=0.75). Thus the concentration of sodium hydroxide solution was not related to the lesion volume. At the same time, the lesion volume was not the same in all the three dose groups (F＝18.97， P<0.01). Student Newman Keuls (SNK) procedure showed a significant difference in volume between group a (0.2 ml) and group b (0.5 ml) (P<0.05), group a (0.2 ml) and group c (1.0 ml) (P<0.05), and group b (0.5 ml) and group c (1.0 ml)(P<0.05). So the lesion volume increased with the increased dose and the necrotic volume was measured according to the injected dose(Fig.4).

Histopathological results

Histopathological examination showed that 5% and 2.5% sodium hydroxide solution produced complete necrosis (Fig.5). The necrotic lesions were oval, localized, and pale under the naked eye. At the center there were necrotic tissues and small fluffed-cotton cavities. Under a microscope necrosis or thrombosis of blood vessels and bile ducts was observed. Near the lesion some bleeding and rarefaced hepatocytes of cytoplasm were noted. But 1% sodium hydroxide solution injection (0.2 ml) caused incomplete necrosis. Under a microscope edema, hyperemia, infiltration of inflammatory cells, swelling of angio-endothelial cell and megakaryocytic reaction were observed in the liver tissues.

Hepatic and renal function changes and side-effect observation

After injection, all the 30 rabbits survived for 7 days. The levels of ALT, creatinine, urea nitrogen in their blood did not change markedly (P>0.05, Table 3). In the side-effect group, when the alkali solution was dropped in the space between the two lobes of the liver, local hyper-echo was found immediately. Seven days later the envelope of the local liver became pale macroscopically. The liver tissues, however, were still normal under the pale envelope, and the rabbits developed no enterocoelia synechia and ascites.

Discussion

HCC is the fifth commonest malignancy worldwide.[15,16]In China, it is also one of the most popular malignancies and its incidence has been increasing in recent years;[17]but most patients with liver cancer are not indicated for surgery. In non-operative therapies, percutaneous ethanol injection therapy (PEIT) has been widely used,[18-21] because it is easy, cheap and repeatable. In some tumors, however, ethanol cannot permeate into the fibers fully and it is not perfect to treat patients. Therefore many researches have focused on finding more effective agents for injection, for instance, acetic acid,[22]hot ethanol, hot saline or ethanol mixed with lipiodol[23-26] and others better than ethanol; but all are still not satisfactory. In 2000, Tamai et al[14] reported alkali injection at different concentrations in HCC as a promising form of local therapy. They found that the necrotic volume of the tumor increased with the increased concentration of sodium hydroxide solution. Liu et al[27] in China also reported similar results in the same year.

After 99.5% ethanol was injected into the liver tumor, the protein of tumor cells degenerated and formed a boundary soon. That boundary impeded ethanol to diffuse fully in the whole tumor, but some tumor cells left alive.[27-30]In this study, alkali solution injection at the concentration of 2.5% and 5% caused rapid, thorough necrosis of normal liver, and the necrotic lesions were localized, oval, or tumor shaped. The lesions showed a low-echo and a clear boundary to the normal liver parenchyma. These findings in this study suggested alkali solution at the concentration of 2.5% and 5%be an ideal agent for intratumor injection. But the relationship was not clear between lesion volume, concentration and dose of the solution, and it was not sure whether there was an interaction between the concentration and the dose. Hence we assessed the necrotic effect of alkali solution at different concentrations, and the relationship not only between the necrotic volume and the concentration but also between the volume and the dose.

In this study, ANOVA showed that the lesion volume produced by the same dose was not significantly different at the concentration of 2.5% and 5%. Considering the effect and safety, we recommend 2.5% sodium hydroxide solution for injection. The lesion volume produced by 2.5% and 5% alkali solution in the normal rabbit liver is only dependent on the dose injected. Hence control of the volume of necrotic lesion by regulating injection dose will be helpful in treating HCC of different size thoroughly while protecting the normal tissues surrounding the tumor.

The lesion volume produced by 2.5% and 5% sodium hydroxide solution is not significant, and it is increased with the increased injection dose. We consider that the well penetration of sodium hydroxide solution in liver tissues and the colliquative necrosis caused by it are related to these results. Because the two concentrations of sodium hydroxide solution cause colliquative necrosis, OH^- can be diluted and stopped to act on the tissue nearby quickly. A high dose ensures a big diffusing volume that can result in a big necrotic lesion. From the ultrasound findings in this study showed that the tissue around the needle tip demonstrated hyper-echo at the moment of injection, and that about 5 seconds later, the hyper-echo turned to be low-echo and even non-echo. These indicated that sodium hydroxide solution could diffuse and permeate easily in the hepatic parenchyma and cause colliquative necrosis rapidly. Because the concentration of sodium hydroxide solution in local lesion is decreased fast, the agent stops to act immediately. We consider that the lesion volume is not related to the concentration but to the dose, as confirmed by the ultrasonographic changes in the whole injection period.

After injection of sodium hydroxide solution, at the concentration of 1%, vague lesion boundary was seen in this study but no obvious local low-echo changes and histopathological results post-injection showed incompleted necrosis in the lesion. Hence we excluded 1% sodium hydroxide solution for incomplete necrosis.

After injection, all the animals survived for more than 7 days. The changes of liver and renal function showed no serious side-effects. Even in the side-effect groups no obvious enterocoelia synechia and ascites were found. These indicate that it is safe to inject a low dose of sodium hydroxide solution at a low concentration in the liver. Further study of the effects of low-dose and low-concentration alkali and its side-effects on other organs is required to assess the clinical value of percutaneous injection of low-concentration sodium hydroxide solution. Our study has demonstrated that injection of percutaneous low-concentration sodium hydroxide solution other than ethanol is helpful in treating liver malignancy.

Funding: None.

Ethical approval: Not needed.

Contributors: JTA wrote the paper and was primarily involved in the experiment design. ZQY proposed the literature search and was involved in experiments monitoring on ultrasound. CMY analyzed the data and jointly wrote the paper. ZSS critically reviewed the paper and provided imputs for the final draft. JTA is the guarantor.

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.

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Received Novermber 12, 2004

Accepted after revision March 2, 2005