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1 Department of Veterinary Medicine, College of Veterinary Medicine, National
Chung Hsing University, Taichung, Taiwan.
2 Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National
Chung Hsing University, Taichung, Taiwan.
3 Department of Radiology, China Medical University Hospital, No. 2, Yuh-Der
Rd., Taichung 404, Taiwan.
Received January 29, 2004;
accepted after revision June 3, 2004.
Supported by the National Science Council and China Medical University
Hospital, Taiwan, under grants NSC90-2314-B-039–016-M08 and
DMR-91–062.
Abstract
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MATERIALS AND METHODS. Thirty New Zealand white rabbits inoculated with VX2 carcinoma in the liver were included in this experiment. These animals were divided into two groups: one for the evaluation of survival time (n = 20) and the other, for tumor size (n = 10). Each group was divided further into control and treatment subgroups. Hypertonic saline was injected directly into the liver tumor of the treatment group under CT guidance 10 days after inoculation. The liver tumor in the control group was injected with normal saline. The group for evaluation of tumor size was sacrificed 14 days later. The other group was raised until they died.
RESULTS. The survival time of the rabbits in the treatment group (38.1 ± 2.3 days) was significantly longer (p < 0.001) than that of the untreated group (29.9 ± 2.9 days). For tumor size, the difference between the treatment group (8.04 ± 2.46 cm2) and the control group (11.08 ± 2.52 cm2) was also significant (p < 0.05).
CONCLUSION. Hypertonic saline injection had the effect of controlling the growth of VX2 carcinoma cells and extending the life of rabbits. It deserves further investigation.
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Percutaneous injection of 95% ethanol provides excellent results in tumors smaller than 3 cm with a complete necrotic rate of 70% [8, 9]. The complications are minor, and the cost is acceptable. Radiofrequency ablation affords advantages over ethanol injection in that tumor control can be achieved with fewer sessions and larger lesions can be treated. Recent data have suggested that radiofrequency achieves similar objective response rates as percutaneous ethanol injection with fewer sessions [10–12]. However, no data on longterm survival after radiofrequency are available to compare with the reported 50% 5-year survival of Child-Pugh's class A patients treated by percutaneous ethanol injection that achieve complete maintained response [13].
Cryoablation is an effective method for the treatment of large liver tumors, but its use has been limited to patients with relatively well-preserved liver function because of its higher treatment-related morbidity and the requirement for laparotomy [14]. The other two interstitial therapies used to treat hepatic tumors are microwave and laser ablation, which have been performed most commonly in Japan and Germany, respectively [15, 16]. The necrotic area achieved with one microwave irradiation treatment usually is small. Multiple treatments are therefore necessary. Some researchers claim that laser ablation is highly effective for the treatment of both hepatocellular carcinoma and colorectal metastasis [17, 18]. Overall, the interest in and enthusiasm for radiofrequency thermal ablation have exceeded that for either microwave or laser ablation. The radiofrequency technique is somehow more aggressive than conventional percutaneous ethanol injection and uses larger needles (14–18 gauge). Major complications may affect up to 10% of patients [10, 12, 19], a figure considerably higher than that reported with percutaneous ethanol injection. A group of researchers who used radiofrequency ablation in a large series of patients with metastatic liver disease have described a high rate (12%) of tumor seeding [20, 21]. Percutaneous ethanol injection therefore should remain the primary treatment option when radiofrequency ablation is not available.
Because no single method is ideal now, it is logical that ongoing searching of new therapeutic methods with simple technique, low cost, fewer sessions, a lower complication rate, and high effectiveness might be beneficial to patients. In this article, we propose a new method for ablation of VX2 tumors in rabbits by injection of a high concentration of hypertonic saline. Hypertonic saline has been used for treatment of benign tumors and has been shown to be effective [22, 23]. To our knowledge, this study is the first one to evaluate direct injection of hypertonic saline for treatment of malignant liver tumor.
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Preparation of VX2 Cell Suspension and VX2 Cell Mass
In this study, the VX2 carcinoma was maintained through serial
transplantation into the hind limb muscle of the New Zealand white rabbit.
After implantation, the tumor enlarged rapidly. For preparation of VX2 tumor
cells suspension, the VX2 tumor was stripped aseptically, mechanically minced,
filtered through iron mesh with 0.08 mm2 pores, and centrifuged at
2,000 rpm for 10 min. Finally, the viable cells were adjusted to a
concentration of 1 x 107 cells/mL. For the VX2 cell mass, the
tumor was excised from the hind limb of the rabbit. The surrounding connective
tissue and fat were removed from the tumor. Then, the tumor was cut into
1-mm3 pieces and readied for implantation.
Effect of NaCl Concentration on Viability of VX2 Cancer Cells
To evaluate the impact of concentrations of NaCl on the viability of VX2
cancer cells, we added different concentrations of NaCl solution (0.9%, 5%,
15%, 25%, and 36.5%) to test tubes with an equal number of VX2 cancer cells
(2.3 x 107). Another test tube of VX2 cancer cells was taken
as a control with the addition of only 10 mL of culture medium. These six test
tubes were placed in an incubator of 37°C with 5% CO2. Then, 1,
3, 5, 7, and 12 hr later, 50 µL of cell suspension was drawn from
each test tube and mixed with 50 µL of trypan blue. Of the
mixture, 15 µL was examined under a light microscope (x100)
for evaluation of cell viability. While the viable cancer cells were not
stained, the dead cells were stained blue.
Blood Sodium and Chloride Concentration After Injection of Hypertonic Saline
The blood concentration of sodium and chloride was checked before and after
injection of different amounts of 36.5% hypertonic saline (1, 2.5, 5, and 8
mL) at different times (5 min, 2 hr, and 6 hr) in four rabbits. The irritation
of 36.5% hypertonic saline on the injected vessel also was evaluated in the
rabbit injected with 5 mL of hypertonic saline by examining the vessel 3 days
later using a light microscope. Another rabbit was examined to assess the
effect of hypertonic saline retention on blood vessels by injecting the same
amount of hypertonic saline and applying a tourniquet for 3 min in the
proximal portion of the injection site so the hypertonic saline remained in
the vessels.
Effect of Hypertonic Saline on Normal Liver Tissue
Twenty-five New Zealand white rabbits were divided into five groups of five
rabbits each. A different amount of 36.5% hypertonic saline—0.5, 1, 1.5,
2, and 3 mL—was injected into the liver of each group after subxiphoid
laparotomy. The necrotic areas were examined 10 days later on CT. One rabbit
in each group was sacrificed at a different time after the injection of
hypertonic saline (10 days–6 weeks) to examine the histopathologic
change under a light microscope.
Implantation of VX2 Cancer Cells
A subxiphoid laparotomy, 3–5 cm in length, was performed with the
rabbit under IV anesthesia to expose the left lobe of the liver to implant the
VX2 carcinoma into the liver. Forty-five rabbits were divided into three
groups. In group 1 (n = 19), 0.1 mL of tumor cells suspension
(106 cells) was injected into the liver with a 27-gauge needle.
Before removal of the needle, the injection site was compressed with a cotton
plug wetted with alcohol for 3 min. In group 2 (n = 16), the same
number of cancer cells suspension was injected into the left lobe of the liver
followed by 0.2 mL of 1% heated agarose. After this injection, we waited for 2
min before removing the needle to allow the agarose to cool down to fix the
injected tumor cells within liver tissue and to seal off the needle track to
prevent regurgitation of tumor cells. In group 3 (n = 10), under
conditions of a sterile laparotomy, a 1-mm3 fragment of VX2
carcinoma was inoculated into both the left and right lobes of the rabbit
liver. The growth of the tumor was evaluated on CT 10 days after
inoculation.
Microscopic Examination
All of the specimens of the injected liver tissue and tumors were fixed in
10% formaldehyde, sectioned, and stained with H and E. The histologic changes
were examined under a light microscope.
Survival Time
The 20 rabbits that were inoculated successfully by direct injection of
cancer cells suspension were divided randomly into treatment and control
groups (10 in each). These rabbits underwent CT 10 days after the inoculation.
The tumors in the treatment group were treated with hypertonic saline, and
those in the control group were treated with same amount of normal saline.
Both groups of rabbits were kept in the same room and environment for
evaluation of survival time.
Assessment of Tumor Size
The 10 New Zealand white rabbits inoculated with 1-mm3 tumor
masses in both the right and left lobes of the liver were included in the
assessment of tumor size. The tumor in the left lobe of the liver was taken as
the treatment group, whereas the tumor in the right lobe of the liver of the
same rabbit was taken as the control group. Ten days after the inoculation,
these animals underwent CT. Two weeks after injection of hypertonic saline and
normal saline in the left and right lobe tumors, respectively, under CT
guidance, all the rabbits were sacrificed and the livers were removed and
examined slice-by-slice at a thickness of 5 mm, the same as that of a CT scan.
The largest cross-sectional areas of the tumors were used for evaluation of
therapeutic response.
CT Examination
Injection therapy was performed under CT guidance (PQ 5000, Picker). The
unenhanced images of the liver were acquired at first with a slice thickness
of 5 mm. Enhanced CT was performed after IV injection of iodinated contrast
agent containing diatrizoate meglumine and diatrizoate sodium (2 mL/kg of body
weight, 76% Urografin, Schering). After identification of the tumor nest,
36.5% hypertonic saline mixed with a small amount of Urografin (20:1) was
injected into the lesions in the treatment group with a 22-gauge needle. The
amount of hypertonic saline was justified to 0.1 mL for 1-mm diameter of
tumor. For example, if the diameter of the VX2 tumor lesion was 1.2 cm, then
1.2 mL of hypertonic saline was used for injection. The same amount of normal
saline was injected into the tumors of the control group. The biggest
cross-sectional area of the tumors was calculated using the formula for the
area of an ellipse:
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Blood Sodium and Chloride Concentration After Injection of Hypertonic Saline
The concentration of sodium and chloride in the blood was within the normal
range after injection of 1 mL of 36.5% hypertonic saline. Although there was
transient elevation of sodium and chloride level in the blood with injection
amounts of 2.5, 5, and 8 mL, they returned to the normal range 6 hr after
injection (Figs. 2 and
3). All of the rabbits showed
normal appetite and physical condition.
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Effect of Hypertonic Saline on Normal Liver Tissue
Different amounts of hypertonic saline created different sizes of necrotic
areas. For 0.5 mL, a necrotic area of 0.13–0.24 cm2, (mean,
0.16 ± 0.04 [SD] cm2) was created. The necrotic area for 1
mL was 0.71–1.04 cm2 (mean, 0.88 ± 0.12
cm2); for 1.5 mL, 1.12–1.53 cm2 (mean, 1.38
± 0.18 cm2); for 2 mL, 1.43–1.76 cm2 (mean,
1.62 ± 0.13 cm2); and for 3 mL, 2.83–3.46
cm2 (mean, 3.18 ± 0.26 cm2). There was good
correlation of the necrotic area with the injection amount in linear
regression analysis (y = 1.5028x–0.3295;
R2 = 0.8683) (Fig.
4).
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Implantation of VX2 Cancer Cells
In group 1, by direct injection of cancer cells suspension followed by
compression with an alcohol cotton plug, six (32%) of 19 rabbits were
inoculated successfully with VX2 tumor in the liver. Ten rabbits showed tumor
seeding outside the liver, and three were not found to have tumor growth in
the liver. These 13 rabbits were regarded as cases of failed inoculation. In
group 2, by direct injection of cancer cells suspension followed by heated
agarose in 16 rabbits, 14 (88%) were inoculated successfully without tumor
seeding. In group 3, 20 VX2 tumor blocks were inoculated in both the left and
right lobes of the liver in 10 rabbits, 19 sites (95%) had tumor growth
without seeding; tumor was not found in one remaining implantation site.
Comparison of Survival of Treatment and Control Groups
The survival time of the 10 rabbits in the control group ranged from 27 to
35 days. The mean was 29.9 ± 2.9 days. The survival time of the other
10 rabbits in the treatment group was 35–43 days. The mean was 38.1
± 2.3 days. The statistical analysis with paired Student's t
test showed the difference in the survival times of the treatment and control
groups was significant (p < 0.00.1)
(Fig. 5).
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Tumor Sizes
Before treatment, the largest cross-sectional area of the tumor in a left
lobe was 1.73 cm2, and the smallest was 0.38 cm2. The
mean was 1.12 ± 0.42 cm2. The largest cross-sectional area
of the tumor in a right lobe was 1.65 cm2, and the smallest was
0.33 cm2. The mean was 1.04 ± 0.37 cm2. The
difference between the two groups was not significant.
Two weeks after the treatment, the largest cross-sectional area of tumor under histologic examination in a right lobe of the liver in the control group was 15.22 cm2, and the smallest one was 7.56 cm2. The mean was 11.08 ± 2.52 cm2, whereas the largest cross-sectional area of tumor in a left lobe of liver in the treatment group after hypertonic saline injection was 13.57 cm2, and the smallest one was 3.98 cm2; the mean was 8.04 ± 2.46 cm2. Statistical analysis with Wilcoxon's paired signed rank test showed the cross-sectional area of tumor in the control group was significantly larger than that of the treatment group (p = 0.015) (Table 1).
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Histologic Findings
Vessel.—Although the vessel of the injection site with
normal passage of hypertonic saline showed no evidence of detectable lesion on
a light microscope, the vessel in which hypertonic saline was retained for 3
min showed damage to the endothelial cells, fewer viable cells, and thrombus
formation in the vessel lumen. Inflammatory cells also were present there.
Normal liver tissue.—Marked necrosis of the liver tissue was found in the injection region (Figs. 6A, 6B, and 6C). The necrotic area was surrounded by fibroblasts and proliferated connective tissue. The dead hepatocytes in the necrotic area were removed by infiltrated macrophages. The hepatocytes adjacent to the necrotic area showed degenerative change and cytoplasmic vacuolation. After 6 weeks of injection, most of the necrotic tissue was removed and replaced by infiltrated fatty cells.
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VX2 tumor.—Fourteen days after injection of 36.5% hypertonic saline, marked coagulative necrosis of the tumor cells was found. The tumor mass was surrounded with proliferated collagen and fibroblasts. Foci of hemorrhagic reaction and calcification also were noticed within the necrotic tumor. There were macrophages and multinuclear giant cells in the necrotic area to move the debris of the dead cancer cells (Figs. 7A, 7B, 7C, 7D, 7E, and 7F).
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Surgical treatment is thought to be the most effective management of small hepatocellular carcinoma. However, poor hepatic functional reserve often precludes surgery. Patients who are not good candidates for surgery, therefore, need other less-invasive therapies. Also, in recent years, the waiting time for patients with hepatocellular carcinoma who are selected as candidates for liver transplantation is increasing progressively, so safe and effective methods to control tumor growth in these transplantation candidates has become a logical and critical issue [7].
Transcatheter hepatic arterial chemoembolization is performed widely as an alternative to resection [25, 26]. However, for large tumors with multiple arterial supplies and small tumors with inadequate angiogenesis, its effect is limited [27]. In contrast, percutaneous ethanol injection can be performed in all patients with small hepatocellular carcinomas except those in whom tumor foci are too numerous or small hepatocellular carcinomas cannot be depicted on sonography or CT [8, 28]. The disadvantage of percutaneous ethanol injection is that it requires a large number of treatment sessions. The number of sessions may be reduced by injecting a larger volume of ethanol during each session than that normally used, but this might increase the frequency of side effects such as portal vein thrombosis and cholangitis. Percutaneous acetic acid injection is as equally effective as percutaneous ethanol injection in the treatment of hepatocellular carcinoma and with the advantage of fewer treatment sessions in each treatment course [29].
Radiofrequency ablation for the treatment of patients with hepatocellular carcinoma has been reported previously and is superior to percutaneous ethanol injection for the enlargement of the necrotic areas. Treatment-related complications with radiofrequency ablation occur in approximately 2–10% of patients [10, 12, 19, 30]. Major complications such as hemoperitoneum, pneumothorax, portal vein thrombosis, capsular hematoma, neoplastic seeding, and infections have been described, and the incidence of these complications has been shown to be significantly higher than that associated with percutaneous ethanol injection [10].
The high complications rate of radiofrequency ablation reported in a study may have been related, at least partly, to the low gauge of the electrode used and to heating of normal structures adjacent to the tumor [10]. Intratumoral fibrosis also may influence heat diffusion (the "oven effect" in reverse) and thereby limit the efficacy of radiofrequency ablation [10]. There is no clear evidence that radiofrequency has a higher effectiveness or impact on survival than conventional percutaneous ethanol injection, and the sole proven benefit is that it requires significantly fewer sessions to achieve response similar to that achieved with conventional percutaneous ethanol injection [10]. One study even showed the complete response rate for small hepatocellular carcinomas (< 3 cm) with a mean of 1.25 radiofrequency ablation sessions was 76%, which resembles previous figures obtained using percutaneous ethanol injection [20]. Therefore, prospective randomized controlled trials are needed to prove whether there is any benefit favoring radiofrequency ablation in terms of antitumoral effect, cost effectiveness, or long-term survival [20].
The ideal therapeutic technique would be simple, effective, economic, and safe. Unfortunately, the currently available methods described cannot fulfill these goals. Continuous searching for other methods is needed and will be beneficial to patients.
Hypertonic saline has long been used for resuscitation of patients in shock and with head injury, for treatment of brain edema, in patients with fluid imbalance, and for induction of sputum. It also has proven to be effective for the treatment of various kinds of lesions such as hepatic hydatid cyst, gastrointestinal ascariasis, hemangioma, renal cyst, and glomus tumor [22, 23, 31–38]. Hypertonic saline has been used for injection into the lumbar intervertebral disks and has caused localized necrosis of the nucleus pulposus cells in a concentration-related fashion [39]. Our experiment showed hypertonic saline can cause necrosis of the liver of healthy rabbits and VX2 tumor. In this experiment, we had no experience in treating large tumors—that is, larger than 3 cm. However, according to the research of Ajito et al. [40], 7.2% hypertonic saline, administered in a dosage of 5 mL/kg of body weight, was safe to healthy beagles for expanding the plasma volume without inducing hypernatremia [40]. Similarly, when 36.5% hypertonic saline is injected in a dosage of 1 mL/kg of body weight, it should be safe. On the basis of this fact, a patient weighing 50 kg should be able to tolerate injection of 50 mL of 36.5% hypertonic saline without marked systemic effect. Therefore, it is expected that a large amount of this agent could be used one time for the treatment of a big hepatic tumor.
A decrease in the number of treatment sessions might reduce the complications rate. Another important benefit of this agent is when it is injected incidentally into blood vessels, it will be diluted rapidly by blood flow without remarkable local or systemic effect. Histologic examination of the normal liver tissue and VX2 tumors treated with hypertonic saline showed evidence of coagulative necrosis with hemorrhage and calcification. The treated tissue was surrounded by fibroblasts and abundant collagen. This reactive tissue barrier, similar to that of percutaneous ethanol injection [41, 42], might prevent the further spread of the tumor.
The diffusibility of hypertonic saline is supposed to be similar to that of ethanol. Figure 7C shows that the injected saline remained in the tumor without leakage into the surrounding liver tissue but that when the liver is not cirrhotic, the tumor has no capsule, or the amount of injected agent is too much, easier diffusion into the surrounding liver tissue is expected to occur for these two agents. Further work, however, is needed to prove this hypothesis.
In this study, the survival time in the treatment group was significantly longer than that of the control group. This result is similar to that of research conducted by Kurohiji et al. [43] using ethanol as the therapeutic agent. The mean survival times in their study were 39.5 ± 5.7 days and 29.8 ± 2.9 days for the treatment and control groups, respectively. It therefore is expected that the effect of 36.5% hypertonic saline should be similar to ethanol in the control of the growth of VX2 cells. Additional comparative studies are needed to clarify this issue.
Regarding control of tumor growth, the effect of hypertonic saline injection was also obvious. Vargas et al. [44] used ethanol to treat the VX2 liver carcinoma. The mean size of the tumors was 0.96 ± 0.65 cm2 before treatment and 4.59 ± 3.4 cm2 after treatment in the experiment group. In the control group, the size of tumor was 0.95 ± 0.45 cm2 before and 6.73 ± 2.1 cm2 after normal saline injection [44]. In our experiment, the tumor was larger after hypertonic saline injection than that in their study with ethanol injection. Several reasons might account for this discrepancy. One is the delay of treatment because the tumor before treatment in our experiment was larger than the tumor in their experiment. Another reason may be that our therapeutic dosage was not enough to infiltrate the tumor cells fully. We have been aware of this experimental problem but cannot resolve it because of the small size of the liver of rabbit. Hence, when a larger amount of hypertonic saline was injected into the tumor lesion, leakage of hypertonic saline into the peritoneal cavity always occurred. The other reason is that a significant amount of the hypertonic saline was injected into the necrotic part of the tumor, which usually is seen even in small VX2 cancer. This made the effective dosage unpredictable.
We conclude that high concentration of hypertonic saline is a potentially effective agent for ablation of liver tumor. The procedure is simple and safe. The experimental result would be better if we can treat the tumor earlier, use a higher dosage of hypertonic saline, and inject it in the viable tumor part.
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), 2.5
(
), 5 (
), and 8 (•) mL.
