Ten-week-old male Sprague-Dawley rats (body weight, 220 to 260 g; Orient Co., Seongnam, Korea) were housed in standard animal laboratories under controlled temperature (23 ± 2℃) with a 12-hour light-dark cycle and ad libitum access to tap water and standard laboratory chow until 12 hours before the experiment when the animals were fasted. The experimental procedures were performed in accordance with the animal care guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences. The experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University.

Thirty rats were divided into three groups: the control group, the 70% hepatectomy group, and the 90% hepatectomy group. The hepatectomy groups were divided into two subgroups according to the performance of splenectomy, yielding a total of five groups: control (n = 6), 70% hepatectomy (n = 6), 70% hepatectomy with splenectomy (n = 6), 90% hepatectomy (n = 6), and 90% hepatectomy with splenectomy (n = 6).

All procedures were performed with the rats under anesthesia induced by isoflurane inhalation (isoflurane concentration, 1.5 to 3%; oxygen flow 0.5 L/min). A transverse incision was made in all animals. For the 70% hepatectomy, the interlobular ligament was dissected, and the left lateral and median lobes were resected according to the methods described by Higgins and Anderson [12]. For the 90% hepatectomy, additional resections of the right superior and right inferior lobes were performed according to procedures reported by Gaub and Iversen [13]. Splenectomy was performed using 5-0 silk ligatures for the vascular pedicles.

All animals received subcutaneous injections of the following: 5 mL of 10% glucose, 0.1 mL analgesics (Ketorolac, Whanin Pharm Co., Seoul, Korea) and 0.1 mL antibiotics (Rosephin, Roche Korea Co., Seoul, Korea). Animals had ad libitum access to 20% glucose solution for drinking and standard laboratory chow. One hour before sacrifice, 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich Co., St. Louis, MO, USA) was injected intraperitoneally (50 mg/kg).

Body weight was measured before the first operation. The remnant liver was removed and weighed immediately after sacrifice. The liver weight index was defined as the remnant liver weight divided by body weight and was expressed as a percentage.

Twenty-four hours after the surgery, the rats were deeply anesthetized using Zoletil 50 (10 mg/kg, i.p.; Virbac Co., Fort Worth, TX, USA) and blood samples were drawn from the aorta and liver tissue was obtained. Animals were euthanized by exsanguination.

MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide blood samples were used for blood counts. We also measured the following serum parameters to evaluate liver function: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, direct bilirubin, lactate dehydrogenase, and ammonia. The serum was stored at -20℃ until measurement.

Normal saline was perfused through the portal vein and the regenerated liver tissue was removed. The livers were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, treated in xylene, and infiltrated and embedded in paraffin. Coronal sections (5-µm-thick) were cut using a paraffin microtome (Thermo Fisher Scientific Inc., Waltham, MA, USA), mounted on coated slides, and then dried at 37℃ overnight on a hot plate. An average of six sections were prepared for each liver.

5-bromo-2'-deoxyuridine (BrdU)-specific immunohistochemistry was performed to detect newly generated hepatocytes in the liver. Slides of paraffin-embedded liver sections were deparaffinized in xylene and rehydrated in graded alcohol solutions followed by a 5 minutes wash under running water. Tissues were denatured by boiling for 10 minutes in 10 mM citric acid (pH 6.0) and then incubating at room temperature for 10 minutes. The sections were first permeabilized by incubating in 0.5% Triton X-100 in phosphate buffered saline (PBS) for 20 minutes, then pretreated in 50% formamide-2x standard saline citrate at 65℃ for 2 hours, denatured in 2 N HCl at 37℃ for 30 minutes, and rinsed twice in 100 mM sodium borate (pH 8.5). Afterwards, the sections were incubated overnight at 4℃ with BrdU-specific mouse monoclonal antibody (1:600; Roche Korea Co.). The sections were then washed three times with PBS and incubated with biotinylated mouse secondary antibody (1:200; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour and with an avidin-peroxidase complex (1:100; Vector Laboratories Inc.) for another hour. For visualization, the sections were incubated in 50 mM Tris-HCl (pH 7.6) containing 0.03% DAB and 0.03% hydrogen peroxide for 5 minutes. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Liver regeneration index was defined as the number of BrdU-positive hepatocytes in 1 mm².

Collected liver tissues were immediately frozen at -70℃. The tissues were homogenized with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 % glycerol, 1% Triton X-100, 1.5 mM MgCl₂·6H₂O, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₂VO₄, and 100 mM NaF, and then centrifuged at 14,000 rpm for 30 minutes. The protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) and 50 µg of protein was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. Rabbit GAPDH antibody (1:5,000; AbFrontier, Seoul, Korea), rabbit HGF antibody (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse transforming growth factor β1 (TGF-β1) antibody (1:1,000; Serotec Ltd., Oxford, UK) were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratorie Inc.) was used as the secondary antibody for TGF-β1 while anti-rabbit antibody (1:3,000; Vector Laboratories Inc.) was used for GAPDH and HGF. All experiments were performed in normal lab conditions and at room temperature except for the membrane transfer. Membrane transfer was performed at 4℃ with a cold pack and pre-chilled buffer. Bands were detected using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology Inc.). To compare protein expression, the detected bands were quantitated densitometrically using Molecular Analyst version 1.4.1 (Bio-Rad Laboratories Inc.).

Data are presented as the mean ± standard error of the mean. Kruskal-Wallis analysis of variance by ranks and the Mann-Whitney test with Bonferroni correction were performed. Data were considered significant when P < 0.05. All statistical analyses were performed using SPSS ver. 13 (SPSS Inc., Chicago, IL, USA).

The differences in liver weight indexes according to the amount of resected liver were significant (P < 0.001). However, the differences between the splenectomy subgroups were not significant (Fig. 1).

The measured values for serum biochemistry are summarized in Table 1. Among the three liver resection groups, total bilirubin, direct bilirubin, AST, and ALT were significantly higher in the 70% hepatectomy group compared to the control group. Serum albumin was significantly lower in the 70% hepatectomy group compared to the control group. Meanwhile, total bilirubin, direct bilirubin, AST, ALT, and ammonia were significantly higher in the 90% hepatectomy group compared to the 70% hepatectomy group. However, the differences between the splenectomy subgroups were not significant in any group. In addition, platelet counts were not significantly different among the groups.

Liver regeneration indexes were significantly different according to the amount of liver resected (P < 0.001) (Fig. 2B). The difference between the splenectomy subgroups was significant only in the 90% hepatectomy group (P < 0.001) (Fig. 2C). Splenectomy did not significantly affect liver regeneration in the 70% hepatectomy group.

The relative expression of HGF was significantly higher in the hepatectomized groups than in the control group (P < 0.001) (Fig. 3) and also in the 90% hepatectomy group compared to the 70% hepatectomy group (P < 0.001). However, the level of HGF expression was not significantly different between the splenectomy subgroups. TGF-β expression was also significantly higher in the 70% and 90% hepatectomized groups than in the control group (P < 0.001). However, the difference was not significant between the 70% and 90% hepatectomy groups. Subgroup analysis showed that TGF-β expression was significantly lower in the splenectomy subgroup of the 90% hepatectomy group (P = 0.005), but not in the 70% hepatectomy groups. To determine the relative balance between HGF and TGF-β levels, the relative expression ratio of HGF to TGF-β was also determined. The HGF to TGF-β ratio was not significantly different between the control group and the 70% hepatectomy group, but it was significantly higher in the 90% group (P = 0.004). The subgroup analysis showed that splenectomy significantly increased the HGF to TGF-β ratio in the 90% hepatectomy group (P = 0.002), but not in 70% hepatectomy group.