A total of 216 male Sprague-Dawley rats (age, 6–8 weeks; weight, 200–220 g), were obtained from the Experimental Animal Center of the Academy of Military Medical Science (Beijing, China). Animals were fed standard chow with water ad libitum and kept at 24°C and 40% humidity with a 12 h light/dark cycle. OJ was induced by ligation of the common bile duct (CBD). To ensure the accuracy of the procedure, an operating microscope for small animal surgery was used (GX.SS.22-3, Binocular Operation Microscope; Shanghai Medical Optical Instruments Factory Co., Ltd, Shanghai, China). The current study was conducted in accordance with the guidelines set by the Guide for the Care and Use of Laboratory Animals (16) and was approved by the Ethical and Research Committee of the Chinese PLA Medical School (Beijing, China).

Prior to surgery, rats were fasted overnight with free access to water. Anesthesia was performed by isoflurane/oxygen inhalation (4% isoflurane for induction, 1.5% for maintenance of anesthesia). Hair on the abdomen of the rats was removed and an upper midline abdominal incision of ~3 cm was made following sterilization. The CBD was exposed and two 8-0 monofilament, non-absorbable nylon sutures (Shanghai Pudong Jinhuan Medical Products Co., Ltd, Shanghai, China) were placed under the bile duct between branches and anastomoses of CBD and pancreas. The sutures were ligated and the CBD between the two sutures was dissected. The abdominal wall was closed with a continuous suture. A second surgery was performed on rats with OJ following 7 days (17).

Rats with OJ were randomly assigned into three groups: C group, sham operation/control group; OPT group, rats with OPT, including portal vein and hepatic artery; and OPV group, rats with OPV and preservation of the hepatic artery. The duration of occlusion was set to 15, 20, 30, 40, 50 and 60 min, and the 7-day survival rate following the second surgery was investigated in the OPT and OPV group (n=10 for each time point in each group, total n=120). The duration of occlusion was set to 30 min for liver injury and liver regeneration studies, and the sample time points were at 6, 24, 72 h and 7 days post hepatectomy (n=24 for C group, n=24 for OPV group and n=36 for OPT group). A total of 12 rats were used for the measurement of liver blood flow.

After a total of 7 days following the surgery inducing OJ in rats, a second surgery was performed. The procedure combined biliary recanalization and partial liver ischemia under portal blood bypass through the caudate lobe (14) and a partial hepatectomy was performed in all rats. Rats with OJ were anesthetized as described above. The abdomen was entered through the incision of the previous procedure and dilated CBDs were isolated. A cannula (8×0.8 mm) was inserted into the duodenal side of the original bile duct and fixed. The second tip of the cannula was inserted into the dilated CBD and fixed.

When the recanalization of the bile duct was completed, the left and median liver lobes, which were resected in following steps, were ligated with 3-0 silk sutures. The pedicle, for the OPT group, or the portal vein, for the OPV group, of the right liver lobe was dissected and clamped with a microvascular clamp. In the C group, the pedicle was dissected but not clamped. The clamping was maintained for 15–60 min. During ischemia, the abdomen was covered with plastic film to prevent evaporation of body fluids. Following occlusion, clamps were removed and liver reperfusion was initiated. The left, median and caudate lobes were resected and the abdomen was closed with 3-0 sutures. Following surgery, all rats were provided with rodent chow and water ad libitum until 6, 24, 72 h and 7 days post hepatectomy.

Serum and liver tissue samples were collected at 6, 24, 72 h and 7 days following partial hepatectomy (n=6 for each time point of each group). Serum samples from rats with OJ that did not undergo the second surgery were also collected for comparison (n=6). All rats were anesthetized as mentioned above and 5 ml blood was obtained from the inferior vena cava for serum testing. Then ≥2 ml blood was drawn to sacrifice the rats weighing 200–220 g by exsanguination. The liver were resected and weighted. The blood samples were centrifuged at 2,500 × g at 4°C for 15 min. The supernatant was stored at −80°C for serum tests. Tissue samples that were taken from the remnant liver were cut into two pieces. One piece was immediately frozen in liquid nitrogen and stored at −80°C for detection of gene and protein expression, the other piece was further sliced and fixed at 4°C for 24 h with 10% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for histopathological study.

A total of 6 normal and 6 rats with OJ were paired according to body weight. Blood flow of the hepatic artery (HAF) and portal vein (PVF) was measured using the Transonic T206 Flow Meter (Transonic Systems Inc., Ithaca, NY, USA) during the second surgery. Following anesthesia and laparotomy, the common portal pedicle of the liver was dissected and a suitable probe was placed around the hepatic artery and portal vein. Blood flow was monitored for 1 min.

Microvascular LBF in rats with OJ was assessed using laser speckle contrast imaging (moorFLPI-2 Full-Field Laser Perfusion Imager; Moor Instruments, Axminster, UK) during the second operation. Following PVF and HAF measurements, LBF of the whole liver was measured as previously described (18). Following ligation of the left and median liver lobes and clamping of the pedicle for the OPT group, or the portal vein for the OPV group, of the right liver lobe, the LBF of the right liver lobe was measured 5 min post-clamping. The sampling frequency was 21 images/sec and the image acquisition rate was 1 frame/sec in the normal resolution mode. The duration of recording was 15 sec. MoorFLPI-2 Review (version 4.0; Moor Instruments) was used to analyze the images and quantify the perfusion in arbitrary laser speckle perfusion units (LSPU).

Serum levels of alanine transaminase (ALT) and direct bilirubin (DBIL) were used as general markers of liver injury, which was measured with a serum analyzer (Cobas-Mira Plus; Roche Diagnostics GmbH, Mannheim, Germany).

Total RNA was extracted from frozen liver tissues using the RNA Simple Total RNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. RNA (4 µg) was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit and Oligo-dT primers (Thermo Fisher Scientific, Inc., Waltham, MA, USA). qPCR was performed with the SYBR Premix Ex Taq II (Takara Bio, Inc., Otsu, Japan) and the StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used were as follows: Tumor necrosis factor (TNF)-α forward, 5′-AAATGGGCTCCCTCTCATCAGTTC-3′ and reverse, 5′-TCTGCTTGGTGGTTTGCTACGAC-3′; interleukin (IL)-1β forward, 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse, 5′-GGGTTCCATGGTGAAGTCAAC-3′; hypoxanthine phosphoribosyltransferase (HPRT) forward, 5′-GCTGAAGATTTGGAAAAGGTG-3′ and reverse, 5′-AATCCAGCAGGTCAGCAAAG-3′. The relative gene expression was normalized to HPRT. Values were calculated using the 2⁻ΔΔ^Cq method as previously described and expressed as fold change vs. normal rat (19).

Formalin-fixed tissues were embedded in paraffin and 5-µm sections were cut. Sections were stained for 3 min with hematoxylin and 2 min with eosin at room temperature for histological examination using a light microscope (magnification, ×400). For immunohistochemistry, sections were incubated with a mouse anti-Ki-67 monoclonal antibody (1:50; cat. no. 550609; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Samples were counterstained with hematoxylin for 2 min at room temperature. The percentage of Ki-67-positive hepatocytes was determined in ten random visual fields using a light microscope (Leica Microsystems GmbH, Wetzlar, Germany) at magnification, ×400 and the Ki-67 labeling index. All histological analyses were performed in a blinded manner with respect to the experimental groups.

Liver regeneration was evaluated using the rate of regeneration following hepatectomy of rats with OJ. Rate of regeneration=actual remnant liver weight at test point/W ×100%, where W represents the weight of the whole liver prior to hepatectomy. W was calculated as weight of the resected liver/76% (the weight of the resected liver lobes account for 76% of the whole liver) (20).

Frozen liver samples were homogenized in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with 1 mM phenyl methane sulfonyl fluoride and 1× phosphatase inhibitor. Homogenates were centrifuged at 10,000 × g for 10 min at 4°C. Supernatants were collected and protein concentrations were determined using the bicinchoninic acid method. Samples containing 50 µg of protein were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: A mouse monoclonal antibody of anti-proliferating cell nuclear antigen (PCNA; 1:200; cat. no. sc-56; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-caspase-3 (Asp175) (5A1E) monoclonal antibody (cat. no. 9664) and rabbit anti-caspase-9 (Asp353) polyclonal antibody (cat. no. 9507) (both 1:1,000; Cell Signaling Technology, Inc.). Following, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (sc-2005) or goat anti-rabbit (sc-2004) secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Blots were developed with Super-Signal chemiluminescent substrate (cat. no. 34580; Thermo Fisher Scientific, Inc.). Blots were analyzed against mouse anti-GAPDH monoclonal antibodies (1:2,000; cat. no. KM9002T; Sungene Biotech Co., Ltd., Tianjin, China) expression, which was used as loading control.

Data are expressed as the mean ± standard deviation. Student's t-test was used for the comparison of two groups and one-way ANOVA with Student-Newman-Keuls test was used for the comparison of three groups. SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA) was used for analyses. P<0.05 was considered to indicate a statistically significant difference.

All rats with OJ survived until the second surgical procedure. The dilated CBDs with a diameter of 8–10 mm were visible during the second laparotomy. To assess the tolerance of rats with OJ to liver ischemia and hepatectomy, the safe limit for the duration of liver ischemia in rats with OJ that underwent OPT and OPV was investigated. In the OPT group, the 7-day survival rate decreased when the occlusion time was >20 min (Fig. 1). The safe limit for the duration of liver ischemia for the OPV group was extended to 40 min without affecting the survival rate. At an occlusion time of 50 min, the 7-day survival rate decreased to 90% (9/10) in the OPV group and 0% (0/10) in the OPT group. The 7-day survival rate of the OPV group was significantly increased compared with the OPT group at 40, 50 and 60 min of occlusion, respectively (P<0.05; Fig. 1). Determined safe limits for the duration of liver ischemia were 20 and 40 min for the OPT and OPV group, respectively. OPV, with preserved HAF in rats with OJ and partial hepatectomy, markedly prolonged the duration of liver ischemia during hepatectomy.

Liver PVF and HAF in rats with OJ were measured with an ultrasonic flow meter and compared with those of normal rats. PVF decreased significantly from 13.83±1.60 to 8.17±0.75 ml/min in rats with OJ compared to the control (P<0.05) and HAF increased significantly from 1.10±0.14 to 1.98±0.38 ml/min in rats with OJ compared with the control (P<0.05; Fig. 2A). As HAF was low compared with PVF, the total liver blood inflow was decreased 7 days following induction of OJ.

During the portal blood occlusion prior to hepatectomy, LBF of the right liver lobe was measured with a laser speckle contrast imager. LBF of the control group was 529.53±91.55 LSPU (Fig. 2B). Following portal occlusion, LBF significantly decreased to 136.89±32.32 LSPU for the OPT and 183.99±49.25 LSPU for the OPV group (P<0.05). OPT and OPV significantly reduced the perfusion of liver microcirculation, with OPT being more effective compared with OPV (P<0.05).

ALT and DBIL levels of rats with OJ prior to the second surgery were 112.6±16.78 IU/l and 158.2±11.38 µmol/l, respectively. Following biliary drainage, portal occlusion and hepatectomy, ALT levels in rats with OJ increased, with the value for the OPT group significantly higher compared with the OPV and control groups at 6 and 24 h of reperfusion (P<0.05; Fig. 3A). At 24 h of reperfusion, the ALT level in the OPV group was significantly higher compared with the control group (P<0.05). Due to the recovery of biliary drainage, DBIL levels in all rats decreased following the second operation, with the value for the OPT group significantly higher compared with the OPV and control groups at 6 h of reperfusion (P<0.05; Fig. 3B). At days 3 and 7 following hepatectomy, ALT and DBIL levels of all rats had decreased and no significant difference was observed between the groups. Rats in the OPT group suffered the most severe liver I/R injury among the three groups and OPV significantly reduced I/R-induced liver injury compared with the OPT method.

The current study assessed mRNA expression of the proinflammatory cytokines IL-1β and TNF-α (Fig. 3C and D) in the remnant liver tissue using RT-qPCR. mRNA expression of IL-1β and TNF-α was significantly upregulated in the OPT group compared with the control and OPV groups at 6 and 24 h following I/R and hepatectomy (P<0.05). The remnant liver in the control and OPV groups expressed similar levels of IL-1β and TNF-α mRNA at all of the time points. The data indicated that OPT induced a more severe inflammatory response and damage compared with OPV.

Hepatic pathological changes in rats with OJ in the C, OPT and OPV groups were assessed at 6, 24 and 72 h and 7 days following different portal occlusion procedures and hepatectomy (Fig. 4). Proliferative encysted cholangioles were observed in all groups, with prevalence at 6 and 24 h following the second surgical procedure. In addition, marked hepatocyte necrosis was detected in the OPT group with many neutrophils invading into the necrotic area at 6 and 24 h post hepatectomy. The necrotic area was enlarged at 72 h following the second surgical procedure. No necrotic area was observed in the C group and hepatocyte necrosis in the OPV group was minimal when compared with the OPT group. OPV was effective in preventing hepatocytic necrosis in rats with OJ, I/R and partial hepatectomy.

Regeneration of the remnant liver following different intraoperative hepatic blood inflow occlusion procedures and partial hepatectomy were evaluated on days 3 and 7 following I/R and hepatectomy. Rates of liver regeneration on day 3 post-hepatectomy of C, OPV and OPT groups were 69.27±4.97, 66.18±4.43 and 59.70±5.03%, respectively (Fig. 5A). The rate of hepatic regeneration of the OPT group was significantly lower compared with the C and OPV groups (P<0.05), indicating that total hepatic pedicle clamping for 30 min prior to hepatectomy inhibited liver regeneration. Preservation of HAF during hepatic blood inflow occlusion in the OPV group did not significantly reduce liver regeneration compared with the control group. On day 7 following the second surgical procedure, rates of liver regeneration of C, OPV and OPT reached 96.17±3.84, 94.23±5.44 and 91.33±9.29%, respectively. No statistical differences were detected among the groups.

In order to further characterize the liver regenerative response, expression of Ki-67 is indicated as a brown nucleus in immunostained tissue sections, a nuclear antigen that is associated with hepatocyte proliferation, was assessed in remnant liver tissues. On day 3 post-operation, Ki-67 labeling indices of the OPT group (26.68±3.27%) were significantly reduced compared with the C group (38.96±4.14%) and the OPV group (31.94±4.38%) (P<0.05; Fig. 5A) with the OPV group also lower than the C group (P<0.05; Fig. 5B and C). On day 7 following hepatectomy, Ki-67 expression was substantially decreased and no significant differences were detected among the groups (Fig. 5B and C).

To further assess the liver regenerative capacity of rats with OJ following hepatectomy with varying liver blood inflow occlusive methods, PCNA, caspase-3 and 9 expression was determined by western blotting (Fig. 6). PCNA is a marker that is associated with hepatocyte proliferation and caspase-3 and −9 are associated with hepatocyte apoptosis (21,22).

PCNA expression increased at 24 h following I/R and hepatectomy in all groups compared with the 6 h measurements. PCNA expression at 24 h in group C was markedly higher compared with OPT and OPV groups. PCNA expression in the OPV group was significantly increased compared with the OPT group at 24 and 72 h following hepatectomy (P<0.05; Fig. 6). The opposite pattern was detected for caspase-3 and 9 expression, where expression in the OPT group was higher compared with the OPV group at 6 and 24 h following I/R and hepatectomy (P<0.05; Fig. 6). The results demonstrated that OPV was effective in improving hepatocyte proliferation and reducing hepatocyte apoptosis in rats with OJ following hepatectomy.