All animal procedures were approved (Permit Number: 2015001) by the Institutional Animal Care and Use Committees of Hunan Cancer Hospital, Changsha, China (Chairman Committee: Jingshi Liu) on 27 March 2015, and were performed in strict accordance with recommendations of the Guide to the Care and Use of Laboratory Animals of the National Institutes of Health.
80 male Sprague-Dawley rats (100±20g; Center of Experimental Animals of Hunan Cancer Hospital, Hunan, China) were used in this experiment. Rats were housed under controlled conditions with a temperature of 25±2℃, relative humidity of 60±10%, room air changes of 12–18 times/h and a 12 h light/dark cycle and were acclimated for 7 days before experiments. They were allowed free access to food and water.
Model Establishment and Experimental Protocol
80 Sprague-Dawley rats were intraperitoneal administrated with 0.19% N- nitrosodiethylamine（DENA）(50 mg kg-1) every 3 days for a total of 16 weeks to make HCC models(15). After sixteen weeks, 58 of these rats were successfully modeled, 48 rats randomly selected from HCC rats by digital random method were stochasticly assigned to 3 groups by digital random method (n=16): surgery without postoperative analgesia (Control); surgery with morphine postoperative analgesia (Morphine); surgery with sufentanil postoperative analgesia (Sufentanil). All animals undergoing surgery were given a standard left hepatolobectomy under isoflurane anesthesia (2-3%). Rats’ abdominal region was shaved and thoroughly cleaned with complex iodine. A 2 cm midline incision was made in the abdomen. After reaching the abdomen cavity, the left lateral leaf of the liver was exposed, and the left leaves were ligated from the root and excised. A implanted osmotic minipumps (volume 2 ml, pump speed 10 μl h-1 for 72h, Alzet, USA) was placed in the abdominal cavity for postoperative analgesia, Morphine Group per mouse was administered in a single dose of 0.25 mg Kg-1 h-1 for 72h (morphine dose was chosen under the document(16) and associated with the average effective dose used clinically), Sufentanil Group per mouse in a dose of 0.25 ug Kg-1 h-1 per mouse was administered for 72h (the dose of sufentanil was calculated in accordance with its analgesic potency in comparison to morphine), Control Group per mouse 0.9% saline was administered 10 ul h-1 per mouse for 72h. Finally, the muscle and skin were closed with sterile sutures. During surgery, the rats' temperatures were maintained using a thermal insulation blanket.
We measured the following parameters in each operated rat to comprehensive evaluate the postoperative analgesic effect on one day before surgery (d0), the first, second and third day after surgery(d1, d2, d3): Mechanical pain threshold assessed using standard von frey monofilaments; Locomotor activity tested using open field test; Body weight, daily food and water consumption. We randomly sacrificed four rats per group on one day before surgery (d0), six rats on the third day after surgery (d3), and all of the remaining rats on the seventh day after surgery (d7) to collect blood samples by cardiac puncturing method. All rats undergoing operation received general anesthesia of isoflurane (2-3%) and started surgery after no movement of clipping the tail, and all the rats were euthanized by the method of cercical vertebra decoupling under anesthesia after collecting blood samples. The rats’ survival situation of each group left after three days of surgery were observed. The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured to assess liver function , and the level of cluster of CD4+, CD8+, Th1,Th2, Th17 and Treg cells in blood were detected to assess immune function using flow cytometry on d0, d3 and d7.
Mechanical pain threshold (MPT)
The abdominal pain threshold was measured using Mechanical pain threshold (MPT) on d0, d1, d2, d3. Detection of MPT along the abdominal incision was assessed using standard von frey monofilaments(17). Rats were placed in test cages prior to the experiment and allowed to fully acclimate to the environment for 3 hours. A 0.1 to 12 g single fiber test needle was used to stimulate the position of the rat's abdominal incision about 0.5 cm perpendicular to the skin surface until the filament was slightly curved in an S shape for 5-6 seconds. The MPT for this region was measured using the Chaplan up-down method(18). If the rat appears to be licking or scratching the stimulated area during the stimulation time or removing the von Frey filament, or a sudden withdrawal or jump occurs, it is recorded as a positive behavioral response.
Locomotor activity—Open field test
The locomotor activity was surveyed using open field test on d0, d1, d2, d3(19). Rats were individually exposed to the same open field (100cm×100cm) for 5min trials with an interval of 30 min between each trial. The open field behavior was videotaped using a camera that was placed above the arena. The videos were subsequently analyzed digitally using Noldus software (Noldus, The Netherlands). Parameters measured were the total distance traveled throughout the arena.
Body weight, daily food and water consumption
During the measured period, rats were housed in individual cages. Body weight, food and water consumption were assessed daily on d0, d1, d2, d3.
Assessment of liver function
Blood samples were collected and sera were obtained by centrifugation in low temperature on d0, d3, d7. Serum AST and ALT were measured using the modified Jaffe rate reaction in the clinical laboratory of The Hunan Cancer Hospital, Changsha, China.
Flow cytometry studies
Fresh heparinized blood samples of rats were collected on d0, d3, d7. Then Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood by standard density gradient separation using Ficoll density gradient. Each specimen is divided into five equal parts in testing CD4+, CD8+, Th1, Th2, Th17 and Treg cells. Isolated cells were washed three times with phosphate buffer saline and used for flow cytometry. A total of 1 × 105 PBMCs prepared for were acquired for each sample. Each sample was surface stained with CD3-PE, CD4-FITC plus PE-Cy7-labeled anti-rat CD8 to detect CD4+T cells, CD8+T cells at room temperature for 15min (avoid light). The subsets detection needed analyze CD4 combined with specific cytokines such as CD4+IFN-γ+ for Th1, CD4+IL-4+ for Th2, and CD4+IL-17+ for Th17. For the Th1, Th2, Th17, samples were surface stained with CD4-FITC at room temperature for 15min (avoid light), and subsequently stimulated for the intracellular cytokines with PE-labeled anti-rat IFN-r, PE-labeled anti-rat IL-4, PE-labeled anti-rat IL-17A respectively according to the manufacturer's instructions. The CD4+Foxp3+ phenotype was recommended for identifying the Treg. Though, samples were surface stained with CD4-FITC at room temperature for 15min (avoid light), and subsequently intracellularly stained with a PE anti-rat Foxp3 staining kit without stimulated according to the manufacturer's instructions. Cells were detected by flow cytometry using a FACS Calibur, and data were analyzed by FlowJo.
Data are shown as mean± SD for normally distributed data. Probability values<0.05 were considered statistically significant. Then the data was transferred to the computer using SPSS 25 software, normally distributed data were analyzed by using a one-way ANOVA followed by a post hoc S-N-K test (Equal variances assumed) and Tamhane T2 test (Equal variances not assumed) to compare the three groups at each time point. When postoperative data and preoperative data in each group were compared, an independent samples-T test was employed. The descriptive findings were compared using Fisher’s exact test with P<0.0001.