Animals
Male Sprague-Dawley rats (250-300g) were from the animal laboratory center of Shanghai Jiaotong University, School of Medicine (Shanghai, China). Animal experiments were subject to approval by the Animal Care and Use Committee of Shanghai Jiaotong University, Shanghai, China. Rats were housed separately and given free access to food and water and exposed to 12-h light and dark cycles with room temperature at 21 ±2℃.
Intrathecal catheter insertion
Before intrathecal catheter insertion, rats were injected with 50mg·kg-1 of pentobarbital intraperitoneally. Skin was dealt with entoiodine. A 4-cm piece of polyethylene (external radius 40mm and internal radius 20mm) was placed through a tiny puncture from the atlanto-occipital membrane to the thoracic spinal cord. Wounds were closed with 3-0 silk sutures. Rats recovered for at least 3 days and were orally given meloxicam for analgesia daily. Rats displaying neurological disability symptoms were excluded.
Hepatic ischemia reperfusion injury model
After administering 50mg·kg-1 pentobarbital intraperitoneally for anesthesia, liver lobes were exposed by midline laparotomy. Right branches of portal veins and hepatic arteries were clamped for 1h for complete interruption of blood flow to the right and caudate lobes. Clamps were removed and livers perfused for 6hs. At baseline and end of refusion, 1- to 2-ml blood samples were collected from cava veins. At the end of reperfusion periods, ischemic liver lobes were collected, washed with cold phosphate-buffered saline and dried. Caudate lobes were fixed in 10% formalin, embedded in paraffin, and cut into 4-μm sections for staining. Remains of other lobes were stored at -80℃ for immunoblots. Rats were euthanized with pentobarbital anesthesia.
Rat groups and experimental protocols
To determine the effects of morphine pretreatment, rats were randomly divided into 4 groups (6 rats per group): (1) IRI, rats with hepatic IRI; (2) MPC1, morphine 0.1μg·mg-1 was administered intrathecally 10 mins before IRI; (3) MPC2, morphine 1μg·mg-1 was intrathecally administered 10 mins before IRI; and (4) MPC3, 10μg·mg-1 was administered intrathecally 10 mins before IRI. To investigate if opioid receptors in the center nerve system were involved in morphine pretreatment effects, the restricted peripheral opioid receptor antagonist naloxone methiodide [14] (NM, 50mg·mg-1, Sigma-Aldrich, St Louis, MO, USA) was used 20 mins before IRI. Another set of rats was randomly divided into 4 groups (6 rats per group): (1) IRI; (2) MPC, optimal protective morphine dosage (lowest Suzuki criteria, see below); (3) MPC+NM group, NM (50mg·mg-1) injected 10 mins before optimal protective morphine dose and 10 mins before IRI; and (4) NM group, NM (50mg·mg-1) administrated intravenously 20 mins before IRI.
Histopathological examination and immunohistochemistry
Liver samples were stained with hematoxylin-eosin (H&E). All histological examinations were evaluated blindly by a pathologist who identified hepatic IRI severity using Suzuki criteria: no changes in sinusoidal congestion, hepatocyte vacuolation and necrosis was 0; severe congestion, vacuolation and more than 60% necrosis was grade 4.
Assessment of apoptosis
Apoptosis of liver samples was examined by transferase-mediated deoxyuridine triphosphate nick end-labeling method (TUNEL, In Situ Cell Detection, Roche Biochemicals, Mannheim, Germany). Positive apoptotic cells were calculated as percent of total hepatocytes in 30 random high-powered fields (magnification: 200X). For apoptotic cell signaling, expression of cleaved caspase-3 was detected by western blots.
Measurement of serum aminotransferase
Blood samples were collected at baseline, ischemia and 6 hs after reperfusion time points. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with commercial kits (Wako Pure Chemical Industries Ltd., Tokyo, Japan) by 7180 autobiochemistry analyzer (Hitachi, Tokyo, Japan).
Enzyme linked immunosorbent assay (ELISA) of TNF-α and IL-1β
Blood samples were collected 6hs after reperfusion and centrifuged at 12,000 x g at 4℃ for 15 mins. Supernatants were transferred to fresh tubes. Serum TNF-α and IL-β was measured using manufacturers' instruction for ELISA kits (R&D System, Minneapolis, MN, USA).
Western blots
Liver samples (50mg) were homogenized with lysis buffer (20mM Tris-HCl, 150mM NaCl, 1 mM EDTA, 1% Triton, 2.5mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1mM Na3VO4, 1μg·mg-1 leupeptin; Cell Signaling Technology, Inc, Danvers, MA, USA). Concentration was determined by bicinchoninic acid protein assay kit (Pierce, Rockford, IL). 10% sodium dodecyl sulfate-polyacrylamide gels separated proteins with molecular weight above 60kDa; 12% gels were used for proteins with molecular weight below 60kDa. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk for 1h. PVDF membranes were incubated at 4℃ overnight with primary antibodies against phospho-Akt (P-Akt, Ser473), total-Akt (T-Akt), phospho-Erk (P-Erk), total Erk (T-Erk), cleaved-caspase-3, total-caspase-3, or GAPDH. All primary antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). PVDF membranes were incubated with horseradish peroxide-conjugated secondary antibody (goat anti-rabbit IgG, Merck Millipore, MA, USA) for 1h. Membranes were soaked in ECL buffer in a dark room. Protein bands were analyzed using computer software (Quantity one, Bio-Rad Company, Hercules, CA, USA). Results were described as the ratio of target to housekeeping protein GAPDH.
Statistical analysis
All results were presented as mean ± standard deviation. Analyses used GraphPad Prism 5.0 (San Diego, USA). Multiple groups were compared with one-way analysis of variance followed by Turkey test. Statistical differences were considered significant if P values were less than 0.05.