Animals
Adult male Sprague-Dawley rats (n= 64), weighing 200 to 250 g, were supplied by the Laboratory Animal Center of Nanjing Drum Tower Hospital (Nanjing, China). All animals were housed in cages and maintained under a 12-h light/12-h dark cycle in a temperature controlled room (22±2°C) with food and water available ad libitum. All methods were carried out in accordance with relevant guidelines and regulations. All animal procedures were approved by the Experimental Animals Welfare and Ethics Committee of Nanjing Drum Tower Hospital and were performed in accordance with the guidelines for the use of laboratory animals. Efforts were made to minimize the number and suffering of animals.
Establishment of remifentanil-induced postoperative hyperalgesia model
After being anesthetized with sevoflurane (Jiangsu Hengrui Pharmaceutical Co., Ltd., China) delivered via a nose mask (induction, 3.5%; maintenance, 3.0%), rats (n= 32) were subcutaneously administered with remifentanil (Yichang Humanwell Pharmaceutical Co., Ltd., China) (0.04 mg/kg, 0.4 ml) at a rate of 0.8 ml/h over a period of 30 min using an infusion pump. This treatment was to induce pronociceptive effects as described in our previous study [12]. Control rats were subcutaneously administered the same volume of NS (0.8 ml/h, 30 min) (n= 32).
The surgical incision to induce postoperative pain was performed as previously described by Brennan et al. [18]. Briefly, after sterilization of the right hind paw with 10% povidoneiodine, a 1-cm longitudinal incision was made through the skin and fascia, starting at 0.5 cm from the edge of the heel and extending toward the toes. The underlying flexor muscle was elevated, incised longitudinally, and retracted, leaving the muscle origin and insertion intact. After suture of the lesion, the wound site was covered with erythromycin ointment. The surgical incision was started approximately 5 min after the infusion of remifentanil or NS. The control group received sham surgery consisting of anesthesia with sevoflurane, sterilization of the hind paw, and erythromycin ointment on the plantar surface, without plantar incision.
Implantation of intrathecal catheter
Intrathecal catheter implantation was conducted according to a method described previously [19]. Briefly, rats were anesthetized, and the polyethylene catheter was inserted in the cisterna magna through an incision advanced 7.0 cm caudally to the lumbar enlargement. Proper location of the intrathecal implantation was confirmed by bilateral hind limb paralysis with injection of 2% lidocaine (Sigma, St. Louis, MO, USA). Afterwards, the catheter was fixed and the incision was sutured.
Administration of recombinant lentiviruses
Recombinant lentiviruses-SIRT2 were purchased from Applied Biological Materials Inc. (GENE, Shanghai, China). The recombinant adenoviruses were amplified using 293T cells and then purified using double CsCl purification. For gene transfer, 1 × 108 pfu recombinant adenoviruses were injected with a microinjection syringe linked with the intrathecal catheter.
Sample collection
At 1 month after administration of recombinant lentiviruses, rats were euthanized. The L4–L5 lumbar spinal cords were removed and the dorsal root ganglia (DRG) from L4–L5 lumbar spinal cords were dissected for detection.
Animal grouping
According to different treatments, rats were divided into eight groups: Group Sham+NS (received sham surgery and NS infusion); Group Inci+NS received (surgical incision and NS infusion); Group Sham+Remi (received sham surgery and remifentanil infusion); Group Inci+Remi (received surgical incision and remifentanil infusion); Group Sham+LV-control (received sham surgery and LV-control); Group Sham+LV-SIRT2 (received sham surgery and LV-SIRT2); Group Inci+Remi+LV-control (received surgical incision and remifentanil infusion treatment after LV-control); Group Inci+Remi+LV-SIRT2 (received surgical incision and remifentanil infusion treatment after LV-SIRT2).
For behavioral tests, 32 rats were randomly divided into Sham+NS, Inci+NS, Sham+Remi, and Inci+Remi groups (n=8 each group) and were tested at 1 d prior to the surgical procedure (baseline value) and at 2 h, 6 h, 24 h, 48 h after the surgical procedure; For Western blot analysis, 32 rats were randomly divided into Sham+NS, Inci+NS, Sham+Remi and Inci+Remi groups (n=8 each group), and specimens were collected at 2 d after the surgical procedure. For immunofluorescence, 16 rats were randomly divided into Sham+LV-control, Sham+LV-SIRT2, Inci+Remi+LV-control, and Inci+Remi+LV-SIRT2 groups (n=4 each group), and specimens were collected at 2 d after the surgical procedure. The observers were blinded to the experimental groups in the experiments.
Nociceptive behavior tests
To assess mechanical allodynia, the paw withdrawal mechanical threshold (PWMT) was measured using a set of von Frey filaments. The animals were placed in plastic boxes (20×20×15 cm) with a wire mesh bottom (1×1 cm) and allowed to acclimatize for 30 min. Each von Frey filament was applied vertically to the plantar surface adjacent to the wound of right hind paw for 6 to 8 s with sufficient force. Positive responses were defined as paw flinching or brisk withdrawal. There was a 5-min interval between withdrawal responses. The PWMT was determined by sequentially increasing and decreasing the stimulus strength as described previously [20]. Each rat was tested five times per stimulus strength, and three or more positive responses of the lowest strength were considered as PWMT. The measurement of PWMT was conducted three times at each time point.
To evaluate thermal hyperalgesia, paw withdrawal thermal latency (PWTL) was measured using a biological research apparatus (Ugo Basile 37370, Comerio, Italy). Rats were placed into glass-floored testing cages and allowed to acclimate for approximately 15 min. Before starting the experiment, the infrared heat intensity of the apparatus was adjusted to give an average PWTL of approximately 10 s, and the cutoff latency was set at 15 s to avoid tissue damage. The infrared source was positioned directly beneath the area adjacent to the wound of the right hind paw. PWTL was defined as the time from onset of the infrared heat stimulus to withdrawal of the paw from the heat source. We repeated the stimulation three times with intervals of approximately 5 min, recorded the latencies, and calculated the mean value.
Western blot analysis
Spinal cord segments (right dorsal part of L4–L5) were homogenized in lysis buffer. The homogenate was centrifuged at 13,000 r/min for 10 min at 4°C, and the supernatant was collected. The protein concentration was determined by the BCA Protein Assay Kit. Samples (50 mg) were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Iba1 (1:600, ab15690, Abcam), and SIRT2 (1:2000, ab211033, Abcam). After washing, the membrane was incubated with the goat anti-mouse secondary antibody (1:5000, ab97040, Abcam) or goat anti-rabbit secondary antibody (1:5000, ab7090, Abcam) conjugated with horseradish peroxidase for 1 h at room temperature. Next, the immune complexes were detected using the electrochemiluminescence system (Millipore Immobilon). β-actin was used as a loading control for total protein. Image-Pro Plus (version 6.0, Media Cybernetics, USA) was used to analyze the density of specific bands.
Immunofluorescence analysis
The L4–L5 spinal cord segments were removed, post-fixed in 4% paraformaldehyde, and then incubated with 30% sucrose. Transverse spinal sections (25 mm) were blocked with 10% goat serum in 0.3% Triton for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies (Iba1, 1:200, Abcam). After washing in phosphate-buffered saline, sections were incubated with secondary antibody (Alexa Fluor 488 for Iba1, 1:1500, ThermoFisher) for 1 h at room temperature. The stained sections were mounted on glass slides, air-dried, covered with coverslips, and examined under a Leica multiphoton confocal microscope (Leica Microsystems, Wetzlar, Germany).
Statistical analysis
Statistical analysis was performed using SPSS 15.0 software. Data were expressed as mean±SD. Two-way analysis of variance with repeated measures followed by post hoc Bonferroni multiple comparisons were used to analyze behavioral data. One-way analysis of variance followed by post hoc Bonferroni multiple comparisons were used to analyze Western blot data. P<0.05 was set as the level of statistical significance.