All experiments were approved by the Institutional Animal Care and Use Committee (Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Permit Number was CIMH 2012-001). All procedures were conducted in accordance with the guidelines specified in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23, revised 1985) as well as the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals. The experiments were performed on 50 male BALB/c mice (Orient Bio, Seoul, Korea) weighing 20 to 25 g and about 6 weeks of age. Mice were housed in groups of five per cage under standard conditions (i.e., a room temperature of 20°C to 25°C, relative humidity of 50% to 60%, and a 12/12 hr light/dark cycle, with the lights on at 08:00) and all experiments were performed at the animal laboratory between 7 am and 11 am. The mice were kept specific pathogen-free and allowed access to food and water ad libitum throughout the study. The mice were acclimatized for at least 1 week before the study.

As an acute pain model, we used the mouse model of plantar incisional pain [11]. Mice were anesthetized with isoflurane 2%-3% that was delivered through a nose cone. After antiseptic preparation of the left hind paw with alcohol, a No. 11 blade was used to make a 5 mm longitudinal incision on the surface of the plantar foot that traversed the skin and fascia. The underlying muscle was elevated with curved forceps. The muscle origin and insertion were left intact. The skin was closed with a single mattress suture by using a TG175-8 needle with 6-0 nylon (Ophthalmic, 1716G: Ethicon, Somerville, NJ). Mice of the control group underwent anesthesia without plantar incision. All surgeries were performed by the same person to minimize experimental variability.

Mice were randomized using block randomization into one of four groups (each n = 8), as follows: saline-treated hind paw plantar incision (the incision group); PGB-treated incision (the PGB-incision group); sham controls undergoing an identical anesthesia procedure but without incision or administration of drugs (the control group); and PGB-treated controls (the PGB-control group). In the PGB-incision group, PGB in saline was administered intraperitoneally at a dose of 30 mg/kg in a volume of 0.1 mL/10 g. In the incision group, the same volume of saline was injected intraperitoneally. For the extension of analgesic effect of PGB without deep sedation, the drugs were administered divided into two doses. The first intraperitoneal injection of PGB or saline was performed 30 minutes before the plantar incision. After the surgery, mice were allowed to recover for 30 minutes before testing for postoperative mechanical thresholds. One hour after the hind paw incision, a second intraperitoneal PGB injection was performed with the same dosage. Mice were euthanized by cervical dislocation under isoflurane anesthesia 12 hours after the plantar incision, and NK cytotoxicity and lymphocyte proliferation were measured by a person who was blinded to the procedures in these animals (Fig. 1).

To determine the mechanical allodynia caused by the incision and the analgesic effect of preabalin on the pain, the mechanical nociceptive thresholds of the incision group and the PGB-incision group were compared with the control group. Mechanical nociceptive thresholds were assessed by PWT 30 minutes before plantar incision, 1 hour after the first and second PGB administrations, and 12 hours after surgery (the second day). The tests were performed by an investigator who was blinded to the drug or procedure that the mice had received. A dynamic plantar aesthesiometer (Ugo Basile, Gemonio, Italy) was used to measure the mechanical thresholds. Animals were placed individually in a test cage with a wire-mesh floor. A rigid-tipped filament was applied to the skin of the mid-plantar area of the hind paw. The filament was exerted with an increasing force, starting below the threshold of detection and increasing to 5 g in 20 seconds, until the animal removed its paw. The PWT was expressed in grams (g). All measurements were performed in triplicate on the same hind paw at 30 second intervals. The mechanical threshold was defined as the mean value.

Mice were euthanized by cervical dislocation under deep isoflurane anesthesia (3%-4%) and the spleen was aseptically excised and stored in tissue culture medium (RPMI 1640; Gibco BRL, Grand Island, NY). A single-cell suspension was prepared by using a 5 mL syringe plunger to pass spleen tissue in fresh wash medium through a 70 µm mesh strainer (BD Falcon, San Diego, CA). The splenocytes were suspended in complete RPMI medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine). Cell density and viability were estimated with a hemocytometer. Trypan blue exclusion was performed to identify the dead cells.

A lactate dehydrogenase (LDH) assay with YAC-1 (mouse lymphoma cells) targets was conducted to determine the effects of incisional pain and of PGB treatment on the tumoricidal activity of NK cells from the spleen of each mouse. Target YAC-1 lymphoma cells were grown in RPMI 1640 medium supplemented with antibiotics and fetal bovine serum. Effector splenocyte suspensions were prepared from each mouse, and various numbers of effector cells were added to the wells of a microtiter plate containing 1 × 10⁴ target YAC-1 cells in 100 µL to achieve final effector to target cell ratios of 80:1, 40:1, and 20:1. The plates were incubated for 4 hours at 37°C in 5% CO₂ at 90% humidity. The cells were then removed by centrifugation, and the supernatants were collected and transferred to a flat-bottom plate. After 100 µL of freshly prepared LDH detection mixture was added to each well, the plate was incubated at room temperature for 30 minutes to permit color development. The absorbance in each well was measured at 490 nm with a plate reader, and the percentage of specific LDH release was determined according to the following equation:

where LDH_experimental = LDH release from coculture of effector cells and target cells; LDH_{effector cells} = LDH release from effector cells cultured on their own; LDH_spontaneous = LDH release from YAC-1 cells cultured on their own (low control); and LDH_maximal = LDH release when YAC-1 cells were lysed in Triton X-100 (high control).

The proliferative response of isolated splenocytes to phytohemagglutinin (PHA) was measured using a bromodeoxyuridine (BrdU) detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany). The splenocytes from each mouse were aliquoted into 96-well micro plates at 2 × 10⁵ cells/well (100 µL/well) and cultured at 37°C in 5% CO₂ at 90% humidity in the presence of PHA for 2 days; 5 µg PHA was added at the start of incubation and then again 24 hours later. For pulse labeling, 10 µL BrdU was added to each well (10 µM final concentration) and the cells were incubated for another 18 hours. The labeling medium was removed using a needle, the cells were air-dried, and 200 µL FixDenant (Roche Diagnostics, Indianapolis, IN) agent was added to each well to fix the cells and denature the genomic DNA, thereby exposing the incorporated BrdU to immunodetection. After removing the fixative, an anti-BrdU antibody (clone BMG 6H8, Fab fragment) was added to each well and the plate was incubated at room temperature for 90 minutes. After washing with phosphate-buffered saline and adding substrate (tetramethylbenzidine), the plate was incubated at 25°C for 20 minutes. The absorbance at 370 nm was then measured by using an automated plate reader. Proliferation was determined as the stimulation index (SI), calculated as follows:

where OD = optical density.

To determine whether the remaining plasma PGB that was not metabolized affected immune response (i.e., NK cytotoxicity and lymphocyte proliferation), plasma concentrations of PGB were measured in 3 separate groups of mice (n = 6). Mice were subjected to intraperitoneal injection of 30 mg/kg PGB, anesthesia, plantar incision, and a second injection of PGB 90 minutes after the first, as described above (Fig. 1). Plasma samples were obtained from the orbital sinus of each mouse 2, 6, or 12 hours (depending on the assigned group) after the first PGB injection and placed in a tube with sodium heparin. Each sample was centrifuged and the plasma was harvested and stored at –80°C until the analysis was performed. Plasma PGB concentrations were measured by validated high-performance liquid chromatography/mass spectrometry (HPLC; 1260 solvent delivery system/6460 tandem mass spectrometer, Agilent, Santa Clara, CA).

Data are reported as means ± standard deviation. All results were analyzed using Sigma-Stat version 3.1 (SPSS Inc., Chicago, IL). Two-way repeated-measures analysis of variance (ANOVA; two-factor repletion) with pairwise multiple-comparisons (Holm-Sidak method) was used to analyze the results of the behavioral experiment. Two-way ANOVA and Bonferroni t-tests were performed to evaluate differences in NK cytotoxicity. One-way ANOVA and multiple comparison tests (Bonferroni t-tests) were used to evaluate differences in the splenocyte proliferative response of the different groups. The Kruskal–Wallis one-way ANOVA by ranks test was applied to compare the serum PGB concentrations at different time points. A value of P < 0.05 was taken to indicate statistical significance.

The PWT was decreased following plantar incision; reduced mechanical thresholds were maintained until the following morning in the incision group, compared with the pre-surgery baseline mechanical threshold (P < 0.001). Double intraperitoneal injection of PGB (30 min before and 1 hr following incision, each at 30 mg/kg) produced significant antinociceptive effects (P < 0.001), and elevation of the PWT, until the second day, compared with the incision group. There were no differences in the serial PWTs of the control and PGB-incision groups, following the first and second dose of PGB (P = 0.867; Fig. 2). During the experimental periods, except anesthesia and recovery periods, the mice maintained normal activity, posture, and appetite.

On the second day, the tumoricidal activity of NK cells against NK-sensitive YAC-1 lymphoma cells from each mouse spleen was assessed. Results are displayed as plots of LDH release (Fig. 3). NK cell activity was significantly higher in the incision group than in the control group (P < 0.001). Perioperative intraperitoneal PGB treatment, in the PGB-incision group, significantly reduced NK activity compared with saline injection in the incision group. However, NK activity in the PGB treatment group (PGB-incision) remained higher vs. the control group (P < 0.001). However, there was no significant difference in the NK cell activity of PGB-control group mice and the control group (P = 0.925; Fig. 3).

The proliferative response of splenic lymphocytes to PHA is presented as a SI plot (Fig. 4). Incision-induced pain in both the incision and PGB-incision groups increased splenic lymphocyte proliferation to a greater degree compared with the control group (P = 0.012 and P = 0.003, respectively). However, there were no changes in the splenocyte proliferation responses of the incision group (24.38 ± 6.76%) and PGB-incision group (25.77 ± 6.94%). Furthermore, PGB pretreatment in the PGB-control group did not induce significant changes in splenocyte proliferation compared with the control group (P = 0.786).

We measured the blood PGB concentrations in a separate groups of mice to rule out the possibility of direct effects of PGB on immune responses. Two hours subsequent to intraperitoneal PGB injection (two doses at 30 mg/kg, with a 90-min interval), plasma concentrations were 7.704 ± 0.963 µg/mL, but decreased to 0.583 ± 0.147 µg/mL (at 6 hr following injection), and finally to 0.013 ± 0.003 µg/mL (at 12 hr following injection; Fig. 5).