There is increasing evidence of the immune system's role in creating and maintaining neuropathic pain.1 Generally, acute pain stimulation enhances the immune response, but various alterations are induced in repetitive noxious stimulation.2,3 In the animal mononeuropathy model, infiltration of various immune cells is identified at the site where the nerve injury is induced.4 – 6 The activity of immune cells including natural killer (NK) cells or T lymphocytes modulates mechanical or thermal hyperalgesia in nociceptive processing.7
NK cells, a population of large granular lymphoid cells, are involved in cytotoxic activity against tumor cells or virally infected cells.8 NK cell activity is important for host defense and immune-surveillance in both humans and experimental animals.9 Pain can induce an instant increase in NK cell cytotoxicity.3 In addition to NK cells, T lymphocyte immune function in response to pathogens is essential for maintaining the integrity of the inflammatory reflex.10 Evaluation of T lymphocyte function is based on the reactivity of mitogenic stimulation such as phytohemagglutinin (PHA), which binds to cell surface receptors and stimulates T lymphocyte activation.11
It also has been demonstrated that chronic administration of analgesics12 and antiepileptics13 – 16 in neuropathic pain alter the immune reactivity in NK cell cytotoxicity or the proliferative ability of splenocytes in experimental animals. Pregabalin [(S)-3-(aminomethyl)-5-methylhexanoic acid] is a new-generation antiepileptic and has clinical and laboratory efficacy for the treatment of neuropathic pain.17 However, it has not been determined whether immunological reactivity such as NK cell activity or splenocyte proliferation could be affected; reactivity could be affected in patients receiving chronic administration of pregabalin.13,14
The purpose of this study was to examine the possible influence of pregabalin treatment on immunomodulation in an animal model. In particular, we assessed the NK tumoricidal activity of isolated splenocytes and the proliferation of splenocytes by PHA mitogenic stimulation in a neuropathic pain model.
All experiments were approved by the Institutional Animal Care and Use Committee (Incheon St. Mary's Hospital, School of Medicine, The Catholic University of Korea CIMH-2010-007). 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.
Specific pathogen-free male BALB/c mice (weighing 20–25 g, 4–6 weeks old) were purchased from OrientBio, Inc. (Seoul, South Korea). The animals were acclimatized for at least 1 week before the experiments and were maintained in an air-conditioned barrier-system room. Mice were housed together in groups of 4 under standard conditions, i.e., a reverse daylight pattern maintained through artificial illumination and a constant temperature and relative humidity of 20°C to 25°C and 50% to 60%, respectively. The mice were allowed unrestricted access to standard rodent chow and tap water.
The mice were divided into 4 groups: saline- or pregabalin-treated control naïve mice, or saline- and pregabalin-treated chronic constriction injury (CCI) mice. NK cell activity and splenocyte proliferation in response to PHA were determined on day 7 postsurgery, after continued saline or pregabalin treatment.
Animal Model Preparation
The neuropathy model was induced by loose ligation of the sciatic nerve.18 Briefly, under isoflurane inhaled anesthesia, the exposed right common sciatic nerve at the level of the midthigh was tied loosely 3 times with blue nonabsorbable 6-0 polypropylene monofilament (Prolene; Ethicon, Somerville, NJ) at a spacing of 0.5 mm. The muscle and skin were closed in layers. All surgery was performed by the same person to minimize experimental variability.
Mechanical allodynia and the antinociceptive effect of pregabalin were assessed on day 0 (presurgery) and day 7 after the surgical procedure, using a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy). Animals were placed in a test cage with a wire mesh floor. A rigid tipped filament was applied to the skin of the midplantar area of the hindpaw. The filament exerted an increasing force, starting below the threshold of detection and increasing to 5 g in 20 seconds, until the animal removed its paw. The paw withdrawal threshold (PWT) was expressed in grams and evaluated with respect to time. The time of pregabalin administration was defined as time zero.
Mice were killed by cervical dislocation after assessing mechanical allodynia on day 7 postsurgery. The spleen was aseptically excised and stored in tissue culture medium (RPMI 1640; Gibco BRL, Grand Island, NY). A single-cell suspension was made 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). 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 number and viability were estimated using a hemocytometer, and trypan blue exclusion to identify dead cells.
NK Cell Cytotoxic Activity Assay
The tumoricidal activity of NK cells from the spleen of each mouse was determined by the measurement of lactate dehydrogenase (LDH) activity using a cytotoxicity detection kit. YAC-1 cells (mouse lymphoma cells) were used for target cells of NK cells. LDH is a stable cytoplasmic enzyme present in all cells and LDH assay is an alternative to the previous radioactive [3H]-thymidine release or the [51Cr]-release assay. This is a colorimetric assay for the quantification of cell death or cell lysis, based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant using a spectrophotometric microplate reader.19
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 LDHexperimental = release from coculture of effector cells and target cells; LDHeffector cells = release from separately cultured effector cells; LDHspontaneous = release from separate cultures of YAC-1 cells (low control); and LDHmaximal = release from total lysis of YAC-1 cells by Triton X-100 (high control).
Mitogenic Stimulation Assay for Splenocyte Proliferation
The immunoreactivity of isolated splenocytes from each mouse was determined by the proliferative responses to mitogen. We used BALB/c splenocytes as responders and PHA as a stimulator. Cell proliferation was measured using an enzyme-linked immunosorbent assay kit for bromodeoxyuridine (BrdU) detection (Roche Molecular Biochemicals, Mannheim, Germany). This is a colorimetric alternative to quantify cell proliferation based on the measurement of BrdU incorporation in newly synthesized cellular DNA.11 Splenocytes from each mouse were counted, and the concentration was adjusted to 2 × 105 cells/100 μL. Aliquots (100 μL, 2 × 105) of cells were added to the wells of a 96-well flat-bottom plate and cultured at 37°C in 5% CO2 at 90% humidity in the presence of PHA for 2 days, with 5 μg of PHA added every 24 hours. For pulse labeling, 10 μL of BrdU was added to each well (10 μM final concentration), and the cells were incubated for 18 hours. The labeling medium was removed using a needle, and the cells were air-dried. Then, 200 μL of FixDenat (Roche Diagnostics, Indianapolis, IN) agent was added per well to fix the cells and denature the genomic DNA, thereby exposing the incorporated BrdU to immunodetection. After incubation for 1 hour at room temperature, the fixative was removed, 100 μL of horseradish peroxidase–conjugated anti-BrdU antibody (clone BMG 6H8, Fab fragment) was added to each well, and the plate was incubated at room temperature for 90 minutes. The wells were then washed 3 times with phosphate-buffered saline, and 100 μL of tetramethylbenzidine substrate was added. The plate was incubated at 25°C for 20 minutes, and the absorbance was measured at 370 nm with an automated plate reader. Proliferation was determined as the stimulation index (SI), calculated as follows:
where OD = optical density.
In Vitro NK Cell Cytotoxic Activity Assay and Splenocyte Proliferation
Splenocytes, as effector cells, were isolated from BALB/c mice to evaluate the direct immunomodulation of pregabalin. Different concentrations of pregabalin (3, 10, and 30 μg/mL) were added to the wells of a microtiter plate. A control well was prepared without adding pregabalin, and the plate was incubated for 24 hours at 37°C in 5% CO2 at 90% humidity. The cells obtained were then washed 1 time with RPMI, and various numbers of effector cells were added to the wells of a microtiter plate containing 2 × 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 cytotoxic effect was measured by LDH assay, the same method as used in vivo, and specific LDH release was determined according to the equation shown above.
The proliferation of splenocytes in response to PHA was evaluated by the level of BrdU in the presence of different concentrations of pregabalin. Pregabalin (3, 10, or 30 μg/mL) was added to the splenocytes adjusted as 2 × 105 cells/100 μL. Aliquots of cells were then added to the wells of a 96-well flat-bottom plate and cultured at 37°C in 5% CO2 at 90% humidity in the presence or absence of PHA for 2 days. Five micrograms of PHA was added every 24 hours for mitogenic stimulation. A cell-proliferation enzyme-linked immunosorbent assay system was used, and the stimulation index was calculated as described above.
Saline (10 mL/kg) or pregabalin (Pfizer Global Research and Development, Groton, CT) in phosphate-buffered saline (pH 7.2) was orally administered by intragastric gavage. In the drug-treated groups, pregabalin was administered at a dosage of 30 mg/kg twice per day, between 8:00 AM and 9:00 AM and between 4:00 PM and 5:00 PM, from day 2 postsurgery.
Data are reported as means ± SD. All results were analyzed using Sigma-Stat version 2.03 (SPSS, Inc., Chicago, IL). Two-way repeated-measures analysis of variance (ANOVA) (2-factor repetition) with all pairwise multiple-comparison procedures was used to analyze the behavioral experiment (Bonferroni post hoc test). The Kruskal-Wallis 1-way ANOVA by ranks was applied to compare splenocyte cytotoxicity. One-way ANOVA was performed to evaluate the difference in the splenocyte proliferative response between the groups. Differences between data of the pregabalin treatment groups were analyzed using the Bonferroni correction for multiple comparisons. Statistical significance was accepted at P < 0.05.
Pregabalin Exhibits an Antinociceptive Effect in CCI Model Mice
We confirmed that pregabalin suppressed the mechanical hypersensitivity that developed in mice after CCI of the sciatic nerve. CCI mice developed marked mechanical allodynia on day 7 postsurgery compared with presurgery (P < 0.001). Oral administration of pregabalin 30 mg/kg produced significant antiallodynic effects (P < 0.001), with elevation of the paw withdrawal threshold to the 240-minute mechanical stimulus (Fig. 1).
Effects of Pregabalin Treatment on NK Cell Activity
On day 7 postsurgery, tumoricidal activity of NK cells against NK-sensitive YAC-1 lymphoma cells from each mouse was assessed. The results are summarized in plots of the percentage of specific LDH release (Fig. 2). NK cell activity was significantly higher in CCI mice than in control naïve (Cont), pregabalin-treated control (Cont + PGB), and pregabalin-treated CCI mice (CCI + PGB) on day 7 (P < 0.001). In CCI mice, pregabalin treatment (CCI + PGB) significantly decreased NK activity, but there was no significant changes in Cont + PGB mice compared with Cont mice (P = 0.178). Specifically, at a 40:1 ratio of NK cells to NK-sensitive YAC-1 lymphoma cells, the NK cell activity was 29.2% ± 20.2% for cells from CCI mice and 8.4% ± 4.7% for cells from control mice. Pregabalin treatment in CCI mice reduced the NK cell activity to 6.8% ± 2.4% (P < 0.001).
Effects of Pregabalin Treatment on Splenocyte Proliferation
The splenocyte proliferative responses are graphically summarized as percent changes of SI in CCI and the CCI + PGB group compared with the control group in Figure 3. In the presence of 5 μg of PHA mitogenic stimulation, the percent changes of SI calculated for splenocytes from CCI were significantly higher than those for splenocytes from control mice (P = 0.023). The SI of pregabalin-treated CCI mice (CCI + PGB) was lower (P < 0.001) than in CCI mice.
In Vitro NK Cell Cytotoxicity Assay and Splenocyte Proliferation
The effect of pregabalin on NK cell cytotoxic activity was evaluated in vitro. At the concentrations of 10 μg/mL pregabalin, NK cell activity was less than at the concentration of 3 μg/mL (P = 0.02) and control (P = 0.02). Its activity was also less at the concentrations of 30 μg/mL than the concentration of 3 μg/mL (P = 0.043) and control (P = 0.042) (Fig. 4).
In Vitro Splenocyte Proliferation
In vitro spleen cells isolated from control mice were cultured in the presence of different concentrations of pregabalin (3, 10, and 30 μg/mL). Pregabalin did not affect splenocyte proliferation in vitro. SI was not significantly different among concentrations (P = 0.927) (Fig. 5).
We evaluated the possible influence of pregabalin treatment on cell-mediated immunity in a neuropathic pain model. CCI-induced neuropathic pain caused immunomodulation, which led to an increase in NK cell activity and splenocyte proliferation in mice. Pregabalin treatment inhibited these activations in CCI mice, but did not induce changes in control naïve mice. In vitro pregabalin suppressed splenic NK cell activity but did not affect splenocyte proliferation.
It has been demonstrated that loss of large-myelinated fibers in a CCI model causes changes in the neurogenic component such as sensory afferent and sympathetic efferent nerve fibers, which activate immune responses.20 Peripheral nerve damage induces the recruitment of immune cells at the injury site of the peripheral nerve.4 – 6 Thus, changes in the profile of neurochemicals4 increase the number and activity of immune cells and affect the plasma catecholamine level, which has an important role in the endocrine-immune network.21,22 During these reactions to injury, the dysregulation of supersystems such as sensory, autonomic, endocrine, and immune responses contributes to an abnormal healing process, resulting in chronic pain.23
The immunological consequences in the present study showed that peripheral neuropathy increased NK cell cytotoxic activity and splenocyte proliferation. The activated immune reactivity was similar to the activation of acute physical stress.2 There might have been insufficient time for inducing painful neuropathy because our results were obtained only 7 days after CCI; therefore, the findings could be regarded as the result of acute pain. However, mechanical allodynia and proinflammatory cytokine responses leading to acute inflammatory changes in mice reach their maxima on the third and seventh day after injury, respectively.24 In mice, patterns of recovery from neuropathic pain differ from rats.25 Variations in gender, age, and genetics also have roles in pain perception and are associated with immune reactions.2 Regarding the type of stressors, CCI could be a weak stressor enhancing the immune responses for mice.26
NK cell activity was inhibited and splenocyte proliferation was suppressed by pregabalin treatment in CCI mice. Reduced NK cell activity can be explained by the alteration of the binding process between effector (NK cell) and target cells (YAC-1 cell) or decreased ability of effector cells to lyse target cells.16 In vitro evaluation of NK cell activity in the presence of pregabalin showed decreased cytotoxicity in a concentration of 10 μg/mL and 30 μg/mL. The concentrations were based on the linear pharmacokinetics of orally administered pregabalin at a dosage of 30 mg/kg and its plasma concentrations. This is a slightly higher concentration than that for control of seizure, but it is much less than the dose to cause ataxia or decreased spontaneous activity in mice. These side effects were shown at dosages of 10- to 30-fold higher than those used to prevent seizure.27 – 29 However, in vitro plasma concentrations of 10 μg/mL and 30 μg/mL are still higher than in vivo plasma concentrations that cause alterations of NK cell activity in mice.
Experimental studies have shown that classic antiepileptic drugs such as phenytoin change peripheral immunological variables by the basal cytotoxic effect of drugs that suppress DNA synthesis during cell proliferation.13 – 16 These findings are in contrast to our in vitro proliferation study in which peripheral immunological variables were not changed by different concentrations of pregabalin, whereas a difference was shown in our in vivo study.
Tsai et al.12 reported that NK cell activity or splenocyte proliferation did not differ when they compared sham-operated rats with CCI rats with thermal hyperalgesia. Although the magnitude of the immune response in sham-operated animals was reproducibly lower compared with animals subjected to CCI, a change in immune reaction could be present.20 The immune responses in sham-operated animals were less than those in CCI, but were definitely altered, suggesting that surgical trauma, stress, anesthesia, and postoperative pain also have a role in modulating immune function.20 We designed this study to investigate the immunological influence of pregabalin on spleen cells, thus we have a pregabalin-treated control group instead of a sham-operated group. The cytotoxicity did not change when pregabalin was administered in control naïve mice. It is speculated that pregabalin has enough efficacy under painful condition to change in immune reaction in mice. The mechanism of the antiallodynic effect of pregabalin and its effects on immunomodulation have not yet been completely evaluated in the literature. In addition to pain, it is also possible that other factors may have been involved. The nature, intensity, duration, frequency of a stressor, and even handling may have a role in activating the neuroendocrine and immune responses8 and in affecting immune cell population.24,30
Because therapeutic efficacy is varied in the management of neuropathic pain, patients often receive long-term treatment with pregabalin. The decreased immunological reactivity of pregabalin should be considered in an immune-compromised state. On the contrary, it could be beneficial in controlling the immune process to cope with an injury from the beginning of the development of neuropathic pain. A neurogenic inflammatory response caused by releasing neuropeptides, which can activate T lymphocytes at the wound, is necessary to initiate the immune defense mechanism to protect and to promote healing.4 – 7 However, the continued, excessive process of inflammation is a cause of repetitive nociception resulting in dysregulation in the neuro-immune system at the peripheral nerve and in the spinal cord. Pregabalin might have efficacy on modulatory interactions of pain and immune response.
In conclusion, neuropathic pain increased immunological reactivity and the antihyperalgesic efficacy of pregabalin modulates this reactivity. Activated NK cell activity and lymphocyte proliferation were suppressed by pregabalin treatment in mice. However, the clinical consequences of the modified immune response observed in the present study cannot be predicted. The mechanism of a possible role of pregabalin on immunomodulation in chronic pain warrants further study.
Ho-Kyung Song, MD, PhD, and Yeon Jang, MD, PhD, are currently affiliated with the Department of Anesthesiology, The Catholic University of Korea, Inchon St. Mary's Hospital, Incheon, Korea; Mi Young Yeom, Bachelor, is currently affiliated with the East-West Medical Research Institute, Inchon St. Mary's Hospital, Incheon, Korea; and Dae Chul Jeong, MD, PhD, is currently affiliated with the Department of Pediatrics, The Catholic University of Korea, Seoul St. Mary's Hospital, Seoul, Korea.
Name: Yeon Jang, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Yeon Jang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Ho-Kyung Song, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Ho-Kyung Song has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Mi Young Yeom, BS.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Mi Young Yeom has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Dae Chul Jeong, MD, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Dae Chul Jeong has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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