Resolvin D1 Inhibits Mechanical Hypersensitivity in Sciatica by Modulating the Expression of Nuclear Factor-κB, Phospho-extracellular Signal–regulated Kinase, and Pro- and Antiinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion

Zhi-hua Liu; Gui-shen Miao; Jun-nan Wang; Cong-xian Yang; Zhi-jian Fu; Tao Sun

doi:10.1097/ALN.0000000000001010

Free

Pain Medicine | April 2016

Resolvin D1 Inhibits Mechanical Hypersensitivity in Sciatica by Modulating the Expression of Nuclear Factor-κB, Phospho-extracellular Signal–regulated Kinase, and Pro- and Antiinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion

Zhi-hua Liu, M.D.; Gui-shen Miao, M.D.; Jun-nan Wang, M.D.; Cong-xian Yang, M.D.; Zhi-jian Fu, M.D., Ph.D.; et al Tao Sun, M.D., Ph.D.

Author Notes

Article Information

Anesthesiology 4 2016, Vol.124, 934-944. doi:https://doi.org/10.1097/ALN.0000000000001010

Abstract

Background: Accumulating evidence indicates that spinal inflammatory and immune responses play an important role in the process of radicular pain caused by intervertebral disk herniation. Resolvin D1 (RvD1) has been shown to have potent antiinflammatory and antinociceptive effects. The current study was undertaken to investigate the analgesic effect of RvD1 and its underlying mechanism in rat models of noncompressive lumbar disk herniation.

Methods: Rat models of noncompressive lumber disk herniation were established, and mechanical thresholds were evaluated using the von Frey test during an observation period of 21 days (n = 8/group). Intrathecal injection of vehicle or RvD1 (10 or 100 ng) was performed for three successive postoperative days. On day 7, the ipsilateral spinal dorsal horns and L5 dorsal root ganglions (DRGs) were removed to assess the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, and transforming growth factor-β1 (TGF-β1) and the activation of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal–regulated kinase (p-ERK) signaling (n = 30/group).

Results: The application of nucleus pulposus to L5 DRG induced prolonged mechanical allodynia, inhibited the production of IL-10 and TGF-β1, and up-regulated the expression of TNF-α, IL-1β, NF-κB/p65, and p-ERK in the spinal dorsal horns and DRGs. Intrathecal injection of RvD1 showed a potent analgesic effect, inhibited the up-regulation of TNF-α and IL-1β, increased the release of IL-10 and TGF-β1, and attenuated the expression of NF-κB/p65 and p-ERK in a dose-dependent manner.

Conclusions: The current study showed that RvD1 might alleviate neuropathic pain via regulating inflammatory mediators and NF-κB/p65 and p-ERK pathways. Its antiinflammatory and proresolution properties may offer novel therapeutic approaches for the management of neuropathic pain.

Abstract

Nucleus pulposus induced a significant inflammatory response in dorsal root ganglia. Resolvin significantly suppressed this inflammatory response and reduced mechanical allodynia for up to 3 weeks. The data suggest that resolvins might serve as novel therapeutic agents for the treatment of neuropathic pain.

What We Already Know about This Topic

Herniated nucleus pulposus (NP) triggers an inflammatory response that plays an important role in the development of mechanical allodynia. Suppression of inflammation might reduce nociception.
Resolvins are endogenous lipid mediators that exert a potent antiinflammatory effect.
To determine whether intrathecal administration of resolvin reduces inflammation and attenuates mechanical allodynia, a model of NP-induced nociception was used in rodents.

What This Article Tells Us That Is New

Nucleus pulposus induced a significant inflammatory response in dorsal root ganglia. Resolvin significantly suppressed this inflammatory response and reduced mechanical allodynia for up to 3 weeks.
The data suggest that resolvins might serve as novel therapeutic agents for the treatment of neuropathic pain.

RADICULAR pain induced by the herniated disk is an unbearable symptom for patients and has become a public health problem.¹ Increasing studies have shown that inflammatory and immune responses occurring in the peripheral and central nervous systems play an important role in the progress of sciatica induced by lumber disk herniation,^2–4 as well as the mechanical compression of nerve roots.^5,6

Resolvins are endogenous lipid mediators and have potent antiinflammatory and proresolution effects.^7–10 Rather than blocking inflammation at an early stage, resolvins promote the timely resolution of inflammation^11–13 by stimulating the cessation of leukocytic infiltration, counterregulation of proinflammatory factors, and uptake of apoptotic neutrophils and cellular debris.¹⁴ Reports have indicated that intrathecal delivery of resolvin D1 (RvD1) could cause pronounced antihyperalgesic effects in a model of adjuvant-induced arthritis (AIA) in rats¹⁵ and also strongly reduce the postoperative pain for several weeks.¹⁶

However, few studies have focused on the effect of RvD1 on radicular pain caused by intervertebral disk herniation. It has been shown that inflammatory cytokines,¹⁷ such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and activation of nuclear factor-κB (NF-κB)/p65¹⁸ and mitogen-activated protein kinase pathways¹⁹ play a vital function in the generation and maintenance of neuropathic pain. Thus, we hypothesized that the regulation of the release of inflammatory cytokines and the inhibition of NF-κB/p65 and phospho-extracellular signal–regulated kinase (p-ERK) in the spinal dorsal horn and dorsal root ganglion (DRG) might be involved in the procedure. The current study aims to evaluate the analgesic effect of RvD1 on sciatica and to uncover its underlying molecular mechanisms.

Materials and Methods

Animals

Adult male Sprague-Dawley rats, weighing 220 to 250 g, were purchased from the Experimental Animal Center of Shandong University (Shandong, China).The rats were housed in groups (three to five per cage), with controlled humidity (40 to 50%), room temperature(22 ± 1°C), 12-h light/dark cycle, and standard rodent chow and water. According to the International Association for the Study of Pain guidelines for pain research in animals, all animal experiments were approved by the Animal Care and Use Committee at the Shandong Provincial Hospital affiliated to Shandong University (No. 2014-002). Rats were distributed into sham group, vehicle group, 10-ng group, and 100-ng group randomly (n = 38/group).

Intrathecal Catheter Implantation

For intrathecal drug administration, polyethylene catheters (PE-10; Smiths Medical, UK) were implanted as described by Kawakami et al.²⁰ and Choi et al.²¹ After L2–L3 intervertebral foramen was exposed, PE-10 catheters were inserted into the epidural space and gently advanced caudally to the lumbar enlargement of the spinal cord. The correct placement was defined by the behavior of dragging or paralysis of bilateral hind limbs after the injection of 2% lidocaine (0.15 ml) after complete recovery from anesthesia. The internalized catheter was fixed with paravertebral muscles, and the externalized catheter was fixed firmly under the skin and sutured at the head. All rats were observed for 3 days before the application of nucleus pulposus (NP) to L5 DRG, and rats with infection, neurological deficit, or catheter prolapse were excluded from the experiments.

Rat Models of Noncompressive Lumbar Disk Herniation

As previously described,^22,23 noncompressive lumbar disk herniation models were established. All rats were anesthetized with 10% chloral hydrate (300 mg/kg, intraperitoneally), and then a midline dorsal incision was performed over the lumbar spine. The multifidus muscles were separated along the L4–L6 spinous processes, and then the right L5 spinal nerve root and the DRG were exposed after laminectomy. A fixed volume of NP from two coccygeal intervertebral disks was applied on the L5 DRG without mechanical compression. Rats in the sham group underwent the same operation without the application of NP.

Chemical Administration

Resolvin D1 was obtained from Cayman Chemical Company (USA). It was provided as a solution in 100% ethanol and stored at −80°C. According to the instruction, within 1 h before the injection, the solvent ethanol was removed by a gentle stream of nitrogen and RvD1 was redissolved immediately in phosphate-buffered saline (PBS), while minimizing the exposure to light.

Intrathecal injection was performed using a 10-μl microinjection syringe connected to the intrathecal catheter. The administration of RvD1 (10 or 100 ng) or vehicle (PBS) was performed in a single volume of 10 μl, and it was followed by administration of 10 µl saline to flush the catheter. Drugs and vehicles were injected for three successive days since the first day after operation.

Assessment of Pain-related Behavior

Animals (n = 8/group) were habituated to the testing environment for 1 h before the behavioral test. As described in detail previously,²⁴ the mechanical thresholds (paw withdrawal thresholds) were evaluated with a series of von Frey filaments (Stoelting, USA) using the “up-down” method, and the results were analyzed through the nonparametric method of Dixon. Tests were performed 1 day before and on days 1, 2, 3, 4, 7, 10, 14, and 21 after surgery. For all assessments of behavioral test, the investigator was blinded to the medication of rats.

RNA Extraction and Quantitative Real-time Polymerase Chain Reaction of Spinal Dorsal Horns

Rats (n = 5/group) were deeply anesthetized with chloral hydrate (300 mg/kg intraperitoneally) followed by perfusion by using 150 ml saline. Ipsilateral lumbar segments (L4–L6) of spinal dorsal horns were then quickly removed, frozen immediately in liquid nitrogen, and stored at −80°C. Total RNA was extracted using TRIzol reagent (Life Technologies, USA). The concentration of RNA was determined by using NanoDrop 2000 (Thermo Scientific, USA), and the purity of RNA was determined by A260/A280. The RNA was then reverse transcribed to complementary DNA using PrimeScript RT reagent kit, and polymerase chain reaction (PCR) amplifications were performed using SYBR Green Premix Ex Tag (TaKaRa Biotechnology Co, Ltd., China), both according to the manufacturer’s instructions. The reactions were conducted using LightCycler^® 480 II (Roche, Switzerland). The mRNA levels of target genes were normalized to the level of housekeeping gene β-actin. The sequences of primers were obtained from Genetimes Technology (China) and are shown in table 1. The investigator was blinded to the medication of rats.

Table 1.

The Primer Sequence for RT-PCR

Full Size | Slide (.ppt)

Western Blot Analysis of Spinal Dorsal Horns

The ipsilateral spinal dorsal horn tissues were harvested on postoperative day 7 (n = 5/group) and stored in liquid nitrogen. Nuclear extracts from the samples were obtained using a nuclear/cytoplasmic isolation kit (Thermo Scientific), and protein levels were evaluated using a BCA Protein Assay kit (Thermo Scientific). Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes (Millipore, USA). For blocking the nonspecific binding sites, the membranes were incubated in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20 for 1 h at room temperature. Membranes were then incubated overnight at 4°C under gentle agitation in primary antibodies: rabbit anti-NF-κB/p65 (1:500; Abcam, USA); rabbit anti-phospho-p44/42 (extracellular signal–regulated protein kinases 1 and 2 [Erk1/2]) and rabbit anti-p44/42 (Erk1/2) (1:500; Cell Signaling Technology, USA); and mouse anti-glyceraldehyde-phosphate dehydrogenase (1:1,000; Abcam). Later, membranes were washed and then incubated in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20 solution containing anti-rabbit and anti-mouse secondary antibodies (1:5,000; Cell Signaling Technology, USA) for 1 h at room temperature. After adequate washing, the protein bands were visualized using an enhanced chemiluminescence assay (Millipore, USA) with LAS-4000 mini (Fuji, Japan). The densities of protein blots were normalized to loading control glyceraldehyde-phosphate dehydrogenase. The investigator was blinded to the drug condition of rats.

Protein Levels in the Spinal Dorsal Horns and DRGs Determined by Enzyme-linked Immunosorbent Assay

Samples were collected on postoperative day 7. Protein levels of the inflammatory cytokines TNF-α, IL-1β, IL-10, and transforming growth factor-β1 (TGF-β1) in the spinal dorsal horns (n = 5/group) and L5 DRGs (n = 5/group) were detected as described previously.^25,26 In brief, samples were homogenized in ice-cold PBS containing 0.05% Tween 20, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM ethylene diamine tetraacetic acid, and 20 IU aprotinin A. After centrifugation at 10,000g for 10 min, the supernatants were separated and stored at −80°C for future analysis. The cytokine levels were evaluated using rat enzyme-linked immunosorbent assay (ELISA) kits (RD Systems, USA; ExCell, China) according to the manufacturers’ instructions.

Immunohistochemistry of Spinal Dorsal Horns and L5 DRGs

Immunohistochemistry was performed as previously described.¹⁵ On postoperative day 7, rats were deeply anesthetized with chloral hydrate and perfused with freshly prepared 4% paraformaldehyde (Sigma, USA). Immunohistochemical analysis was done on paraffin-embedded spinal cords (n = 5/group) and L5 DRGs (n = 5/group). After dewaxing and rehydration, the tissue sections (dissected at 4 μm) were treated with citric acid to retrieve antigen and then blocked in the blocking solution (10% normal goat serum and 0.3% Triton X-100 [Suolaibao, China] in 0.1 M PBS) for 30 min at 37°C. The sections were then incubated in the rabbit anti-NF-κB/p65 antibody (1:4000; Abcam) and rabbit anti-phospho-p44/42 (Erk1/2) (1:400; Cell Signaling Technology) overnight at 4°C. Thereafter, the sections were incubated with goat anti-rabbit secondary antibody for 30 min at 37°C. The immune complexes were visualized in 50 μl diaminobenzidine tetrahydrochloride. The specimens were photographed with a Leica DM4000B microscope and a digital camera (Germany). The investigators were blinded to the medication of rats.

Statistical Analysis

All the data were expressed as mean ± SD and illustrated using GraphPad 5 software (GraphPad Software, USA). Statistical analysis and multiple comparisons were performed using SPSS 20.0 software (IBM, USA). Data of animal behavior were analyzed by two-way ANOVA (group × time) with repeated measures, followed by the Bonferroni post hoc test. Real-time PCR, ELISA, Western blot, and immunohistochemistry data were assessed by ANOVA, followed by Bonferroni post hoc test. Two-tailed P value less than 0.05 was considered the acceptable level of significance.

Results

Alleviation of Mechanical Allodynia after Intrathecal Delivery of RvD1

As presented in figure 1, the paw withdrawal threshold in the vehicle group was decreased significantly after the application of NP to L5 DRG from day 1 (mean ± SD: 6.23 ± 1.97) postsurgery to day 21 (mean ± SD: 6.27 ± 1.21; P < 0.001 on each day) compared with that in the sham group. Compared with the vehicle group, intrathecal injection of RvD1 (10 ng) for three successive days alleviated the mechanical allodynia from day 3 (mean ± SD: 5.58 ± 1.22, P < 0.001) to day 10 (mean ± SD: 6.08 ± 1.85, P = 0.023). Meanwhile, the mechanical allodynia was also alleviated significantly in the RvD1 (100-ng) group from day 2 (mean ± SD: 5.32 ± 1.45, P = 0.018) to day 21 (mean ± SD: 10.55 ± 2.03, P < 0.001) during the observation period.

Fig. 1.

Analgesic effect of resolvin D1 (RvD1) in the model of noncompressive lumbar disk herniation. With the application of nucleus pulposus to L5 dorsal root ganglion, the paw withdrawal threshold (PWT; n = 8/group) was significantly reduced from day 1 postsurgery to the last day in the vehicle group (each P < 0.001). Intrathecal injection of RvD1 (10 or 100 ng) for three successive days alleviated the mechanical allodynia in a dose-dependent manner. The mechanical allodynia was alleviated from day 3 to day 10 in the RvD1 (10 ng) group (each P < 0.001, except day 10: P = 0.023). Meanwhile, the mechanical allodynia was also alleviated significantly in the RvD1 (100 ng) group from day 2 to day 21 (each P < 0.001, except P = 0.018 on day 2). Data are expressed as mean ± SD (**P < 0.01 compared with sham group; #P < 0.05 compared with vehicle group; ##P < 0.01 compared with vehicle group).

Full Size | Slide (.ppt)

RvD1 Regulates the Expressions of TNF-α, IL-1β, IL-10, and TGF-β1 in the Spinal Dorsal Horns and DRGs

The mRNA levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the ipsilateral spinal dorsal horns were examined by real-time PCR (fig. 2A–D). Compared with that in the sham group, the mRNA levels of TNF-α (fig. 2A, mean ± SD: from 1.00 ± 0.41 to 4.86 ± 1.03, P < 0.001), IL-1β (fig. 2B, mean ± SD: from 1.00 ± 0.42 to 7.14 ± 0.95, P < 0.001), and IL-10 (fig. 2C, mean ± SD: from 1.00 ± 0.12 to 1.31 ± 0.09, P = 0.04) were significantly increased in the vehicle group. In contrast, the mRNA level of TGF-β1 (fig. 2D, mean ± SD: from 1.00 ± 0.09 to 0.38 ± 0.09, P < 0.001) was decreased in the vehicle group. Intrathecal delivery of RvD1 (10 or 100 ng) dose-dependently reduced the mRNA levels of TNF-α (for the 10-ng group, mean ± SD: 3.13 ± 0.57, P = 0.012; for the 100-ng group, mean ± SD: 1.94 ± 0.80, P < 0.001) and IL-1β (for the 10-ng group, mean ± SD: 4.21 ± 1.40, P = 0.002; for the 100-ng group, mean ± SD: 2.11 ± 0.95, P < 0.001) and up-regulated the mRNA productions of IL-10 (for the 10-ng group, mean ± SD: 1.74 ± 0.18, P = 0.004; for the 100-ng group, mean ± SD: 2.54 ± 0.22, P < 0.001) and TGF-β1(for the 10-ng group, mean ± SD: 0.56 ± 0.10, P = 0.029; for the 100-ng group: mean ± SD: 0.81 ± 0.07, P < 0.001).

Fig. 2.

Resolvin D1 (RvD1) decreases the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and increases the expression of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in the spinal cord and dorsal root ganglion (DRG). (A–D) The mRNA levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the ipsilateral spinal dorsal horns were quantitated by real-time polymerase chain reaction (n = 5/group). There was a significant increase in the mRNA levels of TNF-α and IL-1β (each P < 0.001) and a decrease in the mRNA level of TGF-β1 (P < 0.001) in the vehicle group. The mRNA expression of IL-10 was increased in rats with the application of nucleus pulposus to the L5 DRG (P = 0.04). Intrathecal administration of RvD1 (10 or 100 ng) dose-dependently decreased the mRNA production of TNF-α (P = 0.012; P < 0.001) and IL-1β (P = 0.002; P < 0.001) and up-regulated the mRNA levels of IL-10 (P = 0.004; P < 0.001) and TGF-β1 (P = 0.029; P < 0.001). The fold change in the sham group was set at 1 for quantifications. (E–H) Enzyme-linked immunosorbent assay (ELISA) were performed for quantification of protein levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the ipsilateral spinal dorsal horns (n = 5/group). There was a significant overexpression of TNF-α and IL-1β (each P < 0.001) and a low expression of IL-10 and TGF-β1 (each P < 0.001). Intrathecal injection of RvD1 (10 or 100 ng) dose-dependently decreased the protein levels of TNF-α (P = 0.022, P < 0.001) and IL-1β (P = 0.017, P < 0.001) and up-regulated the protein production of TGF-β1 (P = 0.01, P < 0.001). The level of IL-10 was also increased in the 100-ng group (P < 0.001) but not significantly in the 10-ng group (P = 0.056). (I–L) Protein levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the DRGs evaluated by ELISA (n = 5/group). In the vehicle group, the protein productions of TNF-α and IL-1β were increased markedly (each P < 0.001), and the levels of IL-10 and TGF-β1 were decreased (each P < 0.001). Intrathecal delivery of RvD1 (10 or 100 ng) down-regulated the expression of TNF-α (P = 0.019, P = 0.001) and IL-1β (P = 0.028, P = 0.001). The protein levels of IL-10 and TGF-β1 were up-regulated in the 100-ng group (P = 0.046, P = 0.026) but not significantly in the 10-ng group (P = 0.816, P = 0.287). Data are presented as mean ± SD (*P < 0.05 vs. sham group; **P < 0.01 vs. sham group; #P < 0.05 vs. vehicle group; ##P < 0.01 vs. vehicle group).

Full Size | Slide (.ppt)

Fig. 2.

Full Size | Slide (.ppt)

To investigate the protein levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the ipsilateral spinal dorsal horns, ELISAs were performed (fig. 2E–H). Compared with the sham group, the protein levels of TNF-α (fig. 2E, mean ± SD: from 139.36 ± 29.40 to 315.44 ± 30.69, P < 0.001) and IL-1β (fig. 2F, mean ± SD: from 277.18 ± 54.67 to 586.81 ± 48.76, P < 0.001) were increased markedly after the application of NP on the L5 DRG. Meanwhile, the protein productions of IL-10 (fig. 2G, mean ± SD: from 311.73 ± 25.49 to 198.27 ± 30.87, P < 0.001) and TGF-β1 (fig. 2H, mean ± SD: from 937.51 ± 114.76 to 372.13 ± 48.34, P < 0.001) were reduced. Intrathecal administration of RvD1 (10 or 100 ng) suppressed the increase of TNF-α (for the 10-ng group, mean ± SD: 260.71 ± 18.80, P = 0.022; for the 100-ng group, mean ± SD: 210.99 ± 20.60, P < 0.001) and IL-1β (for the 10-ng group, mean ± SD: 492.42 ± 31.06, P = 0.017; for the 100-ng group, mean ± SD: 388.03 ± 28.99, P < 0.001) and increased the expression of TGF-β1 (for the 10-ng group, mean ± SD: 560.41 ± 42.11, P = 0.01; for the 100-ng group, mean ± SD: 673.56 ± 86.25, P < 0.001). Whereas the protein level of IL-10 in the 10-ng group (mean ± SD: 259.61 ± 32.72, P = 0.056 > 0.05) was not increased as significantly as in the 100-ng group (mean ± SD: 343.09 ± 40.51, P < 0.001).

In addition, the protein levels of TNF-α, IL-1β, IL-10, and TGF-β1 in the L5 DRGs were determined by ELISA (fig. 2I–L). There was significant increase in the expressions of TNF-α (fig. 2I, mean ± SD: from 16.11 ± 6.88 to 58.30 ± 11.26, P < 0.001) and IL-1β (fig. 2J, mean ± SD: from 11.74 ± 3.42 to 151.27 ± 48.55, P < 0.001) and decrease in the productions of IL-10 (fig. 2K, mean ± SD: from 92.47 ± 15.86 to 46.48 ± 10.79, P < 0.001) and TGF-β1 (fig. 2L, mean ± SD: from 103.95 ± 18.42 to 32.18 ± 22.14, P < 0.001) in the vehicle group compared with those in the sham group. Intrathecal administration of RvD1 (10 or 100 ng) dose-dependently suppressed the overexpressions of TNF-α (for the 10-ng group, mean ± SD: 41.09 ± 6.47, P = 0.019; for the 100-ng group, mean ± SD: 32.49 ± 5.66, P = 0.001) and IL-1β (for the 10-ng group, mean ± SD: 95.21 ± 16.39, P = 0.028; for the 100-ng group, mean ± SD: 62.70 ± 16.72, P = 0.001). Whereas the increase of IL-10 and TGF-β1 in the 10-ng group (mean ± SD: 58.28 ± 10.41, P = 0.816; mean ± SD: 53.81 ± 9.31, P = 0.287) was not as significant as that in the 100-ng group (mean ± SD: 69.37 ± 9.36, P = 0.046; mean ± SD: 65.65 ± 10.17, P = 0.026).

RvD1 Reduces the Expressions of NF-κB/p65 and p-ERK in the Spinal Dorsal Horns and DRGs

Western blot was performed to determine the protein expressions of NF-κB/p65 and p-ERK in the spinal dorsal horns (fig. 3). Significantly higher levels of protein expressions of NF-κB/p65 (fig. 3, A and B, mean ± SD: 1.11 ± 0.28, P < 0.001) and p-ERK (fig. 3, C and D, mean ± SD: 1.13 ± 0.48, P = 0.001) were exhibited in the vehicle group when compared with the sham group (NF-κB/p65, mean ± SD: 0.22 ± 0.11; p-ERK, mean ± SD: 0.13 ± 0.12). Compared with the vehicle group, intrathecal administration of RvD1 (10 or 100 ng) decreased the protein levels of p65 (mean ± SD: 0.69 ± 0.19, P = 0.013; mean ± SD: 0.47 ± 0.13, P < 0.001) and p-ERK (mean ± SD: 0.46 ± 0.23, P = 0.041; mean ± SD: 0.21 ± 0.22, P = 0.02) markedly in a dose-dependent manner.

Fig. 3.

Resolvin D1 (RvD1) reduces the expression of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal–regulated kinase (p-ERK) in the spinal cord. Western blot bar graphs and quantitative data of relative densities show that the application of nucleus pulposus (NP) to L5 dorsal root ganglion (DRG) significantly increased the expression of NF-κB/p65 (A and B, P < 0.001) and p-ERK (C and D, P = 0.001) in the ipsilateral spinal dorsal horns (L4–L6) compared with the sham group (n = 5/group). The intrathecal administration of RvD1 (10 or 100 ng) reduced the protein expression of NF-κB/p65 (P = 0.013, P < 0.001) and p-ERK (P = 0.041, P = 0.02). Quantitative data of relative densities of NF-κB/p65 protein bands (B) were calculated as the ratio of NF-κB/p65 to glyceraldehyde-phosphate dehydrogenase (GAPDH), and p-ERK protein bands (D) were calculated as the ratio of p-ERK to total-extracellular signal–regulated kinase. Values are presented as mean ± SD (**P < 0.01 vs. sham group; #P < 0.05 vs. vehicle group; ##P < 0.01 vs. vehicle group).

Full Size | Slide (.ppt)

Moreover, immunohistochemistry was performed to detect the levels of positive expressions of NF-κB/p65 and p-ERK in spinal dorsal horns (fig. 4) and L5 DRGs (fig. 5). The positive expressions of NF-κB/p65 and p-ERK were significantly increased in the vehicle group when compared with those in the sham group, both in the spinal dorsal horns (fig. 4, A and C, each P < 0.001) and in the DRGs (fig. 5, A and C, each P < 0.001). In the spinal dorsal horns, the score of expression of NF-κB/p65 was increased from 22.8 ± 9.26 (sham group) to 134.2 ± 12.15 (vehicle group) and the score of expression of p-ERK was increased from 10.4 ± 2.7 (sham group) to 99.6 ± 12.3 (vehicle group). Whereas in the DRGs, the score of expression of NF-κB/p65 was increased from 28.0 ± 18.1 (sham group) to 514.0 ± 51.59 (vehicle group) and the score of expression of p-ERK was increased from 22.4 ± 10.59 (sham group) to 519.0 ± 86.48 (vehicle group). Both in the dorsal horns and in the L5 DRGs, intrathecal injection of RvD1 (10 or 100 ng) dose-dependently attenuated the activation of NF-κB/p65 (mean ± SD: 86.6 ± 8.82, P < 0.001; mean ± SD: 50.8 ± 7.4, P < 0.001 in the spinal dorsal horns; mean ± SD: 377.6 ± 57.88, P = 0.001; mean ± SD: 189.2 ± 37.49, P < 0.001 in the DRGs) and inhibited an increase of p-ERK (mean ± SD: 64.0 ± 11.02, P = 0.001; mean ± SD: 36.4 ± 9.1, P < 0.001 in the spinal dorsal horns; mean ± SD: 307.8 ± 39.86, P < 0.001; mean ± SD: 179.6 ± 60.06, P < 0.001 in the DRGs).

Fig. 4.

Resolvin D1 (RvD1) reduces the level of positive images of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal–regulated kinase (p-ERK) in the spinal cord. The immunostaining images of NF-κB/p65 (A) and p-ERK (C) in the spinal dorsal horns (L4–L6) (n = 5/group) were significantly increased in the vehicle group (each P < 0.001). Intrathecal delivery of RvD1 (10 or 100 ng) decreased the positive expression of NF-κB/p65 (each P < 0.001) and p-ERK (P = 0.001, P < 0.001). Panels e to h are magnifications of the boxed areas in panels a to d. The red arrows represent p65 expression in the cytoplasm, and the yellow arrows represent p65 expression in the nuclei. Semiquantitative immunoreactive scores of NF-κB/p65 and p-ERK immunostaining in the spinal dorsal horns are shown in B and D. Data are presented as mean ± SD (*P < 0.05 and **P < 0.01 vs. sham group; #P < 0.05 and ##P < 0.01 vs. vehicle group).

Full Size | Slide (.ppt)

Fig. 5.

Resolvin D1 (RvD1) decreases the level of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal–regulated kinase (p-ERK) in the dorsal root ganglion (DRG). There was a marked increase in the levels of positive images of NF-κB/p65 (A) and p-ERK (C) in the DRGs (n = 5/group) in vehicle group (each P < 0.001). Significant down-regulations of NF-κB/p65 and p-ERK were observed after the administration of RvD1 in the 10-ng group (P = 0.001, P < 0.001) and 100-ng group (each P < 0.001). Panels e to h are magnifications of the boxed areas in panels a to d. The red arrows represent p65 expression in the cytoplasm, and the yellow arrows represent p65 expression in the nuclei. Semiquantitative immunoreactive scores of NF-κB/p65 and p-ERK immunostaining in the DRGs are shown in B and D. Data are presented as mean ± SD (*P < 0.05 and **P < 0.01 vs. sham group; #P < 0.05 and ##P < 0.01 vs. vehicle group).

Full Size | Slide (.ppt)

Discussion

The inflammatory and immune responses of nervous system have been attributed to the pathophysiology of sciatica induced by protuberant NP, as well as mechanical compression.^2,3,27 It has been demonstrated that the application of NP to nerve root and DRG could induce allodynia and hyperalgesia seen in clinical cases.^22,28–30 As a foreign antigen to the systemic circulation, the NP induced a strong autoimmune response and inflammation in the nervous system, resulting in sensitization of nociceptive neurons.^28,31,32 In the current study, the application of NP to L5 DRG, as a nociceptive stimulus to the nervous system, may lead to inflammation and sensitization of primary sensory neurons in the DRG.³³ Continuous and intense nociceptive input from injury site may result in the increase of neurotransmitters and neuromodulators, which induce the excitability of nociceptive neurons in the spinal dorsal horns (i.e., central sensitization).^9,34,35

The proinflammatory cytokines in the peripheral and central systems, such as TNF-α and IL-1β, released rapidly after nerve injury,^2,36 perform a vital function in the generation and maintenance of neuropathic pain.^9,17,37 For example, the application of TNF-α to DRG induced remarkable hyperalgesia^38,39 and treatment with TNF-α inhibitors resulted in a significant alleviation of mechanical allodynia in a rat chronic constrictive injury (CCI) model.⁴⁰ Gene therapy of silencing TNF-α expression relieved neuropathic pain in L5 spinal nerve transaction model rats.⁴¹ IL-1β also participated in the development of neuropathic pain.^42,43 IL-1β knockout or IL-1β antibody treatment^43,44 showed significant alleviation of neuropathic pain, and injection of recombinant IL-1β induced mechanical pain.⁴² Meanwhile, antiinflammatory factors, such as IL-10 and TGF-β1, were important regulators in the regulation of proinflammatory/antiinflammatory responses and played a significant antinociceptive effect in pain conditions.^44–48 Microinjection of IL-10 into the ventrolateral orbital cortex potentially alleviated allodynia in spared nerve injury rats.⁴⁴ The plasmid DNA-encoding IL-10 (pDNA-IL-10) provided long-term pain relief in CCI rats.⁴⁷ Intrathecal administration of TGF-β1 attenuated hyperalgesia in the rat model of CCI⁴⁶ and sciatic nerve ligation.⁴⁹

In the current study, we successfully reproduced mechanical allodynia by implanting the NP on the L5 DRG. Our results indicated that in the model of noncompressive lumbar disk herniation, the protein of proinflammatory cytokines TNF-α and IL-1β was sharply increased, and the antiinflammatory mediators TGF-β1 and IL-10 were suppressed in the L5 DRGs. Meanwhile, there is a significant increase of the mRNA and protein levels of TNF-α and IL-1β, and the release of TGF-β1 is decreased in the ipsilateral spinal dorsal horns. Our results also showed the up-regulation of mRNA level and down-regulation of protein level of IL-10 in the spinal dorsal horns, which was consistent with the findings reported previously.^50,51 The discrepancy between the expression of mRNA and protein of IL-10 may reflect a posttranscriptional regulation of IL-10 synthesis.^50,52 Possibly, the increased level of protein exerts a negative feedback effect on its production by destabilizing its mRNA.^50,53,54

Resolvins, derived from docosahexaenoic acid and eicosapentaenoic acid,^7,9 can actively stimulate the cardinal signs of resolution of inflammation,¹⁴ which has been proved in several inflammation-related diseases, such as asthma, retinopathy, and periodontal diseases.^55,56 Furthermore, it has been found that resolvins serve as a potent analgesic in several inflammatory and neuropathic pain models of rats.^9,10 For example, intrathecal injection of RvE1 could alleviate mechanical allodynia and thermal hyperalgesia and inhibit the expression of TNF-α in the spinal cord in CCI rats.⁵⁷ Resolvins have pronounced effects in alleviating the hyperalgesia and allodynia in arthritis rats and in reducing the production of proinflammatory factors, such as TNF-α, IL-1β, and IL-6.^10,15,58 Intrathecal delivery of RvD1 markedly reduced the postoperative pain in rats.¹⁶ Moreover, RvD1 effectively reversed mechanical allodynia and attenuated the expressions of TNF-α, IL-1β, and IL-6 in the rat model of chronic pancreatitis.⁵⁹ Our data indicated that intrathecal injection of RvD1 down-regulated the overproductions of TNF-α and IL-1β and up-regulated IL-10 and TGF-β1 in the noncompressive lumbar disk herniation rats. These effects were presented both in the spinal dorsal horns and in the DRGs.

The underlying mechanism of RvD1 in antiinflammation and proresolution is uncovered yet. The activation of several cellular signaling pathways, such as NF-κB and p-ERK pathways, might be involved. NF-κB family has been proven to modulate the expression of critical inflammatory mediators in the inflammation and immune process.⁶⁰ In the AIA rats, NF-κB/p65 plays a critical role in the development of arthritis through regulating the expression of cytokines, such as TNF-α and IL-1β.²⁶ Moreover, studies have indicated that NF-κB pathway might be associated with the development of neuropathic pain.^61–63 In our previous work, we observed that NF-κB/p65 was significantly activated in the spinal cord and DRG in the neuropathic pain model,^63–65 and the hyperalgesia and allodynia, as well as the release of inflammatory cytokines TNF-α and IL-1β in the spinal cord, were significantly attenuated by using small interfering RNA technique targeting NF-κB/p65.^18,26 It was reported that resolvin D series could induce the reduction of TNF-α and IL-1β by the inhibition of NF-κB and cyclooxygenase-2 activation in the spinal cord and DRG in AIA rats.¹⁵ In the current study, we detected significant alleviation of mechanical allodynia and a decrease of proinflammatory cytokines after the intrathecal administration of RvD1, which might be associated with the inhibition of NF-κB/p65 in the spinal dorsal horn and DRG.

The phosphorylation of ERK is the specific marker for central sensitization and nociceptive specific in dorsal horn neurons.^9,19,66 A notable up-regulation on the level of p-ERK was detected in rats after the spinal cord injury⁶⁷ and in CCI model rats,⁶⁸ and the inhibition of p-ERK pathway attenuated the pain behavior significantly. RvD1 could reduce the postoperative pain possibly through p-ERK pathway.¹⁶ Spinal administration of RvD1 and E1 reduced the pain behavior and the release of inflammatory cytokines (TNF-α, IL-1β, and IL-6) via inhibition of p-ERK signaling pathway in arthritis rats.¹⁰ In another study, administration of 17(R)-RvD1 and lipoxin A4 attenuated carrageenan-induced mechanical hypersensitivity and spinal release of TNF-α, and it was coupled to the inhibition of p-ERK activation.⁵⁸ Our results suggested that the analgesia effect and regulation of inflammatory mediators of RvD1 might be partially related to the inhibition of p-ERK pathway in the spinal dorsal horn and DRG.

In addition, transient receptor potential (TRP) channels might be related to the effect of RvD1. It has been reported that the analgesic effect of resolvins is attributed to the inhibition of TRP channels,^9,10,69 which have been strongly implicated in the generation of neuropathic and inflammatory pain.^9,69 The inflammatory mediators, such as TNF-α and IL-1β, released after peripheral tissue injury or nerve damage could lead to the hyperactivity of key transduction molecules, such as TRPV1 and A1.^9,70

The current medicines, such as nonsteroidal antiinflammatory drugs and opiates, used in the treatment of sciatic pain caused by a herniated disk are not ideal because of their side effects, such as nausea, vomiting, ulceration, and dependence.^10,71,72 Considering the efficacy without changing basal pain sensitivity and the safety associated with endogenous mediators in treating inflammation and inflammation-associated pain reported by Xu et al.,¹⁰ resolvins may offer a new option in the treatment of inflammatory and pain disorders.^8,10

In conclusion, our current results show that intrathecal injection of RvD1 exhibits marked effect in reducing pain behavior in a rat model of noncompressive lumbar disk herniation. The regulation of inflammatory mediators and the inhibition of NF-κB/p65 and p-ERK pathways might be, at least in part, involved in the underlying mechanisms. Thus, these findings may bring new evidence for the novel treatment option of sciatica and neuropathic pain.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Grant nos. 30801072, 81271232), Science and Technology Development Plan of Shandong Province, China (Grant no. 2014GSF118130), and National Key Clinical Specialist Construction Programs of China.

Dr. Liu was responsible for carrying out the major part of the research, the statistical analyses, and writing the manuscript. Dr. Miao helped conduct the behavioral tests and real-time polymerase chain reaction. Dr. Wang carried out part of the enzyme-linked immunosorbent assay and Western blot. Dr. Yang was in charge of the immunohistochemistry study. Dr. Fu revised the manuscript. Dr. Sun conceived and designed the study. All authors read and approved the final manuscript.

Competing Interests

The authors declare no competing interests.

References

Hoy, D, March, L, Brooks, P, Blyth, F, Woolf, A, Bain, C, Williams, G, Smith, E, Vos, T, Barendregt, J, Murray, C, Burstein, R, Buchbinder, R The global burden of low back pain: Estimates from the Global Burden of Disease 2010 study.. Ann Rheum Dis. (2014). 73 968–74 [Article] [PubMed]

Valat, JP, Genevay, S, Marty, M, Rozenberg, S, Koes, B Sciatica.. Best Pract Res Clin Rheumatol. (2010). 24 241–52 [Article] [PubMed]

Ji, RR, Xu, ZZ, Gao, YJ Emerging targets in neuroinflammation-driven chronic pain.. Nat Rev Drug Discov. (2014). 13 533–48 [Article] [PubMed]

Molinos, M, Almeida, CR, Caldeira, J, Cunha, C, Gonçalves, RM, Barbosa, MA Inflammation in intervertebral disc degeneration and regeneration.. J R Soc Interface. (2015). 12 20141191 [Article] [PubMed]

Winkelstein, BA, Weinstein, JN, DeLeo, JA The role of mechanical deformation in lumbar radiculopathy: An in vivo model.. Spine (Phila Pa 1976). (2002). 27 27–33 [Article] [PubMed]

Hou, SX, Tang, JG, Chen, HS, Chen, J Chronic inflammation and compression of the dorsal root contribute to sciatica induced by the intervertebral disc herniation in rats.. Pain. (2003). 105 255–64 [Article] [PubMed]

Zhang, MJ, Spite, M Resolvins: Anti-inflammatory and proresolving mediators derived from omega-3 polyunsaturated fatty acids.. Annu Rev Nutr. (2012). 32 203–27 [Article] [PubMed]

Serhan, CN Pro-resolving lipid mediators are leads for resolution physiology.. Nature. (2014). 510 92–101 [Article] [PubMed]

Ji, RR, Xu, ZZ, Strichartz, G, Serhan, CN Emerging roles of resolvins in the resolution of inflammation and pain.. Trends Neurosci. (2011). 34 599–609 [Article] [PubMed]

Xu, ZZ, Zhang, L, Liu, T, Park, JY, Berta, T, Yang, R, Serhan, CN, Ji, RR Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions.. Nat Med. (2010). 16 592–7, 1p following 597 [Article] [PubMed]

Lee, HN, Surh, YJ Therapeutic potential of resolvins in the prevention and treatment of inflammatory disorders.. Biochem Pharmacol. (2012). 84 1340–50 [Article] [PubMed]

Serhan, CN Resolution phase of inflammation: Novel endogenous anti-inflammatory and proresolving lipid mediators and pathways.. Annu Rev Immunol. (2007). 25 101–37 [Article] [PubMed]

Uddin, M, Levy, BD Resolvins: Natural agonists for resolution of pulmonary inflammation.. Prog Lipid Res. (2011). 50 75–88 [Article] [PubMed]

Serhan, CN, Chiang, N, Dalli, J, Levy, BD Lipid mediators in the resolution of inflammation.. Cold Spring Harb Perspect Biol. (2015). 7 a016311 [Article]

Lima-Garcia, JF, Dutra, RC, da Silva, K, Motta, EM, Campos, MM, Calixto, JB The precursor of resolvin D series and aspirin-triggered resolvin D1 display anti-hyperalgesic properties in adjuvant-induced arthritis in rats.. Br J Pharmacol. (2011). 164 278–93 [Article] [PubMed]

Huang, L, Wang, CF, Serhan, CN, Strichartz, G Enduring prevention and transient reduction of postoperative pain by intrathecal resolvin D1.. Pain. (2011). 152 557–65 [Article] [PubMed]

Stein, C, Clark, JD, Oh, U, Vasko, MR, Wilcox, GL, Overland, AC, Vanderah, TW, Spencer, RH Peripheral mechanisms of pain and analgesia.. Brain Res Rev. (2009). 60 90–113 [Article] [PubMed]

Sun, T, Luo, J, Jia, M, Li, H, Li, K, Fu, Z Small interfering RNA-mediated knockdown of NF-κBp65 attenuates neuropathic pain following peripheral nerve injury in rats.. Eur J Pharmacol. (2012). 682 79–85 [Article] [PubMed]

Ji, RR, Gereau, RWIV, Malcangio, M, Strichartz, GR MAP kinase and pain.. Brain Res Rev. (2009). 60 135–48 [Article] [PubMed]

Kawakami, M, Tamaki, T, Hayashi, N, Hashizume, H, Nishi, H Possible mechanism of painful radiculopathy in lumbar disc herniation.. Clin Orthop Relat Res. (1998). 241–51

Choi, SS, Kim, YC, Lim, YJ, Lee, CJ, Lee, PB, Lee, SC, Sim, WS, Choi, YL The neurological safety of epidural gabapentin in rats: A light microscopic examination.. Anesth Analg. (2005). 101 1422–6 [Article] [PubMed]

You, C, Zhu, K, Liu, X, Xi, C, Zhang, Z, Xu, G, Yan, J Tumor necrosis factor-α-dependent infiltration of macrophages into the dorsal root ganglion in a rat disc herniation model.. Spine (Phila Pa 1976). (2013). 38 2003–7 [Article] [PubMed]

Kim, SJ, Park, SM, Cho, YW, Jung, YJ, Lee, DG, Jang, SH, Park, HW, Hwang, SJ, Ahn, SH Changes in expression of mRNA for interleukin-8 and effects of interleukin-8 receptor inhibitor in the spinal dorsal horn in a rat model of lumbar disc herniation.. Spine (Phila Pa 1976). (2011). 36 2139–46 [Article] [PubMed]

Chaplan, SR, Bach, FW, Pogrel, JW, Chung, JM, Yaksh, TL Quantitative assessment of tactile allodynia in the rat paw.. J Neurosci Methods. (1994). 53 55–63 [Article] [PubMed]

Manjavachi, MN, Quintão, NL, Campos, MM, Deschamps, IK, Yunes, RA, Nunes, RJ, Leal, PC, Calixto, JB The effects of the selective and non-peptide CXCR2 receptor antagonist SB225002 on acute and long-lasting models of nociception in mice.. Eur J Pain. (2010). 14 23–31 [Article] [PubMed]

Luo, JG, Zhao, XL, Xu, WC, Zhao, XJ, Wang, JN, Lin, XW, Sun, T, Fu, ZJ Activation of spinal NF-κB/p65 contributes to peripheral inflammation and hyperalgesia in rat adjuvant-induced arthritis.. Arthritis Rheumatol. (2014). 66 896–906 [Article] [PubMed]

Risbud, MV, Shapiro, IM Role of cytokines in intervertebral disc degeneration: Pain and disc content.. Nat Rev Rheumatol. (2014). 10 44–56 [Article] [PubMed]

Zhu, X, Cao, S, Zhu, MD, Liu, JQ, Chen, JJ, Gao, YJ Contribution of chemokine CCL2/CCR2 signaling in the dorsal root ganglion and spinal cord to the maintenance of neuropathic pain in a rat model of lumbar disc herniation.. J Pain. (2014). 15 516–26 [Article] [PubMed]

Li, Y, Xi, C, Niu, M, Liu, X, Chi, Z, Wang, X, Yan, J Contralateral neuropathology in dorsal root ganglia in a rat model of noncompressive disc herniation.. Neurosci Lett. (2011). 493 49–54 [Article] [PubMed]

Murata, Y, Nannmark, U, Rydevik, B, Takahashi, K, Olmarker, K Nucleus pulposus-induced apoptosis in dorsal root ganglion following experimental disc herniation in rats.. Spine (Phila Pa 1976). (2006). 31 382–90 [Article] [PubMed]

Bobechko, WP, Hirsch, C Auto-immune response to nucleus pulposus in the rabbit.. J Bone Joint Surg Br. (1965). 47 574–80 [PubMed]

Cuéllar, JM, Borges, PM, Cuéllar, VG, Yoo, A, Scuderi, GJ, Yeomans, DC Cytokine expression in the epidural space: A model of noncompressive disc herniation-induced inflammation.. Spine (Phila Pa 1976). (2013). 38 17–23 [Article] [PubMed]

Takebayashi, T, Cavanaugh, JM, Cüneyt Ozaktay, A, Kallakuri, S, Chen, C Effect of nucleus pulposus on the neural activity of dorsal root ganglion.. Spine (Phila Pa 1976). (2001). 26 940–5 [Article] [PubMed]

DeLeo, JA, Yezierski, RP The role of neuroinflammation and neuroimmune activation in persistent pain.. Pain. (2001). 90 1–6 [Article] [PubMed]

Moalem, G, Tracey, DJ Immune and inflammatory mechanisms in neuropathic pain.. Brain Res Rev. (2006). 51 240–64 [Article] [PubMed]

White, FA, Bhangoo, SK, Miller, RJ Chemokines: Integrators of pain and inflammation.. Nat Rev Drug Discov. (2005). 4 834–44 [Article] [PubMed]

Oh, SB, Tran, PB, Gillard, SE, Hurley, RW, Hammond, DL, Miller, RJ Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons.. J Neurosci. (2001). 21 5027–35 [PubMed]

Zhang, JM, Li, H, Liu, B, Brull, SJ Acute topical application of tumor necrosis factor alpha evokes protein kinase A-dependent responses in rat sensory neurons.. J Neurophysiol. (2002). 88 1387–92 [Article] [PubMed]

Homma, Y, Brull, SJ, Zhang, JM A comparison of chronic pain behavior following local application of tumor necrosis factor alpha to the normal and mechanically compressed lumbar ganglia in the rat.. Pain. (2002). 95 239–46 [Article] [PubMed]

Andrade, P, Hoogland, G, Del Rosario, JS, Steinbusch, HW, Visser-Vandewalle, V, Daemen, MA Tumor necrosis factor-α inhibitors alleviation of experimentally induced neuropathic pain is associated with modulation of TNF receptor expression.. J Neurosci Res. (2014). 92 1490–8 [Article] [PubMed]

Ogawa, N, Kawai, H, Terashima, T, Kojima, H, Oka, K, Chan, L, Maegawa, H Gene therapy for neuropathic pain by silencing of TNF-α expression with lentiviral vectors targeting the dorsal root ganglion in mice.. PLoS One. (2014). 9 e92073 [Article] [PubMed]

Nadeau, S, Filali, M, Zhang, J, Kerr, BJ, Rivest, S, Soulet, D, Iwakura, Y, de Rivero Vaccari, JP, Keane, RW, Lacroix, S Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1β and TNF: Implications for neuropathic pain.. J Neurosci. (2011). 31 12533–42 [Article] [PubMed]

Yano, R, Ma, L, Nagai, J, Ueda, H Interleukin-1β plays key roles in LPA-induced amplification of LPA production in neuropathic pain model.. Cell Mol Neurobiol. (2013). 33 1033–41 [Article] [PubMed]

Shao, Q, Li, Y, Wang, Q, Zhao, J IL-10 and IL-1β mediate neuropathic-pain like behavior in the ventrolateral orbital cortex.. Neurochem Res. (2015). 40 733–9 [Article] [PubMed]

Wang, J, Yu, J, Ding, CP, Han, SP, Zeng, XY, Wang, JY Transforming growth factor-beta in the red nucleus plays antinociceptive effect under physiological and pathological pain conditions.. Neuroscience. (2015). 291 37–45 [Article] [PubMed]

Chen, NF, Huang, SY, Chen, WF, Chen, CH, Lu, CH, Chen, CL, Yang, SN, Wang, HM, Wen, ZH TGF-β1 attenuates spinal neuroinflammation and the excitatory amino acid system in rats with neuropathic pain.. J Pain. (2013). 14 1671–85 [Article] [PubMed]

Soderquist, RG, Sloane, EM, Loram, LC, Harrison, JA, Dengler, EC, Johnson, SM, Amer, LD, Young, CS, Lewis, MT, Poole, S, Frank, MG, Watkins, LR, Milligan, ED, Mahoney, MJ Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.. Pharm Res. (2010). 27 841–54 [Article] [PubMed]

Ledeboer, A, Jekich, BM, Sloane, EM, Mahoney, JH, Langer, SJ, Milligan, ED, Martin, D, Maier, SF, Johnson, KW, Leinwand, LA, Chavez, RA, Watkins, LR Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats.. Brain Behav Immun. (2007). 21 686–98 [Article] [PubMed]

Echeverry, S, Shi, XQ, Haw, A, Liu, H, Zhang, ZW, Zhang, J Transforming growth factor-beta1 impairs neuropathic pain through pleiotropic effects.. Mol Pain. (2009). 5 16 [Article] [PubMed]

George, A, Kleinschnitz, C, Zelenka, M, Brinkhoff, J, Stoll, G, Sommer, C Wallerian degeneration after crush or chronic constriction injury of rodent sciatic nerve is associated with a depletion of endoneurial interleukin-10 protein.. Exp Neurol. (2004). 188 187–91 [Article] [PubMed]

Khan, J, Ramadan, K, Korczeniewska, O, Anwer, MM, Benoliel, R, Eliav, E Interleukin-10 levels in rat models of nerve damage and neuropathic pain.. Neurosci Lett. (2015). 592 99–106 [Article] [PubMed]

Kwilasz, AJ, Grace, PM, Serbedzija, P, Maier, SF, Watkins, LR The therapeutic potential of interleukin-10 in neuroimmune diseases.. Neuropharmacology. (2015). 96pt A 55–69 [Article] [PubMed]

Liou, JT, Liu, FC, Mao, CC, Lai, YS, Day, YJ Inflammation confers dual effects on nociceptive processing in chronic neuropathic pain model.. Anesthesiology. (2011). 114 660–72 [Article] [PubMed]

Okamoto, K, Martin, DP, Schmelzer, JD, Mitsui, Y, Low, PA Pro- and anti-inflammatory cytokine gene expression in rat sciatic nerve chronic constriction injury model of neuropathic pain.. Exp Neurol. (2001). 169 386–91 [Article] [PubMed]

Haworth, O, Cernadas, M, Yang, R, Serhan, CN, Levy, BD Resolvin E1 regulates interleukin 23, interferon-gamma and lipoxin A4 to promote the resolution of allergic airway inflammation.. Nat Immunol. (2008). 9 873–9 [Article] [PubMed]

Connor, KM, SanGiovanni, JP, Lofqvist, C, Aderman, CM, Chen, J, Higuchi, A, Hong, S, Pravda, EA, Majchrzak, S, Carper, D, Hellstrom, A, Kang, JX, Chew, EY, Salem, NJr, Serhan, CN, Smith, LE Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.. Nat Med. (2007). 13 868–73 [Article] [PubMed]

Xu, ZZ, Berta, T, Ji, RR Resolvin E1 inhibits neuropathic pain and spinal cord microglial activation following peripheral nerve injury.. J Neuroimmune Pharmacol. (2013). 8 37–41 [Article] [PubMed]

Abdelmoaty, S, Wigerblad, G, Bas, DB, Codeluppi, S, Fernandez-Zafra, T, El-Awady, el-S, Moustafa, Y, Abdelhamid, Ael-D, Brodin, E, Svensson, CI Spinal actions of lipoxin A4 and 17®-resolvin D1 attenuate inflammation-induced mechanical hypersensitivity and spinal TNF release.. PLoS One. (2013). 8 e75543 [Article] [PubMed]

Quan-Xin, F, Fan, F, Xiang-Ying, F, Shu-Jun, L, Shi-Qi, W, Zhao-Xu, L, Xu-Jie, Z, Qing-Chuan, Z, Wei, W Resolvin D1 reverses chronic pancreatitis-induced mechanical allodynia, phosphorylation of NMDA receptors, and cytokines expression in the thoracic spinal dorsal horn.. BMC Gastroenterol. (2012). 12 148 [Article] [PubMed]

Barnes, PJ, Karin, M Nuclear factor-kappaB: A pivotal transcription factor in chronic inflammatory diseases.. N Engl J Med. (1997). 336 1066–71 [Article] [PubMed]

Meunier, A, Latrémolière, A, Dominguez, E, Mauborgne, A, Philippe, S, Hamon, M, Mallet, J, Benoliel, JJ, Pohl, M Lentiviral-mediated targeted NF-kappaB blockade in dorsal spinal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat.. Mol Ther. (2007). 15 687–97 [PubMed]

Tegeder, I, Niederberger, E, Schmidt, R, Kunz, S, Gühring, H, Ritzeler, O, Michaelis, M, Geisslinger, G Specific inhibition of IkappaB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats.. J Neurosci. (2004). 24 1637–45 [Article] [PubMed]

Sun, T, Yu, E, Yu, L, Luo, J, Li, H, Fu, Z LipoxinA(4) induced antinociception and decreased expression of NF-κB and pro-inflammatory cytokines after chronic dorsal root ganglia compression in rats.. Eur J Pain. (2012). 16 18–27 [Article] [PubMed]

Sun, T, Song, WG, Fu, ZJ, Liu, ZH, Liu, YM, Yao, SL Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-kappaB.. Br J Anaesth. (2006). 97 553–8 [Article] [PubMed]

Miao, GS, Liu, ZH, Wei, SX, Luo, JG, Fu, ZJ, Sun, T Lipoxin A4 attenuates radicular pain possibly by inhibiting spinal ERK, JNK and NF-κB/p65 and cytokine signals, but not p38, in a rat model of non-compressive lumbar disc herniation.. Neuroscience. (2015). 300 10–8 [Article] [PubMed]

Gao, YJ, Ji, RR c-Fos and pERK, which is a better marker for neuronal activation and central sensitization after noxious stimulation and tissue injury?. Open Pain J. (2009). 2 11–7 [Article] [PubMed]

Lang, BC, Zhang, Z, Lv, LY, Liu, J, Wang, TY, Yang, LH, Liao, DQ, Zhang, WS, Wang, TH OECs transplantation results in neuropathic pain associated with BDNF regulating ERK activity in rats following cord hemisection.. BMC Neurosci. (2013). 14 80 [Article] [PubMed]

Wang, H, Li, Y, Dun, L, Xu, Y, Jin, S, Du, J, Ma, L, Li, J, Zhou, R, He, X, Sun, T, Yu, J Antinociceptive effects of oxymatrine from Sophora flavescens, through regulation of NR2B-containing NMDA receptor-ERK/CREB signaling in a mice model of neuropathic pain.. Phytomedicine. (2013). 20 1039–45 [Article] [PubMed]

Park, CK, Xu, ZZ, Liu, T, Lü, N, Serhan, CN, Ji, RR Resolvin D2 is a potent endogenous inhibitor for transient receptor potential subtype V1/A1, inflammatory pain, and spinal cord synaptic plasticity in mice: Distinct roles of resolvin D1, D2, and E1.. J Neurosci. (2011). 31 18433–8 [Article] [PubMed]

Cheng, JK, Ji, RR Intracellular signaling in primary sensory neurons and persistent pain.. Neurochem Res. (2008). 33 1970–8 [Article] [PubMed]

Porreca, F, Ossipov, MH Nausea and vomiting side effects with opioid analgesics during treatment of chronic pain: Mechanisms, implications, and management options.. Pain Med. (2009). 10 654–62 [Article] [PubMed]

Nuckols, TK, Anderson, L, Popescu, I, Diamant, AL, Doyle, B, Di Capua, P, Chou, R Opioid prescribing: A systematic review and critical appraisal of guidelines for chronic pain.. Ann Intern Med. (2014). 160 38–47 [PubMed]

Fig. 1.