Neuropathic pain has been defined as “pain initiated or caused by a primary lesion or dysfunction in the nervous system” and is characterized by spontaneous pain, hyperalgesia, and allodynia.1,2 Until now, the mechanisms of neuropathic pain have not been elucidated completely. Recently, accumulating evidence suggests that glial cells in the spinal cord, including microglia and astrocytes, play an important role in the initiation and maintenance of neuropathic pain. After peripheral nerve injury, nociceptive neurotransmitters and mediators (glutamate, substance P, adenosine triphosphate, fractalkine, etc.) are released from sensitized primary afferent terminals and activate spinal microglia and astrocytes. Activated microglia can release a variety of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which act on their receptors expressed on neighboring spinal astrocytes and activate astrocytes. Activated astrocytes can also release proinflammatory cytokines, which act on their receptors expressed on postsynaptic neurons, leading to postsynaptic hyperexcitability and facilitatory pain transmission.3–5 Moreover, minocycline, a selective inhibitor of microglia, and fluorocitrate, a selective inhibitor of astrocytes, have been demonstrated to block the activation of microglia and astrocytes and to exert antiallodynic and antihyperalgesic effects on neuropathic pain.6–8 Therefore, inhibiting the activation of spinal glial cells may be a potential strategy to alleviate neuropathic pain.
Leukotrienes (LTs) are bioactive lipid mediators involved with inflammatory reactions, which are derived from metabolites of arachidonic acids. Arachidonic acid can be converted into leukotriene A4 (LTA4), an inactive precursor for other leukotrienes. LTA4 can either be converted into LTB4 by LTA4 hydrolase or into LTC4 by LTC4 synthase. LTC4 is further converted to LTD4 and LTE4, which are collectively termed cysteinyl leukotrienes (CysLTs). It has been demonstrated that in spared nerve injury (SNI) rats, the synthesis of LTs and expression of its receptor CysLT1 increase in spinal microglia, which contribute to the generation of neuropathic pain. Moreover, continuous intrathecal administration of CysLT1 receptor antagonists suppresses mechanical allodynia induced by SNI.9 Further study has shown that the CysLT2 receptor is expressed in the rat dorsal root ganglion (DRG) and augments pain behaviors by modulating P2X3.10 In addition, it has been shown that montelukast, a CysLT receptor antagonist, ameliorates ischemia-reperfusion (I/R)-induced vasculitic neuropathic pain in rats.11 These above studies suggest that CysLT receptor antagonists may have therapeutic potential in neuropathic pain. However, the underlying mechanisms of CysLT receptor antagonists are not well known.
Recently, CysLT receptor antagonists have been reported to inhibit microglial activation.12 Since the activation of glial cells plays an important role in the initiation and maintenance of neuropathic pain, we asked whether CysLT receptor antagonists could ameliorate neuropathic pain2 and what the underlying mechanisms are for CysLT receptor antagonists to attenuate neuropathic pain. Therefore, in the present study, we observed the antagonistic effect of montelukast, a selective CysLT1 receptor antagonist, on neuropathic pain in a rat chronic constriction injury (CCI) model, and further explored its underlying mechanism.
All experiments were approved by the Animal Care and Use Committee of Xuzhou Medical College.
Adult male Sprague-Dawley rats weighing 200 to 250 g were from the Experimental Animal Center of Xuzhou Medical College. All rats were housed at a constant room temperature of (23°C ± 1°C) under a 12:12 hours light-dark cycle with free access to food and water.
Chronic Constriction Injury of the Sciatic Nerve
Animals were subjected to CCI as previously described by Bennett and Xie.13 Briefly, rats were anesthetized with chloral hydrate (350 mg/kg intraperitoneal) and the left sciatic nerve was exposed at the midthigh level. Proximal to the sciatic trifurcation, adhering tissue was removed from about 7 mm nerve and 4 ligatures (chromic catgut 4.0) were tied loosely at 1.0 mm intervals. Sham surgery was performed by exposing the left sciatic nerve without ligation.
Rats were randomly divided into 6 groups (6 rats in each group): normal control group (normal), sham group (sham), CCI group (CCI), low dosage of montelukast group (low, 0.5 mg/kg), middle dosage of montelukast group (middle, 1.0 mg/kg), and high dosage of montelukast group (high, 2.0 mg/kg). Montelukast (Hengyuan Pharmaceutical Co. Ltd, Mudanjing, China) or vehicle was administered by intraperitoneal injections once a day for 14 days, starting on the first day after CCI.
von Frey Filament Test for Mechanical Allodynia
Rats were placed in a plastic cage on a wire mesh platform and allowed to acclimate for 15 minutes. von Frey filaments (North Coast Medical, Inc., Gilroy, CA) were applied to stimulate the central region of the plantar surface of left hindpaw in ascending order of force (0.4, 0.6, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, and 26.0 g) and held for <6 seconds. A positive response was noted if there was a withdrawal reflex of the hindpaw during stimulation or immediately after stimulus removal. The first stimulus was always initiated at 2.0 g. When there was a positive response, a filament with the next lower force was used, and when there was no response, a filament with the next higher force was used. After the first change in responses, 4 additional stimulations were determined, and the 50% mechanical withdrawal threshold (MWT) was calculated using the up-down method as previously reported.14
Hargreaves Test for Thermal Hyperalgesia
Thermal hyperalgesia was evaluated using a Hargreaves apparatus (IITC Life Science, Woodland Hills, CA) as previously described.15 Each rat was placed in a Plexiglas chamber on an elevated glass platform and allowed to acclimate to the test chamber. A radiant heat source was then used from underneath the platform to the plantar surface of the hindpaw. The time the rat took to lift its paw was recorded and defined as the thermal withdrawal latency (TWL). A cutoff time of 25 seconds was used to prevent tissue damage.
Enzyme-Linked Immunosorbent Assay
On the 14th day after CCI surgery, 30 minutes after the last dose of drugs, rats were killed by overdose of chloral hydrate (350 mg/kg intraperitoneal). The L4-L6 spinal cord was rapidly removed, frozen in liquid nitrogen, and then stored at −80°C until further processing. Frozen spinal cords were homogenized in normal saline (10 μL/mg tissue). After 4000rpm centrifugation for 15 minutes at 4°C, supernatant was used for enzyme-linked immunosorbent assay (ELISA). The contents of cytokines (TNF-α, IL-1β, and IL-6) were measured by rat-specific ELISA kits (Xitang Bio-tech. Co., Ltd, Shanghai, China) according to the manufacturer’s instructions.
Western Blot Analysis
Western blot was used to quantify the expression of nuclear factor-kappaB (NF-κB)p65 in the nuclear protein and phospho-p38 mitogen-activated protein kinase (p-p38MAPK) in the total protein extracted from the spinal cord. An equal amount of protein was loaded and separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE). The resolved proteins were transferred onto nitrocellulose membranes (Millipore Corporation, Billerica, MA). The membranes were then blocked in 5% nonfat milk for 2 hours at room temperature and incubated overnight at 4°C with rabbit anti-NF-κBp65 (1:1000, Cell Signaling Technology, Inc., Beverly, MA) or rabbit anti-p-p38MAPK (1:1000, Cell Signaling Technology, Inc., Beverly, MA). The blots were then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:500, ZSGB-BIO, Beijing, China) for 2 hours at room temperature. Signals were finally visualized using enhanced BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Biotechnology, Inc., Jiangsu, China).
Transverse spinal cord was cut into 25-μm-thick segments for immunofluorescence. After washing in phosphate-buffered saline (PBS), the tissue sections were incubated in normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 2 hours, followed by rabbit anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:200, Abcam, Cambridge, United Kingdom) or goat anti-Iba-1 polyclonal antibody (1:500, Abcam, Cambridge, United Kingdom) at 4°C for 24 hours. After three 5-minute rinses in PBS, the sections were incubated with donkey anti-rabbit IgG H&L (Alexa Fluor® 488) or donkey anti-goat IgG H&L (Alexa Fluor® 647) (1:500, Abcam, Cambridge, United Kingdom) in the dark for 2 hours at 37°C. Fluorescence was visualized under a confocal microscope (FV1000, Olympus).
For colocalization of p-p38MAPK and microglia or NF-κB and microglia, sections were incubated with rabbit anti-p-p38MAPK (1:800, Cell Signaling Technology, Inc., Beverly, MA) or rabbit anti-p-NF-κB (1:200, Abcam, Cambridge, United Kingdom) and goat anti-Iba-1 polyclonal antibody (1:500, Abcam, Cambridge, United Kingdom) at 4°C for 24 hours. After washing with PBS, the sections were incubated with donkey anti-rabbit IgG H&L (Alexa Fluor® 647) or donkey anti-goat IgG H&L (Alexa Fluor® 488) (1:500, Abcam, Cambridge, United Kingdom) in the dark for 2 hours at 37°C. Fluorescence was visualized under a confocal microscope (FV1000, Olympus Corp., Tokyo, Japan).
All data are expressed as mean ± SD. Statistical analysis was performed using repeated measures 1-way analysis of variance (behavioral data) or 1-way analysis of variance (Western blot and ELISA) followed by Least-significant Difference (if the variance is equal) or Dunnett T3 (if the variance is not equal) test. P < 0.01 was considered statistically significant.
Effects of Montelukast on MWT and TWL in CCI Rats
The effects of different doses of montelukast (0.5, 1.0, and 2.0 mg/kg intraperitoneal) on MWT and TWL in CCI rats are shown in Fig. 1, A and B. Before CCI surgery, the basic value for MWT and TWL in all groups was not significantly different. During the entire observation period, there was no obvious difference between the normal control group and sham group. MWT and TWL in CCI groups decreased markedly on 3, 5, 7, and 14 days after CCI compared with the sham group, indicating the presence of mechanical allodynia and thermal hyperalgesia. However, the reduction of MWT and TWL caused by CCI surgery was inhibited at varying degrees by low, middle, and high doses of montelukast. Furthermore, after 14-day treatment with montelukast, improvement in mechanical and thermal hyperalgesia was obvious. Therefore, further studies to explore the mechanism for montelukast to alleviate neuropathic pain were performed on day 14 after treatment with montelukast.
Effects of Montelukast on the Levels of IL-1β, IL-6, and TNF-α in Spinal Cord of CCI Rats
We tested the possible effect of montelukast on the levels of proinflammatory cytokines in the spinal cord of CCI rats. As shown in Figure 2, no significant differences for the levels of IL-1β (P =0.075, 95% confidence interval, −1263.68 to 65.36), IL-6 (P =0.946), and TNF-α (P =0.418) between the normal control group and sham group were found. Compared with the sham group, the levels of IL-1β, IL-6, and TNF-α were markedly increased in the spinal cord of CCI rats (P < 0.0001). A significant decrease was observed in the levels of IL-1β, IL-6, and TNF-α after montelukast administration following CCI surgery (P < 0.01, P < 0.0001).
Effects of Montelukast on the Activation of NF-κBp65 in Spinal Cord of CCI Rats
Figure 3 showed the protein expression of NF-κBp65 in the spinal cord after CCI. Compared with sham group, CCI resulted in a profound increase of NF-κBp65 protein (P < 0.01). However, compared with the CCI group, the expression of NF-κBp65 protein in low, middle, and high montelukast groups was significantly decreased (P <0.0001).
Effects of Montelukast on the Phosphorylation of p38MAPK in Spinal Cord of CCI Rats
As shown in Figure 4, in contrast to sham-operated rats, remarkably increased p-p38MAPK intensity was observed in the spinal cord of CCI rats (P < 0.01). Montelukast (0.5 and 2.0 mg/kg) attenuated the CCI-induced up-regulation of p-p38MAPK expression in the spinal cord, as indicated by the decreased intensity of p-p38MAPK bands when compared with the CCI group (P < 0.01, P < 0.0001).
Effects of Montelukast on the Activation of Astrocytes and Microglia in Spinal Cord of CCI Rats
As shown in Figure 5, compared with sham-operated rats, the expression of astrocyte marker GFAP and microglia marker Iba-1 was significantly up-regulated in CCI rats. Compared with CCI rats, the expression of Iba-1, but not GFAP, decreased markedly after treatment with high doses of montelukast (2.0 mg/kg) for 14 days.
Effects of Montelukast on the Activation of p38MAPK and NF-κBp65 in Spinal Microglia of CCI Rats
To further explore the relationship between the inhibition of p-p38MAPK or NF-κBp65 and microglia activation by montelukast, double immunofluorescent staining for p-p38MAPK and Iba-1 or p-NF-κBp65 and Iba-1 was performed. As shown in Figure 6, the number of p-p38MAPK and Iba-1 or p-NF-κBp65 and Iba-1 coexpression cells obviously increased in CCI rats compared with sham-operated rats. However, after treatment with high doses of montelukast (2.0 mg/kg), the number of p-p38MAPK and Iba-1 or p-NF-κBp65 and Iba-1 coexpression cells decreased significantly compared with CCI rats.
In the present study, we found that montelukast, a selective CysLT1 receptor antagonist, alleviated the neuropathic pain induced by CCI in rats. Suppression of p38MAPK/NF-κB pathway-mediated neuroinflammation in the spinal cord may be involved in the ameliorative effects of montelukast on neuropathic pain.
Montelukast is a potent and selective compound designed to specifically antagonize leukotriene CysLT1 receptors. We have chosen the well-established CCI model of neuropathic pain in rats because it has both nerve injury and inflammation.16 In our present study, we found that on day 1 after CCI, rats developed obvious mechanical allodynia and thermal hyperalgesia, which remained until day 14 after CCI. However, repeated administration of montelukast (0.5, 1.0, and 2.0 mg/kg intraperitoneal) for 14 days after CCI reversed the established mechanical allodynia and thermal hyperalgesia in rats, which suggests that montelukast induced antinociception in a neuropathic pain model. In accordance with our study, it has been reported that CysLT1 receptor antagonists ameliorate neuropathic pain induced by SNI9 and I/R.11 Therefore, montelukast may be a promising therapeutic intervention to prevent neuropathic pain.
The CCI model induces up-regulation of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in the spinal cord, which is a critical factor in the development and maintenance of hyperalgesia in animal models of neuropathic pain.16–18 Recently, it has also been reported that IL-1ra, IL-1 antibody, and IL-6 antibody attenuate allodynia in models of nerve injury.19–21 The increased proinflammatory cytokines in the spinal cord can promote the transduction of noxious signals by increasing excitatory synaptic transmission, decreasing inhibitory synaptic transmission,22 or promoting the phosphorylation of the N-methyl-D-aspartate receptor.23 Although the role of proinflammatory cytokines in neuropathic pain is undoubtable, there are some differences in the characteristics of cytokine expression. The study by Wang et al.24 has demonstrated that the level of IL-6 (reverse transcriptase polymerase chain reaction and Western blot) obviously increased on day 3 after CCI and peaked on day 14. Lee et al.17 have reported that the IL-1β increased maximally at 3 days and then decreased to control levels by 14 days in the spinal cord, while the level of IL-6 peaked at 7 days and remained elevated over the control level at 28 days. Kuang et al.25 have reported that the expression of TNF-α peaked at 3 days after CCI, but markedly decreased at 7 days after CCI. However, the maximal level of IL-1β expression was observed at 7 days after CCI when the mechanical and heat hyperalgesia were the most obvious. In our present study, we found that the levels of IL-1β, IL-6, and TNF-α increased significantly in the spinal cord on day 14 after CCI when compared with the sham group, which confirmed the effect of proinflammatory cytokines on neuropathic pain. In addition, we found that treatment with montelukast reduced pain behaviors and the levels of IL-1β, IL-6, and TNF-α, suggesting that montelukast is a potent inhibitor of neuroinflammation to peripheral nerve injury. In line with our results, it has been reported that montelukast inhibits the production of proinflammatory cytokines in acute lung injury, chronic renal failure, and renal I/R injury.26–28
NF-κB is a pleiotropic factor also involved in transcriptional control of a wide variety of genes. Several studies have demonstrated that activation of NF-κB occurs in the spinal cord and DRG, which are both involved in the transmission and processing of nociceptive information.29 After activation, NF-κB transfers into the nucleus and regulates the synthesis and release of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β, which may play a pivotal role in neuroinflammation.30 It has been reported that in models of peripheral inflammation-induced pain or pharmacologically induced central pain, intrathecal administration of NF-κB inhibitors results in transitory attenuation of pain.31,32 To explore the mechanism for montelukast to inhibit the production of proinflammatory cytokines, we observed the effect of montelukast on the activation of NF-κB in the spinal cord of CCI rats. Our results showed that, when compared with the sham group, the expression of nuclear NF-κBp65 protein in the lumbar spinal cord was significantly increased, demonstrating that pain stimulation in CCI rats can activate intranuclear translocation of NF-κB. Moreover, montelukast could inhibit the activation of NF-κB and subsequent inflammatory target gene expression, which may be beneficial for improving neuropathic pain.
MAPK plays a critical role in intracellular signal transduction and consists of p38MAPK, extracellular signal-related kinases (ERK1/2), and c-Jun amino terminal kinases (JNK1/2).33 Emerging evidence indicates that nerve injury results in the activation of p38MAPK in spinal cord and pretreatment with a p38MAPK antagonist intrathecally prevents SNL- and CCI-induced allodynia.34,35 In addition, many studies have shown that p38MAPK regulates the production of the proinflammatory cytokines, while proinflammatory cytokines in turn activate p38MAPK, therefore promoting the development of neuropathic pain.34–36 Results from our study indicated that montelukast-mediated inhibition of CCI-induced p38MAPK activation may also be a possible mechanism underlying its inhibitory action on neuropathic pain.
Since both astrocytes and microglia play important roles in the initiation and maintenance of neuropathic pain,3–5 we observed the effect of montelukast on the activation of astrocytes and microglia. Our results showed that compared with sham-operated rats, the expressions of astrocyte marker GFAP and microglia marker Iba-1 increased significantly in CCI rats, which indicated that CCI could induce the obvious activation of astrocytes and microglia. However, compared with the CCI group, the expression of Iba-1, but not GFAP, decreased markedly in the montelukast (2.0 mg/kg) group, which suggests that montelukast could inhibit the activation of microglia but not astrocytes.
Since montelukast not only inhibited the activation of NF-κB and p38MAPK in the spinal cord of CCI rats but also suppressed the activation of microglia, to clarify the relationship between them, double immunofluorescent staining was performed. Our results showed that the number of p-p38MAPK and Iba-1 or p-NF-κBp65 and Iba-1 double-positive cells were obviously up-regulated in CCI rats compared with sham-operated rats. While compared with CCI rats, montelukast rats (2.0 mg/kg) had significantly reduced p-p38MAPK and Iba-1 or p-NF-κBp65 and Iba-1 double-positive cells. These results demonstrated that montelukast could inhibit the activation of NF-κB and p38MAPK in spinal microglia.
In conclusion, our present study suggests that montelukast, a CysLT receptor antagonist, could effectively attenuate neuropathic pain in CCI rats by inhibiting the activation of p38MAPK and NF-κB signaling pathways in spinal microglia. Therefore, montelukast could be developed as a potential therapeutic agent for neuropathic pain.
Name: Chenghua Zhou, MD, PhD.
Contribution: This author helped design the study, analyze the data, and prepare the manuscript.
Attestation: Chenghua Zhou has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xiaotian Shi.
Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.
Attestation: Xiaotian Shi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: He Huang.
Contribution: This author helped prepare the manuscript.
Attestation: He Huang approved the final manuscript.
Name: Yangzi Zhu.
Contribution: This author helped prepare the manuscript.
Attestation: Yangzi Zhu approved the final manuscript.
Name: Yuqing Wu, MD, PhD.
Contribution: This author helped design the study and prepare the manuscript.
Attestation: Yuqing Wu has seen the original study data, approved the final manuscript, and is the archival author.
This manuscript was handled by: Jianren Mao, MD, PhD.
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