Anesthesia & Analgesia:
Pain and Analgesic Mechanisms: Research Report
The Effect of Intrathecal Administration TRPA1 Antagonists in a Rat Model of Neuropathic Pain
Zhang, Wei MD; Liu, Yue MD; Zhao, Xin MD; Gu, Xiaoping PhD; Ma, Zhengliang PhD
From the Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, Nanjing, Jiangsu, People’s Republic of China.
Accepted for publication November 30, 2013.
Published ahead of print May 22, 2014
Funding: This study was supported by Youth Start Fund of Nanjing Health Bureau (QYK10146), Youth Medical Experts Fund of Nanjing (Third Level) and Key Subject of Anesthesiology in Jiangsu Province, China (XK 201140) and National Nature Science Foundation of China (81070892, 81171048, 81171047, 81371207, and 81300951).
The authors declare no conflicts of interest.
Reprints will not be available from the authors.
Address correspondence to Zhengliang Ma, PhD, Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, Nanjing 210008, Jiangsu, People’s Republic of China. Address e-mail to firstname.lastname@example.org.
BACKGROUND: The fact that transient receptor potential ankyrin 1 (TRPA1) on the peripheral terminals could attenuate hyperalgesia is widely accepted, but the effect of spinal TRPA1 in the modulation of hyperalgesia has not been fully demonstrated. In the present study, we investigated the effect of intrathecal (i.t.) administration TRPA1 antagonists on chronic pain and expression of TRPA1 and phosphorylation N-methyl-D-aspartate receptor 2B subunit (p-NR2B) in the spinal cord with chronic compression of the dorsal root ganglia (CCD) in rats.
METHODS: The study was conducted in 2 parts. Part 1: Sixteen rats were divided into 2 groups (n = 8 each): a sham group and CCD group. Paw withdrawal mechanical thresholds (PWMT) were measured at baseline and 1, 3, 7, 10, 14, and 21 days after CCD. Sixteen other rats were used to evaluate expression of TRPA1 and p-NR2B in spinal cord on the seventh and 14th days after CCD; Western blotting was used to evaluate expression levels (n = 4 each). Part 2: 40 rats were divided into 5 groups (n = 8 each): CCD group, CCD + Vehicle group, CCD + HC-030031(10 μg, i.t.) group, CCD + HC-030031(25 μg, i.t.) group, and CCD + HC-030031(50 μg, i.t.) group. PWMTs were measured at baseline and 0.5, 1, 2, 4, and 6 hours after i.t. HC-030031on the third, seventh, 10th, and 14th days after CCD. Another 48 rats were used to evaluate expression of TRPA1 and p-NR2B in spinal cord 2 hours after injection on the seventh and 14th days after CCD in groups CCD, CCD + Vehicle, and CCD+ HC-030031(50 μg, i.t.) using Western blotting (n = 4 each).
RESULTS: Compared with the sham group, PWMT was significantly decreased, and protein expression of TRPA1 and p-NR2B were upregulated, in spinal cord on the seventh and 14th days after CCD operation. TRPA1 antagonists (HC-030031, 50 μg, i.t.) increased the PWMT after CCD and downregulated the protein level of TRPA1 and p-NR2B in spinal cord at 2 hours after the injection on the seventh and 14th days after CCD.
CONCLUSIONS: These data demonstrated that the i.t. administration of TRPA1 antagonists could attenuate neuropathic pain in CCD rats, and this effect could be partially reduced by p-NR2B receptor expression in spinal cord.
The transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable nonselective cation ion channel expressed in the primary afferent sensory neurons. TRPA1 contributes to transduction of potentially harmful stimuli into nociceptive electric signals on terminals of peripheral sensory neurons.1 In the spinal dorsal horn, TRPA1 regulates glutamatergic transmission to spinal interneurons on sensory neurons’ central terminals.2 There is abundant evidence indicating that the TRPA1 channel is relevant for nociceptive transmission involving chemical irritants such as mustard oil, cinnamon oil, allicin, methylparaben, acrolein3–8 and noxious mechanical stimuli.9–14 Blocking the TRPA1 channel could reduce nociception induced by various chemical irritants and noxious mechanical stimulation. Accumulating evidence indicates that TRPA1 channel antagonists or knockout of the TRPA1 channel could reduce pain behavior evoked by these stimuli.
The importance of the TRPA1 ion channel in the maintenance of pain hypersensitivity on central endings of nociceptive primary afferent nerve fibers has been demonstrated in recent studies.9,15,16 On the central terminal of the primary afferent nerve fiber, the TRPA1 ion channel facilitates glutamatergic transmission between the primary afferent terminal and the central neuron in the spinal dorsal horn.17,18 Intrathecal administration (i.t.) of a low-dose TRPA1 channel antagonist had a mechanical antihypersensitivity effect in animals with Complete Freund’s Adjuvant-induced inflammation9 and in diabetic or topical mustard oil-treated animals.19 These findings indicate that the spinal TRPA1 channel contributing to pain hypersensitivity may not be restricted to these particular conditions, and the spinal TRPA1 channel has a more general role in regulation of central pain hypersensitivity.
Previous studies of spinal TRPA1 in central pain hypersensitivity focused on certain chemical irritants and induced inflammatory conditions; it is not completely known whether TRPA1 participates in the generation and maintenance of neuropathic pain. The present study was performed to further investigate the role of TRPA1 in mechanical hyperalgesia caused by neuropathic pain. In a rat model of neuropathic pain induced by chronic compression of the dorsal root ganglia (CCD), we examined the hypothesis that the spinal TRPA1 channel would contribute to maintenance of neuropathic pain after CCD.
All experiments were approved by our Animal Care and Use Committee and were in accordance with guidelines of the Ethics Standards of the International Association for the Study of Pain.20 Experiments were performed with adult male Sprague Dawley rats (220–250 g, obtained from the Laboratory Animal Center of Affiliated Drum Tower Hospital of Medical College of Nanjing University). Animals were kept under standard laboratory conditions with free access to standard laboratory food and tap water. Efforts were made to minimize animal suffering and to reduce the number of animals used.
The study consisted of 2 experiments. Experiment 1 focused on CCD-induced mechanical allodynia and the upregulation of TRPA1 and phosphorylation N-methyl-D-aspartate receptor 2B subunit (p-NR2B) in the spinal cord. Sixteen rats were divided into 2 groups (n = 8 each): sham group and CCD group. Paw withdrawal mechanical thresholds (PWMT) were determined at baseline and days 1, 3, 7, 10, 14, and 21 after CCD. Another 16 rats were used to evaluate expression of TRPA1 and p-NR2B in the spinal cord on the seventh and 14th days after CCD using Western blotting analyses (n = 4 each). Experiment 2 studied the effect of TRPA1 channel antagonists on the behavior of CCD rats and expression of TRPA1 and p-NR2B in the spinal cord. Forty rats were divided into 5 groups (n = 8 each): CCD, CCD + Vehicle, CCD + HC-030031(10 μg, i.t.), CCD + HC-030031(25 μg, i.t.), and CCD + HC-030031(50 μg, i.t.). The TRPA1 channel antagonist HC-030031 was dissolved in 10% dimethylsulfoxide (DMSO), and i.t. administration volume was 20 μL. Administration of HC-030031 was started 3 days after CCD and continued twice per day until day 14. PMWTs were measured at baseline and 0.5, 1, 2, 4, and 6 hours after i.t. HC-030031 on the third, seventh, 10th, and 14th days after CCD. Another 48 rats were studied to evaluate the expression of TRPA1 and p-NR2B in the spinal cord on the seventh and 14th days after CCD in groups CCD, CCD + Vehicle, and CCD+ HC-030031 (50 μg, i.t.) using Western blotting (n = 4 each).
Implantation of the Intrathecal Catheter
Rats that needed i.t. injections had an i.t. catheter implanted. Briefly, rats were implanted with catheters according to the method described by Yaksh and Rudy.21 All operations were performed under sterile conditions. Polyethylene catheters (PE-10, Clay Adams, Parsippany, NJ) were inserted through an incision in the cisterna magna and advanced 7.0 to 7.5 cm caudally to the level of lumbar enlargement. Correct i.t. placement was confirmed by injection of 10 μL of 2% lidocaine through the catheter. Animals with signs of motor dysfunction were excluded from the experiment. Rats were housed individually after surgery and were allowed to recover for 5 days before the next experiment.
The surgical procedure to cause CCD22 was performed under anesthesia with pentobarbital sodium (50 mg/kg, intraperitoneally). Surgery was performed under sterile conditions. SURGIFLO™ (John & Johnson, Somerville, NJ) was used as a surgical matrix to induce focal compression of the L5 dorsal root ganglion (DRG). On the right side, paraspinal muscles were separated from the mammillary process, and intervertebral foraminas of L4 and L5 were exposed. SURGIFLO™, approximately 50 μL, was slowly injected (within 1–2 minutes) into the intervertebral foramen, and 6.0 nylon sutures were used to close the muscle and skin. Rats that had the same surgical procedure without injection of SURGIFLO™ comprised the sham group.
HC-030031, a TRPA1 Channel Antagonist
HC-030031 was purchased from Sigma Aldrich (Sigma, St. Loius, MO). Its degree of purity was higher than 98%. For all the experiments, HC-030031 was dissolved in 10% DMSO.
Observers were not aware of the substance or dosage given in the experiments. The withdrawal threshold to mechanical stimulation was examined in ipsilateral hindpaws. For measurement, a rat was placed into a plastic cage with a wire mesh bottom. Mechanical allodynia was assessed using von Frey filaments (2–15 g bending force; Stoelting, Wood Dale, IL). Rats were placed in individual plastic boxes (20 × 25 × 15 cm) on a metal mesh floor and allowed to acclimate for 30 minutes. Filaments were perpendicular to the area adjacent to the wound with sufficient force to cause slight bending against the paw and were held for 6 to 8 seconds with a 30-second interval between stimulations. A positive response was defined as withdrawal of the hindpaw on stimulus. PWMT was determined by sequentially increasing and decreasing the stimulus strength (“up-and-down” method). A decrease in the paw pressure threshold was defined as mechanical hyperalgesia.
Rats were deeply anesthetized with pentobarbital (60 mg/kg, intraperitoneally), and the right dorsal horn of the L4-L5 segments were extracted rapidly and stored in liquid nitrogen. Tissue samples were homogenized in a lysis buffer. Homogenate was centrifuged at 13,000g for 10 minutes at 4°C, and the supernatant was removed. The protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL), following the manufacturer’s instructions. Samples (50 μg) were separated by SDS–PAGE (10%) and transferred onto a nitrocellulose membrane. Filter membranes were blocked with 5% nonfat milk for 1 hour at room temperature and incubated with the primary antibody (phosphor-Tyr 1472 NR2B, 1:500, Cell Signaling Technology, Beverly, MA) or with goat anti-TRPA1 (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was washed with Tris Buffered Saline Tween (TBST) buffer and incubated for 1 hour with the secondary antibody conjugated with horseradish peroxidase (both 1:5000, Jackson Immuno Research, Inc., West Grove, PA) for 1 hour at room temperature. Immune complexes were detected using the ECL system (Santa Cruz Biotechnology). β-Actin was used as a loading control for total protein. Images of Western blot products were collected and analyzed by Quantity One V4.31 (Bio-Rad, Hercules, CA).
Data were expressed as means ± SD. The results of behavioral tests were analyzed by 2-way analysis of variance (repeated measure), and Western blots were analyzed by 1-way analysis of variance followed by the Least-significant difference (LSD) test for multiple comparisons (statistical software SPSS; version 13; Chicago, IL). Values of P < 0.05 were considered statistically significant.
CCD Induced Mechanical Hyperalgesia and Upregulation of TRPA1 and p-NR2B in the Spinal Cord
As in a previous study,22 mechanical hyperalgesia was observed on the ipsilateral side after CCD and persisted for at least 21 days. Compared with the sham group, PWMTs of ipsilateral hindpaws decreased significantly on day 1 in rats treated with CCD and persisted for 21 days (P < 0.001; Fig. 1), indicating that CCD induced prominent mechanical hyperalgesia.
The levels of TRPA1 and p-NR2B were detected by Western blot in CCD and sham group rats. In CCD rats, the protein levels of TRPA1 and p-NR2B were dramatically increased after CCD surgery. Expression of TRPA1 (P < 0.001; Fig. 2, A and B) and p-NR2B (P = 0.002 D7-CCD vs D7-sham, P = 0.003 D14-CCD vs D14-sham; Fig. 3, A and B) were increased at 7 and 14 days after CCD.
Effect of HC-030031 (i.t.) on the CCD-Induced Mechanical Allodynia
To investigate the role of TRPA1 on radicular pain behaviors, the TRPA1 antagonist HC-030031 (10, 25, and 50 μg, i.t.) was administered twice daily on postoperative days 3, 7, 10, and 14. PWMT was observed at baseline (before injection), 0.5, 1, 2, 4, and 6 hours after administration. The dosage of HC-030031 was selected based on a previous study and on our pilot experiments.9 I.t. administration of HC-030031 attenuated CCD-induced mechanical allodynia dose-dependently. There was no significant difference of PWMT in HC-030031 (10 μg, i.t.) group and HC-030031 (25 μg, i.t.) group compared with the DMSO group. The HC-030031 (50 μg, i.t.) group had significantly increased PWMT compared with DMSO groups at 0.5, 1, 2, and 4 hours after administration (P = 0.011 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4A; P = 0.008 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4B; P = 0.022 HC 50 μg, i.t. vs DMSO at 0.5 hours after administration, P = 0.014 HC 50 μg, i.t. vs DMSO at 4 hours after administration Fig. 4C; P = 0.028 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4D).
Intrathecal Administration of HC-030031 Decreased the Expression of TRPA1 and p-NR2B in the Spinal Cord
To investigate the effect of i.t. administration HC-030031 on TRPA1 and p-NR2B expression, Western blots were used to detect changes in TRPA1 and p-NR2B in the spinal cord. The lumbar enlargement was quickly dissected under deep anesthesia at 2 hours after i.t. administration of 50 μg HC-030031 on the seventh and 14th days after CCD. The protein level of TRPA1 is shown in Fig. 5, A and B. I.t. administration of 50 μg of HC-030031 significantly decreased the TRPA1 protein level in the spinal cord (P = 0.001 D7-HC 50 μg, i.t. vs D7-DMSO, P = 0.002 D14-HC 50 μg, i.t. vs D14-DMSO; Fig. 5, A and B).
Protein levels of p-NR2B are shown in Fig. 6, A and B. p-NR2B protein levels were downregulated in the HC-030031 (50 μg, i.t.) group in the spinal cord of rats on the seventh and 14th days after the operation (P = 0.001 D7-HC 50 μg, i.t. vs D7-DMSO, P = 0.003 D14-HC 50 μg, i.t. vs D14-DMSO; Fig. 6, A and B).
Our study showed that the CCD induced significant mechanical allodynia and upregulation of TRPA1 and p-NR2B protein levels in the spinal cord. This neuropathic pain model induced by SURGIFLO™ could produce a different CCD from the metal rods used in previous rat models of CCD.23,24 This modification of CCD may closely mimic a subset of clinical radicular pain resulting from conditions such as foraminal stenosis and herniated intervertebral disk. Though it was not widely used in neuropathic pain studies, our experiments demonstrated that this model could induce obvious mechanical allodynia on the ipsilateral side after CCD. These findings were in accordance with a previous study by Gu et al.22
Our present study focused on investigation of spinal TRPA1 in neuropathic pain conditions caused by CCD. TRPA1 on peripheral terminals in transduction of noxious stimuli has been well studied,2,5,25–27 but the importance of spinal TRPA1 in modulation of hypersensitivity associated with neuropathic pain conditions still remains poorly understood.28 That the spinal TRPA1 channel contributes to central pain facilitation is supported by studies showing that spinal administration of TRPA1 antagonists could reduce mechanical hypersensitivity in various induced pain hypersensitivity conditions.15 Only secondary mechanical hypersensitivity was attenuated by spinal administration of a TRPA1 antagonist; primary mechanical hypersensitivity was not influenced by spinal administration of a TRPA1 antagonist. These results were found in various pain models, such as formalin-induced, capsaicin-induced, and mustard oil-induced pain models.19 A recent study demonstrated that TRPA1 is expressed on peripheral terminals and on central endings of primary afferent nociceptive nerve fibers within the spinal dorsal horn.28–30 In our present study, we demonstrated that CCD could induce upregulation of TRPA1 protein levels. These results are in accordance with our behavioral data, which indicate that TRPA1 may play an important role in CCD-induced neuropathic pain.
Our study demonstrated upregulation of p-NR2B protein levels in the spinal cord after CCD. Accumulating evidence has suggested that activation of the central glutaminergic system, especially N-methyl-D-aspartate receptor (NMDA) receptors, plays a central role in maintenance of neuropathic pain.31 Tyrosine phosphorylation of NR2B subunits is important for NMDA receptor activation and contributes to nociceptor activity-induced spinal plasticity and development of central sensitization.32–34 It has been reported that tyrosine phosphorylation of NR2B in the dorsal horn is involved in the development of neuropathic35 and inflammatory pain.32 These data and our study suggest that tyrosine phosphorylation of NR2B should be involved in hyperalgesia in the spinal dorsal horn.
In experiment 2, i.t. administration of HC-030031 dose-dependently attenuated CCD-induced hyperalgesia. The i.t. administration of 10 and 25 μg HC-030031 could not depress CCD-induced mechanical allodynia. Compared with a previous study in mice,9 these 2 doses may not be enough for i.t. injection to attenuate hyperalgesia. The i.t. administration of 50 μg HC-030031 downregulates expression TRPA1 and p-NR2B in the spinal cord. Because HC-030031 is a TRPA1 channel antagonist, decreased expression of spinal TRPA1 was not surprising. How HC-030031 modulates NMDA receptor activity is not as clear. Spinal TRPA1 could be active in various induced pain hypersensitivity conditions,15 and several studies demonstrated that the glutaminergic system is involved in transmission of the nociceptive stimulus induced for activation of the TRPA1 channel in the spinal cord.36 These results suggested that spinal TRPA1 participates in the enhancement of glutamatergic transmission of nociceptive signals leading to increased hypersensitivity. HC-030031, the TRPA1 channel antagonist, attenuates activity of NMDA receptors through downregulation of enhanced glutamatergic transmission. Spinal administration of a TRPA1 channel antagonist failed to attenuate mechanical pain hypersensitivity induced by direct chemical activation of NMDA.15 In cinnamaldehyde- (a TRPA1 agonist) induced pain hypersensitivity, the behavior was reduced by the coinjection (i.t.) of camphor (a TRPA1 antagonist) or MK-801 (a NMDA receptor antagonist).36 These results and our findings suggest that the effect of HC-030031 may be partially a result of depression of NMDA in the spinal cord, but the precise mechanism needs further investigation.
The present study demonstrated that CCD-induced hyperalgesia was associated with enhancement of TRPA1 and tyrosine phosphorylation of NR2B in the spinal cord. We determined that the TRPA1 antagonist HC-030031 attenuated hyperalgesia induced by CCD and upregulated TRPA1 and tyrosine phosphorylation of NR2B in the spinal cord. We provide evidence that the antihyperalgesic effect of HC-030031 may depend on its ability to modulate activation of spinal cord NMDA receptors. Our study suggests that the TRPA1 channel may be an effective novel option for treatment of neuropathic pain.
Name: Wei Zhang, MD.
Contribution: This author helped design and conduct the study, analyze the data, prepare the manuscript.
Attestation: Wei Zhang approved the final manuscript. Wei Zhang has seen the original study data, reviewed the analysis of the data, and is the author responsible for archiving the study files.
Name: Yue Liu, MD.
Contribution: This author helped conduct the study and data collection.
Attestation: Yue Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xin Zhao, MD.
Contribution: This author helped data collection and data analysis.
Name: Xiaoping Gu, PhD.
Contribution: This author helped design the study and prepare the manuscript.
Name: Zhengliang Ma, PhD.
Contribution: This author helped design the study and prepare the manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
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