Neuropathic pain is characterized by sensory abnormalities such as hyperalgesia, dysesthesia, and allodynia, and spontaneous pain can arise as a result of this condition.1 Nefopam hydrochloride is a centrally acting antinociceptive compound with both supra-spinal and spinal sites of action.2 It is a nonopioid analgesic, and structurally, nefopam is a cyclic analog of diphenhydramine, with a chemical structure similar to orphenadrine.3 , 4 Previous studies have shown that it induces antinociceptive and antihyperalgesic properties in animal models of nociceptive pain.5–7 In addition, clinical evidence has demonstrated that nefopam is a useful adjuvant to opioids in postoperative pain, by decreasing pain intensity, and reduces morphine consumption.8
The mechanism of action of nefopam is not well understood, although inhibition of monoamine reuptake and synaptosomal reuptake of serotonin, dopamine, and norepinephrine is thought to be involved in its analgesic effects.9 , 10 In addition, involvement of the glutamatergic pathway through modulation of voltage-dependent sodium and calcium channels has been demonstrated.11 , 12 Moreover, the involvement of transient receptor potential subtype 1 (TRPV1) in the analgesic and antihyperalgesic properties of nefopam has been suggested.13 Nefopam, however, does not bind to opioid receptors and is structurally unrelated to nonsteroidal antiinflammatory drugs.14
Several types of potassium (K+) channels are involved in the antinociceptive effects of various drugs and natural products, including adenosine triphosphate (ATP)-sensitive K+ (KATP) channels (members of the inwardly rectifying K+ channel subfamily), some voltage-gated K+ channels, and calcium (Ca2+)-activated K+ (KCa2+) channels. However, little is known about the relationship between potassium channels and nefopam in a neuropathic pain model. The aim of our current study therefore was to examine the ability of different doses of nefopam to attenuate mechanical allodynia in a rat nerve ligation injury model of neuropathic pain. In addition, we evaluated the relationship between K+ channels and nefopam to determine whether the antiallodynic effects of nefopam are mediated by KATP and KCa2+ channels in a neuropathic pain model.
The study protocol was approved by the Institutional Animal Care and Use Committee of Asan Medical Center (Seoul, South Korea). Male Sprague–Dawley rats (Orient Bio, Sungnam, Korea) weighing 180 to 200 g were used. The rats were housed 2 or 3 per cage for 7 days in humidity and temperature controlled (21–23°C) vivaria, with a 12-hour light/dark cycle (07:00 AM hour onset) and free access to food and water at all times. Behavioral testing and analgesiometry were performed according to the ethical guidelines set by the International Association for the Study of Pain,15 and the animals were euthanized after completion of the planned tests.
The animals were anesthetized by sevoflurane, and an approximately 1.5-cm midline incision was made on the left lateral side of each rat at the L5–L6 level. The paravertebral muscles were dissected and retracted, and the L5 and L6 vertebral bodies were exposed partially. The left L6 transverse process was excised partially, and the left L5 and L6 spinal nerves were exposed. Neuropathic pain was induced surgically by tight ligation of the L5 and L6 spinal nerves with a 6-0 black silk distal to the dorsal root ganglion, as previously described.16 At postoperative day 3, an intrathecal catheter was inserted in the animals.17 Under sevoflurane anesthesia, each rat was placed in a stereotaxic head frame. After exposure of the atlantooccipital membrane at the base of the skull, a polyethylene tube was inserted caudally from the cistern magna to the lumbar enlargement. The catheter was externalized through the skin and tunneled subcutaneously to the top of the head. Proper location of the catheter was confirmed by inducing a temporary motor block of both hind limbs by injecting 7 μL of 2% lidocaine through the catheter. Neurologic deficit was evaluated by observing ambulation, weight bearing, and righting and placing/stepping reflexes. Any animals showing postsurgical neurologic deficits were excluded from the study and euthanized.
Nefopam Administration Protocols
To examine the ability of nefopam to prevent the development of mechanical allodynia, 10 mg/kg, 30 mg/kg, 60 mg/kg nefopam hydrochloride (Acupan; Pharmbio, Seoul, Korea) or saline was intraperitoneally administered 20 minutes before spinal nerve ligation (SNL) (pre-nefopam). To examine the ability of intraperitoneal injection of nefopam to attenuate mechanical allodynia after SNL, 7 days after SNL, intraperitoneal injection of saline or nefopam 10 mg/kg, 30 mg/kg, and 60 mg/kg was administered in rats presenting with mechanical allodynia (post-nefopam). The experimenter was blind to the administered drug.
All of the behavioral tests were performed at the same time of day to eliminate the effects of errors associated with diurnal rhythm. For behavioral testing for mechanical allodynia, the rats were placed in individual acryl cages with a wire mesh floor and were allowed to acclimate to the testing environment for 30 minutes. The tactile threshold was measured by applying 8 series of calibrated von Frey filaments (0.41–15.1 g; Stoelting Co., Wood Dale, IL) to the hindpaw ipsilateral to the nerve injury. Sufficient pressure was applied for 6 seconds to cause slight bending of the filament against the midplantar surface of the ipsilateral hindpaw, with brisk withdrawal or flinching of the paw considered a positive response. In the case of a positive response, the next lower force filament was applied, and in the absence of a positive response, the next greater force filament was applied. The 50% withdrawal threshold was determined by use of the up–down method,18 and the cutoff value was defined as the absence of response to a 15.1 g force. Baseline values were determined by performing these behavioral tests 1 day before neuropathic surgery. Behavioral measurements were taken 5, 9, 13, 17, 21, 25, and 29 days after neuropathic pain surgery and drug administration in the pre-nefopam group. In the post-nefopam group, behavioral measurements were taken at 30, 60, 90, and 120 minutes after administration of the study drug, and 8, 9, 13, 17, 21, and 29 days after surgery. The mechanical paw withdrawal threshold (PWT) was reported as the actual threshold in grams (g).
Intrathecal Drug Administration
The study drugs were dissolved in 10 μL normal saline and administered through the intrathecal catheter over 60 seconds with the use of a microinjection syringe, followed by the same volume of solvent flush. The experimenter was blinded to the drug administered.
To evaluate the antiallodynic effects of nefopam on the treatment of neuropathic pain, nefopam or 0.9% saline was administered intraperitoneally 7 days after SNL; 10 (n = 6), 30 (n = 8), or 60 mg/kg (n = 9) of nefopam was dissolved in normal saline (0.9%) so that a total volume of 3 mL/kg body weight was administered intraperitoneally. For control animals (n = 10), 3 mL/kg of 0.9% saline was administered intraperitoneally.
To evaluate the preventive effects of nefopam on the development of neuropathic pain, 10 (n = 5), 30 (n = 9), or 60 mg/kg (n = 10) of nefopam was dissolved in normal saline (0.9%) so that a total volume of 3 mL/kg body weight was administered intraperitoneally 20 minutes before SNL. For control animals (n = 10), 3 mL/kg of 0.9% saline was administered intraperitoneally.
To investigate the relationship between small conductance Ca2+-activated K+ (SKCa2+) channels and the antiallodynic effects of nefopam, apamin, a specific blocker of SKCa2+ channels,19 was administered intrathecally 20 minutes before intraperitoneal administration of 30 mg/kg nefopam 7 days after SNL. Apamin was administered at doses of 0.1 (n = 5) and 3 ng/kg (n = 5) dissolved in 10 μL normal saline20 , 21; the nefopam group (n = 8) received an equal volume of intrathecal saline injections, and the control group (n = 10) received an equal volume of intrathecal saline injection without additional administration of intraperitoneal nefopam. To assess the possible effects of apamin alone on allodynia in a rat neuropathic model with SNL, 0.1 and 3 ng/kg apamin (n = 5 each) were administered intrathecally without additional administration of intraperitoneal nefopam.
To investigate the relationship between large conductance Ca2+-activated K+ (BKCa2+) channels and the antiallodynic effects of nefopam, charybdotoxin, a BKCa2+ channel blocker,19 was administered intrathecally 20 minutes before intraperitoneal administration of 30 mg/kg nefopam 7 days after SNL. Charybdotoxin was administered at doses of 0.01 (n = 5) and 1 ng/kg (n = 5) dissolved in 10 μL normal saline20 , 21; the nefopam group (n = 8) received an equal volume of intrathecal saline injections, and the control group (n = 10) received an equal volume of intrathecal saline injection without additional administration of intraperitoneal nefopam. To assess the possible effects of charybdotoxin alone on allodynia in a rat neuropathic model with SNL, 0.01 and 1 ng/kg of charybdotoxin (n = 5 each) alone were administered intrathecally without additional administration of intraperitoneal nefopam.
To investigate the relationship between KATP channels and the antiallodynic effects of nefopam, glibenclamide, a blocker of KATP channels,22 was administered intrathecally 20 minutes before intraperitoneal administration of 30 mg/kg nefopam 7 days after SNL. Glibenclamide was administered at doses of 0.3 (n = 5) and 3 mg/kg (n = 5) dissolved in 10 μL normal saline; the nefopam group (n = 8) received an equal volume of intrathecal saline injections, and the control group (n = 10) received an equal volume of intrathecal saline injection without additional administration of intraperitoneal nefopam. To assess the possible effects of glibenclamide alone on allodynia in a rat neuropathic model with SNL, 0.3 and 3 mg/kg of glibenclamide (n = 5 each) alone were administered intrathecally without additional administration of intraperitoneal nefopam.
Regarding the results of the KATP channel blocker against nefopam, we further evaluated the effects of KATP agonists on the antiallodynic effects of nefopam. Pinacidil, an opener of the KATP channels,23 was administered intrathecally 20 minutes before intraperitoneal administration of 30 mg/kg nefopam 7 days after SNL. Pinacidil was administered at doses of 10 (n = 5) and 30 μg/kg (n = 5) dissolved in 10 μL normal saline; the nefopam group (n = 8) received an equal volume of intrathecal saline injections, and the control group (n = 10) received an equal volume of intrathecal saline injection without additional administration of intraperitoneal nefopam. To assess the possible effect of pinacidil alone on allodynia in a rat neuropathic model with SNL, 10 and 30 μg/kg of pinacidil (n = 5 each) alone were administered intrathecally without additional administration of intraperitoneal nefopam.
To evaluate the relationship between KATP channels and the preventive effects of nefopam, the KATP channel blocker glibenclamide was administered intrathecally 20 minutes before intraperitoneal administration of nefopam. Then 30 mg/kg of nefopam was administered intraperitoneally 20 minutes before SNL. Glibenclamide was administered at doses of 0.3 and 3 mg/kg dissolved in 10 μL normal saline; the nefopam group received an equal volume of intrathecal saline injections, and the control group received an equal volume of intrathecal saline injection without additional administration of intraperitoneal nefopam (n = 10 for each group).
The data are expressed as mean ± SEM. The differences in PWT between groups were compared at each time point by use of the linear mixed models with various covariance patterns for the repeated observations because sphericity tests were not satisfied. Covariance patterns for each model were selected by the use of likelihood ratio test. The assumption of the models including normality and homoscedasticity was evaluated by residual plot. The Bonferroni correction was used for post-hoc multiple comparisons. The significance level was selected as under 0.05/6 = 0.0083 for comparison among 4 groups and 0.05/3 = 0.0167 for comparison among 3 groups. P < .05 was considered statistically significant. SigmaPlot version 12.0 (Systat Software Inc, Richmond, CA) and SAS version 9.3 (SAS Institute, Cary, NC) were used for data analysis.
Experiment 1: The Antiallodynic Effects of Nefopam
Intraperitoneal administration of nefopam effectively reversed mechanical allodynia in SNL rats, showing a dose-dependent increase in PWT compared with the control group (Figure 1). The greater-dose nefopam groups (30 mg and 60 mg) presented more sustained increase in PWT against mechanical stimuli compared with the 10-mg nefopam group. The antiallodynic effect of nefopam maintained until experimental day 29 (Figure 1).
Experiment 2: The Preventive Effect of Nefopam Against Development of Mechanical Allodynia
Intraperitoneal administration of nefopam effectively suppressed the development of mechanical allodynia in SNL rats, showing a clear increase in PWT compared with the control group (Figure 2); however, the preventive effects of nefopam in the development of neuropathic pain were profound in greater doses (30 and 60 mg/kg of nefopam) and did not show a dose-dependent pattern between nefopam 30 and 60 mg/kg throughout the study period (Figure 2, P > .15).
Experiment 3: The Involvement of SKCa2+ Channels on Nefopam-induced Antiallodynia
Intrathecal pretreatment with the SKCa2+ channel blocker, apamin, suppressed the antiallodynic effect of nefopam 120 minutes after intraperitoneal nefopam injection at the lower dose (0.1 ng/kg). At observation day 8, the both doses (0.1 and 3 ng/kg) of apamin enhanced the antiallodynic effects of 30 mg/kg intraperitoneal nefopam (Figure 3). Both doses of intrathecally administered apamin alone reduced the PWT induced by SNL from 30 minutes onward, thereby aggravating the pain response to mechanical stimuli (Figure 3).
Experiment 4: The Involvement of BKCa2+ Channels on Nefopam-induced Antiallodynia
Intrathecal injection of the BKCa2+ channel blocker, charybdotoxin, suppressed the antiallodynic effect of nefopam 120 minutes after intraperitoneal nefopam injection at the lower dose (0.01 ng/kg), and the greater dose (1 ng/kg) of charybdotoxin enhanced the antiallodynic effect of nefopam at observation day 8 (Figure 4). Intrathecally administered charybdotoxin alone reduced the PWT induced by SNL at either higher or lower doses from 30 minutes onward, thereby aggravating the pain response to mechanical stimuli (Figure 4).
Experiment 5: The Involvement of the KATP Channel Blocker on Nefopam-induced Antiallodynia
Intrathecally administered glibenclamide alone reduced the PWT induced by SNL at either higher or lower doses, thereby aggravating the pain response to mechanical stimuli. The aggravation of allodynia after glibenclamide administration showed a dose-dependent pattern (Figure 5). The 2 doses of intrathecal glibenclamide had different effects in the antiallodynic effects of 30 mg/kg intraperitoneal nefopam. The greater dose of 3 mg/kg glibenclamide induced a significant reversal of the antiallodynic effects of nefopam throughout the observation period. The lower dose of 0.3 mg/kg glibenclamide induced a reversal of the effects of nefopam at 90 and 120 minutes after intraperitoneal injection of nefopam, but in contrast to the greater-dose group, the antiallodynic effects of nefopam were enhanced significantly at observation day 8 and 9 in the lower glibenclamide group (Figure 5).
Experiment 6: The Involvement of the KATP Channel Opener on Nefopam-induced Antiallodynia
Intrathecal administration of the KATP channel opener, pinacidil, produced dose-dependent antiallodynic effects compared with the control group (Figure 6). When administered 20 minutes before intraperitoneal nefopam, both doses of pinacidil produced enhancement of the antiallodynic effects of nefopam at day 8 and 9 (Figure 6).
Experiment 7: The Involvement of the KATP Channel Blocker on the Preventive Effect of Nefopam
Intraperitoneal administration of 30 mg/kg nefopam 30 minutes before SNL effectively suppressed the development of mechanical allodynia in SNL rats. When the KATP channel blocker, glibenclamide, was administered 30 minutes before intraperitoneal nefopam, both dose of glibenclamide (0.3 mg and 3 mg/kg) induced a reversal of preventive effect of 30 mg/kg nefopam at day 5 (Figure 7).
In the current study, we evaluated the relationship between the antiallodynic effects of nefopam and K+ channels. The results suggest that these effects are mediated, in part, by KATP channels. The antiallodynic effects of nefopam were attenuated and enhanced by pretreatment of KATP channel blockers and channel openers, respectively. In contrast, SKCa2+ or BKCa2+ channels did not demonstrate significant influence on the antiallodynic effects of nefopam in a rat nerve ligation injury model of neuropathic pain.
It has been several decades since nefopam has been developed. Since then, in European nations, nefopam has been used widely for the treatment of acute and chronic pain.8 , 24 A number of previous studies have demonstrated the antiallodynic properties of nefopam.8 , 23 , 25–28 In accordance with previous results, intraperitoneal injection of nefopam at day 7 after SNL attenuated the PWT against mechanical stimuli in our study. Furthermore, our results demonstrated that preemptive intraperitoneal injection of nefopam prevented the development of neuropathic pain after SNL.
The precise mechanism underlying the action of nefopam is not clearly understood, but numerous studies have suggested the involvement of voltage-gated Ca2+ and sodium channels, the glutamatergic pathway, TRPV1, 5-HT7 receptors, and inhibition of monoamine uptake.9–13 The preemptive effect of nefopam has been demonstrated only in few numbers of studies, which proposed several possible mechanism of action.6 , 25 The prolonged preemptive effect of nefopam is thought to be a result of inhibition of the central neuroplasticity mechanisms after surgical injuries that lead to pain hypersensitivity. Nefopam is a centrally acting analgesic drug, and the peripheral effect seems to be minor.6 , 26 An injury to nerve may lead to a strong initial burst neuronal activity that may lead to a persistent neuronal abnormality, which may eventually lead to chronic pain and hypersensitivity. An early block of this stage may attenuate the development of neuropathic pain.27 , 28 The results of this study thus may suggest therapeutic implications. Although clinical trials are needed for clarification, preemptive analgesic application of nefopam may have the potential to reduce the risk of developing postoperative chronic pain.
The K+ channels are involved in the antinociceptive effects of certain drugs and have been considered targets for antinociceptive drug development.19 , 29–31 For example, the nonselective KATP channel blocker, glibenclamide, and the mitochondrial KATP channel blocker, 5-hydroxydecanoate, significantly attenuated the antiallodynic effects of R-PIA, and the KATP channel opener, diazoxide, demonstrated dose-dependent antiallodynic effects in an SNL model.22 Our present findings suggest that the antinociceptive effects of nefopam are involved in the opening of KATP channels. KATP channels that are located in peripheral and central neurons have recently been recognized as important mediators in the pathogenesis of neuropathic pain and the opening of KATP channels reduces nociception, making it a target for the treatment of neuropathic pain.32–34 In the current study, the KATP channel blocker, glibenclamide, reversed the antinociceptive effects of nefopam. The intrathecal administration of glibenclamide before intraperitoneal nefopam injection in the preemptive nefopam model also attenuated the antiallodynic effect of nefopam. Furthermore, addition of the KATP channel opener, pinacidil, enhanced the PWT of rats that underwent SNL compared with nefopam treatment alone. Taken together, these findings support that the opening of KATP channels are involved in the antinociceptive mechanism of nefopam. On the other hand, the KCa2+ channel blockers, apamin (SKCa2+ channels) and charybdotoxin (BKCa2+ channels), had no significant effects on the antiallodynic effects of nefopam, suggesting that the KCa2+ channels have a weaker relationship with the antiallodynic action of nefopam compared with KATP channels. Pinacidil alone also demonstrated dose-dependent antiallodynic effects in rats that underwent SNL, which is in accordance with previous studies that showed the administration of KATP channel agonists can enhance antiallodynic effects in neuropathic pain models.19 , 35–37 This finding can arouse one limitation that the enhancement of mechanical thresholds of the SNL animals after administration of pinacidil and nefopam may simply be a synergic effect as we did not investigate in detail about the mechanism of synergy between these 2 drugs. But we could still suggest that KATP channels are involved in the antiallodynic mechanism of nefopam as the effect of the KATP channel antagonist glibenclamide was significant and the experiment with pinacidil was performed to support the results of KATP channel antagonist.
Another interesting finding of our present analyses was that the KATP channel blocker, glibenclamide, significantly suppressed the antiallodynic effects of nefopam at high dose, but at lower dose of glibenclamide, the duration of suppressing the antiallodynic effects of nefopam was much shorter compared with the greater dose. Furthermore, in the later observation period, the low dose of glibenclamide reversibly enhanced the antiallodynic effects of nefopam. These results differ from those in previous studies in which the authors reported that glibenclamide demonstrated a dose-dependent reversal of the antiallodynic effects of KATP channel agonists.22 , 36 In a rat model of acute gout, however, subcutaneously injected glibenclamide reduced nociception and tissue edema, with peak effects occurring 4 hours after subcutaneous injection.38 In that study, the authors concluded that the antinociceptive effects of glibenclamide resulted from inflammasome inhibition, rather than alterations of the KATP channels. The effects of glibenclamide at different doses and at different locations need to be further evaluated to determine the precise dose-response relationship between KATP channel antagonism and pain perception.
A notable limitation of our present study was that we did not examine the effects of selective mitochondrial or sarcolemmal KATP channels and only investigated the effects of glibenclamide, a nonselective KATP channel antagonist. Additional studies with specific inhibitors of mitochondrial and sarcolemmal KATP channels are required to clarify the role of KATP channels on nefopam-induced antinociception. Another limitation is that we did not investigate the molecular mechanisms underlying the involvement of KATP channel in the antiallodynic effect of nefopam. It is known that the antinociceptive effects of certain opioids, certain nonsteroidal antiinflammatory drugs, gabapentin, and pregabalin were linked with KATP channel in the central nervous system.39 , 40 Therefore, the antiallodynic effect of nefopam might be, in part, related with KATP channel by these receptors. In addition, although it is not a direct method, we demonstrated that the antiallodynic effects of nefopam can be enhanced or reversed by KATP channel agonist and antagonist. Further study evaluating the molecular mechanism underlying the antiallodynic effect of nefopam may have strengthened our study results.
In conclusion, the antiallodynic effects of nefopam in a neuropathic pain model are enhanced by a KATP channel agonist and reversed by a KATP channel antagonist, suggesting that the KATP channel is involved in the antiallodynic effects of nefopam. Additional studies are needed to clarify the dose relationship between KATP channel antagonists and pain perception, and to identify the types of KATP channel involved in the underlying mechanism.
Name: Won Uk Koh, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Name: Jin Woo Shin, MD, PhD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Name: Ji-Yeon Bang, MD, PhD.
Contribution: This author helped conduct the study and analyze the data.
Name: Sae Gyeol Kim, MD.
Contribution: This author helped analyze the data and write the manuscript.
Name: Jun-Gol Song, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
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