Acetaminophen Exerts an Analgesic Effect on Muscular Hyperalgesia in Repeated Cold-Stressed Rats through the Enhancement of the Descending Pain Inhibitory System Involving Spinal 5-HT₃ and Noradrenergic α₂ Receptors

Abstract

Musculoskeletal and psychological complaints have increased with the widespread use of visual display terminals, and musculoskeletal pain is known to be closely related to stress. One method of experimentally inducing persistent muscle pain is repeated cold stress (RCS), and animals exposed to such stress exhibit a dysfunction in the descending pain inhibitory system. Acetaminophen (N-acetyl-p-aminophenol; APAP) is widely used to relieve several types of pain, including musculoskeletal pain, and is available as an OTC drug. However, the mechanism underlying its analgesic action has not yet been fully elucidated. In this study, we compared the analgesic effect of APAP on RCS-induced muscular hyperalgesia with those of other analgesics to identify its mechanism of action. The daily oral administration of APAP significantly suppressed the decrease in the mechanical withdrawal threshold caused by RCS, similar to the results for neurotropin but not for the cyclooxygenase inhibitor ibuprofen (IBP). Moreover, the intrathecal administration of antagonists of the 5-hydroxytryptamine (5-HT)₃ receptor or α₂-adrenoceptor significantly abolished the analgesic effect of APAP but not of IBP. These results suggest that the analgesic effect of APAP on RCS-induced muscular pain might be exerted due to the activation of the descending pathways involving the spinal 5-HT₃ receptor or α₂-adrenoceptor.

INTRODUCTION

Neck and shoulder discomfort or tenderness, known as “katakori” in Japanese, is a frequently reported musculoskeletal symptom that can be followed by symptoms such as headache. Visual display terminals, including laptops and smart phones, have become an integral part of modern lifestyles, but the prolonged use of these devices can cause musculoskeletal discomfort, psychological symptoms and asthenopia.¹⁾ Unfavorable muscle loads and mental stress have been shown to induce changes in muscle blood flow and muscle sensitivity associated with the aberration of the autonomic nervous system, and these factors play important roles in the initiation and maintenance of muscle pain.^2–4) The standard treatment for musculoskeletal pain includes medication, such as analgesics, muscle relaxants or Kampo preparations, physiotherapy and acupuncture.

Acetaminophen (N-acetyl-p-aminophenol (APAP), also known as paracetamol) is an analgesic agent that is widely used to relieve several types of pain including headache and neck-shoulder pain and is available as an OTC drug. APAP has distinct properties in terms of its therapeutic activities and adverse effects, and it is categorized differently from non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are well recognized to exert their effects by inhibiting cyclooxygenase (COX) and subsequent prostaglandin synthesis.⁵⁾ On the other hand, several studies have shown that APAP had minimal COX inhibitory and anti-inflammatory actions in peripheral tissues,^6,7) but the mechanism of its analgesic effect has not been fully understood.

The descending pain inhibitory system has been suggested to play an important role in the modulation of pain transmission.⁸⁾ The descending monoaminergic pathways project to the spinal dorsal horn through the rostral ventromedial medulla (RVM, the origin of the serotonergic pathway) or the locus coeruleus (LC, the main origin of the noradrenergic pathway). The transmission of nociceptive information is directly or indirectly inhibited via several types of 5-hydroxytryptamine (5-HT) receptors and α₂-adrenoceptors in the spinal dorsal horn. The release of glutamate, γ-aminobutyric acid (GABA), and glycine from the primary afferent or interneurons can be modified by the activation of the descending system.⁹⁾ Some studies have shown an essential role of the descending serotonergic pathways in the analgesic effect of APAP.^10,11) Therefore, in the present study, we focused on the effect of APAP on musculoskeletal pain.

Stress is known to cause the dysfunction of the descending pain inhibitory system and to be closely related to muscular tenderness. Repeated cold stress (RCS, also called specific alteration of rhythm in temperature (SART) stress) reportedly impairs the function of the descending serotonergic and noradrenergic pathways and causes muscular mechanical hyperalgesia.^12–14) In RCS experiments, animals are transferred repeatedly at short intervals of 30 min between normal (23 °C) and cold (4 °C) rooms. RCS-exposed animals show an autonomic nervous system imbalance and an enhancement of the mechanical response of muscle tissue,^15,16) both of which are compatible with the clinical features of musculoskeletal pain. RCS-induced chronic hyperalgesia in deep tissues, such as muscle, was evaluated using a probe with a large-tip diameter in the Randall–Selitto test, and the withdrawal threshold established using this method was not influenced by surface anesthesia.¹⁴⁾ Neurotropin (NTP), an analgesic for relieving cervical syndrome and shoulder periarthritis, has been shown to inhibit RCS-induced mechanical hyperalgesia by activating the descending pathways involving the spinal 5-HT₃ receptor and the α₂-adrenoceptor.^13,17) On the other hand, the mechanism responsible for the analgesic action of APAP on RCS-induced muscular hyperalgesia has not yet been examined.

Therefore, in this study, to investigate the differences in action between APAP and a COX inhibitor, we produced an RCS-induced chronic muscular hyperalgesia model and evaluated the analgesic effects of the oral administration of APAP and ibuprofen (IBP). Then, we compared the analgesic effects of the oral administration of NTP, which is thought to activate the descending pathways, with those of APAP’s action. Finally, we evaluated the effects of the intrathecal administration of 5-HT₃ receptor or α₂-adrenoceptor antagonists to clarify the involvement of the descending pain inhibitory system in the analgesic effect of APAP or IBP.

MATERIALS AND METHODS

Animals

Male Sprague–Dawley rats were purchased from Japan SLC (Shizuoka, Japan). The rats were housed under conditions of controlled temperature (23 ± 3 °C) except during the period of RCS exposure, controlled humidity (50 ± 20%), and controlled lighting (lights on from 07 : 00 to 19 : 00 h). Eight-week-old animals were used in the study. All the rats were given access to food and tap water ad libitum. All the experimental procedures were conducted with the approval of the Animal Care Committee at Taisho Pharmaceutical Co., Ltd., in accordance with the company’s guidelines for the Care and Use of Laboratory Animals.

Drugs

APAP was purchased from YAMAMOTO CORPORATION (Osaka, Japan) and Lianyungang Kangle Pharmaceutical Co., Ltd. (Jiangsu, China). IBP was purchased from BASF (Ludwigshafen, Germany) and Solara Active Pharma Sciences Ltd. (Tamilnadu, India). NTP was purchased from Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, Japan). Yohimbine hydrochloride and MDL72222 were purchased from Sigma-Aldrich (MO, U.S.A.) and Tocris Bioscience (Bristol, U.K.), respectively.

RCS Exposure

RCS was applied as previously reported according to the following schedule.¹⁴⁾ Rats were placed in a cold room at 4 °C from 19 : 00 h on the first day (defined as Day 0 in this study) to 10 : 00 h the following morning and then were exposed to 23 °C (room temperature) and 4 °C for alternate 30-min periods from 10 : 00 to 17 : 30 h. This procedure was repeated for 5 consecutive days and then stopped on the morning of Day 6 (Fig. 1a). During the stress period, the rats were housed individually in stainless-mesh cages with free access to food and water.

Fig. 1. Effect of RCS Exposure on the Withdrawal Threshold of Deep Tissues in the Randall–Selitto Test in Rats

(a) Schedule for RCS exposure in rats. (b) The withdrawal threshold of the right hind leg muscles was evaluated in rats with or without RCS exposure. The data represent the means ± S.E.M. (n = 8). ** p < 0.01, *** p < 0.001 versus normal group, Student’s t-test.

Randall–Selitto Test

The withdrawal threshold of the deep tissues was assessed using a Randall–Selitto Analgesy–Meter (Ugo Basile, Italy). The rounded tip (tip diameter: 2.6 mm, custom built) of the device was applied to the middle portion of right lower hind leg muscles, including the extensor digitorum longus muscle, through shaved skin. The applied force was increased at a constant rate, and the cut-off point was set at 250 g to avoid tissue damage. The pressure intensity required to produce an escape reaction was defined as the withdrawal threshold. Measurements for each rat were performed eight times at about 20-s intervals, and the withdrawal threshold was calculated as the mean of the last five trials.

Intrathecal Cannulation

Under isoflurane anesthesia, a polyethylene catheter (PE10 intramedic polyethylene tubing; inner diameter, 0.28 mm; outer diameter, 0.61 mm; Becton, Dickinson and Company, NJ, U.S.A.) was inserted through the atlantooccipital membrane into the lumbar enlargement of the spinal cord (close to the L3–L5 segments) of the rats. After the surgery, the rats were housed individually and were allowed to recover for at least 5 d before the start of the experiments. Rats exhibiting no sequential paralysis of their hind paws were used for the following experiments.

Drug Administration

APAP and IBP were suspended in water or 5% (w/v) arabic gum solution, and NTP was suspended in water or 0.5% (w/v) sodium carboxymethyl cellulose solution. For the oral administration experiments from the start of RCS, APAP (100 mg/kg), IBP (10 mg/kg) and NTP (50 neurotropin units (N.U.)/kg) were administered orally twice a day from Day 1 until Day 23. Oral administration was performed at around 10 : 00 and 16 : 00 h, except on the measurement days on which the analgesics were administered 30–60 min before and several hours after the Randall–Selitto test. For the oral administration experiments after the period of RCS, the administration of APAP, IBP and NTP was started on Day 6 and was performed twice a day until Day 23, as above. For normal rats, APAP and IBP were orally administered twice daily for 16 d. For the intrathecal administration of the antagonists, MDL72222 and yohimbine hydrochloride were dissolved in 5% (v/v) dimethyl sulfoxide solution and saline, respectively. Catheterized rats were orally administered APAP or IBP twice a day and intrathecally administered MDL72222 (30 nmol/10 µL) or yohimbine (30 nmol/10 µL) once daily from Day 1 until Day 6. The antagonists were intrathecally administered through the catheter under isoflurane anesthesia. When the withdrawal threshold was measured on Day 6 (1 d after RCS), the oral administration of APAP or IBP and the intrathecal administration of antagonists were performed about 30 and 25 min before the measurement respectively.

Data Analysis

The results of the Randall–Selitto test (4 to 8 rats per group) are presented as the means ± standard error of the mean (S.E.M). The effect of RCS on the withdrawal threshold was analyzed using Student’s t-test compared to the normal group or a paired t-test in the RCS control group. Comparisons of the effects of oral APAP and IBP were performed using Dunnett test. For the oral NTP and the intrathecal antagonist experiments, statistical analyses were performed using Student’s t-test. p < 0.05 was considered as being indicative of statistical significance.

RESULTS

RCS Exposure Induced Chronic Muscular Mechanical Hyperalgesia

RCS exposure reportedly causes persistent muscular mechanical hyperalgesia.¹⁴⁾ We examined the influence of RCS on the mechanical withdrawal threshold of deep tissues. In the no RCS-exposed animals (normal group, Fig. 1b, open circle), the withdrawal threshold measured using the Randall–Selitto device was stable throughout the observation period. In the RCS-exposed animals (RCS group, Fig. 1b, solid circle), however, the withdrawal threshold on Day 6 (1 d after RCS) was significantly lower than that in the normal group. The significant decrease lasted until as long as Day 20 (15 d after RCS). Therefore, we used this model of chronic muscle pain induced by RCS in the following experiments.

Effects of APAP and IBP on RCS-Induced Muscular Mechanical Hyperalgesia and Normal Muscular Mechanical Threshold

We investigated the analgesic effects of APAP and IBP on chronic muscular mechanical hyperalgesia induced by RCS. First, the effects of daily oral administration beginning at the start of RCS exposure (Day 1) were examined. These results are shown in Fig. 2a. RCS exposure induced a significant decrease in the withdrawal threshold in the vehicle-administered animals (control group, open circle) that persisted until Day 23 (18 d after RCS). In the animals administered APAP (APAP group, closed circle), the decrease in the withdrawal threshold caused by RCS was significantly suppressed, compared with that in the control group; the withdrawal threshold recovered to a similar extent as that observed pre-RCS on Day 14. Meanwhile, in the animals that were administered IBP (IBP group, closed triangle), the decrease in the withdrawal threshold was significantly suppressed, compared with that in the control group, but the suppression after RCS was temporary.

Fig. 2. Effects of Orally Administered APAP and IBP on the Withdrawal Threshold in the Randall–Selitto Test in Rats with or without RCS Exposure

(a) The withdrawal threshold in the right hind leg muscles was evaluated in RCS rats administered APAP and IBP from the start of RCS. APAP (100 mg/kg) and IBP (10 mg/kg) were administered twice daily from Day 1 until Day 23. On the measurement days, the first administration was performed about 30 min before the Randall–Selitto test. The data represent the means ± S.E.M. (n = 6). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus pre-RCS in control group, paired-t test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control group, Dunnett test. (b) The withdrawal threshold in the right hind leg muscles was evaluated in RCS rats administered APAP and IBP from post-RCS. APAP and IBP were administered twice daily from Day 6 until Day 23. The data represent the means ± S.E.M. (n = 8). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus pre-RCS in control group, paired-t test. * p < 0.05, ** p < 0.01 versus control group, Dunnett test. (c) The withdrawal threshold in normal rats administered APAP and IBP twice daily for 16 d. The data represent the means ± S.E.M. (n = 8). n.s.: not significant versus control group at the last measurement day (16 d), Dunnett test.

Next, the effects of daily oral administration beginning post-RCS (Day 6) were examined. These results are shown in Fig. 2b. In the control group, a significant decrease in the withdrawal threshold was observed on Day 6 and lasted until Day 20 (15 d after RCS). In the APAP group, the RCS-induced decrease in the withdrawal threshold was slightly prevented by a single administration on Day 6, although the difference was not significant, and daily oral administration resulted in the observance of a significant recovery on Day 8 (3 d after RCS). In contrast, the single and repeated administration of IBP after RCS had no effect on the decreased withdrawal threshold, compared with that in the control group.

Additionally, we investigated the effects of the daily oral administration of APAP and IBP on the withdrawal threshold in normal rats. No significant change in the withdrawal threshold was observed in the normal rats after the repeated administration of APAP and IBP for 16 d (Fig. 2c).

Analgesic Effect of NTP Was Similar to That of APAP on RCS-Induced Muscular Mechanical Hyperalgesia

We next investigated the analgesic effects of NTP on RCS-induced persistent muscular mechanical hyperalgesia. In the experiment examining the effect of daily oral administration beginning at the start of RCS (Day 1), NTP significantly suppressed the RCS-induced decrease in the withdrawal threshold up until Day 14, although its effects were intermittent (Fig. 3a). In the experiment examining the effect of oral administration beginning post-RCS (Day 6), a single administration of NTP on Day 6 significantly reversed the RCS-induced decrease in the withdrawal threshold, and the repeated administration of NTP almost completely restored the withdrawal threshold on Day 11, compared with the control group (Fig. 3b).

Fig. 3. Effects of Orally Administered NTP on the RCS-Induced Decrease in the Withdrawal Threshold in the Randall–Selitto Test in Rats

The withdrawal threshold in the right hind leg muscles was evaluated in rats administered NTP from the start of RCS (a) or post-RCS (b). NTP was administered twice daily at a dose of 50 N.U./kg. The first out of two administrations on the measurement days was performed 30–60 min before the Randall–Selitto test. The data represent the means ± S.E.M. (n = 6). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus pre-RCS in control group, paired-t test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control group, Student’s t-test.

Influence of MDL72222 and Yohimbine on the Analgesic Effect of APAP and IBP

Finally, we examined the involvement of the spinal 5-HT₃ receptor and the α₂-adrenoceptor on the analgesic effect of APAP and IBP. The daily oral administration of APAP or IBP from the start of RCS (Day 1) significantly increased the withdrawal threshold of the animals exposed to RCS to a similar extent (Fig. 4, Veh + APAP, Veh + IBP). The intrathecal administration of MDL72222, a selective 5-HT₃ receptor antagonist, and yohimbine, an α₂-adrenoceptor antagonist, almost completely abolished the analgesic effect of APAP but not that of IBP (Fig. 4, MDL or Yohi + APAP, MDL or Yohi + IBP). MDL72222 and yohimbine separately had no influence on the withdrawal threshold of the RCS-exposed rats (Fig. 4, MDL or Yohi + Veh).

Fig. 4. Effects of Intrathecally Administered Receptor Antagonists on the Analgesic Action of APAP and IBP in the Randall–Selitto Test in Rats

The withdrawal threshold in the right hind leg muscles was evaluated on Day 6 in rats administered APAP and IBP orally and MDL72222 (a) or yohimbine (b) intrathecally. Oral APAP and IBP were administered twice daily at doses of 100 and 10 mg/kg, respectively. Both the intrathecal MDL72222 and yohimbine were administered daily at a dose of 30 nmol/10 µL/site. The daily oral and intrathecal administration was continued from the start of RCS (Day 1), and the last analgesics and antagonists were administered about 30 min and 25 min before the measurement on Day 6. The data represent the means ± S.E.M. (n = 4–8). Veh: vehicle, MDL: MDL72222, Yohi: yohimbine, p.o.: per os (oral), i.t.: intrathecal. n.s.: not significant, * p < 0.05, *** p < 0.001, Student’s t-test.

DISCUSSION

Stress, such as RCS exposure, is known to impair the function of the descending pain inhibitory system,^12,13) and chronic muscle pain conditions are closely related to a stressed condition.^3,18) We first investigated the RCS-induced chronic muscular hyperalgesia model. Surface anesthesia had no influence on the withdrawal threshold measured using a probe with a large tip diameter probe (data not shown), and RCS exposure induced persistent muscular mechanical hyperalgesia (Fig. 1b), as previously reported.¹⁴⁾ Indeed, the significant decrease in the withdrawal threshold lasted for 15 d after RCS, which was also consistent with the findings of our previous study.¹⁹⁾ Therefore, we used this rat model to evaluate the effect of analgesics such as APAP and IBP on chronic muscular pain in the following studies.

APAP and IBP are often used as OTC analgesic drugs to palliate pain, including neck-shoulder pain and headache. However, the analgesic effect and mechanism of APAP on chronic muscular hyperalgesia have never been investigated. We compared the effect of APAP and the COX inhibitor IBP in the Randall–Selitto test. The oral analgesic dosages of APAP and IBP were chosen from the published effective doses in rat models of mechanical hyperalgesia^20–25); high doses were not selected because of reports regarding the risks of repeated administration, such as hepatic and renal toxicity.^26,27)

APAP was effective for chronic muscular pain in both experiments in which administration was started at the beginning of RCS and after RCS, although the analgesic effect of the single administration of APAP after RCS was slight. On the other hand, IBP was temporarily effective when administration was started at the beginning of RCS but was not effective when administration was started after RCS (Figs. 2a, b). The results of the oral IBP experiments are consistent with those of experiments involving the administration of another COX inhibitor, loxoprofen, in our previous study.¹⁹⁾ The considerable difference between APAP and IBP indicates that the analgesic effect of APAP on chronic muscular pain is hardly attributable to the inhibition of COX in peripheral tissues. Furthermore, no significant increase in the withdrawal threshold was observed by the repeated administration of APAP and IBP in the normal rats (Fig. 2c). The withdrawal threshold in RCS-exposed rats was not increased more than the pre-RCS value by the repeated administration (Figs. 2a, b). Our results suggest that APAP exerts an analgesic effect on RCS-induced muscular hyperalgesia and has little effect on the normal withdrawal threshold. Thus, we expect that investigating the involvement of APAP in the pathogenesis of RCS-induced hyperalgesia might help to clarify the distinct analgesic mechanism of APAP.

The leading cause of hyperalgesia as a result of RCS exposure is thought to be the suppression of the descending pain inhibition system.¹²⁾ In a previous report, the RCS-induced decrease in the withdrawal threshold was not observed in rats denervated monoaminergic descending neurons.¹³⁾ NTP reportedly has an antinociceptive effect on RCS-induced mechanical hyperalgesia, which can be attributed to the activation of the descending pain inhibition system involving spinal 5-HT₃ receptors and α₂-adrenoceptors.^13,17) In this study, NTP had an analgesic effect similar to that of APAP on the withdrawal threshold in RCS-exposed rats (Figs. 2a, b, 3); thus, we speculated that APAP also exerted its action by activating the descending pain inhibition system.

We examined the involvement of the descending serotonergic system in the action of APAP on chronic muscular pain. The analgesic effect of oral APAP was significantly inhibited by the intrathecal administration of the selective 5-HT₃ receptor antagonist MDL72222 (Fig. 4a). It has been shown that the descending serotonergic pathway is involved in the analgesic effect of APAP.¹⁰⁾ APAP was inferred to activate the descending serotonergic pathway indirectly, since APAP failed to interact with 5-HT receptors or other receptors of neuromediators involved in the modulation of pain transmission in binding studies.¹¹⁾ Recently, there has been increasing evidence of the role of N-arachidonoylphenolamin (AM404), the APAP metabolite generated in the brain, in the analgesic action of APAP.^28–30) AM404 is an agonist of transient receptor potential vanilloid type 1 (TRPV1), a ligand at cannabinoid receptor type 1 (CB₁), and an inhibitor of cellular anandamide uptake. The activation of TRPV1 and CB₁ receptors by AM404 in the periaqueductal gray (PAG) and RVM, which were important sites for the descending serotonergic system, has been shown to induce APAP’s antinociceptive effect.^29,30) Moreover, the intrathecal administration of the 5-HT₃ receptor antagonist tropisetron was reported to prevent the antinociceptive effect of APAP in normal and inflamed rats.^11,31) Spinal 5-HT₃ receptors have been reported to be densely localized in the superficial dorsal horn.^32,33) From a mechanistic perspective, several studies have suggested that the activation of 5-HT₃ receptors on GABAergic interneurons induces both the release of GABA and antinociception via presynaptic mechanisms in the spinal dorsal horn.^34–36) Thus, in the present study, we assumed that APAP or its metabolites, such as AM404, would reinforce the RCS-induced dysfunctional descending serotonergic pathway and activate the 5-HT₃ receptors expressed on GABAergic neurons at the superficial dorsal horn, increasing the inhibitory synaptic transmission from interneurons.

The involvement of the descending noradrenergic inhibitory system in the action of APAP was also examined. The intrathecal administration of yohimbine, an α₂-adrenoceptor antagonist, significantly abolished the inhibitory action of muscular hyperalgesia by oral APAP (Fig. 4b). This result suggests the involvement of the descending noradrenergic pathways and the spinal α₂-adrenoceptor in APAP’s action. Cannabinoids including AM404 were reported to enhance the N-methyl-D-aspartate-induced excitation of LC neurons via CB₁ receptors in rat brain slices,³⁷⁾ supporting the activation of the descending noradrenergic inhibitory system by APAP. Spinal α₂-adrenoceptors were reported to be densely expressed in the superficial dorsal horn.³⁸⁾ It has been proposed that α₂-adrenoceptors on primary afferent fibers mediate the presynaptic inhibition of glutamatergic transmission in the spinal superficial dorsal horn.^39,40) Several studies have suggested that the augmentation of glutamate release from primary afferent fibers participates in RCS-induced hyperalgesia.^41,42) Therefore, we assumed that APAP or its metabolites, such as AM404, would enhance the descending noradrenergic pathway impaired by RCS and the subsequent activation of spinal α₂-adrenoceptors, thereby reducing excitatory synaptic transmission from primary afferents. However, the α₂-adrenoceptor-dependent signal has never been assumed to participate in the antinociceptive action of APAP in healthy animals and acute pain models. The difference in the pathogenesis of hyperalgesia between RCS and acute pain may be related to whether or not APAP acts through spinal α₂-adrenoceptors.

Interestingly, the inhibition of either the 5-HT₃ receptor or the α₂-adrenoceptor in the spinal cord almost abolished the analgesic effect of oral APAP (Fig. 4). This result indicated that both descending pathways might be indispensable in the analgesic action of APAP on RCS-induced hyperalgesia. Similar to the present study, the denervation of either serotonergic or noradrenergic descending neurons has been reported to override the antinociceptive effect of NTP on RCS-induced hyperalgesia completely.¹³⁾ Several studies have shown that the analgesic effect of intrathecal 5-HT is mediated by noradrenergic neurons and α₂-adrenoceptors in LC or spinal cord.^43,44) PAG and RVM reportedly communicate with noradrenergic sites, including LC via enkephalin or substance P neurons,^45,46) and the electrical stimulation of PAG or RVM increased not only 5-HT, but also noradrenaline levels in the spinal cord to induce an antinociceptive effect.^47,48) As described above, the descending serotonergic and noradrenergic systems have been shown to interact closely in the spinal dorsal horn and at the bulbopontine level to modulate pain transmission. Moreover, a previous study reported that the activation of α₂-adrenoceptors on excitatory interneurons inhibited the synaptic transmission to projection cells.⁴⁹⁾ Since interneurons play an important role in pain modulation, interneurons that express 5-HT₃ receptors or α₂-adrenoceptors might be correlated and may modulate pain transmission in the spinal dorsal horn in a complicated manner. However, the mechanism underlying the interaction of serotonergic and noradrenergic systems in the action of APAP remains to be elucidated; hence, further studies are required.

On the other hand, the intrathecal administration of either MDL72222 or yohimbine did not have any effect on the analgesic action of oral IBP (Fig. 4). These results suggest that IBP exerts its action irrespective of the descending pain inhibitory system and that the analgesic mechanism of IBP differs from that of APAP. The mRNA expression of COX-2, an isoform of COX induced in injured or inflamed locations, was increased in muscle only at the beginning of RCS, compared with a control (data not shown). Together with the results of experiments involving the oral administration of IBP (Figs. 2a, b), these findings suggest that IBP might suppress muscular hyperalgesia by inhibiting COX-2 induced during the early stage of RCS. Moreover, prostaglandins produced by COX are related to not only pain enhancement, but also muscle regeneration. It has been pointed out that the inhibition of prostaglandin synthesis by NSAIDs may interfere with the muscular healing process.^50,51) Therefore, one of the reasons for the temporal effect of IBP during continuous administration may be related to its suppressive action on the physiological action of prostaglandins, which contribute to muscle regeneration.

Additionally, not only muscle mechanical hyperalgesia but also a decrease in blood flow was observed in the lower legs of the RCS-exposed rats (data not shown). A decrease in muscle blood flow reportedly plays an important role in the initiation and maintenance of muscle pain in humans.^2,3) Tenderness and decreased blood flow in the region of pain are common features among patients with neck-shoulder pain.⁵²⁾ Therefore, the chronic muscular hyperalgesia of rats exposed to RCS may partly reflect the pathology of patients with musculoskeletal pain, such as those with neck-shoulder pain. We focused on the analgesic action of APAP and IBP on RCS-induced hyperalgesia in the present study, and the effects of APAP and IBP on muscular blood flow have not yet been examined. Furthermore, no reports have indicated that muscular blood flow is influenced by the oral used of APAP or IBP. Thus, we intend to investigate the involvement of blood flow in the actions of analgesics, including APAP and IBP, in the future.

Other than analgesics such as APAP and IBP, Kampo preparations such as Chikenpaikoukyuho are also used as medication for stiff shoulders and neck-shoulder pain. In our previous study, Chikenpaikoukyuho relieved muscular hyperalgesia in RCS-exposed rats,¹⁹⁾ supporting the hypothesis that RCS-induced muscular mechanical hyperalgesia partially reflects stiff shoulders and neck-shoulder pain in humans. Chikenpaikoukyuho has been said to be especially effective for neck-shoulder pain caused by psychological stress. This Kampo preparation had an analgesic effect on muscular pain that was similar to that of APAP,¹⁹⁾ and its effect was abolished by the intrathecal administration of 5-HT₃ receptor and α₂-adrenoceptor antagonists. Therefore, APAP might also be effective for neck-shoulder pain caused by stress.

Neck and shoulder discomfort or tenderness are related to symptoms such as headache. For instance, chronic tension-type headache sufferers often exhibit pericranial muscle tenderness, including the upper trapezius, sternocleidomastoid and levator scapulae muscles.⁵³⁾ A previous study found a strong positive correlation between tenderness and the frequency of tension-type headache.⁵⁴⁾ APAP or NSAIDs are representatively used to treat tension-type headache,⁵⁵⁾ but not much evidence is available explaining which analgesic drugs should be used. Since APAP had greater analgesic effects than IBP on muscular hyperalgesia in RCS-exposed rats, we propose that APAP might be more effective than IBP for the treatment of headache associated with neck-shoulder tenderness.

In conclusion, APAP effectively suppressed chronic muscular hyperalgesia induced by RCS exposure, suggesting that APAP may be more useful for the treatment of musculoskeletal pain, including neck-shoulder tenderness. The analgesic effect of APAP on RCS-induced hyperalgesia may be due to the enhancement of the descending serotonergic and noradrenergic pain inhibitory pathways involving spinal 5-HT₃ receptors and α₂-adrenoceptors.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Parihar JK, Jain VK, Chaturvedi P, Kaushik J, Jain G, Parihar AK. Computer and visual display terminals (VDT) vision syndrome (CVDTS). Med. J. Armed Forces India, 72, 270–276 (2016).
• 2) Visser B, van Dieën JH. Pathophysiology of upper extremity muscle disorders. J. Electromyogr. Kinesiol., 16, 1–16 (2006).
• 3) Larsson SE, Larsson R, Zhang Q, Cai H, Öberg PÅ. Effects of psychophysiological stress on trapezius muscles blood flow and electromyography during static load. Eur. J. Appl. Physiol. Occup. Physiol., 71, 493–498 (1995).
• 4) Maekawa K, Clark GT, Kuboki T. Intramuscular hypoperfusion, adrenergic receptors, and chronic muscle pain. J. Pain, 3, 251–260 (2002).
• 5) Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New Biol., 231, 232–235 (1971).
• 6) Ghanem CI, Pérez MJ, Manautou JE, Mottino AD. Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity. Pharmacol. Res., 109, 119–131 (2016).
• 7) Flower RJ, Vane JR. Inhibition of prostaglandin synthetase in brain explains the anti-pyretic activity of paracetamol (4-acetamidophenol). Nature, 240, 410–411 (1972).
• 8) Ossipov MH, Dussor GO, Porreca F. Central modulation of pain. J. Clin. Invest., 120, 3779–3787 (2010).
• 9) Yoshimura M, Furue H. Mechanisms for the anti-nociceptive actions of the descending noradrenergic and serotonergic systems in the spinal cord. J. Pharmacol. Sci., 101, 107–117 (2006).
• 10) Tjølsen A, Lund A, Hole K. Antinociceptive effect of paracetamol in rats is partly dependent on spinal serotonergic systems. Eur. J. Pharmacol., 193, 193–201 (1991).
• 11) Pelissier T, Alloui A, Caussade F, Dubray C, Cloarec A, Lavarenne J, Eschalier A. Paracetamol exerts a spinal antinociceptive effect involving an indirect interaction with 5-hydroxytryptamine₃ receptors: in vivo and in vitro evidence. J. Pharmacol. Exp. Ther., 278, 8–14 (1996).
• 12) Ohara H, Kawamura M, Namimatsu A, Miura T, Yoneda R, Hata T. Mechanism of hyperalgesia in SART stressed (repeated cold stress) mice: antinociceptive effect of neurotropin. Jpn. J. Pharmacol., 57, 243–250 (1991).
• 13) Okai H, Okazaki R, Yoshida H, Namba H, Taguchi K, Yamamoto A, Miura T, Kawamura M. Antinoiceptive effects of Neurotropin in repeated cold stressed rats: influences of chemical denervation of monoaminergic descending inhibitory neurons. PAIN RESEARCH, 25, 179–188 (2010).
• 14) Nasu T, Taguchi T, Mizumura K. Persistent deep mechanical hyperalgesia induced by repeated cold stress in rats. Eur. J. Pain, 14, 236–244 (2010).
• 15) Kita T, Hata T, Yoneda R, Okage T. Stress state caused by alteration of rhythms in environmental temperature, and the functional changes in mice and rats. Nihon Yakurigaku Zasshi, 71, 195–210 (1975).
• 16) Wakatsuki K, T-Uchimura Y, Matsubara T, Nasu T, Mizumura K, Taguchi T. Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress. Neurosci. Res., 162, 22–30 (2021).
• 17) Kawamura M, Ohara H, Go K, Koga Y, Ienaga K. Neurotropin induces antinociceptive effect by enhancing descending pain inhibitory systems involving 5-HT₃ and noradrenergic α₂ receptors in spinal dorsal horn. Life Sci., 62, 2181–2190 (1998).
• 18) Bansevicius D, Westgaard RH, Jensen C. Mental stress of long duration: EMG activity, perceived tension, fatigue, and pain development in pain-free subjects. Headache, 37, 499–510 (1997).
• 19) Ito G, Fujimori H, Fujimaru Y, Yamamoto D, Hanawa T. The effects and clinical benefits of Chikenpaikokyuho on shoulder and back pain. Jpn. J. Med. Pharm. Sci., 69, 499–508 (2013).
• 20) Bianchi M, Martucci C, Ferrario P, Franchi S, Sacerdote P. Increased tumor necrosis factor-α and prostaglandin E₂ concentrations in the cerebrospinal fluid of rats with inflammatory hyperalgesia: the effects of analgesic drugs. Anesth. Analg., 104, 949–954 (2007).
• 21) Li T, Wang T, Wang L, Liu R, Zhang L, Zhai R, Fu F. Antinociceptive effects of rotigotine-loaded microspheres and its synergistic interactions with analgesics in inflammatory pain in rats. Eur. J. Pharmacol., 891, 173693 (2021).
• 22) Adams SS, McCullough KF, Nicholson JS. The pharmacological properties of ibuprofen, an anti-inflammatory, analgesic and antipyretic agent. Arch. Int. Pharmacodyn. Ther., 178, 115–129 (1969).
• 23) Craft RM, Hewitt KA, Britch SC. Antinociception produced by nonsteroidal anti-inflammatory drugs in female vs. male rats. Behav. Pharmacol., 32, 153–169 (2021).
• 24) Kaur S, Bijjem KR, Sharma PL. Anti-inflammatory and antihyperalgesic effects of the combination of ibuprofen and hemin in adjuvant-induced arthritis in the Wistar rat. Inflammopharmacology, 19, 265–272 (2011).
• 25) Kalra BS, Shalini, Chaturvedi S, Tayal V, Gupta U. Evaluation of gastric tolerability, antinociceptive and antiinflammatory activity of combination NSAIDs in rats. Indian J. Dent. Res., 20, 418–422 (2009).
• 26) Yamaura K, Shimada M, Nakayama N, Ueno K. Protective effects of goldenseal (Hydrastis canadensis L.) on acetaminophen-induced hepatotoxicity through inhibition of CYP2E1 in rats. Pharmacognosy Res., 3, 250–255 (2011).
• 27) Aprioku J, Nwidu LL, Amadi CN. Evaluation of toxicological profile of ibuprofen in Wistar Albino rats. Am. J. Biomed. Sci., 6, 32–40 (2014).
• 28) Högestätt ED, Jönsson BA, Ermund A, Andersson DA, Björk H, Alexander JP, Cravatt BF, Basbaum AI, Zygmunt PM. Conversion of acetaminophen to the bioactive N-acylphenolamine AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system. J. Biol. Chem., 280, 31405–31412 (2005).
• 29) Mallet C, Barrière DA, Ermund A, Jönsson BA, Eschalier A, Zygmunt PM, Högestätt ED. TRPV1 in brain is involved in acetaminophen-induced antinociception. PLoS ONE, 5, e12748 (2010).
• 30) Klinger-Gratz PP, Ralvenius WT, Neumann E, Kato A, Nyilas R, Lele Z, Katona I, Zeilhofer HU. Acetaminophen relieves inflammatory pain through CB₁ cannabinoid receptors in the rostral ventromedial medulla. J. Neurosci., 38, 322–334 (2018).
• 31) Alloui A, Chassaing C, Schmidt J, Ardid D, Dubray C, Cloarec A, Eschalier A. Paracetamol exerts a spinal, tropisetron-reversible, antinociceptive effect in an inflammatory pain model in rats. Eur. J. Pharmacol., 443, 71–77 (2002).
• 32) Kia HK, Miquel MC, McKernan RM, Laporte AM, Lombard MC, Bourgoin S, Hamon M, Vergé D. Localization of 5-HT₃ receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport, 6, 257–261 (1995).
• 33) Laporte AM, Koscielniak T, Ponchant M, Vergé D, Hamon M, Gozlan H. Quantitative autoradiographic mapping of 5-HT₃ receptors in the rat CNS using [¹²⁵I]iodo-zacopride and [³H]zacopride as radioligands. Synapse, 10, 271–281 (1992).
• 34) Alhaider AA, Lei SZ, Wilcox GL. Spinal 5-HT₃ receptor-mediated antinociception: possible release of GABA. J. Neurosci., 11, 1881–1888 (1991).
• 35) Kawamata T, Omote K, Toriyabe M, Yamamoto H, Namiki A. The activation of 5-HT₃ receptors evokes GABA release in the spinal cord. Brain Res., 978, 250–255 (2003).
• 36) Fukushima T, Ohtsubo T, Tsuda M, Yanagawa Y, Hori Y. Facilitatory actions of serotonin type 3 receptors on GABAergic inhibitory synaptic transmission in the spinal superficial dorsal horn. J. Neurophysiol., 102, 1459–1471 (2009).
• 37) Mendiguren A, Pineda J. Cannabinoids enhance N-methyl-D-aspartate-induced excitation of locus coeruleus neurons by CB₁ receptors in rat brain slices. Neurosci. Lett., 363, 1–5 (2004).
• 38) Stone LS, Broberger C, Vulchanova L, Wilcox GL, Hökfelt T, Riedl MS, Elde R. Differential distribution of α_2A and α_2C adrenergic receptor immunoreactivity in the rat spinal cord. J. Neurosci., 18, 5928–5937 (1998).
• 39) Ueda M, Oyama T, Kuraishi Y, Akaike A, Satoh M. Alpha2-adrenoceptor-mediated inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices. Neurosci. Lett., 188, 137–139 (1995).
• 40) Kawasaki Y, Kumamoto E, Furue H, Yoshimura M. α₂ adrenoceptor-mediated presynaptic inhibition of primary afferent glutamatergic transmission in rat substantia gelatinosa neurons. Anesthesiology, 98, 682–689 (2003).
• 41) Okano K, Ueda M, Kuraishi Y, Satoh M. Effect of repeated cold stress on capsaicin-evoked release of glutamate from rat spinal dorsal horn slices. Neurosci. Res., 29, 319–324 (1997).
• 42) Okano K, Kuraishi Y, Satoh M. Effects of intrathecally injected glutamate and substance P antagonists on repeated cold stress-induced hyperalgesia in rats. Biol. Pharm. Bull., 18, 42–44 (1995).
• 43) Danysz W, Jonsson G, Minor BG, Post C, Archer T. Spinal and locus coeruleus noradrenergic lesions abolish the analgesic effects of 5-methoxy-N,N-dimethyltryptamine. Behav. Neural Biol., 46, 71–86 (1986).
• 44) Nakagawa I, Omote K, Kitahata LM, Collins JG, Murata K. Serotonergic mediation of spinal analgesia and its interaction with noradrenergic systems. Anesthesiology, 73, 474–478 (1990).
• 45) Holden JE, Proudfit HK. Enkephalin neurons that project to the A7 catecholamine cell group are located in nuclei that modulate nociception: ventromedial medulla. Neuroscience, 83, 929–947 (1998).
• 46) Yeomans DC, Proudfit HK. Projections of substance P-immunoreactive neurons located in the ventromedial medulla to the A7 noradrenergic nucleus of the rat demonstrated using retrograde tracing combined with immunocytochemistry. Brain Res., 532, 329–332 (1990).
• 47) Cui M, Feng Y, McAdoo DJ, Willis WD. Periaqueductal gray stimulation-induced inhibition of nociceptive dorsal horn neurons in rats is associated with the release of norepinephrine, serotonin, and amino acids. J. Pharmacol. Exp. Ther., 289, 868–876 (1999).
• 48) Barbaro NM, Hammond DL, Fields HL. Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat. Brain Res., 343, 223–229 (1985).
• 49) Olave MJ, Maxwell DJ. Neurokinin-1 projection cells in the rat dorsal horn receive synaptic contacts from axons that possess α_2C-adrenergic receptors. J. Neurosci., 23, 6837–6846 (2003).
• 50) Veliça P, Bunce CM. Prostaglandins in muscle regeneration. J. Muscle Res. Cell Motil., 29, 163–167 (2008).
• 51) Monda M, Vicidomini C, Viggiano A, Sampaolo S, Di Iorio G, Viggiano A, Viggiano E, De Luca B. Inhibition of prostaglandin synthesis reduces the induction of MyoD expression in rat soleus muscle. J. Muscle Res. Cell Motil., 30, 139–144 (2009).
• 52) Larsson R, Oberg PA, Larsson SE. Changes of trapezius muscle blood flow and electromyography in chronic neck pain due to trapezius myalgia. Pain, 79, 45–50 (1999).
• 53) Fernández-de-Las-Peñas C, Alonso-Blanco C, Cuadrado ML, Gerwin RD, Pareja JA. Myofascial trigger points and their relationship to headache clinical parameters in chronic tension-type headache. Headache, 46, 1264–1272 (2006).
• 54) Jensen R, Rasmussen BK, Pedersen B, Olesen J. Muscle tenderness and pressure pain thresholds in headache. A population study. Pain, 52, 193–199 (1993).
• 55) Bendtsen L, Jensen R. Treating tension-type headache—an expert opinion. Expert Opin. Pharmacother., 12, 1099–1109 (2011).