Co-Administered Low Doses Of Ibuprofen And Dexamethasone Produce Synergistic Antinociceptive Effects On Neuropathic Mechanical Allodynia In Rats

Introduction

It has been well known that neuropathic pain results from conditions that compromise or abolish nerve function, including nerve injury, multiple sclerosis, and diabetes, and remains among the most challenging pain-related disorders to manage.^1,2 A common symptom of neuropathic pain is allodynia, in which previously non-noxious stimuli are perceived as noxious following nerve injury.³ However, traditional analgesics including anticonvulsants, opioids, and nonsteroidal anti-inflammatory drugs (NSAIDs) lack efficacy in treating such pain and/or carry unpleasant side effects.² Thus, neuropathic pain is an intractable disease from which only 40–60% of affected patients achieve even partial relief.⁴

NSAIDs are widely used for the symptomatic relief of all kinds of pain,^2,5 but lack efficacy in treating neuropathic pain.^4,6 Moreover, NSAIDs are associated with adverse side effects, including perforation and bleeding of the gastrointestinal tract, resulting from their inhibitory activity on gastrointestinal prostaglandin E2 synthesis.⁷ Nevertheless, the intrathecal administration of an NSAID, meloxicam was found to attenuate the thermal hyperalgesia and mechanical allodynia induced by spinal nerve ligation.⁸ Moreover, in rats, the intrathecal injection of ketorolac, a cyclooxygenase (COX)-1 inhibitor, or NS-398, a COX-2 inhibitor, attenuated the hyperalgesia and allodynia resulting from a chronic constriction injury of the sciatic nerve⁹ and partial sciatic nerve ligation.¹⁰ These findings suggest that although the COX pathways play an important role in the nociceptive processing of neuropathic pain, the efficacy of COX inhibitors in treating this type of pain has not been established.

Dexamethasone is used to treat a wide variety of inflammatory conditions because it is a synthetic glucocorticoid with anti-inflammatory activity and minimal mineralocorticoid effects.¹¹ A previous study demonstrated that dexamethasone produced potent anti-inflammatory effects on carrageenan-induced inflammation.¹² Additionally, dexamethasone has been used for the treatment of neuropathic pain conditions. Previous reports have indicated that a subcutaneous injection of dexamethasone or epidural injection of betamethasone attenuated mechanical allodynia in rats with spinal nerve ligation.^13,14 However, the prolonged use of dexamethasone can also be associated with serious side effects^15,16 suggesting that it has limited application in treating neuropathic pain conditions.

In our present study, we investigated the synergistic antinociceptive effects of co-administered low doses of ibuprofen and dexamethasone in rats with inferior alveolar nerve injury. Although these drugs were found to be largely ineffective against the pain when administered alone, we confirmed their synergistic antinociceptive effects when given in combination at a low dose by examining the changes in c-fos expression in the trigeminal subnucleus caudalis.

Materials And Methods

Trigeminal Neuropathic Pain Animal Model

Male Sprague-Dawley rats (total number = 163; Central Lab. Animal, Inc., Korea) weighing 200–230 g were used to generate the trigeminal neuropathic pain model. The animals were maintained in a temperature-controlled room (23 ± 1°C) on a 12/12 hr light-dark cycle. To generate the animal model, the rats were anesthetized with a mixture of ketamine (40 mg/kg, intramuscularly; i.m.) and xylazine (4 mg/kg, i.m.) solution. Briefly, under anesthesia, the left lower second molar was extracted and replaced with a mini–dental implant (diameter, 1 mm; length, 4 mm; donated by Megagen, Gyengsan, Korea) to intentionally injure the inferior alveolar nerve, as described previously.^17,18 For the sham group, the operation was identical but without the placement of a dental implant. Only data from the rats presenting with an inferior alveolar nerve injury due to the mal-positioned dental implants were used in the final analyses.

All procedures involving the use of animals were approved by the Institutional Care and Use Committee of the School of Dentistry, Kyungpook National University. The animal experiments were also conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (IASP) for investigations of experimental pain in conscious animals. All experiments were undertaken in a blind manner.

Evaluation Of Mechanical Allodynia

Mechanical allodynia was measured as described previously.^19,20 Briefly, for behavioral observations, each rat was placed in a customized cage in a darkened and noise-free room. The animals were allowed to acclimate for at least 30 mins. Their withdrawal behavior was evaluated over 10 trials at a constant air-puff pressure (4-second duration and 10-second intervals). The intensity and intervals of the air-puff pressure were controlled using a pico-injector (Harvard Apparatus, Holliston, MA). The air-puffs were applied through a 26-gauge metal tube (length, 10 cm) located 1 cm from the skin at 90°. After inferior alveolar nerve injury, we identify the most sensitive areas by air-puff stimulation, as described previously,¹⁸ that included the ipsilateral lower jaw and mouth angle area of the facial region. The air-puff thresholds were determined using the air-puff pressure at which each rat responded in 50% of the trials. The cut-off pressure for the air-puffs was 40 psi, as determined previously.^21,22 The naïve animals did not respond to a pressure lower than 40 psi.

Administration Of Drugs

The present study examined the antinociceptive effects of ibuprofen (2-(4-isobutylphenyl propionic acid) as an analgesic agent. After the intraperitoneal administration of ibuprofen (1, 5, 10, 30 mg/kg) on postoperative day (POD) 3, changes in the air-puff thresholds were investigated (n=6, each group). We also examined the antinociceptive effects of dexamethasone (0.1, 1, 10 mg/kg) after its intraperitoneal injection on POD 3 (n=6, each group). Vehicle was administered intraperitoneally on POD 3 in the control group. Low doses of ibuprofen (1, 5 mg/kg) or dexamethasone (0.1, 1 mg/kg) when administered alone, did not affect the air-puff thresholds and we selected previously employed doses of ibuprofen (5 mg/kg) and dexamethasone (0.01, 0.1, 1 mg/kg) in the combined treatment protocols. We then examined the synergistic antinociceptive effects of these co-administered low doses.

A previous study reported that central hypersensitivity was established within POD 3 and may be independent of the peripheral input after pain has been established.²³ Hence, we examined the synergistic antinociceptive effects of co-administered ibuprofen (5 mg/kg) and dexamethasone (0.01, 0.1, 1 mg/kg) both prior to and after pain establishment on POD 1–3 and POD 7–9, respectively (n=6, each group). We also measured the antinociceptive effects of the intraperitoneal administration of gabapentin (30, 100 mg/kg) in a positive control group of rats with inferior alveolar nerve injury before and after the pain was established (n=6, each group).

Ibuprofen was purchased from Sigma–Aldrich (St. Louis, MO) and dissolved in 5% dimethyl sulfoxide (DMSO). Dexamethasone was purchased from Sigma–Aldrich and dissolved in sterile saline. Gabapentin was purchased from Tokyo Chemical Industry Co., LTD. and dissolved in sterile saline. All chemicals were stored at 4°C before use.

Immunohistochemical Staining Of c-Fos

On POD 3, we applied air-puff pressure (10 sec on/off, 20 psi intensity) for 10 mins after the co-administration of ibuprofen (5 mg/kg) with dexamethasone (1 mg/kg) in rats with inferior alveolar nerve injury. The rats (n = 5 per group) were transcardially perfused with 0.9% saline at 3 hrs after air-puff stimulation followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The caudal medulla was dissected and post-fixed in the same fixative at 4°C for 2 hrs, and then incubated with 30% sucrose in 0.1 M PB overnight. The specimens were frozen and cut into 30 μm thick transverse sections. The free-floating sections were rinsed in phosphate-buffered saline (PBS) and blocked with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature. The sections were incubated at 4°C overnight with the rabbit polyclonal anti-c-fos antibody (1:200; Santa Cruz Biotechnology, Dallas, TX) and then incubated in peroxidase-conjugated anti-rabbit IgG (Vector Laboratories) for 2 hrs at room temperature. After washing, the sections were incubated in a buffer containing diaminobenzidine (DAB, Vector Laboratories) and hydrogen peroxide (pH 7.5, Vector Laboratories) for approximately 1 min. The stained sections were analyzed under BX 41 and U-RFL-T microscopes (Olympus, Tokyo, Japan). Neurons that were immunopositive for c-fos were counted in the superficial laminae (I-II) of the ipsilateral trigeminal subnucleus caudalis on three sections since lamina I and II participate in pain processing in the spinal cord as well as the trigeminal spinal nucleus.^24,25

Statistical Analysis

Statistical analysis of the animal behavioral data was performed by repeated-measures analysis of variance (ANOVA) followed by Holm-Sidak post hoc analysis. The c-fos expression data were analyzed by one-way ANOVA followed by Holm-Sidak post hoc analysis. In all statistical comparisons, P< 0.05 was considered significant. All data are presented as a mean ± SEM.

Results

Effects Of Intraperitoneal Injections Of Ibuprofen Or Dexamethasone Alone On Trigeminal Neuropathic Mechanical Allodynia

Figure 1 illustrates the effects of intraperitoneally administered ibuprofen or dexamethasone alone on neuropathic mechanical allodynia on POD 3. A single intraperitoneal injection of the vehicle or low doses of ibuprofen (1, 5 mg/kg) did not affect the air-puff thresholds. A single 10 mg/kg dose of ibuprofen increased the air-puff thresholds slightly but this was not statistically significant. Only a high dose of ibuprofen (30 mg/kg) significantly attenuated mechanical allodynia compared with the vehicle-treated control group; and these anti-allodynic effects persisted for over 6 hrs after the injection and abated within 24 hrs (Figure 1A and F_(4,25)=122.674, P <0.05).

Figure 1 Effects of the intraperitoneal administration of ibuprofen (A) or dexamethasone (B) on the air-puff thresholds in rats with an inferior alveolar nerve injury. A high dose of ibuprofen (30 mg/kg) or dexamethasone (10 mg/kg) produced significant antiallodynic effects compared with the vehicle-treated control group. These effects persisted for over 6 hrs after the injection and dissipated within 24 hrs. Neither the intraperitoneal injection of the vehicle nor low doses of ibuprofen (1, 5, 10 mg/kg) or dexamethasone (0.1, 1mg/kg) affected the air-puff thresholds. The values shown are the mean ± SEM. There were 6 animals in each group. *p < 0.05, vehicle- vs. drug-treated group.

The dose-dependent effects of intraperitoneally administered dexamethasone on neuropathic mechanical allodynia on POD 3 are illustrated in Figure 1B. Neither the vehicle control nor the low doses of dexamethasone (0.1, 1 mg/kg) had any impact on the neuropathic mechanical allodynia in the rats. Only high dose of dexamethasone (10 mg/kg) significantly attenuated this mechanical allodynia compared with the vehicle-treated control group and again these anti-allodynic effects persisted for over 6 hrs after the injection and had dissipated by 24 hrs (F_(3,20)=50.001, P< 0.05).

Effects Of Co-Administered Low Doses Of Ibuprofen And Dexamethasone On Trigeminal Neuropathic Mechanical Allodynia

Figures 2 and 3 illustrate the effects of co-administered low doses of ibuprofen and dexamethasone on the air-puff thresholds in the experimental rats with inferior alveolar nerve injury. We examined the synergistic antinociceptive effects of this co-treatment on POD 1, 2, 3 (early-phase) before the pain had been established in this model (Figure 2). Vehicle administration did not affect the air-puff thresholds in the animals. In contrast, the co-administration of 5 mg/kg of ibuprofen with 0.01 mg/kg of dexamethasone attenuated the neuropathic mechanical allodynia in the rats, although it was not a statistically significant finding. Moreover, the combined daily treatments with ibuprofen (5 mg/kg) and dexamethasone (0.1 or 1 mg/kg) produced significant anti-allodynic effects compared with the vehicle treatment on POD 1–3 (F_(3,20)=50.001, P <0.05). These anti-allodynic effects appeared immediately after the co-treatment and persisted for over 6 hrs but disappeared by 24 hrs post-injection.

Figure 2 Synergistic antinociceptive effects of co-administered low doses of ibuprofen and dexamethasone on POD 1–3. The early intraperitoneal co-administration of ibuprofen (5 mg/kg) with dexamethasone (0.1, 1 mg/kg) produced significant antiallodynic effects prior to pain establishment in the rat model. These effects appeared immediately and persisted for over 6 hrs. The values shown are the mean ± SEM. There were 6 animals in each group. *p <0.05, vehicle- vs. drug-treated group. Abbreviation: POD, postoperativeday.

Figure 3 Synergistic antinociceptive effects of co-administered low doses of ibuprofen and dexamethasone on POD 7–9. The late intraperitoneal co-administration of ibuprofen (5 mg/kg) with dexamethasone (0.1, 1 mg/kg) produced significant antiallodynic effects after the pain had been established in the rat model. These effects appeared immediately and persisted for over 6 hrs. The values shown are the mean ± SEM. There were 6 animals in each group. *p <0.05, vehicle- vs. drug-treated group. Abbreviation: POD, postoperativeday.

We also examined the synergistic anti-nociceptive effects of these same low doses of ibuprofen (5 mg/kg) and dexamethasone (0.1, 1 mg/kg) after the pain had already been established in the rat model on POD 7–9, and found again that it produced significant anti-allodynic effects (F_(3,20)=44.675, P <0.05) that persisted for over 6 hrs after injection and dissipated within 24 hrs (Figure 3). However, neither the vehicle nor the co-administration of 5 mg/kg of ibuprofen with 0.01 mg/kg of dexamethasone affected the air-puff thresholds in this late treatment period.

Effects Of Co-Administered Low Doses Of Ibuprofen And Dexamethasone On c-Fos Expression

The effects of co-administering low doses of ibuprofen and dexamethasone on c-fos expression at POD 3 are indicated in Figure 4. The inferior alveolar nerve injury in the rats significantly augmented the number of c-fos immunoreactive cells compared with the sham-operated rats on POD 3 (P <0.05). Almost all of the c-fos-positive neurons were observed in the superficial layer (lamina I and II) of the ipsilateral trigeminal subnucleus caudalis and this number was significantly reduced by the co-administration of ibuprofen (5 mg/kg) and dexamethasone (1 mg/kg) compared to the vehicle-treated group (P <0.05). Neither ibuprofen nor dexamethasone treatments alone affected the production of c-fos immunopositive neurons following inferior alveolar nerve injury.

Figure 4 Effects of co-administered ibuprofen and dexamethasone on c-fos expression in the ipsilateral trigeminal subnucleus caudalis of the rat model on POD 3. (A) c-fos immunoreactive neurons in sham rats. (B) c-fos immunoreactive neurons after vehicle treatment in the rats with inferior alveolar nerve injury. (C) c-fos immunoreactive neurons after the intraperitoneal co-administration of ibuprofen (5 mg/kg) with dexamethasone (1 mg/kg) in the rats with inferior alveolar nerve injury. (D) c-fos immunoreactive neurons after the intraperitoneal injection of ibuprofen (5 mg/kg) in the rats with inferior alveolar nerve injury. (E) c-fos immunoreactive neurons after the intraperitoneal injection of dexamethasone (1 mg/kg) in the rats with inferior alveolar nerve injury. (F) Histograms showing the changes in the mean number of c-fos immunoreactive neurons. Inferior alveolar nerve injury in the rats significantly increased the number of c-fos- immunoreactive cells compared with the sham-operated rats. The co-administration of ibuprofen (5 mg/kg) with dexamethasone (1 mg/kg) significantly attenuated the number of c-fos immunopositive neurons compared to the vehicle-treated group. The values shown are the mean ± SEM. There were 5 animals in each group. Scale bar, 50 μm. *p< 0.05, sham- vs. vehicle/drug-treated group; #p< 0.05, vehicle- vs. drug-treated group.

Effects Of Intraperitoneal Treatment With Gabapentin On Trigeminal Neuropathic Mechanical Allodynia

The anti-nociceptive effects of gabapentin on neuropathic mechanical allodynia were also tested in rats with an inferior alveolar nerve injury (Figure 5). Before the pain had been established in this animal model, the daily ntraperitoneal injection of gabapentin at 30 and 100 mg/kg significantly increased the air-puff thresholds compared to the vehicle-treated group on POD 1–3 (F_(2,15)=103.916, P <0.05). Moreover, both doses of gabapentin produced anti-allodynic effects on POD 7–9 when the pain had already been established (F_(2,15)=112.213, P <0.05). Once again, these anti-allodynic effects persisted for over 6 hrs after injection and had dissipated by 24 hrs.

Figure 5 Anti-nociceptive effects of gabapentin on neuropathic mechanical allodynia in the rats with inferior alveolar nerve injury. The daily intraperitoneal administration of gabapentin (30, 100 mg/kg) produced significant anti-allodynic effects both before and after the pain was established in the animals. These effects persisted for 6 hrs and had dissipated within 24 hrs. The values shown are the mean ± SEM. There were 6 animals in each group. *p< 0.05, vehicle- vs. drug-treated group. Abbreviation: POD, postoperativeday.

Discussion

Our present study provides evidence of the synergistic analgesic effects of intraperitoneally co-administered low doses of ibuprofen and dexamethasone on trigeminal neuropathic pain, both before and after the pain was established in a rat model. Significant attenuation of c-fos expression in the ipsilateral trigeminal subnucleus caudalis in the experimental rats confirmed the synergistic antinociceptive effects of this low dose co-treatment and we further found that they were comparable to the effects of gabapentin. A combination of ibuprofen and dexamethasone at low doses thus appears to represent an alternative therapeutic strategy for the treatment of trigeminal neuropathic pain.

COX inhibitors have generally been used as anti-inflammatory, analgesic, and antipyretic drugs because of their effectiveness and minimal side effects.²⁶ Moreover, COX inhibitors are frequently prescribed to treat diabetic neuropathy, postherpetic neuralgia, and back/neck pain with neuropathic involvement⁵ because the involvement of the COX pathways in the sensitization of nociceptive processing at the spinal cord level has been well-described.²⁷ In animal studies, the intrathecal administration of PGE₂ has been reported to produce dose-dependent hyperalgesia in rats.²⁸ This acute anti-hyperalgesic action has been shown to be mediated through the spinal COX-1 or COX-2 pathway in rats.^27,29 Moreover, intrathecal injection of ketorolac, a COX-1 inhibitor, or NS-398, a COX-2 inhibitor, has been found to attenuate the thermal hyperalgesia and cold allodynia produced by chronic constriction injury of the sciatic nerve⁹ and also lessen the tactile allodynia in neuropathic pain following partial sciatic nerve ligation.¹⁰ However, a previous clinical trial showed that blockade of COX pathways by ibuprofen, a non-selective COX 1/2 inhibitor, failed to effectively treat the pain in patients with postherpetic neuralgia.³⁰ Consistently, our present findings in rats indicate that low doses of ibuprofen do not attenuate neuropathic mechanical allodynia and that only a high dose of this drug (30 mg/kg) produces significant analgesic effects. Therefore, the collective evidence to date suggests that patients with neuropathic pain generally do not respond to NSAIDs and that the efficacy of COX inhibitors in the treatment of neuropathic pain has not been established.

Although dexamethasone treatment in rats has been reported to induce potent anti-inflammatory, antihyperalgesic and antiallodynic effects against carrageenan-induced inflammation¹² and chronic arthritis,³¹ the analgesic effects of dexamethasone in neuropathic pain remains controversial. For instance, previous investigations reported that the systemic or intrathecal administration of dexamethasone failed to prevent mechanical and cold allodynia³² or reduce the activation of microglia³³ in rats with a spared nerve injury. Similarly, in our current experimental rat model of neuropathic pain, the intraperitoneal injection of 0.1 or 1 mg/kg of dexamethasone did not produce antinociceptive effects and only a high dose of this agent (10 mg/kg) attenuated the neuropathic mechanical allodynia. Another previous study also demonstrated that only a high dose of dexamethasone produced significant antinociceptive effects in rats with inferior alveolar nerve injury before the pain was established.¹⁷ Moreover, early treatment with dexamethasone (25 mg/kg) decreased the upregulated expression of p38 MAPK and NF-κB in the glial cells following inferior alveolar nerve injury²⁰ and spinal nerve injury³⁴ in rats. These results suggest that early treatment with dexamethasone at high doses is important in the treatment of neuropathic pain, including trigeminal pain. However, dexamethasone has limited application in the treatment of neuropathic pain because of its unwanted side effects at high dosages. The present study demonstrated that combined treatments with ibuprofen (5 mg/kg) and dexamethasone (0.1 or 1 mg/kg) produced significant anti-allodynic effects, which were comparable with those of a high dose (10 mg) of dexamethasone administered alone. Thus, the combined treatment with low doses of ibuprofen and dexamethasone would reduce the unwanted side effects of high dose dexamethasone treatment and can be a good alternative therapeutic tool.

It is widely known that combination treatments with analgesics at lower doses can have beneficial effects against neuropathic pain when compared to monotherapy. Our present investigation in a rat pain model demonstrated that the intraperitoneal co-administration of ibuprofen (5 mg/kg) with dexamethasone (0.1, 1 mg/kg) produced significant anti-allodynic effects that were not seen when these drugs were administered individually. This kind of anti-inflammatory synergism resulting from co-treatments with an NSAID and steroid has been demonstrated in previous studies. Co-administered diclofenac and dexamethasone produced supra-additive anti-inflammatory effects in the carrageenan rat paw edema model³⁵ and synergistic anti-inflammatory activity against lipopolysaccharide-induced inflammation in mice.^36,37 However, there have been no previous reports on the co-administration of ibuprofen with dexamethasone in the treatment of neuropathic pain. Our current study findings have indicated that the co-administration of these drugs at low doses produces significant antinociceptive effects in rats, which were confirmed by the significantly reduced c-fos expression in the ipsilateral trigeminal subnucleus caudalis. Although a previous clinical study demonstrated that a combination of ibuprofen and dexamethasone decreased the pain intensity at 24 and 48 hrs after a lower third molar surgical extraction,³⁸ our current results provide evidence of the synergistic analgesic effects of these drugs on neuropathic pain. Moreover, the efficacy of this combination was found to be comparable with that of gabapentin in our rat model. Hence, the co-administration of low doses of NSAIDs and steroids may minimize the potential side effects of high dose monotherapies using these agents. On the other hand, a previous study reported the potential risk of an ulcerogenic phenomenon following combination treatment with NSAIDs and steroids in rats. Indomethacin (10 mg/kg) administered in combination with prednisolone (10 mg/kg), a corticosteroid, showed the ulcerogenic potential including the deficiency of endogenous prostaglandins, an increase in neutrophil activation, and gastric hypermobility.³⁹ However the present study employed low doses of ibuprofen (5 mg/kg) and dexamethasone (0.01, 0.1, 1 mg/kg) in combined treatment protocols to reduce the side effects produced by co-administration of NSAIDs and steroids in high doses. Therefore, the combination of ibuprofen and dexamethasone at low doses offers an alternative therapeutic strategy for neuropathic pain and provides a rationale for using such drug combinations in patients who are unable to tolerate a high-dose monotherapy.

Time is very important factor in the development of neuropathic pain in the trigeminal area. A previous study reported that central plasticity was established within POD 3 and may be independent of the peripheral input.²³ Moreover, early administration of dexamethasone at high doses produced prolonged anti-allodynic effects before the pain had been established, but failed to reduce mechanical allodynia once the pain had already been established.¹⁷ This finding suggests that early treatment is necessary to overcome neuropathic pain after nerve injury. The present study examined the synergistic antinociceptive effects of co-administered ibuprofen (5 mg/kg) and dexamethasone (0.01, 0.1, 1 mg/kg) both, prior to and after pain establishment. Although co-administered ibuprofen and dexamethasone produced antinociceptive effects before the pain had been established, the effects were reversed within 24 hrs. The synergistic anti-nociceptive effects were also observed after the pain had already been established in the rat model. These results suggest that daily co-administered ibuprofen and dexamethasone can be a good alternative treatment strategy instead of long-term treatment with conventional analgesics for neuropathic pain.