Journal of Acupuncture and Meridian Studies
Volume 3, Issue 1 , Pages 38-42, March 2010

Evaluation of the Analgesic Effect of Dextromethorphan and its Interaction With Nitric Oxide on Sciatic Nerve Ligated Rats

Medical Toxicology Research Center and Pharmacy School, Mashhad, Iran

Received 27 August 2009; accepted 24 December 2009.

Article Outline

Abstract 

The symptoms of neuropathic pain are often intractable because they are poorly relieved by conventional analgesics. This therapeutic area remains one of the least satisfactorily managed by current drugs. Effective therapy for this type of pain is lacking, and the underlying mechanisms are poorly understood. The present study was undertaken to determine the effect of sciatic nerve ligation on inducing neuropathic pain and to understand the mechanisms involved, and the effect of, an L-nitro-arginine methyl ester (L-NAME)/dextromethorphan combination therapy on reducing neuropathic pain. According to our results, L-NAME and dextromethorphan showed analgesic properties, but only 100 mg/kg L-NAME had an additive effect on the analgesic effects of dextromethorphan. Our observations support the idea that N-methyl-D-aspartate/nitric oxide pathways play an important role in the development of such sciatic nerve ligated-evoked pathological pain conditions, thus this combination therapy could be used instead of conventional treatment.

Key Words:  dextromethorphan , L-nitro-arginine methyl ester (L-NAME) , neuropathic pain , nitric oxide , N-methyl-D-aspartic acid , sciatic nerve

 

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1. Introduction 

Neuropathic pain is initiated by a primary lesion or dysfunction of the nervous system, which may be peripheral (peripheral nerve, plexus, nerve root) or central. The painful area within the territory of the injured nerve shows allodynia (hypersensitivity to normally non-painful stimuli), hyperalgesia and hyperpathia, which are hallmark signs of neuropathic pain [1]. There are different anatomical sites where lesions may cause neuropathic pain. The most usual locations are the peripheral nerves, the plexus, dorsal nerve roots, the spinal cord and brain [2].

Although a large number of studies have been done to reveal the mechanism of neuropathic pain but it is still not well established. Different pathological phenomenon might be operating with nerve injury. Chronic nerve constriction leads to a marked demyelination and degeneration of both myelinated A-fibers and unmyelinated C-fibers [3], but the damage of C-fibers is later and to a lesser extent compared with A-fibers after nerve ligatures. This model mimics some aspects of neuropathic pain in humans [4].

Activation of N-methyl-D-aspartic acid (NMDA) receptor is associated with increased intracellular Ca2+ concentration and activation of Ca2+ sensitive protein kinase C, resulting in the production of nitric oxide (NO), which produces persistent enhancement of pain. The mechanism responsible for hyperalgesia in chronic pain is now believed to involve not only NO itself, but also the product of its reaction with superoxide radicals, the peroxynitrites [5].

Several experimental models of peripheral mononeuropathy in rats were developed wherein chronic constriction injury [6] or partial lesion [7] of sciatic nerve or its root [8] was performed. The allodynia and hyperalgesia that develop thereafter have led to considerable advances in understanding neuropathic pain resulting from nerve injury. Changes were observed in pain behavior with a peak occurring at 2-4 weeks post injury [9].

Despite intensive research on the neurobiological mechanism of chronic pain, this therapeutic area remains one of the least satisfactorily managed using current medications. Effective therapy for this type of pain is lacking, and the underlying mechanisms are poorly understood [10]. Chronic pain is often refractory to treatment with conventional analgesics such as opiates and non-steroidal anti-inflammatory drugs 11, 12. The use of opioids in neuropathic pain is controversial because of their limited efficacy in this pain state as compared to other pain states. NMDA receptor antagonists, adenosine analogues, neuronal specific Ca2+ channel blockers and NO modulators are among the pharmacological tools that are being studied to find out new treatment strategies [1].

Several lines of evidence indicate that the NMDA receptor is involved in the induction and maintenance of hypersensitivity states associated with chronic pain, including neuropathic pain. NMDA receptor antagonists block pain transmission in dorsal horn spinal neurons 13, 14 and reduce pain-related behavior in neuropathic animal models 15, 16. The present study is therefore aimed at understanding the role of NO and its interaction with NMDA in chronic constriction injury induced neuropathy in rats.

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2. Materials and Methods 

2.1. Animals and surgery 

Male albino mice weighing 23 ± 2 g were used in all experiments. The animals were housed in groups of six under conditions of constant temperature (22-25°C) and a light-controlled room (12-hour light/dark cycle) and with free access to food and water. The surgical procedure for nerve ligation was performed as follows. Unilateral peripheral mononeuropathy was produced on the right hind limb, based on the method of Seltzer et al [7], except that the animals were anesthetized with ketamine (40 mg/kg) and xylazine (10 mg/kg) and a copper wire was used for ligation. The animal's right sciatic nerve was exposed, and a 2–3 mm long nerve segment was then dissected. Only one ligature with fine metal wire was made around the dissected nerve. All the control animals were sham-operated.

2.2. Analgesic measurement 

Pain sensitivity was assessed using the hot-plate test as described by Eddy and Liembach [17], with minor modifications. Briefly, the animal was placed on a circular surface (diameter 19 cm) maintained at 55 ± 0.2°C and surrounded by a Plexiglass wall (12 cm high). The apparatus (Harvard, England, UK) was equipped with a timer and a thermocoupler to maintain a constant temperature. Licking the forepaws, lifting hindpaws or jumping from the surface was used as the end point for the determination of response latencies [18]. Failure to respond by 45 seconds resulted in the termination of the test (cut-off). Fourteen days after nerve ligation of animals, analgesia achieved following drug administration was measured.

2.3. Drugs 

Chemicals used were dextromethorphan (Alhavi Pharmaceutical Co., Tehran, Iran), L-NAME (Sigma-Aldrich, Munich, Germany), saline, ketamine 5%, xylazine (Chanelle Pharmaceuticals Manufacturing Ltd., Loughrea, Ireland). All drugs were dissolved in saline and were injected intraperitoneally (ip).

2.4. Statistical analysis 

Data was analyzed using analysis of variance followed by Newman-Keuls test. Unpaired t test was used for comparison between control animals (sham-operated) and the sciatic nerve ligated group. Differences between means were considered statistically significant if p < 0.05. Data is represented as the mean ± SEM for six mice.

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3. Results 

Significant allodynia and hyperalgesia were observed following the comparison between control animals (sham-operated) and the sciatic nerve ligated group, 14 days after surgery (Figure 1).

Administration of dextromethorphan (ip) 10, 30 and 45 mg/kg to ligated mice induced significant analgesia as compared with saline treated animals. The analgesic response was dose-dependent and a greater response was obtained using 45 mg/kg of dextromethorphan (Figure 2). Administration of L-NAME (ip) 10, 50 and 100 mg/kg to ligated mice caused a significant dose-dependent analgesia when compared with saline treated animals (Figure 3). Effects of L-NAME 10 mg/kg and 50 mg/kg on the effects of different doses of dextromethorphan after 30, 60 and 90 minutes of administration did not show significant differences to dextromethorphan only treated groups. Latency periods after administration of 100 mg/kg of L-NAME to mice treated with dextromethorphan (ip) 10, 30 and 45 mg/kg in 30, 60 and 90 minutes, increased significantly in comparison with dextromethorphan only treated groups (Figure 4).

  • View full-size image.
  • Figure 2. 

    Latency response of dextromethorphan (Dex) treated group in comparison with saline treated group (n = 6, data shown as mean ± SEM). *p < 0.05. NS = normal saline treated group.

  • View full-size image.
  • Figure 3. 

    Latency response of the L-nitro-arginine methyl ester (L-NAME) treated group in comparison with the saline treated group (n = 6, mean ± SEM). *p < 0.05. NS = normal saline treated group.

  • View full-size image.
  • Figure 4. 

    Effect of L-nitro-arginine methyl ester (L-NAME) on the latency response of dextromethorphan (Dex) in comparison with the Dex-only treated group (n= 6, mean ± SEM). *p < 0.05.

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4. Discussion 

The symptoms of neuropathic pain are often intractable because they are poorly relieved by conventional analgesics 19, 20, 21. Traumatic nerve injury can lead to the development of hyperalgesia or/and allodynia. An increase in the afferent impulse rate from the damaged nerve area enhances the activity of spinal dorsal horn neurons, producing pain [4]. Activation of NMDA receptors in the spinal dorsal horn neurons is critical to the development and maintenance of the hyperalgesia induced by nerve injury 22, 23, 24.

Our study showed that dextromethorphan, an NMDA antagonist, has analgesic properties. This result is in agreement with previous studies using ketamine, a NMDA antagonist, which has been shown to be effective in reducing various types of neuropathic pain symptoms in patients 25, 26, 27, 28.

The activation of neuronal firing by NMDA receptors leads to an increase in intracellular calcium levels 29, 30. Dextromethorphan has been shown to reduce and regulate the influx of intracellular calcium through NMDA receptor-gated channels [31]. This action antagonizes the effects of excitatory amino acids and reduces the release of various peptides, such as glutamate and aspartate, and ultimately may lead to an overall reduction of pain sensation [32].

Glutamate induces thermal hyperalgesia through the activation of NMDA receptors and subsequent production of nitric oxide [4]. As we observed, L-NAME had analgesic effects in sciatic nerve ligated mice. NO is highly involved in analgesic processing in the central nervous system, at both the spinal and supraspinal levels. Previous studies have demonstrated that intrathecal injection of L-arginine and NO-releasing compounds produced hyperalgesia in the tail flick test and formalin pain model 33, 34, 35, illustrating the involvement of NO in the pain pathways. Following the co-administration of dextromethorphan and L-NAME, an increased latency period and more analgesia were observed.

The activation of NMDA receptors in the central nervous system initiates an influx of Ca2+, inducing Ca-dependent intracellular processes 36, 37. Nitric oxide synthase is stimulate in a Ca2+-dependent manner by the activation of NMDA receptors and is responsible for the synthesis of NO from L-arginine 38, 39. In addition, NO has also been shown to enhance the release of excitatory amino acids 40, 41, 42, 43. Furthermore, certain states of pathological pain are reversibly blocked by the administration of nitric oxide synthase inhibitors [44] and NMDA receptor antagonists 45, 46. In conclusion, these observations support the idea that NMDA/NO pathways play an important role in the development of such sciatic nerve ligated-evoked pathological pain conditions as allodynia, hyperalgesia and phantom pain. Further studies are needed to reveal the role of NO and NMDA and their related mechanisms in such pain conditions.

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PII: S2005-2901(10)60006-4

doi:10.1016/S2005-2901(10)60006-4

Journal of Acupuncture and Meridian Studies
Volume 3, Issue 1 , Pages 38-42, March 2010

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