SYM-2081 a kainate receptor antagonist reduces allodynia and hyperalgesia in a freeze injury model of neuropathic pain

Abstract

Cold-freeze injury at −4°C to the rat sciatic nerve produces mechanical allodynia and thermal hyperalgesia [M.A. Kleive, P.S. Jungbluth, J.A. Uhlenkamp, K.C. Kajander, Cold injury to rat sciatic nerve induces thermal hyperalgesia or analgesia, 8th World Congress on Pain, Vancouver, BC, Canada, August 1996 (Abstract).]. The NMDA receptor, an excitatory amino acid (EAA) receptor, appears to be involved in the development of allodynia and hyperalgesia following nerve injury. The role, if any, of the kainate receptor, another EAA receptor, remains unknown. In the current study, we evaluated whether (2S,4R)-4-methylglutamic acid (SYM-2081), a recently developed kainate receptor antagonist, attenuates increased responsiveness following cold injury to the sciatic nerve. During baseline testing, Sprague–Dawley rats were evaluated for frequency of withdrawal from von Frey filaments and latency of withdrawal from a radiant thermal source. Animals were then anesthetized, the left sciatic nerve was exposed, and the nerve was cooled to −4°C for 15 min (n=24). For control rats (n=24), all procedures were identical except that the nerve was maintained at 37°C. Testing resumed on the third day following surgery. On the fifth post-operative day, SYM-2081 (150 or 100 mg/kg), fentanyl citrate (0.04 mg/kg) or vehicle was injected intraperitoneally. Injury to the rat sciatic nerve induced a significant increase in withdrawal frequency and a significant decrease in withdrawal latency (ANOVA, p<0.05). SYM-2081 and fentanyl significantly reduced these responses (p<0.05). These results suggest that kainate and opioid receptors are involved in the mechanical allodynia and thermal hyperalgesia that develop following cold injury to the sciatic nerve.

Introduction

The receptors for excitatory amino acids (EAAs) are the focus of many studies due to increasing evidence for a role in normal and pathological brain function 36, 72, 74. Glutamate, the neurotransmitter at most excitatory synapses, activates a variety of receptor subtypes that can broadly be divided into ionotropic (ligand-gated ion channels) and metabotropic (G-protein-coupled) receptors [78]. Based on pharmacological and molecular biological studies, ionotropic receptors are divided into N-methyl-d-aspartate (NMDA) and non-NMDA subtypes. The non-NMDA receptor group is further divided into α-amino-3-hydroxy-5-methyl isoxazole propionic acid (AMPA) and kainate subtypes 13, 108. It has been established that the subunits GluR1-4, also named GluRA through GluRD, are constituents of the AMPA receptors, whereas GluR5–7, KA1 and KA2 may comprise the kainate-selective (or kainate-preferring) receptor class 12, 53. Until recently, the lack of pharmacological tools to discriminate between AMPA and kainate receptors has greatly hindered progress in understanding the roles of kainate receptors 42, 69.

The pathophysiological mechanisms of neuropathic pain associated with nerve injury remain unclear. It has been hypothesized that EAAs (e.g., glutamate and aspartate) play a role in the development and maintenance of neuropathic pain 28, 70, 114. EAAs are found in the majority of synapses in the central nervous system 27, 29, 55, 106, and are released in dorsal horn of the spinal cord in response to noxious stimulation 60, 93, 95.

One suggested mechanism underlying neuropathic pain is ectopic spontaneous activity from the damaged nerves 3, 34, 58, 62, 64. Prolonged C-fiber afferent stimulation associated with nerve injury produces peripheral sensitization of the receptors 10, 19, 65, 83; and triggers central changes by inducing alterations in spinal cord function such as sensitization 37, 85, 92, 112, wind-up 76, 85 and expansion of the receptive fields of spinal neurons 22, 35, 73.

Central sensitization produces a prolonged and significant enhancement of responses to both noxious and innocuous stimuli 25, 39, 111, 113. This central mechanism may play a significant role in neuropathic pain. A possible mechanism underlying the development of this phenomenon is the preferential loss of large myelinated fibers, which would lead to a presumed loss of central inhibitory controls by large diameter primary afferent neurons 8, 47. This explanation is consistent with previous observations of Noordenbos [81] who proposed that the pain of post-herpetic neuralgia was due to the predominant large fiber loss combined with activity in surviving primary afferent nociceptor. Later, Melzack and Wall [75] proposed that selective damage to pain inhibiting large diameter myelinated sensory neurons was an underlined mechanism in neuropathic pain. Loss of large fiber evoked inhibition could also be brought about if central inhibitory interneurons are impaired. Many neurons in dorsal horn of the spinal cord contain inhibitory neurotransmitters such as GABA and enkephalin 54, 99. Observations in both animal experiments 40, 46, 88, 99 and from the clinical use of the GABA_b agonist baclofen in paroxysmal neurogenic pain conditions [45] indicate that GABA itself and other GABA receptor agonist have antinociceptive properties. Dysfunction of the spinal GABA system may also be involved in the development of allodynia, a symptom often observed in neuropathic pain conditions [50].

Since central sensitization is hypothesized to be associated with NMDA and/or non-NMDA receptor activation, it is reasonable to evaluate both NMDA and non-NMDA antagonists as possible agents for treatment of neuropathic pain. NMDA receptor antagonists, including dizocilpine maleate (MK-801), dextrorphan and ketamine are considered potential agents for therapeutic use in the treatment of neuropathic pain due to their antinociceptive effects following nerve injury in human and animal studies 5, 28, 70, 87, 101, 114. However, studies assessing their potential usefulness have found deleterious adverse effects including hyperlocomotion [17] and motor impairment 24, 50, 51, 110. This provides a rationale for studying antagonists that act at other EAA receptors that may be effective in reducing neuropathic pain without producing these toxic side effects.

Partial peripheral nerve injury often leads to chronic pain states characterized by allodynia, hyperalgesia and causalgia 15, 33, 77, 86, 100, 103. Several animal models have been developed for studying pain following partial nerve injury. In the chronic constriction injury (CCI) model, which was first reported by Bennett and Xie in 1988 [11], four loose ligatures placed around the sciatic nerve produced mechanical allodynia, thermal hyperalgesia and spontaneous pain. Partial denervation of the hindlimb as described by Seltzer et al. [91] also produces mechanical allodynia and thermal hyperalgesia. Kim and Chung [61] found similar results simply by ligating the spinal nerve at the L5 and L6 roots. DeLeo et al. [31] produced touch evoked allodynia simply by repeating freeze–thaw cycle of the sciatic nerve down to −60°C.

Denny-Brown et al. [32], using cold stimuli to injure the sciatic nerve of cats, found that myelin and axis cylinders of the mammalian peripheral nerve are selectively damaged by exposure to cold. The largest axons being the most sensitive and the smallest axons being the least. This damage is similar to that observed in the sciatic nerve following the CCI [11]. More recently, Kleive et al. [63] developed an animal model of nerve injury, in which a cold injury of −4°C for 15 min to the rat's sciatic nerve results in mechanical allodynia and thermal hyperalgesia comparable to levels found in the models of Bennett and Xie, Kim and Chung and DeLeo et al.'s studies. A major advantage of using the cold injury model is the ease of reproducibility and the ability to quantify the degree of nerve injury.

A recently synthesized potent and selective kainate antagonist (2S,4R)-4-methylglutamic acid (SYM-2081) [48] has been demonstrated to be a unique agent due to its specific high affinity for the kainate receptor and acts as a reversible antagonist of kainate receptor function without strong action at the AMPA receptor [115]. In the current study, we evaluated whether SYM-2081 attenuates the mechanical allodynia and thermal hyperalgesia associated with the −4°C cold model of neuropathic pain.