Peripheral and spinal antihyperalgesic activity of SIB-1757, a metabotropic glutamate receptor (mGLUR5) antagonist, in experimental neuropathic pain in rats

Neuroscience Letters 292 (2000) 115±118

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Peripheral and spinal antihyperalgesic activity of SIB-1757, a metabotropic glutamate receptor (mGLUR5) antagonist, in experimental neuropathic pain in rats Ahmet Dogrul a, Michael H. Ossipov b, Josephine Lai b, T. Philip Malan Jr. b, Frank Porreca b,* a

Department of Pharmacology, Faculty of Medicine, Gulhane Medical Military Academy, Ankara, Turkey b Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA Received 31 May 2000; received in revised form 15 August 2000; accepted 18 August 2000

Abstract Recent studies suggest a role of Group 1 metabotropic glutamate receptors in mediating the development of spinal hypersensitivity in some pain states. Here, the possible role of mGluR5 receptors in experimental neuropathic pain elicited by ligation of spinal nerves (L5/L6 spinal nerve ligation, SNL) was explored with SIB-1757, a selective mGluR5 antagonist. SNL-induced tactile allodynia was detected by decreased paw withdrawal thresholds to probing with von Frey ®laments and thermal hyperalgesia by decreased paw withdrawal latencies to radiant heat applied to the plantar aspect of the hindpaw. SIB-1757 was given by either intrathecal (i.th.), subcutaneous (s.c.) or intraplantar (i.pl.) injection. In SNL rats, i.th. SIB-1757 produced a partial reversal of tactile allodynia with a shallow dose-response curve ranging over three-orders of magnitude; SIB-1757 was inactive against allodynia when given systemically. SIB-1757 produced full reversal of thermal hyperalgesia in SNL rats following administration either spinally or locally to the injured paw; administration to the contralateral paw had no effect. SIB-1757 did not produce antinociception in either the SNL or sham-operated rats by any route. These data suggest a signi®cant modulation of thermal hyperalgesia by mGluR5 antagonists, consistent with reports that this receptor may be associated with afferent C-®bers. The less impressive effect seen on tactile allodynia, likely to be mediated by large ®ber input, suggests that the observed modulation may be related to blockade of mGluR5-mediated spinal sensitization. These results do not support the involvement of these receptors in modulation of acute nociception but suggest the possibility of a role for Group I mGluRs in the mediation of aspects of neuropathic pain which may be associated with C-®ber inputs. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metabotropic glutamate receptors; Neuropathic pain; SIB-1757; Tactile allodynia; Thermal hyperalgesia; mGluR5

Experimental peripheral nerve injury has been used to mimic signs of neuropathic pain, notably tactile allodynia and thermal hyperalgesia, in a number of animal pain models [3,12]. An important underlying mechanism driving the development of neuropathic pain is believed to be the spontaneous, persistent afferent discharge from the injured nerves which leads to the development of `sensitization' of the spinal cord. Mao and colleagues have speculated that N-methyl-d-aspartate (NMDA)-receptor driven intracellular activation of protein kinase C may underly spinal sensitization [14]. This central sensitization in turn * Corresponding author. Tel.: 11-520-626-7421; fax: 11-520626-4182. E-mail address: [email protected] (F. Porreca).

is thought to lead to heightened responsiveness to noxious and innocuous input. An important mediator of central sensitization is glutamate released from primary afferent neurons and acting at the NMDA receptor complex on second order neurons to enhance nociceptive transmission to higher centers [22]. Activation of NMDA receptors due to excess glutamate release has been linked to degeneration of spinal neurons after chronic constriction injury (CCI), which is prevented by the non-competitive NMDA antagonist MK-801 [2]. More recently, there has been a growing appreciation for the possible role of metabotropic glutamate receptors (mGluRs), particularly the Group I (mGluR1 and mGluR5) receptors in nociception and pain behavior, although the contribution of individual mGluR subtypes is not yet clear. Evidence exists to show that the

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mGluR5 receptor is expressed in lamina I and II of spinal dorsal horn, an area which contains the spinal cord neurons responsible for the processing of nociceptive signals and which receives the central projections of primary nociceptors [19]. Furthermore, the mGluR5 receptor may be located postsynaptically on lamina I±II neurons and may be involved in local circuits controlling nociceptive signals. Studies employing immunocytochemistry have identi®ed mGluRs on central terminals of unmyelinated and small diameter, myelinated afferent ®bers and also on GABA-containing interneurons in the super®cial laminae of the dorsal horn [11]. Such anatomical localization is consistent with the suggestion that mGluRs may mediate facilitation of afferent input through disinhibition, leading to a sensitization of dorsal horn neurons to sustained high intensity C-®ber input [11,19]. Results of electrophysiological and behavioral studies in rats are consistent with the involvement of Group I mGluRs in the spinal processing of sustained nociceptive input evoked by intraplantar formalin [6] or carrageenan [24] and by repeated cutaneous applications of mustard oil [24]. In the spinal cord, iontophoretic application of a broad-spectrum mGluR antagonist reduced discharges of rat dorsal horn neurons sensitized by a knee joint in¯ammation [16]. Glutamate levels in the super®cial dorsal horn of rats increased after chronic ligature of the sciatic nerve [1]. Spinal injection of antibodies to mGluRs has been shown to block signs of neuropathic pain in rats with CCI of the sciatic nerve without affecting acute nociception [9]. In light of these observations, the present study was designed to explore the contribution of spinal mGluR5 receptors in the manifestation of tactile allodynia and thermal hyperalgesia after spinal nerve ligation (SNL) by using SIB-1757, an antagonist demonstrated to be highly selective for the mGluR5 receptor and to show little or no activity at other mGluR subtypes [20]. Male Sprague±Dawley rats (Harlan; Indianapolis, IN) 200±300 g at time of testing, were maintained in a climate-controlled room on a 12-h light/dark cycle (lights on at 06:00 h) and food and water were available ad libitum. All of the testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain (IASP) and the National Institutes of Health (NIH) guidelines for the handling and use of laboratory animals and received approval from the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona. Nerve injury was induced using the procedure of Kim and Chung [13]. Anesthesia was induced with 2% halothane in O2 at 2 l/min and maintained with 0.5% halothane in O2. After surgical preparation of the rats and exposure of the dorsal vertebral column from L4 to S2, the L5 and L6 spinal nerves were tightly ligated distal to the dorsal root ganglion using 4±0 silk suture. The incision was closed and the animals were allowed to recover for ®ve days. Rats that exhibited motor de®ciency or failure to exhibit subsequent tactile allodynia were excluded from further testing. Sham

surgery was performed by exposing the spinal nerves without ligation. The assessment of tactile allodynia was determined by the application of calibrated von Frey ®laments to the plantar aspect of the hindpaw of rats which were kept in suspended wire-mesh cages. A response was indicated by lifting of the hindpaw paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength (`up and down' method), analyzed using a Dixon non-parametric test and expressed as the mean withdrawal threshold [5]. The method of Hargreaves was employed to assess thermal hyperalgesia [10]. Rats were allowed to acclimate within Plexiglas enclosures on a clear glass plate maintained at 308C. A radiant heat source (i.e. high intensity projector lamp) which was activated with a timer and focused onto the plantar surface of the affected paw of nerve-injured or shamoperated rats. A motion sensor activated by paw withdrawal halted both lamp and timer. A maximal cut-off of 40 s was used to prevent tissue damage. In all tests, baseline data were obtained for the ligated and sham-operated groups before drug or vehicle injections. Rats were tested at 0, 15, 30, 45 and 60 min after injections. Within each of the treatment groups, post-injection means were compared with the baseline values by analysis of variance (ANOVA), followed by post-hoc analysis of least signi®cance difference for multiple comparisons. Comparisons between two means were performed by Student's ttest. A probability level of 0.05 indicated signi®cance. In order to generate a dose-response curve against tactile allodynia, data were converted to % Antiallodynia by the formula: % Antiallodynia ˆ 100 £ (test value 2 control value)/(15 g 2 control value). The A50 and 95% con®dence intervals were determined by linear regression analysis of the log dose-response curve. SIB-1757 was purchased from Tocris Cookson, Inc. (Ballwin, MO). Sham-operated rats displayed paw withdrawal thresholds to probing with von Frey ®laments of 15 ^ 0 g, indicating an absence of tactile allodynia. Rats with L5/L6 SNL demonstrated paw withdrawal thresholds that were signi®cantly (P , 0:05) lower, in the range of 1.2 ^ 0.12 to 1.4 ^ 0.18 g. The i.th. injection of SIB-1757 over the dose range of 0.0003 to 1 mg (3-orders of magnitude) produced a dosedependent antiallodynic effect with a shallow (slope ˆ 15 ^ 2.2) dose-response curve and an A50 of 0.11 mg (0.035±0.36 mg) (Fig. 1). A maximal antiallodynic effect of 66 ^ 10.3% Antiallodynia was detected. The injection of 100 mg of SIB-1757 into the plantar aspect of the injured hindlimb or of 20 mg/kg s.c. did not produce any change in paw withdrawal threshold at any time, indicating a lack of systemic antiallodynic activity. SNL produced reliable thermal hyperalgesia in all groups. Paw withdrawal latencies ranged from 13.3 ^ 0.58± 14.3 ^ 0.73 s, which were signi®cantly (P , 0:05) less than the withdrawal latency of 19.9 ^ 0.97 s displayed by the sham-operated rats. The i.th. injection of 0.01 mg of SIB-

A. Dogrul et al. / Neuroscience Letters 292 (2000) 115±118

Fig. 1. These data show a dose-dependent antiallodynic effect of a wide range of i.th. doses of SIB-1757. Male Sprague±Dawley rats with L5/L6 SNL demonstrating established tactile allodynia received SIB-1757 i.th. The dose-response function with a shallow slope and an A50 of 0.11 mg (0.035±0.36 mg) was constructed from data obtained at the 30 min time point after injection.

1757 produced a reversal of thermal hyperalgesia within 10 min of injection, as indicated by increased paw withdrawal latencies of 18.8 ^ 1.36 s (Fig. 2a). Increasing the dose of SIB-1757 to 1 mg did not produce any further elevation in paw withdrawal latency, nor did it elevate paw withdrawal threshold in sham-operated rats (Fig. 2a). Likewise, the i.pl. injection of 100 mg of SIB-1757 into the injured hindlimb produced a reversal of thermal hyperalgesia whereas the injection of the same dose into the contralateral hindpaw produced no change in paw withdrawal latency (Fig. 2b). The s.c. injection of 20 mg/kg of SIB-1757 also reversed thermal hyperalgesia in SNL rats (Fig. 2c). In contrast, this treatment did not increase withdrawal latencies in shamoperated rats (Fig. 2c). The ®nding that the mGluR5 antagonist SIB-1757 was weakly active against tactile allodynia is consistent with the concept that tactile allodynia is mediated chie¯y through large diameter, myelinated Ab afferent ®bers. These ®bers are not thought to express mGluR5 receptors, but mGluR5 are localized on postsynaptically on second order neurons [11,19]. Furthermore, if the primary afferents mediating tactile allodynia possessed the mGluR5 receptors on peripheral nerve endings, then systemically injected and peripherally (locally) applied SIB-1757 would be expected to have been antiallodynic. Rather, allodynia may have been attenuated as a consequence of diminished central sensitization, mediated by postsynaptic mGluR5 receptors. In contrast to the relative lack of ef®cacy against SNLinduced allodynia, SIB-1757 was active against thermal hyperalgesia after each route of administration tested. These results are consistent with the concept that thermal hyperalgesia is mediated primarily by nociceptive C-®bers [17]. Furthermore, the mGluR5 receptor shows the strongest expression of mGluR subtypes in the lamina I and II of rat and human dorsal horn, which contain the spinal cord neurons responsible for the processing of nociceptive signals and receive the central projections of primary nociceptors [4,11,18,19,21,27]. This anatomical localization corresponds well with a role for the injured nerves in promoting spinal sensitization [11,15,19]. Glutamate acting

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spinally may promote neuronal hyperexcitability through pre- and post-synaptic mechanisms and this action may be blocked by i.th. SIB-1757. Similarly, glutamate release at the site of injury may also contribute to afferent drive and hyperexcitability of the C-®bers through mGluR5 receptors on the peripheral terminals of the C-®bers [25,26]. In that case, locally and/or systemically injected SIB-1757 would be likely to block the action of mGluR5 at the site of nerve injury, thereby also reducing primary afferent drive. Further support for a local mechanism of action at the peripheral terminals of the afferent ®bers is suggested by the fact that SIB-1757 given to the contralateral hindpaw after SNL did not alter thermal hyperalgesia. The results presented here extend the growing appreciation for the role of mGluRs against chronic pain. The i.th. injection of the Group I mGluR agonist dihydroxyphenylglycine has produced thermal hyperalgesia and tactile allodynia [7]. In one study with rats with CCI, it was found that spinal administration of the Group I mGluR antagonist (S)4CPG failed to reverse either tactile allodynia or cold allodynia [8]. In contrast, however, the i.th. injection of antisera to the Group I mGluR subtypes blocked cold allodynia in rats with CCI but did not affect responses to acute thermal or tonic chemical (i.e.; formalin) nociceptive stimuli [9]. It was also demonstrated that knock-down of the Group I receptor

Fig. 2. These data show antihyperalgesic effects of SIB-1757 after i.th. (A), s.c. (B) or intraplantar (C) injections. Paw withdrawal latencies from radiant heat applied to the plantar aspect of the hindpaw were determined before and at several time intervals after injection. Thermal hyperalgesia was indicated by a signi®cantly (P , 0:05) lower response latency when compared to sham-operated rats and reversal of hyperalgesia was indicated by a signi®cant (P , 0:05) elevation in withdrawal latency. In all graphs, open symbols indicate vehicle (50% DMSO/50% water) injection and ®lled symbols indicate SIB-1757 injection. Circles indicate sham-operated rats and squares specify SNL rats. N ˆ 6 for all groups.

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