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Agmatine Reverses Pain Induced by Inflammation, Neuropathy, and Spinal Cord Injury

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Agmatine Reverses Pain Induced by Inflammation, Neuropathy, and Spinal Cord Injury Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury Carolyn A. Fairbanks*†, Kristin L. Schreiber†, Kori L. Brewer‡, Chen-Guang Yu§, Laura S. Stone†, Kelley F. Kitto*†, H. Oanh Nguyen*, Brent M. Grocholski*, Don W. Shoeman*, Lois J. Kehl¶, Soundararajan Regunathanʈ, Donald J. Reisʈ, Robert P. Yezierski§, and George L. Wilcox*†** Departments of *Pharmacology and †Neuroscience and ¶Oral Science, University of Minnesota, Minneapolis, MN 55455; §University of Miami, The Miami Project, Miami, FL 33136; ʈDepartment of Neurology and Neuroscience, Weill–Cornell University Medical College, New York, NY 10021; and ‡East Carolina University School of Medicine, Department of Emergency Medicine, Greenville, NC 27858 Edited by Susan E. Leeman, Boston University School of Medicine, Boston, MA, and approved July 11, 2000 (received for review November 17, 1999) Antagonists of glutamate receptors of the N-methyl-D-aspartate geenan (CARRA), ketamine, dextromethorphan, ifenprodil, ␻ subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) aminoguanidine, N -nitro-L-arginine methyl ester (L-NAME), AG, prevent nervous system plasticity. Inflammatory and neuropathic NMDA, substance P (SP), memantine, and ␣-amino-3-hydroxy-5- pain rely on plasticity, presenting a clinical opportunity for the use methyl-4-isoxazolepropionic acid (AMPA)͞metabotropic agonist of NMDAR antagonists and NOS inhibitors in chronic pain. Agma- quisqualate (QUIS; Sigma); dynorphin (DYN; National Institute tine (AG), an endogenous neuromodulator present in brain and on Drug Abuse), SK&F 86466 (SmithKline Beecham), efaxoran spinal cord, has both NMDAR antagonist and NOS inhibitor activ- (Research Biochemicals), and moxonidine (Solvay Pharma). SP ities. We report here that AG, exogenously administered to ro- and moxonidine were dissolved in acidified saline; CARRA was dents, decreased hyperalgesia accompanying inflammation, nor- dissolved in PBS; and all the other drugs were dissolved in 0.9% malized the mechanical hypersensitivity (allodynia͞hyperalgesia) normal saline. produced by chemical or mechanical nerve injury, and reduced Mechanical Sensitivity Testing. autotomy-like behavior and lesion size after excitotoxic spinal cord Application of von Frey (vF) fila- ments 2.44 (0.4 mN, innocuous) and 3.61 (3.3 mN, noxious) injury. AG produced these effects in the absence of antinociceptive cause mice to lick, withdraw, and͞or shake the paw, actions effects in acute pain tests. Endogenous AG also was detected in representing a behavioral endpoint. In Figs. 1 and 2, nociception rodent lumbosacral spinal cord in concentrations similar to those was tested by multiple vF applications to the plantar (Figs. 1A previously detected in brain. The evidence suggests a unique and 2 C–F, 10 applications) or dorsal (Fig. 2 A and B,6 antiplasticity and neuroprotective role for AG in processes under- applications) hindpaw surfaces. lying persistent pain and neuronal injury. CARRA-Evoked Mechanical Hyperalgesia. Intraplantar injections of gmatine (AG) is formed by the enzymatic decarboxylation CARRA (4%͞50 ␮l), or PBS (50 ␮l) were given to halothane- Aof L-arginine (1). It has been discovered recently in mam- anesthetized mice. After confirmation of hyperalgesia at 3 h mals (2, 3), where it is expressed in the central nervous system. postinjection, AG or SAL was delivered intrathecally (i.t.), In brain, AG meets most of the criteria of a neurotransmitter͞ followed by continued testing for mechanical sensitivity. neuromodulator (4): it is synthesized, stored, and released from CARRA-Evoked Muscle Hyperalgesia. Intramuscular (triceps) injec- specific networks of neurons (5, 6), is inactivated by energy- ͞ ␮ ␮ dependent reuptake mechanisms (7), is degraded enzymatically tions of CARRA (4 mg 75 l), but not PBS (75 l), in (8), and binds with high affinity to ␣ -adrenergic and imidazoline halothane-anesthetized rats produced hyperalgesia, which was 2 indicated by a decrease in forelimb grip tensile force (22) at 12 h (I ) receptors (2, 9). In addition, AG antagonizes N-methyl-D- 1 after CARRA. AG or SAL then was delivered i.t., followed by aspartate receptors (NMDAR) (10) and inhibits all isoforms of continued testing. The data are pooled from three experiments nitric oxide synthase (NOS) (11, 12). NMDAR antagonists and conducted by a blinded observer. NOS inhibitors prevent adaptive changes in neuronal function, including opioid tolerance (13, 14), persistent pain (15–17), and Dynorphin-Induced Allodynia. Intrathecal injections of DYN (3 spinal cord injury (SCI) (18–21). Therefore, AG, which antag- ␮ ͞ nmol) or SAL (5 l) were given to awake mice. After confir- onizes inhibits both NMDAR and NOS, should moderate mation of allodynia at 1 day postinjection, AG or SAL was chronic pain accompanying inflammation, neuropathy or SCI. delivered i.t., followed by continued testing for mechanical We report here that AG, when exogenously administered, sensitivity by a blinded observer. selectively relieves allodynic, hyperalgesic, and autotomy-like states accompanying spinal nerve injury, peripheral inflamma- Nerve Injury-Induced Hypersensitivity. Spinal nerve ligation induces tion, and excitotoxic SCI, respectively. Moreover, as in brain (5, mechanical hypersensitivity of the ipsilateral hindpaw in mice 6), we have detected AG in spinal cord, indicating that AG may be an endogenous modulator of pain pathways. This paper was submitted directly (Track II) to the PNAS office. Methods Abbreviations: AG, agmatine; AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Animals. Institute of Cancer Research mice (25–30 g, Harlan, acid; AR, adrenergic receptor; AUC, area under the curve; CARRA, carrageenan; D␤H, dopamine ␤-hydroxylase; DYN, dynorphin; I1, imidazoline 1 receptor; i.t., intrathecal(ly); L4, ␻ Teklad, Madison, WI), Sprague–Dawley rats [125 g, Harlan L5, L6, lumbar segments 4, 5, and 6; L-NAME, N -nitro-L-arginine methyl ester; MK801, Teklad (Fig. 1D; 400–500 g, Harlan Teklad (Fig. 5C); 200– dizolcipine; NeuN, antineuronal nuclear protein; NMDA, N-methyl-D-aspartate; NMDAR, 250 g, Charles River Breeding Laboratories (Figs. 3 and 4)]. All NMDA receptor; NOS, nitric oxide synthase; o.s., optical sections; QUIS, quisqualate; SAL, experiments were approved by the Institutional Animal Care and saline; SCI, spinal cord injury; SP, substance P. Use Committees. Each group had at least five animals; each **To whom reprint requests should be addressed at: Departments of Neuroscience and Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street SE, animal was used only once. Minneapolis, MN 55455-0217. E-mail: [email protected]. The publication costs of this article were defrayed in part by page charge payment. This Chemicals. The following chemicals were used: MK801 (Merck); article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. LY235959 (Lilly Research Laboratories, Indianapolis); carra- §1734 solely to indicate this fact. 10584–10589 ͉ PNAS ͉ September 12, 2000 ͉ vol. 97 ͉ no. 19 Downloaded by guest on September 27, 2021 (24, 25). In halothane-anesthetized mice, the left paraspinal Electrophysiology. Spinal L3-L5 laminectomy permitted extracel- muscle (at the level of the lumbrosacral spinal nerves L4–S2) and lular single-neuron recordings in urethane-anaesthetized (1.2 the L6 transverse process were removed. The L5 spinal nerve was g͞kg i.p.) male rats (450 g, 38°C) via the center barrel (2 M⍀ ligated (6-0 silk thread) distal to the dorsal root ganglion and carbon) of seven-barreled glass microiontophoretic electrodes: proximal to the L4-L5 spinal nerve confluence. Control groups 50 mM NMDA (pH 8.0) and 15 mM AG (pH 6.5), both in 150 included naı¨ve or sham-operated (no nerve ligation) mice. After mM NaCl, administered with automatic current balancing. Re- induction of allodynia͞hyperalgesia was confirmed, AG or SAL cordings were made from dorsal horn neurons responding to was injected followed by continued testing for mechanical sen- noxious or to both innocuous (brush) and noxious (pinch, sitivity by blinded observers. squeeze) stimuli applied to the ipsilateral hindpaw receptive fields. Peristimulus time histograms of action potentials evoked SCI. QUIS (125 nmol) or QUIS ϩ AG (1, 5, and 10 nmol) was by iontophoretic application of NMDA (20 neurons) or natural injected (1 ␮l) in anesthetized rats 900 ␮m below the dorsal stimulation (vide infra) of the cutaneous receptive field involved surface of the spinal cord in laminae IV–VI. detection (window discriminator) and accumulation (computer). Histology. After 3 days, rats were perfused transcardially Receptive fields of three spinal neurons were activated by (saline, then 10% formalin) and cords were removed for histol- cutaneous stimulation (vF 5.18, 140 mN, 0.5–1 Hz or no. 1 ogy. Spinal cord cross-sections (75 ␮m) were examined with light paintbrush, 1–5 min on, 1–5 min off, repeatedly for 15–30 min). microscopy, and reconstructions of the lesion were made with an overhead projector and camera lucida. Areas of tissue damage Rotarod Assay. After two training sessions, mice walked for 300 s eight sections rostral and eight sections caudal (1,275 ␮m total on an accelerating (4–40 rpm) rotarod (Ugo Basile, Varese, Ϯ ϭ length) to the lesion epicenter were measured by using a Italy). We compared the latency to fall before (291 5.4 s, n computer-aided imaging system (26) (IMAGE 1; Universal Imag- 48) and after delivery (i.t.) of saline or drug (MK801, L-NAME, AG) by the formula: % motor
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