Advertisement

The Effects of Minocycline or Riluzole Treatment on Spinal Root Avulsion–Induced Pain in Adult Rats

  • Daniel J. Chew
    Correspondence
    Address reprint requests to Daniel J. Chew, PhD, John Van Geest Centre for Brain Repair, E.D. Adrian Building, Forvie Site, Robinson Way, Cambridge, Cambridgeshire CB2 0PY, United Kingdom
    Affiliations
    Centre for Neuroscience and Trauma, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
    Search for articles by this author
  • Thomas Carlstedt
    Affiliations
    Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, London, United Kingdom
    Search for articles by this author
  • Peter J. Shortland
    Affiliations
    Centre for Neuroscience and Trauma, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
    Search for articles by this author

      Abstract

      Spinal root avulsion produces tactile and thermal hypersensitivity, neurodegeneration, and microglial and astrocyte activation in both the deafferented and the adjacent intact spinal cord segments. Following avulsion of the fifth lumbar spinal root, immediate and prolonged treatment with riluzole or minocycline for 2 weeks altered the development of behavioral hypersensitivity. Riluzole delayed the onset of thermal and tactile hypersensitivity and partially reversed established pain behavior. Minocycline effectively prevented and reversed both types of behavioral change. Histologic analysis revealed that both drugs reduced microglial staining in the spinal cord, with minocycline being more effective than riluzole. Astrocyte activation was ameliorated to a lesser extent. Surprisingly, neither drug provided a neuroprotective effect on avulsed motoneurons.

      Perspective

      Immediate treatment of spinal root avulsion injuries with minocycline or riluzole prevents the onset of evoked pain hypersensitivity by reducing microglial cell activation. When treatment is delayed, minocycline, but not riluzole, reverses pre-established hypersensitivity. Thus, these drugs may provide a new translational treatment option for chronic avulsion injury pain.

      Key words

      Adult spinal root avulsions (SRAs) commonly occur with major trauma associated with road traffic accidents and acts of violence. They often affect the brachial plexus but can also affect the lumbosacral/cauda equina regions.
      • Carlstedt T.
      Root repair review: Basic science background and clinical outcome.
      • Hoang T.X.
      • Havton L.A.
      Novel repair strategies to restore bladder function following cauda equina/conus medullaris injuries.
      The injuries damage the dorsal and ventral roots, and patients who suffer complete plexus injuries complain of excruciating pain often described as “a constant crushing, or burning, pain,” which is felt in the insensate limb, and “severe pain, which shoots into the dermatome of the injured spinal nerve.”
      • Berman J.S.
      • Birch R.
      • Anand P.
      Pain following human brachial plexus injury with spinal cord root avulsion and the effect of surgery.
      • Kato N.
      • Htut M.
      • Taggart M.
      • Carlstedt T.
      • Birch R.
      The effects of operative delay on the relief of neuropathic pain after injury to the brachial plexus: A review of 148 cases.
      Pharmacologic management of the pain is often unsatisfactory and is based on empirical measures,
      • Berman J.S.
      • Birch R.
      • Anand P.
      Pain following human brachial plexus injury with spinal cord root avulsion and the effect of surgery.
      • Carvalho G.A.
      • Nikkhah G.
      • Samii M.
      Pain management after post-traumatic brachial plexus lesions. Conservative and surgical therapy possibilities.
      • Gordon D.B.
      • Love G.
      Pharmacologic management of neuropathic pain.
      rather than following a mechanism-based approach. Anticonvulsant, antidepressant, opioids, and cannabinoid drugs are effective in some patients but often fail to provide permanent pain relief, and all have serious adverse effects associated with their use that reduce patient compliance.
      • Berman J.S.
      • Symonds C.
      • Birch R.
      Efficacy of two cannabis based medicinal extracts for relief of central neuropathic pain from brachial plexus avulsion: Results of a randomised controlled trial.
      • Hutchinson M.R.
      • Bland S.T.
      • Johnson K.W.
      • Rice K.C.
      • Maier S.F.
      • Watkins L.R.
      Opioid-induced glial activation: Mechanisms of activation and implications for opioid analgesia, dependence, and reward.
      • Hutchinson M.R.
      • Coats B.D.
      • Lewis S.S.
      • Zhang Y.
      • Sprunger D.B.
      • Rezvani N.
      • Baker E.M.
      • Jekich B.M.
      • Wieseler J.L.
      • Somogyi A.A.
      • Martin D.
      • Poole S.
      • Judd C.M.
      • Maier S.F.
      • Watkins L.R.
      Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia.
      • Mika J.
      • Wawrzczak-Bargiela A.
      • Osikowicz M.
      • Makuch W.
      • Przewlocka B.
      Attenuation of morphine tolerance by minocycline and pentoxifylline in naive and neuropathic mice.
      The molecular mechanisms of neuropathic pain associated with avulsion injury are incompletely understood. Traditionally, it was thought that avulsion of primary afferents resulted in disinhibition of the spinal gate,
      • Melzack R.
      • Wall P.D.
      Pain mechanisms: A new theory.
      which in turn led to pain generated centrally by deafferented spinal cord neurons.
      • Brill S.
      • Aryeh I.G.
      Neuromodulation in the management of pain from brachial plexus injury.
      Electrophysiologic recordings of neurons within the deafferented human and rodent spinal cord models of avulsion injury support this idea.
      • Dalal A.
      • Tata M.
      • Allegre G.
      • Gekiere F.
      • Bons N.
      • Albe-Fessard D.
      Spontaneous activity of rat dorsal horn cells in spinal segments of sciatic projection following transection of sciatic nerve or of corresponding dorsal roots.
      • Guenot M.
      • Bullier J.
      • Rospars J.P.
      • Lansky P.
      • Mertens P.
      • Sindou M.
      Single-unit analysis of the spinal dorsal horn in patients with neuropathic pain.
      Additionally, an imbalance of GABAergic/glutaminergic levels in the dorsal horn produced by either excitotoxic or ischemic cell death
      • Meisner J.G.
      • Marsh A.D.
      • Marsh D.R.
      Loss of GABAergic interneurons in laminae I-III of the spinal cord dorsal horn contributes to reduced GABAergic tone and neuropathic pain after spinal cord injury.
      may contribute to the pain sensations. In the avulsed cord, approximately 10% of dorsal horn cells and 25 to 40% of motoneurons are lost.
      • Bergerot A.
      • Shortland P.J.
      • Anand P.
      • Hunt S.P.
      • Carlstedt T.
      Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
      • Chew D.J.
      • Leinster V.H.
      • Sakthithasan M.
      • Robson L.G.
      • Carlstedt T.
      • Shortland P.J.
      Cell death after dorsal root injury.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      Other evidence suggests that changes in supraspinal pathways may also contribute to pain hypersensitivity.
      • Chen Y.
      • Oatway M.A.
      • Weaver L.C.
      Blockade of the 5-HT3 receptor for days causes sustained relief from mechanical allodynia following spinal cord injury.
      • Oatway M.A.
      • Chen Y.
      • Weaver L.C.
      The 5-HT3 receptor facilitates at-level mechanical allodynia following spinal cord injury.
      • Ramer M.S.
      Endogenous neurotrophins and plasticity following spinal deafferentation.
      However, at least a third of patients report allodynia and hyperalgesia with avulsion injury that can be ameliorated by anesthetic blockade of nonavulsed roots, suggesting that noninjured afferents also contribute to the pain.
      • Bertelli J.A.
      • Ghizoni M.F.
      Pain after avulsion injuries and complete palsy of the brachial plexus: The possible role of nonavulsed roots in pain generation.
      • Htut M.
      • Misra P.
      • Anand P.
      • Birch R.
      • Carlstedt T.
      Pain phenomena and sensory recovery following brachial plexus avulsion injury and surgical repairs.
      Avulsion injuries also produce glial changes in the spinal cord,
      • Chew D.J.
      • Carlstedt T.
      • Shortland P.J.
      A comparative histological analysis of two models of nerve root avulsion injury in the adult rat.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      • Colburn R.W.
      • Rickman A.J.
      • DeLeo J.A.
      The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior.
      • Iwasaki R.
      • Matsuura Y.
      • Ohtori S.
      • Suzuki T.
      • Kuniyoshi K.
      • Takahashi K.
      Activation of astrocytes and microglia in the c3-t4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.
      • Penas C.
      • Casas C.
      • Robert I.
      • Fores J.
      • Navarro X.
      Cytoskeletal and activity-related changes in spinal motoneurons after root avulsion.
      and it is now well recognized that microglia and astrocytes are important contributors to the generation and maintenance of neuropathic pain conditions.
      • Chiang C.Y.
      • Sessle B.J.
      • Dostrovsky J.O.
      Role of astrocytes in pain.
      • Graeber M.B.
      • Christie M.J.
      Multiple mechanisms of microglia: A gatekeeper's contribution to pain states.
      • Inoue K.
      • Tsuda M.
      Microglia and neuropathic pain.
      • Milligan E.D.
      • Watkins L.R.
      Pathological and protective roles of glia in chronic pain.
      • Nesic O.
      • Lee J.
      • Johnson K.M.
      • Ye Z.
      • Xu G.Y.
      • Unabia G.C.
      • Wood T.G.
      • McAdoo D.J.
      • Westlund K.N.
      • Hulsebosch C.E.
      • Regino Perez-Polo J.
      Transcriptional profiling of spinal cord injury-induced central neuropathic pain.
      • Raghavendra V.
      • Tanga F.
      • DeLeo J.A.
      Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
      Gliosis has a significant role in the development of peripheral nerve injury pain,
      • Ledeboer A.
      • Sloane E.M.
      • Milligan E.D.
      • Frank M.G.
      • Mahony J.H.
      • Maier S.F.
      • Watkins L.R.
      Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.
      • Raghavendra V.
      • Tanga F.
      • DeLeo J.A.
      Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
      as well as “at-level” and “below-level” spinal cord injury pain.
      • Crown E.D.
      • Gwak Y.S.
      • Ye Z.
      • Johnson K.M.
      • Hulsebosch C.E.
      Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury.
      • Gwak Y.S.
      • Kang J.
      • Unabia G.C.
      • Hulsebosch C.E.
      Spatial and temporal activation of spinal glial cells: Role of gliopathy in central neuropathic pain following spinal cord injury in rats.
      • Hains B.C.
      • Waxman S.G.
      Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
      Thus, multiple mechanisms may contribute, with some playing a more prominent role than others; for example, neuronal death and glial cell activation. Therefore, drugs that are neuroprotective and/or anti-inflammatory may be of benefit in the search for new novel treatments for avulsion injury pain.
      Two drugs that meet the criteria for neuroprotection and glial cell modulation are riluzole and minocycline. Riluzole is used to treat patients with motoneuron disease and has been shown to be beneficial in prolonging tracheostomy-free survival in amyotrophic lateral sclerosis patients by up to 21 months.
      • Cifra A.
      • Mazzone G.L.
      • Nistri A.
      Riluzole: What it does to spinal and brainstem neurons and how it does it.
      • Wilson J.R.
      • Fehlings M.G.
      Riluzole for acute traumatic spinal cord injury: A promising neuroprotective treatment strategy.
      In preclinical models of spinal cord injury and peripheral nerve injury, riluzole has been shown to ameliorate behavioral hypersensitivity.
      • Coderre T.J.
      • Kumar N.
      • Lefebvre C.D.
      • Yu J.S.
      A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain.
      • Hama A.
      • Sagen J.
      Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      Similarly, minocycline has shown to be neuroprotective in ventral root avulsion injury and to ameliorate pain in models of peripheral nerve injury and spinal cord injury.
      • Guasti L.
      • Richardson D.
      • Jhaveri M.
      • Eldeeb K.
      • Barrett D.
      • Elphick M.R.
      • Alexander S.P.
      • Kendall D.
      • Michael G.J.
      • Chapman V.
      Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.
      • Hains B.C.
      • Waxman S.G.
      Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
      • Hoang T.X.
      • Akhavan M.
      • Wu J.
      • Havton L.A.
      Minocycline protects motor but not autonomic neurons after cauda equina injury.
      • Hua X.Y.
      • Svensson C.I.
      • Matsui T.
      • Fitzsimmons B.
      • Yaksh T.L.
      • Webb M.
      Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.
      • Ledeboer A.
      • Sloane E.M.
      • Milligan E.D.
      • Frank M.G.
      • Mahony J.H.
      • Maier S.F.
      • Watkins L.R.
      Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.
      • Raghavendra V.
      • Tanga F.
      • DeLeo J.A.
      Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
      Thus, the current study explored the effects of immediate or delayed administration of 2 drugs currently used in clinical conditions or clinical trials for neurodegenerative central nervous system (CNS) diseases, riluzole and minocycline,
      • Hawryluk G.W.
      • Rowland J.
      • Kwon B.K.
      • Fehlings M.G.
      Protection and repair of the injured spinal cord: A review of completed, ongoing, and planned clinical trials for acute spinal cord injury.
      using a recently developed model of avulsion injury pain.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.

      Methods

      Surgery

      All experimental procedures were carried out in accordance with the UK Scientific Procedures Act (1986) and guidelines set out by the International Association for the Study of Pain guidelines for the care and use of animals.
      • Zimmermann M.
      Ethical guidelines for investigations of experimental pain in conscious animals.
      Thirty male Wistar rats (Charles River, Margate, Kent, UK) weighing 150 to 175 g were anesthetized with 4% isoflurane (Abbott, Berkshire, UK) in 1.5 L/min of O2 and subjected to L5 SRA under aseptic surgical conditions. The method, as described previously,
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      involves removal of the L5 lateral vertebral process and exposure of the L5 spinal nerve root. This mixed nerve root is then hooked with curved watchmaker forceps and pulled caudally from the cord creating a ventral and dorsal root avulsion, without the added trauma of a laminectomy. The lesion is complete and no animals were lost due to the surgical procedure.
      Animals were split into the following groups (n = 6/group): group 1: SRA + immediate vehicle treatment for 2 weeks; group 2: SRA + immediate riluzole treatment for 2 weeks; group 3: SRA + immediate minocycline treatment for 2 weeks; group 4: SRA + delayed riluzole treatment for 2 weeks; and group 5: SRA + delayed minocycline treatment for 2 weeks.

      Drug Treatment Regimen

      Minocycline hydrochloride (catalogue no. M9511, Sigma-Aldrich, Gilliangham, Dorset, UK) was dissolved in .9% saline (50 mg/mL), and delivered intraperitoneally (i.p.) to the animal at 40 mg/kg. This was selected in accordance with the previous published literature on doses of minocycline that were effective in reducing pain hypersensitivity.
      • Guasti L.
      • Richardson D.
      • Jhaveri M.
      • Eldeeb K.
      • Barrett D.
      • Elphick M.R.
      • Alexander S.P.
      • Kendall D.
      • Michael G.J.
      • Chapman V.
      Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.
      • Hoang T.X.
      • Akhavan M.
      • Wu J.
      • Havton L.A.
      Minocycline protects motor but not autonomic neurons after cauda equina injury.
      • Hua X.Y.
      • Svensson C.I.
      • Matsui T.
      • Fitzsimmons B.
      • Yaksh T.L.
      • Webb M.
      Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.
      Riluzole (Rilutek; Sigma-Aldrich) was dissolved in .1 M HCl (10 mg/mL) and delivered i.p. at 4 mg/kg, in accordance with our own
      • Iwasaki R.
      • Matsuura Y.
      • Ohtori S.
      • Suzuki T.
      • Kuniyoshi K.
      • Takahashi K.
      Activation of astrocytes and microglia in the c3-t4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.
      • Ralston 3rd, H.J.
      • Ralston D.D.
      The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: A quantitative electron microscope study.
      and previous
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      studies. Vehicle-treated rats were injected with the same volume of .1 M HCl, buffered in saline, to a pH of between 3.5 and 4.5. No adverse behavioral effects were noted with i.p. vehicle injections. For groups 1 to 3, animals were injected i.p. immediately after SRA and every day for 7 days postoperatively (p.o.), and then every other day for another week (ie, on days 9, 11, and 13 p.o.). In groups 4 and 5, drugs were administered using the same treatment protocol but starting on day 7 postinjury (ie, every day for days 7–14 p.o., and then every other day for days 16, 18, and 20 p.o.). Behavioral effects were assessed 24 hours after drug administration. The treatment protocol chosen is based on our previously published neuroprotective strategies in ventral root avulsion studies.
      • Bergerot A.
      • Shortland P.J.
      • Anand P.
      • Hunt S.P.
      • Carlstedt T.
      Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
      Injections were performed by a person not associated with behavioral assessment.

      Behavioral Analysis

      For 2 weeks prior to baseline recordings, animals were acclimatized to the experimenter (D.J.C.), the acrylic glass test boxes, and the stimuli. Each test was performed in the morning, with mechanical testing preceding thermal testing. The experimenter was blinded throughout to the animal's surgical and drug status. Each animal's weight was assessed daily. Tactile and thermal flexion withdrawal thresholds were determined using the automated plantar anesthesiometer and automated thermal plantar test apparatus (Ugo Basile, Varese, Italy), as described previously.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      Ipsilateral and contralateral hind paws were stimulated, repeated 3 times, and averaged. Behavioral tests were performed on days 0, 1, 3, 5, 7, 9, 11, and 14 for the immediate treatment groups, and on days 0, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 for the delayed treatment groups. Postoperative survival times were 2 weeks for groups 1 to 3 and 4 weeks for groups 4 to 5.

      Immunohistochemistry

      Rats in groups 1 to 3 were overdosed with sodium pentobarbitone at 14 days postinjury and perfused with saline followed by cold 4% paraformaldehyde. The L4 to L5 spinal cord was removed, postfixed for 2 hours at 4 °C, and then cryoprotected in 30% sucrose overnight prior to cryostat sectioning. Slides containing 10 to 15 transverse sections of the spinal cord (18 μm, each separated by 180–240 μm) were prepared for histologic assessment. Sections were incubated for 1 hour in 10% normal donkey serum at room temperature, followed by 1 of the following primary antibodies for 24 hours at room temperature: microglia (rabbit anti-Iba1, 1:1,000, concentration 50 μg/100 μL; Wako, Dusseldorf, Germany), astrocyte processes (mouse anti–glial fibrillary acidic protein [GFAP], 1:1,000, concentration 1 mg/1 mL; Chemicon, Chandlers Ford, UK), and neurons (rabbit anti-NeuN, 1:1,000, concentration 1 mg/mL; Chemicon). Slides were then washed 3 times in phosphate-buffered saline, and goat anti-mouse or anti-rabbit Alexa Fluor-594 secondary antibodies (1:1,000, 2 mg/mL; Invitrogen, Paisley, UK) were then applied for a further 1 hour at room temperature; slides were then washed in phosphate-buffered saline and coverslip mounted using glycerol phosphate-buffered saline. Optimum antibody dilutions were established through serial dilution assessments and have been used as described previously.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      Histologic controls for antibody specificity included omission of the primary antibody and staining with secondary antibodies only. This revealed no specific staining in spinal cord tissue.
      Antibody characterization: the rabbit polyclonal Iba1 antiserum was generated against the synthetic peptide PTGPPAKKAISELP (Wako) and recognizes a single band of 17 kDa in Western blotting consistent with the molecular weight of Iba1. It does not cross-react with neurons or astrocytes. The monoclonal GFAP antibody reacts with GFAP from human, pig, chicken, and rat. In tissue sections this antibody stains astrocytes and Bergman glia cells (manufacturer's technical information). It recognizes a single 50-kDa band that corresponds to GFAP on Western blot analysis. The NeuN antibody is a polyclonal version of the monoclonal NeuN A60 clone raised against the N-terminus that specifically recognizes the DNA binding, neuron-specific protein in most CNS and peripheral nervous system neurons (manufacturer's technical information). According to the manufacturer, it recognizes 2 or 3 bands between 45 and 62 kDa on Western blot of rat brain that may represent multiple phosphorylation states of the protein.
      The staining pattern of these antibodies in the rat spinal cord is consistent with previous reports.
      • Chew D.J.
      • Carlstedt T.
      • Shortland P.J.
      A comparative histological analysis of two models of nerve root avulsion injury in the adult rat.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      • Cho D.C.
      • Cheong J.H.
      • Yang M.S.
      • Hwang S.J.
      • Kim J.M.
      • Kim C.H.
      The effect of minocycline on motor neuron recovery and neuropathic pain in a rat model of spinal cord injury.
      • Iwasaki R.
      • Matsuura Y.
      • Ohtori S.
      • Suzuki T.
      • Kuniyoshi K.
      • Takahashi K.
      Activation of astrocytes and microglia in the c3-t4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.
      The specificity of these antibodies has also been detailed in the Journal of Comparative Neurology antibody database (http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291096-9861/homepage/jcn_antibody_database.htm).

      Quantitative Analysis

      Identical methods to that described in Chew et al
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      were used. Briefly, 6 sections from the L4 and L5 spinal segments of each animal were selected, averaged per animal to yield a single value of percentage positive density staining, and then these values averaged for the group. Images of the ipsilateral and contralateral dorsal and ventral horns were captured at ×20 magnification with a digital camera (Hamamatsu, Welwyn Garden City, UK) attached to a Leica DMRD microscope (Leica, Milton Keynes, UK). Using Leica Qwin software, the percentage density of positive staining was calculated for astrocytes and microglia by placing a 200,000-pixel
      • Bergerot A.
      • Shortland P.J.
      • Anand P.
      • Hunt S.P.
      • Carlstedt T.
      Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
      frame area in the middle of the dorsal and ventral horns (laminae III-IV and VII-VIII, equivalent to ∼40,000 μm2 per image). The image is thresholded to give a binary read out of staining (either positive or negative pixels), and the percentage number of positive pixels compared to total is quantified by the software. Total cell profile counts per section were made for the dorsal and ventral horn neurons using NeuN. As each section is separated by 180 to 240 μm, double counting of neurons is not likely.

      Statistical Analysis

      Results are expressed as means ± standard errors of the mean, with statistical comparisons between groups made using 2-way analysis of variance, to determine F-ratio significance. Post hoc analysis was made by Fisher's protected least significant difference, using SPSS, version 15.0 (SPSS Inc, Chicago, IL). The levels of significance are indicated as P < .05, P < .005, and P < .001 above each data point. Statistical comparisons are made between SRA-vehicle, SRA-riluzole, and SRA-minocycline, for ipsilateral versus contralateral (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6) and vehicle versus riluzole or minocycline (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5).
      Figure thumbnail gr1
      Figure 1Effects of riluzole and minocycline treatment on withdrawal threshold and latency after L5 SRA. Administration of vehicle does not prevent the development of ipsilateral behavioral hypersensitivity to tactile (A) or thermal (B) stimulation. Tactile hypersensitivity is established by day 1 compared to contralateral. Thermal hypersensitivity is established by day 9. Riluzole treatment reduces the tactile hypersensitivity compared to vehicle-treated animals during the second week (days 7–11) (C), but this effect is not maintained at day 14. Minocycline treatment significantly prevents the development of tactile hypersensitivity over 14 days (C). Riluzole treatment prevents the development of thermal hypersensitivity against contralateral values but was only significantly different from vehicle treatment at day 9 (D). Minocycline treatment significantly prevents the development of thermal hypersensitivity over 14 days (D). Riluzole, minocycline, and vehicle treatment had no effects on mechanical thresholds in the contralateral limb (E). Riluzole had no effect on contralateral thermal thresholds compared to vehicle-treated animals, but minocycline significantly elevated thresholds during the second week compared to vehicle treatments (F). Significant differences are indicated for vehicle versus treatment (#P < .05, ##P < .01, ###P < .001) and for ipsilateral versus contralateral (∗P < .05, ∗∗P < .01, ∗∗∗P <.001). n = 6 per group.
      Figure thumbnail gr2
      Figure 2Effects of drug treatments on dorsal horn neuronal profiles. (A) Micrographs of NeuN staining indicate a loss of dorsal horn neurons within both the L5 and L4 segments. Scale = 100 μm. This loss is significant compared to contralateral levels (B). Treatment with riluzole and minocycline produce no significant neuroprotection compared to vehicle treatment in either L4 or L5. Data expressed as mean ± standard error of the mean. Significant differences for ipsilateral versus contralateral sides (∗P < .05, ∗∗P < .01) are shown. n = 6 in each group.
      Figure thumbnail gr3
      Figure 3Effect of drug treatment on ventral motor neuronal profiles. (A) Micrographs of NeuN staining indicate a loss of ventral horn neurons within both the L5 and L4 segments 14 days after SRA. Scale = 100 μm. When calculated, this reduction represents ∼70% loss of motor neurons within L5 and ∼40% in L4 segment (B). This loss is not ameliorated by the administration of either riluzole or minocycline.
      Figure thumbnail gr4
      Figure 4Effects of drug treatment on microglia activity 14 days post L5 SRA. Increased Iba1 labeling can be seen in the superficial dorsal and lateral ventral horns after SRA, in both the ipsilateral L5 and L4 spinal cord segments (A), and treatment with both riluzole and minocycline reduced this. Scale = 100 μm. Compared to vehicle-treated animals, riluzole significantly reduced Iba1 staining in the L4 and L5 dorsal horns (B), but only in the L4 ventral horn (C). Minocycline significantly reduces Iba1 staining in all areas shown compared to vehicle-treated animals (B and C). Significant differences are indicated for ipsilateral versus contralateral sides (∗P < .05, ∗∗P < .01, ∗∗∗P <.001), and vehicle versus treatment (#P < .05, ##P < .01, ###P < .001). n = 6 per group.
      Figure thumbnail gr5
      Figure 5Effects of drug treatment on astrocyte staining 14 days post L5 SRA. Increase in GFAP labeling can be seen surrounding motoneurons and in the superficial dorsal horn, Lissauer tract, and the dorsal root entry zone, in both L5 and L4 spinal cord segments. Scale = 100 μm (A). Riluzole treatment has little effect on GFAP staining intensity in either the dorsal (B) or ventral (C) horns of L4 and L5. Minocycline treatment significantly ameliorates GFAP staining in the ventral horns of L4 and L5 (C) but has no significant effect in the dorsal horns (B). Significant differences are indicated for ipsilateral versus contralateral (∗P < .05, ∗∗P < .01, ∗∗∗P <.001) and vehicle versus treatment (#P < .05, ##P < .01, ###P < .001). n = 6 per group.
      Figure thumbnail gr6
      Figure 6Effects of delayed riluzole or minocycline treatment on L5 SRA-established hypersensitivity. Delayed minocycline treatment permanently reversed established tactile hypersensitivity by day 9 postinjury (2 days after treatment onset) (A). Delayed minocycline treatment also reversed thermal hypersensitivity by day 9 postinjury, with only transient return of hypersensitivity on days 17 and 19 (B). After cessation of treatment on day 19, minocycline's beneficial effects are still apparent for both sensory modalities. Delayed riluzole treatment transiently reverses mechanical hypersensitivity (C) and thermal hypersensitivity (D). Significant differences are indicated for ipsilateral versus contralateral (∗P < .05, ∗∗P < .01, ∗∗∗P <.001). n = 6 per group. A vehicle control group was not performed in these experiments.

      Results

      Preoperative baseline values for both thermal and tactile stimulation were in accordance with previously published values
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      and were not significantly different across groups.

      Group 1: Vehicle Treatment

      Vehicle treatment starting immediately after L5 SRA for 14 days did not prevent or reverse the development of ipsilateral hypersensitivity to tactile (Fig 1A) or thermal (Fig 1B) stimulation. Contralateral values were not significantly different from preoperation levels (or naïve animals
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      ), indicating no contralateral hypersensitivity. Significant ipsilateral tactile hypersensitivity was established against contralateral values by day 1 (15.9 ± 2.0 g, P < .05) and maintained for 14 days (11.6 ± 1.4 g, P < .001, Fig 1A). Significant ipsilateral thermal hypersensitivity was established by day 9 (5.83 ± .8 s, P < .01) compared to contralateral values and maintained up to day 14 (6.01 ± .3 s, P < .05, Fig 1B).

      Group 2: Immediate Riluzole Treatment

      Riluzole treatment had a transient and limited effect on tactile and thermal hypersensitivity. Riluzole delayed the development of tactile hypersensitivity during days 7 to 11 after injury (Fig 1C). Withdrawal thresholds were significantly higher for treatment at day 7 (17.7 ± 1.2, P < .005) and day 9 (20.1 ± 1.5, P < .005) compared to vehicle-treated animals. Riluzole produced a transient effect on thermal hypersensitivity, with significant reduction occurring on day 9 (7.57 ± .6, P < .05) postinjury (Fig 1D) compared to vehicle-treated animals. No effects were seen on the contralateral limb with riluzole administration compared to vehicle-treated animals (Figs 1E and 1F).

      Group 3: Minocycline Treatment

      Minocycline treatment produced a more robust response than that which was shown with riluzole. Immediate treatment after L5 SRA for 14 days ameliorated the development of thermal and tactile hypersensitivity compared to vehicle treatment (Figs 1C and 1D). Withdrawal thresholds were significantly higher than vehicle levels from day 7 for tactile stimulation (22.0 ± 1.3, P < .001) and from day 5 for thermal stimulation (10.9 ± 1.3, P < .001), and this was maintained up to 14 days postinjury. No effect was seen on the contralateral withdrawal thresholds to tactile stimulation (Fig 1E). An elevated contralateral thermal threshold was observed from 7 to 14 days (P < .005) compared to vehicle controls (Fig 1F). However, when the ipsilateral and contralateral sides of minocycline-treated animals were compared, there were no significant differences between them for thermal thresholds.

      Immunohistologic Analysis of L4 and L5 Spinal Cord in Riluzole-, Minocycline-, and Vehicle-Treated Rats

      Dorsal Horn Neuronal Profile Counts

      Fourteen days after SRA-vehicle treatment, L5 segmental NeuN loss was seen in the superficial lamina directly below the Lissauer tract, the dorsal root entry zone, and the lateral superficial laminae, as reported previously.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      NeuN profile counts showed a smaller loss in the adjacent L4 segment (Fig 2A). In spinal cord tissue from 14-day SRA-vehicle-treated animals, the mean L5 ipsilateral dorsal horn neuronal profile counts per section (376 ± 8) was significantly (P < .005) lower compared to the contralateral side (410 ± 5, Fig 2B). The L4 ipsilateral dorsal horn neuronal count (389 ± 4) was also significantly (P < .05) reduced compared to the contralateral side (408 ± 5). Neither minocycline nor riluzole treatment produced a significant improvement in ipsilateral NeuN numbers in the deafferented L5, nor adjacent intact L4 dorsal horn, compared to vehicle treatment. Minocycline-treated animals showed a trend toward neuroprotection of L4 dorsal horn neurons compared to the contralateral side, such that neuron number was no longer statistically significant (P = .06). However, these counts were not statistically higher than those with vehicle treatment.

      Ventral Horn Neuronal Profile Counts

      In vehicle-treated animals, L5 SRA produced a 69 ± 4% loss of motoneurons in L5 spinal cord and a 50 ± 5% loss in the L4 ventral horns compared to the contralateral side at 14 days postinjury (Fig 3). Surprisingly, neither riluzole nor minocycline administered for 14 days postinjury produced any significant neuroprotection of motoneurons in the ventral horn of the L4 to L5 spinal cord. Motoneuron loss in riluzole-treated animals was 69 ± 3% and 47 ± 2% in the L5 and L4 spinal cord, respectively. In minocycline-treated animals, the respective values were 71 ± 5% and 43 ± 11%.

      Microglial Reactivity in the Dorsal and Ventral Horns

      Fourteen days following SRA and vehicle treatment, microglial staining was greatly increased in the ipsilateral L4 to L5 dorsal and ventral horns (Fig 4A). Within the deafferented L5 dorsal horn, Iba1 expression reached 9.39 ± .7%, significantly higher than contralateral (3.38 ± .4%, P < .001) (Fig 4B). Iba1 expression was confined to the gray matter in the L4 cord but was also significantly raised (8.85 ± .9, P < .001) (Fig 4B). Microglial expression in vehicle-treated rats was also significantly increased in the ipsilateral ventral horns of both deafferented L5 (9.34 ± .5%) and adjacent L4 (8.95 ± .7%) cord, compared to the contralateral side (P < .001) (Fig 4C), with particularly intense staining associated with the lateral motoneuron pools (Fig 4A).
      Immediate riluzole treatment for 14 days significantly reduced dorsal horn Iba1 staining, compared to vehicle-treated animals, in ipsilateral areas L4 (5.12 ± .6%, P < .005) and L5 (5.67 ± .6%, P < .001). However, amelioration was only partial, and staining intensity still remained significantly higher than the respective contralateral sides (Fig 4B). In the ventral horn, however, riluzole treatment had no significant ameliorative effect on Iba1 staining in the L5 cord (8.44% ± 1.0) (Fig 4C). A significant reduction was observed in the L4 ventral horn (6.43 ± .4%, P < .05) compared to vehicle-treated animals, but again treatment had only partial ameliorative effects, and levels remained significantly elevated compared to contralateral (Figs 4A and 4C).
      Immediate minocycline treatment for 14 days effectively reduced microglial staining in all areas of the ipsilateral cord (Fig 4A). Quantitative analysis revealed that Iba1 staining was significantly reduced, compared to vehicle, in the L4 (5.12 ± .6%, P <.001) and L5 (5.67 ± .6%, P < .001) dorsal horns to levels statistically similar to contralateral (Fig 4B). Likewise, in the L4 and L5 ventral horns, injury-induced upregulation of Iba1 staining intensity was abolished (P < .001) (Fig 4C).

      Astrocyte Reactivity in the Dorsal and Ventral Horns

      Fourteen days after SRA with vehicle treatment, GFAP staining was significantly increased in the L4 (8.97 ± .8%, P < .001) and L5 (8.87 ± .8%, P < .001) dorsal horns and the L4 (7.76 ± .4%, P < .001) and the L5 (7.93 ± .5%, P < .005) ventral horns ipsilateral to the injury (Fig 5). Immediate treatment with riluzole maintained for 14 days produced no significant changes in GFAP staining in the dorsal and ventral horns after SRA compared to vehicle-treated animals (Figs 5B and 5C). Similarly, treatment with minocycline for 14 days following SRA did not significantly reduce GFAP staining compared to vehicle-treated animals in the dorsal horn (Fig 5B). However, in the ipsilateral ventral horn, GFAP staining density was significantly reduced through minocycline treatment compared to vehicle in both L4 (6.07 ± .2%, P < .05) and L5 (6.64 ± .3%, P < .005) segments. Levels were still elevated compared to the contralateral side (Fig 5C).

      Effects of a Delayed Drug Treatment Regimen on L5 SRA-Induced Hypersensitivity

      Our results so far suggest that both riluzole and minocycline could prevent the onset of behavioral hypersensitivity when administered immediately after injury. Effects persist through maintained administration of minocycline, but not riluzole. From a therapeutic perspective, because many patients present with established pain, it was of interest to see whether or not these drugs could ameliorate or reverse hypersensitivity once it has already become established. Therefore, a second set of behavioral experiments were performed to see the effects of these compounds on established tactile and thermal hypersensitivity.

      Minocycline Delayed Treatment

      Minocycline given daily from 7 to 13 days and every other day from 14 to 21 days after L5 SRA reversed established ipsilateral hypersensitivity to both tactile and thermal (Figs 6A and 6B) stimulation. After cessation of treatment after day 19, the reflex withdrawal latencies were maintained for a further 7 days at levels indistinguishable from contralateral (Figs 6A and 6B). Similarly, minocycline treatment reversed thermal hypersensitivity by 2 days posttreatment compared to contralateral levels and, although fluctuating on days 17 and 19, is maintained even after cessation of administration (Fig 6B).

      Riluzole Delayed Treatment

      The effects of delayed riluzole administration were less obvious than with minocycline. When given daily for 7 days and every other day for another 7 days, riluzole partially reversed tactile (Fig 6C) and thermal (Fig 6D) hypersensitivity, but this was not maintained. For tactile and thermal thresholds, the effects were only apparent during the second week of treatment and were not maintained after withdrawal of treatment. Ameliatorive effects for tactile withdrawal thresholds were noted on days 15 and 19, and for thermal withdrawal thresholds on day 15. As soon as treatment ended, behavioral hypersensitivity returned.

      Discussion

      Our results suggest that immediate treatment of avulsion injury with minocycline or riluzole prevents onset of pain hypersensitivity by reducing microglial cell activation, with minocycline the more effective.
      Riluzole is a benzothiazole drug that has a complex pharmacology.
      • Doble A.
      The pharmacology and mechanism of action of riluzole.
      It is thought to act by reducing presynaptic glutamate release and by increasing glutamate uptake by glial transporters,
      • Cifra A.
      • Mazzone G.L.
      • Nistri A.
      Riluzole: What it does to spinal and brainstem neurons and how it does it.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      thereby reducing overstimulation of CNS neurons. Riluzole inactivates sodium channels that contribute to spontaneous firing of CNS neurons, a mechanism that is thought to underlie spontaneous pain associated with avulsion injuries.
      • Chow D.S.
      • Teng Y.
      • Toups E.G.
      • Aarabi B.
      • Harrop J.S.
      • Shaffrey C.I.
      • Johnson M.M.
      • Boakye M.
      • Frankowski R.F.
      • Fehlings M.G.
      • Grossman R.G.
      Pharmacology of riluzole in acute spinal cord injury.
      • Guenot M.
      • Bullier J.
      • Rospars J.P.
      • Lansky P.
      • Mertens P.
      • Sindou M.
      Single-unit analysis of the spinal dorsal horn in patients with neuropathic pain.
      Riluzole also inhibits potassium, calcium, and other neurotransmitter pathways, and induces trophic factor production, promoting neuroprotection.
      • Caumont A.S.
      • Octave J.N.
      • Hermans E.
      Specific regulation of rat glial cell line-derived neurotrophic factor gene expression by riluzole in C6 glioma cells.
      • Mizuta I.
      • Ohta M.
      • Ohta K.
      • Nishimura M.
      • Mizuta E.
      • Kuno S.
      Riluzole stimulates nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis in cultured mouse astrocytes.
      • Peluffo H.
      • Estevez A.
      • Barbeito L.
      • Stutzmann J.M.
      Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.
      These mechanisms may also explain its neuroprotective properties by reducing excitotoxicity. Since avulsion injury produces neurodegeneration,
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      we surmised that riluzole would be neuroprotective and reduce behavioral hypersensitivity.
      When administered immediately after injury and for 14 days, riluzole was able to slow the development of thermal hypersensitivity and delay the onset of tactile hypersensitivity. The differential effects on thermal versus tactile hypersensitivity may reflect the observation that riluzole has preferential effects on small fibers rather than large ones,
      • Leinster V.H.
      • Robson L.G.
      • Shortland P.J.
      Differential effects of riluzole on subpopulations of adult rat dorsal root ganglion neurons in vitro.
      or that the dosing regimen was different during the first week compared to the second. A more frequent dosing regimen during the second week may have produced a clearer behavioral response.
      • Coderre T.J.
      • Kumar N.
      • Lefebvre C.D.
      • Yu J.S.
      A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain.
      • Hama A.
      • Sagen J.
      Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain.
      In humans, riluzole is taken twice a day, every day, for life. These results are consistent with prior reports whereby riluzole has been able to prevent the onset of behavioral hypersensitivities to thermal and tactile stimuli
      • Coderre T.J.
      • Kumar N.
      • Lefebvre C.D.
      • Yu J.S.
      A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      in animal models of peripheral nerve injury.
      We,
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      and others,
      • Iwasaki R.
      • Matsuura Y.
      • Ohtori S.
      • Suzuki T.
      • Kuniyoshi K.
      • Takahashi K.
      Activation of astrocytes and microglia in the c3-t4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.
      have demonstrated that glial cells contribute to behavioral hypersensitivity after avulsion injuries. This paper now shows that riluzole reduces microglial activity, with no significant effect on astrocytes. Microglia are known to produce proinflammatory mediators and cytokines that stimulate and sensitize spinal cord neurons.
      • Chiang C.Y.
      • Sessle B.J.
      • Dostrovsky J.O.
      Role of astrocytes in pain.
      • Graeber M.B.
      • Christie M.J.
      Multiple mechanisms of microglia: A gatekeeper's contribution to pain states.
      • Inoue K.
      • Tsuda M.
      Microglia and neuropathic pain.
      • Milligan E.D.
      • Watkins L.R.
      Pathological and protective roles of glia in chronic pain.
      The effects of riluzole were primarily restricted to the dorsal horn, with some efficacy in the L4 ventral horn. Riluzole had no significant effect on the astrocytic response, and this may well be due to the dosing regimen in this study. Conclusions are difficult to draw as there are few reports of its effects on spinal cord glia. It has been shown to partially reduce GFAP expression in cultured striatal astrocytes.
      • Carbone M.
      • Duty S.
      • Rattray M.
      Riluzole neuroprotection in a Parkinson's disease model involves suppression of reactive astrocytosis but not GLT-1 regulation.
      However, conversely, riluzole has a robust effect of elevating GLT-1 expression and activation of astrocytes, and generally stimulating activity.
      • Carbone M.
      • Duty S.
      • Rattray M.
      Riluzole elevates GLT-1 activity and levels in striatal astrocytes.
      • Peluffo H.
      • Estevez A.
      • Barbeito L.
      • Stutzmann J.M.
      Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.
      Delaying administration of riluzole until behavioral hypersensitivity was established was less effective at reversing both thermal and mechanical hypersensitivity, and the effects were only transient; once treatment stopped, pain behavior resumed. Positive reversal of behavioral hypersensitivity that outlasts the treatment period with riluzole has been reported in animal models of peripheral nerve injury and spinal cord injury.
      • Coderre T.J.
      • Kumar N.
      • Lefebvre C.D.
      • Yu J.S.
      A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain.
      • Hama A.
      • Sagen J.
      Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      • Wu Y.
      • Satkunendrarajah K.
      • Teng Y.
      • Chow D.S.
      • Buttigieg J.
      • Fehlings M.G.
      Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury.
      Two likely explanations for the discrepancies between animal studies are the concentration of riluzole used, and frequency of administration. Those studies that were able to reverse pain behavior used concentrations of 6 to 12 mg/kg, administered every 12 hours for 7 to 14 days, based on its effectiveness in human studies.
      • Le Liboux A.
      • Cachia J.P.
      • Kirkesseli S.
      • Gautier J.Y.
      • Guimart C.
      • Montay G.
      • Peeters P.A.
      • Groen E.
      • Jonkman J.H.
      • Wemer J.
      A comparison of the pharmacokinetics and tolerability of riluzole after repeat dose administration in healthy elderly and young volunteers.
      • Wokke J.
      Riluzole.
      This study used a dosing regimen based on previous neuroprotective data.
      • Bergerot A.
      • Shortland P.J.
      • Anand P.
      • Hunt S.P.
      • Carlstedt T.
      Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
      • Nogradi A.
      • Vrbova G.
      The effect of riluzole treatment in rats on the survival of injured adult and grafted embryonic motoneurons.
      Although riluzole has a long half-life in rats (10 hours in naïve and 30 hours in spinal cord injured),
      • Wu Y.
      • Satkunendrarajah K.
      • Teng Y.
      • Chow D.S.
      • Buttigieg J.
      • Fehlings M.G.
      Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury.
      it appears that intermittent dosing may not be an effective treatment plan for alleviating neuropathic pain.
      One important fact for consideration is the afferent terminal degeneration that occurs after avulsion injury.
      • Ralston 3rd, H.J.
      • Ralston D.D.
      The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: A quantitative electron microscope study.
      • Ralston 3rd, H.J.
      • Ralston D.D.
      The distribution of dorsal root axons to laminae IV, V, and VI of the macaque spinal cord: A quantitative electron microscopic study.
      Many of riluzole's beneficial effects are exerted at the presynaptic terminals, such as by increasing glutamate transporter (GLAST and GLT-1) activity.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      Within a week after injury, many avulsed terminals will have degenerated, with myelinated fibers degenerating more rapidly than unmyelinated ones. This may account for the observations of only a transient effect on mechanical hypersensitivity after immediate administration. Moreover, at later stages after injury, glutamate transporters may be downregulated so that riluzole is less effective. It is clear from the present results that the earlier and more frequently that riluzole is administered, the more effective it is at modulating behavioral hypersensitivity. This is consistent with a human study in which riluzole appears to have equivocal effects in chronic neuropathic pain.
      • Galer B.S.
      • Twilling L.L.
      • Harle J.
      • Cluff R.S.
      • Friedman E.
      • Rowbotham M.C.
      Lack of efficacy of riluzole in the treatment of peripheral neuropathic pain conditions.
      Minocycline treatment immediately after injury was able to prevent avulsion-induced hypersensitivity. This effect is most likely mediated by its well-documented effects on activated microglia,
      • Milligan E.D.
      • Watkins L.R.
      Pathological and protective roles of glia in chronic pain.
      as histologic assessment showed significant amelioration of staining throughout the dorsal and ventral horns of the L4 to L5 spinal cord, implying decreased activation. Activated glial cells release proinflammatory mediators, chemokines, and trophic factors, which can sensitize the L4 afferents or spinal cord neurons.
      • McMahon S.B.
      • Malcangio M.
      Current challenges in glia-pain biology.
      By suppressing microglia function, particularly in the adjacent segment to the injury, this study confirms their role in the pathology of avulsion-pain, as evoked hind-paw hypersensitivity is mediated through the “spared” L4 afferents.
      • Bertelli J.A.
      • Ghizoni M.F.
      Pain after avulsion injuries and complete palsy of the brachial plexus: The possible role of nonavulsed roots in pain generation.
      • Bertelli J.A.
      • Ghizoni M.F.
      The possible role of regenerating axons in pain persistence after brachial plexus grafting.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      Future work will determine if this is indeed the mechanism by which minocycline ameliorates behavioral hypersensitivity. The amelioration of avulsion-induced behavioral hypersensitivity by minocycline is consistent with its effects seen in other conditions such as peripheral neuropathic pain and spinal cord injury.
      • Guasti L.
      • Richardson D.
      • Jhaveri M.
      • Eldeeb K.
      • Barrett D.
      • Elphick M.R.
      • Alexander S.P.
      • Kendall D.
      • Michael G.J.
      • Chapman V.
      Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.
      • Hains B.C.
      • Waxman S.G.
      Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
      • Ledeboer A.
      • Sloane E.M.
      • Milligan E.D.
      • Frank M.G.
      • Mahony J.H.
      • Maier S.F.
      • Watkins L.R.
      Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.
      • Raghavendra V.
      • Tanga F.
      • DeLeo J.A.
      Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
      Minocycline exerts its pharmacologic effects via inhibition of p38-MAPK phosphorylation and microglial reactivity, blockade of caspase and metallomatrix proteins, and production of endocannabinoid, pathways that are involved in neuroprotection and analgesia.
      • Domercq M.
      • Matute C.
      Neuroprotection by tetracyclines.
      • Guasti L.
      • Richardson D.
      • Jhaveri M.
      • Eldeeb K.
      • Barrett D.
      • Elphick M.R.
      • Alexander S.P.
      • Kendall D.
      • Michael G.J.
      • Chapman V.
      Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.
      • Hains B.C.
      • Waxman S.G.
      Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
      • Tikka T.
      • Fiebich B.L.
      • Goldsteins G.
      • Keinanen R.
      • Koistinaho J.
      Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia.
      Inhibition of p38-MAPK by minocycline may contribute to the reduced microgliosis seen after avulsion injury. Downregulation of GFAP after minocycline treatment has also been reported after spinal cord injury.
      • Cho D.C.
      • Cheong J.H.
      • Yang M.S.
      • Hwang S.J.
      • Kim J.M.
      • Kim C.H.
      The effect of minocycline on motor neuron recovery and neuropathic pain in a rat model of spinal cord injury.
      It is unclear whether inhibition of astrogliosis is direct or indirect, and why it is more robust around motoneurons. The development of astrogliosis around axotomized motoneurons may play an important role in synaptic stability, plasticity, and regeneration.
      • Aldskogius H.
      • Liu L.
      • Svensson M.
      Glial responses to synaptic damage and plasticity.
      • Emirandetti A.
      • Graciele Zanon R.
      • Sabha Jr., M.
      • de Oliveira A.L.
      Astrocyte reactivity influences the number of presynaptic terminals apposed to spinal motoneurons after axotomy.
      If astrocyte reactivity is prevented, excessive synaptic pruning around axotomized motoneurons may result, preventing any reestablishment of the microenvironment. This may also contribute to the lack of motoneuron protection seen in the current study. Effects of minocycline on astrocytes, similar to riluzole, therefore needs further study.
      Minocycline effectively reverses an established neuropathic hypersensitivity, presumably by reducing the influence of microglial activity. Importantly, this is the first demonstration that minocycline maintains efficacy outside the treatment period. The biological half-life of minocycline, when administered orally in humans, is 18 to 23 hours.
      • Langevitz P.
      • Livneh A.
      • Bank I.
      • Pras M.
      Benefits and risks of minocycline in rheumatoid arthritis.
      When given i.p. in rats, the peritoneal cavity acts as a slow release “depot,” and the compound can remain at a steady concentration in circulation for more than 8 hours.
      • Fagan S.C.
      • Edwards D.J.
      • Borlongan C.V.
      • Xu L.
      • Arora A.
      • Feuerstein G.
      • Hess D.C.
      Optimal delivery of minocycline to the brain: Implication for human studies of acute neuroprotection.
      Although delayed minocycline reversed pain-like behavior in an animal model of spinal cord injury, the effect was lost when treatment ceased.
      • Hains B.C.
      • Waxman S.G.
      Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
      Previously, a limited postoperative therapeutic window has been shown for minocycline,
      • Mei X.P.
      • Xu H.
      • Xie C.
      • Ren J.
      • Zhou Y.
      • Zhang H.
      • Xu L.X.
      Post-injury administration of minocycline: An effective treatment for nerve-injury induced neuropathic pain.
      whereas others show no amelioration of nerve injury pain after a delayed treatment.
      • Raghavendra V.
      • Tanga F.
      • DeLeo J.A.
      Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
      This suggests that the contributions of different glial cell subpopulations to hypersensitivity development and maintenance may be injury model–specific and time dependent.
      It is well documented that both riluzole and minocycline exert neuroprotective effects on motoneurons and other CNS neurons.
      • Bergerot A.
      • Shortland P.J.
      • Anand P.
      • Hunt S.P.
      • Carlstedt T.
      Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
      • Domercq M.
      • Matute C.
      Neuroprotection by tetracyclines.
      • Hoang T.X.
      • Akhavan M.
      • Wu J.
      • Havton L.A.
      Minocycline protects motor but not autonomic neurons after cauda equina injury.
      • Nogradi A.
      • Vrbova G.
      The effect of riluzole treatment in rats on the survival of injured adult and grafted embryonic motoneurons.
      • Wu Y.
      • Satkunendrarajah K.
      • Teng Y.
      • Chow D.S.
      • Buttigieg J.
      • Fehlings M.G.
      Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury.
      Therefore, it was surprising that immediate treatment with either drug provided no significant neuroprotection. This may be due to the specifics of the models used previously versus here: In models limiting injury to the ventral roots, only motor axons are damaged and sensory axons remain intact; in SRA, both sensory afferents and motor axons are damaged, leading to far greater neurodegeneration, loss of regulatory processes such as glial transporters, loss of vascular integrity, and variability in levels of necrosis versus apoptosis.
      • Chew D.J.
      • Carlstedt T.
      • Shortland P.J.
      A comparative histological analysis of two models of nerve root avulsion injury in the adult rat.
      • Chew D.J.
      • Murrell K.
      • Carlstedt T.
      • Shortland P.J.
      Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
      • Hoang T.X.
      • Akhavan M.
      • Wu J.
      • Havton L.A.
      Minocycline protects motor but not autonomic neurons after cauda equina injury.
      • Sung B.
      • Lim G.
      • Mao J.
      Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
      Alternatively, minocycline and riluzole may have other adverse effects brought on by glial inhibition. Glial cells produce trophic factors and neuronal support.
      • Caumont A.S.
      • Octave J.N.
      • Hermans E.
      Specific regulation of rat glial cell line-derived neurotrophic factor gene expression by riluzole in C6 glioma cells.
      • Peluffo H.
      • Estevez A.
      • Barbeito L.
      • Stutzmann J.M.
      Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.
      This suggests that a delicate balance should be defined between the need for pain treatment and side effects of preventing endogenous neuroprotection.
      The present results suggest that both these drugs can ameliorate behavioral hypersensitivity associated with avulsion injury. Both minocycline and riluzole are undergoing clinical trials for spinal cord injury,
      • Casha S.
      • Zygun D.
      • McGowan M.D.
      • Bains I.
      • Yong V.W.
      • Hurlbert R.J.
      Results of a phase II placebo-controlled randomized trial of minocycline in acute spinal cord injury.
      • Chow D.S.
      • Teng Y.
      • Toups E.G.
      • Aarabi B.
      • Harrop J.S.
      • Shaffrey C.I.
      • Johnson M.M.
      • Boakye M.
      • Frankowski R.F.
      • Fehlings M.G.
      • Grossman R.G.
      Pharmacology of riluzole in acute spinal cord injury.
      • Wilson J.R.
      • Fehlings M.G.
      Riluzole for acute traumatic spinal cord injury: A promising neuroprotective treatment strategy.
      and the promising results obtained here suggest that their use may provide a new treatment option for avulsion injuries, a very specific type of spinal cord injury. Further benefits may ensue if these therapies are combined with surgical root reimplantation, the main clinical treatment shown to provide functional benefit to patients.
      • Carlstedt T.
      Root repair review: Basic science background and clinical outcome.
      • Carlstedt T.
      • Grane P.
      • Hallin R.G.
      • Noren G.
      Return of function after spinal cord implantation of avulsed spinal nerve roots.

      References

        • Aldskogius H.
        • Liu L.
        • Svensson M.
        Glial responses to synaptic damage and plasticity.
        J Neurosci Res. 1999; 58: 33-41
        • Bergerot A.
        • Shortland P.J.
        • Anand P.
        • Hunt S.P.
        • Carlstedt T.
        Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury.
        Exp Neurol. 2004; 187: 359-366
        • Berman J.S.
        • Birch R.
        • Anand P.
        Pain following human brachial plexus injury with spinal cord root avulsion and the effect of surgery.
        Pain. 1998; 75: 199-207
        • Berman J.S.
        • Symonds C.
        • Birch R.
        Efficacy of two cannabis based medicinal extracts for relief of central neuropathic pain from brachial plexus avulsion: Results of a randomised controlled trial.
        Pain. 2004; 112: 299-306
        • Bertelli J.A.
        • Ghizoni M.F.
        Pain after avulsion injuries and complete palsy of the brachial plexus: The possible role of nonavulsed roots in pain generation.
        Neurosurgery. 2008; 62 (discussion 1113-1104): 1104-1113
        • Bertelli J.A.
        • Ghizoni M.F.
        The possible role of regenerating axons in pain persistence after brachial plexus grafting.
        Microsurgery. 2010; 30: 532-536
        • Brill S.
        • Aryeh I.G.
        Neuromodulation in the management of pain from brachial plexus injury.
        Pain Physician. 2008; 11: 81-85
        • Carbone M.
        • Duty S.
        • Rattray M.
        Riluzole elevates GLT-1 activity and levels in striatal astrocytes.
        Neurochem Int. 2012; 60: 31-38
        • Carbone M.
        • Duty S.
        • Rattray M.
        Riluzole neuroprotection in a Parkinson's disease model involves suppression of reactive astrocytosis but not GLT-1 regulation.
        BMC Neurosci. 2012; 13: 38
        • Carlstedt T.
        Root repair review: Basic science background and clinical outcome.
        Restor Neurol Neurosci. 2008; 26: 225-241
        • Carlstedt T.
        • Grane P.
        • Hallin R.G.
        • Noren G.
        Return of function after spinal cord implantation of avulsed spinal nerve roots.
        Lancet. 1995; 346: 1323-1325
        • Carvalho G.A.
        • Nikkhah G.
        • Samii M.
        Pain management after post-traumatic brachial plexus lesions. Conservative and surgical therapy possibilities.
        Orthopade. 1997; 26: 621-625
        • Casha S.
        • Zygun D.
        • McGowan M.D.
        • Bains I.
        • Yong V.W.
        • Hurlbert R.J.
        Results of a phase II placebo-controlled randomized trial of minocycline in acute spinal cord injury.
        Brain. 2012; 135: 1224-1236
        • Caumont A.S.
        • Octave J.N.
        • Hermans E.
        Specific regulation of rat glial cell line-derived neurotrophic factor gene expression by riluzole in C6 glioma cells.
        J Neurochem. 2006; 97: 128-139
        • Chen Y.
        • Oatway M.A.
        • Weaver L.C.
        Blockade of the 5-HT3 receptor for days causes sustained relief from mechanical allodynia following spinal cord injury.
        J Neurosci Res. 2009; 87: 418-424
        • Chew D.J.
        • Carlstedt T.
        • Shortland P.J.
        A comparative histological analysis of two models of nerve root avulsion injury in the adult rat.
        Neuropathol Appl Neurobiol. 2011; 37: 613-632
        • Chew D.J.
        • Leinster V.H.
        • Sakthithasan M.
        • Robson L.G.
        • Carlstedt T.
        • Shortland P.J.
        Cell death after dorsal root injury.
        Neurosci Lett. 2008; 433: 231-234
        • Chew D.J.
        • Murrell K.
        • Carlstedt T.
        • Shortland P.J.
        Segmental spinal root avulsion in the adult rat: A model to study avulsion injury pain.
        J Neurotrauma. 2013; 30: 160-172
        • Chiang C.Y.
        • Sessle B.J.
        • Dostrovsky J.O.
        Role of astrocytes in pain.
        Neurochem Res. 2012; 37: 2419-2431
        • Cho D.C.
        • Cheong J.H.
        • Yang M.S.
        • Hwang S.J.
        • Kim J.M.
        • Kim C.H.
        The effect of minocycline on motor neuron recovery and neuropathic pain in a rat model of spinal cord injury.
        J Korean Neurosurg Soc. 2011; 49: 83-91
        • Chow D.S.
        • Teng Y.
        • Toups E.G.
        • Aarabi B.
        • Harrop J.S.
        • Shaffrey C.I.
        • Johnson M.M.
        • Boakye M.
        • Frankowski R.F.
        • Fehlings M.G.
        • Grossman R.G.
        Pharmacology of riluzole in acute spinal cord injury.
        J Neurosurg Spine. 2012; 17: 129-140
        • Cifra A.
        • Mazzone G.L.
        • Nistri A.
        Riluzole: What it does to spinal and brainstem neurons and how it does it.
        Neuroscientist. 2013; 19: 137-144
        • Coderre T.J.
        • Kumar N.
        • Lefebvre C.D.
        • Yu J.S.
        A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain.
        J Neurochem. 2007; 100: 1289-1299
        • Colburn R.W.
        • Rickman A.J.
        • DeLeo J.A.
        The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior.
        Exp Neurol. 1999; 157: 289-304
        • Crown E.D.
        • Gwak Y.S.
        • Ye Z.
        • Johnson K.M.
        • Hulsebosch C.E.
        Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury.
        Exp Neurol. 2008; 213: 257-267
        • Dalal A.
        • Tata M.
        • Allegre G.
        • Gekiere F.
        • Bons N.
        • Albe-Fessard D.
        Spontaneous activity of rat dorsal horn cells in spinal segments of sciatic projection following transection of sciatic nerve or of corresponding dorsal roots.
        Neuroscience. 1999; 94: 217-228
        • Doble A.
        The pharmacology and mechanism of action of riluzole.
        Neurology. 1996; 47: S233-S241
        • Domercq M.
        • Matute C.
        Neuroprotection by tetracyclines.
        Trends Pharmacol Sci. 2004; 25: 609-612
        • Emirandetti A.
        • Graciele Zanon R.
        • Sabha Jr., M.
        • de Oliveira A.L.
        Astrocyte reactivity influences the number of presynaptic terminals apposed to spinal motoneurons after axotomy.
        Brain Res. 2006; 1095: 35-42
        • Fagan S.C.
        • Edwards D.J.
        • Borlongan C.V.
        • Xu L.
        • Arora A.
        • Feuerstein G.
        • Hess D.C.
        Optimal delivery of minocycline to the brain: Implication for human studies of acute neuroprotection.
        Exp Neurol. 2004; 186: 248-251
        • Galer B.S.
        • Twilling L.L.
        • Harle J.
        • Cluff R.S.
        • Friedman E.
        • Rowbotham M.C.
        Lack of efficacy of riluzole in the treatment of peripheral neuropathic pain conditions.
        Neurology. 2000; 55: 971-975
        • Gordon D.B.
        • Love G.
        Pharmacologic management of neuropathic pain.
        Pain Manag Nurs. 2004; 5: 19-33
        • Graeber M.B.
        • Christie M.J.
        Multiple mechanisms of microglia: A gatekeeper's contribution to pain states.
        Exp Neurol. 2012; 234: 255-261
        • Guasti L.
        • Richardson D.
        • Jhaveri M.
        • Eldeeb K.
        • Barrett D.
        • Elphick M.R.
        • Alexander S.P.
        • Kendall D.
        • Michael G.J.
        • Chapman V.
        Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.
        Mol Pain. 2009; 5: 35
        • Guenot M.
        • Bullier J.
        • Rospars J.P.
        • Lansky P.
        • Mertens P.
        • Sindou M.
        Single-unit analysis of the spinal dorsal horn in patients with neuropathic pain.
        J Clin Neurophysiol. 2003; 20: 143-150
        • Gwak Y.S.
        • Kang J.
        • Unabia G.C.
        • Hulsebosch C.E.
        Spatial and temporal activation of spinal glial cells: Role of gliopathy in central neuropathic pain following spinal cord injury in rats.
        Exp Neurol. 2012; 234: 362-372
        • Hains B.C.
        • Waxman S.G.
        Activated microglia contribute to the maintenance of chronic pain after spinal cord injury.
        J Neurosci. 2006; 26: 4308-4317
        • Hama A.
        • Sagen J.
        Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain.
        J Neurotrauma. 2011; 28: 127-134
        • Hawryluk G.W.
        • Rowland J.
        • Kwon B.K.
        • Fehlings M.G.
        Protection and repair of the injured spinal cord: A review of completed, ongoing, and planned clinical trials for acute spinal cord injury.
        Neurosurg Focus. 2008; 25: E14
        • Hoang T.X.
        • Akhavan M.
        • Wu J.
        • Havton L.A.
        Minocycline protects motor but not autonomic neurons after cauda equina injury.
        Exp Brain Res. 2008; 189: 71-77
        • Hoang T.X.
        • Havton L.A.
        Novel repair strategies to restore bladder function following cauda equina/conus medullaris injuries.
        Prog Brain Res. 2006; 152: 195-204
        • Htut M.
        • Misra P.
        • Anand P.
        • Birch R.
        • Carlstedt T.
        Pain phenomena and sensory recovery following brachial plexus avulsion injury and surgical repairs.
        J Hand Surg Br. 2006; 31: 596-605
        • Hua X.Y.
        • Svensson C.I.
        • Matsui T.
        • Fitzsimmons B.
        • Yaksh T.L.
        • Webb M.
        Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.
        Eur J Neurosci. 2005; 22: 2431-2440
        • Hutchinson M.R.
        • Bland S.T.
        • Johnson K.W.
        • Rice K.C.
        • Maier S.F.
        • Watkins L.R.
        Opioid-induced glial activation: Mechanisms of activation and implications for opioid analgesia, dependence, and reward.
        ScientificWorldJournal. 2007; 7: 98-111
        • Hutchinson M.R.
        • Coats B.D.
        • Lewis S.S.
        • Zhang Y.
        • Sprunger D.B.
        • Rezvani N.
        • Baker E.M.
        • Jekich B.M.
        • Wieseler J.L.
        • Somogyi A.A.
        • Martin D.
        • Poole S.
        • Judd C.M.
        • Maier S.F.
        • Watkins L.R.
        Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia.
        Brain Behav Immun. 2008; 22: 1178-1189
        • Inoue K.
        • Tsuda M.
        Microglia and neuropathic pain.
        Glia. 2009; 57: 1469-1479
        • Iwasaki R.
        • Matsuura Y.
        • Ohtori S.
        • Suzuki T.
        • Kuniyoshi K.
        • Takahashi K.
        Activation of astrocytes and microglia in the c3-t4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.
        J Hand Surg Am. 2013; 38: 841-846
        • Kato N.
        • Htut M.
        • Taggart M.
        • Carlstedt T.
        • Birch R.
        The effects of operative delay on the relief of neuropathic pain after injury to the brachial plexus: A review of 148 cases.
        J Bone Joint Surg Br. 2006; 88: 756-759
        • Langevitz P.
        • Livneh A.
        • Bank I.
        • Pras M.
        Benefits and risks of minocycline in rheumatoid arthritis.
        Drug Saf. 2000; 22: 405-414
        • Le Liboux A.
        • Cachia J.P.
        • Kirkesseli S.
        • Gautier J.Y.
        • Guimart C.
        • Montay G.
        • Peeters P.A.
        • Groen E.
        • Jonkman J.H.
        • Wemer J.
        A comparison of the pharmacokinetics and tolerability of riluzole after repeat dose administration in healthy elderly and young volunteers.
        J Clin Pharmacol. 1999; 39: 480-486
        • Ledeboer A.
        • Sloane E.M.
        • Milligan E.D.
        • Frank M.G.
        • Mahony J.H.
        • Maier S.F.
        • Watkins L.R.
        Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.
        Pain. 2005; 115: 71-83
        • Leinster V.H.
        • Robson L.G.
        • Shortland P.J.
        Differential effects of riluzole on subpopulations of adult rat dorsal root ganglion neurons in vitro.
        Neuroscience. 2010; 166: 942-951
        • McMahon S.B.
        • Malcangio M.
        Current challenges in glia-pain biology.
        Neuron. 2009; 64: 46-54
        • Mei X.P.
        • Xu H.
        • Xie C.
        • Ren J.
        • Zhou Y.
        • Zhang H.
        • Xu L.X.
        Post-injury administration of minocycline: An effective treatment for nerve-injury induced neuropathic pain.
        Neurosci Res. 2011; 70: 305-312
        • Meisner J.G.
        • Marsh A.D.
        • Marsh D.R.
        Loss of GABAergic interneurons in laminae I-III of the spinal cord dorsal horn contributes to reduced GABAergic tone and neuropathic pain after spinal cord injury.
        J Neurotrauma. 2010; 27: 729-737
        • Melzack R.
        • Wall P.D.
        Pain mechanisms: A new theory.
        Science. 1965; 150: 971-979
        • Mika J.
        • Wawrzczak-Bargiela A.
        • Osikowicz M.
        • Makuch W.
        • Przewlocka B.
        Attenuation of morphine tolerance by minocycline and pentoxifylline in naive and neuropathic mice.
        Brain Behav Immun. 2009; 23: 75-84
        • Milligan E.D.
        • Watkins L.R.
        Pathological and protective roles of glia in chronic pain.
        Nat Rev Neurosci. 2009; 10: 23-36
        • Mizuta I.
        • Ohta M.
        • Ohta K.
        • Nishimura M.
        • Mizuta E.
        • Kuno S.
        Riluzole stimulates nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis in cultured mouse astrocytes.
        Neurosci Lett. 2001; 310: 117-120
        • Nesic O.
        • Lee J.
        • Johnson K.M.
        • Ye Z.
        • Xu G.Y.
        • Unabia G.C.
        • Wood T.G.
        • McAdoo D.J.
        • Westlund K.N.
        • Hulsebosch C.E.
        • Regino Perez-Polo J.
        Transcriptional profiling of spinal cord injury-induced central neuropathic pain.
        J Neurochem. 2005; 95: 998-1014
        • Nogradi A.
        • Vrbova G.
        The effect of riluzole treatment in rats on the survival of injured adult and grafted embryonic motoneurons.
        Eur J Neurosci. 2001; 13: 113-118
        • Oatway M.A.
        • Chen Y.
        • Weaver L.C.
        The 5-HT3 receptor facilitates at-level mechanical allodynia following spinal cord injury.
        Pain. 2004; 110: 259-268
        • Peluffo H.
        • Estevez A.
        • Barbeito L.
        • Stutzmann J.M.
        Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.
        Neurosci Lett. 1997; 228: 207-211
        • Penas C.
        • Casas C.
        • Robert I.
        • Fores J.
        • Navarro X.
        Cytoskeletal and activity-related changes in spinal motoneurons after root avulsion.
        J Neurotrauma. 2009; 26: 763-779
        • Raghavendra V.
        • Tanga F.
        • DeLeo J.A.
        Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy.
        J Pharmacol Exp Ther. 2003; 306: 624-630
        • Ralston 3rd, H.J.
        • Ralston D.D.
        The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: A quantitative electron microscope study.
        J Comp Neurol. 1979; 184: 643-684
        • Ralston 3rd, H.J.
        • Ralston D.D.
        The distribution of dorsal root axons to laminae IV, V, and VI of the macaque spinal cord: A quantitative electron microscopic study.
        J Comp Neurol. 1982; 212: 435-448
        • Ramer M.S.
        Endogenous neurotrophins and plasticity following spinal deafferentation.
        Exp Neurol. 2012; 235: 70-77
        • Sung B.
        • Lim G.
        • Mao J.
        Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats.
        J Neurosci. 2003; 23: 2899-2910
        • Tikka T.
        • Fiebich B.L.
        • Goldsteins G.
        • Keinanen R.
        • Koistinaho J.
        Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia.
        J Neurosci. 2001; 21: 2580-2588
        • Wilson J.R.
        • Fehlings M.G.
        Riluzole for acute traumatic spinal cord injury: A promising neuroprotective treatment strategy.
        World Neurosurg. 2013;
        • Wokke J.
        Riluzole.
        Lancet. 1996; 348: 795-799
        • Wu Y.
        • Satkunendrarajah K.
        • Teng Y.
        • Chow D.S.
        • Buttigieg J.
        • Fehlings M.G.
        Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury.
        J Neurotrauma. 2013; 30: 441-452
        • Zimmermann M.
        Ethical guidelines for investigations of experimental pain in conscious animals.
        Pain. 1983; 16: 109-110