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FTY720 Improves Functional Recovery after Spinal Cord Injury by Primarily Nonimmunomodulatory Mechanisms

  • Yusuke Norimatsu
    Affiliations
    Department of Orthopedics, Jichi Medical University School of Medicine, Tochigi, Japan

    Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Tsukasa Ohmori
    Correspondence
    Address reprint requests to Tsukasa Ohmori, M.D., Ph.D., or Yoichi Sakata, M.D., Ph.D., Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
    Affiliations
    Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Atsushi Kimura
    Affiliations
    Department of Orthopedics, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Seiji Madoiwa
    Affiliations
    Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Jun Mimuro
    Affiliations
    Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Atsushi Seichi
    Affiliations
    Department of Orthopedics, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Yutaka Yatomi
    Affiliations
    Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
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  • Yuichi Hoshino
    Affiliations
    Department of Orthopedics, Jichi Medical University School of Medicine, Tochigi, Japan
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  • Yoichi Sakata
    Correspondence
    Address reprint requests to Tsukasa Ohmori, M.D., Ph.D., or Yoichi Sakata, M.D., Ph.D., Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
    Affiliations
    Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan
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      Spinal cord injury (SCI) is an incapacitating injury that can result in limited functional recovery. We have previously shown increases in the lysophospholipid mediator, sphingosine-1-phosphate (S1P), in the spinal cord after contusion injury. To apply S1P receptor modulation to the treatment of SCI, we examined the therapeutic effects of FTY720, an S1P receptor agonist, on locomotor recovery after SCI in mice. Oral administration of FTY720 shortly after contusion SCI significantly improved motor function recovery, as assessed by both Basso Mouse Scale scores and Rotarod Performance test results. FTY720 induced lymphopenia and reduced T-cell infiltration in the spinal cord after SCI but did not affect the early infiltration of neutrophils and the activation of microglia. In addition, plasma levels and mRNA expression of inflammatory cytokines in the spinal cord after SCI were not attenuated by FTY720. Vascular permeability and astrocyte accumulation were both decreased by FTY720 in the injured spinal cord. The therapeutic effects of FTY720 were not solely dependent on immune modulation, as confirmed by the demonstration that FTY720 also ameliorated motor function after SCI in mice with severe combined immunodeficiency. Finally, the S1P1 receptor agonist, SEW2871, partly mimicked the therapeutic effect of FTY720. Our data highlight the importance of immune-independent functions of FTY720 in decreasing vascular permeability and astrogliosis in the injured spinal cord and promoting locomotor function recovery after SCI.
      Spinal cord injury (SCI) is a debilitating injury often sustained in accidents and ultimately results in marked poor neuropathological features and limited functional recovery, despite adequate existing surgical and medical treatments.
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      Update on the treatment of spinal cord injury.
      SCI primarily arises from mechanical disruption of the spinal cord, leading to rapid death of neurons and glia.
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      Neuronal repair and replacement in spinal cord injury.
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      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      In addition to initial tissue damage, much of the post-traumatic degeneration of the spinal cord is due to a multifactorial secondary injury (eg, neuronal and glial apoptosis, inflammation, glial scar formation, local edema/ischemia, and oxidative stress).
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      Neuronal repair and replacement in spinal cord injury.
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      • Popovich P.G.
      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      The theoretical aim of SCI treatment is to counteract the mechanisms of secondary injury and/or to prevent their pathological consequences, because central nervous system (CNS) neurons have a limited capacity to self-repair and regenerate.
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      Molecular targets for axon regeneration: focus on the intrinsic pathways.
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      Repair and neurorehabilitation strategies for spinal cord injury.
      Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid mediator that produces a variety of cellular responses, including proliferation, survival, motility, and cytoskeletal reorganization, through the action of the S1P subfamily of G-protein–coupled receptors.
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      Structural and functional characteristics of S1P receptors.
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      Biological effects of lysophospholipids.
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      Sphingosine 1-phosphate: synthesis and release.
      We have previously shown that the S1P concentration in the spinal cord was significantly increased in the location of a contusion injury and that such changes stimulated S1P1-mediated migration of in vivo–transplanted neural stem/progenitor cells.
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      Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury.
      Furthermore, in areas of brain infarction, S1P2 receptor antagonism also enhances the migration of endogenous neural stem/progenitor cells.
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      Antagonism of sphingosine 1-phosphate receptor-2 enhances migration of neural progenitor cells toward an area of brain.
      Because S1P receptors are ubiquitously expressed in many organs, including the CNS, and S1P produces a variety of responses related to the function of the nervous system,
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      Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology.
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      Effects of LPA and S1P on the nervous system and implications for their involvement in disease.
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      Sphingosine 1-phosphate in neural signalling and function.
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      Multifaceted roles of sphingosine-1-phosphate: how does this bioactive sphingolipid fit with acute neurological injury?.
      we hypothesize that targeting these receptors may become a candidate therapy for various refractory CNS disorders, including SCI.
      FTY720 is an orally available S1P receptor modulator, known clinically as fingolimod.
      • Brinkmann V.
      FTY720 (fingolimod) in multiple sclerosis: therapeutic effects in the immune and the central nervous system.
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      FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function.
      It acts as a broad S1P receptor agonist after phosphorylation by sphingosine kinase in vivo
      • Brinkmann V.
      • Cyster J.G.
      • Hla T.
      FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function.
      and has recently had clinical efficacy in phase 3 clinical trials for multiple sclerosis.
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      A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis.
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      • Kappos L.
      Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis.
      Also, FTY720 protects a rodent model of ischemia-reperfusion after cerebral ischemia
      • Wei Y.
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      • Lo E.H.
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      Fingolimod provides long-term protection in rodent models of cerebral ischemia.
      • Hasegawa Y.
      • Suzuki H.
      • Sozen T.
      • Rolland W.
      • Zhang J.H.
      Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats.
      and improves functional outcomes in a rat model of SCI.
      • Lee K.D.
      • Chow W.N.
      • Sato-Bigbee C.
      • Graf M.R.
      • Graham R.S.
      • Colello R.J.
      • Young H.F.
      • Mathern B.E.
      FTY720 reduces inflammation and promotes functional recovery after spinal cord injury.
      The efficacy of FTY720 in CNS disorders is believed to derive from immunological modulation.
      • Wei Y.
      • Yemisci M.
      • Kim H.H.
      • Yung L.M.
      • Shin H.K.
      • Hwang S.K.
      • Guo S.
      • Qin T.
      • Alsharif N.
      • Brinkmann V.
      • Liao J.K.
      • Lo E.H.
      • Waeber C.
      Fingolimod provides long-term protection in rodent models of cerebral ischemia.
      • Hasegawa Y.
      • Suzuki H.
      • Sozen T.
      • Rolland W.
      • Zhang J.H.
      Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats.
      • Lee K.D.
      • Chow W.N.
      • Sato-Bigbee C.
      • Graf M.R.
      • Graham R.S.
      • Colello R.J.
      • Young H.F.
      • Mathern B.E.
      FTY720 reduces inflammation and promotes functional recovery after spinal cord injury.
      However, other secondary responses that worsen functional outcome in CNS disorders have been identified, and the effect of the drug on these disorders has not been elucidated. In this study, we examine the therapeutic effects of FTY720 on a mouse model of SCI and highlight the nonimmunological mechanism by which FTY720 improves secondary injuries after SCI.

      Materials and Methods

      Animals

      Female C57BL/6N and CB-17/Icr-scid/scid [SCID (severe combined immunodeficiency)] mice (aged 9 to 12 weeks) were obtained from Japan SLC, Inc. (Shizuoka, Japan) and CLEA Japan, Inc. (Tokyo, Japan), respectively. All animal procedures were approved by the Institutional Animal Care and Concern Committee of Jichi Medical University (Tochigi, Japan), and animal care was performed in accordance with the guidelines of the committee.

      Mouse Model of Contusion SCI

      Contusion SCI was induced using an Infinite Horizon Impactor (Precision Systems and Instrumentation, Lexington, KY).
      • Kimura A.
      • Ohmori T.
      • Ohkawa R.
      • Madoiwa S.
      • Mimuro J.
      • Murakami T.
      • Kobayashi E.
      • Hoshino Y.
      • Yatomi Y.
      • Sakata Y.
      Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury.
      After anesthesia with isoflurane, the spinal cord segment was exposed by removing the dorsal part of the vertebra, and a contusion injury of the tenth thoracic spinal cord was induced at a force of 60 kdyne. Postoperative care was performed as previously described.
      • Kimura A.
      • Ohmori T.
      • Ohkawa R.
      • Madoiwa S.
      • Mimuro J.
      • Murakami T.
      • Kobayashi E.
      • Hoshino Y.
      • Yatomi Y.
      • Sakata Y.
      Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury.

      Drug Administration

      FTY720 was kindly provided by Novartis Pharma AG (Basel, Switzerland) and dissolved in distilled water. SEW2871, a specific agonist for the S1P1 receptor,
      • Sanna M.G.
      • Liao J.
      • Jo E.
      • Alfonso C.
      • Ahn M.Y.
      • Peterson M.S.
      • Webb B.
      • Lefebvre S.
      • Chun J.
      • Gray N.
      • Rosen H.
      Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate.
      was obtained from Calbiochem (San Diego, CA). SEW2871 was dissolved in 100% dimethyl sulfoxide and diluted with a 25% (v/v) aqueous solution of Tween-20, because of its low solubility in pure water. FTY720 (3, 0.3, or 0.03 mg/kg), SEW2871 (10 mg/kg), or a corresponding control solvent was given orally to mice every 24 hours, immediately after SCI.

      Assessment of Functional Recovery from SCI

      Recovery was scored by the Basso Mouse Scale (BMS) open-field locomotor rating scale, developed specifically for mice and consisting of scores ranging from 0 (complete paralysis) to 9 (normal mobility).
      • Basso D.M.
      • Fisher L.C.
      • Anderson A.J.
      • Jakeman L.B.
      • McTigue D.M.
      • Popovich P.G.
      Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.
      Scoring was performed by two independent evaluators blinded to the group (Y.N. and A.K.), and analysis was performed on a mean value of two scores.
      Recovery of motor function was also quantified by the Rotarod Performance test (MK-610A; Muromachi Kikai Co., Tokyo). The Rotarod treadmill consists of a computer-controlled stepper motor-driven drum (diameter, 30 mm) with either constant or accelerating speed modes; it automatically records the amount of time spent by the animal on the drum at the point of falling. We measured ride performance in acceleration speed mode (30 rpm/300 seconds) to assess motor function at the indicated times after SCI. Two trials were performed by each mouse, and analysis was performed on the average.

      Blood Collection and Isolation of White Blood Cells

      Whole blood (180 μL) was drawn from the right jugular vein using a 29-gauge syringe containing 20 μL of sodium citrate. After performing a complete blood cell count by automatic analyzer (MEK-6358 Celltac α; Nihon Kohden Corp, Tokyo, Japan), platelet-poor plasma was isolated by centrifugation and stored at −80°C. White blood cells isolated from the buffy-coat fraction by hemolysis were incubated with one of a panel of biotin-conjugated antibodies against cell-specific cell surface antigens (B220, CD3e, CD11b, and Gr-1 for B cells, T cells, macrophages, and granulocytes, respectively; BD Biosciences, San Jose, CA). Samples were then incubated with allophycocyanin-conjugated streptavidin, and antibody binding was analyzed by flow cytometry (FACSAria cell sorter; BD Biosciences).

      ELISA, Quantitative RT-PCR, and Test for Blood Coagulation

      Plasma levels of IL-6, IL-10, interferon-γ, monocyte chemotactic protein-1, and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (eBioscience Inc., San Diego, CA). The ELISA kit for IL-1β was obtained from R&D Systems Inc. (Minneapolis, MN). Real-time quantitative RT-PCR was performed as previously described.
      • Kimura A.
      • Ohmori T.
      • Ohkawa R.
      • Madoiwa S.
      • Mimuro J.
      • Murakami T.
      • Kobayashi E.
      • Hoshino Y.
      • Yatomi Y.
      • Sakata Y.
      Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury.
      The oligonucleotide primer pairs used for RT-PCR were as follows: 5′-CCCAAGCAATACCCAAAGAA-3′ (sense) and 5′-GCTTGTGCTCTGCTTGTGAG-3′ (antisense) for IL-1β (GenBank NM_008361.3); 5′-CCGGAGAGGAGACTTCACAG-3′ (sense) and 5′-TCCACGATTTCCCAGAGAAC-3′ (antisense) for IL-6 (GenBank NM_031168.1); 5′-TAGCCAGGAGGGAGAACAGA-3′ (sense) and 5′-TTTTCTGGAGGGAGATGTGG-3′ (antisense) for TNF-α (GenBank NM_013693.2); 5′-ACAACTTTGGCCGACTTCAC-3′ (sense) and 5′-GGGTTCACTGGCACTTTGAT-3′ (antisense) for IL-18 (GenBank NM_008360.1); 5′-GTCGGTGTGAACGGATTT-3′ (sense) and 5′-CGTGAGTGGAGTCATACTGGAA-3′ (antisense) for glyceraldehyde-3-phosphate dehydrogenase (GenBank NM_008084.2). Plasma prothrombin time and activated partial thromboplastin time were measured using an automatic coagulation analyzer (CA500; Sysmex, Kobe, Japan).

      Infiltration of Peripheral Blood Cells and Microglia in the Spinal Cord

      Mice anesthetized with isoflurane were perfused with PBS, and the section of spinal cord (10 mm thick) was isolated. The extracted spinal cord was homogenized in 500 μL of dissociation medium (Dulbecco's modified Eagle's medium/Ham's F-12 containing 1.3 mg/mL trypsin, 0.7 mg/mL hyaluronidase, 0.2 mg/mL kynurenic acid, and 200 U/mL DNase) and incubated at 37°C for 30 minutes. After neutralizing trypsin with a trypsin inhibitor (Invitrogen Corp., Carlsbad, CA), the cells were washed twice with PBS, then simultaneously labeled with phosphatidylethanolamine-conjugated anti-mouse CD45 (clone 30-F11; BD Biosciences) and lineage-specific antibodies (Gr-1, CD11b, B220, and CD3e). Total numbers of CD45-positive cells and cells positive for each lineage marker were determined using Flow-Count Fluorospheres (Beckman Coulter, Miami, FL) and flow cytometry. To assess infiltration of a T-cell subset in the injured spinal cord, the cells were stained with mouse T-lymphocyte subset antibody cocktail (BD Biosciences) after the removal of myelin debris by Percoll-saline solution.
      • Marcondes M.C.
      • Furtado G.C.
      • Wensky A.
      • Curotto de Lafaille M.A.
      • Fox H.S.
      • Lafaille J.J.
      Immune regulatory mechanisms influence early pathology in spinal cord injury and in spontaneous autoimmune encephalomyelitis.

      Tissue Bleeding

      Mice were sacrificed at 3 days after SCI and perfused with heparinized saline to remove intravascular blood. Segments (10 mm thick) of cord encompassing the lesion were homogenized in distilled water (250 μL) and processed to measure tissue bleeding, as previously described.
      • Choudhri T.F.
      • Hoh B.L.
      • Solomon R.A.
      • Connolly Jr, E.S.
      • Pinsky D.J.
      Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice.
      Briefly, 20 μL of supernatant containing hemoglobin was incubated with 80 μL of Drabkin's reagent (Sigma-Aldrich, St. Louis, MO), and the hemoglobin concentration was assessed by measuring the optical density of the solution at a 550-nm wavelength.

      Vascular Permeability

      To measure vascular permeability, 100 μL of saline containing 2.5% Evans blue dye (Sigma-Aldrich) was i.v. injected into each mouse. Animals were then anesthetized with isoflurane and perfused through the heart with 100 to 150 mL of PBS containing heparin sulfate (0.01 U/mL). Spinal cords (10 mm centered on the lesion site) were removed and dissociated in 150 μL of dimethylformamide using a homogenizer. After incubation at 37°C for 24 hours, Evans blue fluorescence was quantified by using a microplate spectrofluorometer (Gemini EM; Molecular Devices, Inc., Sunnyvale, CA) (excitation, 620 nm; emission, 680 nm). Sample values were compared with Evans blue dye standards in the same solvent. Results were expressed as a percentage of total injected Evans blue dye.

      Histological Analysis

      Mice anesthetized with isoflurane were perfused with 50 mL of PBS, followed by 50 mL of 4% paraformaldehyde. The isolated spinal cord was fixed with 4% paraformaldehyde in PBS for 2 hours at 4°C, incubated with PBS containing sucrose (10% to 30%), and then frozen in the presence of optimal cutting temperature (OCT) compound (Sakura Finetek Inc., Torrance, CA). Sections were prepared from frozen tissues at −25°C and attached to polylysine-coated glass slides. To assess for myelin sparing, staining with eriochrome cyanine was performed as previously described,
      • Rabchevsky A.G.
      • Fugaccia I.
      • Sullivan P.G.
      • Scheff S.W.
      Cyclosporin A treatment following spinal cord injury to the rat: behavioral effects and stereological assessment of tissue sparing.
      and the area of myelin sparing in the SCI lesion epicenter was quantified. The accumulation of astrocytes was assessed by immunostaining with anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (Dako Denmark, Glostrup). Antibody binding was detected by the Dako REAL EnVision Detection System (Dako Denmark). Image analysis was performed using Scion Image for Windows (Scion Corp, Frederick, MD).

      Statistical Analysis

      Unless otherwise stated, values are expressed as the mean ± SEM. Statistical analysis was performed by a Student's t-test or a two-way repeated-measures analysis of variance with a post hoc Bonferroni test, as indicated in the figures.

      Results

      FTY720 Improves Functional Outcome after SCI in Mice

      We first examined whether FTY720, an S1P receptor–activating drug, improves locomotor performance after contusion SCI in mice. We administered FTY720 or control solvent daily immediately after SCI and assessed the mouse hind limb locomotor function by BMS locomotor scoring and the Rotarod Performance test result for up to 4 weeks. All mice became almost completely paraplegic and were unable to stay on the rotary drum after the SCI but gradually began to display partial recovery of locomotor activity (Figure 1A). The administration of FTY720 significantly improved recovery of hind limb motor function assessed by both BMS score and Rotarod Performance test result after SCI, and the effect continued to the end of the analysis (Figure 1A). The beneficial effects of FTY720 were dose dependent and became significant at 7 to 10 days after SCI (Figure 1A and Table 1). We next assessed whether FTY720 exerted therapeutic effects in the early stage after SCI. We measured the recovery rate of BMS score and the Rotarod Performance test result between each of the observation points and found statistical significance in the recovery rate from 0 to 14 days, but not 14 to 28 days, suggesting that FTY720 improves the early phase of secondary injury after SCI (Figure 1B). The improvement of motor function was accompanied with histological amelioration, as evidenced by changes in the myelin-sparing area (Figure 2, A and B).
      Figure thumbnail gr1
      Figure 1FTY720 promotes functional locomotor recovery after SCI in mice. A: FTY720 (3 mg/kg) or saline was given orally every 24 hours after a contusion SCI. The recovery of motor function was quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) at the indicated times after SCI. Data represent mean ± SEM (n = 9 in each group). Drug efficacy was statistically significant by two-way repeated-measures analysis of variance (P < 0.0001). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control experiment (post hoc Bonferroni test). B: The changes in recovery rate of motor function quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) were separately assessed in the early (0 to 14 days) or late (14 to 28 days) phase after SCI. Data represent the mean ± SEM (n = 9 in each group). The therapeutic effect was observed in the early phase after SCI. **P < 0.01, ***P < 0.001 compared with the control experiment (two-tailed Student's t-test).
      Table 1FTY720 Promotes Functional Locomotor Recovery after SCI in Mice
      Time after SCI (days)BMS scoreP value
      13710142128
      FTY720 (mg/kg)0.111 ± 0.2201.167 ± 0.3542.889 ± 1.173.389 ± 0.9283.889 ± 0.8583.833 ± 1.1464.333 ± 0.901NS
       Control, 0
        30.222 ± 0.2641.444 ± 0.5274.056 ± 1.467
      P < 0.05;
      4.278 ± 1.1765.444 ± 1.044
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      5.278 ± 0.833
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      5.667 ± 1.118
      P < 0.05;
      0.0272
        0.30 ± 01.250 ± 0.4632.750 ± 0.6553.250 ± 0.9264.125 ± 1.3024.125 ± 1.3564.250 ± 1.1650.9255
        0.030 ± 01.188 ± 0.2592.625 ± 0.5183.063 ± 0.6233.688 ± 0.7534.188 ± 1.0674.313 ± 0.7990.8895
      Rotarod performance test score (seconds)
      03710142128
      FTY720 (mg/kg)ND73.39 ± 26.3781.67 ± 35.1384.67 ± 42.0499.22 ± 28.5289.83 ± 24.3790.39 ± 32.32NS
       Control, 0
        3ND91.06 ± 27.75109.50 ± 52.18139.78 ± 44.81
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      162.00 ± 4.33
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      150.17 ± 46.47
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      150.63 ± 43.60
      P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      0.0257
        0.3ND61.38 ± 10.8786.12 ± 31.04103.81 ± 34.28114.63 ± 47.61122.19 ± 72.00109.88 ± 57.980.6002
        0.03ND61.56 ± 15.0093.38 ± 22.51103.88 ± 30.0696.50 ± 35.15104.81 ± 41.29103.69 ± 31.060.7884
      Daily doses of control and FTY720 are indicated.
      ND, data could not be detected because all mice became completely paraplegic and were unable to stay on the rotary drum; NS, not significant.
      low asterisk P < 0.05;
      low asterisklow asterisk P < 0.01 compared with the control experiment (post hoc Bonferroni test).
      Figure thumbnail gr2
      Figure 2FTY720 ameliorates the myelin-sparing area in the spinal cord after SCI. A: Representative micrographs showing eriochrome cyanine staining of tissue sections at 28 days after SCI, with or without FTY720. Scale bar = 500 μm. B: Quantification of myelin sparing in the dorsal column in serial tissue sections rostral and caudal to the lesion epicenter. Data represent the mean ± SEM (n = 6 in each group). Drug efficacy was statistically significant by two-way repeated-measures analysis of variance (P < 0.0001). **P < 0.01, ***P < 0.001 compared with the control experiment (post hoc Bonferroni test).

      Failure of FTY720 to Ameliorate Early Inflammatory Response after SCI

      Early inflammation after the SCI is one of a series of important downstream events termed secondary injuries, which culminate in progressive degenerative events in the spinal cord after SCI.
      • Hagg T.
      • Oudega M.
      Degenerative and spontaneous regenerative processes after spinal cord injury.
      • Beattie M.S.
      Inflammation and apoptosis: linked therapeutic targets in spinal cord injury.
      Because FTY720 acts as an immunosuppressant through its actions on the S1P receptor, we next examined whether FTY720 modulates inflammatory responses after SCI. As shown in Figure 3A, we confirmed that FTY720 induces lymphopenia, with CD3e-positive T lymphocytes and B220-positive B lymphocytes disappearing from peripheral blood after treatment with FTY720. However, mRNA expression levels of inflammatory mediators, including IL-1β, IL-6, IL-18, and TNF-α, in contused spinal cord were not changed by FTY720 (Figure 3B). Furthermore, plasma levels of IL-6, IL-10, IL-1β, monocyte chemotactic protein-1, interferon-γ, and TNF-α, which represent systemic inflammation, were unaffected by treatment with FTY720 (Figure 4).
      Figure thumbnail gr3
      Figure 3Failure of FTY720 to suppress early inflammatory responses in the spinal cord after SCI. FTY720 (3 mg/kg) or saline was given orally every 24 hours after contusion SCI. A: Surface antigens on peripheral blood cells (Gr-1, CD11b, B220, and CD3e) were assessed by flow cytometry at 3 days after SCI. The results shown are representative of at least five independent experiments. B: mRNA expression of IL-1β, IL-6, IL-18, and TNF-α in contused spinal cord at the indicated number of days after SCI. Black bars indicate control mice without SCI; white bars, mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SEM (n = 4 to 10 in each group).
      Figure thumbnail gr4
      Figure 4FTY720 does not ameliorate systemic inflammatory response after SCI. FTY720 (3 mg/kg) or saline was given orally every 24 hours after contusion SCI. Plasma levels of IL-1β, IL-6, IL-10, interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, and TNF-α were measured by ELISA on the indicated number of days after SCI. Black bars indicate control mice without SCI; white bars, mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SEM (n = 10 in each group).

      FTY720 Reduces Infiltration of T Cells, but not Neutrophils and Microglia, at the Site of SCI

      We next assessed infiltration of peripheral blood cells and activation of microglia in the spinal cord after SCI. Blood cells constitutively expressing CD45 antigen in the spinal cord were assessed by flow cytometry. In this method, CD45/CD11b double-positive cells are clearly characterized into blood-derived cells or microglial cells by the expression pattern of CD45 (Figure 5A). Gr-1– and CD11b-positive neutrophils rapidly accumulated within the lesion at day 1 after SCI and were gradually cleared (Figure 5, A and B). In contrast, T-lymphocyte entry and microglial accumulation were delayed for several weeks after injury (Figure 5B). FTY720 significantly suppressed the late infiltration of CD3e-positive T lymphocytes but not the early accumulation of peripheral neutrophils and microglial activation in the injured spinal cord (Figure 5B). The reduction of T-cell infiltration in the injured spinal cord by FTY720 was observed only at 14 days after SCI. The administration of FTY720 also significantly reduced the infiltration of CD4-positive helper T cells but not the CD8-positive cytotoxic T-cell population (Figure 5B).
      Figure thumbnail gr5
      Figure 5FTY720 reduces infiltration of T cells, but not neutrophils and microglia, in contused spinal cord. FTY720 (3 mg/kg) or saline was given orally every 24 hours after contusion SCI. A: CD45 expression (vertical) and lineage marker (horizontal; Gr-1, CD11b, B220, and CD3e) in cells obtained from the spinal cord were examined by flow cytometry. Representative data at 1 day after SCI, with or without FTY720, are shown. The data obtained from mice without SCI are also shown as control. CD45+ and CD11b+ cells are clearly characterized, and noted, as blood-derived macrophage or microglial cells by the expression pattern of CD45. B: The number of each cell type was quantified at the indicated days after SCI. Black bars indicate control mice without SCI; white bars, mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SD (n = 5 in each group). *P < 0.05, **P < 0.01 compared with the control experiment (two-tailed Student's t-test).

      Attenuation of Vascular Permeability and Astrocyte Accumulation in the Injured Spinal Cord by FTY720

      Because sphingolipids may affect thrombin generation in vitro,
      • Deguchi H.
      • Yegneswaran S.
      • Griffin J.H.
      Sphingolipids as bioactive regulators of thrombin generation.
      we next focused on the effect of FTY720 on the blood coagulation cascade and extravasation of blood in contused spinal cord after SCI. Blood coagulation (assessed by the measurement of prothrombin time and activated partial thromboplastin time at the indicated times after SCI) was not modified by treatment with FTY720 (Figure 6A). Furthermore, FTY720 failed to improve the amount of extravasated blood in the contused spinal cord (Figure 6B).
      Figure thumbnail gr6
      Figure 6FTY720 decreases vascular permeability after SCI. A: Blood coagulation was assessed by the measurement of prothrombin time (PT) and activated partial thromboplastin time (APTT) at the indicated times after SCI. Black bars indicate control mice without SCI; white bars, mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SEM (n = 5 in each group). B: Tissue bleeding in contused spinal cord was measured at 3 days after SCI. White bars indicate mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SEM (n = 7 to 8). Right panel: Representative macroscopic findings showing bleeding in the spinal cord at 3 days after SCI, with or without FTY720, are shown. Scale bar = 5 mm. C: Vascular permeability in the spinal cord was quantified at 1 or 3 days after SCI, as described in Materials and Methods. White bars indicate mice with SCI treated with saline; and gray bars, mice with SCI treated with FTY720. Data represent the mean ± SEM (n = 5 in each group). **P < 0.01 compared with the control experiment (two-tailed Student's t-test). Right panel: Representative macroscopic findings of the spinal cord after the injection of Evan's blue dye at 3 days after SCI, with or without FTY720, are shown. Scale bar = 5 mm.
      Microvascular permeability after contusion injury may affect local edema and functional deterioration. We assessed vascular permeability in the spinal cord at 1 or 3 days after SCI by measuring extravasation of Evans blue dye injected i.v. (Figure 6C). The diapedesis of Evans blue was markedly suppressed with FTY720 at 3 days after SCI.
      We next assessed astrocyte accumulation in the injured spinal cord, a condition that can lead to glial scar formation. The area of GFAP immunostaining that represents astrocytic reactivity in the injured spinal cord was significantly reduced after treatment with FTY720 (Figure 7, A and B). These data suggest that treatment with FTY720 negatively regulates vascular permeability and astrocyte accumulation in the injured spinal cord.
      Figure thumbnail gr7
      Figure 7FTY720 regulates astrocyte accumulation in the injured spinal cord. A: Representative micrographs showing anti-GFAP staining of tissue sections at 28 days after SCI, with or without FTY720. Scale bar = 200 μm. B: Quantification of the GFAP-positive area in serial tissue sections in the lesion epicenter. Data represent the mean ± SEM (n = 6). *P < 0.05 compared with the control experiment (two-tailed Student's t-test).

      FTY720 Promotes Functional Recovery after SCI Even in SCID Mice

      To investigate whether FTY720 improves functional outcome of SCI through immune modulation, we next used mice homozygous for the scid spontaneous mutation that is characterized by an absence of functional T and B cells and hypogammaglobulinemia (SCID mice). FTY720 significantly improved recovery of hind limb motor function, assessed by both BMS score and the Rotarod Performance test result after SCI (Figure 8A). The recovery rate of motor function showed statistical significance from 0 to 14 days but not from 14 to 28 days (Figure 8B). FTY720 reduced vascular permeability and astrocyte accumulation in the lesion after SCI in SCID mice (Figure 8, C and D). These data clearly indicate the importance of immune-independent functions of FTY720 in the amelioration of functional deficits after SCI in mice.
      Figure thumbnail gr8
      Figure 8FTY720 promotes functional recovery after SCI in SCID mice. FTY720 (3 mg/kg) or saline was given orally every 24 hours in SCID mice after a contusion SCI. A: The recovery of motor function was quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) at the indicated times after SCI. Data represent the mean ± SEM (n = 10 in each group). Drug efficacy was statistically significant, as calculated by two-way repeated-measures analysis of variance (P < 0.0001). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control experiment (post hoc Bonferroni test). B: The changes in recovery rate of motor function quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) were separately assessed in the early (0 to 14 days) or late (14 to 28 days) phase after SCI. Data represent the mean ± SEM. The therapeutic effect was observed in the early phase after SCI. *P < 0.05, **P < 0.01 compared with the control experiment (two-tailed Student's t-test). C: Vascular permeability in the spinal cord was quantified 3 days after SCI, as described in Materials and Methods. Data represent the mean ± SEM (n = 5). *P < 0.05 compared with the control experiment (two-tailed Student’s t-test). D: Quantification of the GFAP-positive area in serial tissue sections in the lesion epicenter at 28 days after SCI. Data represent the mean ± SEM (n = 6). *P < 0.05 compared with the control experiment (two-tailed Student's t-test).

      SEW2871, an S1P1 Receptor Agonist, Partially Mimics the Effects of FTY720

      An active phosphorylated metabolite of FTY720 activates all S1P receptors (S1P1, S1P3, S1P4, and S1P5), except for S1P2.
      • Mandala S.
      • Hajdu R.
      • Bergstrom J.
      • Quackenbush E.
      • Xie J.
      • Milligan J.
      • Thornton R.
      • Shei G.J.
      • Card D.
      • Keohane C.
      • Rosenbach M.
      • Hale J.
      • Lynch C.L.
      • Rupprecht K.
      • Parsons W.
      • Rosen H.
      Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.
      To distinguish whether S1P1 is specifically involved in the actions of FTY720 after SCI, we used SEW2871, an S1P1 receptor–specific agonist. Similar to our results with FTY720, the administration of SEW2871 ameliorated motor function after SCI (Figure 9A). As was the case with FTY720, the improvement in recovery rate was observed from day 0 to day 14 after SCI (Figure 9B), and vascular permeability and astrocyte accumulation were also significantly reduced (Figure 9, C and D). However, the efficacy of SEW2871 was considerably lower than that of FTY720 (Figure 1), suggesting that the therapeutic effect of FTY720 after SCI requires other S1P receptors in addition to S1P1.
      Figure thumbnail gr9
      Figure 9SEW2871 mimics the effects of FTY720. SEW2871 (10 mg/kg) was administered orally to mice every 24 hours after SCI. A: The recovery of motor function was quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) at the indicated times after SCI. Data represent the mean ± SEM (n = 12). Drug efficacy was statistically significant by two-way repeated-measures analysis of variance (P < 0.0001). *P < 0.05 compared with the control experiment (post hoc Bonferroni test). B: The changes in recovery rate of motor function quantified by BMS score (top panel) and Rotarod Performance test result (bottom panel) were separately assessed in the early (0 to 14 days) or late (14 to 28 days) phase after SCI. Data represent the mean ± SEM. The therapeutic effect was observed in the early phase after SCI. *P < 0.05 compared with the control experiment (two-tailed Student's t-test). C: Vascular permeability in the spinal cord was quantified 3 days after SCI, as described in Materials and Methods. Data represent the mean ± SEM (n = 5). **P < 0.01 compared with the control experiment (two-tailed Student’s t-test). D: Quantification of the GFAP-positive area in serial tissue sections in the lesion epicenter at 28 days after SCI. Data represent the mean ± SEM (n = 5). *P < 0.05 compared with the control experiment (two-tailed Student's t-test). DMSO, dimethyl sulfoxide.

      Discussion

      We demonstrated herein a therapeutic effect of the S1P receptor agonist, FTY720, in a mouse model of SCI and examined the mechanism by which FTY720 improves secondary degeneration after SCI. FTY720 is phosphorylated by sphingosine kinase,
      • Brinkmann V.
      • Cyster J.G.
      • Hla T.
      FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function.
      and this phosphorylated form acts as a nonselective S1P receptor agonist for four of the five S1P receptors (S1P1, S1P3, S1P4, and S1P5) in vivo.
      • Mandala S.
      • Hajdu R.
      • Bergstrom J.
      • Quackenbush E.
      • Xie J.
      • Milligan J.
      • Thornton R.
      • Shei G.J.
      • Card D.
      • Keohane C.
      • Rosenbach M.
      • Hale J.
      • Lynch C.L.
      • Rupprecht K.
      • Parsons W.
      • Rosen H.
      Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.
      The most recognized systemic effect of FTY720 administration is an immunosuppressive action, mediated by the S1P1 receptor, which prevents lymphocyte egress from lymphoid organs.
      • Mandala S.
      • Hajdu R.
      • Bergstrom J.
      • Quackenbush E.
      • Xie J.
      • Milligan J.
      • Thornton R.
      • Shei G.J.
      • Card D.
      • Keohane C.
      • Rosenbach M.
      • Hale J.
      • Lynch C.L.
      • Rupprecht K.
      • Parsons W.
      • Rosen H.
      Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.
      FTY720 has been effective in several animal models of autoimmune disorders and allograft survival
      • Nikolova Z.
      • Hof A.
      • Baumlin Y.
      • Hof R.P.
      Combined FTY720/cyclosporine treatment promotes graft survival and lowers the peripheral lymphocyte count in a murine cardiac allotransplantation model.
      • Furukawa H.
      • Suzuki T.
      • Jin M.B.
      • Yamashita K.
      • Taniguchi M.
      • Magata S.
      • Ishikawa H.
      • Ogata K.
      • Masuko H.
      • Shimamura T.
      • Fukai M.
      • Hayashi T.
      • Fujita M.
      • Nagashima K.
      • Omura T.
      • Kishida A.
      • Todo S.
      Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720: effect of monotherapy and combined treatment with conventional drugs.
      • Matsuura M.
      • Imayoshi T.
      • Okumoto T.
      Effect of FTY720, a novel immunosuppressant, on adjuvant- and collagen-induced arthritis in rats.
      ; reperfusion injury, including cerebral ischemia
      • Wei Y.
      • Yemisci M.
      • Kim H.H.
      • Yung L.M.
      • Shin H.K.
      • Hwang S.K.
      • Guo S.
      • Qin T.
      • Alsharif N.
      • Brinkmann V.
      • Liao J.K.
      • Lo E.H.
      • Waeber C.
      Fingolimod provides long-term protection in rodent models of cerebral ischemia.
      • Hasegawa Y.
      • Suzuki H.
      • Sozen T.
      • Rolland W.
      • Zhang J.H.
      Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats.
      ; and renal injury.
      • Troncoso P.
      • Ortiz M.
      • Martinez L.
      • Kahan B.D.
      FTY 720 prevents ischemic reperfusion damage in rat kidneys.
      The main biological activity responsible for these actions is believed to be immunological, but our data suggest that nonimmunological role(s) of FTY720 are also important in the treatment of SCI.
      SCI induces a complex cascade of secondary injury that further promotes tissue destruction and results in clinical deterioration.
      • Donnelly D.J.
      • Popovich P.G.
      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      The cascade of secondary injury for SCI involves vascular disturbances, ischemia, propagation of free radicals, excitotoxicity, and inflammation, which leads to necrotic and apoptotic death of neurons and glia and subsequently to demyelination and axonal damage.
      • Donnelly D.J.
      • Popovich P.G.
      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      • Di Giovanni S.
      Molecular targets for axon regeneration: focus on the intrinsic pathways.
      A complex inflammatory response is believed to be a prominent component of secondary tissue damage, which increases functional loss.
      • Bareyre F.M.
      Neuronal repair and replacement in spinal cord injury.
      • Donnelly D.J.
      • Popovich P.G.
      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      • Alexander J.K.
      • Popovich P.G.
      Neuroinflammation in spinal cord injury: therapeutic targets for neuroprotection and regeneration.
      FTY720 reportedly attenuated cytokine production and inflammatory response in a mouse model of multiple sclerosis and cerebral infarction in previous studies.
      • Wei Y.
      • Yemisci M.
      • Kim H.H.
      • Yung L.M.
      • Shin H.K.
      • Hwang S.K.
      • Guo S.
      • Qin T.
      • Alsharif N.
      • Brinkmann V.
      • Liao J.K.
      • Lo E.H.
      • Waeber C.
      Fingolimod provides long-term protection in rodent models of cerebral ischemia.
      • Fujino M.
      • Funeshima N.
      • Kitazawa Y.
      • Kimura H.
      • Amemiya H.
      • Suzuki S.
      • Li X.K.
      Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment.
      However, we saw no decrease of any accumulation of neutrophils or microglia nor changes in the expression of inflammatory cytokines in the earlier phases after SCI. The discrepancy between previous reports is probably due to differences in disease models or animal species. Early inflammation in the spinal cord after traumatic SCI is mainly initiated by a transient increase in inflammatory cytokines and neutrophil accumulation within the lesion, which are rapidly cleared. On the other hand, lymphocyte entry after SCI is delayed in humans and mice.
      • Donnelly D.J.
      • Popovich P.G.
      Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.
      The modulation of late T-cell infiltration by FTY720 in mouse models of SCI would not explain the early therapeutic response to FTY720. More important, FTY720 maintained its efficacy, even in SCID mice, confirming the importance of immune-independent mechanisms of action of FTY720 in this mouse model of SCI.
      Because S1P receptors exist in many types of cells, including the CNS, and S1P plays a role in many cellular processes,
      • Herr D.R.
      • Chun J.
      Effects of LPA and S1P on the nervous system and implications for their involvement in disease.
      • Milstien S.
      • Gude D.
      • Spiegel S.
      Sphingosine 1-phosphate in neural signalling and function.
      it is not surprising that various aspects of the response to SCI have been targeted by FTY720. Among the secondary injury events other than inflammation, we observed that FTY720 decreased vascular permeability and astrocyte accumulation in the injured spinal cord. These changes were also observed in SCID mice, suggesting that they are not dependent on modulating lymphocyte function. In addition, S1P1 receptors are a likely target, because the administration of SEW2871 produced similar effects. Increased vascular permeability can lead to destruction of the blood-brain barrier in the spinal cord and causes local edema that adversely affects functional outcomes after SCI.
      • Sharma H.S.
      A select combination of neurotrophins enhances neuroprotection and functional recovery following spinal cord injury.
      Furthermore, astrocyte accumulation is the main cellular component of the glial scar after CNS injury.
      • Sofroniew M.V.
      Molecular dissection of reactive astrogliosis and glial scar formation.
      Although the formation of the glial scar has had both beneficial and detrimental effects,
      • Okada S.
      • Nakamura M.
      • Katoh H.
      • Miyao T.
      • Shimazaki T.
      • Ishii K.
      • Yamane J.
      • Yoshimura A.
      • Iwamoto Y.
      • Toyama Y.
      • Okano H.
      Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury.
      the proliferation of astrocytes modifies the extracellular matrix by secretion of laminin, fibronectin, and proteoglycans, which inhibit the regeneration process after SCI.
      • Blackburn D.
      • Sargsyan S.
      • Monk P.N.
      • Shaw P.J.
      Astrocyte function and role in motor neuron disease: a future therapeutic target?.
      • Muller H.W.
      • Junghans U.
      • Kappler J.
      Astroglial neurotrophic and neurite-promoting factors.
      FTY720 might counteract these secondary injuries and, thereby, prevent their pathological consequences.
      The issue remaining unresolved is whether FTY720 behaves as an agonist or antagonist for S1P receptors in the treatment of SCI. Several reports
      • Lee M.J.
      • Thangada S.
      • Claffey K.P.
      • Ancellin N.
      • Liu C.H.
      • Kluk M.
      • Volpi M.
      • Sha'afi R.I.
      • Hla T.
      Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.
      • Garcia J.G.
      • Liu F.
      • Verin A.D.
      • Birukova A.
      • Dechert M.A.
      • Gerthoffer W.T.
      • Bamberg J.R.
      • English D.
      Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.
      implicate the importance of the S1P1 receptor in endothelial function, including endothelial barrier function, migration, cell survival, and angiogenesis. It is possible that the modification of vascular permeability in the injured spinal cord may result from S1P1 stimulation by phosphorylated FTY720, as is the case with allergen-induced plasma leakage.
      • Ble F.X.
      • Cannet C.
      • Zurbruegg S.
      • Gerard C.
      • Frossard N.
      • Beckmann N.
      • Trifilieff A.
      Activation of the lung S1P(1) receptor reduces allergen-induced plasma leakage in mice.
      On the other hand, the reduction of vascular leakage in this study may be dependent on unknown compensatory mechanisms, because FTY720 also inhibits vascular permeability independently of S1P1.
      • Dudek S.M.
      • Camp S.M.
      • Chiang E.T.
      • Singleton P.A.
      • Usatyuk P.V.
      • Zhao Y.
      • Natarajan V.
      • Garcia J.G.
      Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor.
      It is also possible that phosphorylated FTY720 acts as a functional antagonist for the S1P1 receptor to inhibit astrocyte accumulation in the injured spinal cord. It was reported that S1P receptors bound to FTY720 are irreversibly internalized and degraded.
      • Oo M.L.
      • Chang S.H.
      • Thangada S.
      • Wu M.T.
      • Rezaul K.
      • Blaho V.
      • Hwang S.I.
      • Han D.K.
      • Hla T.
      Engagement of S1P-degradative mechanisms leads to vascular leak in mice.
      • Sensken S.C.
      • Bode C.
      • Graler M.H.
      Accumulation of fingolimod (FTY720) in lymphoid tissues contributes to prolonged efficacy.
      Recently, in a mouse model of autoimmune encephalomyelitis, phosphorylated FTY720 induced the loss of the S1P1 receptor on astrocytes through functional antagonism, and the functional loss of S1P1 receptors from the astrocyte cell surface is a primary locus for FTY720 efficacy.
      • Choi J.W.
      • Gardell S.E.
      • Herr D.R.
      • Rivera R.
      • Lee C.W.
      • Noguchi K.
      • Teo S.T.
      • Yung Y.C.
      • Lu M.
      • Kennedy G.
      • Chun J.
      FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation.
      Although specific S1P1 receptor modulation by SEW2871 reproduced some treatment effects, including reducing vascular permeability and astrocyte accumulation, treatment with SEW2871 led to weaker improvement in locomotor function compared with FTY720, suggesting a requirement for the additional activity of other S1P receptors in obtaining full efficacy with FTY720 in SCI. Additional studies will be required using mice genetically engineered to perturb other S1P receptors.
      Our present study focused mainly on secondary injury after SCI, but direct beneficial effects of FTY720 on other neurological cells, including neurons and oligodendrocytes, cannot be excluded. S1P receptors are also expressed on many CNS cell types and have influenced CNS cell proliferation, morphological characteristics, and migration.
      • Brinkmann V.
      Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology.
      • Herr D.R.
      • Chun J.
      Effects of LPA and S1P on the nervous system and implications for their involvement in disease.
      • Milstien S.
      • Gude D.
      • Spiegel S.
      Sphingosine 1-phosphate in neural signalling and function.
      FTY720 easily crosses the blood-brain barrier, resulting in high concentrations of free phosphorylated FTY720 in the spinal fluid.
      • Foster C.A.
      • Howard L.M.
      • Schweitzer A.
      • Persohn E.
      • Hiestand P.C.
      • Balatoni B.
      • Reuschel R.
      • Beerli C.
      • Schwartz M.
      • Billich A.
      Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis.
      FTY720 rescues oligodendrocyte progenitors from death induced by growth factor depletion and exposure to activated microglia
      • Coelho R.P.
      • Payne S.G.
      • Bittman R.
      • Spiegel S.
      • Sato-Bigbee C.
      The immunomodulator FTY720 has a direct cytoprotective effect in oligodendrocyte progenitors.
      and directly enhances remyelination through S1P3 and S1P5 receptors.
      • Miron V.E.
      • Ludwin S.K.
      • Darlington P.J.
      • Jarjour A.A.
      • Soliven B.
      • Kennedy T.E.
      • Antel J.P.
      Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar slices.
      Furthermore, S1P acts as a chemoattractant for neural progenitor cells toward CNS injury,
      • Kimura A.
      • Ohmori T.
      • Ohkawa R.
      • Madoiwa S.
      • Mimuro J.
      • Murakami T.
      • Kobayashi E.
      • Hoshino Y.
      • Yatomi Y.
      • Sakata Y.
      Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury.
      • Kimura A.
      • Ohmori T.
      • Kashiwakura Y.
      • Ohkawa R.
      • Madoiwa S.
      • Mimuro J.
      • Shimazaki K.
      • Hoshino Y.
      • Yatomi Y.
      • Sakata Y.
      Antagonism of sphingosine 1-phosphate receptor-2 enhances migration of neural progenitor cells toward an area of brain.
      suggesting that neurogenesis could be directly modified by FTY720 treatment.
      Some limitations in this study merit discussion. Our data suggest that FTY720 has pleiotropic actions, in addition to modulating early inflammation and lymphocyte sequestration, to improve neuronal outcomes after SCI. However, we could not completely exclude the importance of the immunosuppressive actions of FTY720 and the role of marginal immune responses in SCID mice to improve functional outcomes. In addition, we could not show evidence that these pleiotropic actions modulate vascular permeability and astrocyte migration to directly improve motor function after SCI.
      In summary, our data suggest that targeting S1P receptors with FTY720 is an attractive therapeutic approach for SCI. FTY720 is an oral drug that has shown efficacy in clinical trials for human multiple sclerosis
      • Kappos L.
      • Radue E.W.
      • O'Connor P.
      • Polman C.
      • Hohlfeld R.
      • Calabresi P.
      • Selmaj K.
      • Agoropoulou C.
      • Leyk M.
      • Zhang-Auberson L.
      • Burtin P.
      A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis.
      • Cohen J.A.
      • Barkhof F.
      • Comi G.
      • Hartung H.P.
      • Khatri B.O.
      • Montalban X.
      • Pelletier J.
      • Capra R.
      • Gallo P.
      • Izquierdo G.
      • Tiel-Wilck K.
      • de Vera A.
      • Jin J.
      • Stites T.
      • Wu S.
      • Aradhye S.
      • Kappos L.
      Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis.
      ; it was recently approved by the Food and Drug Administration. The safety profile of FTY720 in the treatment of multiple sclerosis encourages us to apply it in a clinical trial for SCI. However, further evaluation using larger animals, such as nonhuman primates, will be necessary to confirm its efficacy in treating SCI. Furthermore, strategies targeted at modulating the S1P concentration in the injured CNS may lead to new therapeutic approaches toward repairing various CNS disorders.

      Acknowledgments

      We thank Dr. Volker Brinkmann (Novartis Pharma AG) for the gift of the FTY720-related compounds and helpful discussion and Naoko Matsumoto and Masanori Ito for their excellent technical assistance.

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