FTY720 Improves Functional Recovery after Spinal Cord Injury by Primarily Nonimmunomodulatory Mechanisms
2012; Elsevier BV; Volume: 180; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.12.012
ISSN1525-2191
AutoresYusuke Norimatsu, Tsukasa Ohmori, Atsushi Kimura, Seiji Madoiwa, Jun Mimuro, Atsushi Seichi, Yutaka Yatomi, Yuichi Hoshino, Yoichi Sakata,
Tópico(s)Spinal Cord Injury Research
ResumoSpinal 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 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.1Baptiste D.C. Fehlings M.G. Update on the treatment of spinal cord injury.Prog Brain Res. 2007; 161: 217-233Crossref PubMed Scopus (145) Google Scholar SCI primarily arises from mechanical disruption of the spinal cord, leading to rapid death of neurons and glia.2Bareyre F.M. Neuronal repair and replacement in spinal cord injury.J Neurol Sci. 2008; 265: 63-72Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Donnelly D.J. Popovich P.G. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.Exp Neurol. 2008; 209: 378-388Crossref PubMed Scopus (745) Google Scholar 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).2Bareyre F.M. Neuronal repair and replacement in spinal cord injury.J Neurol Sci. 2008; 265: 63-72Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Donnelly D.J. Popovich P.G. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.Exp Neurol. 2008; 209: 378-388Crossref PubMed Scopus (745) Google Scholar 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.4Di Giovanni S. Molecular targets for axon regeneration: focus on the intrinsic pathways.Expert Opin Ther Targets. 2009; 13: 1387-1398Crossref PubMed Scopus (51) Google Scholar, 5Ruff R.L. McKerracher L. Selzer M.E. Repair and neurorehabilitation strategies for spinal cord injury.Ann N Y Acad Sci. 2008; 1142: 1-20Crossref PubMed Scopus (38) Google Scholar 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.6Sanchez T. Hla T. Structural and functional characteristics of S1P receptors.J Cell Biochem. 2004; 92: 913-922Crossref PubMed Scopus (410) Google Scholar, 7Rivera R. Chun J. Biological effects of lysophospholipids.Rev Physiol Biochem Pharmacol. 2008; 160: 25-46PubMed Google Scholar, 8Yatomi Y. Ozaki Y. Ohmori T. Igarashi Y. Sphingosine 1-phosphate: synthesis and release.Prostaglandins Other Lipid Mediat. 2001; 64: 107-122Crossref PubMed Scopus (166) Google Scholar 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.9Kimura 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.Stem Cells. 2007; 25: 115-124Crossref PubMed Scopus (161) Google Scholar Furthermore, in areas of brain infarction, S1P2 receptor antagonism also enhances the migration of endogenous neural stem/progenitor cells.10Kimura 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.Stroke. 2008; 39: 3411-3417Crossref PubMed Scopus (79) Google Scholar 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,11Brinkmann V. Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology.Pharmacol Ther. 2007; 115: 84-105Crossref PubMed Scopus (467) Google Scholar, 12Herr D.R. Chun J. Effects of LPA and S1P on the nervous system and implications for their involvement in disease.Curr Drug Targets. 2007; 8: 155-167Crossref PubMed Scopus (86) Google Scholar, 13Milstien S. Gude D. Spiegel S. Sphingosine 1-phosphate in neural signalling and function.Acta Paediatr Suppl. 2007; 96: 40-43Crossref PubMed Scopus (32) Google Scholar, 14Singh I.N. Hall E.D. Multifaceted roles of sphingosine-1-phosphate: how does this bioactive sphingolipid fit with acute neurological injury?.J Neurosci Res. 2008; 86: 1419-1433Crossref PubMed Scopus (27) Google Scholar 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.15Brinkmann V. FTY720 (fingolimod) in multiple sclerosis: therapeutic effects in the immune and the central nervous system.Br J Pharmacol. 2009; 158: 1173-1182Crossref PubMed Scopus (381) Google Scholar, 16Brinkmann V. Cyster J.G. Hla T. FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function.Am J Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (422) Google Scholar It acts as a broad S1P receptor agonist after phosphorylation by sphingosine kinase in vivo16Brinkmann V. Cyster J.G. Hla T. FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function.Am J Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (422) Google Scholar and has recently had clinical efficacy in phase 3 clinical trials for multiple sclerosis.17Kappos 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.N Engl J Med. 2010; 362: 387-401Crossref PubMed Scopus (2144) Google Scholar, 18Cohen 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.N Engl J Med. 2010; 362: 402-415Crossref PubMed Scopus (1863) Google Scholar Also, FTY720 protects a rodent model of ischemia-reperfusion after cerebral ischemia19Wei 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.Ann Neurol. 2011; 69: 119-129Crossref PubMed Scopus (228) Google Scholar, 20Hasegawa 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.Stroke. 2010; 41: 368-374Crossref PubMed Scopus (204) Google Scholar and improves functional outcomes in a rat model of SCI.21Lee 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.J Neurotrauma. 2009; 26: 2335-2344Crossref PubMed Scopus (58) Google Scholar The efficacy of FTY720 in CNS disorders is believed to derive from immunological modulation.19Wei 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.Ann Neurol. 2011; 69: 119-129Crossref PubMed Scopus (228) Google Scholar, 20Hasegawa 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.Stroke. 2010; 41: 368-374Crossref PubMed Scopus (204) Google Scholar, 21Lee 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.J Neurotrauma. 2009; 26: 2335-2344Crossref PubMed Scopus (58) Google Scholar 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. 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. Contusion SCI was induced using an Infinite Horizon Impactor (Precision Systems and Instrumentation, Lexington, KY).9Kimura 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.Stem Cells. 2007; 25: 115-124Crossref PubMed Scopus (161) Google Scholar 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.9Kimura 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.Stem Cells. 2007; 25: 115-124Crossref PubMed Scopus (161) Google Scholar FTY720 was kindly provided by Novartis Pharma AG (Basel, Switzerland) and dissolved in distilled water. SEW2871, a specific agonist for the S1P1 receptor,22Sanna 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.J Biol Chem. 2004; 279: 13839-13848Crossref PubMed Scopus (540) Google Scholar 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. 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).23Basso 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.J Neurotrauma. 2006; 23: 635-659Crossref PubMed Scopus (1055) Google Scholar 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. 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). 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.9Kimura 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.Stem Cells. 2007; 25: 115-124Crossref PubMed Scopus (161) Google Scholar 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). 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.24Marcondes 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.Am J Pathol. 2005; 166: 1749-1760Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar 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.25Choudhri 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.Stroke. 1997; 28: 2296-2302Crossref PubMed Scopus (175) Google Scholar 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. 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. 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,26Rabchevsky 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.J Neurotrauma. 2001; 18: 513-522Crossref PubMed Scopus (130) Google Scholar 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). 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. 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).Table 1FTY720 Promotes Functional Locomotor Recovery after SCI in MiceTime after SCI (days)BMS scoreP value13710142128FTY720 (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 ± 0.7990.8895Rotarod performance test score (seconds)03710142128FTY720 (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 ± 31.060.7884Daily 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. P < 0.05; P < 0.01 compared with the control experiment (post hoc Bonferroni test). Open table in a new tab 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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.27Hagg T. Oudega M. Degenerative and spontaneous regenerative processes after spinal cord injury.J Neurotrauma. 2006; 23: 264-280Crossref PubMed Scopus (43) Google Scholar, 28Beattie M.S. Inflammation and apoptosis: linked therapeutic targets in spinal cord injury.Trends Mol Med. 2004; 10: 580-583Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar 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 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). Because sphingolipids may affect thrombin generation in vitro,29Deguchi H. Yegneswaran S. Griffin J.H. Sphingolipids as bioactive regulators of thrombin generation.J Biol Chem. 2004; 279: 12036-12042Crossref PubMed Scopus (45) Google Scholar 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
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