Matrix Metalloproteinase-3 Promotes Early Blood–Spinal Cord Barrier Disruption and Hemorrhage and Impairs Long-Term Neurological Recovery after Spinal Cord Injury
2014; Elsevier BV; Volume: 184; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2014.07.016
ISSN1525-2191
AutoresJee Youn Lee, Hae Young Choi, Hyun‐Jong Ahn, Bong Gun Ju, Tae Young Yune,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoAfter spinal cord injury (SCI), blood–spinal cord barrier (BSCB) disruption by matrix metalloproteinases (MMPs) leads to BSCB permeability and blood cell infiltration, contributing to permanent neurological disability. Herein, we report that MMP-3 plays a critical role in BSCB disruption after SCI in mice. MMP-3 was induced in infiltrated neutrophils and blood vessels after SCI, and NF-κB as a transcription factor was involved in MMP-3 expression. BSCB permeability and blood cell infiltration after injury were more reduced in Mmp3 knockout (KO) mice than in wild-type (WT) mice, which was significantly inhibited by Mmp3 siRNA or a general inhibitor of MMPs, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. The level of tight junction proteins, such as occludin and zonula occludens-1, which decreased after SCI, was also higher in Mmp3 KO than in WT mice. Exogenously, MMP-3 injection into the normal spinal cord also induced BSCB permeability. Furthermore, MMP-9 activation after injury was mediated by MMP-3 activation. Finally, improved functional recovery was observed in Mmp3 KO mice compared with WT mice after injury. These results demonstrated the role of MMP-3 in BSCB disruption after SCI for the first time and suggest that the regulation of MMP-3 can be considered a therapeutic target to inhibit BSCB disruption and hemorrhage, and thereby enhance functional recovery after acute SCI. After spinal cord injury (SCI), blood–spinal cord barrier (BSCB) disruption by matrix metalloproteinases (MMPs) leads to BSCB permeability and blood cell infiltration, contributing to permanent neurological disability. Herein, we report that MMP-3 plays a critical role in BSCB disruption after SCI in mice. MMP-3 was induced in infiltrated neutrophils and blood vessels after SCI, and NF-κB as a transcription factor was involved in MMP-3 expression. BSCB permeability and blood cell infiltration after injury were more reduced in Mmp3 knockout (KO) mice than in wild-type (WT) mice, which was significantly inhibited by Mmp3 siRNA or a general inhibitor of MMPs, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. The level of tight junction proteins, such as occludin and zonula occludens-1, which decreased after SCI, was also higher in Mmp3 KO than in WT mice. Exogenously, MMP-3 injection into the normal spinal cord also induced BSCB permeability. Furthermore, MMP-9 activation after injury was mediated by MMP-3 activation. Finally, improved functional recovery was observed in Mmp3 KO mice compared with WT mice after injury. These results demonstrated the role of MMP-3 in BSCB disruption after SCI for the first time and suggest that the regulation of MMP-3 can be considered a therapeutic target to inhibit BSCB disruption and hemorrhage, and thereby enhance functional recovery after acute SCI. CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Traumatic spinal cord injury (SCI) is a devastating condition that results in permanent disability. Currently, treatment options are limited, but significant advances have been made in understanding the pathophysiological features of SCI. Initial mechanical injury, followed by secondary injury, is known to contribute to pathophysiological features, leading to cell death and functional disability after SCI.1Yu F. Kamada H. Niizuma K. Endo H. Chan P.H. Induction of mmp-9 expression and endothelial injury by oxidative stress after spinal cord injury.J Neurotrauma. 2008; 25: 184-195Crossref PubMed Scopus (103) Google Scholar The blood–spinal cord barrier (BSCB) is the functional equivalent of the blood-brain barrier (BBB), providing a specialized microenvironment for the cellular constituents of the spinal cord. The barrier function of BSCB is based on the specialized system of nonfenestrated endothelial cells and their accessory structures, including basement membrane, pericytes, and astrocytic end feet processes, which provide its regulatory and protective functions.2Bartanusz V. Jezova D. Alajajian B. Digicaylioglu M. The blood-spinal cord barrier: morphology and clinical implications.Ann Neurol. 2011; 70: 194-206Crossref PubMed Scopus (264) Google Scholar When BSCB is damaged by injury, blood cells cross into injured parenchyma and contribute to secondary injuries, such as inflammation.3Hawkins B.T. Davis T.P. The blood-brain barrier/neurovascular unit in health and disease.Pharmacol Rev. 2005; 57: 173-185Crossref PubMed Scopus (1977) Google Scholar, 4Zlokovic B.V. The blood-brain barrier in health and chronic neurodegenerative disorders.Neuron. 2008; 57: 178-201Abstract Full Text Full Text PDF PubMed Scopus (2322) Google Scholar, 5Abbott N.J. Ronnback L. Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier.Nat Rev Neurosci. 2006; 7: 41-53Crossref PubMed Scopus (3760) Google Scholar These secondary damages induce apoptosis of neurons and glia, leading to permanent neurological deficits.6Noble L.J. Donovan F. Igarashi T. Goussev S. Werb Z. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events.J Neurosci. 2002; 22: 7526-7535PubMed Google Scholar Matrix metalloproteinases (MMPs) are known to degrade extracellular matrix and other extracellular proteins7Sternlicht M.D. Lochter A. Sympson C.J. Huey B. Rougier J.P. Gray J.W. Pinkel D. Bissell M.J. Werb Z. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis.Cell. 1999; 98: 137-146Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar and are essential for remodeling of extracellular matrix and wound healing.8Werb Z. ECM and cell surface proteolysis: regulating cellular ecology.Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar However, excessive proteolytic activity of MMPs can be detrimental, leading to numerous pathological conditions, including BBB/BSCB disruption after injury.6Noble L.J. Donovan F. Igarashi T. Goussev S. Werb Z. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events.J Neurosci. 2002; 22: 7526-7535PubMed Google Scholar, 9Rosenberg G.A. Estrada E.Y. Dencoff J.E. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain.Stroke. 1998; 29: 2189-2195Crossref PubMed Scopus (739) Google Scholar, 10Liu W. Hendren J. Qin X.J. Shen J. Liu K.J. Normobaric hyperoxia attenuates early blood-brain barrier disruption by inhibiting MMP-9-mediated occludin degradation in focal cerebral ischemia.J Neurochem. 2009; 108: 811-820Crossref PubMed Scopus (162) Google Scholar, 11Gurney K.J. Estrada E.Y. Rosenberg G.A. Blood-brain barrier disruption by stromelysin-1 facilitates neutrophil infiltration in neuroinflammation.Neurobiol Dis. 2006; 23: 87-96Crossref PubMed Scopus (201) Google Scholar, 12Mun-Bryce S. Rosenberg G.A. Gelatinase B modulates selective opening of the blood-brain barrier during inflammation.Am J Physiol. 1998; 274: R1203-R1211PubMed Google Scholar For example, MMP-9 plays a key role in abnormal vascular permeability after SCI, and blocking of MMP-9 improves functional recovery.6Noble L.J. Donovan F. Igarashi T. Goussev S. Werb Z. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events.J Neurosci. 2002; 22: 7526-7535PubMed Google Scholar After SCI, MMP-12 up-regulation increases BSCB permeability, followed by blood cell infiltration, thereby hindering recovery of motor function.13Wells J.E. Rice T.K. Nuttall R.K. Edwards D.R. Zekki H. Rivest S. Yong V.W. An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice.J Neurosci. 2003; 23: 10107-10115Crossref PubMed Google Scholar After brain focal ischemia, the degradation of tight junction (TJ) proteins is also blocked by inhibiting MMP-2 and MMP-9 activities.14Ishrat T. Sayeed I. Atif F. Hua F. Stein D.G. Progesterone and allopregnanolone attenuate blood-brain barrier dysfunction following permanent focal ischemia by regulating the expression of matrix metalloproteinases.Exp Neurol. 2010; 226: 183-190Crossref PubMed Scopus (126) Google Scholar Several studies suggest that MMP-3 is involved in BBB disruption after injury. For example, lipopolysaccharide (LPS)–induced BBB disruption is reduced in Mmp3 knockout (KO) mice when compared with wild-type (WT) mice.11Gurney K.J. Estrada E.Y. Rosenberg G.A. Blood-brain barrier disruption by stromelysin-1 facilitates neutrophil infiltration in neuroinflammation.Neurobiol Dis. 2006; 23: 87-96Crossref PubMed Scopus (201) Google Scholar In addition, cyclooxygenase-1 and cyclooxygenase-2 modulate LPS-induced BBB disruption through MMP-3 and MMP-9.15Aid S. Silva A.C. Candelario-Jalil E. Choi S.H. Rosenberg G.A. Bosetti F. Cyclooxygenase-1 and -2 differentially modulate lipopolysaccharide-induced blood-brain barrier disruption through matrix metalloproteinase activity.J Cereb Blood Flow Metab. 2010; 30: 370-380Crossref PubMed Scopus (55) Google Scholar Recently, BBB disruption in a MPTP mouse model of Parkinson's disease is suppressed by MMP-3 deletion.16Chung Y.C. Kim Y.S. Bok E. Yune T.Y. Maeng S. Jin B.K. MMP-3 contributes to nigrostriatal dopaminergic neuronal loss, BBB damage, and neuroinflammation in an MPTP mouse model of Parkinson's disease.Mediators Inflamm. 2013; 2013: 370526Crossref PubMed Scopus (65) Google Scholar However, no direct evidence for the role of MMP-3 in the BBB/BSCB disruption after accidental trauma, such as SCI and/or ischemic stroke, has been presented yet. Thus, we examined the precise role of MMP-3 in BSCB disruption after SCI through four different approaches: i) Mmp3 KO mice, ii) Mmp3 siRNA, iii) a general inhibitor of MMPs, and iv) exogenous MMP-3 injection. Finally, we determined the effect of MMP-3 on functional recovery and histological outcome after SCI by comparing Mmp3 KO mice with WT control. We used adult (13- to 16-week-old, 28 to 30 g) male WT (n = 171), Mmp3 KO (n = 60), and IkkβΔmye (n = 21) mice in this study. Mmp3 KO mice were generously donated by Dr. Tong Hyup Joh (Cornell University, Ithaca, NY). Myeloid cell type–specific IκB kinase (Ikk)-β–deficient (IkkβΔmye) mice were donated by Dr. Sung Joong Lee (Seoul University, Seoul, Republic of Korea). Because the genetic background of these mice has been maintained in C57BL/6, the same strain from Samtako (Osan, Republic of Korea) was used as WT. Adult male mice (18 to 22 g; Samtako) were anesthetized with 4% chloral hydrate, and a laminectomy was performed at the T9 to T10 level, exposing the cord beneath without disrupting the dura. The exposed dorsal surface of the cord then was subjected to moderate contusion injury (50 kilodyne force per 500- to 600-μm displacement) using the Infinite Horizons impactor (Infinite Horizons Inc., Lexington, KY). For sham–operated on controls, animals underwent a T9 to T10 laminectomy without contusion injury. Surgical interventions and postoperative animal care were performed in accordance with the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care Committee of the Kyung Hee University (Seoul, Republic of Korea). Mouse Mmp3 siRNA and control nontargeting siRNA (control siRNA) were purchased from Thermo Fisher Scientific (Lafayette, CO). Each targeted siRNA was a mixture of four siRNAs formulated to enhance the effectiveness of the mixture in knocking down the target gene. The control siRNA was a mixture of siRNAs with scrambled sequences, confirmed by microarray to have minimal targeting of known genes in human, mouse, and rat cells. siRNAs were dissolved in distilled water (pH 7.4) and injected bilaterally (2 μL per site; final, 0.01 or 0.05 nmol per cord) into the spinal cord at 30 minutes after SCI, as previously reported.17Li A.J. Wang Q. Dinh T.T. Ritter S. Simultaneous silencing of Npy and Dbh expression in hindbrain A1/C1 catecholamine cells suppresses glucoprivic feeding.J Neurosci. 2009; 29: 280-287Crossref PubMed Scopus (44) Google Scholar An active MMP-3 recombinant enzyme (Calbiochem, La Jolla, CA) was dissolved in phosphate-buffered saline (PBS) and then injected bilaterally (5 μL per site; final, 20 or 200 pmol per cord) in normal spinal cord. Bilateral intraspinal injection was performed at the T9 to T10 level, according to the following stereotaxic coordinate: lateral ± 0.3 mm, 0.5-mm depth. Control groups received injections of equal volumes of control siRNA or PBS. A general inhibitor of MMPs, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH; Enzo Life Sciences International, Plymouth Meeting, PA) was dissolved in 10% dimethyl sulfoxide (5 μL of 5 mmol/L NNGH) and administrated by intraspinal injection (lesion center, 0.5-mm depth) using glass pipette at 30 minutes after SCI, as previously described.18Leong D.J. Gu X.I. Li Y. Lee J.Y. Laudier D.M. Majeska R.J. Schaffler M.B. Cardoso L. Sun H.B. Matrix metalloproteinase-3 in articular cartilage is upregulated by joint immobilization and suppressed by passive joint motion.Matrix Biol. 2010; 29: 420-426Crossref PubMed Scopus (60) Google Scholar Intraspinal injection into the spinal cord was performed using a pulled glass capillary pipette (30-μm external tip diameter), as previously described.19Yune T.Y. Park H.G. Lee J.Y. Oh T.H. Estrogen-induced Bcl-2 expression after spinal cord injury is mediated through phosphoinositide-3-kinase/Akt-dependent CREB activation.J Neurotrauma. 2008; 25: 1121-1131Crossref PubMed Scopus (75) Google Scholar Tissue preparation was performed as previously described.20Lee J.Y. Kim H.S. Choi H.Y. Oh T.H. Yune T.Y. Fluoxetine inhibits matrix metalloprotease activation and prevents disruption of blood-spinal cord barrier after spinal cord injury.Brain. 2012; 135: 2375-2389Crossref PubMed Scopus (122) Google Scholar At specific time points after SCI, animals were anesthetized with chloral hydrate (500 mg/kg) and perfused via cardiac puncture initially with 0.1 mol/L PBS, pH 7.4, and subsequently with 4% paraformaldehyde in 0.1 mol/L PBS, pH 7.4. A 20-mm section of the spinal cord, centered at the lesion site, was dissected out, post-fixed by immersion in the same fixative for 4 hours, and placed in 30% sucrose in 0.1 mol/L PBS, pH 7.4. The segment was embedded in optimal cutting temperature compound for frozen sections, and longitudinal or transverse sections were then cut at 10 or 20 μm. For molecular and biochemical works, animals were perfused with 0.1 mol/L PBS, and segments of spinal cord (8 mm), including the lesion site, were isolated and frozen at −80°C. Frozen sections were processed for immunohistochemistry (IHC) with antibodies against MMP-3 (Santa Cruz Biotechnology, Santa Cruz, CA), ED-1 (Serotec, Raleigh, NC), CD11b (Serotec), NeuN (Millipore, Billerica, MA), CC1 (Oncogene, Cambridge, MA), glial fibrillary acidic protein (GFAP; Millipore), myeloperoxidase (MPO; Neomarkers, Fremont, CA), CD31 (BD Biosciences, San Jose, CA), NF-κB (Santa Cruz Biotechnology), occludin (Invitrogen, Carlsbad, CA), claudin-5 (Abcam, Cambridge, MA), zonula occludens (ZO-1; Invitrogen), and neurofilament 200 kDa (NF200; Sigma, St. Louis, MO), as previously described.21Yune T.Y. Lee J.Y. Jung G.Y. Kim S.J. Jiang M.H. Kim Y.C. Oh Y.J. Markelonis G.J. Oh T.H. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury.J Neurosci. 2007; 27: 7751-7761Crossref PubMed Scopus (220) Google Scholar The sections were incubated with primary antibodies, followed by biotin-conjugated secondary antibodies (Dako, Carpinteria, CA). The avidin-biotin complex method was used to detect labeled cells using a Vectastain kit (Vector Labs, Burlingame, CA). Diaminobenzidene served as the substrate for peroxidase. For double labeling, fluorescein isothiocyanate (FITC) or cy3-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used. Also, nuclei were labeled with DAPI, according to the manufacturer's protocol (Molecular Probes, Eugene, OR). In all controls, reaction to the substrate was absent if the primary antibody was omitted or if the primary antibody was replaced by a nonimmune, control antibody. Serial sections were also stained for histological analysis with Cresyl violet acetate. Total RNA was isolated from spinal cord segments (8 mm), centered at the lesion site by using TRIzol reagent (Invitrogen), according to the manufacturer's instruction. cDNA was synthesized from 5 μg of the total RNA using MMLV-RT (Invitrogen), and real-time PCR was performed using SYBR Green PCR master mix (Invitrogen), as previously described.22Hong J. Cho I.H. Kwak K.I. Suh E.C. Seo J. Min H.J. Choi S.Y. Kim C.H. Park S.H. Jo E.K. Lee S. Lee K.E. Lee S.J. Microglial Toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death.J Biol Chem. 2010; 285: 39447-39457Crossref PubMed Scopus (51) Google Scholar The primers used for real-time PCR were synthesized by Genotech (Daejeon, Republic of Korea), and the sequences of the primers are as follows (5′ to 3′): MMP-3, 5′-GGCTTCAGTACCTTCCCAGG-3′ (forward) and 5′-GCAGCAACCAGGAATAGGTT-3′ (reverse); MMP-12, 5′-GAAACCCCCATCCTTGACAA-3′ (forward) and 5′-TTCCACCAGAAGAACCAGTCTTTAA-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase, 5′-AACTTTGGCATTGTGGAAGG-3′ (forward) and 5′-GGAGACAACCAGGTCCTCAG-3′ (reverse). Spinal cord tissues were processed for Western blot analysis, as described previously.21Yune T.Y. Lee J.Y. Jung G.Y. Kim S.J. Jiang M.H. Kim Y.C. Oh Y.J. Markelonis G.J. Oh T.H. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury.J Neurosci. 2007; 27: 7751-7761Crossref PubMed Scopus (220) Google Scholar Antibodies used were polyclonal antibodies against MMP-3 (Santa Cruz Biotechnology), ZO-1 (Invitrogen), occludin (Invitrogen), claudin-5 (Invitrogen), and β-tubulin (Sigma). The primary antibodies were detected with a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch). Immunoreactive bands were visualized by chemiluminescence using Supersignal (Pierce, Rockford, IL). β-Tubulin was used as an internal control. The gels shown in figures are representative of results from three separate experiments. The activities of MMP-2 and MMP-9 in the injured spinal cord were examined 1 day after injury by gelatin zymography, as described.20Lee J.Y. Kim H.S. Choi H.Y. Oh T.H. Yune T.Y. Fluoxetine inhibits matrix metalloprotease activation and prevents disruption of blood-spinal cord barrier after spinal cord injury.Brain. 2012; 135: 2375-2389Crossref PubMed Scopus (122) Google Scholar For some experiments, spinal cord total lysates (50 μg, at 1 day after SCI) were incubated with or without recombinant active MMP-3 (final concentration, 100 ng/mL; Calbiochem) and NNGH (final concentration, 60 μmol/L; Enzo Life Sciences International) for 2 hours at 37°C; then, gelatin zymography was performed. The permeability of BSCB was investigated with Evans Blue Dye extravasation, as described.20Lee J.Y. Kim H.S. Choi H.Y. Oh T.H. Yune T.Y. Fluoxetine inhibits matrix metalloprotease activation and prevents disruption of blood-spinal cord barrier after spinal cord injury.Brain. 2012; 135: 2375-2389Crossref PubMed Scopus (122) Google Scholar, 23Gibson J.G. Evans W.A. Clinical studies of the blood volume, I: clinical application of a method employing the azo dye “Evans blue” and the spectrophotometer.J Clin Invest. 1937; 16: 301-316Crossref PubMed Google Scholar MMP-3 enzymatic activity was measured in total lysates (100 μg) from spinal cord using a 5-carboxyfluorescein (5-FAM)/QXL520 fluorescence resonance energy transfer peptide (AnaSpec Inc., San Jose, CA), as previously described.15Aid S. Silva A.C. Candelario-Jalil E. Choi S.H. Rosenberg G.A. Bosetti F. Cyclooxygenase-1 and -2 differentially modulate lipopolysaccharide-induced blood-brain barrier disruption through matrix metalloproteinase activity.J Cereb Blood Flow Metab. 2010; 30: 370-380Crossref PubMed Scopus (55) Google Scholar Neutrophil isolation was performed, as previously described, with some modifications.24Kang J. Jiang M.H. Min H.J. Jo E.K. Lee S. Karin M. Yune T.Y. Lee S.J. IKK-beta-mediated myeloid cell activation exacerbates inflammation and inhibits recovery after spinal cord injury.Eur J Immunol. 2011; 41: 1266-1277Crossref PubMed Scopus (22) Google Scholar, 25Yuan Y. Fleming B.P. A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies.Microvasc Res. 1990; 40: 218-229Crossref PubMed Scopus (71) Google Scholar In brief, blood was collected from mice by cardiac puncture into a 15-mL conical tube containing acid citrate dextrose solution. Immediately after collection, whole blood was lysed in 10 mL of red blood cell (RBC) lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L EDTA) for 20 minutes on ice to remove red blood cells. The RBC-lysed blood solution was passed through a 100-μm Nylon mesh (BD Biosciences), and then subjected to centrifugation for 5 minutes at 400 × g (Hanil, Seoul, Republic of Korea). The cell pellet was lysed further in 10 mL of RBC lysis buffer for 10 minutes on ice and resuspended in 30 mL of 0.1 mol/L PBS. The sample was centrifuged for 5 minutes at 400 × g and layered onto the discontinuous Percoll gradient solution (3 mL of 80%, 3 mL of 60%, and 3 mL of 50%). Neutrophils were recovered from 60%/80% Percoll interface and after high centrifugation (2500 × g, for 30 minutes at 4°C) (Sorvall RC-5 C Plus; Thermo Fisher Scientific, Lafayette, CO). Cells were washed twice with 0.1 mol/L PBS and incubated in Dulbecco’s modified Eagle’s medium containing 5% bovine serum. Isolated neutrophils were stimulated by LPS (50 ng/mL) for 3 hours. For control, 0.9% NaCl solution (saline) was treated. Flow cytometry was performed, as previously described, with minor modifications.20Lee J.Y. Kim H.S. Choi H.Y. Oh T.H. Yune T.Y. Fluoxetine inhibits matrix metalloprotease activation and prevents disruption of blood-spinal cord barrier after spinal cord injury.Brain. 2012; 135: 2375-2389Crossref PubMed Scopus (122) Google Scholar, 26Stirling D.P. Yong V.W. Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry.J Neurosci Res. 2008; 86: 1944-1958Crossref PubMed Scopus (135) Google Scholar Spinal cords (one spinal cord per sample) were mechanically disrupted with a small glass Dounce homogenizer, and cell suspensions were obtained by passing the solution through a wire mesh screen (Sigma). Samples were subjected to centrifugation at 4°C at 1100 rpm (200 × g) for 10 minutes (low brake). Pellets were resuspended in fetal bovine serum staining buffer (BD Biosciences) and were subjected to centrifugation at 3000 rpm (1300 × g) for 7 minutes, slow brake at 4°C. Pellets were then resuspended in fetal bovine serum staining buffer. Spinal cord samples were split into several tubes, and unstained cells and isotype-matched control samples were generated from 10 μL of each sample (mix of all spinal cord samples) to control for non-specific binding and autofluorescence. The following isotype control antibodies were used: phycoerythrin-labeled rat IgG2b,κ and FITC-labeled rat IgG2b,κ (Pharmingen, San Diego, CA). Cell counts were performed by adding 10 μL of trypan blue to 10 μL of each sample to optimize antibody dilutions. After blocking with Fc block (BD Biosciences) for 10 minutes at 4°C, we stained the cells by using antibodies directly conjugated with fluorochromes. The antibodies used in the current study were anti–Gr-1 FITC, anti-CD11b FITC, and anti-CD45 phycoerythrin (BD Biosciences). All samples were then immediately analyzed with a Becton Dickinson LSR Benchtop Flow Cytometer (BD Biosciences). Forward scatter was adjusted to minimize cellular debris, and propidium iodide exclusion was used to determine cell viability. A minimum of 250,000 cells from spinal cord samples were analyzed. The hemoglobin content of spinal cord tissue subjected to the experimental procedures below was quantified with a spectrophotometric assay, as described.27Simard J.M. Woo S.K. Norenberg M.D. Tosun C. Chen Z. Ivanova S. Tsymbalyuk O. Bryan J. Landsman D. Gerzanich V. Brief suppression of Abcc8 prevents autodestruction of spinal cord after trauma.Sci Transl Med. 2010; 2: 28ra29Crossref PubMed Scopus (60) Google Scholar, 28van Kampen E., Zijlstra W.G. Standardization of hemoglobinometry, II: the hemiglobincyanide method.Clin Chim Acta. 1961; 6: 538-544Crossref PubMed Scopus (913) Google Scholar In brief, mice were perfused with heparinized saline to remove intravascular blood, and 5-mm segments of cord, including the lesion site, were homogenized and sonicated on ice with a pulse ultrasonicator for 1 minute. After centrifugation at 13,000 × g for 30 minutes, the supernatant was collected and Drabkin's reagent (Sigma Diagnostics, Charleston, WV) was added and incubated for 15 minutes. This reaction converts hemoglobin to cyanomethemoglobin, which has an absorbance at 540 nm, and whose concentration can then be assessed by the OD of the solution at a 550-nm wavelength. To generate a standard absorbance curve, blood was obtained from control mice by cardiac puncture after anesthesia, and incremental aliquots of this blood were added to freshly homogenized spinal cord tissue obtained from WT mice; then, similar procedures were performed alongside every spinal cord tissue assay. The volume of extravascular blood was determined by comparison to the standard curve. Locomotor outcome after spinal cord contusion injury was assessed using the Basso Mouse Scale (BMS).29Basso 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 (1031) Google Scholar Mice were scored in an open-field environment by randomized investigators who were blind as to the experimental conditions (H.Y.C. and T.Y.Y.). Consensus scores for each animal were averaged at each time point for a maximum of 9 points for the BMS score and 11 points for the subscore, which assess finer aspects of locomotion. Lesion volume was assessed by using mice used for behavioral analysis. Serial longitudinal sections (10 μm thick) through the dorsoventral axis of the spinal cord were stained with Cresyl violet acetate, and the lesion area was determined by MetaMorph software version 7.1 (Molecular Devices, Sunnyvale, CA), as previously described.30Lee J.Y. Chung H. Yoo Y.S. Oh Y.J. Oh T.H. Park S. Yune T.Y. Inhibition of apoptotic cell death by ghrelin improves functional recovery after spinal cord injury.Endocrinology. 2010; 151: 3815-3826Crossref PubMed Scopus (75) Google Scholar To assess the spared myelin, serial transverse sections (20 μm thick) were stained with Luxol fast blue and quantitative analysis of spared WM area was performed, as previously described.31Donnelly D.J. Longbrake E.E. Shawler T.M. Kigerl K.A. Lai W. Tovar C.A. Ransohoff R.M. Popovich P.G. Deficient CX3CR1 signaling promotes recovery after mouse spinal cord injury by limiting the recruitment and activation of Ly6Clo/iNOS+ macrophages.J Neurosci. 2011; 31: 9910-9922Crossref PubMed Scopus (173) Google Scholar WT and Mmp3 KO mice were anesthetized at 28 days after injury, and frozen sections were prepared as described above. For quantitative analysis of axonal densities, serial transverse sections collected every millimeter rostral and caudal 3 mm to the lesion site were stained with NF200 antibody. Axonal densities were determined within preselected fields (40 × 40 μm, 1600 μm2) at specific sites within the vestibulospinal tract for NF200-positive axons, as previously described.21Yune T.Y. Lee J.Y. Jung G.Y. Kim S.J. Jiang M.H. Kim Y.C. Oh Y.J. Markelonis G.J. Oh T.H. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury.J Neurosci. 2007; 27: 775
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