REGULAR PAPER

Masayuki Hashimoto Æ Masao Koda Æ Hidetoshi Ino

Katsunori Yoshinaga Æ Atsushi Murata

Masashi Yamazaki Æ Kensuke Kojima Æ Kan Chiba

Chisato Mori Æ Hideshige Moriya

Gene expression profiling of cathepsin D, metallothioneins-1and -2, osteopontin, and tenascin-C in a mouse spinalcord injury model by cDNA microarray analysis

Received: 29 March 2004 / Revised: 27 July 2004 / Accepted: 3 August 2004 / Published online: 9 December 2004� Springer-Verlag 2004

Abstract The purpose of this study was to use a cDNAmicroarray to identify new genes involved in healing ofspinal cord injury. C57BL/6 mice (7–8 weeks, male)were subjected to spinal cord compression injury (SCI)at the T7/8 level (20 g, 5 min; SCI group). For thecontrol group, mice underwent only laminectomy. Micewere killed at 1, 3 and 7 days. cDNA transcribed frommRNA was hybridized to NIA mice 15K microarrays ateach time point. We found 84 genes showing significantexpressional changes, including higher and lowerexpression levels in the SCI groups than in the control[more than 1.0 or less than �1.0 using log ratio (base 2)].Five genes were selected for further quantitative geneexpression analysis by real-time reverse transcription(RT)-PCR. For histological examination, we applied insitu hybridization and fluorescence immunohistochem-istry. Cathepsin D, metallothionein-1 (MT-1), metallo-thionein-2 (MT-2), osteopontin (OPN), and tenascin-Cwere selected for quantitative and histological analysis.

Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7 daysand also of MT-1, MT-2, and tenascin-C expression at1 day. Tenascin-C was re-up-regulated at 7 days. Thesevalues agreed with those of real-time RT-PCR analysis.By double labeling with in situ hybridization and fluo-rescence immunohistochemistry, MT-1, MT-2 andtenascin-C expression was observed in neurons and glialcells at 1 day, whereas at 7 days the main MT-2 andtenascin-C expression was found in fibronectin-positivefibroblasts. The main cathepsin D and OPN expressionwas observed in activated macrophages/microglia at 3and 7 days. The five genes picked up by microarray geneexpression profiling were shown to exhibit temporal andspatial changes of expression after SCI. This system ispotentially useful for identifying genes that are involvedin the response to SCI.

Keywords Microarray Æ In situ hybridization ÆReal-time RT-PCR Æ Æ Gene expression

Introduction

The vulnerability of the spinal cord makes it difficult torecover from damage caused by mechanical trauma, andspinal cord injury (SCI) often results in permanentmotor and/or sensory loss. Contusion of the spinal cordcan damage blood vessels and cause hemorrhage aroundthe injured area. Such hemorrhage in the spinal cordparenchyma launches the signal transduction cascadesthat are associated with acute injury. The invasion ofneutrophils starts at around the same time as up-regu-lation of inflammatory cytokine (IL-1a, IL-1b IL-6, andTNF-a) [39], about 6 h after SCI, which changes thecondition of the spinal cord tissue. Neutrophils demar-cate the necrotic area and their number reaches a peaklevel at 1 day in a contusion model [11] and a spinal cordlesion model [47].

M. Hashimoto Æ M. Koda (&) Æ K. YoshinagaA. Murata Æ M. Yamazaki Æ H. MoriyaDepartment of Orthopedic Surgery,Graduate School of Medicine,Chiba University, 1-8-1 Inohana, Chuo-ku,260-8677 Chiba, JapanE-mail: masaokoda@faculty.chiba-u.jpTel.: +81-43-2262117Fax: +81-43-2262116

H. InoDepartment of Neurobiology,Graduate School of Medicine,Chiba University, Chiba, Japan

K. Kojima Æ K. ChibaDepartment of Pharmacogenomics,Graduate School of Pharmaceutical Sciences,Chiba University, Chiba, Japan

C. Mori Æ M. KodaDepartment of Bioenvironmental Medicine,Graduate School of Medicine,Chiba University, Chiba, Japan

Acta Neuropathol (2005) 109: 165–180DOI 10.1007/s00401-004-0926-z

Scavenger cells (macrophages/microglia) appear at1 day, and the number of activated macrophages/microglia reaches a peak at 2 or 3 days in the contusionmodels [11, 23] and 4 days in the lesion model [47].Pathological proliferation of macrophages/microgliamay contribute to subsequent exacerbation of the initialdamage [1, 5]. Activated macrophages/microglia pro-duce free radicals, nitric oxide, and arachidonic acidderivatives. Many of these molecules participate ininhibiting axon regeneration in the damaged region [18].

After the wave of inflammatory cells subsides, theresidual tissue is surrounded by glial scars. Astrocytesincrease by 2 days after SCI and the levels are main-tained up to 14 days [3]. Astrocytes produce tenascin,brevican, and neurocan [18], which are potent inhibitorsof neurite outgrowth. Inducible nitric oxide synthetase(iNOS), which exerts a harmful influence on neurons[14], is synthesized in glial scars. Regeneration of spinalcord tracts is inhibited by glial scar formation.

Considering the above points, the post-lesion phaseof cell infiltration is divided into three stages. The firstcomprises infiltration of neutrophils. At this time, celldeath dramatically increases [33]. The second stagecomprises infiltration of macrophages/microglia. Sig-nals from macrophages/microglia often exert harmfuleffects on surrounding tissues. The third stage is glialscar formation. Astrocytes surround damaged tissuesand produce scar-associated compounds, which aredetrimental to regeneration of tracts. In the presentstudy, we focused on three time points after SCI (1, 3and 7 days), which represent the above three stages.

Several methods are in current use for the investiga-tion of gene expression. Exhaustive studies of geneexpression patterns are provided by cDNA microarrays,because of the possibility to compare tens of thousandsof genes at a time. In the central nervous system, therehave been some comparative analyses of mRNA levelsin response to experimental traumatic brain injury [34],fluid percussion brain injury [24], spinal cord ischemia[13] and spinal cord contusion injury [12].

The NIA mouse 15K cDNA microarray [50] wasderived from NIA mouse cDNA library 52374 expressedsequence taqs (EST). From this cDNA collection, amicroarray representing about 15,000 unique genes wasassembled. There have no reports that address thetemporal changes in gene expression after SCI using theNIA mouse 15K microarray.

In the present study, we first screened for geneswhose expression was changed 1, 3 and 7 days afterSCI by microarray analysis. From these data, we se-lected five genes [cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), andtenascin-C] that have typical time course expressionand have a close relation with the focused event ofinfiltration of inflammation cells (neutrophils, macro-phages/microglia and astrocytes). Changes of geneexpression were further examined for these five genesby quantitative real-time reverse transcription (RT)-PCR. Cells expressing these genes were identified by

in situ hybridization and fluorescence immunohisto-chemistry double labeling.

Materials and methods

Animal model for SCI

Male C57BL/6 mice (n=45, 8–9 weeks old, averageweight 20 g; SLC, Hamamatsu, Japan) were deeplyanesthetized with 1–1.2% halothane in 0.5 l/min oxygenand laminectomized at the T7/8 level. SCI was producedby extradural compression using a rod (20 g, 15 cmlong, with a 2·1mm plastic plate at the end) for 5 min(SCI group). As the control group, laminectomy alonewas performed (n=35 mice). Food and water were givenad libitum. All animals were treated and cared for inaccordance with the Chiba University School of Medi-cine guidelines pertaining to the treatment of experi-mental animals.

Hind limb motor function rating

Hind limb function was assessed using a scale withscores from 0 to 13 [17]: 0, no hind limb movements;1–3, faint to obvious movements of any joints; 4–5,stepping and forward propulsive movement; 6–8, weightbearing ability; 9–13, ability to walk on a 2–0.5 cm bar.Animals in the control and SCI groups were testedblindly two times everyday after operation.

Tissue preparation

The animals were killed at 1, 3 and 7 days post opera-tively. For microarray and real-time RT-PCR analyses,spinal cords were removed for over three spinal seg-ments (T7–9) including the injury epicenter (T8) underpentobarbital anesthesia (50 mg/kg), and were frozenimmediately in liquid nitrogen. Total RNAs were ex-tracted using a TRIzol reagent (Gibco Life Technolo-gies, Rockville, MD) as previously described [27].

For histological evaluation, the animals were per-fused intracardially with 4% paraformaldehyde in PBS(pH 7.4) and spinal cords (T7–9) were collected. Theprocedure for making cryoprotected sections was asdescribed previously [20].

RNA labeling

From total RNAs, poly(A)+mRNAs were extractedwith the Oligotex-dT30 mRNA purification kit (Takara,Tokyo, Japan) according to the manufacturer’s proto-col. Mixtures containing total RNAs (1 lg/ll; 150 ll),2· binding buffer and Oligotex-dT30 were incubated for3 min at 70�C. After incubation for 10 min at room

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temperature, poly(A)+RNAs were centrifuged at15,000 rpm for 5 min. After removal of supernatants,poly(A)+RNAs were suspended in wash buffer, appliedto spin columns and centrifuged at 15,000 rpm for 30 s.Supernatants were suspended again in the wash buffer,applied to spin columns and centrifuged at 15,000 rpmfor 30 s. After removal of the wash buffer, poly(-A)+RNAs were suspended with H2O incubated at70�C, applied to spin columns and centrifuged at15,000 rpm for 30 s. Poly(A)+RNA was finally sus-pended with 20 ll H2O.

Mixtures (15.4 ll) including 1–2 lg poly(A)+RNAand 2.25–4.5 lg oligo(dT)12–18 primer (Sigma, St Louis,MO) were incubated for 3 min at 70�C and put on icefor 3 min. To the mixtures were added 3 ll 0.1 M DTT,0.6 ll dNTP mix (Amersham), 6 ll 5· Superscript IIbuffer and 3 ll 1 mM cyanine 3 (Cy3)- or cyanine 5(Cy5)-dUTP (Perkin-Elmer Life Sciences, Boston, MA).After incubation at 42�C for 2 min, 2.0 ll Superscript II(200 U/ll; Invitrogen, Carlsbad, CA) were added to themixtures, incubated at 42�C for 90 min, and furtherincubated for 30 min after adding 0.5 ll Superscript II(200 U/ll). To the mixtures, 5 ll 0.5 M EDTA (pH 8.5)and 1 ll 10 N NaOH were added, incubated at 65�C for30 minutes, and then 25 ll 1 M TRIS-HCl (pH 7.5) wasadded. The mixtures were purified with the MiniElutePCR Cleanup Kit (Qiagen, Hilden, Germany) accordingto the manufacturer’s protocol.

Preparation of microarray

The NIA mouse 15K cDNA clone set [50] was con-structed by the NIA (National Institute of Aging) andprovided to K.C. through the RIKEN DNA bank(Tsukuba, Japan). Plasmid DNAs were prepared in 96-well format (Montage Mlasmid Miniprep96 kit; Milli-pore, Billerica, MA). To amplify cDNA inserts, PCRprimers (5’-CCAGTCACGACGTTGTAAAACGAC-3’; 5’-GTGTGGAATTGTGAGCGGATAACAA-3’)were designed from sequences flanking the multiplecloning site of pSPORT1 plasmid vector (Invitrogen),which was used for cDNA cloning. For each cDNAclone, about 10 ng of plasmid DNA was used in100 ll PCR reaction containing 10 mM TRIS-HCl(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.5 lM eachprimer, 0.8 mM dNTPs, and 3.25 U Taq polymerase.Amplification was done in an EZC96 (Asahi Techno-glass, Funabashi, Japan) under the following condi-tions: initial denaturation at 95�C for 1 min; 38 cyclesof denaturation at 94�C for 30 s, annealing at 65�C for45 s, and extension at 72�C for 3 min. PCR productswere purified in 96-well format (Montage PCR96

Cleanup kit; Millipore). To examine the quality andquantity of PCR products, they were run on 1%agarose gels. PCR products were dissolved in TRIS-EDTA (TE), and spotted onto amino silane-coatedslide glass using an original pin-type arrayer (AsahiTechnoglass).

Hybridization to microarrays

After denaturing at 95�C for 2 min, hybridization mix-tures [13.5 ll Cy3- or Cy5-dUTP labeled cDNA, 2 ll(20 lg/ll) salmon sperm DNA, 5 ll (10 lg/ll) yeasttRNA, 1.25 ll (10 lg/ll) poly-d(A), 4.8 ll 20· SSC and0.84 ll 10% SDS] were hybridized onto the array at65�C overnight in a humidity box. Then the microarrayswere washed with 2· SSC containing 0.1% SDS for10 min, with 0.2· SSC containing 0.1% SDS for 10 mintwice and with 99.5% ethanol for 3 min and dried bycentrifugation. The microarrays were scanned on aScanArray Lyte (Perkin-Elmer Life Sciences). Resultswere digitalized with the QuantArray (ver 3.0; Perkin-Elmer Life Sciences) software. The experiments wereduplicated using the dye swap method.

Data analysis

Scanning parameters (light intensity and detection lev-els) were set such that the brightest spots were just belowsaturation. Spots with obvious defects and spots lowerthan average background levels were excluded. Afterbackground subtraction, the two channels were nor-malized to channel-specific median signal intensity. Theratio of SCI/control was log-transformed (base 2), andgenes whose log ratio was inverted after dye swap trialwere omitted from analysis. The representative micro-array data was plotted in two-dimensional scatter plotsand Spearman’s correlations were calculated. Weaccepted only those mRNA values with log ratio changethat was higher than 1.0 for up-regulated genes andlower than �1.0 for down-regulated genes.

Cluster analysis

The similarity of up-regulated or down-regulated geneswas analyzed by hierarchical clustering analysis basedon cosine correlation between the expression vectors.The clustering of arrays was performed by the Gene-Math software (Applied Maths BVBA, Sint-Martens-Latem, Belgium), with the procedure that uses CompleteLinkage.

Real-time RT-PCR

Real-time RT-PCR was carried out to confirm the re-sults obtained by the microarray analysis at each timepoint using a Smartcycler (Cepheid, Sunnyvale, CA).Expression levels of each mRNA were analyzed by SybrGreen methods (Applied Biosystems, Foster City, CA)according to the manufacturer’s instructions, with onemodification. Reaction volume was adjusted to 25 ll(12.5 ll 2· Sybr Green, 0.2 lM forward and reverseprimers and 10 ll template cDNA). Primer pairs usedfor real-time RT-PCR are summarized in Table 1 and

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b-actin was used for control analysis. The size ofresulting products was 164 bp for cathepsin D, 137 bpfor MT-1, 225 bp for MT-2, 170 bp for OPN, 196 bp fortenascin-C, and 214 bp for b-actin. The PCR parameterswere 95�C for 10 min and 40 cycles of 95�C for 15 s and60�C (b-actin; 62�C) for 60 s. At the end of each pro-gram melt-curve analysis was done. All experimentswere done for three samples and duplicated.

Preparation of cRNA probes for in situ hybridization

OPN

pCR II plasmids (Invitrogen) containing rat OPNcDNA [a kind gift from Shintaro Nomura, OsakaUniversity, Japan; nucleotides (nt) 259–1025] were line-arized by digestion with BamHI or XbaI. Antisense andsense RNA probes were transcribed in vitro from thelinearized plasmids using a digoxigenin (DIG) RNAlabeling mixture (Roche Diagnostics, Basel, Switzer-land) and SP6 or T7 RNA polymerase (Takara, Tokyo,Japan) as previously described [23]. The homology be-tween the PCR product amplified from mouse cDNAwith the above primer pairs and rat OPN cDNA wasscored as more than 90%.

MT-1 and MT-2

PCR products amplified with the above primer pairswere subcloned into pGEM-T easy vector (Promega,Madison, WI) and linearized by PstI or SphI. Antisense/sense digoxigenin-labeled cRNA probes were tran-scribed as above.

Cathepsin D

PCR product (nt 287–984; 698 bp) amplified with5’-AGGCTATTGTGGACACAGGG-3’ and 5’-CCTG-CTTCAGAGTACTGCCC-3’ primers was subclonedinto pGEM-T easy vector and linearized with SalI orNcoI. Antisense/sense digoxigenin-labeled cRNA probeswere transcribed as above.

Tenascin-C

PCR products amplified with the above primer pairswere subcloned into pGEM-T easy vector and linearizedwith PstI or SphI. Antisense/sense digoxigenin-labeledcRNA probes were transcribed as above. The foursubcloned PCR products were verified by sequencing.

In situ hybridization

In situ hybridization was performed as described previ-ously with some modifications before hybridization [23,26, 27]. Sections were rehydrated in 0.3% Triton X-100in PBS for 120 min, washed with PBS three times for15 min, and deproteinated in a 1–10 lg/ml solution ofproteinase-K (Roche Diagnostics) in PBS for 10 min at37�C. The sections were then fixed in 4% paraformal-

Table 1 Primer pairs and PCRproducts (MT metallothionein) Name (accession no.) Primers PCR product (bp)

Cathepsin D (BC019682) Sense 5¢-CTGAGTGGCTTCATGGGAAT-3¢ 164Antisense 5¢-CCTGACAGTGGAGAAGGAGC-3¢

MT-1 (BG077818) Sense 5¢-CTCCGTAGCTCCAGCTTCAC-3¢ 136Antisense 5¢-AGGAGCAGCAGCTCTTCTTG-3¢

MT-2 (BG063925) Sense 5¢-ACTTGTCGGAAGCCTCTTTG-3¢ 225Antisense 5¢-CGACTATCCCTTCAAACCGA-3¢

Osteopontin (X13986) Sense 5¢-TCTGATGAGACCGTCACTGC-3¢ 170Antisense 5¢-AGGTCCTCATCTGTGGCATC-3¢

TenascinC (XM_12428) Sense 5¢-TGTGTGCTTCGAAGGCTATG-3¢ 196Antisense 5¢-GCAGACACACTCGTTCTCCA-3¢

b-Actin (X03765) Sense 5¢-TAAAGACCTCTATGCCAACAC-3¢ 214Antisense 5¢-CTCCTGCTTGCTGATCCACAT-3¢

Fig. 1 Hind limb motor function rating of mice (n=12). A score 0–13 was assigned assessing the hind limb function according to [17].The mice subjected to SCI recover to an average score of about 4after 7 days. They could walk with alternative stepping and hadpropulsive movements of hind limbs but no weight bearing (SCIspinal cord injury)

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Table 2 Up-regulated genes after SCI (SCI spinal cord injury, Aapoptosis, C cell cycle, D DNA replication, E energy/metabolism, Iinflammation, L lipid metabolism, M matrix/structural proteins, P protein synthesis/translation control, S signal transduction, T tran-scription/chromatin, unk unknown)

Function Clone name Accession no. Clone description 1 day 3 days 7 days

A H3083G02 BG070106 Mus museulus 24p3 gene 1.22 0.46 0.48A H3136E12 BG087187 M. musculusuncoupling protein-2 0.89 0.87 1.17A H3015E08 BG064105 Human Tis11d gene 1.3 0.66 0.87A H3117012 BG085800 M. musculus5-lipoxygenase-activating protein 0.35 0.99 1.09C H3024E04 BG064846 M. musculuscell division cycle 2 homolog A 0.3 0.85 1.19D H3139A05 BG087390 M. musculustopoisomerase (DNA) II alpha 0.11 0.7 1.03E H3028F05 BG065221 Mouse kidney ornithine decarboxylase 1.46 0.36 0.27E H3085G03 BG083209 Mouse cytochrome beta-558 0.09 0.79 1.67E H3023H12 BG064797 M. musculuslactate dehydrogenase 1 1.25 1.22 0.91H H3082D09 BG070071 M. musculussmall heat shock protein (HSP25) gene 1.49 0.93 0.39H H3020C02 BG077818 Mouse metallothionein-I 1.48 0.73 0.05H H3013D11 BG063925 Mouse metallothionein II 1.65 0.82 0.29I H3016D10 BG064176 Murine L-34 galactoside-binding lectin 1.12 1.13 2.8I H3150E06 BG088241 M. musculuslymphocyte specific 1 -0.08 0.54 1.29I H3082C12 BG070062 Mouse minopontin 1.87 1.55 3.81I H3003A10 BG076479 M. musculuscytotoxic T lymphocyte-associated protein 2 alpha 1.49 1 1.17L H3144F06 BG075211 Mouse adipose differentiation-related protein 0.98 1.16 2.72M H3013F05 BG063941 M. musculus syndecan 1 1.17 0.38 0.31M H3017F05 BG077621 M. musculuslamin A 1.18 0.49 0.38M H3112C01 BG085352 M. musculusalpha-1 type IV collagen 1.2 0.53 2.14M H3124A01 AW550270 M. musculustenascin C 2.26 0.58 1.11M H3119G01 BG073184 M.museulus vimentin 1.17 1.12 1.15M H3010E09 AW538759 1.75 0.32 0.26M H3116A10 BG085662 M. muscuiusfibronectin 1.18 1.21 1.67M H3138E02 BG087341 Mouse collagen alpha-2(IV) 0.55 0.67 1.3M H3111D11 BG072504 M. musculusalpha-1 (XVIII) collagen gene 0.3 0.68 1.91M H3014F03 AW542365 M. musculusvillin 2 1.27 0.49 0.47M H3135G11 BG087124 M. musculus Fe gamma receptor III (Fegr3) mRNA, Fegr3-b allele 0.96 0.86 1.46M H3126C03 BG086439 M. musculusannexin A2 1.4 1.15 0.93.M H3017H12 BG077550 M. musculus transgelin 1.29 0.85 1.31M H3136G08 AW553642 Homo sapienscaldesmon 0.97 0.74 1.16M H3025E04 BG064933 M. musculusnidogen 1 0.15 0.64 1.05M H3129G04 BG086718 M. musculusvillin 2 1.47 0.96 0.28M H3022F08 BG078017 Mous nonmuscle tropomyosin 5 1.14 0.86 0.99M H3134H12 AW553287 M. musculus osteoblast specific factor 2 0.35 0.77 1.28M H3019A12 BG077732 AC005290, complete sequence 2.12 1.15 2.5M H3019A08 BG077728 M. musculusactin-related protein complex 1b 1.31 01.12 1.61M H3119A08 BG073116 M. musculusMHC locus class III regions Hsc70 t gene 1.09 0.8 1.32P H3009F01 BG076993 H. sapiens KIAA1398 protein 1.4 0.37 -0.13P TO007B09 BG063430 M. musculuseIF4A 1.08 0.39 0.72P H3107F01 BG085008 M. musculuspoly A binding protein, cytoplasmic 1 1.28 0.79 0.68P H3022D05 BG064656 Mouse ctla-2-beta, homolog. to cysteine protease proregion 1.41 0.67 1.19P H3133G06 BG087028 M. musculusHI 9 and muscle-specific Nctc genes -0.25 0.8 1.37P H3037F12 C77656 M. musculus dipeptidyl peptidase I precursor 0.54 0.63 1.34P H3044C12 BG066632 Moesin homolog (mice, teratocarcinoma F9 cells) 1.38 0.94 1.11P H3010F04 BG077092 M. musculusRNA binding motif protein 3 1.46 1.11 0.57P H3014A12 BG063978 M. musculuscapping protein (actin filament), gelsolin-like 0.71 1.04 1.92S H3095B05 BG071140 H. sapiensstabilin-1 0.13 0.65 1.61S H3025D09 BG064928 M. musculusmitogen-responsive 96 kDa phosphoprotein p96 1.35 0.62 1.14S H3087H09 BG083409 M. musculuscalcium binding protein A6 2.12 1.15 2.5S H3138H09 BG087382 M. musculuscathepsin D 1.31 1.12 1.61S H3058C09 BG080846 M. musculusJun oncogene .09 0.8 1.32S H3015D10 BG064095 Homo sapiens uridine monophosphate kinase 1.4 0.37 -0.13S H3051F10 BG080268 M. musculusGRO1 oncogene 1.08 0.39 0.72S H3008B11 BG063519 M. musculusfos-related antigen 1 1.28 0.79 0.68S H3028F03 BG078496 M. musculuscathepsin L 1.41 0.67 1.19S H3012G07 BG063787 Rattus norvegicus atypical PKC specific binding protein -0.25 0.8 1.37S H3009E11 BG076991 M. musculuscytokine inducible SH2-containing protein 3 0.54 0.63 1.34S H3057E07 BG080700 M. musculusfibroblast growth factor regulated protein 2 1.38 0.94 1.11S H3154G07 BG088567 R. norvegicus Insulin-like growth factor-binding protein 1.46 1.11 0.57S H3001B08 BG063022 Mouse lyn B protein tyrosine kinase 0.71 1.04 1.92S H3056G04 BG067655 0.13 0.65 1.61T H3005D09 BG076751 H. sapiensfollistatin-like 3 1.35 0.62 1.14T H3029B11 BG065272 M. musculus high mobility group protein I 1.49 0.89 0.74T H3139C11 BG087418 M. musculusMS4A11 0.52 1.12 2.79T H3053E12 BG067364 M. musculustranscription factor LRG-21 1.02 0.47 0.54

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dehyde in PBS, pH 7.4 for 10 min. The sections wereacidified in 0.2 M HCl for 10 min, immersed in 100 mMtriethanolamine-HCl pH 8.0 for 10 min, and acetylatedwith 0.25% acetic anhydride in 100 mM triethanol-amine-HCl pH 8.0 for 10 min. After washing three timesin PBS for 20 min, the sections were prehybridized with50% formamide in 2· SSC for 60 min at room tem-perature. The sections were hybridized with DIG-labeledcRNA probes in the hybridization buffer for 28 h at

50�C. After washing and blocking with a blockingsolution for 60 min, the sections were reacted with analkaline phosphatase-labeled anti-digoxigenin antibody(1:1,000; Roche) at 4�C overnight. Signal was visualizedwith nitro blue tetrazolium (Sigma, St Louis, MO) and5-bromo-4-chloro-3-indolyl phosphate (Sigma) in analkaline phosphatase buffer for 2 h. The reaction wasstopped by incubating with 10 mM TRIS-HCl pH 8.0and twice with TE for 5 min.

Double staining by in situ hybridizationand fluorescence immunohistochemistry

After in situ hybridization, sections were washed threetimes with PBS for 10 min, and then reacted overnightat 4�C with a primary antibody. The primary antibodieswere anti-neuronal nuclei (NeuN; 1:400; ChemiconInternational, Temecula, CA), anti-APC (Ab7/CC-1;1:400; Oncogene Research Products, Cambridge, MA),anti-mouse glial fibrillary acidic protein (GFAP; 1:400;Sigma), and anti-cellular fibronectin (1:400; Chemicon).After washing with PBS three times for 10 min, thesections were reacted with Alexa Fluor 488-conjugatedgoat anti-mouse IgG antibody (Molecular Probes, Eu-gene, OR) for the anti-NeuN, CC-1, GFAP and fibro-nectin antibodies, and mounted on slides withfluorescence mounting medium (DakoCytomation,Copenhagen, Denmark).

Fluorescence immunohistochemistry

OPN and tenascin-C were detected by in situ hybridiza-tion. Fluorescence immunohistochemistry with anti-mouse CD11b (Mac-1; 1:400; Serotec, Oxford, England)was performed on adjacent sections. Because the CD11bantigen was destroyed in the harsh conditions for insitu hybridization, double staining was impossible.

Table 2 (Contd.)

Function Clone name Accession no. Clone description 1 day 3 days 7 days

unk H3 056 DO 5 BG667620 1.02 0.63 0.16unk H3010H01 BG063804 Human KIAA0062 1.55 0.4 0.49unk H3082H09 BG082965 H. sapiens mRNA fragment 1.36 0.54 0.53unk H3130C01 BG074047 1.29 0.94 1.71unk H3065B06 BG068407 1.06 0.86 0.44unk H3067H02 BG068648 1.08 0.76 0.53unk H3134E06 BG074388 1.55 0.68 0.65unk H3012H07 BG063885 1.99 1.07 0.8unk H3012F07 BG063776 0.44 0.65 1.03unk H3056F03 BG067642 H. sapiens cDNA: FLJ22334 fis, clone HRC05837 0.57 0.64 1.15unk H3064C11 BG068331 1.14 0.48 0.35unk H3053C11 BG067341 1.14 0.45 -0.18unk H3115H03 BG085649 H. sapiens KIAA0963 protein 0.56 0.43 1.29unk H3010C11 AW538705 1.18 0.64 1.07unk H3056H08 BG067670 2.26 0.78 0.98unk H3096D08 BG071239 1.28 0.37 0.12unk H3044G12 BG066678 1.55 1.22 3.47unk H3054D12 BG067439 2.17 0.69 0.51

Fig. 2 Partial microarray images of the SCI group (left panel) andcontrol group (right panel) in the same array. Many spots in the leftpanel are up-regulated compared with the right panel, indicatingthat many genes are up-regulated after SCI

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Fluorescence immunohistochemistry was performed aspreviously described [26]. After incubation with anti-mouse CD11b overnight at 4�C, sections were reactedwith biotinylated rat IgG (Vector Laboratories, Burlin-game, CA) for 2 h. After washing three times in PBS, thesections were reacted with Alexa Fluor 488-conjugatedstreptavidin (Molecular Probes) for 1 h and mounted asdescribed above.

Results

Hind limb motor function rating

SCI induced paraplesia of hind limbs in the SCI groups1 day after spinal cord injury (Fig. 1). The average scoredecreased to 0.25 (0–3) at day 1, gradually improved to1.9 (0–4) at day 3, and 4 (2–6) at day 7 (Fig. 1). Thecontrol group mice showed only slight impairment, thatis, the average score decreased only one to three pointsfrom day 1 to day 7 (average 10.4–10.6 for 7 days).

Microarray analysis

Of the 15,488 genes represented on the array, 867 geneswere acceptable for the following analysis based on the

results of dye swap trials. Of these 867 genes, 84 geneswere up-regulated greater than 1.0 in the log-trans-formed ratio (base 2) of SCI/control at the same timepoints (Table 2). There were no down-regulated genesshowing values lower than �1.0. Representative arraydata shows that more genes were up-regulated in the SCIgroup than control group (Fig. 2). The expression levelsof the 867 genes detected on the microarray at each timepoint are plotted on two axes in the scatter plots (Fig. 3).Each time point showed a correlation coefficient (R2)from 0.57 to 0.80.

We selected five genes that showed typical expressionprofiles in the array analysis (Table 3) for detailedanalysis. Detailed expression profile of these genes hasnot been reported in SCI of rodents, although theirexpression in the CNS is known. In addition, these fivegenes are thought to have a relationship with inflam-matory events caused by infiltration of neutrophils,macrophages/microglia and astrocytes, respectively.Cathepsin D (BC019682) was up-regulated at 3 and7 days after SCI. As shown previously, cathepsin D waspresent in the vesicles of macrophage endosomes [6].OPN was up-regulated at all time points. The relation-ship between OPN and macrophages/microglia infiltra-tion has been described previously [23]. OPN mRNAlevels peaked at 3 days with peaks of macrophage/mi-croglia numbers. MT-1 (BG077818) and MT-2(BG063925) were up-regulated at 1 day. A link has beenestablished between hepatic metallothionein and in-creased leukocyte numbers [44]. Tenascin-C (XM12428)was up-regulated at 1 and 7 days. Reports have shownthat tenascin-C is expressed by astrocytes [19]. Tenascin-C has a close relationship with astroglial scars.

The clustering analysis of the 84 genes showed thatthe clusters of these five genes were scattered, except forMT-1 and MT-2 (Table 4), which showed similarexpression profile in the microarray data (Table 3).

Real-time RT-PCR

The temporal changes in the gene expression of thefive selected genes after SCI were further examined byreal-time RT-PCR analysis (Table 5). Up-regulation ofexpression was observed at 3 and 7 days for cathepsinD, at 1 day for MT-1 and MT-2, at 1 and 7 days fortenascin-C, and 1, 3 and 7 days for OPN. These resultsagree with those obtained from microarray analysis.

Fig. 3 Representative scatter plots among SCI signal (vertical axis)and control signal (horizontal axis) at 1, 3 and 7 days afteroperation. R2 value for each pair-wise comparison is indicated inthe respective box

Table 3 Cathepsin D, MT-1, MT-2, OPN, tenascin-C geneexpression after SCI by microarray analysis. Five genes were se-lected for detailed analysis based on their expression showing atypical time course of upregulation (MT metallothionein,OPNosteopontin)

Gene Log ratio (SCI/control)

I day 3 days 7 days

Cathepsin D 0.52 1.12a 2.79*MT-1 1.48* 0.73 0.05MT-2 1.65* 0.82 0.29OPN 1.87* 1.55* 3.81*Tenascin-C 2.26* 0.58 1.11*

aSignificant values with log ratio change that was higher than 1.0for up-regulated genes

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Table 4 Cluster analysis ofmRNA expression levelssignificantly changed afterspinal cord injury

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In situ hybridization

To reveal in which cells the five genes were expressedafter SCI, we analyzed their expression by in situhybridization.

Cathepsin D

In the control mice, positive hybridization signal ofcathepsin D was observed in neurons of the anterior andposterior horns and small round cells in the white matter(data not shown). After SCI, cathepsin D expression wasdetected in the same cells at 1 day (Fig. 4A), and after3 days the number of positive cells increased greatly,especially in the contusion area (Fig. 4B). These cellshad granules in the cytoplasm, which were not obvious

in the small round cells at 1 day, and some of thembecame large cells like phagocytes (Fig. 4C). Using flu-orescence immunohistochemistry on adjacent sections,cathepsin D-expressing cells in the contusion area at 7days were shown to be Mac-1+ macrophages/microglia(Fig. 4C, D). Expression of cathepsin D mRNA grad-ually increased until day 7. The temporal changes shownby these in situ hybridization data were almost coinci-dent with the microarray data.

MT-1

MT-1 mRNA was expressed in glial cells of the whitematter and in neurons of the gray matter at 1 day afterSCI (Fig. 5A). By in situ hybridization and fluorescenceimmunohistochemistry double labeling, MT-1-express-ing cells were shown to be NeuN-positive neurons(Fig. 5C, D) and GFAP-positive astrocytes (Fig. 5E, F).MT-1-positive cells were not found in the injured area at7 days after SCI, but were observed in the area sur-rounding the injured site. These cells were GFAP-posi-tive astrocytes (Fig. 5B).

MT-2

MT-2 mRNA expression was up-regulated in survivingresidual motoneurons and interneurons of the graymatter near the injured site (Fig. 6A, C, D) at 1 dayafter SCI. At 3 days, in addition to surviving neurons,MT-2 expression was also detected in small round cells

Table 5 Real-time RT-PCR analysis of five genes mRNA expres-sion after SCI compared with control. RNAs extracted from thelesioned spinal cord. Ratio of the SCI/control group values wascalculated after the normalization by b-actin expression (n=3)

Gene Ratio (SCI/ Control)

I day 3 days 7 days

Cathepsin D 0.40 1.58 2.58*MT-1 1.35 0.51 0.20MT-2 4.65* 1.46 0.48**OPN 1.39 3.75* 5.92*Tenascin-C 1.65 1.30 1.75

*P<0.05, **P<0.01

Fig. 4 A–C Cathepsin DmRNA expression in the SCIgroup shown by in situhybridization. Cathepsin D isup-regulated in survivingneurons and round cells ofvarious size in the contusionarea at 3 days (A). Cathepsin Dexpression is highly up-regulated in the posterior areaof the spinal cord (B) andlateral funiculus of the spinalcord (C) at 7 days. Cathepsin Dexpression is up-regulated incells of various size, which havemany granules in the cytoplasm(C). D Immunohistochemistryof Mac-1 in an adjacent sectionto C. Cathepsin D is expressedin Mac-1-positivemacrophages/microglia. BarsA–D 50 lm

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invading contusion area and meningeal fibroblasts (datanot shown). At 7 days, MT-2 expression was mainlyseen in meningeal fibroblasts and fibroblasts in andaround the spinal cord parenchyma (Fig. 6B). A smallamount of MT-2 expression was seen in GFAP-positiveastrocytes. In situ hybridization and immunohisto-chemistry double labeling confirmed MT-2 expression infibroblasts of the fibroblast scar tissue (Fig. 6E, F) andGFAP-positive astrocytes (data not shown).

OPN

OPN mRNA was sporadically expressed in the whitematter in small round cells at 1 day after SCI (Fig. 7A).After 3–7 days, the number of positive cells increased inthe contusion area (Fig. 7B, C); these cells had granulesin the cytoplasm. Fluorescence immunohistochemistry

using adjacent sections revealed that OPN-positive cellswere Mac-1-positive macrophages/microglia (Fig. 7C,D).

Tenascin-C

Tenascin-C mRNA expression was up-regulated in smallround cells in the white matter and in various-sizedneurons at 1 day after SCI (Fig. 8A). At 7 days afterSCI, the distribution of positive cells was dramaticallychanged; they were found around the scar area in thespinal cord parenchyma. In addition, meningeal cells inthe outer edge of the spinal cord showed tenascin-Cexpression (Fig. 8B). In situ hybridization and fluores-cence immunohistochemistry double labeling showedthat, at 1 day, the tenascin-C-expressing cells wereNeuN-positive neurons (Fig. 8C, D), but at 7 day were

Fig. 5 A, B MT-1 mRNAexpression in the SCI groupshown by in situ hybridizationat 1 day (A) and 7 days (B).MT-1 expression is up-regulated in cells of various sizein the anterior horn at 1 day. At7 days, MT-1-positive cells arerestricted to small cells aroundthe contusion area apart fromthe lesion epicenter. C, DDouble labeling by in situhybridization for MT-1 (C) andimmunohistochemistry forNeuN (D). MT-1-expressingcells (C, arrows) are NeuN-positive neurons (D,arrowheads). E, F Doublelabeling by in situ hybridizationfor MT-1 (E) andimmunohistochemistry forGFAP (F). MT-1-expressingcells (E, arrows) are GFAP-positive astrocytes (F,arrowheads) (MTmetallothionein, GFAP glialfibrillary acidic protein). BarsA, B 50 lm; C–F 20 lm

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fibronectin-positive fibroblasts (Fig. 8E, F). A smallamount of expression of tenascin-C was seen in theGFAP-positive astrocytes (data not shown). By micro-array and real-time RT-PCR analyses, the two-phaseup-regulation of tenascin-C expression observed at 1and 7 days was consistent with the histological data.

Discussion

In this study we identified a number of potentially sig-nificant genes that are expressed following SCI. UsingNIA mouse 15K array analysis, we obtained detailedexpression profiles of five genes out of these genes thatshowed typical up-regulation at 1, 3, and 7 days. Toverify the microarray data, we analyzed the expressionlevels by real-time RT-PCR, which showed good cor-relation with the microarray data. We also used histo-

logical techniques to determine which cells expressedthese genes in the spinal cord before and after SCI.

Cathepsin D

Cathepsin D is a representative aspartic protease inlysosomes and is widely distributed in various tissues,with particularly high levels in CNS neurons [58]. Thesedata are consistent with the present in situ hybridizationstudy, showing that cathepsin D was expressed in theneurons of the laminectomy control (data not shown)and 1 day after SCI (Fig. 4A). Cathepsin D-deficientmice show manifest seizures and become blind near theterminal stage (postnatal day 26). Histological observa-tion of these mice shows autophagosome/autolysosome-like bodies straggled in the cytoplasm of CNS neurons[30]. It was concluded that cathepsin D has a protective

Fig. 6 A, B MT-2 mRNAexpression in the SCI groupshown by in situ hybridizationat 1 day (A) and 7 days (B).MT-2 expression is up-regulated in large cells in theanterior horn at 1 day (A) andmeningeal cells and scar tissuein the parenchyma of theinjured spinal cord at 7 days(B). C, D Double labeling by insitu hybridization for MT-2 (C)and immunohistochemistry forNeuN (D). MT-2-expressingcells (C, arrows) are NeuN-positive neurons (D,arrowheads). E, F Doublelabeling by in situ hybridizationfor MT-2 (E) andimmunohistochemistry forfibronectin (F). MT-2-expressing cells (E) arefibronectin-positive fibroblasts(F) (NeuN neuronal nuclei).Bars A 50 lm; B 100 lm; C–F20 lm

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role in CNS neurons. On the other hand, the up-regu-lation of cathepsin D in the neurons possibly means thatit plays a pathological or age-related role. It has beenreported that cathepsin D is up-regulated in the cho-linergic neurons of nucleus basalis in the brain in Alz-heimer’s disease [38] and in regions of the brain of agedrats [29]. Further investigation is needed to clarify therole of neuronal cathepsin D expression in SCI.

In the present study, a dramatic up-regulation wasobserved in macrophages/microglia invading the con-tusion site, and these cells contained many granules orvacuoles in their cytoplasm at 3 days. The up-regulatedcathepsin D parallels the invasion of macrophages/mi-croglia, since in the spinal cord of laminectomy controlthere were only few macrophages/microglia expressingcathepsin D (data not shown).

Cathepsin D expression after SCI has been reportedby Banik et al. [4], who focused on myelinolysis using anexperimental SCI model. Some early studies show thatthe A1 protein of myelin is specifically cleaved bycathepsin D [8], and treatment of experimental allergicencephalomyelitis with an inhibitor of cathepsin D(pepstatin) has the effect of delaying myelin basic proteindegradation [7]. Furthermore, cathepsin D mediatesprogrammed cell death induced by TNF-a [16]. The up-regulation of cathepsin D in the macrophages/microgliaindicates a pathological condition as in neurons. Inhi-bition of cathepsin D activity after SCI may become animportant factor for treatment of myelinolysis and de-layed neurodegeneration.

MT-1 and MT-2

MTs are cysteine-rich heavy metal binding proteins oflow molecular weight (6–7 kDa), normally isolated fromtissues as zinc-MTs. Some early studies suggested thatMTs play a role scavenging free radicals, which areproduced under various stress conditions [2]. Expressionof MT-1 and -2 is predominantly localized to astrocytes[6, 21], whereas MT-3 is abundant in glutamatergicneurons [37]. MT-1 is expressed in cortical neurons aftercerebral ischemia, shown by in situ hybridization [10]. Inthe present study, MT-1 was expressed in NeuN-positiveneurons and GFAP-positive astrocytes. MT-2 was ex-pressed not only in neurons and astrocytes, but was alsoup-regulated at 7 days in fibronectin-positive fibroblaststhat formed the scar tissue occuping the spinal cordparenchyma at this time point. MT has been reported tobe expressed in differentiating fibroblasts in vitro [46];there is no report in vivo. Interestingly, quantitativestudy of MT-2 showed no up-regulation of the mRNAlevel at 7 days.

Treatment with Zinc-MT-2 prevents demyelinationand axonal damage in experimental autoimmuneencephalomyelitis [41]. Fibroblast growth factors(FGFs) up-regulate MT expression and increase thefrequency of cadmium-resistant variants of Swiss-Web-ster 3T3 cells [25]. These data suggest that MT-2expression in the fibroblast scar may play a protectiverole for injured axons. MT-1 also has a protective roleagainst oxidative or apoptotic stress. MT-1-transgenic

Fig. 7 A–C OPN mRNAexpression in the SCI groupshown by in situ hybridizationat 1 day (A) and 7 days (B, C).OPN expression is up-regulatedin small round cells at 1 day (A)and in small to large granule-containing cells in the contusionarea at 7 days (B, C). DFluorescenceimmunohistochemistry forMac-1 in an adjacent section toC. OPN-positive cells in thecontusion area are Mac-1-positive macrophages/microglia(OPN osteopontin). Bars A, B100 lm; C, D 50 lm

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mice show resistance against mild focal ischemia [6] andcryolesion-induced oxidative stress [22]. MT-1 preventsapoptosis of a T cell line [53]. Mouse embryonic cells nullfor MT due to loss of functional MT-1 and MT-2 geneswere more susceptible to apoptotic death [31]. Thus, theup-regulation of MT-1 and MT-2 in spinal cord neuronsat 1 day may also protect against oxidative stress.

OPN

OPN is a noncollagenous extracellular matrix proteinthat is expressed in various tissues. In the CNS, OPN isexpressed in brain macrophages/microglia after middlecerebral artery occlusion [56] and in spinal cord mac-rophages/microglia after SCI [23]. Out of the five genesselected in the present study, only OPN expression wasup-regulated at all the time points in the microarraydata. The scores of quantitative real-time RT-PCRanalysis were 3.75 and 5.93 at 3 and 7 days after SCI,respectively. These data are compatible with theNorthern blot data we found previously in a rat model[23]. OPN-expressing cells were Mac-1-positive macro-phages/microglia as previously described in the ratmodel [23].

OPN expression in injured tissues plays several rolessuch as promotion of wound healing [36], chemotactic

Fig. 8 A, B, Tenascin-C mRNA expression in the SCI group shownby in situ hybridization at 1 day (A) and 7 days (B). Tenascin-Cexpression is upregulated in large cells in the anterior horn and smallround cells in the white matter at 1 day (A) and in meningeal cellsand scar tissue in the parenchyma of the injured spinal cord at7 days (B). C, D Double labeling by in situ hybridization fortenascin-C (C) and immunohistochemistry for NeuN (D). Tenascin-C-expressing cells (C, arrows) are NeuN-positive neurons at 1 day(D, arrowheads). E, F Double labeling by in situ hybridization fortenascin-C (E) and immunohistochemistry for fibronectin (F).Tenascin-C-expressing cells (E) are fibronectin-positive fibroblastsat 7 days (F). Bars A 100 lm; B 50 lm; C–F 20 lm

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activity on cells such as astrocytes [56], macrophages[57], vascular smooth muscle cells [60], osteoclasts [52]and endothelial cells [48], cell adhesion [42], inhibition ofiNOS [43] and anti-apoptotic activity [45]. We previ-ously reported that the up-regulation of OPN expressionwas synchronized with the increase of activated macro-phages/microglia. Thus one role of OPN expression inthe present model may be chemotaxis of macrophages/microglia to recruit them into the injured area. OPNbecomes opsonin for phagocytosis of macrophages/mi-croglia [42]. These functions facilitate cleaning of debrisin damaged tissues and promote wound healing.

Tenascin-C

The tenascins constitute a family of extracellular glyco-proteins. Tenascin-C shows a highly regulated expres-sion pattern during embryogenesis. In the adultorganism, re-expression of tenascin-C occurs in patho-logical conditions [55]. Although tenascin-C expressionis up-regulated at the mRNA level after spinal cord stabinjury [51] and at the protein level after unilaterallydorsal column transection [62], there has been no reportthat tenascin-C is up-regulated after spinal cord contu-sion injury.

In the present study, tenascin-C expression had twopeaks. In situ hybridization analysis showed thattenascin-C expression occurred in neurons, astrocytes,and fibroblasts. At 1 day, tenascin-C expression wasfound in surviving neurons of various sizes in the graymatter and small glial cells in the white matter. At7 days after SCI, tenascin-C expression was found inmeningeal fibroblasts. In previous reports, tenascin-Cexpression was observed in the scar tissue from 1 day to7 day [15], in GFAP-positive astrocytes, meningeal cells,macrophages, and Schwann cells after thoracic dorsalcolumn injury [62], in Golgi epithelial cells and astro-cytes in the cerebellum after stab injury [32], and inneurons and glial cells in the spinal cord postnatally [61].These lines of evidence suggest that tenascin-C is ex-pressed in many kinds of cells existing in the spinal cord.

Between days 1 and 7, tenascin-C expression shiftedfrom neurons to fibroblasts. The number of neuronsdecreases after spinal cord hemisection [59], after ische-mic change [28], and after SCI [54]. In the present study,tenascin-C-expressing neurons may decrease with timeas a result of apoptotic and necrotic cell death as pre-viously reported. Formation of scar tissue was not evi-dent in the injury area until 3 days (data not shown). At7 days, many tenascin-C-positive scar tissues showingfibronectin immunoreactivity were formed in the con-tusion area. In an immunoblot study of tenascin-C, therelative protein level increases from 1 day and reaches apeak at 8 days [51]. In an immunohistochemical study oftenascin-C, tenascin-C-expressing cells are identified asGFAP-positive astrocytes at 2 days and fibronectin-po-sitive fibroblasts at 8 days [51]. These reports parallel thepresent study. In vitro studies showed that TGF-b

induces tenascin-C synthesis in astrocytes [49] andfibroblasts [40]. Tenascin-C knockout mouse showsreduced expression of fibronectin in both skin and cor-neal wounds [35]. On inhibition of the TGFb-1 signalingcascade, tenascin-C production is probably reduced,which is one of the methods to elucidate tenascin-Cfunction in SCI.

In conclusion, we reported here the gene expressionpattern of five microarray-screened genes that arethought to be typical in each stage of cell infiltrationafter SCI. These data may be helpful in clarifying thepathological reaction after SCI and to establish newtherapeutic approach for SCI.

Acknowledgements This work was supported by grants-in-aid toKatsunori Yoshinaga for scientific research from the Ministry ofEducation, Science and Culture of Japan. We thank Dr. ShintaroNomura, Osaka University, Osaka, Japan, for kindly providing theplasmid containing OPN cDNA, and Drs. Masamichi Tahara andMasatoshi Komiyama, Chiba University Graduate School ofMedicine, for their technical advice and useful discussions.

References

1. Abe Y, Nakamura H, Yoshino O, Oya T, Kimura T (2003)Decreased neural damage after spinal cord injury in tPA-defi-cient mice. J Neurotrauma 20:43–57

2. Abel J, Ruiter N de (1989) Inhibition of hydroxyl-radical-generated DNA degradation by metallothionein. Toxicol Lett47:191–196

3. Baldwin SA, Broderik R, Blades DA, Scheff SW (1998)Alterations in temporal/spatial distribution of GFAP- and vi-mentin-positive astrocytes after spinal cord contusion with theNew York University spinal cord injury device. J Neurotrauma15:1015–1026

4. Banik NL, Hogan EL, Powers JM, Smith KP (1986) Proteo-lytic enzymes in experimental spinal cord injury. J Neurol Sci73:245–256

5. Blight AR (1985) Delayed demyelination and macrophageinvasion: a candidate for secondary cell damage in spinal cordinjury. Cent Nerv Syst Trauma 2:299–315

6. Blum JS, Fiani ML, Stahl PD (1991) Proteolytic cleavage ofricin A chain in endosomal vesicles. Evidence for the action ofendosomal proteases at both neutral and acidic pH. J BiolChem 266:22091–22095

7. Boehme DH, Umezawa H, Hashim G, Marks N (1978)Treatment of experimental allergic encephalomyelitis with aninhibitor of cathepsin D (pepstatin). Neurochem Res 3:185–194

8. Brostoff SW, Reuter W, Hichens M, Eylar EH (1974) Specificcleavage of the A1 protein from myelin with cathepsin D. J BiolChem 249:559–567

9. Campagne MV, Thibodeaux H, Bruggen N van, Cairns B,Gerlai R, Palmer JT, Williams SP, Lowe DG (1999) Evidencefor protective role of metallothionein-1 in focal cerebralischemia. Proc Natl Acad Sci USA 96:12870–12875

10. Campagne MV, Thibodeaux H, Bruggen N van, Cairns B,Lowe DG (2000) Increased binding activity at an antioxidant-responsive element in the metallothionein-1 promoter and rapidinduction of metallothionein-1 and �2 in response to cerebralischemia and reperfusion. J Neurosci 20:5200–5207

11. Carlson SL, Parish ME, Springer JE, Doty K, Dossett L (1998)Acute inflammatory response in spinal cord following impactinjury. Exp Neurol 151:77–88

12. Carmel JB, Galante A, Soteropoulos P, Tolias P, Recce M,Young W, Hart RP (2001) Gene expression profiling of acutespinal cord injury reveals spreading inflammatory signals andneuron loss. Physiol Genomics 7:201–213

178

13. Carmel JB, Kakinohana O, Mestril R, Young W, Marsala M,Hart RP (2004) Mediators of ischemic preconditioning identi-fied by microarray analysis of rat spinal cord. Exp Neurol185:81–96

14. Cassina P, Peluffo H, Pehar M, Martinez-Palma L, Ressia A,Beckman JS, Estevez AG, Barbeito L (2002) Peroxynitritetriggers a phenotypic transformation in spinal cord astrocytesthat induces motor neuron apotosis. J Neurosci Res 67:21–29

15. Deckner M, Lindholm T, Cullheim S, Risling M (2000) Dif-ferential expression of tenascin-C, tenascin-R, tenascin/J1, andtenascin-X in spinal cord scar tissue and in the olfactory sys-tem. Exp Neurol 166:350–362

16. Deiss LP, Galinka H, Berissi H, Cohen O, Kimchi A (1996)Cathepsin D protease mediates programmed cell death inducedby interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J15:3861–3870

17. Farooque M, Hillered L, Holtz A, Olsson Y (1996) Effects ofmethylprednisolone on extracellular lactic acidosis and aminoacids after severe compression injury of rat spinal cord. JNeurochem 66:1125–1130

18. Fawcett JW, Asher RA (1999) The glial scar and central ner-vous system repair. Brain Res Bull 49:377–391

19. Ferhat L, Chevassus au Louis N, Jorquera I, Niquet J,Khrestchatisky M, Ben-Ari Y, Represa A (1996) Transientincrease of tenascin-C in immature hippocampus: astroglial andneuronal expression. J Neurocytol 25:53–66

20. Fu Y, Hashimoto M, Ino H, Murakami M, Yamazaki M,Moriya H (2003) Spinal root avulsion–induced up-regulation ofosteopontin expression in the adult rat spinal cord. ActaNeuropathol 107:8–16

21. Gasull T, Giralt M, Garcia A, Hidalgo J (1994) Regulation ofmetallothionein-I+II levels in specific brain areas and liver inthe rat: role of catecholamines. Glia 12:135–143

22. Giralt M, Penkowa M, Lago N, Molinero A, Hidalgo J (2002)Metallothionein-1+2 protect the CNS after a focal brain in-jury. Exp Neurol 173:114–128

23. Hashimoto M, Koda M, Ino H, Murakami M, Yamazaki M,Moriya H (2003) Upregulation of osteopontin expression in ratspinal cord microglia after traumatic injury. J Neurotrauma20:287–296

24. Hellmich HL, Frederickson CJ, DeWitt DS, Saban R, ParsleyMO, Stephenson R, Velasco M, Uchida T, Shimamura M,Prough DS (2004) Protective effects of zinc chelation in trau-matic brain injury correlate with upregulation of neuroprotec-tive genes in rat brain. Neurosci Lett 355:221–225

25. Herschman HR (1985) A 12-O-tetradecanoylphorbol-13-ace-tate (TPA)-nonproliferative variant of 3T3 cells is resistant toTPA-enhanced gene amplification. Mol Cell Biol 5:1130–1135

26. Ikeda O, Murakami M, Ino H, Yamazaki M, Nemoto T, KodaM, Nakayama C Moriya H (2001) Acute up-regulation ofbrain-derived neurotrophic factor expression resulting fromexperimentally induced injury in the rat spinal cord. ActaNeuropathol 102:239–245

27. Ikeda O, Murakami M, Ino H, Yamazaki M, Koda M,Nakayama C, Moriya H (2002) Effects of brain-derived neu-rotrophic factor (BDNF) on compression-induced spinal cordinjury: BDNF attenuates down-regulation of superoxidedismutase expression and promotes up-regulation of myelinbasic protein expression. J Neuropathol Exp Neurol 61:142–153

28. Kato H, Kanellopoulos GK, Matsuo S, Wu YJ, Jacquin MF,Hsu CY, Kouchoukos NT, Choi DW (1997) Neuronal apop-tosis and necrosis following spinal cord ischemia in the rat. ExpNeurol 148:464–474

29. Kenessy A, Banay-Schwartz M, DeGuzman T, Lajtha A (1989)Increase in cathepsin D activity in rat brain in aging. J NeurosciRes 23:454–456

30. Koike M, Nakanishi H, Saftig P, Ezaki J, Isahara K, OhsawaY, Schulz-Schaeffer W, Watanabe T, Waguri S, Kametaka S,Shibata M, Yamamoto K, Kominami E, Peters C, Figura Kvon, Uchiyama Y (2000) Cathepsin D deficiency induces lyso-

somal storage with ceroid lipofuscin in mouse CNS neurons. JNeurosci 15:6898–6906

31. Kondo Y, Rusnak JM, Hoyt DG, Settineri CE, Pitt BR, LazoJS (1997) Enhanced apoptosis in metallothionein null cells. MolPharmacol 52:195–201

32. Laywell ED, Dorries U, Bartsch U, Faissner A, Schachner M,Steindler DA (1992) Enhanced expression of the developmen-tally regulated extracellular matrix molecule tenascin followingadult brain injury. Proc Natl Acad Sci USA 89:2634–2638

33. Liu XZ, Xu XM, Hu R, Du C, Zhang SX, McDonald JW,Dong HX, Wu YJ, Fan GS, Jacquin MF, Hsu CY, Choi DW(1997) Neuronal and glial apoptosis after traumatic spinal cordinjury. J Neurosci 17:5395–5406

34. Long Y, Zou L, Liu H, Lu H, Yuan X, Robertson CS, Yang K(2003) Altered expression of randomly selected genes in mousehippocampus after traumatic brain injury. J Neurosci Res71:710–720

35. Matsuda A, Yoshiki A, Tagawa Y, Matsuda H, Kusakabe M(1999) Corneal wound healing in tenascin knockout mouse.Invest Ophthalmol Vis Sci 40:1071–1080

36. McKee MD, Nanci A (1996) Secretion of osteopontin bymacrophages and it’s accumulation at tissue surfaces duringwound healing in mineralized tissues : a potential requirementfor macrophage adhesion and phagocytosis. Anat Rec 245:394–409

37. Montoliu C, Monfort P, Carrasco J, Palacios O, Capdevila M,Hidalgo J, Felipo V (2000) Metallothionein-III prevents glu-tamate and nitric oxide neurotoxicity in primary cultures ofcerebellar neurons. J Neurochem 75:266–273

38. Mufson EJ, Counts SE, Ginsberg SD (2002) Gene expressionprofiles of cholinergic nucleus basalis neurons in Alzheimer’sdisease. Neurochem Res 27:1035–1048

39. Pan JZ, Ni L, Sodhi A, Aguanno A, Young W, Hart RP (2002)Cytokine activity contributes to induction of inflammatorycytokine mRNAs in spinal cord following contusion. J Neu-rosci Res 68:315–322

40. Pearson CA, Pearson D, Shibahara S, Hofsteenge J, Chiquet-Ehrismann R (1988) Tenascin: cDNA cloning and induction byTGF-beta. EMBO J 7:2977–2982

41. Penkowa M, Hidalgo J (2003) Treatment with metallothioneinprevents demyelination and axonal damage and increases oli-godendrocyte precursors and tissue repair during experimentalautoimmune encephalomyelitis. J Neurosci Res 72:574–586

42. Reinholt FP, Hultenby K, Oldberg A, Heinegard D (1990)Osteopontin—a possible anchor of osteoclasts to bone. ProcNatl Acad Sci USA 87:4473–4475

43. Rollo EE, Laskin DL, Denhardt DT (1996) Osteopontininhibits nitric oxide production and cytotoxicity by activatedRAW264.7 macrophages. J Leukoc Biol 60:397–404

44. Sasagawa S, Matsubara J, Satow Y (1993) Stress-relatedinduction of hepatic metallothionein synthesis and increase inperipheral polymorphonuclear leukocytes in mice. Immunop-harmacal Immunotoxicol 15:217–226

45. Scatena M, Almeida M, Chaisson ML, Fausto N, Nicosia RF,Giachelli CM (1998) NF-jB mediates avb3 integrin-inducedendothelial cell survival. J Cell Biol 141:1083–1093

46. Schmidt C, Beyersmann D (1999) Transient peaks in zinc andmetallothionein levels during differentiation of 3T3L1 cells.Arch Biochem Biophys 364:91–98

47. Schnell L, Fearn S, Klassen H, Schwab ME, Perry VH (1999)Acute inflammatory responses to mechanical lesions in theCNS: differences between brain and spinal cord. Eur J Neurosci11:3648–3658

48. Senger DR, Ledbetter SR, Claffey KP (1996) Stimulation ofendothelial cell migration by vascular permeability factor/vas-cular endothelial growth factor through cooperative mecha-nisms involving the avb3 integrin osteopontin, and thrombin.Am J Pathol 149:293–305

49. Smith GM, Hale JH (1997) Macrophage/microglia regulationof astrocytic tenascin: synergistic action of transforminggrowth factor-beta and basic fibroblast growth factor. J Neu-rosci 17:9624–9633

179

50. Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X,Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H,Wood III WH, Becker KG, Ko MSH (2000) Genome-wideexpression profiling of mid-gestation placenta and embryousing a 15000 mouse developmental cDNA microarray. ProcNatl Acad Sci USA 97:9127–9132

51. Tang X, Davies JE, Davies SJA (2003) Changes in distribution,cell association, and protein expression levels of NG2, neuro-can, phosphacan, brevican, versican V2, and tenascin-C duringacute to chronic maturation of spinal cord scar tissue. J Neu-rosci Res 71:427–444

52. Terai K, Takano-Yamamoto T, Ohba Y, Hiura K (1999) Roleof osteopontin in bone remodeling caused by mechanical stress.J Bone Miner Res 14:839–849

53. Tsangaris GT, Tzortzatou-Stathopoulou F (1998) Metallothi-onein expression prevents apoptosis: a study with antisensephosphorothioate oligodeoxynucleotides. Anticancer Res18:2423-2434

54. Wada S, Yone K, Ishidou Y, Nagamine T, Nakahara S,Niiyama T, Sakou T (1999) Apoptosis following spinal cordinjury in rats and preventative effect of N-methyl-D-aspartatereceptor antagonist. J Neurosurg 91(Suppl 1):98–104

55. Wallner K, Li C, Shah PK, Fishbein MC, Forrester JS, Kaul S,Sharifi BG (1999) Tenascin-C is expressed in macrophage-richhuman coronary atherosclerotic plaque. Circulation 99:1284–1289

56. Wang XK, Louden C, Yue TL, Ellison JA, Barone FC (1998)Delayed expression of osteopontin after focal stroke in the rat.J Neurosci 18:2075–2083

57. Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H,Ashkar S (2002) Phosphorylation-dependent interaction ofosteopontin with its receptors regulates macrophage migrationand activation. J Leukoc Biol 72:752–761

58. Whitaker JN, Rhodes RH (1983) The distribution of cathepsinD in rat tissues determined by immunohistochemistry. Am JAnat 166:417–428

59. Yick LW, Wu W, So KF, Wong SY (1998) Time course ofNOS expression and neuronal death in Clarke’s nucleus fol-lowing traumatic injury in adult rat spinal cord. Neurosci Lett241:155–158

60. Yue TL, McKenna PJ, Ohlstein EH, Farach-Carson MC(1994) Osteopontin-stimulated vascular smooth muscle cellmigration is mediated by b3 integrin. Exp Cell Res 214:459–464

61. Zhang Y, Anderson PN, Campbell G, Mohajeri H, SchachnerM, Lieberman AR (1995) Tenascin-C expression by neuronsand glial cells in the rat spinal cord: changes during postnataldevelopment and after dorsal root or sciatic nerve injury. JNeurocytol 24:585–601

62. Zhang Y, Winterbottom JK, Schachner M, Lieberman AR,Anderson PN (1997) Tenascin-C expression and axonalsprouting following injury to the spinal dorsal columns in theadult rat. J Neurosci Res 49:433–450

180