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Research Report

Immunohistochemical study of flotillin-1 in the spinal cord ofLewis rats with experimental autoimmune encephalomyelitis

Heechul Kima, Meejung Ahna, Changjong Moona, Yoh Matsumotob,Chang Sung Kohc, Taekyun Shina,⁎aDepartment of Veterinary Medicine, Cheju National University, Jeju 690-756, South KoreabDepartment of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183, JapancDepartment of Biomedical Laboratory Sciences, Shinshu University School of Health Sciences, 3-1-1 Asahi, Matsumoto 390-8621, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +82 64 756 3354.E-mail address: shint@cheju.ac.kr (T. Shin

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.07.054

A B S T R A C T

Article history:Accepted 14 July 2006Available online 21 August 2006

We analyzed flotillin-1 expression in the spinal cords of Lewis rats with experimentalautoimmune encephalomyelitis (EAE). Western blot analysis showed that flotillin-1expression increased significantly in the spinal cords from rats at the peak stage of EAEcompared with the levels in control animals (p<0.05) and declined thereafter.Immunohistochemistry demonstrated that flotillin-1 was expressed constitutively inthe gray matter (particularly in the dorsal horn) of the normal rat spinal cord and insome neurons and glial cells. In EAE lesions, flotillin-1 immunoreactivity was detectedin some macrophages and astrocytes, in which cathepsin D, a lysosomal marker, waslocalized. In the spinal cord cells in EAE, there was increased expression of flotillin-1above the constitutive expression of flotillin-1 in normal spinal cords. Taking all thesefindings into consideration, we postulate that expression of flotillin-1 begins to increasewhen EAE is initiated and that flotillin-1 contributes to the formation of phagosomes inaffected cells in EAE.

© 2006 Elsevier B.V. All rights reserved.

Keywords:AstrocytesCathepsin DExperimental autoimmuneencephalomyelitisFlotillin-1Macrophages

1. Introduction

Flotillin-1 and the associated protein flotillin-2 (epidermalsurface antigen) are important structural proteins in lipid rafts(Bickel et al., 1997). These proteins have been included in anexpanding list of proteins, including protein kinase C alpha,Ras, Rap Src-like kinase, and glycosylphosphatidyinositol-linked receptors, that are colocalized at caveolae (Harris et al.,2002; Razani et al., 2002).

Flotillins are particularly enriched in the detergent-insoluble glycolipid-enriched membrane domains that areinvolved in both signal transduction and vesicular trafficking

).

er B.V. All rights reserved

(Lang et al., 1998; Hooper, 1999), as well as in proteintyrosine nitration in the brain (Dremina et al., 2005). In thediseased brain, flotillins are associated with the productionof beta-amyloid in flotillin-enriched lipid rafts, which mayreflect the progression of Alzheimer's disease pathology (Leeet al., 1998; Kokubo et al., 2000). Flotillin-1 was localizedpredominantly to catecholaminergic nerves in the substantianigra in a rat model of Parkinson's disease (Jacobowitz andKallarakal, 2004). In addition to their constitutive neuronalexpression and increased expression in the brains of animalmodels of Alzheimer's (Kokubo et al., 2000) and Parkinson's(Jacobowitz and Kallarakal, 2004) diseases, flotillins are

.

Fig. 1 – Western blot analysis of flotillin-1 expression inspinal cords from rats with experimental autoimmuneencephalomyelitis (EAE). Samples of spinal cord werecollected from normal control rats, and from rats at the peakof EAE [14 days post-immunization (p.i.); stage G.3], andduring recovery from EAE (day 21 p.i.; stage R.0).(A) Representative photographs of Western blots witharrowheads indicating the expression of flotillin-1 (48 kDa)and beta-actin (45 kDa). (B) Semiquantitative analysis offlotillin-1 immunoreactivity in the spinal cord normalized tothe intensity of beta-actin expression in the sameimmunoblot. The controlwas assigned a value of 1. Flotillin-1expression was significantly greater in spinal cords from ratskilled at the peak of EAE (p<0.05) than in spinal cords fromcontrol rats, and the expression level declined to controllevels during recovery from EAE. Data (mean±SEM) are fromthree experiments.

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abundant within the phagosomes of phagocytes, whichsuggests that flotillins are involved in phagocytic processes(Dermine et al., 2001; Garin et al., 2001; Li et al., 2003). Basedon previous studies of flotillins in the diseased brain (Bickelet al., 1997; Kokubo et al., 2000), it is possible that flotillinsare involved in the pathogenesis of immune-mediatedautoimmune diseases within the CNS.

A group of proteases in the endosomal/lysomal proteolyticsystem are called cathepsins, which is derived from the Greekterm meaning “to digest” (Nakanishi, 2003). Cathepsin D is amajor component of lysosomes and functions as a highlyactive endopeptidase with an optimum pH between 3.0 and5.0 (Steinfeld et al., 2006). Cathepsins often leak into thecytoplasm following the endocytosis of oxidizable substratesthat destabilize the lysosomal membranes through lipidperoxidation (Nakanishi, 2003). Cathepsin D expression isincreased in macrophages in chronic obstructive pulmonarydisease (Bracke et al., 2005). In addition, cathepsin D mediatescellular processes, such as apoptosis in macrophages duringsilicosis (Thibodeau et al., 2004).

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune disease model that is character-ized by the infiltration of T cells and macrophages into thesubarachnoid space during the early stage and the activationof microglia and astrocytes during the peak (symptomatic)stage of the disease (Shin et al., 1995). Cell activation events,such as increased expression of cell adhesion molecules onvascular endothelial cells (Shin et al., 1995), activation ofmitogen-activated protein kinases (Shin et al., 2003), astro-cyte proliferation, activation of brain macrophages, andapoptosis of inflammatory cells, are typical findings in bothEAE and multiple sclerosis lesions (Schonrock et al., 1998).The activation of neurons and phagocytes in EAE lesions isassociated with an increase in the expression of caveolins, astructural protein in lipid rafts (Shin et al., 2005). However,while flotillins also occur within lipid rafts, flotillins differfrom caveolins in that the former are more abundant withinthe CNS (Bickel et al., 1997) and are involved in thematuration of phagosomes in macrophages (Li et al., 2003).The pattern of flotillin expression in animals with EAE isunknown.

This study elucidated the pattern of flotillin-1 expression inspinal cords from rats with EAE and determined the pheno-types of the cells that are involved.

2. Results

2.1. Increased expression of flotillin-1 in the spinal cord inEAE

Western blot analysis was used to examine flotillin-1expression in the spinal cord. Flotillin-1 was expressedconstitutively in spinal cords from normal control rats, butits expression was significantly greater (approximately five-fold increase vs. control; p<0.05) in spinal cords in EAE at thepeak stage (Fig. 1). Flotillin-1 immunoreactivity during therecovery stage of EAE declined to the level of normal controlrats (Fig. 1).

2.2. Localization of flotillin-1 in spinal cord sections fromEAE-affected rats

2.2.1. Flotillin-1 immunoreactivityImmunohistochemically, flotillin-1 was detected in a few glialcells in the spinal cords of normal rats (Fig. 2A). In the peakstage of EAE (G3, day 10 post-immunization (p.i.)), manyflotillin-1-positive glial cells were detected in the white(Fig. 2B) and gray (data not shown) matter of the spinal cord.In addition, during the peak stage of EAE, there was massiveinfiltration of inflammatory cells in the parenchyma, wheresome round cells were positive for flotillin-1 (Fig. 2C). In therecovery stage of EAE (R0, day 21 p.i.), there were fewerinflammatory cells than in the peak stage; a few glial cellswere positive for flotillin-1 (Fig. 2D).

Furthermore, diffuse flotillin-1 immunoreactivity wasdetected in the dorsal horn lamina in the spinal cords ofnormal rats (Fig. 3A). In the peak stage of EAE (G3, day 14 p.i.),flotillin-1 immunoreactivity in the dorsal horn lamina wasincreased compared with normal control rats (Fig. 3B); theimmunoreactivity decreased slightly in the recovery stage ofEAE (Fig. 3C). In addition, some flotillin-1-positive ependymalcells were detected in the spinal cords of normal control ratsand rats with EAE (data not shown).

Fig. 2 – Immunohistochemical staining of flotillin-1 in the spinal cord of normal control rats (A) and rats at the peak (day 14 p.i.)(B, C) and recovery stages of EAE (day 21 p.i.) (D). Flotillin-1 was weakly detected in some glial cells in the spinal cord of normalcontrol rats (A, arrow). In the peak stage of EAE (B andC), glial cells showed increased immunoreactivity for flotillin-1 (B, arrows),and flotillin-1-positive inflammatory cellswere detected in the spinal cord parenchyma (C, arrows). In the recovery stage of EAE,some flotillin-1 immunoreactive cells remained (D, arrows). Counterstained with hematoxylin. Scale bars=50 μm.

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2.2.2. Identification of flotillin-1-positive cells in the spinalcords of normal controls and rats with EAEThe pattern of flotillin-1 immunofluorescence in the spinalcords of normal control rats and rats with EAE was similar tothat seen with single immunoperoxidase staining (Fig. 2).

Very little flotillin-1 (Fig. 4A, red) was immunodetected inGFAP-positive astrocytes (Fig. 4B, green) in the spinal cords ofnormal control rats (Fig. 4C, merge).

In the spinal cord in EAE (day 14 p.i.), flotillin-1-positiveastrocytes increased (Figs. 4D–F). In addition, flotillin-1 (Fig.4G, red) was abundant in ED1-positive cells (Fig. 4H, green; Fig.4I, merge). This suggests that themajority of macrophages arepositive for flotillin-1 in EAE lesions at the peak stage. Inaddition, some flotillin-1 was also localized in ED2-positiveresident macrophages (Fig. 5). In frozen sections reacted withflotillin-1 antibody and R73, some T cells were positive forflotillin-1 (data not shown).

The intensity of the flotillin-1 immunoreactivity in thenormalcontrol rats and EAE-affected rats is summarized in Table 1.

2.2.3. Immunofluorescence of cathepsin D in spinal cords inEAETo examine the relationship between flotillin-1 and cathepsinD, double labelingwas performed (Figs. 6A–I). In the peak stage

of EAE (day 14 p.i.), cathepsinDwas colocalized in ED1-positivemacrophages (Figs. 6A–C) and in GFAP-positive astrocytes(Figs. 6D–F). Furthermore, cathepsin D immunoreactivity wascolocalized in flotillin-1-positive cells (Figs. 6G–I). This sug-gests that flotillin-1 is closely associated with cathepsin D inmacrophages and astrocytes in EAE.

3. Discussion

This is the first demonstration that the level of flotillin-1expression increases within the CNS during autoimmuneinflammation. Previously, we found that expression of caveo-lins was enhanced in the spinal cord of EAE-affected rats andthat different caveolin isotypes were expressed differently invascular endothelial cells and astrocytes (Shin et al., 2005).The results of the present study indicate that the expression offlotillin-1 increases significantly in the spinal cord of EAE-affected rats, which suggests that flotillin-1 is responsive tothe progression of EAE.

Flotillin-1 is associated with lipid rafts, which are impor-tant sites for signal transduction and vesicular trafficking(Bickel et al., 1997). Therefore, we speculate that enhancedexpression of flotillin-1 in the dorsal horn lamina in EAE-

Fig. 3 – Immunohistochemical staining of flotillin-1 in thedorsal horn lamina in the spinal cords of normal control rats(A) and rats at the peak (day 14 p.i.) (B) and recovery stages ofEAE (day 21 p.i.) (C). Flotillin-1 was constitutivelyimmunostained in the dorsal horn lamina in the normalcontrols; its immunoreactivity increased in the dorsal hornlamina in the peak stage (B), and it declined slightly in therecovery stage of EAE (C). Counterstained with hematoxylin.Scale bars=200 μm.

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affected spinal cord reflects a role for this protein in the activetrafficking of various molecules into CNS neurons. Thissuggests that flotillin-1 is involved in the activation ofneuronal cells in EAE, although the precise roles of flotillin-1in neuronal cells remain to be determined.

There is a consensus of opinion that flotillin-1 is associatedwith the formation of phagosomes in macrophage cell lines(Dermine et al., 2001; Garin et al., 2001; Ng Yan Hing et al.,2004). In EAE lesions,macrophages participate in the clearanceof apoptotic cells and cell debris, through the formation ofphagosomes including lysosomes. We also confirmed thatflotillin-1 was colocalized with cathepsin D, a lysosomalmarker, as shown in a previous paper (Girardot et al., 2003).Therefore, these data indicate that flotillin-1, a marker forlipid rafts, accumulates in lysosomes in macrophages andastrocytes in EAE lesions.

The lysosomal enzyme activity initiates activation of thedeath signal during apoptosis in macrophages (Thibodeau

et al., 2004). In addition, flotillin-1 and cathepsin D areassociated with cellular apoptosis in neuronal cells andmacrophages, respectively (Thibodeau et al., 2004; Cheunget al., 2004). In this study, we found that flotillin-1 andcathepsin D were localized mainly in ED1-positive macro-phages at the peak stage of EAE. In addition, flotillin-1 wascolocalized in cathepsin D-positive cells in the perivascularcuffs in EAE lesions. It has been reported that the majorityof inflammatory T cells and ED1-positive macrophages areeliminated via apoptosis with expression of death signalsduring the peak stage of EAE (Moon et al., 2000). Consider-ing these results, it is highly possible that the increasedexpression of flotillin-1 in ED1-positive macrophages stimu-lates lysosomal enzyme activity in the spinal cord of ratswith EAE, leading to apoptosis of ED1-positive macrophages.

A previous study suggested that resident macrophages actas antigen-presenting cells and are involved in re-stimulationof myelin basic protein (MBP)-sensitized T cells after theyinfiltrate across the blood–brain barrier in EAE (Polfliet et al.,2002). In the CNS, amajor population of residentmacrophagesconsist of perivascular and meningeal macrophages (Polflietet al., 2002) that are positive for ED2 (Dijkstra et al., 1994). Sincesome ED2-positive macrophages also contain flotillin-1, wecannot rule out the possibility that both ED1-positive hema-togenous and ED2-positive residentmacrophages are involvedin antigen processing in rat EAE.

Although the role of flotillins in T cells is largely known, theactivation of CD4+ T cells is associated with the increasedexpression of flotillin-1 in surface membranes (Slaughter etal., 2003). In this study, only a few T cells were found to bepositive for flotillin-1 in EAE lesions. This suggests that, inEAE, T cells in the activation stage in peripheral lymphoidorgans express flotillin-1 and stop its expression in the targetorgan, i.e., the spinal cord.

Collectively, our results suggest that a lipid raft protein,flotillin-1, plays an important role in immune and neuronalcells within the CNS during EAE, possibly via the activation ofsignal transduction and lysosomal activity.

4. Experimental procedures

4.1. Animals

Lewis rats were obtained from Harlan (Indianapolis, IN) andwere bred in our animal facility. Female rats (7–12 weeks old;160–200 g) were used in this study. All experiments followedaccepted ethical guidelines.

4.2. Induction of experimental autoimmuneencephalomyelitis

The footpads of both hind feet of rats in the EAE group wereinjected with 100 μl of an emulsion that contained equal partsof myelin basic protein (1 mg/ml) and complete Freund'sadjuvant (CFA) supplemented with Mycobacterium tuberculosisH37Ra (5 mg/ml) (Difco, Detroit, MI). Control rats wereimmunized with CFA only. After immunization, the ratswere observed daily for clinical signs of EAE. The progressionof EAE was divided into seven clinical stages: Grade 0 (G.0), no

Fig. 4 – Immunofluorescent colocalization of flotillin-1 (A, D, G; red) with either anti-GFAP (B, E; green) or ED1 (H; green) in thespinal cords of normal control rats (A–C) and rats at the peak stage of EAE (day 14 p.i.) (D–I). In the normal controls, someflotillin-1-immunopositive glial cells were colocalized in GFAP-positive astrocytes in the spinal cords (A–C). In the EAE-affectedspinal cords, many flotillin-1-positive glial cells were positive for GFAP (D–F). In addition, some flotillin-1-positive cells werecolocalized in ED1-positive macrophages or activated microglia (G–I). C, F, and I are merged images. Scale bars=20 μm. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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signs; G.1, floppy tail; G.2, mild paraparesis; G.3, severeparaparesis; G.4, tetraparesis; G.5, moribund condition ordeath; and R.0, recovery.

4.3. Antibodies

Rabbit polyclonal anti-flotillin-1 (clone H104) and mouse anti-flotillin-1 antibodies (IgG1, clone 18, molecular weight 48 kDa)were obtained from Santa Cruz Biotechnology (Santa Cruz, CA)and BD Biosciences (San Jose, CA), respectively. Goat anti-cathepsin D (IgG, clone R-20, catalog no. sc-6487), a lysosomalmarker (Girardot et al., 2003), was obtained from Santa Cruz.Mouse monoclonal anti-beta-actin and mouse anti-GFAP wereobtained from Sigma (St. Louis, MO). ED1 (mouse monoclonalanti-rat macrophages; ED1 recognizes a lysosomal membrane-related antigen on both monocytes and macrophages (Dijkstra etal., 1994)) and ED2 (mouse monoclonal anti-rat macrophages; ED2recognizes an antigen on a subset of mature rat macrophagesincluding the perivascular and meningeal macrophages in the

CNS (Dijkstra et al., 1994)) were obtained from Serotec (London,UK). R73 (monoclonal anti-T cell receptor αβ) was obtained fromBlackthorn (Bicester, Bucks, UK).

4.4. Tissue sampling

The rats were sacrificed under ether anesthesia. Spinal cordswere dissected from each group at 12–14 and 21 days post-immunization (n=5 rats/group); these periods coincided with thepeak (G.3, day 12–14 p.i.) and recovery (R.0, day 21 p.i.) stages ofEAE. Samples of the spinal cordswereprocessed for embedding inparaffinwax after fixation in 4%paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). Paraffin sections (5 μm thick) wereused for all immunostaining, except for the T cell marker R73. ForT cell immunostaining, pieces of the spinal cords were snap-frozen in optimal cutting temperature (OCT) compound (Sakura,Tokyo, Japan), and sections (8-μm thick) were cut using a cryostat(Leica, Nussloch, Germany). Additional samples of the spinalcords were snap-frozen and stored for immunoblot analysis.

Fig. 5 – Immunofluorescence colocalizationof flotillin-1 (A)with ED2 (B) in the spinal cords of rats in thepeak stageof EAE (day14p.i.). Some flotillin-1 (A, red; arrows) was immunostained in ED2-positive resident macrophages (B, green; arrows) (C, merge;arrows). Scale bars=20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

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4.5. Western blot analysis

Spinal cord tissue was homogenized in lysis buffer (40 mMTris, 120 mM NaCl, 0.1% Nonidet 40, 2 mM Na3VO4, 1 mMphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/mlleupeptin) with 20 strokes in a homogenizer. The homogenatewas transferred into microtubes that were centrifuged at14,000 rpm for 20 min, after which the supernatant washarvested.

For the immunoblot assay, supernatant that contained20 μg of protein was loaded into individual lanes of 10%sodium dodecyl (lauryl) sulfate-polyacrylamide gels, electro-phoresed, and immunoblotted onto nitrocellulose mem-branes (Schleicher and Schuell, Keene, NH). The residualbinding sites on the membrane were blocked by incubationwith 5% nonfat milk in Tris-buffered saline (TBS; 10 mM Tris–HCl, pH 7.4, and 150 mM NaCl) for 1 h. Subsequently, themembrane was incubated for 2 h with mouse monoclonalanti-flotillin-1 (1:3000 dilution) antibody. The membraneswere washed three times in TBS that contained 0.1% Tween20 before being incubated with horseradish-peroxidase-con-jugated anti-mouse IgG (Vector, Burlingame, CA) for 1 h.Bound antibodies were detected using enhanced chemilumi-nescence reagents (Amersham, Arlington Heights, IL) accord-ing to the manufacturer's instructions. After imaging, themembranes were stripped and reprobed using anti-beta-actin antibody (Sigma). The optical density (per mm2) of eachband was measured with a scanning laser densitometer (GS-700, Bio-Rad, Hercules, CA), and these values are presentedas means±SEM. The ratios of the density of each flotillinband relative to that of the beta-actin band were comparedusing Molecular Analyst software (Bio-Rad).

The data were analyzed using one-way ANOVA followed bythe Student–Newman–Keuls post hoc test for multiple compa-risons. In all cases, p<0.05 was taken as statistically significant.

4.6. Immunohistochemistry

Paraffin-embedded spinal cord sections (5 μm) were depar-affinized, treated with citrate buffer (0.01 M, pH 6.0) in amicrowave for 3 min, and then treated with 0.3% hydrogen

peroxide in methyl alcohol for 20 min to block endogenousperoxidase activity. After three washes with PBS, the sectionswere incubated with 10% normal goat serum and thenincubated for 1 h at room temperature with mouse mono-clonal flotillin-1. Immunoreactivity was visualized using theavidin–biotin peroxidase complex (Vector Elite; Vector). Theperoxidase reaction was developed using a diaminobenzidinesubstrate kit (Vector).

To examine the cell phenotype of flotillin-1 expression,double immunofluorescence was applied using cell-type-specific markers, including ED1 for monocyte-like macro-phages, ED2 for resident macrophages, anti-GFAP for astro-cytes, and R73 for T cells. First, paraffin sections were reactedsequentially with primary rabbit anti-flotillin-1, secondarybiotinylated anti-rabbit IgG (Vector, Burlingame, CA), andtetramethyl rhodamine isothiocyanate (TRITC)-labeled strep-tavidin (Zymed, San Francisco, CA). Then, the slides wereincubated with second primary reagents, including ED1, ED2,and anti-GFAP, and this was followed with fluoresceinisothiocyanate (FITC)-labeled goat anti-mouse IgG (1:50 dilu-tion; Sigma). To label flotillin-1 and R73, frozen sections wereused for the procedure described above.

To see the colocalization of cathepsin D with ED1, anti-GFAP, or anti-flotillin-1 in EAE lesions, sections immunos-tainedwith one of theprimary reagents, e.g., ED1, anti-GFAP, orrabbit anti-flotillin-1, were reacted with biotinylated anti-mouse mouse IgG or anti-rabbit IgG (Vector, Burlingame, CA)followed by TRITC-labeled streptavidin (Zymed, San Francisco,CA), and then secondary primary antisera (anti-cathepsin D,×400 dilution) was reacted and followed by FITC-labeled rabbitanti-goat IgG (Sigma).

To reduce or eliminate lipofuscin autofluorescence, thesections were washed in PBS (three times for 1 h) at RT, dippedbriefly in distilled H2O, treated with 10 mM CuSO4 inammonium acetate buffer (50 mM CH3COONH4, pH 5.0) for20min, dipped briefly again in distilledH2O, and then returnedto PBS. The double immunofluorescence-stained specimenswere examined with an FV500 laser confocal microscope(Olympus, Tokyo, Japan).

To evaluate the immunoreactivity of flotillin-1 in the spinalcords, two blinded observers examined three different

Table 1 – Immunoreactivity of flotillin-1 in the spinal cords of normal and CFA-immunized rats and in rats with EAE

Antibody and cell type Normal CFA EAE G3 (day 14 p.i.) a EAE R0 (day 21 p.i.)

Flotillin-1b

Dorsal horn lamina Weakc Weak Intense ModerateNeurons +d + + +Astrocytes + + +++ ++Macrophagese NDf ND +++ ++R73-positive T cells ND ND ++ +Ependymal cells Weak Weak Intense ModerateVessels Weak Weak Moderate Weak

a Rat spinal cords were obtained at days 14 (EAE G3) and 21 (EAE R0) post-immunization (p.i.).b Three different sections from three animals in each group were examined by two blinded observers.c The intensity of flotillin-1 in dorsal horn lamina, ependymal cells, and vessels is arbitrarily classified as weak, moderate, and intense by twoblind observers.d The presence of immunoreactive cells in the spinal cords of each group is expressed as negative (−), <10 cells positive (+), 10–30 cells (++), and>30 cells (+++) under ×20 magnification of an objective lens.e Macrophages included activated microglia and/or ED1-positive cells.f ND: no inflammatory cells were observed in the spinal cords of normal rats.

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sections from three rats per group. The intensity of flotillin-1immunoreactivity in the dorsal horn lamina, ependymal cells,and vessels in the spinal cords was arbitrarily classified asweak, moderate, and intense. Flotillin-1 immunoreactivity in

Fig. 6 – Immunofluorescence colocalization of cathepsin D (A, D, Gcords of rats in the peak stage of EAE (day 14 p.i.). Arrows indicate(A–C), GFAP-positive astrocytes (D–I), and flotillin-1-positive cells

astrocytes, neurons, macrophages, and T cells was quantifiedas − (immunonegative), + (<10 immunopositive cells), ++ (10–30 immunopositive cells), and +++ (>30 immunopositive cells)(Table 1).

) with ED1 (B), anti-GFAP (E), or anti-flotillin-1 (H) in the spinalcathepsin D immunoreactivity in ED1-positive macrophages

. C, F, and I are merged images. Scale bars=20 μm.

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Acknowledgments

This work was supported by a Korea Research FoundationGrant from the Korean Government (MOEHRD) (KRF-2005-041-E00402).

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