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Increased phosphorylation of cyclic AMP responseelement-binding protein in the spinal cord of Lewisrats with experimental autoimmune encephalomyelitis
Heechul Kima,1, Changjong Moonb,1, Meejung Ahna, Yongduk Leea, Seungjoon Kima,Yoh Matsumotoc, Chang-Sung Kohd, Moon-Doo Kime, Taekyun Shina,⁎aDepartment of Veterinary Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, South KoreabDepartment of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South KoreacDepartment of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183, JapandDepartment of Biomedical Laboratory Sciences, Shinshu University School of Health Sciences, 3-1-1 Asahi, Matsumoto 390-8621, JapaneDepartment of Psychiatry, Cheju National University College of Medicine, Jeju 690-756, South Korea
Article history:Accepted 31 May 2007Available online 21 June 2007
To investigate whether the phosphorylation of cyclic AMP response element-bindingprotein (CREB) is implicated in the pathogenesis of experimental autoimmuneencephalomyelitis (EAE), the change in the level of CREB phosphorylation was analyzedin the spinal cord of Lewis rats with EAE. Western blot analysis showed that thephosphorylation of CREB in the spinal cord of rats increased significantly at the peak stageof EAE compared with the controls (pb0.05) and declined significantly in the recoverystage (pb0.05). Immunohistochemistry showed that the phosphorylated form of CREB (p-CREB) was constitutively immunostained in few astrocytes and dorsal horn neurons in thespinal cord of normal rats. In the EAE-affected spinal cord, p-CREB was mainly found inED1-positive macrophages at the peak stage of EAE, and the number of p-CREB-immunopositive astrocytes was markedly increased in the spinal cord with EAEcompared with the controls. Moreover, p-CREB immunoreactivity of sensory neurons,which are closely associated with neuropathic pain, was significantly increased in thedorsal horns at the peak stage of EAE. Based on these results, we suggest that theincreased phosphorylation of CREB in EAE lesions was mainly attributable to theinfiltration of inflammatory cells and astrogliosis, possibly activating gene transcription,and that its increase in the sensory neurons in the dorsal horns is involved in thegeneration of neuropathic pain in the rat EAE model.
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1. Introduction
Cyclic AMP response element-binding protein (CREB) is atranscription factor (Brindle and Montminy, 1992) that wasoriginally shown to be phosphorylated at serine residue 133(Ser-133) by an activated cAMP-dependent protein kinase Aand mitogen activated protein kinases (MAPKs). The phos-phorylation of this residue allows the recruitment of CREB-binding protein (CBP) or its paralogue, p300 (Johannessen etal., 2004; Roach et al., 2005). CREB regulates responses togrowth factors, inflammatory mediators, and some cell-activating agents by binding to cAMP response elements(CRE) (Caivano and Cohen, 2000; Liu et al., 2004; Ma andEisenach, 2002; Mayr and Montminy, 2001). The phosphor-ylation of CREB participates in a wide range of cellularevents in the nervous system, including neuronal dif-ferentiation (Bender et al., 2001), neuronal survival (Ryu etal., 2005), neuropathic pain (Ma and Quirion, 2001), and in-flammation (Etienne-Manneville et al., 1999). Little is knownabout the phosphorylation of CREB in autoimmune centralnervous system (CNS) disease models such as experimentalautoimmune encephalomyelitis (EAE), which is characterizedby reactive gliosis as well as infiltration of autoimmune Tand bystander cells (Schonrock et al., 1998; Shin et al., 1995,2003).
Our previous studies have shown that the MAPK pathway,which is upstream of CREB, is activated in autoimmuneinflammation in the nervous system (Ahn et al., 2004; Moon
Fig. 1 – Histopathological examination of normal and EAE-affectspinal cords of the normal control rats. (B) On day 12 after the inpresent in the spinal cord of EAE-affected rats. (C, D) The majoritED1-positive macrophages (C, arrows) and R73 (TCR αβ)-positiveC and D: immunostained with either ED1 (C) or R73 (D) and coun80 μm; in panels C and D, 40 μm.
et al., 2005; Shin et al., 2003) and that the resulting pro-inflammatory cytokines are associated with the induction ofEAE paralysis (Tanuma et al., 1997). These factors areassociated with the activation of CREB in each cell type.
The aim of the present study was to determine whetherCREB is implicated in the course of EAE, which is an animalmodel of human multiple sclerosis.
2. Results
2.1. Clinical progression of experimental autoimmuneencephalomyelitis and histopathological findings
EAE-affected rats immunized with myelin basic protein (MBP)developed floppy tails (grade 1, G.1) on days 9–11 post-immu-nization (p.i.) and exhibited progressive hind limb paralysis(G.2 or G.3) on days 12–14 p.i. All of the rats recovered on day21 p.i. (recovery 0, R.0).
Histopathological examination showed no infiltrating cellsin the spinal cord of the normal controls (Fig. 1A). With hindlimb paralysis, inflammatory cells infiltrated the parenchymaof the spinal cord in EAE-affected rats (day 12 p.i.; Fig. 1B). Inthe EAE lesions, most of the inflammatory cells were ED1-positive macrophages (Fig. 1C) and monoclonal anti-T cellreceptor αβ (R73)-positive T cells (Fig. 1D). The clinicalobservations and histological findings related to EAE largelycorresponded to those described previously (Shin et al., 1995,2003).
ed rat spinal cords. (A) There are no inflammatory cells in thejection of myelin basic protein, many inflammatory cells arey of the inflammatory cells within the EAE lesions areT cells (D, arrows). A and B: hematoxylin–eosin staining.
terstained with hematoxylin. Scale bars: in panels A and B,
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2.2. Activation of CREB and ATF-1 in the spinal cord in EAE
Antibody to the phosphorylated form of CREB (p-CREB) detectsendogenous levels of CREB only when CREB is phosphorylatedat Ser-133 and also detects the phosphorylated form of CREB-related protein activating transcription factor-1 (p-ATF-1).Therefore, we used different normalized bands for the ana-lysis of p-CREB and p-ATF-1; the intensity of p-CREB was nor-malized to CREB, and the intensity of p-ATF-1 was normalizedto beta-actin.
The level of p-CREB in the spinal cord was semiquantita-tively evaluated during the course of EAE, using Western blotanalysis. The expression of p-CREB immunoreactivity wasdetected at low levels in the spinal cord of normal controlrats (density, 0.35±0.06 OD/mm2; n=5); it significantlyincreased in the spinal cord during the peak stage of EAE(G.3, day 12 p.i.; 1.57±0.15 OD/mm2; n=5; pb0.05 vs. controls)and subsequently declined in the recovery stage (R.0, day21 p.i.; 0.34± 0.07 OD/mm2; n=5; pb0.05 vs. peak stage of EAE)(Fig. 2).
The expression of p-ATF-1 immunoreactivity was alsodetected at low levels in the spinal cord of normal control rats(0.26±0.04 OD/mm2; n=5); it significantly increased in thespinal cord in the peak stage of EAE (G.3, day 12 p.i.; 0.84±0.13OD/mm2; n=5; pb0.05 vs. controls). The expression was
Fig. 2 – Western blot analysis of p-CREB and p-ATF-1 expressionwere collected from normal control rats, rats at the peak stage of(day 21 p.i.; stage R.0). (A) Representative photographs of Wester(∼43 kDa), p-ATF-1 (∼35 kDa), CREB (∼43 kDa), and beta-actin (∼4immunoreactivity in spinal cords, normalized to the intensity ofexpression is significantly greater in spinal cords from rats sacrifcontrol rats, and the expression level declines to control levels dufrom five experiments. *p<0.05 vs. normal controls and the recoimmunoreactivity in spinal cords, normalized to the intensity ofdetected in the normal controls, and its expression is significantcontrols.
slightly lower in the recovery stage of EAE (R.0, day 21 p.i.;0.53±0.1 OD/mm2; n=5) but was still higher than the controllevel (pb0.05 vs. controls) (Fig. 2).
2.3. Localization of p-CREB in spinal cord sections fromEAE-affected rats
2.3.1. p-CREB immunoreactivityImmunohistochemically, p-CREB was detected in a few glialcells in the normal rat spinal cord (Fig. 3A). In the peak stageof EAE (G.3, day 12 p.i.), many p-CREB-positive glial cellswere detected in the white (Fig. 3B) and gray (data notshown) matter of the spinal cord. In addition, during thepeak stage of EAE, there was massive infiltration of inflam-matory cells in the parenchyma, where some round cellswere positive for p-CREB (Fig. 3C). In the recovery stage ofEAE (R.0, day 21 p.i.), there were fewer inflammatory cellsthan in the peak stage, and a few glial cells were positive forp-CREB (Fig. 3D).
In addition, p-CREB was detected in neurons in the dorsalhorn lamina of the normal rat spinal cord (mean number±SEM: 39.57±7.62) (Fig. 4A). In the peak stage of EAE (G.3, day 12p.i.), p-CREB-positive neurons in the dorsal horn lamina wereincreased compared with normal control rats (66.57±7.62;pb0.05 vs. controls) (Fig. 4B); the immunoreactivity decreased
in the spinal cord of rats with EAE. Spinal cord samplesEAE (day 12 p.i.; stage G.3), and rats during the recovery stagen blots; arrowheads indicate the expression of p-CREB5 kDa). (B) Semiquantitative analysis of p-CREBCREB expression in the same immunoblot. The p-CREBiced at the peak of EAE (G.3, p<0.05) than in spinal cords fromring the recovery stage (R.0) from EAE. Data (mean±SEM) arevery stage of EAE. (C) Semiquantitative analysis of p-ATF-1beta-actin expression in the same immunoblot. p-ATF-1 isly increased in EAE-affected spinal cords. *p<0.05 vs. normal
Fig. 4 – Immunohistochemical staining of p-CREB in the dorsal horn lamina in the spinal cords of normal control rats (A) andrats at the peak (day 12 p.i.) (B) and recovery stages of EAE (day 21 p.i.) (C). (A) p-CREB is constitutively immunostained in thedorsal horn lamina in the normal controls. (B, C) The p-CREB immunoreactivity is significantly increased in the dorsal hornlamina in the peak stage (B) and has declined in the recovery stage of EAE (C). (D) Tissue from a rat in the peak stage of EAEstained without primary antisera shows no staining. Counterstained with hematoxylin. Scale bars: 100 μm.
Fig. 3 – Immunohistochemical staining of p-CREB in the spinal cords of normal control rats (A) and rats at the peak (day 12 p.i.)(B, C) and recovery stages of EAE (day 21 p.i.) (D). (A) p-CREB is weakly detected in some glial cells (arrow) in the spinal cord ofnormal control rats. (B, C) In the peak stage of EAE, glial cells show increased immunoreactivity for p-CREB (B, arrows), andp-CREB-positive inflammatory cells are detected in the subarachnoid space (B, arrowheads) and parenchyma (C, arrowheads).(D) In the recovery stage of EAE, some p-CREB immunoreactive cells remain (arrow). Counterstained with hematoxylin. Scalebars: 40 μm.
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Fig. 5 – Immunofluorescent co-localization of p-CREB (A, D; red) with anti-GFAP (B, E; green) in the spinal cords of normal controlrats (A–C) and rats at the peak stage of EAE (day 12 p.i.) (D–F). (A–C) In the normal controls, some p-CREB-immunopositive glialcells are co-localized in GFAP-positive astrocytes in the spinal cords (arrows). (D–F) In the EAE-affected spinal cords, manyp-CREB-positive glial cells were positive for GFAP (arrows). C and F are merged images. Scale bars: 50 μm.
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significantly in the recovery stage of EAE (43.86±5.2; pb0.05 vs.peak stage of EAE) (Fig. 4C). Some p-CREB-positive ependymalcells were detected in the spinal cords of normal control ratsand rats with EAE (data not shown).
2.3.2. Identification of p-CREB-positive cells in the spinal cordsof normal controls and rats with EAEThe patterns of p-CREB immunofluorescence in the spinalcords of normal control rats and rats with EAE were similar tothose seen with single immunoperoxidase staining (Figs. 3and 4). Very little p-CREB (Fig. 5A, red) was immunodetected inGFAP-positive-astrocytes (Fig. 5B, green) in the control spinalcord (Fig. 5C, merge). In the EAE spinal cord (day 12 p.i.),p-CREB-positive astrocytes increased (Figs. 5D–F). p-CREB
Fig. 6 – Immunofluorescent co-localization of p-CREB (A) with ED(day 12 p.i.). A large amount of p-CREB (A, red; arrows) was imm(C, merge; arrows). Scale bars: in A–C, 20 μm.
(Fig. 6A, red) was abundant in ED1-positive cells (Fig. 6B,green; Fig. 6C, merge), suggesting that the majority of macro-phages were positive for p-CREB in EAE lesions at the peakstage.
3. Discussion
This study is the first to show that a gene transcription factor,CREB, is phosphorylated in host and inflammatory cells in thespinal cord of animals with EAE, particularly during the peakstage, suggesting that CREB phosphorylation is closely asso-ciated with autoimmune inflammatory attack in the spinalcord.
1 (B) in the spinal cord of rats in the peak stage of EAEunostained in ED1-positive macrophages (B, green; arrows)
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With regard to the phosphorylation of CREB in the peakstage of EAE, the activation of CREB has been associated withsignal enzymes such asMAPKs (Shin et al., 2003) aswell as pro-inflammatory cytokines, including interferon-γ (IFN-γ) andtumor necrosis factor-α (TNF-α) (Renno et al., 1995; Tanuma etal., 1997). The MAPKs transduce a variety of extracellularstimuli through a cascade of protein phosphorylation, leadingto the activation of gene transcription factors (Seger andKrebs,1997). Two potential in vivo substrates for the MAPK pathwayare CREB and the closely related ATF-1. MAPKs phosphorylateCREB at Ser-133 (Johannessen et al., 2004). Moreover, severalfurther lines of evidence are consistent with the hypothesisthat CREB and ATF-1 become phosphorylated at the relevantsites in response to growth factors, inflammatory mediators,and some cell-activating and -damaging stimuli (Caivano andCohen, 2000; Kingsley-Kallesen et al., 1999; Zaman et al., 1999).This study confirms that the phosphorylation of CREB in EAElesions occurs in the inflammatory cells, astrocytes, andneurons, which corresponds to the increased phosphorylationof MAPKs in rats with EAE as reported in our previous study(Shin et al., 2003). Thus, we postulate that the activation ofMAPKs affects the phosphorylation of CREB in affected cells inthe spinal cord with EAE through the activation of CREB andATF-1 signaling pathways.
In response to stimuli, macrophages undergo a series ofprocesses suchas chemotaxis, phagocytosis, and the release ofinflammatory mediators (Rotshenker, 2003). IFN-γ has beenshown to activate the CREB signaling pathway in murineperitoneal macrophages (Liu et al., 2004), and the activation ofCREB/ATF-1 promotes the production of pro-inflammatorycytokines such as TNF-α and interleukin-1β (IL-1β) in lipopo-lysaccharide (LPS)-stimulated macrophages (Caivano andCohen, 2000). The inflammatory mediators (IFN-γ and TNF-α)described above have been closely associated with EAE lesions(Renno et al., 1995; Tanuma et al., 1997). Considering the dataobtained from the present study, we postulate that pro-inflammatory cytokines, including IFN-γ, activate ED1-posi-tive macrophages with an increased phosphorylation of CREB,possibly leading to the further production of pro-inflammatorycytokines, including TNF-α.
With regard to the inflammatory stimuli involved in thephosphorylation of CREB in astrocytes, which are an impor-tant cell type in EAE, it is known that LPS induces an increasein CREB phosphorylation via the MAPK pathway in culturedastrocytes (Buzas et al., 2002). In addition, LPS has beenassociated with the induction of IL-1 and TNF-α in culturedhuman astrocytes (Velasco et al., 1991). In the present study,we found that the phosphorylation of CREB increased in reac-tive astrocytes in the peak stage of EAE, suggesting that CREBactivationmay be involved in the gene transcription of inflam-matory mediators in autoimmune-stimulated astrocytes aswell as in macrophages.
Although the phosphorylation of CREB has been immuno-detected at intense levels in macrophages and astrocytes inEAE, it was also found in the dorsal horn neurons in the peakstage of EAE in the present study. This finding implies thatincreased phosphorylation of CREB plays an important role inthe sensory function during the course of EAE. There is ageneral consensus that the phosphorylation of CREB in dorsalhorn neurons is possibly involved in the generation and
maintenance of neuropathic pain caused by partial sciaticnerve ligation (Ma and Quirion, 2001) and spinal cord injury(Crown et al., 2006).
It was recently shown that, in both active and passive EAE,there was an initial increase in tail withdrawal latency(hypoalgesia) that peaked several days prior to the peak inmotor deficits during the acute disease phase (Aicher et al.,2004). Considering these results, it is highly possible thatincreased CREB phosphorylation in the dorsal horn neurons inthe spinal cord of rats with EAE leads to the generation andmaintenance of neuropathic pain.
When all the findings are taken into consideration, theysuggest that increased phosphorylation of CREB occurs in thespinal cord in the peak stage of EAE and contributes to theactivation of inflammatory cells such as macrophages, to theoccurrence of reactive astrogliosis, and partially to the gene-ration of neuropathic pain during the course of rat EAE andpossibly in human multiple sclerosis.
4. Experimental procedures
4.1. Animals
Lewis rats were obtained from Harlan (Indianapolis, IN) andwere bred in our animal facility. Male rats (7–8 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 MBP (1 mg/ml) and complete Freund's adjuvant (CFA)supplemented with Mycobacterium tuberculosis H37Ra (5 mg/ml) (Difco, Detroit, MI). Control rats were immunized with CFAonly. After immunization, the rats were observed daily forclinical signs of EAE. The progression of EAE was divided intoseven clinical stages: Grade 0 (G.0), no signs; G.1, floppy tail;G.2, mild paraparesis; G.3, severe paraparesis; G.4, tetrapar-esis; G.5, moribund condition or death; and R.0, recovery (Shinet al., 1995).
4.3. Antibodies
Rabbit polyclonal anti-p-CREB (Ser-133) and anti-CREB anti-bodies were obtained from Cell Signaling Technology (Beverly,MA).Thep-CREBantibodyalsodetects thephosphorylated formof CREB-related protein ATF-1 (as characterized by the manu-facturer). Mouse monoclonal anti-beta-actin and mouse anti-GFAP were obtained from Sigma (St. Louis, MO). ED1 (mousemonoclonal anti-rat macrophages) was obtained from Serotec(London, UK). R73 (mouse monoclonal anti-T cell receptor αβ)was obtained from Blackthorn (Bicester, Bucks, UK).
4.4. Tissue sampling
The rats were sacrificed under ether anesthesia. The spinalcords were dissected from each group at 12–14 and 21 days p.i.
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(n=5 rats/group); these periods coincided with the peak (G.3,day 12–14 p.i.) and recovery (R.0, day 21 p.i.) stages of EAE.Samples of the spinal cords were processed for embedding inparaffin wax after fixation in 4% paraformaldehyde in phos-phate-buffered saline (PBS, pH 7.4). Paraffin sections (5 μmthick) were used for all immunostaining, except for the T cellmarker R73. For T cell immunostaining, pieces of the spinalcords were snap-frozen in optimal cutting temperaturecompound (Sakura, Tokyo, Japan), and sections (8 μm thick)were cut using a cryostat (Leica, Nussloch, Germany). Addi-tional spinal cord samples were snap-frozen and stored forimmunoblot analysis.
4.5. Western blot analysis
The spinal cord tissue was homogenized in modified radio-immunoprecipitation assay (RIPA) buffer (20 mM Tris, pH 7.5,150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate, 1% NP-40, 10 mM NaF, 1 mMEDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, 10 μg/mlaprotinin, and 10 μg/ml leupeptin) with 20 strokes in a homo-genizer. The homogenate was transferred to microtubes andcentrifuged at 14,000 rpm for 20 min, and the supernatant washarvested.
For the immunoblot assay, supernatant samples containing40 μg of protein each were loaded into individual lanes of 10%sodium dodecyl (lauryl) sulfate-polyacrylamide gels, electro-phoresed, and immunoblotted onto nitrocellulosemembranes(Schleicher and Schuell, Keene, NH). The residual binding siteson the membrane were blocked by incubation with 5% nonfatmilk in Tris-buffered saline (TBS; 10 mM Tris–HCl, pH 7.4, and150 mM NaCl) for 1 h. Subsequently, the membrane wasincubated for 2 h with rabbit polyclonal anti-p-CREB (1:1000dilution) antibody. The membranes were washed three timesin TBS containing 0.1% Tween 20 and then incubated withhorseradish peroxidase-conjugated anti-rabbit IgG (Vector,Burlingame, CA) for 1 h. Bound antibodies were detectedusing chemiluminescent substrate (WEST-oneTM Kit; iNtRONBiotech Inc., Kyungki, Korea) according to the manufacturer'sinstructions. After imaging, themembranes were stripped andreprobed using anti-CREB and anti-beta-actin antibodies. Theoptical density (per mm2) of each band was measured with ascanning laser densitometer (GS-700, Bio-Rad, Hercules, CA),and these values are presented as means± SEM. The ratios ofthe density of each p-CREB or p-ATF-1 band relative to that ofthe CREB or beta-actin band, respectively, were comparedusing Molecular Analyst software (Bio-Rad).
The data were analyzed using one-way ANOVA followedby the Student–Newman–Keuls post hoc test for multiplecomparisons. In all cases, pb0.05 was taken as statisticallysignificant.
4.6. Immunohistochemistry
Paraffin sections were used for the immunoperoxidase stain-ing of p-CREB and rat macrophages, and frozen sections wereused for the detection of T cells.
Briefly, paraffin-embedded spinal cords (5-μm sections)were deparaffinized by treatment with citrate buffer (0.01 M,pH 6.0) in amicrowave for 10min. To identify T cells, the frozen
spinal cord sections were air-dried and fixed in 4% parafor-maldehyde buffered with 0.1 M PBS (pH 7.2) for 20 min. Afterthree washes with PBS, the sections were treated with 0.3%hydrogen peroxide in methyl alcohol for 20 min to blockendogenous peroxidase activity.
After three washes with PBS, the sections were incubatedwith 10% normal goat or horse serum and then with theprimary antigens, including rabbit polyclonal anti-p-CREB(1:200 dilution), ED1 (1:800 dilution), and R73 (1:1,000 dilution).Immunoreactivity was visualized using the avidin–biotinperoxidase reaction (Vector Elite kit, Vector, Burlingame, CA).Peroxidase was developed using a diaminobenzidine sub-strate kit (Vector). Sections were counterstained with hema-toxylin before mounting. As a control, the primary antiserawere omitted for a few test sections in each experiment.
To examine the cell phenotype in p-CREB expression,double immunofluorescence was applied using cell type-specific markers: ED1 for monocyte-like macrophages andanti-GFAP for astrocytes. First, paraffin sections were reactedsequentially with rabbit anti-p-CREB (1:100 dilution), biotiny-lated anti-rabbit IgG (Vector) (1:200 dilution), and tetramethylrhodamine isothiocyanate (TRITC)-labeled streptavidin(Zymed, San Francisco, CA) (1:1,000 dilution). The slides werethen incubated with ED1 (1:200 dilution) and anti-GFAP (1:200dilution), followed by fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (1:50 dilution; Sigma).
To minimize lipofuscin autofluorescence, the sectionswere washed in PBS (3×1 h) at RT, dipped briefly in distilledH2O, treated with 10 mM CuSO4 in ammonium acetatebuffer (50 mM CH3COONH4, pH 5.0) for 20 min, dipped brieflyagain in distilled H2O, and then returned to PBS. The doubleimmunofluorescence-stained specimens were examinedunder an FV500 laser confocal microscope (Olympus, Tokyo,Japan).
To semiquantify the immunostaining for p-CREB-positiveneurons in the dorsal horn lamina, the number of p-CREB-positive dorsal horn neurons was counted in seven spinalcords from each group.
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