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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Glycogen Synthase Kinase-3 Inhibition Reduces Secondary Damage in Experimental Spinal Cord Trauma

Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy (S.C., T.G., E.M., C.C., C.M., R.D.P.); The William Harvey Research Institute, Centre for Experimental Medicine, Nephrology and Critical Care, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London, United Kingdom (M.C., C.T.); Istituto di Ricovero e Cura a Carattere Scientifico Centro Neurolesi "Bonino-Pulejo", Messina, Italy (S.C., E.M., P.B., T.G.); and Department of Experimental Pharmacology, University of Naples "Federico II", Naples, Italy (E.E.)

Received February 13, 2006; accepted April 5, 2006.

	Abstract

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Glycogen synthase kinase-3 (GSK-3) has recently been identified as an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and plays an important role in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3 inhibition on the degree of experimental spinal cord trauma induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury (SCI) in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective GSK-3 inhibitor, significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase activity); 3) inducible nitric-oxide synthase, nitrotyrosine, and cyclooxygenase-2 expression; and 4) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiments, TDZD-8 significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with TDZD-8 reduces the development of inflammation and tissue injury associated with spinal cord trauma.

The cardinal features of inflammation, namely, infiltration of inflammatory cells (not only polymorphonuclear neutrophils but also macrophage and lymphocytes), release of inflammatory mediators, and activation of endothelial cells leading to increased vascular permeability, edema formation, and tissue destruction, have been widely characterized in animal models of SCI (Popovich et al., 1997). Both necrotic and apoptotic mechanisms of cell death after SCI have been extensively and well described in animal SCI models (Profyris et al., 2004). One of the mechanisms of neuronal apoptosis intensely studied involves GSK-3 (glycogen synthase kinase-3), which is also implicated in neurotrophic base of apoptosis. For instance, overexpression of GSK-3 induces apoptosis in neuronal cells in culture, and specific inhibitors of GSK-3 are able to ameliorate this apoptotic response (Pap and Cooper, 1998).

GSK-3 is an isoform of GSK-3, which was initially identified as an enzyme that negatively regulates the activity of glycogen synthase (Cohen, 1985). Recently, GSK-3 has been discovered to be involved in the regulation of growth and development, mostly because the activation of this enzyme contributes to proapoptotic signaling. Lithium, an inhibitor of GSK-3, was found to protect cultured neurons against glutamate-induced apoptosis in a phosphatidyl-inositol-3-kinase (PI-3K)-dependent manner (Shimomura et al., 2003). Moreover, recently it has been demonstrated that lithium, in combination with Avidin: Biotinylated enzyme Complex (ABC; Vector Laboratories, Segrate, Italy), improves the regenerative response after central nervous system injury and enhances the recovery of forelimb function compared with a single application of ABC alone (Yick et al., 2004). A large body of evidence supports the hypothesis that pharmacological inhibitors of GSK-3 could be used to treat several diseases, including Alzheimer's disease and other neurodegenerative diseases. More than 30 inhibitors of GSK-3 have been identified. Seven of these have been cocrystallized with GSK-3, and all localize within the ATP-binding pocket of the enzyme. GSK-3, as part of a multiprotein complex that contains proteins such as axin, presenilin, and -catenin, contains many additional target sites for specific modulation of its activity.

In this study, we used the GSK-3 inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) to investigate the role of this kinase in the modulation of secondary injury in the spinal cord. In particular, we have determined the following endpoints of the inflammatory response: 1) histological damage; 2) motor recovery; 3) neutrophil infiltration; 4) NF-B expression; 5) nitrotyrosine, iNOS, and COX-2 expression; 6) apoptosis [terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining]; and 7) Bax and Bcl-2 expression.

	Materials and Methods

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Animals. Male adult CD1 mice (25-30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care complied with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the European Economic Community regulations (O.J. of E.C. L358/1 12/18/1986).

SCI. Mice were anesthetized using chloral hydrate (400 mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away exposing T5-T8 vertebrae. The spinal cord was exposed via a four-level T6-T7 laminectomy, and SCI was produced by extradural compression of the spinal cord using an aneurysm clip with a closing force of 24 g. In all injured groups, the spinal cord was compressed for 1 min. Sham animals were only subjected to laminectomy. After surgery, 1.0 ml of saline was administered subcutaneously to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were individually housed in a temperature-controlled room at 27°C for a survival period of 10 days. Food and water were provided to the mice ad libitum. During this time period, the animals' bladders were manually voided twice a day until the mice were able to regain normal bladder function.

Experimental Groups. Mice were randomly allocated into the following groups. 1) SCI + vehicle group: mice were subjected to SCI plus intraperitoneal administration of vehicle (N = 30); 2) SCI + TDZD-8 group: mice were subjected to SCI plus intraperitoneal administration of TDZD-8 at the dose of 1 mg/kg (10% DMSO) 1 h before and 3 and 6 h after SCI (N = 30); 3) Sham + vehicle group: mice were subjected to the surgical procedures as the above groups except that the aneurysm clip was not applied (N = 30); and 4) Sham + TDZD-8 group: identical to Sham + vehicle group except for the administration of TDZD-8 1 h before and 3 and 6 h after SCI (N = 30).

Ten mice from each group were sacrificed at different time points (see Fig. 1) to collect samples for the evaluation of the parameters as described below. In the experiments investigating the motor score, the animals were treated with TDZD-8 or with SB415286 (1 mg/kg 10% DMSO) 1 h before and 3 and 6 h after SCI and daily until day 9.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Mice were sacrificed at different time points to evaluate the various parameters. n = 10 mice from each group for each time point. See Materials and Methods for further explanations.

Western Blot Analysis for IB-, Phospho-NF-B p65 (Serine 536), NF-B p65, Bax, and Bcl-2. The levels of IB-, phospho-NF-B p65 (serine 536), Bax, and Bcl-2 were quantified in whole extracts 24 h after SCI by Western blot analysis. Proteins were transferred onto nitrocellulose membranes according to the manufacturer's instructions. In brief, the membranes were saturated by incubation at room temperature for 1 h with 10% (w/v) nonfat dry milk in PBS and then incubated overnight at 4°C with anti-IB- (1:1000) or anti-Bax (1:100) or anti-Bcl-2 (1:100) or anti-phospho-NF-B p65 (serine 536) (1:1000). Nuclear fractions were incubated with anti-NF-B p65 (1:1000; Santa Cruz, DBA, Milan, Italy). Membranes were washed three times with 1% (w/v) Tween 20 in PBS and then incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The immune complexes were visualized using the SuperSignal West Pico chemiluminescence substrate (Pierce, Milan, Italy). Thereafter, the relative expression of the proteins was quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst; IBM, White Plains, NY).

Electrophoretic Mobility-Shift Assay. Double-stranded oligonucleotides containing the NF-B recognition sequence (5'-GAT CGA GGG GAC TTT CCC TAG-3') were end-labeled with -[³²P]ATP (ICN Biomedicals). Aliquots of whole extracts collected 24 h after SCI (20 µg of protein for each sample) were incubated for 30 min with radiolabeled oligonucleotides (2.5-5.0 x 10⁴ cpm) in 20 µl of reaction buffer containing 2 µg of poly(dI-dC), 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM ethylenediaminotetraacetic acid, 1 mM DL-dithiothreitol, 1 mg/ml bovine serum albumin, and 10% glycerol. The specificity of the DNA/protein binding was determined for NF-B by competition reaction in which a 50-fold molar excess of unlabeled wild-type, mutant, or Sp-1 oligonucleotide was added to the binding reaction 10 min before the addition of radiolabeled probe. Protein-nucleic acid complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5x Tris borate ethylenediaminotetraacetic acid buffer at 150 V for 2 h at 4°C. The gel was dried and autoradiographed with intensifying screen at -80°C for 20 h. Thereafter, the relative bands were quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst; IBM).

Myeloperoxidase Activity. Twenty-four hours after SCI, myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was determined as described previously (Mullane et al., 1985). At the specified time after SCI, spinal cord tissues were obtained and weighed and each piece homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine and 0.1 mM H₂O₂. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide per min at 37°C and was expressed in milliunits per gram of wet tissue.

Immunohistochemical Localization of Nitrotyrosine, iNOS, COX-2, Bax, and Bcl-2. Twenty-four hours after SCI, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8-mm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA), respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody [1:500 in PBS (v/v)], with anti-iNOS polyclonal antibody rat [1:500 in PBS (v/v)], anti-COX-2 monoclonal antibody [1:500 in PBS (v/v)], anti-Bax rabbit polyclonal antibody [1:500 in PBS (v/v)], or with anti-Bcl-2 polyclonal antibody rat. Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for nitrotyrosine, iNOS, COX-2, Bax, and Bcl-2, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out. Immunohistochemical photographs (n = 5 photos from each sample collected from each mice in each experimental group) were assessed by densitometry as described previously (Shea, 1994) by using Optilab Graftek software on a Macintosh personal computer.

TUNEL Assay. TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instructions (Apotag, HRP kit; DBA). In brief, sections were incubated with 15 µg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H₂O₂ for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase buffer containing deoxynucleotidyl transferase and biotinylated dUTP in terminal deoxynucleotidyltransferase buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.

Light Microscopy. Spinal cord biopsies were taken at 24 h after trauma. The biopsies were fixed for 24 h in paraformaldehyde solution (4% in PBS, 0.1 M) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (5-µm thickness) were deparaffinized with xylene, stained with hematoxylin/eosin (H&E) and Luxol fast blue staining (used to assess demyelination), and studied using light microscopy (Dialux 22; Leitz). All the histological studies were performed in a blinded fashion.

Grading of Motor Disturbance. The motor function of mice subjected to compression trauma was assessed daily for 10 days after injury. Recovery from motor disturbance was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) (Basso et al., 1995) hind limb locomotor rating scale (Joshi and Fehlings, 2002a,b).

Materials. Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Milan, Italy). TDZD-8 was obtained from Axxora Corporation (Bingham, Nottingham, UK). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter, Milan, Italy) or 10% DMSO.

Statistical Evaluation. All values in the figures and text are expressed as mean ± standard error of the mean (S.E.M.) of N observations. For the in vivo studies, N represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. A p value of less than 0.05 was considered significant. BBB scale data were analyzed by the Mann-Whitney test and considered significant when p value was less than 0.05.

	Results

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TDZD-8 Reduces the Severity of Spinal Cord Trauma. The severity of the trauma at the level of the perilesional area, assessed as the presence of edema as well as alteration of the white matter (Fig. 2b), was evaluated at 24 h after injury. A significant damage to the spinal cord was observed in the spinal cord tissue of control mice subjected to SCI compared with sham-operated mice (Fig. 2a). It is noteworthy that a significant protection against the spinal cord injury was observed in TDZD-8-treated mice (Fig. 2c). Myelin structure was detected by Luxol fast blue staining (Fig. 2, d-f). In sham animals (Fig. 2d), myelin structure was clearly stained by Luxol fast blue in both lateral and dorsal funiculi of the spinal cord. At 24 h after the injury, a significant loss of myelin in lateral and dorsal funiculi was observed in control mice subjected to SCI (Fig. 2e). In contrast, in TDZD-8-treated mice, myelin degradation was attenuated in the central part of lateral (Fig. 2f) and dorsal funiculi. To evaluate whether histological damage to the spinal cord was associated with a loss of motor function, the modified BBB hind limb locomotor rating scale score was evaluated. Whereas motor function was only slightly impaired in sham mice, mice subjected to SCI had significant deficits in hind limb movement (Fig. 3). A significant amelioration of hind limb motor disturbances was observed in TDZD-8-treated mice (Fig. 3). In addition, to confirm that the protective effects of TDZD-8 on motor function are related to the GSK-3, we have also investigated whether SB415286, another GSK-3 selective inhibitor, attenuates the motor dysfunction induced by SCI. As shown in Fig. 3, the treatment with SB415286 also significantly leads to an amelioration of hind limb motor disturbances. Please note that no significant difference was found between the TDZD-8 or SB415286 treatment (Fig. 3).

View larger version (144K): [in this window] [in a new window]   Fig. 2. Effect of TDZD-8 on histological alterations of the spinal cord tissue 24 h after injury. No histological alteration (a) and no modification of the myelin structure (d) were observed in the spinal cord tissues from sham-operated mice. Twenty-four hours after trauma, a significant damage to the spinal cord from untreated SCI-operated mice at the perilesional area was assessed by the presence of edema as well as alteration of the white matter (b). It is noteworthy that a significant protection from the SCI was observed in the tissue collected from TDZD-8 SCI-treated mice (c). Myelin structure was observed by Luxol fast blue staining. At 24 h after the injury in untreated SC-operated mice (e), a significant loss of myelin was observed. In contrast, in TDZD-8 SCI-treated mice, myelin degradation was attenuated (f). This figure is representative of at least three experiments performed on different experimental days. wm, white matter; gm, gray matter.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effect of TDZD-8 and SB415286 on hind limb motor disturbance after spinal cord injury. The degree of motor disturbance was assessed every day until 10 days after SCI by Basso, Beattie, and Bresnahan criteria. Treatment with TDZD-8 or with SB415286 reduces the motor disturbance after SCI. Values shown are mean ± S.E. mean of 10 mice for each group. *, p < 0.01 versus SCI.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of TDZD-8 on MPO activity. After the injury, MPO activity in spinal cord of untreated SCI-operated mice was significantly increased at 24 h after the damage in comparison to sham mice. Treatment with TDZD-8 significantly reduced the SCI-induced increase in MPO activity. Data are means ± S.E. mean of 10 mice for each group. *, p < 0.05 versus vehicle. , p < 0.01 versus SCI.

TDZD-8 Modulates Expression of Nitrotyrosine, iNOS, and COX-2 after SCI. Twenty-four hours after SCI, nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in the spinal cord sections to determine the localization of "peroxynitrite formation" and/or other nitrogen derivatives produced during SCI. Immunohistological staining for iNOS and COX-2 in the spinal cord was also determined 24 h after injury. Sections of spinal cord from sham-operated mice did not stain for nitrotyrosine, iNOS, or COX-2 (data not shown), whereas spinal cord sections obtained from SCI control mice exhibited positive staining for iNOS (Figs. 5a and 6), nitrotyrosine (Figs. 5c and 6), and COX-2 (Figs. 5e and 6). The positive staining was localized in various cells in the gray matter. TDZD-8 treatment (1 h before and 3 and 6 h after SCI induction) of mice subjected to SCI reduced the degree of positive staining for iNOS (Figs. 5b and 6), nitrotyrosine (Figs. 5d and 6), and COX-2 (Figs. 5f and 6) in the spinal cord.

View larger version (158K): [in this window] [in a new window]   Fig. 5. Immunohistochemical localization of nitrotyrosine, iNOS, and COX-2. Administration of TDZD-8 to SCI-operated mice produced a marked reduction in the immunostaining for nitrotyrosine (b), iNOS (d), and COX-2 (f) in spinal cord tissue compared with positive nitrotyrosine (a), iNOS (c), and COX-2 (e) staining obtained from the spinal cord tissue of mice 24 h after the injury. This figure is representative of at least three experiments performed on different experimental days.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for iNOS, nitrotyrosine, COX-2, Bax, and Bcl-2 from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as percentage of total tissue area. *, p < 0.01 versus sham; , p < 0.01 versus SCI. ND, not detectable.

Effect of TDZD-8 on IB- Degradation, Phosphorylation of Ser536 on p65, Expression of NF-B p65, and NF-B Translocation.

IB- appearance in the spinal cord homogenates was investigated by immunoblot analysis at 24 h after SCI. A basal level of IB- was detected in the spinal cord from shamoperated animals (Fig. 7, a and a1), whereas in SCI control mice, IB- levels were substantially reduced (Fig. 7, a and a1). TDZD-8 prevented the SCI-induced IB- degradation, and the IB- expression remained unchanged 24 h after SCI in TDZD-8-treated mice (Fig. 7, a and a1). In addition, SCI caused a significant increase in the phosphorylation of Ser536 at 24 h (Fig. 7, b and b1). The treatment with the GSK-3 inhibitor TDZD-8 significantly reduced the phosphorylation of p65 on Ser536 (Fig. 7, b and b1). Moreover, the levels of the NF-kB p65 subunit protein in the nuclear fractions of the spinal cord tissue were also significantly increased at 24 h after SCI compared with the sham-operated mice (Fig. 7, c and c1). TDZD-8 treatment significantly reduced the levels of NF-kB p65 protein as shown in Fig. 7, c and c1. To detect NF-B/DNA binding activity, whole extracts of spinal cord from each mouse were analyzed by EMSA. A low basal level of NF-B/DNA binding activity was detected in tissue from sham-operated mice (Fig. 8, a and a1). Twenty-four hours after SCI, the DNA binding activity was significantly increased in whole extracts obtained from vehicle-treated SCI control mice (Fig. 8, a and a1). Treatment of mice with TDZD-8 caused a significant inhibition of SCI-induced NF-B/DNA binding activity, as revealed by EMSA (Fig. 8, a and a1). The specificity of NF-B/DNA binding complex was demonstrated by the complete displacement of the NF-B/DNA binding in the presence of a 50-fold molar excess of unlabeled NF-B probe (wild type, 50x) in the competition reaction. In contrast, a 50-fold molar excess of unlabeled mutated NF-B probe (mutant, 50x) or Sp-1 oligonucleotide (Sp-1, 50x) had no effect on this DNA-binding activity (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Representative Western blots of IB- degradation (a and a1), phospho Ser536 on NF-B p65 (b and b1), and total NF-B p65 subunit protein (c and c1). A basal level of IB- was detected in the spinal cord from sham-operated animals (a and a1), whereas in SCI control mice, IB- levels were substantially reduced (a and a1). TDZD-8 prevented the SCI-induced IB- degradation, and the IB- band remained unchanged 24 h after SCI in TDZD-8-treated mice (a and a1). In addition, SCI caused a significant increase in the phosphorylation of Ser536 at 24 h (b and b1). The treatment with the GSK-3 inhibitor TDZD-8 significantly reduced the phosphorylation of p65 on Ser536 (b and b1). Moreover, the levels of the NF-B p65 subunit protein in the nuclear fractions of the spinal cord tissue were also significant increased at 24 h after SCI compared with the sham-operated mice (c and c1). The levels of NF-kB p65 protein were significantly reduced in the nuclear fractions of the spinal cord tissues from animals that had received TDZD-8 as shown in c and c1. Immunoblotting in a, b, and c are representative of one spinal cord of five analyzed. The results in a1, b1, and c1 are expressed as mean ± S.E. mean from five blots. *, p < 0.01 versus sham, , p < 0.01 versus SCI.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Effect of TDZD-8 on NF-B/DNA binding activity in mice spinal cord. Whole extracts from injured (SCI) or non-inflamed (sham) mice spinal cord were prepared as described under Materials and Methods and incubated with 32P-labeled NF-B probe. Representative EMSA (a) of NF-B shows the effect of TDZD-8 (SCI + TDZD-8) NF-B/DNA binding activity evaluated in spinal cord tissue 24 h after the injury. The intensity of retarded bands (measured by Phosphor-Imager; Molecular Devices, Sunnyvale, CA) in SCI-operated mice was significantly increased versus sham group (a1). TDZD-8 treatment significantly reduced the SCI-induced elevation of NF-B/DNA binding activity. The results in a1 are expressed as mean ± S.E. mean from EMSA. *, p < 0.01 versus sham, , p < 0.01 versus SCI.

View larger version (117K): [in this window] [in a new window]   Fig. 9. Representative TUNEL coloration in rat spinal cord tissue section. The number of apoptotic cells (see arrows) increased at 24 h after SCI (a), associated with a specific apoptotic morphology characterized by the compaction of chromatin into uniformly dense masses in perinuclear membrane, the formation of apoptotic bodies, and the membrane blebbing (see particles, a1). In contrast, tissues obtained from TDZD-8-treated mice (b) demonstrated a small number of apoptotic cells or fragments. Panel c demonstrates the positive staining in the Kit positive control tissue (normal rodent mammary gland). Figure is representative of at least three experiments performed on different experimental days.

View larger version (28K): [in this window] [in a new window]   Fig. 10. Representative Western blot of Bax levels (A) and Bcl-2 (B). Western blot analysis was realized in spinal cord tissue collected at 24 h after injury. A, sham: basal level of Bax was present in the tissue from sham-operated mice. SCI: Bax band is more evident in the tissue from spinal cord-injured mice. SCI + TDZD-8: Bax band is disappeared in the tissue from spinal cord-injured mice that received TDZD-8. B, sham: basal level of Bcl-2 was present in the tissue from sham-operated mice. SCI: Bcl-2 band is disappeared in the tissue from spinal cord-injured mice. SCI + TDZD-8: Bcl-2 band is more evident in the tissue from spinal cord-injured mice that received TDZD-8. a1 and b1, the intensity of retarded bands (measured by PhosphorImager; Molecular Devices) in all the experimental groups. Immunoblotting in A and B is representative of one spinal cord tissues of five to six analyzed. The results in A1 and B1 are expressed as mean ± S.E.M. from five to six spinal cord tissues. *, p < 0.01 versus sham; , p < 0.01 versus SCI.

Moreover, samples of spinal cord tissue were taken at 24 h after SCI to determine the immunohistological staining for Bax and Bcl-2. Sections of spinal cord from sham-operated mice did not stain for Bax (Fig. 11a), whereas spinal cord sections obtained from SCI control mice exhibited a positive staining for Bax (Fig. 11b). TDZD-8 treatment reduced the degree of positive staining for Bax in the spinal cord of mice subjected to SCI (Fig. 11c).

View larger version (143K): [in this window] [in a new window]   Fig. 11. Immunohistochemical expression of Bax and Bcl-2. No positive staining for Bax was observed in the tissue section from sham-operated mice (a). SCI caused at 24 h an increase in the release of Bax expression (b). Treatment with TDZD-8 significantly inhibited the SCI-induced increase in Bax expression (c). On the contrary, positive staining for Bcl-2 was observed in the spinal cord tissues of sham-operated mice (d). At 24 h after SCI, significantly less staining for Bcl-2 was observed (e). The TDZD-8 treatment significantly prevents the loss of Bcl-2 expression induced by SCI (f). Figure is representative of at least three experiments performed on different experimental days.

	Discussion

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SCI induces lifetime disability, and no suitable therapy is available to treat victims or to minimize their suffering. The inhibition of GSK-3 activity is believed to be beneficial in a number of experimental models of neurodegenerative diseases, diabetes type II, bipolar disorders, stroke, cancer, sepsis, and chronic inflammatory disease. In this report, we demonstrate that pharmacological inhibition of GSK-3 exerts beneficial effects in a mice model of spinal cord injury. We demonstrate here that SCI induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy resulted in edema and loss of myelin in lateral and dorsal funiculi. This histological damage was associated to the loss of motor function. SCI induced an inflammatory response in the spinal cord, characterized by increased IB- degradation, enhanced NF-B/DNA binding, amplified expression of proinflammatory mediators iNOS, COX-2, and nitrotyrosine, and increased MPO activity. Our results show that TDZD-8, a potent and selective GSK-3 inhibitor, reduced 1) the degree of spinal cord damage; 2) neutrophil infiltration; 3) NF-B/DNA binding; 4) IB- degradation; 5) expression of iNOS, nitrotyrosine, and COX-2; and 6) apoptosis.

The role of GSK-3 in the regulation of spinal cord injury is of special interest because several transcription factors important to the regulation of secondary damage serve as substrates for GSK-3. Among these is the transcription factor NF-B, whose function is strikingly altered by GSK-3 (Hoeflich et al., 2000; Buss et al., 2004). NF-B plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation. These include the genes for TNF-, IL-1, iNOS, and COX-2, to name but a few (Verma, 2004). The discovery in 1997 that inhibition of the NF-B activation may be useful in conditions associated to local or systemic inflammation (Ruetten and Thiemermann, 1997) stimulated the search for agents that prevent the activation of NF-B. The extent to which GSK-3 activates or blocks NF-B signaling remains unclear. In 2000, Hoeflich et al. first demonstrated that deletion of GSK-3 had no effect on the TNF--induced IB- degradation or on the nuclear translocation of the subunit p65 but prevented the activation of NF-B by an unknown mechanism. On the other hand, other studies provided evidence for an inverse association between GSK-3 activity and NF-B signaling. Sanchez et al. (2003) reported that overexpression of GSK-3 in astrocytes was associated to the inhibition of NF-B activation. Another recent report showed that GSK-3-dependent phosphorylation of a specific serine residue (Ser468) on p65 blocks the activation of NF-B and that inhibition of GSK-3 was related to increased p65 activity (Buss et al., 2004). We report here that SCI caused a significant increase in the phosphorylation of Ser536 on p65 in the spinal cord tissues at 24 h, whereas GSK-3 inhibitor TDZD-8 treatment significantly reduced this phosphorylation. Moreover, we also demonstrate that selective and potent GSK-3 inhibitor TDZD-8 inhibited the IB- degradation as well as the NF-B translocation. Taken together, the balance between proinflammatory and prosurvival roles of NF-B may depend on the phosphorylation status of p65, and GSK-3 may play a central role in this process. However, the reasons for the apparent discrepancies in the modulatory effects of GSK-3 on NF-B activity remain to be fully clarified. In the pathological processes of acute SCI, the upregulation of COX-2, a key enzyme in the synthesis of prostaglandins, has also been proposed to be involved. It is known that COX-1 and COX-2 mRNA and protein are present in the spinal cord tissue and that COX-2 protein is expressed in white matter astrocytes during basal conditions (Beiche et al., 1998). In particular, COX-2 has been found in neurons of all laminae and in the white matter glial cells. Many conditions in which inflammation and pain play an important role are associated to COX-2 expression, which may be widespread (Vanegas and Schaible, 2001). Rao et al. (2004) have recently shown that the inhibition of GSK-3 leads to an activation of COX-2 via induction of NF-B-dependent pathways. However, it has been also demonstrated that inhibition of GSK-3 down-regulates the expression of COX-2, induced by TNF or hypertonic stress, in cells via the induction of a NF-B-COX-2-dependent pathway (Rao et al., 2004). The results of this study are in accordance with the latter study, by demonstrating that the increase of COX-2 expression is reduced in the spinal cord from mice subjected to SCI and treated with TDZD-8. COX-2 is one of the genes regulated by NF-B, and there is good evidence that an enhanced formation of prostanoids after COX-2 induction contributes to the pathophysiology of inflammation as well as in this and in other models of inflammation (Malmberg and Yaksh, 1995).

It has also been demonstrated that an enhanced formation of NO by iNOS contributes to the inflammatory process (Abramson et al., 2001; Vanegas and Schaible, 2001). This study demonstrates that TDZD-8 attenuates the expression of iNOS in the tissue from SCI-treated mice compared with SCI+vehicle-operated mice. Therefore, the inhibition of both COX-2 and iNOS expression by TDZD-8 described in the present study is likely to be due to the inhibition of NF-B activation by TDZD-8 mediated by GSK-3. Furthermore, we have found that the tissue damage induced by SCI in SCI+vehicle-operated mice was associated to an intense immunostaining of nitrotyrosine formation, which suggests that an alteration of the tissue had also occurred because of the formation of highly reactive nitrogen derivatives. Peroxynitrite, one of a number of toxic factors produced in the spinal cord tissues after SCI (Xu et al., 2001), probably contributes to secondary neuronal damage through pathways resulting from the chemical modification of cellular proteins and lipids. Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for the detection of the endogenous formation "footprint" of peroxynitrite (Beckman, 1996). There is, however, recent evidence that certain other reactions can also induce tyrosine nitration; e.g., the reaction of nitrite with hypochlorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine (Endoh et al., 1994). Increased nitrotyrosine staining is considered, therefore, as an indication of "increased nitrosative stress" rather than a specific marker of the peroxynitrite generation. There is a large amount of evidence that implicated reactive oxygen species in the secondary neuronal damage of SCI (Xu et al., 2001). Generation of free radicals and nitric oxide by activated macrophages has also been implicated in causing oligodendrocyte apoptosis (Merrill et al., 1993). In an effort to prevent or diminish levels of apoptosis, we have demonstrated that the treatment with TDZD-8 attenuates the degree of apoptosis, measured by TUNEL detection kit, in the spinal cord after the damage. There is evidence that direct overexpression of GSK-3 is known to induce apoptosis in neuronal cells in culture, and specific inhibitors of GSK-3 are able to ameliorate this apoptotic response (Pap and Cooper, 1998). Recent studies from our laboratory have shown that up-regulation of GSK-3 activity can also lead to cell death and aberrant neuronal migration in primary neuronal populations (Tong et al., 2001). Dong et al. (2003) used Wld^s (Wallerian degeneration, slow) mice and Bax-deficient (Bax-/-) mice in a lateral cord hemisection model of SCI to test the hypothesis that the protracted wave of apoptotic death of oligodendrocytes may be dependent on axonal degeneration and Bax activation. The Bax gene plays an important role in developmental cell death (Chittenden et al., 1995) and in central nervous system injury (Bar-Peled et al., 1999). Apoptosis and the neuronal cell loss that occurs during normal nervous system development and in response to trophic factor deprivation is attenuated in Bax-/-mice (Deckwerth et al., 1996). Nesic-Taylor et al. (2005) showed that administering Bcl-xL fusion protein (Bcl-xL FP, the most robustly expressed antiapoptotic Bcl-2 molecule in adult central nervous system) into injured spinal cords significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death. Because Bcl-xL fusion protein increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI. It is known that pathways which inhibit GSK-3 activity, such as PI-3K or Wnt signaling, often lead to the induction of the NF-B cell survival pathway (Bournat et al., 2000). Indeed, GSK-3 is a major target of Akt/protein kinase B (van Weeren et al., 1998), which is activated by the PI-3K-mediated signaling pathway. Cellular factors that have been implicated in the regulation of astrocyte apoptosis include the PI-3K pathway (Kim et al., 2001). Inhibition of this signaling cascade has been shown to lead to cell death in several paradigms (Carbott et al., 2002), and this has been attributed, at least in part, to the reduction in activity of PI-3K's major physiologic target, Akt. Loss of Akt activity, in turn, results in the transduction of several proapoptotic signals, including sequestration of Bcl-2 and enhanced activation of an Akt substrate, GSK-3 (Pap and Cooper, 1998). We identified proapoptotic transcriptional changes, including up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2, using Western blot assay and by immunohistochemical staining. We report in the present study for the first time that the treatment with TDZD-8 in an SCI experimental model documents features of apoptotic cell death after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI. In particular, we demonstrated that the treatment with TDZD-8 lowers the signal for Bax in treated group compared with SCI+vehicle-operated mice spinal cord, whereas on the contrary, the signal is much more expressed for Bcl-2 in TDZD-8-treated mice than in SCI+vehicle-operated mice. These results are in agreement with recent evidence that has clearly demonstrated that lithium treatment up-regulates the expression of Bcl-2 in axotomized rubrospinal tract via inhibiting GSK-3 (Yick et al., 2004). Based on this evidence, we clearly have shown that TDZD-8 interferes in the apoptotic process induced by SCI. However, it is not possible to exclude that the antiapoptotic effect observed after TDZD-8 treatment may be partially dependent on the attenuation of the inflammatory-induced damage. Further studies are needed to clarify this mechanism.

Finally, in this study, we demonstrated that TDZD-8 treatment significantly reduced the SCI-induced spinal cord tissue alteration as well as improved the motor function. Taken together, the results of the present study enhanced our understanding of the role of GSK-3 in the pathophysiology of spinal cord cell and tissue injury after trauma. Our results imply that inhibitors of the activity of GSK-3 may be useful in the therapy of spinal cord injury, trauma, and inflammation.

	Acknowledgements

 
We thank Giovanni Pergolizzi and Carmelo La Spada for excellent technical assistance during this study, Caterina Cutrona for secretarial assistance, and Valentina Malvagni for editorial assistance with the manuscript.

	Footnotes

 
This study was supported by Programmi di Ricerca di Interesse Nazionale 2003.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.102863.

ABBREVIATIONS: SCI, spinal cord injury; GSK-3, glycogen synthase kinase-3; PI-3K, phosphatidyl-inositol-3-kinase; TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione; NF-B, nuclear factor-B; iNOS, inducible nitric-oxide synthase; COX, cyclooxygenase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DMSO, dimethyl sulfoxide; SB415286, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione; PBS, phosphate-buffered saline; MPO, myeloperoxidase; EMSA, electrophoretic mobility shift assay; BBB, Basso, Beattie, and Bresnahan; TNF, tumor necrosis factor.

Address correspondence to: Prof. Salvatore Cuzzocrea, Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi 98100 Messina Italy. E-mail: salvator{at}unime.it

	References

Top Abstract Materials and Methods Results Discussion References

 

Abramson SB, Attur M, Amin AR, and Clancy R (2001) Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis. Curr Rheumatol Rep 3: 535-541.[Medline]

Amar AP and Levy ML (1999) Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery 44: 1027-1039.[CrossRef][Medline]

Bar-Peled O, Knudson M, Korsmeyer SJ, and Rothstein JD (1999) Motor neuron degeneration is attenuated in Bax-deficient neurons in vitro. J Neurosci Res 55: 542-556.[CrossRef][Medline]

Bartholdi D and Schwab ME (1995) Methylprednisolone inhibits early inflammatory processes but not ischemic cell death after experimental spinal cord lesion in the rat. Brain Res 672: 177-186.[CrossRef][Medline]

Basso DM, Beattie MS, and Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12: 1-21.[Medline]

Beckman JS (1996) Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 9: 836-844.[CrossRef][Medline]

Beiche F, Klein T, Nusing R, Neuhuber W, and Goppelt-Struebe M (1998) Localization of cycloogygenase-2 and prostaglandin E₂ receptor EP3 in the rat lumbar spinal cord. J Neuroimmunol 89: 26-34.[CrossRef][Medline]

Bournat JC, Brown AM, and Soler AP (2000) Wnt-1 dependent activation of the survival factor NF-kB in PC12 cells. J Neurosci Res 61: 21-32.[CrossRef][Medline]

Buss H, Dorrie A, Schmitz ML, Frank R, Livingstone M, Resch K, and Kracht M (2004) Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity. J Biol Chem 279: 49571-49574.[Abstract/Free Full Text]

Carbott DE, Duan L, and Davis MA (2002) Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells. Apoptosis 7: 69-76.[CrossRef][Medline]

Carlson SL, Parrish ME, Springer JE, Doty K, and Dossett L (1998) Acute inflammatory response in spinal cord following impact injury. Exp Neurol 151: 77-88.[CrossRef][Medline]

Chittenden T, Harrington EA, O'Connor R, Flemington C, Lutz RJ, Evan GI, and Guild BC (1995) Induction of apoptosis by the Bcl-2 homologue Bak. Nature (Lond) 374: 733-736.[CrossRef][Medline]

Cohen P (1985) The role of protein phosphorylation in the hormonal control of enzyme activity. Eur J Biochem 151: 439-448.[Medline]

de Castro R Jr, Hughes MG, Xu GY, Clifton C, Calingasan NY, Gelman BB, and McAdoo DJ (2004) Evidence that infiltrating neutrophils do not release reactive oxygen species in the site of spinal cord injury. Exp Neurol 190: 414-424.[CrossRef][Medline]

Deckwerth TL, Elliott JL, Knudson CM, Johnson EM Jr, Snider WD, and Korsmeyer SJ (1996) Bax is required for neuronal death after trophic factor deprivation and during development. Neuron 17: 401-411.[CrossRef][Medline]

Dong H, Fazzaro A, Xiang C, Korsmeyer SJ, Jacquin MF, and McDonald JW (2003) Enhanced oligodendrocyte survival after spinal cord injury in Bax-deficient mice and mice with delayed Wallerian degeneration. J Neurosci 23: 8682-8691.[Abstract/Free Full Text]

Endoh M, Maiese K, and Wagner J (1994) Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. Brain Res 651: 92-100.[CrossRef][Medline]

Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett JR (2000) Requirement for glycogen synthase kinase-3 in cell survival and NF-B activation. Nature (Lond) 406: 86-90.[CrossRef][Medline]

Joshi M and Fehlings MG (2002a) Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 1. Clip design, behavioral outcomes and histopathology. J Neurotrauma 19: 175-190.[CrossRef][Medline]

Joshi M and Fehlings MG (2002b) Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 2. Quantitative neuroanatomical assessment and analysis of the relationships between axonal tracts, residual tissue and locomotor recovery. J Neurotrauma 19: 191-203.[CrossRef][Medline]

Kim MS, Cheong YP, So HS, Lee KM, Kim TY, Oh J, Chung YT, Son Y, Kim BR, and Park R (2001) Protective effects of morphine in peroxynitrite-induced apoptosis of primary rat neonatal astrocytes: potential involvement of G protein and phosphatidylinositol 3-kinase (PI3 kinase). Biochem Pharmacol 61: 779-786.[CrossRef][Medline]

La Rosa G, Cardali S, Genovese T, Conti A, Di Paola R, La Torre D, Cacciola F, and Cuzzocrea S (2004) Inhibition of the nuclear factor-kB activation with pyrrolidine dithiocarbamate attenuates inflammation and oxidative stress after experimental spinal cord trauma in rats. J Neurosurg Spine 1: 311-321.[Medline]

Malmberg AB and Yaksh TL (1995) Cyclooxygenase inhibition and the spinal release of prostaglandin E2 and amino acids evoked by paw formalin injection: a microdialysis study in unanesthetized rats. J Neurosci 15: 2768-2776.[Abstract]

Merrill JE, Ignarro LJ, Sherman MP, Melinek J, and Lane TE (1993) Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol 151: 2132-2141.[Abstract]

Mullane KM, Kraemer R, and Smith B (1985) Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods 14: 157-167.[CrossRef][Medline]

Nesic-Taylor O, Cittelly D, Ye Z, Xu GY, Unabia G, Lee JC, Svrakic NM, Liu XH, Youle RJ, Wood TG, et al. (2005) Exogenous Bcl-xL fusion protein spares neurons after spinal cord injury. J Neurosci Res 79: 628-637.[CrossRef][Medline]

Pap M and Cooper GM (1998) Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 273: 19929-19932.[Abstract/Free Full Text]

Popovich PG, Wei P, and Stokes BT (1997) Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. J Comp Neurol 377: 443-464.[CrossRef][Medline]

Profyris C, Cheema SS, Zang D, Azari MF, Boyle K, and Petratos S (2004) Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol Dis 15: 415-436.[CrossRef][Medline]

Rao R, Hao CM, and Breyer MD (2004) Hypertonic stress activates glycogen synthase kinase 3beta-mediated apoptosis of renal medullary interstitial cells, suppressing an NFB-driven cyclooxygenase-2-dependent survival pathway. J Biol Chem 279: 3949-3955.[Abstract/Free Full Text]

Ruetten H and Thiemermann C (1997) Effect of calpain inhibitor I, an inhibitor of the proteolysis of IkB, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat. Br J Pharmacol 121: 695-704.[Medline]

Sanchez JF, Sniderhan LF, Williamson AL, Fan S, Chakraborty-Sett S, and Maggirwar SB (2003) Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling. Mol Cell Biol 23: 4649-4662.[Abstract/Free Full Text]

Shea TB (1994) Technical report. An inexpensive densitometric analysis system using a Macintosh computer and a desktop scanner. Biotechniques 16: 1126-1128.[Medline]

Shimomura A, Nomura R, and Senda T (2003) Lithium inhibits apoptosis of mouse neural progenitor cells. Neuroreport 14: 1779-1782.[CrossRef][Medline]

Tong N, Sanchez JF, Maggirwar SB, Ramirez SH, Guo H, Dewhurst S, and Gelbard HA (2001) Activation of glycogen synthase kinase 3 beta (GSK-3) by platelet activating factor mediates migration and cell death in cerebellar granule neurons. Eur J Neurosci 13: 1913-1922.[CrossRef][Medline]

Vanegas H and Schaible HG (2001) Prostaglandins and cyclooxygenases in the spinal cord. Prog Neurobiol 64: 327-363.[CrossRef][Medline]

van Weeren PC, de Bruyn KM, de Vries-Smits AM, van Lint J, and Burgering BM (1998) Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 273: 13150-13156.[Abstract/Free Full Text]

Verma IM (2004) Nuclear factor (NF)-kappaB proteins: therapeutic targets. Ann Rheum Dis 63 (Suppl 2): ii57-ii61.[Free Full Text]

Xu J, Kim GM, Chen S, Yan P, Ahmed SH, Ku G, Beckman JS, Xu XM, and Hsu CY (2001) iNOS and nitrotyrosine expression after spinal cord injury. J Neurotrauma 18: 523-532.[CrossRef][Medline]

Yick LW, So KF, Cheung PT, and Wu WT (2004) Lithium chloride reinforces the regeneration-promoting effect of chondroitinase ABC on rubrospinal neurons after spinal cord injury. J Neurotrauma 21: 932-943.[CrossRef][Medline]

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