Glycogen Synthase Kinase-3β inhibition reduces secondary

Transcription

JPET Fast Forward. Published on April 6, 2006 as DOI: 10.1124/jpet.106.102863
JPETThis
Fast
Forward.
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onformatted.
April 6,The
2006
DOI:10.1124/jpet.106.102863
article
has not been
copyedited and
final as
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may differ from this version.
JPET #102863
Glycogen Synthase Kinase-3β inhibition reduces secondary damage
in experimental spinal cord trauma.
Salvatore Cuzzocrea, Tiziana Genovese, Emanuela Mazzon, Concetta Crisafulli, Rosanna Di
Paola, Carmelo Muià, Marika Collin, Emanuela Esposito, Placido Bramanti and Christoph
Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine,
University of Messina, Italy: (SC, TG, EM, CC, CM RDP).
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, UK:
(MC, CT).
IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy (S.C. EM, PB, TG).
Department of Experimental Pharmacology, University of Naples “Federico II”, Via D. Montesano
49, Naples, Italy (E.E.)
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Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 12, 2016
Thiemermann
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #102863
Running title: GSK-3β inhibition reduces spinal cord injury.
Author for correspondence: 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; Tel.: (39) 090 2213644, Fax.:
(39) 090 2213300; email: salvator@unime.it
Number of text pages: 35
Number of tables: 0
Number of figures: 11
Number of references: 40
Number of words in the Abstract: 189
Number of words in the Introduction: 572
Number of words in the Discussion: 1767
List of non-standard abbreviations:
SCI: spinal cord injury
GSK-3β: glycogen synthase kinase-3β
PMN: polymorphonuclear leukocytes
H&E: Haematoxylin/Eosin
BBB: Basso, Beattie, and Bresnahan
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ABSTRACT
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-3β inhibition on the degree of experimental spinal cord trauma induced by the application of
(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
TDZD-8, a potent and selective GSK-3β inhibitor, significantly reduced the degree of (1) spinal
cord
inflammation
and
tissue
injury
(histological
score),
(2)
neutrophil
infiltration
(myeloperoxidase activity), (3) iNOS, nitrotyrosine, and COX-2 expression (4) and apoptosis
(TUNEL 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.
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vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury
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INTRODUCTION
An excessive post-traumatic inflammatory reaction may play an important role in the
secondary injury processes, which develop after spinal cord injury (SCI) (Bartholdi and Schwab,
1995). The primary traumatic mechanical injury to the spinal cord causes the death of a number of
neurons that to date can neither be recovered nor regenerated. However, neurons continue to die for
hours after spinal cord injury (SCI), and this represents a potentially avoidable event (Amar and
Levy, 1999). This secondary neuronal death is determined by a large number of cellular, molecular,
the evolution of the secondary damage is the local inflammatory response in the injured spinal cord.
Recent evidence, however, suggests that leukocytes, especially neutrophils which are the first
leukocytes to arrive within the injured spinal cord (Carlson et al., 1998), may also be directly
involved in the pathogenesis and extension of spinal cord injury in rats. Several authors have
demonstrated that neutrophils are especially prominent in a `marginal zone' around the main area of
injury and infarction at 24 h (de Castro et al., 2004).
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 then, have
been well and extensively descripted in animal SCI models (Profyris et al., 2004). One of the
mechanisms of neuronal apoptosis intensely studied involves GSK-3β (glycogen synthase kinase3β), which is also implicated in neurotrophic base of apoptosis. For instance, overexpression of
GSK-3β induces apoptosis in neuronal cells in culture, and specific inhibitors of GSK-3β are able to
ameliorate this apoptotic response (Pap and Cooper, 1998).
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and biochemical cascades. One such cascade that has been proposed to contribute significantly to
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GSK-3β 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 pro-apoptotic 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 chondroitinase ABC, improve the regenerative response after CNS
ChABC 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 co-crystallized with GSK-3β and all localize within the ATPbinding pocket of the enzyme. GSK-3, as part of a multi-protein complex that contains proteins
such as axin, presenilin and beta-catenin, contains many additional target sites for specific
modulation of its activity.
In this study, we used the GSK-3β inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5dione (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-κB
expression, (5) nitrotyrosine, iNOS and COX-2 expression, (6) apoptosis (TUNEL staining), (7)
Bax and Bcl-2 expression.
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injury and enhances the recovery of forelimb function compared with a single application of
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METHODS
Animals
Male adult CD1 mice (25-30g, Harlan Nossan, Milan, Italy) were housed in a controlled
environment and provided with standard rodent chow and water. Animal care was in compliance
with Italian regulations on protection of animals used for experimental and other scientific purpose
(D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).
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 T6T7 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. Following surgery, 1.0 cc of saline
was administered subcutaneously in order 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.
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Spinal cord injury (SCI)
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Experimental groups
Mice were randomly allocated into the following groups:
(i)
SCI + vehicle group. Mice were subjected to SCI plus intraperitoneal administration of
vehicle (N=30);
(ii)
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 prior, 3 and 6 h after SCI (N=30);
(iii)
Sham + vehicle group. Mice were subjected to the surgical procedures as the above
(iv)
Sham + TDZD-8 group. Identical to Sham + vehicle group except for the administration
of TDZD-8 1 h prior, 3 and 6 h after SCI (N= 30).
Ten mice from each group were sacrificed at different time points (see figure 1) in order to collect
samples for the evaluation of the parameters as described below. In the experiments investigating
the motor score, the animals were treated with 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione
(TDZD-8) or with 3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione
(SB415286) (1 mg/kg 10% DMSO) 1 h prior, 3 and 6 h after SCI and daily until day 9.
Preparation of extracts of whole spinal cord
All the extraction procedures were performed on ice using ice-cold reagents. Spinal cord tissues
from each mouse were suspended in 6 ml of a high-salt extraction buffer (20 mM pH 7.9 HEPES,
420 mM NaCl, 1.5 mM MgCl2, 0.2 mM Ethylenediaminetetraacetic acid (EDTA), 25% glycerol,
0.5 mM phenylmethylsulphonylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A,
5 µg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and homogenized at the highest
setting for 2 min in a Polytron PT 3000 tissue homogenizer. The homogenates were chilled on ice for
15 min and then vigorously shaken for few minutes in the presence of 20 µl of 10% Nonidet P-40. The
pellets were suspended in the supplied complete lysis buffer containing 10mM dithiothreitol, lysed
for 30 min on ice and then centrifuged (10 min, 14,000 x g) to yield the nuclear fraction.
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groups except that the aneurysm clip was not applied (N=30);
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Protein concentration in the supernatant was determined by the Bio-Rad protein assay kit
(Bio-Rad), and stored at –80°C.
Western blot analysis for IκB-α, phospho-NF-κB p65 (serine 536), NF-kB p65, Bax and Bcl-2
The levels of IκB-α, phospho-NF-κB 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
incubation at room temperature for 1 hr with 10 % (w/v) non-fat dry milk in PBS and then
incubated overnight at 4°C with anti-IκB-α (1:1000) or anti-Bax (1:100) or anti-Bcl-2 (1:100) or
anti phospho-NF-κB p65 (serine 536) (1:1000). Nuclear fractions were incubated with anti- NF-kB
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 perpxidase-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.). Subsequently, the
relative expression of the proteins was quantified by densitometric scanning of the X-ray films with
GS-700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst, IBM).
Electrophoretic mobility-shift assay (EMSA)
Double-stranded oligonucleotides containing the NF-κB recognition sequence (5’-GAT
CGA GGG GAC TTT CCC TAG-3’) were end labeled with γ-[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 reaction
buffer containing 2 µg poly dI-dC, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM
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membranes, according to the manufacturer's instructions. Briefly, the membranes were saturated by
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ethylenediaminotetraacetic acid, 1 mM DL-dithiothreitol, 1 mg/ml bovine serum albumin, 10%
glycerol. The specificity of the DNA/protein binding was determined for NF-κB 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 addition of radiolabeled probe. Protein-nucleic
acid complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 ×
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. Subsequently, the relative bands were
Rad) and a computer program (Molecular Analyst, IBM).
Myeloperoxidase activity
Twentyfour hours after SCI myeloperoxidase (MPO) activity, an indicator of
polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described
(Mullane et al., 1985). At the specified time following SCI, spinal cord tissues were obtained and
weighed and each piece homogenized in a solution containing 0.5 % (w/v) hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30
min at 20,000 x g 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/g of wet tissue.
Immunohistochemical localization of nitrotyrosine, iNOS, COX-2, Bax and Bcl-2
At the 24 h after SCI, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8
mm sections were prepared from paraffin embedded tissues. After deparaffinisation, 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. Non-specific
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quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (Bio-
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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 antinitrotyrosine 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
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 previously described (Shea, 1994) by using Optilab Graftek software on a
Macintosh personal computer.
Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling (TUNEL) Assay.
TUNEL assay was conducted by using a TUNEL detection kit according to the
manufacturer’s instructions (Apotag, HRP kit DBA, Milano, Italy). Briefly, 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 (TdT) buffer
containing deoxynucleotidyl transferase and biotinylated dUTP in TdT 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.
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biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To verify the
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Light microscopy
Spinal cord biopsies were taken at 24 h following 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 (thickness 5
µm) were deparaffinized with xylene, stained with Haematoxylin/Eosin (H&E) and Luxol Fast Blue
staining (used to assess demyelination) and studied using light microscopy (Dialux 22 Leitz). All
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 non-pyrogenic saline (0.9% NaCl; Baxter, Italy) or 10% DMSO.
Statistical evaluation
All values in the figures and text are expressed as mean ± standard error of the mean (SEM) 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 ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value of
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the histological studies were performed in a blinded fashion.
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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.
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Results
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 (Figure 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
when compared with sham-operated mice (Figure 2a). Notably, a significant protection against the
spinal cord injury was observed in TDZD-8 treated mice (Figure 2c). Myelin structure was detected
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 (Figure 2e). In contrast, in TDZD-8 treated mice myelin degradation was
attenuated in the central part of lateral (Figure 2f) and dorsal funiculi. In order to evaluate if
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. While motor function was only slightly
impaired in sham mice, mice subjected to SCI had significant deficits in hind limb movement
(Figure 3). A significant amelioration of hind limb motor disturbances was observed in TDZD-8
treated mice (Figure 3). In addition, in order to confirm that the protective effects of TDZD-8 on
motor function are related to the GSK-3β, we have also investigated whether SB415286, an other
GSK-3β selective inhibitor, attenuates the motor dysfunction induced by SCI. As shown in Figure
3 also the treatment with SB415286 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 (Figure 3).
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by Luxol fast blue staining (Figure 2d,e,f). In sham animals (Figure 2d), myelin structure was
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Effects of TDZD-8 on neutrophil infiltration
The abovementioned histological pattern of spinal cord injury appeared to be correlated with
the influx of leukocytes into the spinal cord. Therefore, we investigated the role of TDZD-8 on the
neutrophil infiltration by measuring tissue myeloperoxidase (MPO) activity. MPO activity was
significantly elevated in the spinal cord at 24 h after injury in control mice subjected to SCI when
compared with sham-operated mice (Figure 4). In TDZD-8 treated mice, the MPO activity in the
spinal cord at 24 h after injury was significantly attenuated in comparison to that observed in SCI
TDZD-8 modulates expression of nitrotyrosine, iNOS and COX-2 after SCI
Twenty-four h 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 (Figures 5a, 6), nitrotyrosine (Figures 5c, 6) and COX-2 (Figures 5e,
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 (Figures 5b, 6), nitrotyrosine (Figures 5d, 6) and COX-2 (Figures 5f, 6) in the
spinal cord.
Effect of TDZD-8 on IκB-α degradation, phosphorylation of Ser536 on p65, expression of NF-κB
p65 and NF-κB translocation.
By Western Blot analysis and translocation of NF-κB, one of the major transcription factors
involved in the signal transduction of inflammation (La Rosa et al., 2004), we evaluated both IκB-α
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controls (Figure 4).
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degradation, phosphorylation of Ser536 on the NF-κB subunit p65, total NF-κB p65 to investigate
the cellular mechanisms by which treatment with TDZD-8 may attenuate the development of SCI.
IκB-α appearance in the spinal cord homogenates was investigated by immunoblot analysis at 24 h
after SCI. A basal level of IκB-α was detected in the spinal cord from sham-operated animals
(Figure 7a,a1), whereas in SCI control mice IκB-α levels were substantially reduced (Figure
7a,a1). TDZD-8 prevented the SCI-induced IκB-α degradation and the IκB-α expression remained
unchanged 24 h after SCI in TDZD-8 treated mice (Figure 7a,a1). In addition, SCI caused a
GSK-3β inhibitor TDZD-8 significantly reduced the phosphorylation of p65 on Ser536 (Figure
7b,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 24h after SCI compared to the sham-operated mice
(Figure 7c,c1). TDZD-8 treatment significantly reduced the levels of NF-kB p65 protein as shown
in Figure 7c,c1. To detect NF-κB/DNA binding activity, whole extracts of spinal cord from each
mouse were analyzed by EMSA. A low basal level of NF-κB/DNA binding activity was detected in
tissue from sham-operated mice (Figure 8a,a1). 24 h after SCI, the DNA binding activity was
significantly increased in whole extracts obtained from vehicle-treated SCI control mice (Figure
8a,a1). Treatment of mice with TDZD-8 caused a significant inhibition of SCI-induced NFκB/DNA binding activity as revealed by EMSA (Figure 8a,a1). The specificity of NF-κB/DNA
binding complex was demonstrated by the complete displacement of the NF-κB/DNA binding in
the presence of a 50-fold molar excess of unlabeled NF-κB probe (W.T. 50x) in the competition
reaction. In contrast a 50-fold molar excess of unlabeled mutated NF-κB probe (Mut. 50x) or Sp-1
oligonucleotide (Sp-1 50x) had no effect on this DNA-binding activity (data not shown).
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significant increase in the phosphorylation of Ser536 at 24 h (Figure 7b,b1). The treatment with the
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Effects of TDZD-8 on apoptosis in spinal cord after injury
To test whether spinal cord damage was associated to cell death by apoptosis, we measured
TUNEL-like staining in the perilesional spinal cord tissue. Almost no apoptotic cells were detected
in the spinal cord from sham-operated mice (data not shown). At 24h after the trauma tissues
from SCI control mice demonstrated a marked appearance of dark brown apoptotic cells and
intercellular apoptotic fragments (Figure 9a, a1). In contrast, tissues obtained from mice treated
with TDZD-8 (Figure 9b) demonstrated a small number of apoptotic cells or fragments.
At 24 h after SCI the appearance of Bax in spinal cord homogenates was investigated by
Western blot. A basal level of Bax was detected in the spinal cord from sham-operated animals
(Figure 10a,a1). Bax levels were appreciably increased in the spinal cord from control mice
subjected to SCI (Figure 10 a,a1). On the contrary, TDZD-8 treatment (1 h prior and 3 and 6 h after
SCI induction) prevented the SCI-induced Bax expression (Figure 10 a,a1)
By Western blot analysis were also analyzed whole extracts from spinal cord of each mice to
detect Bcl-2 expression . A low basal level of Bcl-2 expression was detected in spinal cord from
sham-operated mice (Figure 10b,b1). 24 h after SCI, the Bcl-2 expression was significantly
reduced in whole extracts obtained from spinal cord of SCI control mice (Figure 10b,b1).
Treatment of mice with TDZD-8 (1 h prior and 3 and 6 h after SCI induction) significantly reduced
the SCI-induced inhibition of Bcl-2 expression (Figure 10b,b1).
Moreover, samples of spinal cord tissue were taken at 24 h after SCI in order to determine
the immunohistological staining for Bax and Bcl-2. Sections of spinal cord from sham-operated
mice did not stain for Bax (Figure 11a) whereas spinal cord sections obtained from SCI control
mice exhibited a positive staining for Bax (Figure 11b). TDZD-8 treatment reduced the degree of
positive staining for Bax in the spinal cord of mice subjected to SCI (Figure 11c).
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Western blot analysis and immunohistochemistry for Bax and Bcl-2
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In addition, spinal cord sections from sham-operated mice demonstrated Bcl-2 positive
staining (Figure 11d) while in SCI control mice the staining significantly reduced (Figure 11e).
TDZD-8 treatment attenuated the loss of positive staining for Bcl-2 in the spinal cord from SCIsubjected mice (Figure 11f).
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DISCUSSION
Spinal cord injury (SCI) induces lifetime disability, and no suitable therapy is available to
treat victims or to minimize their suffering. The inhibition of GSK-3β 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-3β 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
funiculi. This histological damage was associated to the loss of motor function. SCI induced an
inflammatory response in the spinal cord, characterized by increased IκB-α degradation, enhanced
NF-κB/DNA binding, amplified expression of pro-inflammatory mediators iNOS, COX-2 and
nitrotyrosine, and increased MPO activity. Our results show that TDZD-8, a potent and selective
GSK-3β inhibitor, reduced (1) the degree of spinal cord damage, (2) neutrophils infiltration, (3) NFκB/DNA binding, (4) IκB-α degradation, (5) expression of iNOS, nitrotyrosine, and COX-2, and
(6) apoptosis.
GSK-3β role 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 GSK3β. Among these is the transcription factor NF-κB, whose function is strikingly altered by GSK-3β
(Hoeflich et al., 2000; Buss et al., 2004). NF-κB 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-α, IL-1β, iNOS and COX-2, to name but a few (Verma , 2004). The discovery in 1997 that
inhibition of the NF-κB activation may be useful in conditions associated to local or systemic
inflammation (Ruetten and Thiemermann, 1997) stimulated the search for agents which prevent the
activation of NF-κB. The extent to which GSK-3β activates or blocks NF-κB signaling remains
unclear. In 2000 Hoeflich and colleagues first demonstrated that deletion of GSK-3β had no effect
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dura via a four-level T5-T8 laminectomy resulted in edema and loss of myelin in lateral and dorsal
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on the TNF-α-induced IκB-α degradation or on the nuclear translocation of the subunit p65, but
prevented the activation of NF-κB by an unknown mechanism (Hoeflich et al., 2000). On the other
hand, other studies provided evidence for an inverse association between GSK-3β activity and NFκB signaling. Sanchez and colleagues reported that over-expression of GSK-3β in astrocytes was
associated to the inhibition of NF-κB activation (Sanchez et al., 2003). Another recent report
showed that GSK-3β-dependent phosphorylation of a specific serine residue (Ser468) on p65 blocks
the activation of NF-κB and that inhibition of GSK-3β was related to increased p65 activity (Buss
on p65 in the spinal cord tissues at 24 h, whereas GSK-3β inhibitor TDZD-8 treatment
significantly reduced this phosphorylation. Moreover, we also demonstrate that selective and potent
GSK-3β inhibitor TDZD-8, inhibited the IκB-α degradation as well as the NF-κB translocation.
Taken together, the balance between pro-inflammatory and pro-survival roles of NF-κB may
depend on the phosphorylation status of p65, and GSK-3β may play a central role in this process.
However, the reasons for the apparent discrepancies in the modulatory effects of GSK-3β on NFκB activity remain to be fully clarified. In the pathological processes of acute SCI the up-regulation
of COX-2, a key enzyme in the synthesis of prostaglandins (PGs), 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 and
colleagues have recently shown that the inhibition of GSK-3β leads to an activation of COX-2 via
induction of NF-κB-dependent pathways (Rao et al., 2004). However, it has been also demonstrated
that inhibition of GSK-3β down-regulates the expression of COX-2, induced by TNF or hypertonic
stress, in cells via the induction of a NF-κB-COX-2 dependent pathway (Rao et al., 2004). The
19
et al., 2004). We report here that SCI caused a significant increase in the phosphorylation of Ser536
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result 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 TDZD8. COX-2 is one of the genes regulated by NF-κB and there is good evidence that an enhanced
formation of prostanoids following 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 (Vanegas and Schaible, 2001; Abramson et al., 2001). This study
when 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-κB activation by TDZD-8 mediated by GSK-3β. 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 that suggests that a alteration of the tissue had also
occurred, due to 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), likely
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 ROS in the secondary
neuronal damage of spinal cord injury (SCI) (Xu et al., 2001). Generation of free radicals and nitric
20
demonstrates that TDZD-8 attenuates the expression of iNOS in the tissue from SCI treated mice
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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 demonstrates
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-3β is
known to induce apoptosis in neuronal cells in culture, and specific inhibitors of GSK-3β are able to
ameliorate this apoptotic response (Pap and Cooper, 1998). Recent studies from our laboratory have
shown that up-regulation of GSK-3β activity can also lead to cell death and aberrant neuronal
(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 (Dong et al., 2003). The Bax gene
plays an important role in developmental cell death (Chittenden et al., 1995) and in CNS 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 and colleagues 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 FP
increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically
relevant in preserving sensory and motor functions after SCI (Nesic-Taylor et al., 2005). It’s known
that pathways which inhibit GSK-3β activity, such as PI-3K or Wnt signaling, often lead to the
induction of the nuclear factor kB (NF-kB) cell survival pathway (Bournat et al., 2000). Indeed,
GSK-3β is a major target of Akt/PKB (van Weeren et al., 1998), which is activated by the PI-3K
mediated signaling pathway. Cellular factors which have implicated in the regulation of astrocyte
apoptosis include 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,
21
migration in primary neuronal populations (Tong et al., 2001). Dong and colleagues, used Wlds
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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 pro-apoptotic signals including sequestration of
Bcl-2 and enhanced activation of an Akt substrate, GSK-3β (Pap and Cooper, 1998). We identified
proapoptotic
transcriptional changes,
including upregulation
of proapoptotic Bax
and
downregulation of antiapoptotic Bcl-2, using western blot assay and by immunohystochemical
staining. We report in the present study for the first time that the treatment with TDZD-8 in SCI
experimental model documents features of apoptotic cell death after SCI, suggesting that protection
demonstrated that the treatment with TDZD-8 lowers the signal for Bax in treated group when
compared with SCI+vehicle operated mice spinal cord, while on the contrary, the signal is much
more express for Bcl-2 in TDZD-8 treated mice than in SCI+vehicle operated mice. These results
are in agreement with a recent evidence that have clearly demonstrated that lithium treatment
upregulates the expression of Bcl-2 in axotomized rubrospinal tract via inhibiting GSK-3β (Yick et
al., 2004). Base on this evidence we clearly have shown that TDZD-8 interfere in the apoptotic
process induced by SCI. However is not possible to exclude that antiapoptotic effect observed
after TDZD-8 treatment it may be partially dependent on the attenuation of the inflammatoryinduced damage. Further studies are needed in order to clarify this mechanisms.
Finally, in this study we demonstrate that TDZD-8 treatment significantly reduced the SCI-induced
spinal cord tissues alteration as well as improve the motor function. Taken together, the results of
the present study enhance our understanding of the role of GSK-3β in the pathophysiology of spinal
cord cell and tissue injury following trauma. Our results imply that inhibitors of the activity of
GSK-3β may be useful in the therapy of spinal cord injury, trauma and inflammation.
22
from apoptosis may be a prerequisite for regenerative approaches to SCI. In particular, we
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Acknowledgments
The authors would like to thank Giovanni Pergolizzi and Carmelo La Spada for their
excellent technical assistance during this study, Mrs Caterina Cutrona for secretarial
assistance and Miss Valentina Malvagni for editorial assistance with the manuscript.
23
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Footnotes
This study was supported by PRIN 2003.
31
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Legends for Figures
Figure 1. Mice were sacrificed at different time points in order to evaluate the various parameters.
n=10 mice from each group for each time point. See material and methods for further explanations.
Figure 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. 24 h after trauma a significant damage to the
presence of edema as well as alteration of the white matter (b). Notably, 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 no-treated SCI
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 3 experiments
performed on different experimental days. wm: White matter; gm: gray matter.
Figure 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 vs.
SCI.
Figure 4: Effects of TDZD-8 on myeloperoxidase (MPO) activity. Following the injury
myeloperoxidase (MPO) activity in spinal cord of no-treated 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 vs. vehicle. °p<0.01 vs. SCI.
32
spinal cord, from no-treated SCI operated  mice at the perilesional area, was assessed by the
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Figure 5: Immunohistochemical localization of nitrotyrosine, iNOS, 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, when
compared to positive nitrotyrosine (a), iNOS (c) and COX-2 (e) staining obtained from the spinal
performed on different experimental days.
Figure 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 % of total tissue area.
*P<0.01 vs. Sham; °P<0.01 vs. SCI. ND: not detectable.
Figure 7. Representative Western blots of IκB-α degradation (aa1), phospho Ser536 on NF-κB
p65 (b,b1) and total NF-kB p65 subunit protein (c,c1).
A basal level of IκB-α was detected in the spinal cord from sham-operated animals (a,a1), whereas
in SCI control mice IκB-α levels were substantially reduced (a,a1). TDZD-8 prevented the SCIinduced IκB-α degradation and the IκB-α band remained unchanged 24 h after SCI in TDZD-8
treated mice (a,a1). In addition, SCI caused a significant increase in the phosphorylation of Ser536
at 24 h (b,b1). The treatment with the GSK-3β inhibitor TDZD-8 significantly reduced the
phosphorylation of p65 on Ser536 (b,b1). Moreover, the levels of the NF-kB p65 subunit protein in
the nuclear fractions of the spinal cord tissue were also significant increased at 24h after SCI
33
cord tissue of mice 24 h after the injury. This figure is representative of at least 3 experiments
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compared to the sham-operated mice (c,c1). The levels of NF-kB p65 protein were significantly
reduced in the nuclear fractions of the spinal cord tissues animals from animals that had received
TDZD-8 as shown in (c,c1 ). Immunoblotting in panel a, b, c are representative of one spinal cord
out of 5 analyzed. The results in panel a1, b1, c1 are expressed as mean ± s.e.mean from 5 blots
*P<0.01 versus SHAM, °P<0.01 versus SCI.
Figure 8. Effect of TDZD-8 on NF-κB/DNA binding activity in mice spinal cord
described in Materials and Methods and incubated with 32P-labelled NF-κB probe. Representative
EMSA (a) of NF-κB shows the effect of TDZD-8 (SCI + TDZD-8) NF-κB/DNA binding activity
evaluated in spinal cord tissue 24 h after the injury. The intensity of retarded bands (measured by
phosphoimager) in SCI-operated mice was significantly increased vs. sham group (a1). TDZD-8
treatment significantly reduced the SCI-induced elevation of NF-κB/DNA binding activity. The
results in panel a1 are expressed as mean ± s.e.mean from EMSA. *P<0.01 versus SHAM, °P<0.01
versus SCI
Figure 9: Representative TUNNEL 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 as well as 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. Section c demonstrate the positive staining in the Kit
positive control tissue (normal rodent mammary gland). Figure is representative of at least 3
experiments performed on different experimental days.
34
Whole extracts from injured (SCI) or non-inflamed (sham) mice spinal cord were prepared as
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Figure 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 inured mice which 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
(a1 and b1) The intensity of retarded bands (measured by phosphoimager) in all the experimental
groups. Immunoblotting in panel A and B is representative of one spinal cord tissues out of 5-6
analyzed. The results in panel A1 and B1 are expressed as mean ± s.e.m. from 5-6 spinal cord
tissues. *P<0.01 vs. Sham; °P<0.01 vs. SCI.
Figure 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 24h 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 3 experiments performed on different
experimental days.
35
evident in the tissue from spinal cord inured mice which received TDZD-8.

Glycogen Synthase Kinase-3β inhibition reduces secondary

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