Article

Activation of JAK/STAT signaling in neruons following spinal cord injury in mice

Department of Neurosurgery, Nagoya University, Graduate School of Medicine, Nagoya, Japan.
Journal of Neurochemistry (Impact Factor: 4.28). 03/2006; 96(4):1060-70. DOI: 10.1111/j.1471-4159.2005.03559.x
Source: PubMed

ABSTRACT

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway is one of the most important in transducing signals from the cell surface to the nucleus in response to cytokines. In the present study, we investigated chronological alteration and cellular location of JAK1, STAT3, phosphorylated (p)-Tyr1022/1023-JAK1, p-Tyr705-STAT3, and interleukin-6 (IL-6) following spinal cord injury (SCI) in mice. Western blot analysis showed JAK1 to be significantly phosphorylated at Tyr1022/1023 from 6 h after SCI, peaking at 12 h and gradually decreasing thereafter, accompanied by phosphorylation of STAT3 at Tyr705 with a similar time course. ELISA analysis showed the concentration of IL-6 in injured spinal cord to also significantly increase from 3 h after SCI, peaking at 12 h, then gradually decreasing. Immunohistochemistry revealed p-Tyr1022/1023-JAK1, p-Tyr705-STAT3, and IL-6 to be mainly expressed in neurons of the anterior horns at 12 h after SCI. Pretreatment with a JAK inhibitor, AG-490, suppressed phosphorylation of JAK1 and STAT3 at 12 h after SCI, reducing recovery of motor functions. These findings suggest that SCI at the acute stage produces IL-6 mainly in neurons of the injured spinal cord, which activates the JAK/STAT pathway, and that this pathway may be involved with neuronal response to SCI.

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Available from: Nobuteru Usuda, Nov 04, 2014
Activation of JAK/STAT signalling in neurons following spinal
cord injury in mice
Katsuaki Yamauchi,* Koji Osuka,* Masakazu Takayasu, Nobuteru Usuda,à Ayami Nakazawa,à
Norimoto Nakahara,§ Mitsuhiro Yoshida ,* Chihiro Aoshima,* Masahito Hara* and Jun Yoshida*
*Department of Neurosurgery, Nagoya University, Graduate School of Medicine, Nagoya, Japan
Department of Neurosurgery, Okazaki Municipal Hospital, Aichi, Japan
àDepartment of Anatomy II, Fujita Health University, School of Medicine, Aichi, Japan
§Department of Biomedicine, Nagoya University, Graduate School of Medicine, Nagoya, Japan
Abstract
The Janus kinase (JAK)/signal transducer and activator of
transcription (STAT) signalling pathway is one of the most
important in transducing signals from the cell surface to the
nucleus in response to cytokines. In the present study, we
investigated chronological alteration and cellular location
of JAK1, STAT3, phosphorylated (p)-Tyr1022/1023-JAK1,
p-Tyr705-STAT3, and interleukin-6 (IL-6) following spinal cord
injury (SCI) in mice. Western blot analysis showed JAK1 to be
significantly phosphorylated at Tyr1022/1023 from 6 h after
SCI, peaking at 12 h and gradually decreasing thereafter,
accompanied by phosphorylation of STAT3 at Tyr705 with a
similar time course. ELISA analysis showed the concentration
of IL-6 in injured spinal cord to also significantly increase from
3 h after SCI, peaking at 12 h, then gradually decreasing.
Immunohistochemistry revealed p-Tyr1022/1023-JAK1,
p-Tyr705-STAT3, and IL-6 to be mainly expressed in neurons
of the anterior horns at 12 h after SCI. Pretreatment with a
JAK inhibitor, AG-490, suppressed phosphorylation of JAK1
and STAT3 at 12 h after SCI, reducing recovery of motor
functions. These findings suggest that SCI at the acute stage
produces IL-6 mainly in neurons of the injured spinal cord,
which activates the JAK/STAT pathway, and that this pathway
may be involved with neuronal response to SCI.
Keywords: interleukin-6, Janus kinase 1, phosphorylation,
signal transducer and activator of transcription 3, spinal cord
injury.
J. Neurochem. (2006) 96, 1060–1070.
Pro-inflammatory cytokines such as tumour necrosis factor-a
(TNF-a), interleukin (IL)-1b, and IL-6 contribute to secon-
dary damages in spinal cord injury (SCI) by initiating a
complex cascade leading to specific signalling molecules. It
has been reported that expression of mRNAs for TNF-a,
IL-1b and IL-6 is rapidly up-regulated after SCI, with peaks
at 1, 6 and 3 h, respectively (Hayashi et al. 2000). Most
inflammatory responses induced by TNF-a are mediated at
the transcriptional level by nuclear factor-jB (NF-jB)
(Baeuerle and Henkel 1994). In injured spinal cords, NF-jB
is activated within 0.5 h and this persists for at least 72 h,
expression being found in macrophages/microglia, endothel-
ial cells, and neurons (Bethea et al. 1998). TNF- a induces
apoptosis mediated in part by nitric oxide via up-regulation
of inducible nitric oxide synthase (iNOS) (Yune et al . 2003)
and neuronal co-localization of iNOS and NF-jB has been
demonstrated, suggesting these to be important effectors in
trauma-induced neuropathology after SCI (Bethea et al.
1998). Signals for IL-6 mRNA have also been observed in
motoneurons at 24–72 h after injury, with the suggestion that
these molecules may be involved in promoting axonal
sprouting in the injured spinal cord (Hayashi et al. 2000).
Received March 2, 2005; revised manuscript received September 20,
2005; accepted September 21, 2005.
Address correspondence and reprint requests to Koji Osuka, Depart-
ment of Neurosurgery, Nagoya University, Graduate School of Medicine,
65 Tsurumai Showa-ku Nagoya, 466–8560, Japan.
E-mail: kosuka@d6.dion.ne.jp
Abbreviations used: CNTF, ciliary neurotrophic factor; DMSO,
dimethylsulfoxide; ECL, enhanced chemiluminescence; GFAP, glial
fibrillary acidic protein; gp130, glycoprotein 130; IL-6, interleukin-6;
iNOS, inducible nitric oxide synthase; JAK, Janus kinase; NF-jB,
nuclear factor-jB; PVDF, polyvinylidene difluoride; SCI, spinal cord
injury; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; STAT, signal transducer and activator of transcription;
TNF-a, tumour necrosis factor-a.
Journal of Neurochemistry, 2006, 96, 1060–1070 doi:10.1111/j.1471-4159.2005.03559.x
1060 Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
2006 The Authors
Page 1
The resultant signalling induced by these pro-inflammatory
cytokines plays important roles in pathophysiological chan-
ges after SCI, but detailed signalling pathways induced by
IL-6 remain to be elucidated after SCI in vivo.
The Janus kinase (JAK)/signal transducer and activator of
transcription (STAT) signalling pathways is one of the most
important transducing signals from the cell surface to the
nucleus in response to cytokines (Hirano et al. 1997;
Cattaneo et al. 1999). Binding of cytokines to the cell
membrane receptor induces dimerization of glycoprotein (gp)
130, followed by activation of JAK1 in the cell membrane,
which subsequently phosphorylates STAT3 at Tyr705 in the
cytoplasm. Signals from the phosphorylated STAT3 then
translocate into the nucleus, where the molecule is further
phosphorylated at Ser727, followed by transcription of target
genes. The JAK/STAT signalling pathway activated by
cytokines may also be involved in the neuronal or glial
differentiation of precursor cells in developing brain (Bonni
et al. 1997; Cattaneo et al. 1999). With cerebral ischaemia,
STAT3 is phosphorylated in neurons and endothelial cells
within 24 h of post-ischaemic reperfusion (Suzuki et al.
2001). Other authors have reported activation of STAT3 in
reactive astrocytes of ischaemic regions (Justicia et al. 2000;
Choi et al. 2003). Following axotomy in rat regenerating
hypoglossal neurons, activation of JAK and STAT3 were
confirmed in hypoglossal motoneurons (Yao et al. 1997;
Schwaiger et al. 2000). In the intact spinal cord, STAT3 is
mainly localized in motor neurons and dendrite-like struc-
tures in the anterior horn (Stromberg et al. 2000). However,
chronological or topographical alterations of JAK or STAT3
under pathophysiological circumstances, for example after
SCI, have hitherto remained unclear.
The purpose of the present study was thus to investigate
the correlation between IL-6 and elements in the JAK/STAT
signalling pathway and examine where this occurs in the
mouse spinal cord compression model using western blot-
ting, immunohistochemistry and ELISA techniques. We also
explored the role of this signalling pathway after SCI by
assessing recovery of hindlimb motor functions of mice
subjected to SCI in vivo with or without a JAK inhibitor.
Materials and methods
Animals
Female mice (C57BL/6NCrj, Charles River Japan Inc., Yokohama
Japan) between 8 and 10 weeks of age (weight 20–23 g), were
housed two or three per cage and kept at a temperature of 24C with
free access to water and food before and after surgery. All
experiments were carried out in accordance with the Nagoya
University Institutional Animal Care and Use Committee.
Spinal cord injury (SCI) model
The mice were anaesthetized with 1.5% halothane and maintained
on 1.25% halothane in an oxygen/nitrous oxide (30/70%) gas
mixture. Temperature was monitored with rectal probes and
maintained between 36.5 and 37.5C with heating pads and
lamps. The mice were fixed with a stereotaxic apparatus
and subjected to severe spinal cord compression as described by
Farooque (2000). In brief, laminectomy at Th8 vertebra was
performed with the dura intact. Compression (10 g/mm
2
) was
applied to the exposed spinal cord for 5 min. After compression
the skin incision was closed. The epicenters (2 mm in length) of
SCI were used as samples for analysis and were collected after 0
(immediately after SCI), 1, 3, 6, 12, 18, 24, 48, 72, 120 and 168 h
after SCI, from mice killed by decapitation under deep anaesthesia.
Those killed at the times of 0 and 1 h after SCI were maintained
on halothane throughout. Control mice underwent all surgical
manipulations, including laminectomy, without spinal cord com-
pression. The removed spinal cord tissues were frozen in liquid
nitrogen and kept at )80C until use.
Western blot analysis
One sample was prepared for analysis from each animal.
Manipulations were carried out on ice. Spinal cord tissue samples
for western blot analysis (n ¼ 4) were homogenized using a
homogenizer in 300 lL of buffer and phosphatase inhibitor
solution: 50 mmol/L Tris base/HCl (pH 7.5), 0.2 mmol/L EGTA
(pH 7.5), 0.2 mmol/L EDTA (pH 8.0), 0.2 mmol/mL phenyl-
methylsulfonyl fluoride, 1 mg/mL pepstatine, 0.2 mg/mL aproti-
nine, 2 mg/mL leupeptine, 0.1 mmol/L dithiothreitol, 1 mmol/L
sodium orthovanadate (Na
3
VO
4
), 50 mmol/L sodium fluoride,
2 mmol/L sodium pyrophosphate (Na
4
P
2
O
7
10 H
2
O), and 2%
sodium dodecyl sulfate (SDS). The homogenates were then
centrifuged at 18 800 g at 4C for 15 min and protein
concentrations of the supernatants were determined by the
method of Bradford using bovine serum as the standard. Crude
supernatant samples containing 10 mg of protein were subjected
to 7.5% SDS–polyacrylamide gel electrophoresis (PAGE), and
proteins were transferred to polyvinylidene difluoride (PVDF)
membranes and incubated with primary antibodies against
phosphorylated (p)-Tyr1022/1023-JAK1 and p-Tyr1007/1008-
JAK2 (Cell Signaling Technology, Beverly, MA, USA) at a
dilution of 1 : 250 overnight at 4C, p-Tyr705-STAT3 (Cell
Signaling Technology) at a dilution of 1 : 500 for 1 h at 25C,
and actin (Sigma, St Louis, MO, USA) at a dilution of 1 : 300
for 1 h at room temperature. After washing, the membranes
were incubated with goat anti-rabbit polyclonal IgG conjugated
to horseradish peroxidase (Sigma) at a dilution of 1 : 3000
for 30 min at room temperature. Reactions were developed
with enhanced chemiluminescence (ECL) or ECL plus
(Amersham Biosciences, Little Chalfont, UK). Phosphorylated-
Tyr1022/1023-JAK1, p-Tyr1007/1008-JAK2 or p-Tyr705-STAT3
immunoblots were stripped from PVDF membranes and re-blotted
with primary antibodies against JAK1 (BD Biosciences Pharm-
ingen, Franklin Lakes, NJ, USA), JAK2 (Chemicon, Temecula,
CA, USA) and STAT3 (BD Biosciences Pharmingen) at a
dilution of 1 : 500 for 60 min at room temperature. Exposure
to the secondary antibody, goat anti-mouse monoclonal or anti-
rabit polyclonal IgG (Sigma), was at a dilution of 1 : 3000 for
30 min, followed by colour development. Band intensities
were quantitated by densitometric scanning using the NIH
IMAGE program.
JAK/STAT activation following spinal cord injury 1061
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Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
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ELISA analysis for interleukin-6
Spinal cord tissue samples for ELISA (n ¼ 4) were prepared using
the following buffer: 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L
sodium chloride, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl
fluoride, 1% Triton X-100, 10 mg/mL aprotinine, and 1 mg/mL
pepstatin. Homogenates were centrifuged at 18 800 g at 4C for
15 min and protein concentrations of the supernatants were
determined as described above. The concentrations of interleukin-
6 (IL-6) were measured using a sandwich ELISA kit (Genzyme
Technology, Minneapolis, MN, USA) according to the manufac-
turers instructions. The IL-6 level was expressed in picograms per
millilitre.
Immunohistochemistry
Control and experimental mice 12 h after SCI were perfused with
ice-cold 100 mL of 4% paraformaldehyde in 0.1 mol/L phosphate
buffer (pH 7.4). The epicenters of SCI were removed and preserved
in the fixation solution for 3 h and rinsed with 0.1 mol/L lysine
hydrochloride in 0.1 mol/L phosphate-buffered saline for 3 h. Serial
axial cryostat sections (10 lm) were collected on silane-coated
slides for staining by the ABC technique at room temperature. The
staining sequence was as follows: 2% goat serum for 30 min;
primary polyclonal antibodies against p-Tyr1022/1023-JAK1 or
p-Tyr705-STAT3 (the same as for western blot), JAK1 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), STAT3 (Cell Signaling
Technology) or IL-6 (Genzyme Technology) each at a dilution of
1 : 500 overnight; biotinylated anti-rabbit or goat IgG for 1 h; and
avidin–biotinylated peroxidase complex for 1 h. The solution for
antibody dilution was made with 20 mmol/L sodium phosphate
(pH 7.4) in 150 mmol/L sodium chloride containing 2% normal
goat serum. Sera for the blocking step, biotinylated antibodies and
avidin–biotinylated peroxidase complex were purchased from
Vector Laboratories (Burlingame, CA, USA). Reaction products
were demonstrated by incubation in 0.05% 3,3¢-diaminobendizine
tetrachloride and 0.01% H
2
O
2
in 50 mmol/L Tris-HCl (pH 7.5) for
10 min. To amplify the reaction where necessary, development was
carried out for 15 min with the same reagents in 100 mmol/L
sodium phosphate (pH 7.2), containing 0.025% cobalt chloride and
0.02% nickel ammonium.
Immunofluorescent staining
To study the cellular localization of IL-6 and p-Tyr705-STAT3,
double staining of IL-6/neuron-specific class III b-tubulin (TuJ-1),
p-Tyr705-STAT3/TuJ-1, p-Tyr705-STAT3/glial fibrillary acidic pro-
tein (GFAP) and p-Tyr705-STAT3/CD11b was performed. Tissue
samples at 12 and 48 h after SCI were prepared according to the
methods described above. Non-specific immunoreactions were
blocked with fetal bovine serum. After permeabilization in 0.1%
Triton X-100, 100 mmol/L sodium phosphate (pH 7.4), and
150 mmol/L sodium chloride, samples were treated with primary
antibodies against IL-6 (Genzyme Technology; at a dilution of
1 : 500)/TuJ-1 (mouse monoclonal antibody; Genzyme Technology;
at a dilution of 1 : 50), p-Tyr705-STAT3 (Cell Signaling Technol-
ogy; at a dilution of 1 : 500)/TuJ-1, p-Tyr705-STAT3/GFAP (mouse
monoclonal antibody; Sigma; at a dilution of 1 : 400), and
p-Tyr705-STAT3/CD11b (a kind gift from Professor K. Ikenaka,
National Institute for Physiological Sciences, Japan, at a dilution of
1 : 10) overnight at 4 C. IL-6 and p-Tyr705-STAT3 were detected
with Alexa 488-labelled IgG (Molecular Probes, Eugene, OR, USA;
at a dilution of 1 : 200) and TuJ-1, GFAP, and CD11b with
Alexa 546-labelled IgG (at a dilution of 1 : 200). The samples were
then examined with an Apo Tome (Zeiss, Jena, Germany).
Glycoprotein (gp) 130 dimer analysis
We investigated whether dimerization of gp130 could be detected
under non-reducing conditions. Control (n ¼ 4) and spinal cord tissue
samples 12 h after SCI (n ¼ 4) were prepared as described above,
under both reducing and non-reducing conditions. Reducing and non-
reducing samples were, respectively, prepared by adding SDS sample
buffer with and without b-mercaptoethanol. Crude samples (10 mg)
were subjected to 7.5% SDS–PAGE, and proteins were transferred to
PVDF membranes and incubated with a primary antibody to gp130
(Genzyme Technology) diluted at 1 : 500 for 60 min at room
temperature. Membranes were then incubated with anti-goat poly-
clonal IgG conjugated with horseradish peroxidase (Sigma) at a
dilution of 1 : 3000 for 30 min at room temperature. Reaction
products were demonstrated with ECL plus (Amersham Biosciences).
Intraperitoneal administration of AG-490
JAK inhibitor, AG-490 (40 lg/g), was freshly dissolved in 45%
dimethylsulfoxide (DMSO) and slowly injected into the peritoneal
cavities of mice 20 min before SCI (SCI + AG-490, n ¼ 6).
Control mice received the same dose of vehicle (45% DMSO) at
the same time (SCI + DMSO, n ¼ 4). Severe SCI was induced as
described above. At 12 h after SCI, the animals were killed and the
spinal cord tissues were removed, frozen in liquid nitrogen and kept
at )80C until used for western blot (p-Tyr1022/1023-JAK1, JAK1,
p-Tyr705-STAT3, STAT3 and actin) and ELISA (IL-6) analysis as
described above.
Behavioural study
To investigate the effects of AG-490 on SCI, we measured recovery
of hindlimb motor functions using the Basso–Beattie–Bresnahan
motor rating scale in mice treated with AG-490 (SCI + AG-490,
n ¼ 5) or DMSO alone (SCI + DMSO, n ¼ 5), as described
previously (Basso et al. 1995). Two investigators observed each
animal for 5 min and provided a defined score (0–21) for each
hindlimb at 1, 2, 4, 7, 10 and 14 days after SCI. The scores from the
two hindlimbs were averaged to obtain a single value per animal for
each time point.
Statistical analysis
Data are expressed as mean ± SEM values. Statistical analysis was
performed by one-way
ANOVA
followed by the Bonferroni–Dunn
method for multiple comparisons. Behavioural recovery patterns and
the effects of AG-490 were compared between groups using the
Mann–Whitney non-parametric analysis of variance. Statistical
significance was concluded at the p < 0.05 level.
Results
Activation of JAK1 and STAT3 following SCI
We first investigated activation of JAK/STAT signalling
following SCI by western blot analysis. Blotting with
1062 K. Yamauchi et al.
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
2006 The Authors
Page 3
antibodies to JAK1, JAK2 and STAT3 detected a single
protein band in crude samples of injured spinal cord tissue at
all time points, as well as in controls (Fig. 1c). Densitometric
analysis of band of p-Tyr1022/1023-JAK1/JAK1 revealed
increases immediately after SCI, peaking at 12 h and
gradually decreased thereafter (Fig. 1a). Statistical signifi-
cance was achieved from 6 to 18 h after SCI, compared with
the control value (p<0.05). In contrast, that of p-Tyr1007/
1008-JAK2/JAK2 showed no significant change (data not
shown). Phosphorylated-Tyr705-STAT3 was hardly detected
in controls, but became evident immediately after SCI.
Densitometric analysis of band of p-Tyr705-STAT3/STAT3
revealed increases immediately after SCI, peaking at 12 h
and gradually decrease thereafter (Fig. 1b). Expression was
significantly elevated from 6 to 168 h after SCI (p<0.05).
Similar levels of actin were detected in crude samples with or
without SCI (Fig. 1c), confirming that equivalent amounts of
proteins were loaded and that SCI modulates JAK1 and
STAT3 through the phosphorylation at Tyr1022/1023 and
Tyr705, respectively.
Immunohistochemistry of JAK1, STAT3,
p
-Tyr1022/
1023-JAK1 and
p
-Tyr705-STAT3 following SCI
To investigate where phosphorylation of JAK1 and STAT3
occurs in injured spinal cord, we performed immunohisto-
chemistry for p-Tyr1022/1023-JAK1 and p-Tyr705-STAT3.
Tissue damage of spinal cord was more severe in dorsal than
in ventral sites at 12 h after SCI (Farooque 2000). Small
numbers of deformed and shrunken neurons remained and
JAK1 and STAT3 were mainly detected on these neurons in
anterior horn of spinal cord, even 12 h after SCI (Figs 2b and
f, respectively). Immunoreactivity of p-Tyr1022/1023-JAK1
and p-Tyr705-STAT3 was hardly detected in control samples
(Figs 2c and g, respectively), while markedly attenuated in
cytoplasm of neurons at 12 h after SCI (Figs 2d and h,
respectively). Immunofluorescence staining also revealed
that cells immunoreactive for p-Tyr705-STAT3 (Fig. 3b)
were positive for neuron-specific class III b-tubulin (TuJ-1,
Fig. 3a) to a great extent at 12 h after SCI (Fig. 3c), this
becoming weak at 48 h (Figs 3d–f). Immunoreactivity for
p-Tyr705-STAT3 was apparent within the nuclei at 12 h after
SCI, suggesting nuclear translocation of STAT3 signals
(Fig. 3c). GFAP-positive cells and microglia were hardly
detected at 12 h after SCI (Figs 3g and i, respectively). In
contrast, co-localization of p-Tyr705-STAT3 signals with the
activated astrocyte and microglia marker/CD11b became
apparent at 48 h (Figs 3h and j, respectively). These data
suggest that p-Tyr705-STAT3 is mainly present in neurons in
acute stages and in activated astrocytes and microglia with
long-term SCI.
Expression and cellular localization of IL-6 in the spinal
cord following SCI
We quantified the expression of IL-6 using a specific ELISA.
In controls, the mean concentration of IL-6 in the spinal cord
tissue was 13.80 ± 2.94 pg/mL. Increase was evident from
3 h after SCI, to a peak at 12 h, with gradual decreasing
thereafter. A significant increase was noted from 3 to 24 h
after SCI. The maximum concentration of IL-6 in injured
Fig. 1 Phosphorylation of Janus kinase 1 (JAK1) at Tyr1022/1023,
JAK2 at Tyr1007/1008, and signal transducer and activator of tran-
scription 3 (STAT3) at Tyr705 in injured spinal cord. At 0, 1, 3, 6, 12,
18, 24, 48, 72, 120 or 168 h after spinal cord injury (SCI), as indicated
below the panel, crude samples were subjected to western blotting
with anti-actin (a-Actin), anti-JAK1 (a-JAK1), anti-phosphospecific
JAK1 (a-p-Tyr1022/1023-JAK1), anti-JAK2 ( a-JAK2), anti-phospho-
specific JAK2 (a-p-Tyr1007/1008-JAK2), anti-STAT3 (a-STAT3), and
anti-phosphospecific STAT3 at Tyr705 (a-p-Tyr705-STAT3) antibod-
ies (c). The histogram shows the amount of a-p-Tyr1022/1023-JAK1
relative to that of a-JAK1 and the amount of a-p-Tyr705-STAT3 rel-
ative to that of a-STAT3 in the membrane (a, b, respectively). Phos-
phorylation of JAK2 showed no significant change after SCI.
Mean ± SE values from data of four animals are shown. CNT, control
spinal cord without compression. * p<0.05 denotes a significant dif-
ference between control and SCI samples by analysis of variance
followed by the Bonferroni–Dunn test.
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Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
Page 4
spinal cord tissues was more than eight times the control
level (Fig. 4). Immunohistochemistry revealed IL-6 in the
cytoplasm of neurons in the anterior horns of spinal cords at
12 h after SCI (Fig. 5a), but not in controls (data not shown).
Double labelling for IL-6 (green) and TuJ-1 (red) showed
most neurons to be immunopositive for IL-6 at 12 h after
SCI (Fig. 5b).
Expression of gp130 following SCI
Because the above results suggested that IL-6 might induce
signal transduction in the JAK/STAT pathway after SCI and
binding to its receptor induces dimerization of gp130, we
investigated this latter parameter by western blot analysis.
Under reducing conditions, blotting with antibodies against
gp130 homogeneously detected a 130-kDa band in both
controls and at 12 h after SCI (Fig. 6a). In contrast, under
non-reducing conditions not only a 130-kDa but also a 260-
kDa band of gp130, considered to be a dimerization product,
were detected at 12 h after SCI. However, only the 130-kDa
band was detected in controls (Fig. 6b).
Inhibition of JAK1 and STAT3 activation
To further investigate signal transduction through the JAK/
STAT pathway after SCI, we examined whether pretreatment
with AG-490, a well-known inhibitor of JAK, might
suppress phosphorylation of JAK1 at Tyr1022/1023 and
STAT3 at Tyr705 at 12 h after SCI. Immunoblot analysis
revealed equal levels of actin, JAK1 and STAT3 in crude
samples (Fig. 7c). Densitometric analysis of bands of
p-Tyr1022/1023-JAK1/JAK1 and p-Tyr705-STAT3/STAT3
revealed significant suppression in those treated with
AG-490 (SCI + AG-490) compared with DMSO alone
(SCI + DMSO) (Figs 7a and b, respectively). Interestingly,
the IL-6 level of those treated with AG-490 (SCI + AG-490)
was significantly higher than with DMSO alone (SCI +
DMSO) in injured spinal cord tissue (Fig. 7d).
Behavioural recovery
The recovery of hindlimb locomotor function was evaluated
over 2 weeks after severe SCI using the Basso-Beattie-
Bresnahan rating scale. The Basso-Beattie-Bresnahan
scores of the mice treated with AG-490 (SCI + AG-490)
gradually improved from 0.2 ± 0.2 on day 1 to 6.2 ± 0.7 on
day 14, while the scores of those treated with DMSO alone
(SCI + DMSO) gradually improved from 1.8 ± 0.2 on day 1
to 9.4 ± 0.9 on day 14 after SCI. Mice treated with AG-490
had worse hindlimb motor function during the entire
observation period of 14 days. The difference was statisti-
cally significant at each time point (Fig. 8).
Discussion
In this study, we have explored the changes in expression and
the cellular location of IL-6 and elements in the JAK/STAT
signalling pathway after severe SCI. The present study
revealed that phosphorylation of JAK1 at Tyr1022/1023 and
STAT3 at Tyr705 occurred immediately after spinal SCI,
peaked at 12 h and decreased thereafter. The concentration of
IL-6 in injured spinal cord significantly increased from 3 h,
peaked at 12 h after SCI, and gradually decreased thereafter,
which was coincident with phosphorylation of JAK1 at
Tyr1022/1023 or STAT3 at Tyr705. Immunohistochemistry
Fig. 2 Immunohistochemical staining of Janus kinase 1 (JAK1, a, b),
phosphospecific (p )-Tyr1022/1023-JAK1 (c, d), signal transducer and
activator of transcription 3 (STAT3, e, f) and p-Tyr705-STAT3 (g, h) in
anterior horns of spinal cord. Mice subjected to laminectomy without
compression (a, c, e, g) or 12 h after spinal cord injury (SCI) (b, d, f, h)
were perfused with 4% paraformaldehyde. Slices (10 lm) were
immunostained with antibodies recognizing either JAK1 (a, b),
p-Tyr1022/1023-JAK1 (c, d) STAT3 (e, f) or p-Tyr705-STAT3 (g, h) by
the ABC method. Immunoreactivity against JAK1 and STAT3 was
preserved in the cytoplasm of neuron in the grey matter at 12 h after
SCI (b, f, respecti vely) compared with control (a, e, respectively). Note
that p-Tyr1022/1023-JAK1 (d) and p-Tyr705-STAT3 (h) are well
stained in the cytoplasm of neurons in the anterior horn even 12 h after
SCI, while samples without compression lack immunoreactivity (c, g).
Higher magnifications of representative neurons are superimposed in
each figure. Scale bars, 20 lm.
1064 K. Yamauchi et al.
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
2006 The Authors
Page 5
showed p-Tyr1022/1023-JAK1, p-Tyr705-STAT3 and IL-6
to be mainly expressed in neurons in the anterior horns of
injured spinal cords at 12 h. Immunofluorescence revealed p-
Tyr705-STAT3 to be expressed in astrocytes and microglia in
the long term after SCI. Pretreatment with AG-490, a JAK
inhibitor, could significantly suppress phosphorylation of
JAK1 at Tyr1022/1023 and STAT3 at Tyr705, while
increasing the concentration of IL-6 in injured spinal cord
at 12 h after SCI, compared with the control group. AG-490
reduced the functional recovery of hindlimbs after SCI, a
finding important for understanding processes of secondary
degeneration.
Pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-a,
demonstrate increased expression at both mRNA and protein
levels in injured spinal cords (Bartholdi and Schwab 1997;
Streit et al. 1998; Hayashi et al. 2000; Pan et al. 2002).
These pro-inflammatory cytokines activate secondary cyto-
toxic events and may be involved in cell death after SCI.
Recently, immunoreactivity of IL-1b, IL-6 and TNF-a was
observed in neurons in humans as early as 0.5 h after SCI
(Yang et al. 2004). After SCI in mice, TNF-a immuno-
reactivity can be detected at 1 h in neurons, which preceded
the production of IL-6 (Pan and Kastin 2001). TNF-a
activates the JAK/STAT signalling cascade by acting through
Fig. 3 Immunofluorescence staining of phosphospecific (p)-Tyr705-
STAT3 and neuron-specific class III b-tubulin (TuJ-1), glial fibrillary
acidic protein (GFAP) and a microglia marker (CD11b) in anterior horns
of spinal cords. Merged images for TuJ-1/p-Tyr705-STAT3 at 12 h (c)
and 48 h (f), for GFAP/p-Tyr705-STAT3 at 12 h (g) and 48 h (h), and for
CD11b/p-Tyr705-STAT3 at 12 h (i) and 48 h (j) after spinal cord injury
(SCI) are shown. Mice subjected to SCI were perfused with 4% para-
formaldehyde at 12 h (a, b, c, g, i) or 48 h (d, e, f, h, j) and 10-lm sections
were immunostained with antibodies recognizing p-Tyr705-STAT3,
TuJ-1, GFAP and CD11b. Immunoreactivity against p-Tyr705-STAT3
was detected with Alexa 488-labelled IgG (green) and against TuJ1,
GFAP or CD11b with Alexa 546-labelled IgG (red). Note much stronger
immunoreactivity for both p-Tyr705-STAT3 and TuJ-1 at 12 h (c) than at
48 h (f) after SCI. Immunoreactivity for p-Tyr705-STAT3 is evident in the
nuclei of neurons immunopositive for TuJ-1 (b, c) at 12 h after SCI.
Common immunoreactivity for p-Tyr705-STAT3 and GFAP (g) or
CD11b (i) was essentially limited to 12 h after SCI, weak staining being
detected at 48 h (h, j) after SCI. Scale bars, 20 l m.
JAK/STAT activation following spinal cord injury 1065
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
Page 6
TNFR1 (Guo et al. 1998; Miscia et al. 2002). The early
inflammatory responses after SCI may be initiated by
neutrophils that infiltrate the lesion site after injury. These
neutrophils are able to release reactive oxygen as well as
cytokines and chemokines. Spinal cord trauma induced severe
oxidative stress within 1 h after SCI (Kamencic et al. 2001).
Oxidative damage contributes to the pathogenesis of several
diseases. This oxidative stress accompanies the activation of
the JAK/STAT pathway (Simon et al. 1998). These TNF-a
and oxidative stresses may be involved with the activation of
the JAK/STAT pathway immediately after SCI before induc-
tion of IL-6.
Besides IL-6, the JAK/STAT signalling pathway is known
to be activated by a variety of IL-6 family cytokines, including
IL-11, leukaemia inhibiting factor, ciliary neurotrophic factor
(CNTF), oncostatin M and cardiotrophin-1 (Taga 1996;
Hirano et al. 1997; Taga and Kishimoto 1997; Heinrich et al.
1998; Cattaneo et al. 1999). These cytokines bind to the cell
membrane receptor and induce dimerization of gp130,
followed by activation of JAK in cell membranes, which
subsequently phosphorylates STAT3 at Tyr705 resulting in
translocation into the nucleus. We have confirmed the peak
expression of IL-6 to be coincident with maximum activation
of JAK1 or STAT3 in neurons, with translocation of
phosphorylated STAT3 at Tyr705 to the nucleus. Furthermore,
Fig. 5 Immunohistochemical staining of interleukin-6 (IL-6) in the
anterior horns of spinal cord. Mice subjected to spinal cord injury (SCI)
were perfused with 4% paraformaldehyde 12 h after SCI. Slices
(10 lm) were immunostained with antibodies recognizing IL-6 by the
ABC method (a). Double staining for IL-6 (green) and neuron-specific
class III b-tubulin (TuJ-1, red) in the anterior horn of spinal cord (b).
Note mainly the increase in immunoreactivity against IL-6 in the
cytoplasm of neurons in the anterior horn at 12 h after SCI (arrows).
Scale bars, 20 lm.
Fig. 6 Western blotting analysis of glycoprotein (gp) 130 dimerization
under reducing (a) and non-reducing (b) conditions. Interleukin-6 binds
to its receptor and consequently induces dimerization of gp130, which
results in activation of the Janus kinase/signal transducer and acti-
vator of transcription (JAK/STAT) signalling pathway. Crude samples
from control spinal cord and 12 h after spinal cord injury (SCI) were
subjected to western blotting with anti-actin (a-Actin) and anti-gp130
(a-gp130) antibodies. Note the 260-kDa band was detected under
non-reducing conditions 12 h after SCI (b). CNT; control spinal cord
without compression. The experiments were performed four times with
similar results.
Fig. 4 Serial changes in the concentrations of interleukin-6 (IL-6) in
injured spinal cord. At 0, 1, 3, 6, 12, 18, 24, 48, 72, 120 or 168-h after
spinal cord injury (SCI), as indicated below the panel, the concentra-
tions of IL-6 in injured spinal cord were measured using an ELISA kit.
Mean ± SE values of data from four animals are shown. CNT, control
spinal cord without compression. * p<0.05 denotes a significant dif-
ference between control and SCI samples by analysis of variance
followed by the Bonferroni–Dunn test.
1066 K. Yamauchi et al.
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
2006 The Authors
Page 7
western blot analysis under non-reducing conditions con-
firmed dimerization of gp130, suggesting that IL-6 could be
one of the activators of JAK/STAT signalling in neurons after
SCI. Oyesiku et al. (1997) have earlier reported an increase in
CNTF-receptor a mRNA in motoneurons in the ventral horn
and CNTF elevation in white matter within 24 h after spinal
cord hemisection. SCI induced the expression of CNTF in
reactive astrocytes (Lee et al. 1998). Leukaemia inhibiting
factor is a potent pro-inflammatory factor, which also appears
to play important roles in regulating inflammatory reactions
after SCI (Kerr and Patterson 2004). The available data thus
suggest that not only IL-6 but also other IL-6 family cytokines
may be involved in signalling after SCI.
STAT3 significantly remained phosphorylated at Tyr705
until 168 h after SCI, while the concentration of IL-6 and
phosphorylation of JAK1 at Tyr1022/1023 decreased to
almost the control level. In ischaemic brain tissue, STAT3 is
known to be highly induced in reactive microglial cells
(Planas et al. 1996) and Justicia et al. (2000) have reported
that phosphorylated STAT3 translocates into nuclei of
reactive microglia until 15 days after transient cerebral
ischaemia. In injured spinal cord, infiltration of microglia
Fig. 8 Recovery of hindlimb motor functions after spinal cord injury
(SCI) was assessed using the Basso–Beattie–Bresnahan (BBB) motor
rating scale. Mice treated with AG-490 (SCI + AG-490, n ¼ 5) or
DMSO alone (SCI + DMSO, n ¼ 5) were examined for hindlimb motor
function by two investigators at 1, 2, 4, 7, 10 and 14 days after SCI.
The scores were averaged for each animal at each time point.
Mean ± SE values from data for five animals are shown. An asterisk
indicates a significant difference between the two group s by the
Mann–Whitney U-test (p<0.05).
Fig. 7 Effects of intraperitoneal injection of AG-490 on phosphoryla-
tion of Janus kinase 1 (JAK1) at Tyr1022/1023 (a) and signal trans-
ducer and activator of transcription 3 (STAT3) at Tyr705 (b), and the
concentrations of interleukin-6 (IL-6) in injured spinal cord (d). Mice
were pretreated with AG-490 (40 mg/g) or DMSO 20 min before spinal
cord injury (SCI). Crude samples were subjected to western blotting
with anti-JAK1 (a-JAK1), anti-phosphospecific JAK1 at Tyr1022/1023
(a-p-Tyr1022/1023-JAK1), anti-STAT3 (a-STAT3), and anti-phospho-
specific STAT3 at Tyr705 (a-p-Tyr705-STAT3) antibodies. Two rep-
resentative western blot analyses are shown (c). The histogram shows
the amount of a-p-Tyr1022/1023-JAK1 relative to that of a-JAK1 (a)
and the amount of a-p-Tyr705-STAT3 relative to that of a-STAT3 (b).
The concentrations of IL-6 were measured by ELISA. Mean ± SE
values are shown (d). Numbers of animals are given in parentheses.
Asterisks indicate a significant difference by the Mann–Whitney U-test
(p<0.05).
JAK/STAT activation following spinal cord injury 1067
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1060–1070
Page 8
increases from 12 h, peaking at 48 h after SCI (Morino et al.
2003). Our data showed that microglia can hardly be detected
at 12 h after SCI, while activated microglia infiltrating within
injured spinal cords and expressing phosphorylated STAT3
signals are frequent after 48 h, suggesting that phosphory-
lation of STAT3 in the chronic stage may be induced by such
activation of microglia.
After transient forebrain ischaemia, phosphorylated
STAT3 was earlier observed in reactive astrocytes of the
hippocampus, persisting for 14 days with reperfusion (Choi
et al. 2003). In addition, reactive astrocytes in the penumbra
area were found to be immunoreactive for phosphorylated
STAT3 for 7 days following transient focal cerebral isch-
aemia (Justicia et al. 2000). After SCI, astrocyte responses
are up-regulated within 24 h and persist for 30 days
(Farooque et al. 1995; Baldwin et al. 1998). Recently,
reactive astrocytes were found to protect neurons and
oligodendrocytes and preserve motor functions after SCI
(Faulkner et al. 2004), possibly because of their production
of pro-inflammatory cytokines (Lieberman et al. 1989). Our
finding that they are immunoreactive for phosphorylated
STAT3 after SCI suggests a role for phosphorylation of
STAT3 in astrocyte activation, especially in chronic stages,
within injured spinal cords.
In order to further confirm that SCI itself induces the signal
transduction from JAK1 to STAT3 in injured spinal cord, we
investigated the effects of a JAK inhibitor. Although AG-490
can specifically inhibit all JAKs, it does not affect other
tyrosine kinases (Meydan et al. 1996; Nielsen et al. 1997;
Negoro et al. 2000; Xuan et al. 2001). The present pretreat-
ment with AG-490 significantly suppressed the phosphory-
lation of both JAK1 and STAT3 at 12 h after SCI, suggesting
that the latter was induced by JAK1 after SCI. Stringent
mechanisms for signal attenuation are necessary for
appropriate cellular responses to cytokine stimulation.
SH2-containing phosphorylates (SHP), protein inhibitors of
activated STATs (PIAS), and suppressors of cytokine signal-
ling (SOCS) play some roles in negative regulation (Heinrich
et al. 1998). Pretreatment with AG-490 here significantly
inhibited activation of the JAK/STAT signalling pathway in
SCI, while increasing the concentration of IL-6 in injured
spinal cord compared with DMSO alone, suggesting that
there may be an autoregulation of these two targets.
Activation of JAK/STAT signalling has already been
reported in cerebral ischaemia (Planas et al. 1996; Suzuki
et al. 2001; Wen et al. 2001; Choi et al. 2003) or cardiac
ischaemia (Xuan et al. 2001). Xuan et al. (2001) found that
up-regulation of the JAK/STAT signalling pathway could
reduce myocardiac damage through transcriptional activation
of iNOS. Tuna et al. (2001) have demonstrated SCI to
produce iNOS and this is attenuated with anti-IL-6 antibody
treatment, although the responsible mechanisms were not
clarified. In cerebral ischaemia, STAT3 mediated by gra-
nulocyte colony-stimulating factor (G-CSF) can promote
neuroprotective effects (Schabitz et al. 2003). It has previ-
ously been reported that co-administration of IL-6 and
soluble IL-6 receptor (sIL-6R) can induce neurological
improvement and prevent degeneration of spinal motor
neurons in the wobbler mouse (Ikeda et al. 1996), and IL-6
gene knockout (IL-6
–/–
) mice exhibit more severe damage
and loss of spinal cord neurons, compared with wild-type
counterparts after virus-induced injury (Pavelko et al. 2003).
Other authors have provided evidence that IL-6 is essential to
modulate sensory functions in vivo (Zhong et al. 1999). Our
data showed that pretreatment of AG-490 reduced the
functional recovery of hindlimbs after SCI. Considering
these findings, activation of JAK/STAT signalling induced by
IL-6 in neurons may contribute to neuronal protection after
SCI.
To the best of our knowledge, this is the first demonstra-
tion of time-dependent induction of IL-6 and activation of the
JAK/STAT signalling pathway following severe SCI. Further
studies using IL-6 gene knockout mice may allow us to
decipher the roles of JAK/STAT signalling in responses to
injury and hopefully provide novel therapeutic approaches,
considering the time window of activated JAK/STAT signal-
ling.
Acknowledgements
This research was supported by grants from the General Insurance
Association of Japan. We thank Kenmei Mizutani for expert
technical assistance, Dr Malcolm Moore for critical reading of the
manuscript, and Dr Kazuyoshi Hattori (Department of Neurosur-
gery, Gifu Social Insurance Hospital) for useful advice.
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  • Source
    • "The 12-day amitriptyline administration enhanced the spinal protein level of IL-6 in naı¨venaı¨ve animals. IL-6 is an important pronociceptive cytokine that is known to be upregulated after nervous system injuries (Kreutzberg, 1996; Gruol and Nelson, 1997; Yamauchi et al., 2006; Dominguez et al., 2008). Intrathecal administration of IL-6 in naı¨venaı¨ve animals caused allodynia (DeLeo et al., 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: Neuropathic pain is a severe clinical problem, often appearing as a co-symptom of many diseases or manifesting as a result of damage to the nervous system. Many drugs and agents are currently used for the treatment of neuropathic pain, such as tricyclic antidepressants (TCAs). The aims of this paper were to test the effects of two classic TCAs, doxepin and amitriptyline, in naïve animals and in a model of neuropathic pain and to determine the role of cytokine activation in the effects of these drugs. All experiments were carried out with Albino-Swiss mice using behavioral tests (von Frey test and the cold plate test) and biochemical analyses (qRT-PCR and Western blot). In the mice subjected to chronic constriction injury (CCI), doxepin and amitriptyline attenuated the symptoms of neuropathic pain and diminished the CCI-induced increase in the levels of spinal interleukin-6 and -1β mRNA, but not the protein levels of these cytokines, measured on day 12. Unexpectedly, chronic administration of doxepin or amitriptyline for 12 days produced allodynia and hyperalgesia in naïve mice. The treatment with these drugs did not influence the spinal levels of IL-1β and IL-6 mRNA, however, the protein levels of these pronociceptive factors were increased. The administration of ondansetron (5-HT3 receptor antagonist) significantly weakened the allodynia and hyperalgesia induced by both antidepressants in naïve mice; in contrast, yohimbine (α2 adrenergic receptors antagonist) did not influence these effects. The allodynia and hyperalgesia induced in naïve animals by amitryptyline and doxepin may be associated with an increase in the levels of pronociceptive cytokines resulting from 5-HT3-induced hypersensitivity. Our results provide new and important information about the possible side effects of antidepressants. Further investigation of these mechanisms may help to guide decisions about the use of classic TCAs for therapy. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Mar 2015 · Neuroscience
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    • "In rodent models of SCI, levels of pro-inflammatory interleukin such as IL-6 peak acutely in the injured areas and lead to activation of the JAK1-STAT3 signaling pathway, which contributes to development of neuropathic pain [5], [6]. Moreover, in previous work, conditional ablation of STAT3 increased motor deficits after spinal cord injury [7]. "
    [Show abstract] [Hide abstract] ABSTRACT: Spinal cord injury or amyotrophic lateral sclerosis damages spinal motor neurons and forms a glial scar, which prevents neural regeneration. Signal transducer and activator of transcription 3 (STAT3) plays a critical role in astrogliogenesis and scar formation, and thus a fine modulation of STAT3 signaling may help to control the excessive gliogenic environment and enhance neural repair. The objective of this study was to determine the effect of STAT3 inhibition on human neural stem cells (hNSCs). In vitro hNSCs primed with fibroblast growth factor 2 (FGF2) exhibited a lower level of phosphorylated STAT3 than cells primed by epidermal growth factor (EGF), which correlated with a higher number of motor neurons differentiated from FGF2-primed hNSCs. Treatment with STAT3 inhibitors, Stattic and Niclosamide, enhanced motor neuron differentiation only in FGF2-primed hNSCs, as shown by increased homeobox gene Hb9 mRNA levels as well as HB9+ and microtubule-associated protein 2 (MAP2)+ co-labeled cells. The increased motor neuron differentiation was accompanied by a decrease in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes. Interestingly, Stattic and Niclosamide did not affect the level of STAT3 phosphorylation; rather, they perturbed the nuclear translocation of phosphorylated STAT3. In summary, we demonstrate that FGF2 is required for motor neuron differentiation from hNSCs and that inhibition of STAT3 further increases motor neuron differentiation at the expense of astrogliogenesis. Our study thus suggests a potential benefit of targeting the STAT3 pathway for neurotrauma or neurodegenerative diseases.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "It is secreted mostly by macrophages, but also by microglial cells (Kreutzberg, 1996; Milligan et al., 2003). Peripheral nerve damage leads to an increase in IL-6 levels at dorsal spinal level, particularly in layers I-II (DeLeo et al., 1996; Murphy et al., 1995), both in microglial cells (Dominguez et al., 2008) and in neurons (Yamauchi et al., 2006). IL-6 is not only known as peripheral immune modulator. "
    [Show abstract] [Hide abstract] ABSTRACT: Glia plays a crucial role in the maintenance of neuronal homeostasis in the central nervous system. The microglial production of immune factors is believed to play an important role in nociceptive transmission. Pain may now be considered a neuro-immune disorder, since it is known that the activation of immune and immune-like glial cells in the dorsal root ganglia and spinal cord results in the release of both pro- and anti-inflammatory cytokines, as well as algesic and analgesic mediators. In this review we presented an important role of cytokines (IL-1alfa, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-15, IL-18, TNFalpha, IFNgamma, TGF-beta 1, fractalkine and CCL2); complement components (C1q, C3, C5); metaloproteinases (MMP-2,-9) and many other factors, which become activated on spinal cord and DRG level under neuropathic pain. We discussed the role of the immune system in modulating chronic pain. At present, unsatisfactory treatment of neuropathic pain will seek alternative targets for new drugs and it is possible that anti-inflammatory factors like IL-10, IL-4, IL-1alpha, TGF-beta 1 would fulfill this role. Another novel approach for controlling neuropathic pain can be pharmacological attenuation of glial and immune cell activation. It has been found that propentofylline, pentoxifylline, minocycline and fluorocitrate suppress the development of neuropathic pain. The other way of pain control can be the decrease of pro-nociceptive agents like transcription factor synthesis (NF-kappaB, AP-1); kinase synthesis (MEK, p38MAPK, JNK) and protease activation (cathepsin S, MMP9, MMP2). Additionally, since it is known that the opioid-induced glial activation opposes opioid analgesia, some glial inhibitors, which are safe and clinically well tolerated, are proposed as potential useful ko-analgesic agents for opioid treatment of neuropathic pain. This review pointed to some important mechanisms underlying the development of neuropathic pain, which led to identify some possible new approaches to the treatment of neuropathic pain, based on the more comprehensive knowledge of the interaction between the nervous system and glial and immune cells.
    Full-text · Article · Mar 2013 · European journal of pharmacology
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