Journal of Neurochemistry, 2006, 97, 1337–1348
doi:10.1111/j.1471-4159.2006.03808.x
Direct evidence for spinal cord microglia in the development of a
neuropathic pain-like state in mice
Minoru Narita, Takuya Yoshida, Mayumi Nakajima, Michiko Narita, Mayumi Miyatake,
Tomoe Takagi, Yoshinori Yajima and Tsutomu Suzuki
Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan
Abstract
The present study was undertaken to further investigate the
role of glial cells in the development of the neuropathic painlike state induced by sciatic nerve ligation in mice. At 7 days
after sciatic nerve ligation, the immunoreactivities (IRs) of the
specific astrocyte marker glial fibrillary acidic protein (GFAP)
and the specific microglial marker OX-42, but not the specific
oligodendrocyte marker O4, were increased on the ipsilateral
side of the spinal cord dorsal horn in nerve-ligated mice
compared with that on the contralateral side. Furthermore, a
single intrathecal injection of activated spinal cord microglia,
but not astrocytes, caused thermal hyperalgesia in naive mice.
Furthermore, 5-bromo-2¢-deoxyuridine (BrdU)-positive cells
on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly
co-localized with another microglia marker, ionized calciumbinding adaptor molecule 1 (Iba1), but neither with GFAP nor
a specific neural nuclei marker, NeuN, in the spinal dorsal
horn of nerve-ligated mice. The present data strongly support
the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain-like state, and that the proliferated and activated microglia directly contribute to the
development of a neuropathic pain-like state in mice.
Keywords: activation, microglia, neuropathic pain, proliferation, spinal cord.
J. Neurochem. (2006) 97, 1337–1348.
In many clinical pain syndromes, painful sensations are
greatly amplified. Neuropathic pain is well characterized by
spontaneous burning pain, hyperalgesia (exaggerated pain in
response to painful stimuli) and allodynia (pain evoked by
normally innocuous stimuli). Neuropathic pain is the most
difficult pain to manage in the pain clinic field, because this
pain is often refractory to general analgesics such as
acetaminophen, non-steroidal anti-inflammatory drugs and
opioids. Many studies have focused on the long-term
changes in functions of the spinal dorsal horn neurons,
which contain some receptors, protein kinases and peptides,
following nerve injury. However, the mechanisms that
underlie neuropathic pain are still largely unclear.
In the CNS, there are two categories of cells: neurons and
adjacent glial cells. For years, glial cells were thought to be
passive cells that had few responses to synaptic activation,
and were overlooked as merely supportive cells in the CNS.
However, a growing body of evidence has recently suggested
that glial cells communicate with one another and with
neurons primarily through chemical signals (Araque et al.
1999; Haydon 2001; Zonta et al. 2003). Glial cells dynamically modulate the function of neurons under both physiological and pathological conditions (Temburni and Jakob
2001). There are three general types of glial cells in the CNS:
oligodendrocytes, astrocytes and microglia. Oligodendrocytes are responsible for myelinization in the CNS, and can
be considered to be the central equivalent of Schwann cells,
which are the myelinating cells in the peripheral nervous
system. Astrocytes are the principal type of glial cell
and participate in a wide variety of physiological and
Received July 11, 2005; revised manuscript received November 9, 2005;
accepted December 19, 2005.
Address correspondence and reprint requests to Minoru Narita PhD
and Tsutomu Suzuki PhD, Department of Toxicology, Hoshi University
School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara,
Shinagawa-ku, Tokyo 142–8501, Japan. E-mail: [email protected] or
[email protected]
Abbreviations used: aACM, activated ACM; ACM, astrocyte-conditioned medium; BDNF, brain-derived neurotrophic factor, BrdU,
5-bromo-2¢-deoxyuridine; CGRP, calcitonin-gene related peptide,
DMEM, Dulbecco’s modified Eagle’s medium; GFAP, glial fibrillary
acidic protein; Iba1, ionized calcium-binding adaptor molecule 1; IL,
interleukin; i.p., intraperitoneal, IR, immunoreactivity; i.t., intrathecal;
MAG, myelin-associated glycoprotein, MAP2a/b, microtubule-associated protein 2a/b; NGS, normal goat serum; PBS, phosphate-buffered
saline; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; PPF,
propentofylline, s.c., subcutaneous, TNFa, tumor necrosis factor a.
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1338 M. Narita et al.
pathological processes (Araque et al. 1999; Haydon 2001).
Microglia are found throughout the parenchyma of the CNS.
Accumulating evidence suggests that microglia, in synergy
with astrocytes, monitor and maintain the physiological
homeostasis of their microenvironment (Kreutzberg 1996;
Aloisi 2001; von Bernhardi and Ramirez 2001; Kempermann
and Neumann 2003).
At the level of the spinal cord, pathological pain is
classically viewed as being created and maintained solely by
neurons. However, a growing body of evidence suggests that
spinal astrocytes and microglia are activated under the pain
state. Therefore, it has been recognized that astrocytes and
microglia are also implicated in the exaggerated pain state
created by such diverse manipulations as subcutaneous
inflammation, neuropathy and spinal immune activation
(Hashizume et al. 2000; Sweitzer et al. 2001; Watkins et al.
2001a,b; Watkins and Maier 2002; Raghavendra et al. 2003,
2004; Tsuda et al. 2003; Ledeboer et al. 2005). It was
previously reported that the immunoreactivity (IR) levels of
the specific astrocyte marker glial fibrillary acidic protein
(GFAP) and the specific microglial marker OX-42 were
elevated under the neuropathic pain-like state caused by L5
spinal nerve transection in rats (Sweitzer et al. 2001). One
question that has yet to be addressed is whether or not the upregulation of glial markers is due solely to increased
expression in cells already in the spinal cord, or if cells are
proliferating or being recruited. Therefore, using 5-bromo-2¢deoxyuridine (BrdU), a cell proliferation marker, we investigated whether either astroglial or microglial proliferation
could be induced under the neuropathic pain-like state.
Tsuda and colleagues previously reported that tactile
allodynia can be caused by an intrathecal (i.t.) injection of
activated microglia treated with ATP in rats (Tsuda et al.
2003). However, it has not yet been documented that either
activated astrocytes or microglia could induce thermal
hyperalgesia. Recently, it has been reported that activated
protein kinase C (PKC) within the spinal cord plays an
important role in the induction of the neuropathic pain-like
state in mice (Malmberg et al. 1997; Narita et al. 2000; Ji
and Woolf 2001; Yajima et al. 2003). Furthermore, we
previously reported that treatment with phorbol 12,13dibutyrate (PDBu), a PKC activator, induces a robust
activation of astrocytes, as detected by a stellate morphology
and an increase in the level of GFAP-like IR in cultures
(Miyatake et al. 2005). Microglia are activated by ATP
(Watkins et al. 2001a; Tsuda et al. 2003). Considering this
background, the present study was also undertaken to
investigate whether thermal hyperalgesia could be caused
by an i.t. injection of either activated spinal astrocytes or
microglia treated with either PDBu or ATP in mice.
Materials and methods
The present study was conducted in accordance with the Guiding
Principles for the Care and Use of Laboratory Animals, as adopted by
the Committee on Animal Reseach of Hoshi University, which is
accredited by the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
Animals
Male ICR mice weighing 20–30 g (Tokyo Laboratory Animals
Science Co., Ltd, Tokyo, Japan) were used in this study. Animals
were housed in a room maintained at 23 ± 1C with an alternating
12-h light–dark cycle. Food and water were available ad libitum.
Each animal was used only once.
Neuropathic pain model
The mice were anesthetized with either intraperitoneal (i.p.)
injection of pentobarbital (70 mg/kg) or isoflurane. We produced a
partial sciatic nerve injury model by tying a tight ligature with an 8–
0 silk suture around approximately 1/3–1/2 the diameter of the
sciatic nerve located on the right-hand side (ipsilateral side)
observed under a light microscope (SD30; Olympus, Tokyo, Japan).
This method is similar to the approach described in rats by Seltzer
et al. (1990) and in mice by Malmberg et al. (1997). In shamoperated mice, the nerve was exposed without ligation.
Latency of paw withdrawal in response to a thermal stimulus
To assess the sensitivity to thermal stimulation, each of the hind paws
of mice was tested individually using a thermal stimulus apparatus
(model 33, Analgesia Meter; IITC/Life Science Instruments, Woodland Hills, CA, USA). The intensity of the thermal stimulus was
adjusted to achieve an average baseline paw withdrawal latency of
approximately 8–10 s in naive mice. Only quick hind paw
movements (either with or without licking of hind paws) away from
the stimulus were considered to be a withdrawal response. Paw
movements associated with either locomotion or weight shifting
were not counted as a response. Each paw was measured alternately
after more than 3 min. The latency of paw withdrawal in response to
a thermal stimulus was determined as the average of three
measurements per paw. Before testing the behavioral responses to
the thermal stimulus, mice were habituated for at least 1 h in an
acrylic cylinder (15-cm high with 8-cm inner diameter). Under these
conditions, the latency of paw withdrawal in response to the thermal
stimulus was tested. The measurement of latency of paw withdrawal
in response to a thermal stimulus was performed before the surgery
and 1, 3, 5 and 7 days after the surgery.
In the experiment of a single i.t. treatment with either spinal cord
astrocytes or microglia, the measurement of paw withdrawal latency
was performed just before and just after the injection. The latency
of paw withdrawal in response to a thermal stimulus was determined
as the average of both paws.
Measurement of paw withdrawal in response to a tactile
stimulus
To quantify the sensitivity to tactile stimulus, paw withdrawal
response to tactile stimulus was measured using two different
bending forces (0.02 g and 0.16 g) of von Frey filaments (North
Coast Medical Inc., Morgan Hill, CA, USA). Each von Frey
filament was applied to the plantar surface of the hind paw for 3 s
and repeated three times with 5 s intervals. Each of the mouse hind
paws was tested individually. The paw withdrawal responses to the
tactile stimulus were evaluated by scoring as follows: 0, no
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Neuropathic pain and microglia 1339
response; 1, a slow and/or slight response against the stimulus; 2, a
quick withdrawal response away from the stimulus without flinching
and licking; 3, an intense withdrawal response away from the
stimulus with brisk flinching and/or licking. The paw withdrawal
response to each filament was determined as the average of two
scores per paw. Paw movements associated with either locomotion
or weight shifting were not counted as a response. Each paw was
measured alternately after more than 3 min. Before testing the
behavioral responses to tactile stimulus, mice were habituated for at
least 1 h on an elevated nylon mesh floor. Under these conditions
the paw-withdrawal response to the tactile stimulus was tested. The
measurement of paw-withdrawal response to the tactile stimulus was
performed just before the surgery and on the next day after the
measurement of the thermal threshold (days 2, 4, 6 and 8).
for seven consecutive days after the surgery. Each single injection of
either spinal cord astrocytes or spinal cord microglia was performed in
naive mice.
Intrathecal injection
The i.t. injection was performed as described by Hylden and Wilcox
(1980) using a 25-lL Hamilton syringe with a 30 1/2-gauge needle.
The needle was inserted into the intervertebal space between the L5
and L6 level of the spinal cord. A reflexive flick of the tail was
considered to be an indicator of the accuracy of each injection. The
injection volume was 4 lL for i.t. injection.
Groups of mice were given repeated i.t. treatment with minocycline
(1 nmol/mouse; Sigma-Aldrich Co., St Louis, MO, USA), which is
an antibiotic used in severe human infection and has the ability to
inhibit microglial activation and proliferation in the culture. Minocycline was given 1 h before sugery and post-measurement once a day
BrdU injection
Five-bromo-2¢-deoxyurindine (BrdU, 100 mg/kg i.p.; Sigma-Aldrich
Co.) was injected once a day for four consecutive days after sciatic
nerve ligation.
Drug treatment
Propentofylline, a novel xanthine derivative, is known to modulate
gilal activity under pathlogical conditions. It has been reported that
propentofylline (PPF) depresses the activation of microglia and
astrocytes, which are associated with neuronal damage during
ischemic injury (DeLeo et al. 1988). Therefore, we used PPF as a
glial modulating agent in the present study. Groups of mice were
repeatedly treated with PPF subcutaneously (s.c.) (3 mg/kg; SigmaAldrich Co.) 1 h before the surgery and once a day for seven
consecutive days after the surgery.
Sample preparation
Seven days after nerve ligation, mice were deeply anesthetized with
isoflurane and intracardially perfusion-fixed with freshly prepared 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4).
After perfusion, the lumber spinal cord was quickly removed and
postfixed in 4% paraformaldehyde for 2 h, and permeated with 20%
sucrose in 0.1 M PBS for 1 day and 30% sucrose in 0.1 M PBS for
Fig. 1 Immunofluorescent staining for O4-,
GFAP- and OX-42-like immunoreactivities
(IRs) on the contralateral (left-hand) and
ipsilateral (right-hand) sides of the dorsal
horn of the L5 spinal cord in nerve-ligated
mice. Glial fibrillary acidic protein (GFAP)and OX-42-like IRs, but not O4-like IR, were
increased on the ipsilateral (a-ii, b-ii and c-ii)
superficial dorsal horn of the L5 spinal cord
in nerve-ligated mice as compared with that
of the contralateral side (a-i, b-i and c-i).
The samples were prepared at 7 days after
sciatic nerve ligation in mice. Scale bars:
50 lm.
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1340 M. Narita et al.
2 days with agitation. Then, the L5 spinal cord segments were frozen
in an embedding compound (Sakura Finetechnical, Tokyo, Japan) on
isopentane using liquid nitrogen and stored at )30C until used.
Frozen spinal cord segments were cut with a freezing cryostat (Leica
CM 1510; Leica Microsystems AG, Wetzlar, Germany) at a 10-lm
thickness and thaw-mounted on a poly-L-lysine-coated glass slides.
Immunohistochemistry
The spinal cord sections were blocked in 10% normal goat serum
(NGS) in 0.01 M PBS for 1 h at room temperature (23C). For the
BrdU staining assay, spinal cord sections were immersed in citrate
buffer (10 mM, 90C) before blocking. The primary antibodies were
diluted in 0.01 M PBS containing 10% NGS [1 : 15–20 GFAP
(Nichirei Co., Tokyo, Japan), 1 : 250 OX-42 (Serotec Ltd, Oxford,
UK), 1 : 350 O4 (Chemicon International Inc., CA, USA), 1 : 150
BrdU (Abcam Ltd, Cambridgeshire, UK), 1 : 200 NeuN (Chemicon
Fig. 2 Effect of repeated injection of propentofylline (PPF) on thermal
hyperalgesia observed in the ipsilateral (a) and contralateral (b) sides
of either sham-operated or nerve-ligated mice. Groups of mice were
repeatedly treated with either PPF (3 mg/kg, s.c) or saline 1 h prior to
the surgery (day 0). During days 1–7 after sciatic nerve ligation, the
measurement of thermal hyperalgesia was performed 15 h after the
PPF injection. Each point represents the mean ± SEM of 5–8 mice.
**p < 0.01 and ***p < 0.001 vs. Sham/Vehicle group. ##p < 0.01 and
### p < 0.001 vs. Ligation/Vehicle group. d, Ligation/Vehicle; s,
Ligation/PPF 3 mg/kg; j, Sham/Vehicle; h, Sham/PPF 3 mg/kg.
International Inc.), 1 : 150 ionized calcium-binding adaptor molecule 1 (Iba1; Wako Pure Chemical Ltd, Osaka, Japan)] and
incubated for two nights at 4C. The samples were then rinsed
and incubated with the appropriate secondary antibody conjugated
with Alexa 488 and Alexa 546 for 2 h at room temperature. The
slides were then coverslipped with PermaFluor Aqueous mounting
medium (ImmunonTM; ThermoShandon, Pittsburgh, PA, USA).
Fluorescence of the immunolabelings was detected using the light
microscope (Olympus AX-70; Olympus) and photographed with a
digital camera (CoolSNAP HQ; Olympus). In the cultured cell
experiments, the density of either GFAP- or OX-42-like IR labeling
was shown as a pseudo-color image by using a computer-assisted
imaging analysis system (NIH Image). In the BrdU staining assay,
the number of BrdU-positive cells was counted.
Preparation of spinal cord astrocytes
Purified spinal cord astrocytes were grown as follows: spinal
cords were obtained from newborn ICR mice (Tokyo Laboratory
Animals Science Co., Ltd), minced, and treated with trypsin
(0.025%, Invitrogen Co., Carlsbad, CA, USA) dissolved in a PBS
solution containing 0.02% L-cystein monohydrate (Sigma-Aldrich
Co.), 0.5% glucose (Wako Pure Chemical Ltd) and 0.02% bovine
serum albumin (Wako Pure Chemical Ltd). After enzyme
treatment at 37C for 15 min, cells were dispersed by gentle
agitation through a pipette and plated on a flask. One week after
seeding, the flask was shaken (90 shakes/min) for 12 h at 37C to
remove non-astroglial cells. The cells were seeded at a density of
1 · 105 cells/cm2. The cells were maintained for 3–10 days in
Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Co.)
supplemented with 5% fetal bovine serum (Invitrogen Co.), 5%
heat-inactivated (56C, 30 min) horse serum (Invitrogen Co.),
10 U/mL penicillin and 10 lg/mL streptomycin in a humidified
atmosphere of 95% air and 5% CO2 at 37C. At days 4–7
in vitro, the cells were treated with an either normal medium or a
PKC activator, PDBu (100 nM, Sigma-Aldrich Co.), for 1 day.
The former cells were used as astrocytes and the latter cells were
used as activated astrocytes in the present study.
For the preparation of an astrocyte-conditioned medium (ACM)
and activated ACM (aACM), astrocytes and activated astrocytes
were grown to confluence (see above). Cells were washed once with
DMEM and then covered with an equal volume of serum-free
medium for 24 h at 37C and 5% CO2 in the presence of indicated
treatments. The supernatants were collected 1 day after changing to
the serum-free medium culture and centrifuged for 20 min at
1000 g. The final supernatants were used as either ACM or aACM.
Finally, DMEM with fibronectin, astrocytes with ACM plus
fibronectin or activated astrocytes with aACM plus fibronectin were
injected intrathecally in naive mice.
Preparation of spinal cord microglia
Purified spinal cord microglia were grown as follows: spinal
cords were obtained from newborn ICR mice (Tokyo Laboratory
Animals Science Co., Ltd), minced, and treated with trypsin
(0.025%, Invitrogen Co.) dissolved in a PBS solution containing
0.02% L-cystein monohydrate (Sigma-Aldrich Co.), 0.5% glucose
(Wako Pure Chemical Ltd) and 0.02% bovine serum albumin
(Wako Pure Chemical Ltd). After enzyme treatment at 37C for
15 min, cells were dispersed by gentle agitation through a pipette
and plated on a flask. Ten days after seeding, medium was
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Neuropathic pain and microglia 1341
Fig. 3 Effect of the repeated injection of
propentofylline (PPF) on tactile allodynia
observed in the ipsilateral (a and c) and
contralateral (b and d) sides of either shamoperated or nerve-ligated mice. Tactile stimulus was performed by two different
bending forces of filaments (0.02 g in a and
b; 0.16 g in c and d). Groups of mice were
repeatedly treated with either PPF (3 mg/
kg, s.c) or saline 1 h prior to the surgery
(day 0). During days 1–7 after the nerve
ligation, the measurement of allodynia was
performed 15 h after the PPF injection.
Each point represents the mean ± SEM
of 5–8 mice. *p < 0.05, **p < 0.01 and
***p < 0.001 vs. Sham/Vehicle group.
#p < 0.05, ##p < 0.01 and ###p < 0.001
vs. Ligation/Vehicle group. d, Ligation/Vehicle; s, Ligation/PPF 3 mg/kg; j, Sham/
Vehicle; h, Sham/PPF 3 mg/kg.
(a)
Fig. 4 Increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR) by
in vitro treatment with a protein kinase C
(PKC) activator phorbol 12,13-dibutyrate
(PDBu) for 1 day in mouse spinal purified
astrocytes. Mouse spinal purified astrocytes
were incubated with either normal medium
(a) or PDBu (100 nM, b). The cells were
stained with a mouse polyclonal antibody to
GFAP. These panels show pseudo-color
images of GFAP-like IR in the spinal cord
astrocytes (a and b). Scale bar: 50 lm. (c)
No effect of a single i.t. injection of
Dulbecco’s modified Eagle’s medium
(DMEM + fibronectin, h), Astrocytes (1 ·
106 cells/mL) with either Astrocyte-conditioned medium (ACM) plus fibronectin
(astrocytes + ACM + fibronectin, d) or activated Astrocytes (1 · 106 cells/mL) with
activated ACM (aACM) plus fibronectin
(activated astrocytes + aACM + fibronectin, s) on the latency of paw withdrawal in
response to a thermal stimulus in normal
mice. Each point represents the mean ±
SEM of 10–11 mice.
(b)
(c)
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1342 M. Narita et al.
collected after shaking the flask. The collected medium was then
centrifuged for 20 min at 3000 g. The supernatant was used as
spinal cord microglia. Spinal cord miroglia were grown to
confluence (see above). After treating with either PBS or ATP,
mediums containing cells were centrifuged for 10 min 3000 g.
The final pellet was dissolved, in turn injected intrathecally as
either microglia or activated microglia.
with PDBu (100 nM). In the present study, PDBu caused the
astroglial proliferation, as characterized by the increase in
GFAP-like IR levels, and astroglial hypertrophy, as detected
by a stellate morphology of GFAP-like IR in the purified
spinal cord astrocytes, as compared with that of control
astrocytes (Figs 4a and b).
Results
Changes in GFAP-, OX-42- and O4-like IRs by sciatic
nerve ligation in the dorsal horn of the mouse L5 spinal
cord
GFAP-, OX-42- and O4-like IRs were detected on the
ipsilateral side of the L5 spinal dorsal horn of sham-operated
mice (data not shown). Seven days after nerve ligation, O4like IR was not affected by sciatic nerve ligation (Figs 1a-i
and a-ii). In contrast, GFAP-like IR in laminae II of the
ipsilateral side of the L5 spinal dorsal horn was increased as
compared with that of the contralateral side in nerve-ligated
mice (Figs 1b-i and b-ii). Furthermore, OX-42-like IR on the
ipsilateral dorsal horn of the L5 spinal cord was also
dramatically elevated as compared with that on the contralateral side in nerve-ligated mice (Figs 1c-i and c-ii).
Effect of repeated s.c. injection of a glial modulating
agent PPF on thermal hyperalgesia and tactile allodynia
induced by sciatic nerve ligation in mice
Partial sciatic nerve ligation caused a marked decrease in the
latency of paw withdrawal in response to thermal stimulus
only on the ipsilateral side in nerve-ligated mice. The thermal
hyperalgesia observed on the ipsilateral side in nerve-ligated
mice was abolished by repeated injection of a glial modulating agent PPF (3 mg/kg, s.c.) just before the ligation and
once a day for seven consecutive days after nerve ligation
(F1,10 ¼ 43.931, p < 0.001 vs. Ligation/Vehicle; Fig. 2a).
The mice with sciatic nerve ligation also revealed a marked
increase in the paw withdrawal response to a tactile stimulus
only on the ipsilateral side. Under these conditions, repeated
injection of PPF (3 mg/kg s.c.) abolished the increase in the
paw withdrawal response to an innocuous tactile stimulus
induced by nerve ligation in mice (F1,10 ¼ 85.035, p < 0.001
vs. Ligation/Vehicle, Fig. 3a; F1,10 ¼ 6.539, p < 0.05 vs.
Ligation/Vehicle, Fig. 3c).
Repeated s.c. injection of PPF at the dose used in the
present study failed to affect thermal and tactile thresholds on
the contralateral side in nerve-ligated mice and on both sides
in sham-operated mice (Figs 2 and 3).
Morphological changes in purified spinal cord astrocytes
by treatment with a PKC activator PDBu
We investigated whether morphological changes in the
purified spinal cord astrocytes could be caused by treatment
Fig. 5 Effect of repeated i.t. injection of minocycline on thermal
hyperalgesia observed on the ipsilateral (a) and contralateral (b) sides
of either sham-operated or nerve-ligated mice. Groups of mice were
repeatedly treated with either minocycline (1 nmol/mouse i.t.) or
vehicle 1 h prior to the surgery (day 0). During days 1–7 after nerve
ligation, repeated i.t. injection of either minocycline (1 nmol/mouse) or
vehicle was performed after the measurement of thermal hyperalgesia. Each point represents the mean ± SEM of 5–7 mice. **p < 0.01,
***p < 0.001 vs. Sham/Vehicle group. ##p < 0.01 and ###p < 0.001
vs. Ligation/Vehicle group. d, Ligation/Vehicle; s, Ligation/Minocycline 1 nmol/mouse; j, Sham/Vehicle; h, Sham/Minocycline 1 nmol/
mouse.
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Neuropathic pain and microglia 1343
Time-course changes in thermal thresholds following a
single i.t. treatment with spinal cord astrocytes
We next investigated whether a single i.t. treatment with
spinal cord astrocytes (1 · 106 cells/mL) could cause a
hyperalgesic response in naive mice. A single i.t. injection of
spinal cord astrocytes activated by PDBu failed to produce
thermal hyperalgesia in naive mice (Fig. 4c). Furthermore, a
single i.t. injection of spinal cord astrocytes (1 · 106 cells/
mL) without PDBu did not cause any changes in the latency
of thermal stimulus (Fig. 4c).
Effect of repeated i.t. injection of an inhibitor of
microglial activation, minocycline, on thermal
hyperalgesia and tactile allodynia induced by sciatic
nerve ligation in mice
Partial sciatic nerve ligation caused a marked decrease in the
latency of paw withdrawal after a thermal stimulus only on
the ipsilateral side in nerve-ligated mice. The thermal
hyperalgesia observed on the ipsilateral side in nerve-ligated
mice was significantly prevented by repeated i.t. injection of
minocycline (1 nmol/mouse) just before the ligation and
once a day for seven consecutive days after nerve ligation
(F1,10 ¼ 37.173, p < 0.001 vs. Ligation/Vehicle, Fig. 5a).
The mice with sciatic nerve ligation also revealed a
marked increase in the paw withdrawal response to tactile
stimulus only on the ipsilateral side in nerve-ligated mice.
Under these conditions, repeated i.t. injection of minocycline
(1 nmol/mouse) prevented the increase in the paw withdrawal response to an innocuous tactile stimulus induced by
nerve ligation in mice (F1,10 ¼ 8.323, p < 0.05 vs. Ligation/
Vehicle, Fig. 6a; F1,10 ¼ 5.093, p < 0.05, Fig. 6c).
Repeated i.t. injection of minocycline at the dose used in
the present study failed to affect thermal and tactile
thresholds on the contralateral side in nerve-ligated mice
and on both sides in sham-operated mice (Figs 5 and 6).
Time-course changes in thermal thresholds following an
exogenous i.t. treatment with spinal cord microglia
To clarify the direct involvement of activated spinal cord
microglia in the development of a neuropathic pain-like state
in mice, we investigated whether a single i.t. treatment with
spinal cord microglia could cause thermal hyperalgesia in
naive mice. It is of interest to note that a single i.t. injection
of spinal cord microglia activated by ATP (50 lM;
1 · 106 cells/mL), which has been well known as a
microglial activator, significantly decreased paw withdrawal
latency to thermal stimulus at 6 h after the injection in naive
mice (p < 0.01, vs. pre-injection, Fig. 7d). In contrast, a
single i.t. injection of spinal cord microglia without ATP
(1 · 106 cells/mL), medium and medium plus ATP failed to
cause thermal hyperalgesia (Figs 7c, e and f).
Localization of BrdU-like IR in the ipsilateral dorsal horn
of the L5 spinal cord in the mouse
A growing body of evidence suggests that the activated
microglia has the ability to differentiate and proliferate itself
(Hanisch 2002; Streit 2002). We demonstrated that the
number of BrdU-positive cells on the ipsilateral dorsal horn
Fig. 6 Effect of repeated i.t. injection of
minocycline on tactile allodynia observed
on the ipsilateral (a and c) and contralateral
(b and d) sides of either sham-operated or
nerve-ligated mice. Tactile stimulus was
performed by two different bending forces
of filaments (0.02 g in a and b; 0.16 g in c
and d). Groups of mice were repeated i.t.
injection of either minocycline (1 nmol/
mouse) or vehicle 1 h prior to the surgery
(day 0). During days 1–7 after nerve
ligation, repeated i.t. injection of either
minocycline (1 nmol/mouse) or vehicle was
performed after the measurement of
allodynia. Each point represents the
mean ± SEM of 5–8 mice. *p < 0.05,
**p < 0.01 and ***p < 0.001 vs. Sham/Vehicle group. #p < 0.05, ##p < 0.01 and
###p < 0.001 vs. Ligation/Vehicle group. d,
Ligation/Vehicle; s, Ligation/Minocycline
1 nmol/mouse; j, Sham/Vehicle; h, Sham/
Minocycline 1 nmol/mouse.
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 1337–1348
1344 M. Narita et al.
(a)
(c)
(b)
(d)
(e)
(f)
Fig. 7 Increase in the OX-42-like immunoreactivity by in vitro treatment with ATP for 1 h in mouse purified spinal microglia. Mouse
purified spinal microglia were incubated with either normal medium (a)
or ATP (50 lM, b). The cells were stained with a rat polyclonal antibody
to OX-42. These panels show pseudo-color images in the spinal cord
microglia (a and b). Scale bar: 50 lm. Time-course changes in the
latency of paw withdrawal to the thermal stimulus induced by a single
i.t. injection of (c) microglia (1 · 106 cells/mL), (d) activated microglia
(1 · 106 cells/mL) with ATP, (e) medium or (f) medium plus ATP in
normal mice. Each column represents the mean ± SEM of 7–10 mice.
**p < 0.01 vs. Pre-injection.
in nerve-ligated mice was dramatically increased as compared with that on the contralateral side and that on the
ipsilateral side in sham-operated mice (**p < 0.01 vs.
Contralateral Side/Ligation; ##p < 0.01 vs. Ipsilateral side/
Sham; Fig. 8b). In contrast, the number of BrdU-positive
cells on the ipsilateral side was not changed as compared
with that on the contralateral side in sham-operated mice
(Fig. 8b). In addition, BrdU-like IR was highly co-localized
with another specific microglia marker Iba1, but not with a
neuron-specific nuclear protein marker, NeuN, and GFAP
(Fig. 9).
stimuli. Our recent research suggests that the activation of
spinal PKC, which can modulate synaptic plasticity, plays a
substantial role in the development of a neuropathic pain-like
state in mice (Yajima et al. 2003). In our preliminary study,
we found that the neuronal cell body and dendrite marker
microtubule-associated protein 2a/b (MAP2a/b)-like IR
within the spinal dorsal horn was not affected by sciatic
nerve ligation (M. Narita, T. Yoshida, M. Nakajima, M.
Narita, M. Miyatake, T. Takagi, Y. Yajima and T. Suzuki,
Department of Toxicology, Hoshi University School of
Pharmacy and Pharmaceutical Sciences, Tokyo, Japan,
unpublished observation), indicating that neurons within
the spinal dorsal horn may not be degenerated under the
neuropathic pain-like state.
CNS synapses are encapsulated by glial cells. Glial cells
express receptors for many neurotransmitters and neuromodulators, synthesize and release numerous transmitters, and
produce transporters that either uptake or release transmitters
from the extracellular and synaptic space, respectively
(Araque et al. 1999; Haydon 2001). Therefore, glial cells
Discussion
Several lines of evidence have demonstrated that synaptic
plasticity characterized by long-term potentiation is caused
by chronic pain (Narita et al. 2000; Ji and Woolf 2001; Julius
and Basbaum 2001; Yajima et al. 2002). It has been shown
that many plastic changes in neurons in the pain pathway can
amplify the transmission of information regarding noxious
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 1337–1348
Neuropathic pain and microglia 1345
(a)
(b)
Fig. 8 Immunofluorescent staining for 5-bromo-2¢-deoxyuridine
(BrdU)-like immunoreactivity (IR) on the L5 spinal cord in nerve-ligated
mice (a). BrdU positive cells were significantly increased on the ipsilateral dorsal horns of the L5 spinal cord in nerve-ligated mice as
compared with that on the contralateral side and that on the ipsilateral
side in sham-operated mice (b). Each column represents the
mean ± SEM of 3–4 mice (**p < 0.01 vs. Contralateral side/Ligation;
##p < 0.01 vs. Ipsilateral side/Sham). Repeated i.p. injection of BrdU
was performed once a day for four consecutive days after nerve ligation. The samples were prepared 7 days after nerve ligation in mice.
Scale bars: 100 lm.
are well positioned to regulate neuronal function. It has been
recognized that spinal cord glial cells, astrocytes and
microglia, are activated by procedures that enhance pain
(Watkins et al. 2001a; Raghavendra et al. 2003; Tsuda et al.
2003, 2004, 2005; Watkins and Maier 2003; Raghavendra
et al. 2004). Furthermore, astrocytes and microglia are
activated by pain-related substances such as substance P,
calcitonin-gene related peptide (CGRP), ATP and excitatory
amino acid from primary afferent terminals, in addition to
virus and bacteria (Norenberg 1994; Watkins et al. 2001a).
In the present study, we found that at 7 days after sciatic
nerve ligation, O4-like IR was not changed in the ipsilateral
dorsal horn of the spinal cord compared with that on the
contralateral side. O4 occurs in pro-oligodendrocytes, but not
in O-2A-progenitor cells, and is a marker for oligodendrocyte
cell bodies. Anti-O4 can be used for studies on myelinization/demyelinization. In our preliminary study, we found
that another oligodendrocyte marker myelin-associated
glycoprotein (MAG) IR was not affected by nerve ligation
(M. Narita, T. Yoshida, M. Nakajima, M. Narita, M.
Miyatake, T. Takagi, Y. Yajima and T. Suzuki, Department
of Toxicology, Hoshi University School of Pharmacy and
Pharmaceutical Sciences, Tokyo, Japan, unpublished observation). These findings indicate that demyelinization may not
be caused by the neuropathic pain-like state in mice.
In contrast, GFAP- and OX-42-like IRs were elevated in
the ipsilateral dorsal horn of the spinal cord compared with
that on the contralateral side at 7 days after sciatic nerve
ligation. Moreover, we demonstrated that thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation
were abolished by repeated treatment with the glial modulating agent PPF. It has also been documented that treatment
with PPF prevented tactile allodynia induced by spinal nerve
transection in rats (Sweitzer et al. 2001). Our findings
provide further evidence that astroglial and microglial
activation is caused under the neuropathic pain-like state in
mice.
It is generally recognized that morphological changes in
glial cells reflect their active state. Microglia are the earlyresponding glial cells in the CNS after injury. Products
released from activated microglia lead to astroglial activation, which in turn maintains a long-term pathological state
(Svensson et al. 1993; Meller et al. 1994; Kreutzberg 1996;
Popovich et al. 1997; Watkins et al. 1997; Sweitzer et al.
2001; Raghavendra et al. 2003). In the present study, we
demonstrated that an exogenous single i.t. injection of
activated spinal cord astrocytes treated with PDBu did not
cause any changes in the latency of paw withdrawal in
response to thermal stimulus. These findings suggest that
astroglial activation within the spinal dorsal horn may not be
directly involved in the development of the neuropathic painlike state in mice. Although we cannot completely exclude
the possibility that a long-term activation of astrocytes
followed by the early activation of microglia modulates the
neuropathic pain-like state, the pathological significance of
astroglial activation does not seem to be strongly linked to
the neuropathic pain-like state. In conclusion, either a
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 1337–1348
1346 M. Narita et al.
(a-i)
(a-ii)
(a-iii)
(b-i)
(b-ii)
(b-iii)
(c-i)
(c-iii)
(c-ii)
(c-iv)
complete time-course study or a cell concentration-dependent
response is still needed to prove whether astroglial activation
could be occasionally responsible for the development of the
neuropathic pain-like state.
In the present study, thermal hyperalgesia and tactile
allodynia induced by sciatic nerve ligation were significantly
inhibited by repeated i.t. treatment with minocycline, which
is an antibiotic used in severe human infection and which can
inhibit microglial activation and proliferation (Tikka and
Koistinaho 2001; Tikka et al. 2001; Demercq and Matute
2004). Raghavendra and colleagues (Raghavendra et al.
2003) previously reported that minocycline prevented tactile
allodynia induced by nerve injury. In addition, an exogeneous single i.t. injection of activated spinal cord microglia
treated with ATP caused a significant thermal hyperalgesia in
naive mice. Tsuda and colleagues (Tsuda et al. 2003)
recently reported that tactile allodynia was observed upon
i.t. treatment with activated microglia treated with ATP in
naive rat. Collectively, these findings suggest that microglial
activation within the spinal cord may be a major factor in
directly inducing the development of a neuropathic pain-like
state in mice.
(c-v)
Fig. 9 BrdU-positive cells (a-i and b-i) were
not detected on the specific neuronal nuclei
marker NeuN (a-ii) and glial fibrillary acidic
protein (GFAP)-like immunoreactivities
(IRs) (b-ii) at the ipsilateral side of the spinal
dorsal horn in nerve-ligated mice (a-iii and
b-iii). In contrast, there is apparent
co-localization of BrdU (c-i) with Iba1 (c-ii),
another specific microglia marker, in the
dorsal horn of the spinal cord of nerveligated mice. Double labeling with BrdU
(c-iii, c-iv and c-v) shows that some of the
Iba1-like IR cells were either proliferated or
generated. Scale bars: 50 lm (except for
c-iv and c-v); scale bars in c-iv and c-v:
10 lm.
The key point of the present study was to investigate
whether either astroglial or microglia proliferation could be
caused under the neuropathic pain-like state induced by sciatic
nerve ligation by using BrdU, a cell proliferation marker. At
7 days after nerve ligation, BrdU-positive cells on the
ipsilateral side of the spinal dorsal horn were significantly
increased compared with the contralateral side in nerve-ligated
mice. In addition, BrdU-positive cells were highly co-localized
with another specific microglia marker, Iba1, but not with the
neuron-specific nuclear markers, NeuN and GFAP, within the
ipsilateral dorsal horn of the spinal cord. These findings
strongly suggest that microglia are, at least in part, either
proliferated or generated by sciatic nerve ligation in mice.
When microglia sense trauma, ischemia, tumors and
peripheral inflammation, they are spontaneously activated
(Nakajima and Kohsaka 2001; Watkins and Maier 2003).
This condition is a graded phenomenon, characterized by
specific morphological changes (hypertrophy), proliferation
and changes in functional activities (migration to areas of
damage, phagocytosis and production/release of pro-inflammatory substances). Activated microglia remove dead cells
or dangerous debris either by releasing toxic factors (like
2006 The Authors
Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 1337–1348
Neuropathic pain and microglia 1347
tumor necrosis factor TNFa and interleukin IL-1b) or by
phagocytosis; whereas these cells repair injured cells by
releasing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and IL-6 (Hide et al. 2000; Nakajima
et al. 2001; Shigemoto-Mogami et al. 2001). MAP2a/b-like
IR was not affected by sciatic nerve ligation (unpublished
observation). Considering this background, the present data
support the idea that activated microglia alter neuronal
excitability and sculpt neuronal growth and connection
through pro-inflammatory cytokines and trophic factors
released from these cells rather than its phagocytosis in the
spinal cord. Activated spinal cord glial cells lead to
hyperalgesia and allodynia by releasing soluble factors that
act on neurons in the pain pathways. However, the soluble
factors that are released from activated glial cells are unclear.
Therefore, it would be important to identify which soluble
factors (pro-inflammatory cytokines, chemokines and trophic
factors) are released from not only activated microglia but
also activated astrocytes under the neuropathic pain-like state
in order to better understand the mechanisms involved.
In conclusion, the present study suggests that spinal cord
glial cells, astrocytes and microglia, are activated under the
development of a neuropathic pain-like state in mice. Furthermore, the proliferated and activated microglia, but not
astrocytes, in the spinal dorsal horn are directly implicated in
the expression of a neuropathic pain-like state in mice.
Acknowledgements
This work was supported in part by a research grant from Ministry
of Education, Culture, Sports, Science and Technology and Ministry
of Health, Labor and Welfare of Japan. We wish to thank Ms Seiko
Hashimoto, Mr Hiroyuki Nozaki and Ms Yui Kazama for their
expert technical assistance.
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