Crosstalk between spinal astrocytes and neurons in nerve injury-induced neuropathic pain.

Abstract

Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-alpha-aminoadipate (L-AA) reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal "crosstalk". The current study suggests that neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that the intervention of such interaction may offer some novel pain therapeutic strategies.

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Crosstalk between Spinal Astrocytes and Neurons in
Nerve Injury-Induced Neuropathic Pain
Wei Wang., Wen Wang., Xiaopeng Mei, Jing Huang, Yanyan Wei, Yayun Wang, Shengxi Wu*, Yunqing
Li*
Department of Anatomy, Histology and embryology; K. K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an, People’s Republic of China
Abstract
Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced
neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a
lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes
reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was
used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated
neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at
the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively.
Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-a-aminoadipate (L-AA)
reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration
of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA
shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are
closely related with the maintenance of neuropathic pain through a reciprocal ‘‘crosstalk’’. The current study suggests that
neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that
the intervention of such interaction may offer some novel pain therapeutic strategies.
Citation: Wang W, Wang W, Mei X, Huang J, Wei Y, et al. (2009) Crosstalk between Spinal Astrocytes and Neurons in Nerve Injury-Induced Neuropathic Pain. PLoS
ONE 4(9): e6973. doi:10.1371/journal.pone.0006973
Editor: Shawn Hochman, Emory University, United States of America
Received May 11, 2009; Accepted August 11, 2009; Published September 11, 2009
Copyright: ? 2009 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the grants from National Program of Basic Research of China (G2006CB500808), Program for New Century Excellent Talents
in University (NCET-06-0931). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: shengxi@fmmu.edu.cn (SW); deptanat@fmmu.edu.cn (YL)
. These authors contributed equally to this work.
Introduction
Peripheral nerve injury often results in neuropathic pain [1]
which is essentially intractable to currently available anti-
nociceptive therapies. This is in part due to poor understanding
on how neuropathic pain is induced and maintained [2].
According to classic pain research, the pain pathway has been
described simply as a serial chain of neuronal elements, but
immerging research has implicated spinal cord glial cells as key
players in the induction and maintenance of neuropathic pain [3].
Due to the immune cell nature of microglia and their ability of
releasing the ‘‘proinflammatory’’ cytokines such as interlukin-1
(IL-1), tumor necrosis factor (TNF) and adenosine triphosphate
(ATP) when activated [4,5,6,7,8], microglia receive intensive focus
in pain research, especially persistent pain after inflammation or
nerve injury [9,10,11,12]. Thanks to decades of pain researchers’
endeavors, the underlying mechanisms for microglia-neuron
interaction and its contribution to neuropathic pain have almost
been clarified [13]. However, the spinal astrocytes, which are
more dominant than microglia, are majorly investigated for its
supportive and tropic functions for neurons [14,15] instead of their
contributions to neuropathic pain. A few studies suggested the
activationofspinal astrocytes
[3,13,16,17], but it is not clear whether there exists astrocyte-
duringneuropathicpain
neuron crosstalk just as in the case of microglia, and how this
happens [18].
C-fos, as a protooncogene, has been shown to act as an inducible
transcriptional factor and suggested to regulate the expression of
the downstream response genes [6,19,20]. Fos protein is rapidly
expressed in the spinal dorsal horn neurons following noxious
peripheral nerve stimulation [21] and correlates well with
nociceptive behaviors [19]. Thus, Fos expression is used to
identify nociceptive input activated or excited population of
neurons [22,23,24].
Astrocytes can sensitively and rapidly respond to various
physiological or noxious stimuli with the increased expression of
glial fibrillary acidic protein (GFAP), thus be used as a marker for
astrocytic activation [25]. Our previous research indicates that
GFAP upregulation correlates well with nerve injury-induced
neuropathic pain [26]. However, astrocytes cannot generate
action potential and are not able to contribute to the nociceptive
transmission alone and directly [14]. Hence, a hypothesis is that
the activated astrocytes and neurons are ‘‘crosstalking’’ so as to
contribute to the neuropathic pain states [27]. However, there still
lacks direct evidence for this hypothesis.
To achieve this goal, it is necessary to shut-down the activated
astrocytes or neurons to elucidate their individual contribution to
neuropathic pain. In the current study, L-a-aminoadipate (L-AA)
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was used to inactivate astrocytes based on the fact that its
disruption of the astrocytic network in amygdala is confined to
astrocytes [28,29]. C-fos antisense oligodeoxynucleotides (ASO)
was used in our previous studies and the corresponding
nociceptive behavioral changes were observed as a consequence
of knocking down its protein product [24,30,31,32], thus, it was
employed in this study to shut down activated neurons.
In the current study, we observed the temporal activation of
neurons and astrocytes along with lumbar 5 spinal nerve ligation
(SNL) induced neuropathic pain behaviors by using Fos or GFAP
expression, respectively. Then, the effect of intrathecal (i.t.)
injection of c-fos ASO or L-AA on pain behaviors as well as
neuronal or astrocytic activation was identified morphologically.
The possible ‘‘crosstalk’’ between astrocytes and neurons was
investigated by checking the effect of inhibited activation of one
type of cells on the other.
Results
SNL-induced mechanical allodynia was reversed by c-fos
ASO or L-AA treatment
SNL-induced neuropathic pain is characterized by ipsilateral
mechanical allodynia [33]. Previous studies indicated that SNL
produced rapidly appearing (,3 d) and persistent (.3 w)
ipsilateral mechanical allodynia shown by decrease of paw
withdrawal threshold (PWT) [17,26].
C-fos ASO treatment significantly reversed mechanical allody-
nia in 3 d post SNL. The ASO treatment and post operative day
(POD) had significant effect on the SNL-induced ipsilateral
allodynia [Fig. 1A; two way ANOVA (between subjects factors:
Group and Day): Group: F (2,326)=127.314, P=0.000; Day: F
(7,326)=209.909, P=0.000; Group*Day: F (14,326)=9.676,
P=0.000]. The effect of SNL on the allodynia of saline and SO
groups was incubated with time. SNL rats in saline and c-fos SO
group demonstrated significant ipsilateral allodynia 1 d after the
surgery (PWT=6.861.3 g and 7.460.6 g on POD 1 for Saline
and SO groups, respectively; Post hoc test, P,0.05, vs correspond-
ing baseline for saline and SO group, respectively) and the
ipsilateral allodynia reached a high level on POD 3 and lasted for
the whole observing window. There was no significant difference
between saline and SO group at every corresponding time point.
Treatment with ASO significantly reversed the ipsilateral
allodynia on POD 1 (PWT=6.861.3 g, 7.460.6 g, 20.462.3 g
at POD 1 for Saline, SO and ASO group, respectively; Post hoc
test, P,0.01, vs Saline or SO group), and such reversal effect
existed for the whole observing window (Post hoc test, P,0.01, for
2, 3, 4, 5 and P,0.05 for 6 and 7 days after SNL, vs Saline or SO
group). But the preventing effect weakened gradually. On POD 4,
PWT in the Saline group was 3.460.4 g, while it was 10.861.6 g
in the ASO group. And the difference of PWT between groups
was much less but still significant 7 d after SNL (Fig. 1A). Thus,
SNL itself produced a significant allodynia which was incubated
with time; SO treatment did not change the neuropathic pain
performance after SNL, while ASO treatment reversed the SNL-
induced neuropathic pain with a long-lasting effect.
Treatment with the astrocyte inactivator L-AA, reversed
mechanical allodynia only at the late stage. The L-AA treatment
and POD had significant effect on the SNL-induced ipsilateral
allodynia [Fig. 1B; two way ANOVA (between subjects factors:
Group and Day): Group: F (1,216)=157.194, P=0.000; Day: F
(7,216)=94.389,
P=0.000;Group*Day:
p=0.000]. Treatment with L-AA significantly reversed the
ipsilateral allodynia on POD 3 (PWT=4.760.8 g and 12.461.4 g
for Saline and L-AA groups, respectively; Post hoc test, P,0.01), and
F (7,216)=11.567,
such reversal effect existed since POD 3 (Post hoc test, P,0.01 for
POD 4 and 5 and P,0.05 for POD 6 and 7, vs saline group). On
POD 7, the PWTs for saline and L-AA groups were 3.460.5 g and
8.861.6 g, respectively. Inactivation of astrocytes by L-AA
selectively reversed mechanical allodynia 7 d post nerve injury.
In the sham-operated animals, PWTs was not influenced
compared with that of naı ¨ve rats. Furthermore, intrathecal
injection of c-fos ASO, SO or L-AA on sham-operated rats did
not change their PWTs (data not shown).
SNL enhanced Fos and GFAP expression in the ipsilateral
spinal dorsal horn
Immunofluorescent histochemistry was employed to demonstrate
the Fos expression from different groups (Fig. 2A, A’). To rule out
Figure 1. Effects of intrathecal administration of c-fos ASO (A)
or L-AA (B) on SNL-induced mechanical allodynia. A, SNL rats in
saline and c-fos SO group demonstrated significant ipsilateral allodynia
since 1 d after the surgery. Intrathecal administration of c-fos ASO
(50 mg/10 ml, starting 4 h prior to the surgery and once a day for a
further 3 d following surgery) significantly reversed the ipsilateral
allodynia. The preventing effect of ASO weakened gradually but still
significant 7 d after SNL B, Intrathecal administration of astroglial toxin
L-AA (100 nmol/10 ml, injected daily from 1 d before till 3 d after the
surgery) reversed mechanical allodynia only at the late stage. The data
are presented as the mean 6 S.E.M. * and ** indicate statistically
significant difference with P,0.05 and P,0.01 compared to saline
control of each time point, respectively.
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the possible grouping and treatment bias, the number of Fos-
immunoreactive (IR) neuronswasnormalized tothat innaı ¨verats at
corresponding time points. SNL induced a significant increase of
Fos expression in the ipsilateral but not the contralateral spinal
dorsal horn (Fig. 2A, A’). A time-course study showed that the Fos
expression reached the highest level 2 h after SNL, then declined
gradually, to normal level by 1 w (Fig. 2D, 5D). Almost all of the
Fos-IR cells were neurons as indicated by NeuN staining (95% of
Fos-IR neurons were also positive for NeuN, Fig. 3). SNL-induced
Fos expression was restricted to the neuronal population as the ratio
of Fos positive NeuN-IR cells to Fos-IR did not vary too much
among different groups nor at different time points. Thus, increased
Fos expression was reliably used as an marker for activated neurons
but not astrocytes. Meanwhile, most of the Fos-IR neurons were
observed in laminae I and II with a few in laminae III and IV and
some in laminae V and VI of the dorsal horn (Fig. 2A), suggesting
most of them were nociceptive neurons.
Similar to our previous observation [26], SNL induced a
marked expression of GFAP in the ipsilateral but not the
contralateral dorsal horn (Fig. 2B). A time course study showed
that GFAP up-regulation was not evident on POD 1, but became
significant on POD 3, and reached a peak on POD 7 after SNL
(Fig. 4D, 5D). Double immunofluorescent labeling indicated that
few GFAP-IR cells were positive for Fos, while most of them were
overlapped nuclei of activated neurons (Fig. 4). These activated
astrocytes were mainly distributed in the superficial laminae of
dorsal horn ipsilateral to the injury, suggesting a role in the
nociceptive transmission. The Fos-IR neurons were surrounded
with many GFAP-IR astrocytic processes, forming many close
contacts (Fig. 3B’).
C-fos ASO treatment suppressed SNL-induced neuronal
and astrocytic activations in different manners
Besides its effect on the SNL-induced ipsilateral mechanic
allodynia, c-fos ASO significantly inhibited SNL-induced Fos
protein expression in the ipsilateral dorsal horn. Statistical analysis
revealed a significant effect of treatment as well as POD on the
SNL induced dorsal horn Fos expression [Fig. 4D; two way
ANOVA (between subjects factors: Group and Day): Group: F (2,
36)=117.717, P=0.000; Day: F (3, 36)=85.247, P=0.000;
Group*Day: F (6, 36)=20.944, P=0.000]. Inhibition of ASO
on SNL-induced Fos expression occured right after the nerve
injury (at 2 h after SNL). The Fos expression level in ASO group
was almost the same as baseline and this effect lasted during the
whole observing window (Fig. 4D).
Meanwhile, c-fos ASO also inhibited the SNL-induced GFAP
up-regulation but such effect was different from that on Fos
expression. According to our previous study [26], SNL-enhanced
GFAP expression occurs later than POD 3. Statistical analysis
revealed a significant effect of treatment as well as POD on the
SNL-induced GFAP up-regulation [Fig. 4D; two way ANOVA
(between subjects factors: Group and Day): Group: F (2,
36)=6.930, P=0.004; Day: F (3, 36)=103.403, P=0.000;
Group*Day: F(6, 36)=2.204, P=0.078]. ASO treatment rendered
inhibitory effect on the enhanced GFAP on POD 3 and 7. Our
changing course study of GFAP expression in the ipsilateral SDH
of Saline, as well as for c-fos SO and ASO groups indicated that
there were no obvious differences at 2 h and POD 1 when no
obvious astrocytic activation could be detected (Fig. 4D). While the
mean immunofluorescence density of the GFAP signal was
significantly suppressed by ASO on POD 3 and 7, when the
astrocytic activation in the ipsilateral dorsal horn was at a very
high level in the saline control group (Fig. 4C).
L-AA treatment down-regulated SNL-induced astrocytic
and neuronal activations in different manners
Intrathecal injection of an astrocytic toxin, L-AA, produced
analgesic effect on SNL-induced mechanical allodynia. As
demonstrated above, this effect was significant during the late
stage of the mechanic allodynia. Such behavioral change was
Figure 2. Immunofluorescent staining for Fos and GFAP in the spinal dorsal horn after SNL. A–A’, SNL induced a significant up-
regulation of Fos expression in the ipsilateral dorsal horn 2 h after SNL. B–B’, GFAP immunoreactive astrocytes were significantly activated 1 w after
nerve injury in the ipsilateral compared to the contralateral dorsal horn. Scale bar=200 mm.
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accompanied with relevant attenuating effect on the SNL-
enhanced GFAP expression. Statistical analysis revealed a
significant effect of treatment as well as POD on the SNL-induced
GFAP expression [Fig. 5D; two way ANOVA (between subjects
factors: Group and Day): Group: F (1, 24)=94.917, P=0.000;
Day:F (3, 24)=157.417,
24)=28.865, P=0.000]. L-AA treatment rendered inhibitory
effect on the enhanced-GFAP on POD 3 and 7 compared to the
saline control). Our changing course study of GFAP expression in
the ipsilateral superficial dorsal horn of Saline and L-AA groups
indicated that there were no obvious differences at 2 h and POD 1
when no obvious astrocytic activation could be detected (Fig. 5D).
The effect of L-AA appeared to be astrocytic specific, because it
only reduced the number of GFAP-expressing astrocytes, but not
that of NeuN-expressing neurons (Fig. 5B).
P=0.000;Group*Day: F(3,
To our surprise, although the total number of NeuN-positive
neurons did not change under either SNL or SNL + L-AA
treatment, the activation of neurons was suppressed by L-AA
treatment, possibly via an indirect way other than L-AA’s direct
impairment to neurons. Statistical analysis revealed a significant
effect of treatment as well as POD on the SNL induced SDH Fos
expression [Fig. 5D; two way ANOVA (between subjects factors:
Group and Day): Group: F (1, 24)=5.284, P=0.035; Day: F (3,
24)=35.764, P=0.000; Group*Day: F (3, 24)=1.359, P=0.291].
L-AA treatment rendered inhibitory effect on the enhanced Fos
expression on POD 1 and 3. Studies on Fos expression in the
ipsilateral superficial dorsal horn of saline controls and L-AA
groups indicated that there were no obvious differences at 2 h
when significant astrocytic activation could not be detected
(Fig. 5D). The Fos expression of the L-AA group dropped to the
Figure 3. Double immunofluorescence for Fos (red)/NeuN (green) and Fos (red)/GFAP (green) in the ipsilateral dorsal horn 3 d after
SNL. A’ and B’ are the magnified images of the rectangles indicated in the above top panels in A and B, respectively. Double immunofluorescence
indicated that few GFAP-IR astrocytes were Fos positive (shown by double arrowheads in B’), while most of them were nuclei of activated neurons
(shown by double arrowheads in A’). These activated neurons and astrocytes were mainly distributed in the superficial layer of dorsal horn, and the
Fos-positive neurons were surrounded with many GFAP-positive astrocytic processes formed close relationship with each other. The figures shows
representative photomicrographs obtained from a minimum of three independent experiments. Fos and NeuN double labeled neurons observed in
the ipsilateral superficial dorsal horn were counted and the proportion of double labeling neurons in total Fos positive cells were calculated. The
statistical results of these comparisons are presented in C. Scale bar=200 mm in A, B and 80 mm in A’ and B’.
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normal level on POD 3, while this could only be observed on POD
7 in saline control animals. These data indicated that this astroglial
toxin did not change the peak value but shorten the duration of
neuronal activation (Fig. 5C, D).
In the sham-operated animals, neuronal and astrocytic
activation was not influenced compared with that of naı ¨ve rats.
Furthermore, intrathecal injection of c-fos ASO, SO or L-AA on
sham-operated rats did not change the expression of Fos and
GFAP (data not shown).
Discussion
In this study, we examined the relationship between nerve
injury-induced mechanical allodynia, neuronal and astrocytic
activation, and there interactions. Our findings established that:
(1) Inhibiting either neuronal or astrocytic activity prevented SNL
induced neuropathic pain; (2) Astrocytic activation is downstream
to the activation of neurons; (3) Astrocytic activation can enhance
neuronal activation. Concluding from the above results, we
hypothesize that there is a positive feedback circuit for neuronal-
glial interaction under neuropathic pain states.
Neuronal and astrocytic activation emerges mainly in
induction and maintenance of allodynia, respectively
Allodynia is one of the clinically common symptoms which
affect the patients’ life quality severely. It is usually described in
two different stages: induction (or initiation, usually occurs within
3 d after nerve injury in animal models) and maintenance (or
duration, usually occurs later than 3 d post nerve injury).
Figure4.TheexpressionsofFosandGFAP intheipsilateraldorsalhorninsaline,c-fosSOandASOgroupsafterSNL.Comparedtosaline
or c-fos SO treated group, c-fos ASO produced a significantinhibitory effect on Fos protein expression (A–A0), as well as on astrocytic activation(B–B0, C–
C0) in thespinal dorsal horn. D, Statistics of thetime coursestudy.Thebaseline level ofFos andGFAP was setas 100%,respectively. Thechangingcourse
of Fos (red curves) or GFAP (green curves) was determined with comparison to the relative baseline value. The relative increase in the number of Fos-
positive neurons reached a peak 2 h in the SO and saline group, then fell back quickly to the normal level in 1 w. However, Fos expression was
maintained at the baseline level by ASO treatment. The GFAP expression presented a different changing course tendency: increasing since day 3 and
keepingahighlevel 1 wafterSNL.Interestingly,theincreaseofGFAP expression wasinterferedwithbyASO treatment onday3 and7.#,P,0.05and#
#, P,0.01, as compared to the baseline value; *, P,0.05 and **, P,0.01, as compared to the saline control in the same time point. Scale bar=200 mm.
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