OVERVIEW
Role of Astrocytes in Pain
C.-Y. Chiang • B. J. Sessle • J. O. Dostrovsky
Received: 30 January 2012 / Revised: 18 April 2012 / Accepted: 7 May 2012 / Published online: 26 May 2012
� Springer Science+Business Media, LLC 2012
Abstract Over the last decade, a series of studies has
demonstrated that glia in the central nervous system play
roles in many aspects of neuronal functioning including
pain processing. Peripheral tissue damage or inflammation
initiates signals that alter the function of the glial cells
(microglia and astrocytes in particular), which in turn
release factors that regulate nociceptive neuronal excit-
ability. Like immune cells, these glial cells not only react at
sites of central and/or peripheral nervous system damage
but also exert their action at remote sites from the focus of
injury or disease. As well as extensive evidence of mi-
croglial involvement in various pain states, there is also
documentation that astrocytes are involved, sometimes
seemingly playing a more dominant role than microglia.
The interactions between astrocytes, microglia and neurons
are now recognized as fundamental mechanisms
underlying acute and chronic pain states. This review
focuses on recent advances in understanding of the role of
astrocytes in pain states.
Keywords Astrocyte � Intracellular calcium �Gliotransmitter � Chemokine � Inflammatory pain �Neuropathic pain
Introduction
It has been clearly demonstrated that glia in the central
nervous system play roles in many aspects of neuronal
functioning including pain processing. Peripheral tissue
damage or inflammation initiates signals that alter the
function of the glial cells (microglia and astrocytes in
particular), causing the release of factors that regulate
nociceptive neuronal excitability. The aim of this article is
to focus on the recent advances in the role of astrocytes in
pain at the spinal cord and medullary levels.
Overview of Pain Research
Starting from the 1920s, electrophysiological studies have
revealed that peripheral nerve fibers are composed of fast
conducting thick myelinated Aa and Ab fibers, and slow
conducting thin myelinated Ac and Ad and non-myelinated
C fibers; normally, many of these Ad and C fibers are
involved in pain and aversive reactions. Until fairly
recently, research focused on the normal functional char-
acteristics of the nociceptors in the peripheral nervous
system and the nociceptive neurons in the central nervous
system (CNS). Nociceptive neurons are found primarily in
the superficial laminae I–II and deep laminae V–VI of the
Special Issue: Leif Hertz/overview.
C.-Y. Chiang (&) � B. J. Sessle � J. O. Dostrovsky
Department of Oral Physiology, Faculty of Dentistry,
University of Toronto, 124 Edward Street,
Toronto, ON M5G 1G6, Canada
e-mail: [email protected]
B. J. Sessle
e-mail: [email protected]
J. O. Dostrovsky
e-mail: [email protected]
B. J. Sessle � J. O. Dostrovsky
Department of Physiology, Faculty of Medicine,
University of Toronto, 1 King’s Circle,
MSB, Toronto, ON M5S 1A8, Canada
123
Neurochem Res (2012) 37:2419–2431
DOI 10.1007/s11064-012-0801-6
spinal and medullary dorsal horns, and also in some regions
of the thalamus and cortex. In addition, there have been
many studies of the endogenous analgesic descending
system, which modulates nociceptive inputs particularly at
the spinal and medullary levels [1].
During the 1960–1980s, the discovery in the brain of
specific receptors of amino acids (GABA, glutamate, etc.)
and peptides/proteins (opioid peptides, neurotrophic fac-
tors, kinases, etc.) has tremendously enriched and pro-
moted research into mechanisms underlying various pain
states. Following tissue damage and the activation of
nociceptive afferent endings in the tissue, the local com-
plex process of inflammation which is associated with
release of serotonin, bradykinin, prostaglandins, growth
factors and cytokines etc. can also lead to the increased
responsiveness of the peripheral nociceptor endings; this
sensitization of nociceptive afferent fiber endings (periph-
eral sensitization) is presumed to be partially responsible
for the increased pain sensitivity (primary hyperalgesia) in
injured tissue [2]. Congruent with this peripheral process, a
prolonged nociceptive neuronal hypersensitivity in associ-
ation with an increased synaptic activity in the CNS (e.g.,
the spinal and medullary dorsal horns) has been docu-
mented as increases in spontaneous activity, mechanore-
ceptive field size and responses to mechanical and thermal
stimuli and decreases in activation threshold that are
coherent with the development of hyperalgesia (an exag-
gerated response to noxious stimulation) and allodynia
(pain produced by a normally non-noxious stimulus, e.g.,
touch) [3–5]. This phenomenon of central neuronal
hypersensitivity is termed ‘‘central sensitization’’ and is
now widely accepted as an essential mechanism underlying
various pain states, including acute and chronic inflam-
matory pain and neuropathic pain [6–8].
Despite the remarkable advances in our understanding
of the underlying neuronal mechanisms, most novel drugs
derived from these cellular and molecular investigations
have proven to be of limited clinical effectiveness in the
treatment of pain conditions, suggesting that other impor-
tant factors e.g., neuron-glia (or neuroimmune) interactions
may also be involved [8–14]. Indeed, concomitant with the
studies on neuronal mechanisms, a tremendous develop-
ment of in vitro studies on glia has also been carried out by
means of various neurobiological techniques as well as the
latest in vivo non-invasive methods such as positron
emission tomography, magnetic resonance imaging scan-
ning, magnetic resonance spectroscopy etc. Astrocytes (as
well as microglia and the oligodendrocytes, which are not
included in this review) have been comprehensively
investigated; their functions cover a wide range of
dynamic structure–function characteristics: water and ion
balance, metabolic specialization and cerebral oxidative
metabolism, cell–cell communication, and roles of astro-
cytes in various diseases. The prominent features of
astrocytes relevant to neurotransmission and global brain
function have been recently reviewed in depth [10, 11, 15–
23]; in particular, the studies of Hertz et al. [24, 25] have
played a prominent role in elucidating many of the meta-
bolic and physiological processes of astrocytes. Some
features of astrocytes are briefly summarized in Box 1.
In the early 1990s, pioneering work [26, 27] demon-
strated that the NMDA receptor antagonist MK801 could
block sciatic chronic constriction injury-induced hyperal-
gesia and increased glial fibrillary acidic protein (GFAP)
expression (which is used as a marker of enhanced
astrocytic activity, although the function of GFAP
remains unclear in relation to the activity state of astro-
cytes). In addition, it was shown that intraplantar zymo-
san-induced thermal and mechanical hyperalgesia and
mechanical allodynia were blocked by fluorocitrate, a
selective inhibitor of aconitase which is an enzyme in the
astrocytic Krebs cycle [28, 29]. These findings revealed a
predictive relationship between glial activation and
exaggerated pain states. Subsequently, this analgesic
effect of fluorocitrate (or fluoroacetate) has been repeat-
edly verified in various experimental pain models of acute
and chronic inflammatory pain as well as neuropathic pain
(see below), and was followed in the last decade by the
discovery of a remarkable series of complicated mecha-
nisms related to neuron-glial interactions involved in pain
processing [8, 10, 13, 30–39]. The remainder of this
article will especially focus on recent advances in the role
of astrocytes in pain at the spinal and medullary dorsal
horns, leaving those of satellite glial cells (the astrocyte-
like cells in the spinal dorsal root/trigeminal ganglion) to
other specific reviews [40–43].
To explore the role of astrocytes in pain, two strategies
have been usually adopted: (1) most studies have used
conventional approaches by which the inflammatory and/or
injury-evoked increased afferent impulses initiate signals
that alter the function of glial cells in the peripheral or
central nervous system, and when recruited, these glial
cells in turn modulate neuronal function. By means of
multiple technologies including electrophysiological, neu-
rochemical, immunocytochemical, optical recording toge-
ther with Ca2? sensitive dyes, and behavioral tests
performed in wild and gene-knockout rodents, these studies
have systematically investigated the individual changes in
astrocytes, microglia, neurons, transmitters, mediators, and
their interactions in different pain states. (2) A few studies
have used specific chemicals to interfere with or block one
of the essential functions of astrocytes (but not microglia or
neurons) to demonstrate their involvement in different pain
states.
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Glia-Neuron Interactions in Pain States
Role of Astrocytes in Different Pain States and Their
Activation and Time Courses Relative to Microglia
Although the features underlying the processes and time
courses of astrocytic and microglial activation in inflam-
matory and neuropathic pain are variable and dependent on
the different experimental pain models [44], a general
scheme still can be outlined.
1. Acute inflammatory pain: Following subcutaneous
injection of the inflammatory irritant formalin, first
GFAP-immunoreactive astrocytes are detected in the
spinal dorsal horn, then OX42-immunoreactive
microglia are observed a few minutes later, and Fos/
NeuN-immunoreactive (neuron marker) neurons are
found slightly later [45]. Similarly, a prompt (within
15 min) onset of central sensitization in the medullary
dorsal horn induced by application of the inflammatory
irritant mustard oil to the tooth pulp can be blocked by
pre-emptive intrathecal superfusion of the astrocytic
aconitase inhibitor fluoroacetate or the NMDA recep-
tor antagonist MK-801 [46, 47]. Following paw
incision, GFAP expression in laminae I-II and ionized
calcium binding adaptor protein (Iba-1; microglia
marker) in deep laminae of the spinal lumbar 5
segment are increased on day 1, and then in the entire
dorsal horn by day 4, and dissipate by day 7 in parallel
with the accompanying allodynia [48].
2. Chronic inflammatory pain: In the acute phase
0.5–24 h after injection of Complete Freund’s Adju-
vant (an immunopotentiator) into a jaw muscle (mas-
seter), significant increases occur in NMDA receptor
NR1 serine 896 phosphorylation and GFAP levels in
the medullary dorsal horn (also known as trigeminal
subnucleus caudalis) and its junctional region with the
more rostral subnucleus interpolaris (the interpolaris/
caudalis transition zone) which is an important struc-
ture for processing orofacial deep as well as cutaneous
nociceptive inputs; local injection of fluorocitrate or
the microglial inhibitor minocycline at day 1 into the
transition zone significantly attenuates the masseter
hyperalgesia that occurs bilaterally in this pain model
[49]. Also, following Complete Freund’s Adjuvant-
induced peripheral inflammation, a significant increase
in microglial markers (macrophage antigen complex-1,
Box 1 Functional features of astrocytes
1. The membrane of astrocytes expresses a variety of neurotransmitter receptors including N-methyl D-aspartate (NMDA) and non-NMDA,
metabotropic glutamatergic, neurotrophic tyrosine kinase, neurokinin-1, purinergic, adrenergic, cytokine, and aquaporin receptors. Most
metabotropic receptors when activated may induce the phospholipase-dependent accumulation of inositol 1, 4, 5-triphosphate that
stimulates the release of Ca2? from intracellular inositol 1, 4, 5-triphosphate-sensitive internal stores [16, 118, 142]
2. Glutamate and adenosine-50-triphosphate (ATP) can readily elicit astroglial transients and oscillatory waves of cytoplasmic Ca2?, which
may propagate to adjoining and/or distant astrocytes and neurons through two pathways: (1) the cytoplasmic diffusion of Ca2?-mobilizing
second messengers inositol 1, 4, 5- triphosphate through the gap junction channels between astroglia, and (2) extracellular diffusion of ATP
that is released through astrocytic connexins (Cx) e.g., Cx-43 and pannexin-1 hemichannels and/or vesicular exocytosis and then acts on
nearby glia and neurons to modulate neurotransmission [15, 30, 119, 120, 143]. A disruption of astrocytic networks (Cx-30-/- and Cx-
43-/-) causes combined pre- and post-synaptic alterations including enhanced neuronal excitability, release probability and insertion of
postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [62]
3. In exaggerated pain states reflected in hyperalgesia and allodynia, the activated astrocytes (or properly termed ‘‘reactive astrocytes’’) may
release gliotransmitters such as glutamate, ATP, D-serine, or neurotrophic factors, cytokines (e.g., interleukin (IL)-1beta) and chemokine
chemoattractant ligands (CCL)-2. Notably, glutamate is a potent excitatory neurotransmitter, and ATP through its metabolic product
adenosine indirectly inhibits the function of local cells i.e., neurons and glial cells [16, 18, 22, 30–34, 144–148]
4. Astrocytes readily uptake K? in the extracellular space through Na?-K?-Cl- cotransporters (NKCC). They also regulate NKCC in cerebral
microvessel endothelial cells by secreting interleukin-6, thus participating in the maintenance of cerebral ionic homeostasis across the
blood- brain barrier [149, 150]. Astrocytes also efficiently uptake extrasynaptic glutamate by Na?-dependent electrogenic uptake systems.
An increase in the intracellular concentration of Na? stimulates glycolysis in astrocytes i.e., glucose utilization and lactate production
mediated by an activation of Na?/K? ATPase [25, 151]
5. Glutamate, while being an important excitatory neurotransmitter, is also an important metabolic agent in the astrocytes where it stimulates
glycolysis, i.e., glucose utilization and lactate production which are then degraded to carbon dioxide and water. Of the extrasynaptic
glutamate uptaken by astrocytes, about one-third enters the tricarboxylic acid cycle (Krebs cycle) to generate de novo glutamate, and two-
thirds transform to glutamine (a glutamate precursor) which is released into the extracellular space and then is taken up by glutamatergic
presynaptic terminals to replenish the glutamate pool for neurotransmission. This process is termed the ‘‘glutamate-glutamine shuttle (or
cycle)’’ [24]
6. Astrocytes bidirectionally exchange information with the pre- and post-synaptic neuronal elements, responding to synaptic activity and, in
turn, regulating synaptic transmission (thereby also being an integral part of the ‘‘tripartite synapse’’) and their ramified end-feet closely
contact local vessels [152]. Such a well-organized complex structure can modulate uptake of K?, uptake and release of glucose-derived
transmitters (glutamate and GABA) and various neuroactive molecules to monitor nearby neuronal excitability as well as glial functions and
particularly, to play a critical role in coupling blood flow to meet functional demands of neural activity [16, 17, 21, 22]
Neurochem Res (2012) 37:2419–2431 2421
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OX-42 and CD14) as well as an enhanced expression
of proinflammatory cytokines such as IL-1b, IL-6 and
tumor necrosis factor (TNFa) occurs in the spinal cord,
brainstem and forebrain during all phases (acute,
subacute and chronic) of inflammation, whereas up-
regulation of astroglial markers (GFAP and S100b, a
calcium-binding peptide) is observed only at the
subacute and chronic phases of inflammation, thus
indicating that microglial activation precedes astro-
cytic activation [50]. Consistently, an early expression
of phosphorylated p38 mitogen-activated protein
kinase (MAPK) in the microglia and late induction
of phosphorylated nuclear factor (NF)jB in astrocytes
in the medullary dorsal horn both play an important
role in trigeminal neuropathic pain resulting from mal-
positioned dental implants [51]; both p38 MAPK and
phosphorylated extracellular signal-regulated protein
kinase (ERK) (but not c-Jun N-terminal kinase, JNK)
are mostly up-regulated and co-localized with neurons
and microglia, but rarely with astrocytes, during the
3–10 days following occlusal interference [52 and
personal communication]. Also, GFAP co-localized
with glutamine synthetase antibody-immunoreactive
astrocytes is significantly increased for 7–14 days after
pulp exposure in a chronic dental pulpitis pain model
[53].
3. Neuropathic pain: In a neuropathic pain model
involving lumbar 5 spinal nerve transection in adult
rats, the spinal S100b mRNA and protein are steadily
increased from day 4 to 28, implicating the late
involvement of astrocytes [54]. In the same pain
model, mechanical allodynia is induced from day 1 to
7 which correlates with Iba-1 increases in the spinal
lumbar 5 segment, whereas GFAP increases from day
4 to 7, thus suggesting that astrocytes (as reflected in
GFAP expression) play a role in the maintenance of
chronic pain while microglia activation (reflected in
Iba-1 expression) closely correlates with the early
phase in this neuropathic pain model [48]. Also, in rats
with injury to the inferior alveolar nerve, GFAP-
labelled astrocytes and nociceptive neuronal excitabil-
ity reflecting central sensitization increases in the
ipsilateral medullary dorsal horn and is associated with
nociceptive behaviour at postoperative day 7; all these
changes can be prevented following intrathecal fluo-
roacetate administration [55]. Similarly, in rats with
upper cervical nerve injury, GFAP-labelled astrocytes
(most also showed glutamine synthetase immunoreac-
tivity) and ERK-immunoreactive neurons occur on day
7 in superficial laminae of both medullary and upper
cervical dorsal horns in association with behavioral
allodynia and heat hyperalgesia that can be prevented
by post-operative administration of fluoroacetate or the
MEK1/2 inhibitor PD98059 [56]. Interestingly, this
spinal/medullary microglial and astrocytic response
profile after nerve injury may vary either during
development of animals [57] or depending on the
lesion site (e.g., sciatic nerve versus infraorbital nerve
chronic constriction injury) in adult animals [58].
Clarifying the temporal profiles of microglial and
astrocytic activation in various pain models will be
helpful in determining the optimal time for drug
administration to counteract the glial changes contrib-
uting to the pain states.
Another important finding is that none of the glial
modulatory drugs affected normal pain processing, indi-
cating that glial activation may be only involved in path-
ological pain states [35, 43]. This leads to the next
consideration of whether direct activation of glia can
generate nociceptive neuronal hypersensitivity associated
with mechanical and thermal hyperalgesia and tactile
allodynia.
Direct Activation of Astrocytes in the Dorsal Horn
by i.t Exogenous Chemicals
Given such a complicated neuron-glia interaction and that
both elements share most of the same receptors in the
spinal and medullary dorsal horns, it is challenging to
selectively target a set of astrocytes or microglia in in vivo
conditions. One approach was provided by Watkins’ group
who showed that i.t. application of an immune stimulus
(HIV envelope glycoprotein, gp120) produces robust
thermal hyperalgesia and mechanical allodynia which are
blocked by spinal pretreatment with fluorocitrate and CNI-
1493; the latter compound is thought to preferentially
disrupt the function of microglia and astrocytes, because
they possess CXCR4 or CCR5 chemokine receptors which
can bind to gp120 and then initiate a cascade of mediators
and release of proinflammatory cytokines, nitric oxide and
prostaglandins to activate neurons as well as glial cells
[59].
Regarding astrocyte activation, a recent study has
demonstrated astrocyte-to-neuron responses in rat spinal
cord slices mediated by gliotransmitters (glutamate), i.e., in
experiments involving patch-clamp and confocal micro-
scopic calcium imaging techniques, application of ben-
zoylbenzoyl ATP (a potent P2X7 receptor agonist) was
shown to trigger Ca2? elevations in astrocytes through
purinergic receptors and induce slow inward currents in
lamina II neurons which were blocked by d-AP5 (amino-5-
phosphonovaleric acid, a competitive NMDA receptor
antagonist) [60]. These findings suggest that astrocyte-
released glutamate evokes NMDA receptor-mediated epi-
sodes of synchronous activity in groups of superficial
2422 Neurochem Res (2012) 37:2419–2431
123
laminae I–II neurons. In accordance with these findings,
the development of thermal hyperalgesia and mechanical
allodynia in a model of inflammation produced by in-
traplantar zymosan is also accompanied by a significant
increase of spontaneous slow inward currents in dorsal
horn neurons. It thus appears that NMDA receptor-medi-
ated astrocyte-to-neuron signaling may contribute to the
control of central sensitization in pathological pain states
[60]. Another study showed that spinal intrathecal injection
of astrocytes, which had been briefly (15 min) pretreated
by TNFa, induced a substantial decrease in paw withdrawal
thresholds, indicating the development of mechanical
allodynia. This allodynia was prevented when the astrocyte
cultures had been pre-treated with a JNK inhibitor or by a
MCP-1 (also termed chemotactic cytokine ligand 2
[CCL2]) neutralizing antibody. Finally, pretreatment of
astrocytes with MCP-1 siRNA attenuated astrocyte-
induced mechanical allodynia [61].
These findings demonstrate that direct activation of
either astrocytes or microglia not only produces exagger-
ated pain states including neuronal hypersensitivity asso-
ciated with behavioral hyperalgesia and allodynia, but also
suggest that they are involved in a particular phase of the
pathological pain process [48, 54]. Thus, astrocytes may
play a role in the initiation of acute inflammatory pain [45–
47, 60] and the maintenance of chronic inflammatory and
neuropathic pain [48, 50, 51, 53–56, 61], and microglia are
involved in early or all phases of pathological pain [48, 50,
51, 59]. However, these findings are not conclusive and
further stringent validation is still needed in additional
experiments such as using antisense, transgenic or gene-
knocked animals as previously reported [61–67]. For
instance, a recent study showed that liposaccharide-
induced hypersensitivity thought to be mediated by toll-
like receptors can be observed in wild-type but not P2X7
knock-out mice [68], indicating that P2X7 receptors are
essential elements for immune antigen-induced neuronal
hypersensitivity.
Signaling Pathways Underlying Inflammatory
and Neuropathic Pain States
Peripheral inflammation and nerve injury activate several
signaling pathways (e.g., protein kinases A and C, calcium/
calmodulin-dependent protein kinase) in primary sensory
and dorsal horn neurons that then activate MAPKs
including p38 MAPK, ERK, and JNK in spinal and med-
ullary dorsal horn neurons and microglia or astrocytes or
both, leading to the glial production of pro-inflammatory
mediators (e.g., IL-1b, TNFa) that sensitize dorsal horn
neurons as well as induce behavioral hyperalgesia and
allodynia [32, 56, 69]. For instance, after nerve injury the
activation of NR2B-containing NMDA receptor-mediated
astrocytic JNK activation in the spinal dorsal horn releases
IL-1b which involves positive feedback mechanisms to
enhance and prolong neuropathic pain [70]. Also, local
injection of Complete Freund’s Adjuvant into the masseter
muscle caused an upregulation of GFAP expression
(astrocytes), IL-1b and phosphorylation of serine 896 of
the NR1 subunit of the NMDA receptor in the trigeminal
interpolaris/caudalis transition zone which is an important
structure for processing orofacial deep as well as cutaneous
nociceptive inputs; these changes can be significantly
reduced by pre-emptive local injection of lidocaine into the
masseter muscle [71]. A recent study has demonstrated that
Janus kinase-signal transducers and activators of the tran-
scription 3 signaling pathway are critical transducers of
astrocyte proliferation (a critical process in reactive as-
trogliosis) and are involved in the maintenance of tactile
allodynia [72].
The proinflammatory cytokines IL-1b, IL-6 and TNFaand reactive oxygen species play a central role in the
pathogenesis of various pain states. Activated astrocytes
can release pro-inflammatory cytokines (e.g., IL-1b),
which powerfully modulate synaptic transmission in the
spinal cord by enhancing excitatory synaptic transmission
via phosphorylation of the NMDA receptor NR-1 subunit
[73] and suppress inhibitory synaptic transmission [74].
Although IL-1b may also be expressed in microglia as well
as astrocytes in some brain areas, this significant role of
astrocytic IL-1b in the spinal cord is supported by a recent
report that an up-regulation of IL-1b occurs in astrocytes in
the medullary dorsal horn by 30 min after Complete Fre-
und’s Adjuvant-induced inflammation in the masseter
muscle [75] and IL-1b up-regulation has been also
observed in the medullary dorsal horn in animals with
hyperalgesia induced by mental nerve transection [76].
Cytokines including IL-1b also exert their actions partially
through the activation of the transcription factor NFjB in
astrocytes, which in turn regulates the transcription of
many inflammatory mediators (including cytokines and
chemokines) thus causing a positive feedback loop [77]. A
recent study using transgenic mice has reported that a
functional inactivation of the NFjB pathway in GFAP-
labelled cells in the spinal cord leads to a reduction in pain
behavior and inflammation produced by sciatic nerve
chronic constriction injury [78].
A short exposure of astrocyte cultures to pro-inflam-
matory TNFa dramatically increases the expression and
release of MCP-1 in a JNK-dependent manner, and
accordingly, i.t. administration of TNFa induces MCP-1
expression in spinal dorsal horn astrocytes in association
with behavioral allodynia that can be reversed by a MCP-1
neutralizing antibody [61]. TNFa enhances spontaneous
release of neurotransmitters from primary afferent fibers by
modulation of tetrodotoxin-sensitive sodium channels
Neurochem Res (2012) 37:2419–2431 2423
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following sciatic nerve transection [79] and also increases
sEPSC frequency in spinal outer lamina II neurons, an
effect that may involve TRPV1 activation since it can be
abolished in TRPV1 knock-out mice [80]. TNFa is mainly
formed in and released from microglia mediated by phos-
phorylation of p38 MAPK following activation of toll-like
receptors [81] and/or P2X7 and P2Y12 receptors in
microglia upon tissue inflammation and/or nerve injury
[33, 82–85]. Our recent study has also demonstrated that
P2X7 receptor activation is also involved in the central
sensitization in the medullary dorsal horn produced by
mustard oil application to the tooth pulp [86]. In addition,
in primary cultures of spinal cord astrocytes, application of
ATP through its action on astrocytic P2Y1 receptors can
mobilize a release of arachidonic acid and prostaglandin
E2. Inhibition of this prostaglandin E2 release can reduce
behavioral signs of pain after spinal cord injury [87].
Recent studies have particularly emphasized the impor-
tance of chemokines in inflammatory and neuropathic pain
states. The chemokine MCP-1 has been implicated in neu-
ron- and astrocyte-to-microglia signaling. MCP-1 is
released from the central terminals of nociceptors after
peripheral injury. Whereas the activation of microglia sur-
rounding MCP-1-expressing spinal cord dorsal horn neu-
rons peaks by day 14 after a sciatic nerve chronic
constriction injury, astrocyte activation becomes detectable
later, progresses more slowly and also remains increased
until the end of the observation period (150 days) [88],
suggesting that astrocyte activation plays an important role
in maintaining persistent pain states. Recent evidence also
indicates that nerve injury and inflammation activate the
JNK in spinal astrocytes, leading to a substantial increase in
the expression and release of pro-inflammatory chemokines
(e.g., MCP-1) in the spinal dorsal horn which enhance and
prolong persistent pain states. For example, MCP-1 rapidly
induces central sensitization by increasing the activity of
NMDA receptors in dorsal horn neurons [for reviews see
10, 33] and enables T-leukocytes and macrophage migra-
tion into the parenchyma of the CNS, which propagates the
immune response and further induces microglial and
astrocytic immuno-competence [11]. Furthermore, the
matrix metalloproteinase-9-induced pro-IL-1b cleavage
leads to p38 MAPK activation in microglia during the onset
and early stages of neuropathic pain, whereas matrix
metalloproteinase-2-induced pro-IL-1b cleavage leads to
astrocyte activation during the ongoing and later stages of
neuropathic pain [89]. Therefore, spinal and medullary i.t.
administration of inhibitors of the IL-1b, TNFa, JNK,
MCP-1, matrix metalloproteinase-2 or NFjB signaling can
each attenuate processes related to inflammatory, neuro-
pathic pain [90, for more references, see 33].
Microglial activation utilizes two signaling pathways
that also involve astrocytic processes [36]: (1) Through
their pattern recognition receptors, such as toll-like recep-
tors, microglia can detect and differentiate viral, bacterial
and fungal structures and others. Activation of toll-like
receptor-4 in microglia initiates immune-like processes,
such as release of pro-inflammatory cytokines and che-
mokines [32, 33, 48, 91], leading to a positive excitatory
feedback loop in the pain processes and a decrease in
opioid analgesic efficacy as well as phagocytosis and the
release of anti-inflammatory factors e.g., IL-10 [8, 10, 12,
14]. Importantly, selective acute antagonism of the toll-like
receptor-4 results in reversal of neuropathic pain as well as
potentiation of opioid analgesia [14, 92]. Like microglia,
activation of astrocytes also leads to an inflammatory
response producing diverse inflammatory mediators
(TNFa, IL-1b, prostaglandins, NO, and reactive oxygen
species), all of which serve immune surveillance functions.
For instance, hours after lipopolysaccharide application to
astrocytic cultures, a prompt increase in CDK11p58
expression in astrocytes promotes the astrocyte-induced
inflammatory response via p38 MAPK and JNK activation
[93]. (2) Microglia-to-neuron or to-astrocyte interactions
through their constitutive chemokine signaling pathways,
i.e., the ligand-receptor pairs CD200-CD200R, and
CX3CL1 (also termed fractalkine)-CX3CR1 normally
promote a ‘‘calming’’ environment. Any disrupted signal-
ing (‘off’ signaling) due to impairment of neuronal integ-
rity, as might be produced for example by nerve injury,
causes glial activation. For example, after spinal nerve
injury, but not after peripheral inflammation, the activated
microglia in the spinal dorsal horn release cathepsin S that
enzymatically cleaves neuronal fractalkine (CX3CL1); this
in turn activates CX3CR1 receptors on microglia, leading
to a further release of proinflammatory cytokines from
microglia, thus establishing a positive feedback loop which
boosts chronic pain states [10]. The neuron-glia interaction
mechanisms underlying inflammatory and neuropathic pain
are diagrammatically shown in Fig. 1. Further clarification
of neuronal mechanisms common to both inflammatory
pain and neuropathic pain promises to shed light on the
mechanisms underlying the transition from acute pain to
persistent pain [39].
Glucose Metabolism in Astrocytes
Glucose is used as an energy substrate by both neurons and
astrocytes but can generate the carbon skeleton of gluta-
mate only in astrocytes, not in neurons, because neurons
lack the enzyme pyruvate carboxylase that allows net
synthesis of the oxaloacetate, a link in the tricarboxylic
acid cycle (also termed Krebs cycle) in astrocytes. As
outlined by Hertz and Zielke [24], the newly produced
oxaloacetate condenses with acetyl coenzyme A derived
2424 Neurochem Res (2012) 37:2419–2431
123
from pyruvate, to generate citrate, which is then metabo-
lized in the tricarboxylic acid cycle to form a-ketoglutarate,
a direct precursor of glutamate.
Fluoroacetate and its metabolite fluorocitrate are potent
inhibitors of glial cells especially astrocytes at low doses
[94, 95]. Fluoroacetate combines with acetyl coenzyme A
to form fluoroacetyl coenzyme A, which can substitute for
acetyl coenzyme A in the tricarboxylic acid cycle and
reacts with citrate synthase to produce fluorocitrate, a
metabolite which then binds very tightly to aconitase,
thereby halting the astrocytic tricarboxylic cycle [96].
Intrathecal administration of low dose fluorocitrate can
block persistent thermal and mechanical hyperalgesia
produced by zymosan [29] and by formalin [97], and these
striking blocking effects have been repeatedly confirmed in
various pain models, such as acute inflammatory pain
induced by capsaicin, mustard oil, snake and scorpion
venoms, acute incision surgery, tetanic stimulation of
peripheral nerves and glycine disinhibition-induced allo-
dynia [47, 98–103]; chronic inflammatory pain induced by
carrageenan, Complete Freund’s Adjuvant and traumatic
dental occlusion [44, 49, 89, 104–106]; nerve injury and
A B
C
Acute inflammatory pain
Peak: 0.5-24 h; Duration: few days
D
ERK/p38
ATPCytokines
IL-1β
p38
Glu
Chronic inflammatory pain
BDNF
ERK
Peak: day 3-14; Duration: 21-28 days
Trk BRp38IL-1β
Glu
SP
ATPCGRP
ATPCCL2
Cytokines
Mature IL-1β
Pro-IL-1βTNFα
NF B
p38
CatSFKN
GluSP
ERK
ERK/p38
Neuropathic pain (maintenance phase)
Peak: day 7-14; Duration: months
BDNF
ATP
FKN
PGE2
COX-2iNOS
NO
CGRPBDNF
CCL2
Caspase-1
MMP2
ATPCCL2
Cytokines
Mature IL-1β
Pro-IL-1βTNFp38
FKN
GluSP
JNK
ERK
Neuropathic pain (initiation+induction phase)
Peak: day 0.5-3; Duration: within a week
ATP
CCR2
PGE2
COX-2iNOS
NO
CGRPBDNF
CCL2
Nav1.3
P2Y12R
Caspase-1TLR4
Glu
ATPCytokines
IL-1β
Glu
Ca2+ Ca2+P2X7R
P2X4R
IL-1R
ERKEnhanced inputs
p38
P2X3R
Ca2+
ATP
GluSP
CGRP
Ca2+Gln PGE2, NO
IL-1β
ATCA
GSGluR
Ca2+ PGE2, NO
ERKCX3CR1
FKN
IL-1β
Glu
GDNFNGF
LPS, OpioidMMP9
LPS, Opioid
TNFα
MMP2
MMP9 MMP2
LPS
CDK11
NO
Presynaptic Postsynaptic
Astrocyte Microglia
/ERK /ERK
Fig. 1 Diagrams showing some neuron-glia interactions in acute
inflammatory pain (a), chronic inflammatory pain (b), and in the
initiation/induction phase (c) and maintenance phase (d) of neuro-
pathic pain in the spinal or trigeminal dorsal horn. Different colorsrepresent different pathways of individual mediators; the different
thickness of arrows/fonts represents their importance in individual
cases. For figure clarity, some receptors have not been linked with
appropriate agonists or mediators, and peptidergic (e.g., substance P
[SP], calcitonin gene-related peptide [CGRP]) mechanisms are
omitted in the diagrams. A adenosine, ATP adenosine-50-triphosphate,
BDNF brain-derived neurotrophic factor, Caspase-1 an enzyme that
proteolytically cleaves other proteins, such as pro-IL-1b into mature
IL-1b, CatS lysosomal cysteine protease Cathepsin S, CCL2 chemo-
kine (C–C motif) ligand 2, also known as monocyte chemotactic
protein-1 (MCP-1), CCR2 receptor for CCL2, CDK11 cyclin-
dependent kinase 11, COX-2 cyclooxygenase-2, CX3CR1 receptor
for Fractalkine, Cytokine immunomodulating agents including IL-1b,
IL-6 and TNFa, ERK extracellular signal-regulated kinase, FKNchemokine Fractalkine or neurotactin, GDNF glial cell line-derived
neurotrophic factor, Gln glutamine, Glu glutamate, GS glutamine
synthetase, IL interleukin, IL-1R interleukin-1 receptor, iNOS cyto-
kine-inducible nitric oxide synthase (NOS-2), JNK c-Jun N-terminal
kinase, LPS lipopolysaccharides, MMP matrix metalloproteinase,
Nav1.3 voltage-gated sodium channel, type III a-isoform, NGF nerve
growth factor, NFjB nuclear factor-jB is a transcription factor, NOnitric oxide, P2XR purinergic 2X receptor, P2YR purinergic 2Y
receptor, P38 p38-mitogen activated protein kinase (MAPK), PGE2prostaglandin E2, R receptor, TCA tricarboxylic acid, TLRs toll-like
receptors, TNF tumor necrosis factor, TrkBR neurotrophic tyrosine
kinase B receptor or BDNF/NT-3 growth factor receptor
Neurochem Res (2012) 37:2419–2431 2425
123
neuropathic pain [33, 55, 56, 65, 107] as well as devel-
opment of morphine tolerance [108, for review, see 8].
Nonetheless, it should be noted that fluorocitrate or fluo-
roacetate, at an appropriate dose, only blocks the exag-
gerated pain state reflected in central sensitization i.e.,
hypersensitivity of nociceptive neurons, but not the normal
nociceptive processing in CNS. Intracerebral application of
higher doses of fluorocitrate ([1 nmol) or fluoroacetate
([1 mmol in our lab) can severely affect astrocytic ultra-
structure and glio-neuronal interactions due to accumula-
tion of citrates in the brain that may chelate free calcium
ions [95].
The Astrocyte-Neuron ‘‘Glutamate-Glutamine Shuttle’’
A major function of astrocytes is the uptake of extracellular
glutamate (and GABA) in the synaptic region through
astrocytic transporters such as glutamate transporter-1, and
glutamate aspartate transporter with the cotransport of Na?/
H? and counter transport of K? [109]. The cytosolic gluta-
mate can be transformed by glutamine synthetase to gluta-
mine (an important precursor for glutamate and GABA),
which is released and then trafficked through transporters
into glutamatergic neuronal terminals to replenish the glu-
tamate transmitter pool [110]. This glutamate-glutamine
shuttle (or cycle) as noted by Hertz and Zielke [24], is a
crucial mechanism in nociceptive neuronal central sensiti-
zation and is specific to astrocytes. Therefore, any chemicals
that specifically disrupt transformation from glutamate to
glutamine in the glutamate-glutamine shuttle e.g., methio-
nine sulfoximine, a selective inhibitor of glutamine syn-
thetase, or that specifically block glutamatergic presynaptic
uptake of glutamine e.g., methylamino-isobutyric acid, a
selective inhibitor of the neuronal system A transporter of
glutamine, may affect the availability of glutamate and
consequently the production of central sensitization in acute
and chronic inflammatory pain states [43, 53, 111, 112]. In
particular, this transporter inhibitor-induced attenuation of
trigeminal central sensitization can be readily restored by
application of exogenous glutamine [112]. It is also note-
worthy that i.t. administration of either of these 2 inhibitors
does not affect the normal basal nociceptive processing of
the dorsal horn neurons [43, 46, 111, 112].
There is also evidence that reduction in the expression
and glutamate uptake activity of glutamate transporters
plays a crucial role in both the induction and maintenance
of neuropathic pain following peripheral nerve injury [113]
and in taxol-induced hyperalgesia [114]. Similarly, glial
glutamate transporters in astrocytes are down-regulated in
spinal pathological pain models and up-regulation or
functional enhancement of these transporters prevents
pathological pain [115]. For example, propentofylline
exerts a protective action against post-ischemic damage in
the CNS, because it causes a potent dose-dependent
induction of glutamate transporter-1 mRNA and protein in
astrocytes and decreases MCP-1 release from astrocytes
[116]. Conversely, i.t. injection of threo-beta-benzylox-
yaspartate (a glutamate uptake blocker) can produce
spontaneous nociceptive behavior [117].
Astrocytic Ca21 Wave Propagation and Gap Junctions
Another important feature of astrocytes is that glutamate
and ATP can readily elicit astrocytic transients and oscil-
latory waves of cytoplasmic Ca2?, which may propagate to
adjoining and/or distant astrocytes through gap junctions
and hemichannels; this reactive astrocytic network may
release gliotransmitters (glutamate, ATP, D-serine) to
modulate synaptic transmission. The intercellular Ca2?
wave propagation may involve two pathways: (1) the
cytoplasmic diffusion of Ca2?-mobilizing second messen-
ger inositol 1, 4, 5-triphosphate through the gap junction
channels between astrocytes, and/or (2) extracellular dif-
fusion of ATP that is released through Cx-43 and pann-
exin-1 hemichannels and/or vesicular exocytosis [60, 118–
121]. Gap junctions also occur between neurons and
astrocytes that are formed by proteins Cx-30/Cx-43 and
inactivation of these proteins attenuates hippocampal syn-
aptic transmission [62].
Carbenoxolone is a potent blocker of the hemichannel
protein Cx-43 and plasmalemmal channel protein pannex-
in-1 and can produce analgesia in different behavioral pain
models by inhibiting the coupling between astrocytes or
between astrocytes and neurons [118, 120, 122]. In a for-
malin-induced orofacial pain model, the expression of
many heterotypic Cx-43/Cx-32 (astrocyte/neuron) gap
junctions is up-regulated in the medullary dorsal horn
along with nociceptive behavior, and both are significantly
attenuated by carbenoxolone pretreatment [123]. Also, in
an acute inflammatory pain model produced by mustard oil
application to the tooth pulp, medullary superfusion of
carbenoxolone can completely block central sensitization
induced in the medullary dorsal horn [124], suggesting that
spatio-temporal features of central sensitization, such as the
increased mechanoreceptive field size in particular, may
result from the intercellular Ca2? wave-mediated asyn-
chronous excitation of neurons and non-neuronal cells,
mainly involving astrocytic gap junctions and hemichan-
nels [16, 18, 23, 120]. Interestingly, in sciatic inflammatory
neuropathic or chronic constriction injury pain models, a
low dose of carbenoxolone can reverse ‘mirror image’
(contralateral) mechanical allodynia, while leaving ipsi-
lateral mechanical allodynia unaffected, thus suggesting
that gap junctions may be involved in mediating the ‘mirror
2426 Neurochem Res (2012) 37:2419–2431
123
image’ allodynia [125]. In addition, we have observed that
carbenoxolone can equipotently depress bilateral orofacial
allodynia as well as medullary dorsal horn central sensiti-
zation produced by unilateral injury of the infraorbital
nerve [126]. An earlier study has reported that bilateral
allodynia associated with upregulation of GFAP-labelled
satellite glial cells and TNFa expression bilaterally in the
dorsal root ganglia and dorsal horns occurs in an unilateral
nerve injury pain model [127]. These previous findings
indicate that carbenoxolone blocks the maintenance of
neuropathic pain as well as the development of acute
inflammatory pain.
There are also several other interesting findings relevant
to carbenoxolone and gap junctions: (1) In double gene-
knockout mice, a disruption of astrocytic networks
(Cx-30-/- and Cx-43-/-) causes combined pre- and post-
synaptic alterations including enhanced neuronal excit-
ability, release probability and insertion of postsynaptic
AMPA receptors, suggesting that gap junctions may have
inhibitory influences on neurotransmission in normal con-
ditions [62]; (2) Carbenoxolone completely reverses the
inhibitory effects of cromakalim (a KATP channel opener)
on the hyperalgesia and allodynia after sciatic nerve injury
[128]; (3). Carbenoxolone also potently blocks P2X7
receptors [122]. These observations suggest that carben-
oxolone by blocking gap junctions may exert an excitatory
effect under normal conditions but may depress neuro-
transmission in some pain states but confirmation of this
interpretation requires further investigations.
Astrocyte-Neuron ‘‘Serine Shuttle’’
Allodynia and hyperalgesia rely on activation of the
NMDA receptor, a specific subtype of the ionotropic glu-
tamate receptor, which is composed of a voltage-dependent
ion channel non-selective to cations normally blocked by
magnesium, and other (allosteric, glycine etc.) binding
sites. Astrocytes may regulate NMDA receptor activation
through their specific release of D-serine (a co-agonist of
the NMDA receptor), which acts on the strychnine-insen-
sitive glycine binding site on the NMDA receptor [129].
For instance, D-serine is necessary for the acquisition of
specific pain-related negative emotion involving the rostral
anterior cingulate cortex but is not involved in formalin-
induced acute nociceptive behaviors and electric foot
shock-induced conditioned place avoidance [130]. In an
intrathecally strychnine-induced orofacial allodynia model,
fluorocitrate but not minocycline blocks allodynia as well
as reduces the over-expression of GFAP (but not OX-42)
and c-fos in the superficial layers of the medullary dorsal
horn. Intrathecal D-amino acid oxidase, which selectively
induces degradation of D-serine, blocks the allodynia which
can be restored by intrathecal exogenous D-serine; inter-
estingly, this exogenous D-serine also restores the allodynia
blocked by fluorocitrate [131]. Other studies have provided
evidence that in normal rats, intrathecal D-serine signifi-
cantly enhances the C-fiber (but not Ab-fiber) and Ad-fiber
evoked responses in nociceptive wide dynamic range
neurons in the spinal dorsal horn, and co-administering
D-serine with the glycine binding site antagonist 7-chloro-
kynurenic acid completely blocks the facilitation of
D-serine on C-fiber evoked responses [132]. However, a
recent study showed that a D-amino acid oxidase inhibitor
produces analgesia via blockade of spinal hydrogen per-
oxide production rather than by interacting with spinal
D-serine [133].
Concluding Perspectives
Recent studies provide evidence that by means of Ca2?
signaling and gliotransmitters and/or neuroactive mole-
cules, activated astrocytes are involved in key functions of
the brain under physiological conditions, such as astrocyte-
neuron lactate transport which is required for long-term
memory formation [134] and the control of breathing
through their pH-dependent release of ATP [135]; the
physiological functions of astrocytic Ca2? signaling and
gliotransmitters have been recently reviewed [19, 20, 30,
136]. In the case of pain mechanisms, the importance of
astrocytic Ca2? signaling in normal nociceptive processing
remains unclear. For instance, why does a temporary dis-
ruption of the astrocytic metabolic cycle and/or the gen-
eration of astrocytic [Ca2?]i waves only attenuate the
exaggerated pain component but does not affect basal
nociceptive neuronal responses to noxious stimuli and
animal’s nocifensive behavior? Does the mechanism
underlying the initiation, location, duration and integration
of gliotransmitters of astrocytes that have been demon-
strated in vitro also operate in in vivo states? What factors
cause the discrepant results between wild and gene-
knockout rodents? Additional studies of other astrocytic
mechanisms such as those involving the astrocyte-neuron
lactate transport, ion/water balance [137–139], and neuro-
vascular coupling [17, 140] may provide a clearer under-
standing of the role of astrocytes and other non-neural
processes in acute, chronic inflammatory and neuropathic
pain conditions.
Virtually all pharmacological agents developed to alle-
viate acute or chronic pain have targeted neuronal processes
underlying pain. Since astrocytes as well as microglia can
each modulate the central nociceptive mechanisms in
pathological pain states, they may provide new targets for
the development of novel drugs to control pain. However,
this will be especially challenging since glia originate from
Neurochem Res (2012) 37:2419–2431 2427
123
the immune system which is ubiquitously distributed in the
whole body and participates in many functions. Thus side-
effects of glial inhibitors specifically developed to coun-
teract pain may be difficult to avoid and thus specifically
targeting gene therapy-based approaches may be an
appropriate or even obligatory choice [141].
Acknowledgments This work was supported by the NIH Grant DE-
04786 to B.J.S. and CIHR Grant MOP-82831 to J.O.D.
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