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: zhen.jiang@utoronto.ca

B. J. Sessle

e-mail: barry.sessle@utoronto.ca

J. O. Dostrovsky

e-mail: j.dostrovsky@utoronto.ca

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

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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

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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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