European Journal of Pharmacology 716 (2013) 120–128

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Review

Astrocytes—Multitaskers in chronic pain

Rikke Rie Hansen n, Marzia Malcangio
Wolfson Centre for Age-Related Diseases, King's College London, Hodgkin Building, Guy's Campus, London, SE1 1UL, United Kingdom

art ic l e i nf o a b s t r a c t

Article history: Treatment of chronic pain remains a clinical challenge and sufficient pharmacological management is
Accepted 4 March 2013 difficult to achieve without concurrent adverse drug effects. Recently the concept of chronic pain as a
Available online 22 March 2013 solely neuron-mediated phenomenon has evolved and it is now appreciated that also glial cells are of
Keywords: critical importance in pain generation and modulation. Astrocytes are macroglial cells that have close
Astrocyte structural relationships with neurons; they contact neuronal somata and dendrites and enwrap synapses,
Pain where small astrocytic processes have been shown to be highly motile. This organization allows
c-Jun N-terminal kinase astrocytes to directly influence and coordinate neurons located within their structural domains.
ATP Moreover, astrocytes form astroglial networks and calcium wave propagations can spread through
neighbouring astrocytes. ATP, which is released from astrocytes in response to elevated intracellular
calcium concentrations, can contribute to the central mechanisms in chronic pain via purinergic
receptors. In this review we highlight the structural organization and the functionalities of astrocytes
that allow them to undertake critical roles in pain processing and we stress the possibility that astrocytes
contribute to chronic pain not via a single pathway, but by undertaking various roles depending on the
pain condition.
& 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
2. Defining astrocyte activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3. Structural organization of astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4. Reactive astrocytes in preclinical models of chronic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5. Calcium wave propagation in astrocytic networks and implications for ATP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6. ATP signalling in spinal cord pain processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7. Monitoring astrocytic response: c-Jun N-terminal kinase signalling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
8. Clinical implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
9. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

1. Introduction require activity exclusively within neuronal networks and pain

Pain constitutes a complex sensory and emotional experience
which varies not only with the type and severity of injury, but also
with emotional states, previous experiences and future life expec-
tations (Lumley et al., 2011). Pain was originally considered to
n
Corresponding author. Tel.: þ44 207 848 8154; fax: þ44 207 7848 6816.
E-mail addresses: rikke.hansen@kcl.ac.uk,
marzia.malcangio@kcl.ac.uk (M. Malcangio).
modulation by physiological or psychological processes was
thought to rely mostly on neuron-based control systems (Tawfik
and DeLeo, 2009). However, as a result of extensive investigation
within the last decade these concepts have been challenged and it
is now appreciated that glial cells, including microglia and astro-
cytes, are of critical importance in pain generation and modulation
(Clark and Malcangio, 2012; Milligan and Watkins, 2009; Scholz
and Woolf, 2007; Sofroniew and Vinters, 2010).
In this review we highlight the complex organization of
astrocytes and selected astrocytic functionalities that mediate

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.03.023
 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128
their contribution to chronic pain. We then suggest that astrocytes
contribute to chronic pain not via a single pathway, but by
undertaking various roles depending on the conditions of the
individual pain state.
The experience of pain in its simplest form consists of a
harmful stimulus to peripheral tissues such as skin and joints that
is transduced and transmitted by thin myelinated Aδ-fibres and
unmyelinated C-fibres from the periphery to the superficial
laminae of the dorsal horn of the spinal cord. Here the release of
neurotransmitters from the central terminals of Aδ- and C-fibres
activate second order neurons at the first pain synapse which relay
the nociceptive signal to higher brain centres ultimately resulting
in a coordinated activation of specific central nervous system
(CNS) areas, known as the “Pain Matrix”, and the pain is perceived
(Julius and Basbaum, 2001; Tracey and Mantyh, 2007). The
experience of pain is determined by a balance between pro-
nociceptive and anti-nociceptive inputs, which under normal
physiological conditions allow us to recognise and react to pain.
However, this nociceptive balance can be altered in response to
injury or disease and in the worst case lead to development of
chronic pain where pain persists beyond the initial cause or is
maintained by an underlying chronic condition, for example
osteoarthritis or cancer.
Chronic pain patients comprise a heterogeneous group that
includes patients presenting with different pain syndromes,
including inflammatory, neuropathic, and cancer-induced pain
(Finnerup and Jensen, 2006; Mercadante, 1997; Reid et al., 2012).
Pharmacological management with non-steroidal anti-inflamma-
tory drugs (NSAIDs), opioid-related medications, anticonvulsants
and antidepressants remains the mainstay for alleviating pain, but
these drugs are limited to targeting specific pathological mechan-
isms and often lack sufficient efficacy or are poorly tolerated
(Finnerup et al., 2010; Mercadante and Arcuri, 1998; Reid et al.,
2012). Thus, significant challenges remain in the treatment of
chronic pain. These can in part be met by investigation of novel
contributors to nociceptive signalling and the accompanying
prospect of novel mechanism of action analgesics.
A characteristic feature of chronic pain is central sensitization
which manifests as pain hypersensitivity, including allodynia and
hyperalgesia, and is the result of increased excitability and
synaptic efficacy in the central pain pathways (Sandkühler, 2009;
Woolf, 2011). In the spinal cord the first pain synapse, formed by
the central terminals of primary afferent sensory neurons and the
dorsal horn neurons, undergoes plastic and neurochemical
changes triggered by the nociceptive afferent inputs. Besides
neurons, glial cells contribute to the generation and maintenance
of central sensitization (Basbaum et al., 2009; McMahon and
Malcangio, 2009; Tawfik and DeLeo, 2009). Specifically, damage
to peripheral nerves and tissues can lead to an altered state of glial
cells. Microglia switch from a surveillant state to an enhanced
response state (McMahon and Malcangio, 2009) and astrocytes
respond to the peripheral damage by transition into a reactive
state (Garrison et al., 1991; Svensson and Brodin, 2010; Watkins
et al., 2001). Much effort has been given to investigate the role of
microglia in chronic pain mechanisms, while astrocytes only more
recently have attracted intense attention.
Astrocytes originate from neuroepithelial stem cells which also
give rise to other glial cells, namely oligodendrocytes and poly-
dendrocytes, as well as neurons. Astrocytes are the most abundant
cell type in the central nervous system (Aldskogius and Kozlova,
1998), where they form close connections to neurons and capil-
laries. They are innate immune neuroglia involved in the regula-
tion of almost all aspects of neuronal functioning, including
extracellular ion homeostasis, neurotransmitter reuptake and
release, control of the blood–brain–barrier, control of synaptic
strength, physical compliance, and metabolic regulation (Butt,
121
2011; Kimelberg, 2010). Given this multifunctional role it seems
reasonable that alterations in any of these functions may lead to
perturbation of CNS functioning, including perturbation of noci-
ceptive neurotransmission.
The aim of this review is not to outline the astrocyte-related
mechanisms suggested to be involved in chronic pain which have
been covered in recent reviews (Gao and Ji, 2010; Milligan and
Watkins, 2009; Svensson and Brodin, 2010). Rather the aim is to
illustrate the complexity of astrocytes which allows them to
undertake roles in nociceptive signalling.
2. Defining astrocyte activation
Astrocytes are active cells that under normal conditions under-
take supportive roles for neurons and are important for the control
of neuronal signalling (Kimelberg, 2010; Svensson and Brodin,
2010). Astrocytes respond to peripheral and CNS tissue and nerve
damage and undergo changes to a reactive state observed by an
alteration in morphology characterized by hypertrophy and
increased expression of glial fibrillary acidic protein (GFAP)
(Garrison et al., 1991; Svensson and Brodin, 2010; Watkins et al.,
2001). Reactive astrocytes are believed to be active players in the
nociceptive signalling leading to abnormal pain perception (Gao
and Ji, 2010; Ren and Dubner, 2008).
In light of the many physiological mechanisms undertaken by
the astrocyte the role of the reactive astrocyte could be equally
manifold (Kimelberg, 2010). Astrocytes respond to a wide range of
neurotransmitters and release a wide range of mediators, includ-
ing neurotransmitters such as glutamate and ATP, and neuromo-
dulators such as cytokines, chemokines and growth factors (Coco
et al., 2003; Lee et al., 2011; Mothet et al., 2005; Parpura et al.,
1994). So, as is the case for microglia, different stimuli might
engage different astrocytic receptors leading to distinct signalling
cascades and resulting in distinct morphological changes and
releases of mediators (Ransohoff and Perry, 2009). A future
challenge is thus to define more fully the range of response
states of the astrocytes including more comprehensive studies of
receptors expressed and factors released. Here, as a fulfilling
definition is still elusive, the term reactive astrocyte will be used
to indicate astrocytes presenting an altered state in response to
injury.
3. Structural organization of astrocytes
Astrocytes are characterized by their stellate shape. Morpholo-
gically they consist of a cell soma, large processes, finer processes
(filopodia and lamellipodia) and characteristic endfeets that
contact the basal lamina around blood vessels (Reichenbach
et al., 2009; Ventura and Harris, 1999). Astrocytes are heteroge-
neous in nature and differences are observed in morphology,
receptor expression, and level and type of ion channel expression
dependent on the tissue from which they originate (Oberheim
et al., 2006; Wilkin et al., 1990). Three-dimensional reconstruc-
tions of electron micrographs of hippocampal brain slices
demonstrated that astroglial processes intimately enwrap and
interact with different compartments of neurons, including the
neuronal soma, dendritic spines, and synapses (Halassa et al.,
2007).
Using transgenic mice in which enhanced green fluorescent
protein (eGFP) is expressed selectively within astrocytes, double
labeling techniques and patch pipette-mediated single-neuron dye
filling Halassa and colleagues (2007) showed that cortical astro-
cytes occupy non-overlapping domains. A single astrocyte was
found on average to enwrap four neuronal cell somata and
122 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128

estimated to make contacts with 300–600 dendrites thus enabling
a single astrocyte to make contact to hundreds of neurons (Halassa
et al., 2007). This anatomical organization has led to the following
two suggestions: first, that a single astrocyte has the potential to
coordinate small clusters of neurons if signals are transmitted
from the astrocyte to the neuronal cells bodies; and second, that
signals transmitted from a single astrocyte to dendrites, spines and
synapses has the potential to regulate the function of hundreds of
neurons; consequently make possible for a single astrocyte to
coordinate the function of many different neurons within its
domain (Halassa et al., 2007).
The somata and larger processes of astrocytes are stationary,
whilst small processes can be highly motile at active synapses.
Two different types of motility have been reported; a gliding of
thin lamellipodia-like process endings along the neuronal surfaces
and filipodia-like extensions budding out from processes into the
surrounding environment. This motility is suggested to allow
astrocytes to obtain placement at sites of gliotransmitter release
leading to direct shaping of the neuronal environment (Hirrlinger
et al., 2004). As the finest processes account for most of the
astrocytic volume (Reichenbach et al., 2009), this motility is
presumably of great importance for the astrocytes potential to
modulate neuronal signalling.
Together, the astrocytes unique anatomical organization and
motility provide a framework that allows astrocytes to directly
influence and possibly coordinate neurons located within the
domain of a single astrocyte. Notably, much knowledge has been
obtained from in vivo studies of cortical astrocytes and astrocytic
cell cultures isolated from various regions of the brain, and the
anatomical organization described above applies to brain–astro-
cytes. In contrast astrocyte reactivity is in most preclinical pain
models evaluated at the spinal cord level, and thus caution must
be taken when transposing knowledge based on investigations
carried out in tissues other than the spinal cord.
Temporal activation of microglia and astrocytes are observed in
many pain models (Hald et al., 2009; Wang et al., 2011; Zhang and
De Koninck, 2006), which has led to the suggestion that microglia
activation precedes astrocyte activation and is important for
initiation of chronic pain and necessary for activation of astrocytes,
while astrocytes are important for maintenance of chronic pain.
However, findings from models of neuropathic and cancer-induced
bone pain indicate that this temporal sequence does not apply to
all models of chronic pain, thus suggesting that astrocyte activa-
tion does not follow a defined sequence.
A comparative study by Honore and colleagues (2000)
designed to investigate spinal cord neurochemical changes
between different mouse models of pain has provided evidence
that pain models show unique spinal cord neurochemical signa-
tures, where activation of astrocytes is dependent on the specific
pain model. Indeed, no reactive astrocytes were found in a model
of inflammatory pain, moderate activation in models of neuro-
pathic pain, and strong activation in a model of cancer-induced
bone pain. In accordance a similar finding was reported in another
study comparing cancer-induced bone pain and neuropathic pain
(Hald et al., 2009).
A comparative study in neuropathic pain models based on
various lesions of the L5 spinal nerve showed that glial activation
is dependent on the site of the lesion (proximal or distal to the
dorsal root ganglion). Reactive astrocytes were observed in both

4. Reactive astrocytes in preclinical models of chronic pain

Amongst the first reports suggesting a role for astrocytes in

neuropathic pain was a finding by Garrison and colleagues (1991),
describing an elevated GFAP staining intensity in the ipsilateral
site of the spinal cord after a chronic constriction injury (CCI) of
the sciatic nerve in rats. The increase in GFAP expression was
attributed to hypertrophy of astrocytes and shown to correlate
with the degree of observed hyperalgesia (Garrison et al., 1991).
Since then reactive astrocytes have been identified in the spinal
cord in a selected range of pain models, including models of
inflammatory, neuropathic and cancer-induced pain, demonstrat-
ing both transient and persistent activation of astrocytes (Chang
et al., 2010; Gao et al., 2009a; Hald et al., 2009; Ma and Quirion,
2002; Sweitzer et al., 1999; Wang et al., 2011; Zhang et al., 2005;
Zhuang et al., 2006). Interestingly, reactive astrocytes are in some
models not only found in the dorsal horn ipsilateral to the
peripheral damage, but also in the dorsal horn contralateral to
the injury, and spread across an area larger than that expected
from the site of injury has been reported (Hald et al., 2008; Ma and
Quirion, 2002), indicating that astrocytes can be involved in the
sensitization of neighbouring neurons. Although the spinal cord is
the preferred tissue for examination of glial activation in pain
models, supraspinal activation can also be observed implying that
reactive astrocytes are involved in modulation of nociceptive Fig. 1. Differential activation of spinal cord astrocytes in mouse models of femoral
signalling at higher levels (Tanga et al., 2004; Wei et al., 2008). bone cancer pain. Spinal cord glial fibrillary acidic protein (GFAP) expression is not
Transition of astrocytes into a reactive state is most likely to be increased in the contralateral or ipsilateral dorsal horn of sham-operated (A) and
cancer-bearing BALB/cJ mice (B) inoculated with 4T1 mammary cancer cells. In
an orchestrated event that relies on a highly organised reciprocal contrast increased GFAP spinal cord expression is seen in the ipsilateral dorsal horn
communication between neurons and astrocytes, between micro- of cancer-bearing C3H/HeN mice (C) inoculated with NCTC 2472 osteosarcoma
glia and astrocytes, and also between astrocytes themselves. cells. Scale, 200 mm. Figure is modified from Hansen et al. (2011).
 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128
conditions, whereas microglial activation one week after injury
was observed only when the lesion was performed distal to the
dorsal root ganglion (Colburn et al., 1999). However, microglia
activation has been reported after lesion proximal to the dorsal
root ganglions in investigations performed 3 days after lesion
(Zhuang et al., 2005) suggesting that microglia may show different
temporal patterns of activation which are dependent on the lesion.
The model dependent activation of astrocytes is even more
evidently demonstrated in models of cancer-induced bone pain.
Initial findings suggested that a significant astrogliosis was a
hallmark of cancer-induced bone pain; this without a preceding
or concomitant activation of microglia (Hald et al., 2009; Honore
et al., 2000; Schwei et al., 1999). However, with the development
of an increasing range of different models of cancer-induced bone
pain employing different rodents, different types of cancer cells,
and different sites of bone inoculation, it has become apparent
that microglia activation can be observed in some models (Wang
et al., 2011; Zhang et al., 2005), and that reactive astrocytes,
defined by an increase in GFAP expression, are not present in all
models of cancer-induced bone pain (Hansen et al., 2011). Reactive
astrocytes were not found in a modified version of the original
Mantyh model of sarcoma-induced bone pain (Schwei et al., 1999)
employing a different mouse strain and different cancer cells
(Hansen et al., 2011) (Fig. 1), revealing that astrocyte activation is
not a hallmark of cancer-induced bone pain, but more likely
dependent on the species and the cancer cell type.
The morphological changes observed in preclinical models
indicate an involvement of astrocytes in chronic pain mechanisms
at spinal cord level. There are supportive, although not conclusive,
pharmacological evidence obtained with use of glial inhibitors.
Direct investigations on the astrocyte specific contribution to
chronic pain are hindered by the lack of astrocyte selective drugs.
However, some evidence has been provided by astrocyte modula-
tors. Fluoroacetate and its metabolite fluorocitrate are general
inhibitors of glial cells, but will at low doses mainly disrupt
astrocytic metabolism by blockade of the glial specific enzyme
aconitase (Clark et al., 2007; Gao and Ji, 2010). Intrathecal
administered fluorocitrate and fluoroacetate have proven effective
in preclinical models of both inflammatory and neuropathic pain
(Milligan et al., 2003; Okada-Ogawa et al., 2009). Also, intrathecal
administration of L-alpha-aminoadipate, another relative specific
astrocyte inhibitor (Khurgel et al., 1996), resulted in attenuation of
mechanical allodynia in models of neuropathic pain (Mei et al.,
2010; Wang et al., 2009; Zhang et al., 2011). Interestingly, fluor-
ocitrate did not reverse mechanical allodynia in a model lacking
activation of glial cells following injection of intramuscular acidic
saline (Ledeboer et al., 2006). Together, this evidence clearly
suggests that reactive astrocytes might provide new targets for
Fig. 2. Calcium wave propagation in astrocytic networks. An astrocyte, initially activated by a neurotransmitter such as glutamate or ATP, recruits phospholipase C (PLC) that
hydrolyses phosphatidylinositol bisphosphate to the intracellular secondary messenger inositol triphosphate (IP3). IP3 in turn activates its receptor (IP3R) located in the
endoplasmatic reticulum (ER) resulting in an additional release of calcium. Calcium wave propagation occurs via two mechanisms: IP3 can passively diffuse through gap
junctions to adjacent astrocytes and there trigger release of intracellular calcium from the ER; and ATP released from the astrocyte in response to the elevated calcium
concentration can stimulate adjacent astrocytes initiating IP3 production.
123
the treatment of specific chronic pain states. Yet, the important
question of how to approach analgesia through modulation of
astrocyte functionality still remains to be elucidated. To accom-
plish this better understanding of the roles of reactive astrocytes in
pain models is needed.
5. Calcium wave propagation in astrocytic networks and
implications for ATP signalling
An important characteristic of astrocytes are the formation of
astroglial networks. Gap junctions are formed at apposing astro-
cyte plasma membranes (Haydon, 2001; Orthmann-Murphy et al.,
2008). These gap junctions are composed of six gap junction
proteins, termed connexins, and form hemichannels allowing
direct intercellular movements of ions, metabolites and second
messengers between the astrocytes. The main functional connexin
in astrocytes is connexin-43 (C  43) (Thompson and Macvicar,
2008).
Astrocytes show both spontaneous and activity-evoked oscilla-
tions in their intracellular calcium concentrations, and increases in
intracellular calcium concentration are seen in response to stimu-
lation of a wide range of neurotransmitters (Fellin, 2009;
Hassinger et al., 1996; Haydon, 2001). These calcium oscillations
are believed to be important for astrocytic modulation as they
have been found to propagate between adjacent astrocytes, thus
creating a route of signal transduction which is independent of
neuronal signalling (Fellin, 2009; Guthrie et al., 1999; Hassinger
et al., 1996). The calcium oscillations led to the acknowledgement
of astrocytes as co-players in neuronal signalling (Araque et al.,
1999), and have also been suggested to contribute to nociceptive
signalling in chronic pain (Donnelly-Roberts et al., 2008; Milligan
et al., 2003; Obata et al., 2010; Spataro et al., 2004; Watkins et al.,
2003).
Calcium ions are omnipresent and function as the most
ubiquitous intracellular messenger. As cytosolic calcium concen-
trations are at the nanomolar level calcium gradients are created
between the cytosol and cellular compartments (Nedergaard et al.,
2010). Astrocytic calcium signalling relies primarily upon release
of calcium from dynamic intracellular calcium stores associated
with the endoplasmatic reticulum (ER) (Nedergaard et al., 2010).
An initial increase in intracellular calcium often results from
activation of a G-protein coupled receptor, which recruit phos-
pholipase C (PLC) that hydrolyses phosphatidylinositol bispho-
sphate (PIP2) to the intracellular secondary messenger inositol
triphosphate (IP3). IP3 in turn activates its receptor (IP3R) located
in the ER resulting in an additional increase in the intracellular
calcium concentration as calcium is released from the ER store.
124 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128
This increased calcium concentration can then trigger astrocytic
release of neurotransmitters such as glutamate, ATP and D-serine
(Coco et al., 2003; Haydon, 2001; Mothet et al., 2005; Parpura
et al., 1994).
Two concurrent mechanisms are suggested to underlie the
propagation of calcium waves (Fellin, 2009; Haydon, 2001).
Calcium wave propagation can be abolished by IP3 antagonists,
gap junction inhibitors, and inhibitors of the ER calcium-ATPase,
demonstrating that as IP3 passively diffuses through gap junctions
to adjacent astrocytes, it also triggers the release of intracellular
calcium from the ER of the adjacent astrocytes (Leybaert et al.,
1998; Haydon, 2001). Also, calcium waves can independently of
gap-junctions propagate across the intercellular space at distances
up to 120 mm (Hassinger et al., 1996). ATP has been identified as
the main transmitter involved in this type of calcium wave
propagation (Haydon and Carmignoto, 2006). It is believed that
the initial increase in intracellular calcium concentrations med-
iates release of ATP which subsequently can activate purinergic
receptors on adjacent astrocytes, thereby initiating increase in
calcium concentrations and subsequent release of ATP from the
adjacent astrocytes (Haydon and Carmignoto, 2006) (Fig. 2).
The propagation of calcium waves is suggested to contribute to
the spread of central sensitization underlying chronic pain as
inhibition of astrocytic function with fluorocitrate reverses con-
tralateral mirror image pain in models of neuropathic pain
(Donnelly-Roberts et al., 2008; Milligan et al., 2003; Obata et al.,
2010; Watkins et al., 2003); i.e. block of calcium wave propagation
reverses mechanical allodynia observed in the hind paw contral-
ateral to the site of injury. Also, decoupling of gap-junction
function by a gap-junction decoupler has been found to reverse
mirror image mechanical allodynia in a model of sciatic nerve
inflammation-induced neuropathic pain (Spataro et al., 2004).
ATP released from astrocytes in response to the original stimuli
and the subsequent ATP released from neighbouring astrocytes
within the astrocytic network might enhance nociceptive signal-
ling, thus allowing astrocytes an important role in pain processing
(Wu et al., 2012). Pharmacological evidence supporting a role of
ATP in chronic pain is substantial (Burnstock, 2009; Donnelly-
Roberts et al., 2008; Jarvis, 2010; Wirkner et al., 2007), but the
importance of the astrocytic pool of ATP is still not known.
6. ATP signalling in spinal cord pain processing
ATP has long been recognized as a neuromodulator and has an
established role in nociceptive signalling (Burnstock, 2009;
Donnelly-Roberts et al., 2008; Falk et al., 2012). ATP acts as an
agonist for two classes of purinergic receptors: ligand gated ion
channels, the P2X receptors, and G-protein-coupled P2Y receptors
(Burnstock, 2009). The role of ATP in nociceptive signalling has
been reviewed elsewhere (Donnelly-Roberts et al., 2008; Jarvis,
2010; Wirkner et al., 2007) and here only a brief overview will be
given of some of the mechanisms suggested to enhance nocicep-
tive signalling at the spinal cord level. ATP released from reactive
astrocytes is at the spinal cord level assumed to act on purinergic
receptors expressed on neurons, microglia and astrocytes
themselves.
Activation of P2X2/3 and P2X3 receptors expressed on the
central terminals of primary afferent neurons can transiently
enhance the release of presynaptic glutamate (Nakatsuka et al.,
2003; Vulchanova et al., 1998). In astrocytes, a rapid non-vesicular
release of glutamate from astrocytes occurs in response to P2X7
receptor activation (Duan et al., 2003), but also in a P2X7 receptor
independent manner involving activation of astrocytic P2Y1
receptors (Fellin et al., 2006; Zeng et al., 2008). P2X7 receptors
expressed on both microglia and astrocytes have furthermore been
associated with release of the cytokines interleukin-1beta (IL-1β)
and tumor necrosis factor-alpha (TNF-α) (Chu et al., 2010; Clark
et al., 2010; Mingam et al., 2008; Zhang et al., 2005). The astrocytic
expression of P2X7 receptors has been questioned (Bennett et al.,
2009; Chu et al., 2010; Duan et al., 2003), but a current proposal is
that although the P2X7 receptor is not abundantly expressed in
astrocytes under normal physiological conditions, thus hindering
detection, it becomes upregulated in pathological states (Cotrina
and Nedergaard, 2009). Consistently, an increased expression of
P2X7 receptor protein can be detected in cultured astrocytes after
treatment with IL-1β (Narcisse et al., 2005). Activation of micro-
glial P2X4 receptors has been associated with release of brain-
derived neurothrophic factor (BDNF), which via phosphorylation
of the NMDA receptor subunit NR1 expressed on spinal neurons
contributes to the development of mechanical allodynia (Coull
et al., 2005; Ulmann et al., 2008).
In summary, both calcium wave propagation and release of ATP
add an extra dimension to the astrocytic control of neuronal
signalling, and it is possible that astrocytic released ATP contri-
butes to central sensitisation in chronic pain.
7. Monitoring astrocytic response: c-Jun N-terminal kinase
signalling
Mitogen activated protein kinases (MAPKs) are a family of
protein kinases that play a pivotal role in intracellular signal
transduction and mediate cellular responses to a variety of
signalling molecules (Krishna and Narang, 2008). Eukaryotic cells
possess multiple MAPK pathways that are activated by a distinct
set of stimuli, which allow the cell to respond in a coordinated way
to multiple and divergent inputs (Krishna and Narang, 2008).
Mounting evidence demonstrate a pronociceptive role of MAPKs in
chronic pain processing (Gao et al., 2009a, 2009b; White et al.,
2011).
Three major members of the MAPK family are p38, extracellular
signal-regulated kinases (ERKs), and c-Jun N-terminal kinases
(JNKs) (Widmann et al., 1999). These MAPKs demonstrate differ-
ential activation in glial cells in various models of chronic pain
following peripheral damage; p38 has been found activated in
microglia; ERK in both microglia and astrocytes, while JNK has
been found activated primarily in astrocytes in the spinal cord
(Chang et al., 2010; Ma and Quirion, 2002; Svensson et al., 2003;
Wang et al., 2011; Zhuang et al., 2005, 2006).
JNK exist in three isoforms, JNK1, JNK2 and JNK3 (Krishna and
Narang, 2008). The JNK1 isoform is primarily expressed in astro-
cytes and is found phosphorylated, and thus activated, in models of
both neuropathic, inflammatory and cancer-induced pain (Gao
et al., 2009a, 2009b, 2010; Katsura et al., 2008 Ma and Quirion,
2002; Zhuang et al., 2006). Although clear co-localization is
observed between JNK1 and astrocytes, phosphorylated JNK has
been found only in a portion of spinal astrocytes (Zhuang et al.,
2006). This implies that astrocytic responses are heterogeneous and
that subpopulations may exist within the same spinal cord areas.
In spinal cord astrocytes phosphorylated JNK has been
observed as early as one day after spinal nerve ligation (SNL)
and found to persist for up to 21 days (Zhuang et al., 2006). This
increase in phosphorylated JNK has been confirmed and also seen
in a model of partial sciatic nerve ligation (PSNL) (Gao et al.,
2009b; Ma and Quirion, 2002). Astrocytic JNK activation has also
been shown in the carrageenan and complete Freunds adjuvant
(CFA) models of inflammatory pain (Gao et al., 2010; Svensson
et al., 2007), and increased spinal cord phosphorylated JNK1 levels
were reported in a model of skin cancer-induced pain associated
with reactive astrocytes (Gao et al., 2009a). Interestingly, bilateral
activation of JNK1 was observed in the spinal cord in CFA-induced
 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128
pain models associated with ipsi- and contralateral mechanical
allodynia; and both ipsi- and contralateral allodynia could be
attenuated by intrathecal inhibition of JNK suggesting that the
JNK pathway is critical for the development of bilateral mechanical
allodynia (Gao et al., 2010). However, no JNK activation was
reported in a model of spinal cord injury (Crown et al., 2006),
although reactive astrocytes are present (Gwak et al., 2012),
indicating that direct injury to the CNS may result in a dissimilar
astrocytic response.
The involvement of spinal cord JNK1 in chronic pain is
substantiated pharmacologically. Intrathecal administration of
D-JNKI-1, an inhibitory peptide highly selective for JNK1, attenu-
ated mechanical allodynia after spinal nerve ligation (Gao et al.,
2009b; Zhuang et al., 2006). Mechanical allodynia was also
attenuated in the models of CFA- and skin cancer-induced pain
(Gao et al., 2009a, 2010), while no effect was seen on heat
hypersensitivity in either of the latter models. A similar pattern
was found in the CFA model after intrathecal administration of
another JNK inhibitor, SP600125, and moreover the differential
effect was confirmed in JNK1 knockout mice where CFA-induced
mechanical allodynia was attenuated, while heat hyperalgesia was
retained (Gao et al., 2010). These latter findings are consistent with
the suggestion that heat hypersensitivity is mediated peripherally,
whereas mechanical allodynia is spinally mediated. Importantly,
JNK inhibition was not found to reduce the number of GFAP-
positive astrocytes or their morphological appearance in the spinal
nerve ligation model (Zhuang et al., 2006). This finding indicates
the complex involvement of astrocytes in nociceptive signalling
where multiple pathways can lead to the transition of astrocytes
into a reactive state.
A diversity of stimuli has been found to activate the JNK
pathway. These include stress related stimuli, cytokines, growth
factors and activation of the toll-like receptors (TLR), including
Fig. 3. Activation and signal transduction in the c-Jun N-terminal kinase (JNK)
pathway. The JNK pathway can be activated by a diversity of stimuli. These lead in a
coordinated way to equally diverse events as JNK can phosphorylate a wide range
of substrates, including transcription factors, kinases and receptors, and thus direct
a wide range of cellular responses. bFGF: basic fibroblast growth factor, GPCR:
G-protein coupled receptor, IL-1β: Interleukin-1beta, IL-6: Interleukin 6, MCP-1:
Monocyte chemotactic protein-1, NO: Nitric oxide, PGE2: Prostaglandin E2, RTK:
Receptor tyrosine kinases, TLR: toll-like receptor, TNF-α: Tumor necrosis factor-
alpha.
125
IL-1β, IL-6, TNF-α, basic fibroblast growth factor (bGFG) and
transforming growth factor (TGF) (Krishna and Narang, 2008).
The downstream events of JNK activation is equally diverse as JNK
can phosphorylate a wide range of substrates, including transcrip-
tion factors, other kinases and protein-like cytoskeletal proteins,
and thereby direct a wide range of cell responses (Gao and Ji,
2008; Ji et al., 2009; Krishna and Narang, 2008) (Fig. 3). Thus,
activation of the JNK pathway in astrocytes can possibly contribute
to chronic pain through numerous mechanisms. In support, JNK
inhibitors decrease the expression of cyclooxygenase-2 (COX-2)
and inducible nitric oxide synthase (iNOS), and the release of
prostaglandin E2 (PGE2), nitric oxide (NO) and IL-6 in astrocytic
cultures stimulated with a mixture of cytokines (Falsig et al.,
2004); bFGF has been linked to the release of astrocytic nerve
growth factor (NGF) and bFGF (Ji et al., 2006), while TNF-α
stimulation has been linked to induction of monocyte chemotactic
protein-1 (MCP-1/CCL2) expression (Gao et al., 2009b).
In summary evidence suggests that JNK1 plays an important
role in nociceptive signalling and thus constitute a putative target
for chronic pain (Gao et al., 2009b). In light of the great diversity of
stimuli and outcomes associated with JNK activation it is possible
that several JNK mediated pathways are engaged in the various
preclinical models of pain. Thus, further investigations into the JNK
pathway in nociceptive signalling could contribute to a better
understanding of the response states of astrocytes.
8. Clinical implications
Evidence for involvement of astrocytes in human chronic pain
states is sparse and the first clinical trial with a glial modulator
failed to provide proof of concept (Landry et al., 2012). The trial
was completed in 2009, where propentofylline, an atypical
methylxanthine, failed to alleviate postherpetic neuralgia in com-
parison to placebo treatment. This was a disappointment as
studies in preclinical models of acute and chronic pain had
demonstrated a potent analgesic property of propentofylline
(Sweitzer et al., 2001, 2006; Tawfik et al., 2007). The precise
mechanism of action of propentofylline is unknown, however it is
a phosphodiesterase inhibitor endowed with neuroprotective and
anti-inflammatory effects both in vitro and in vivo. It is not known
if these effects are mediated by neurons, microglia, astrocytes or
other cells, though propentofylline is largely thought to control
glial activation (Landry et al., 2012).
The design of the clinical trial and also the suitability of human
postherpetic neuralgia as a model for testing propentofylline
efficacy were questioned due to lack of preclinical evidence for
glial activation (Watkins et al., 2012). However, a dramatic activa-
tion of spinal astrocytes has subsequently been demonstrated in a
rat model of virally-induced post herpetic neuralgia where inter-
estingly intrathecal administration of L-alpha-aminoadipate, but
not the microglia specific inhibitor minocycline, attenuated
mechanical allodynia (Zhang et al., 2011). Although the transla-
tional impact of these pre-clinical findings remains to be estab-
lished as the presence of reactive astrocytes in the CNS of
postherpetic neuralgia patients has not been fully ascertained,
the preclinical findings again point to a contribution of spinal
astrocytes.
Another consideration is the temporal appearance of activated
microglia and reactive astrocytes in preclinical models of chronic
pain. Findings have suggested that pre-emptive treatment with
glial inhibitors is more effective than treatment of established pain
due to inhibition of both microglia and astrocytic components
that promote pronociceptive phenotypes (Mika et al., 2009;
Raghavendra et al., 2003; Sweitzer and DeLeo, 2011; Sweitzer
et al., 2001; Tawfik et al., 2007). In a crossover study using the rat
126 R. Rie Hansen, M. Malcangio / European Journal of Pharmacology 716 (2013) 120–128
L5 spinal nerve transection model of neuropathic pain Sweitzer
and colleagues (2001) demonstrated that preventive treatment
with propentofylline for up to day 7 after injury was more
effective than treatment initiated after establishment of nocicep-
tion. How this temporal pattern of glial activation applies to
human pain conditions is unknown, but it should be considered
in planning of clinical trials.
There is limited evidence for reactive astrocytes in pain
patients. Examination of cerebrospinal fluid (CSF) levels in patients
presenting with sciatica caused by lumbar disc herniation revealed
an increased level of S100β, a calcium-binding protein found
predominantly in the cytoplasm of astrocytes and Schwann cells
of the CNS, whilst GFAP levels were unchanged (Brisby et al.,
1999). Centrally reactive astrocytes have been shown in the spinal
cord posterior horn of a patient with long standing complex
regional pain syndrome. Notably, reactive astrocytes were
observed as most prominent at the level pertaining to the original
injury but did extend throughout the entire length of the spinal
cord (Del Valle et al., 2009). A recent study by Shi and colleagues
(2012) compared neurochemical changes in the spinal dorsal horn
of HIV-infected patients who had HIV-associated distal sensory
peripheral neuropathy and clinically documented pain syndromes
to HIV-patients who did not experience pain. Glial changes were
observed only in HIV-patients suffering from chronic pain, and
intriguingly only astrocytes and not microglia were found to be in
a reactive state, thus identifying astrocytes as a potential drug
target in HIV-associated chronic pain. Although the above pilot
findings are interesting and provide initial evidence that reactive
astrocytes found in pre-clinical models can translate to the human
conditions, these need to be confirmed in larger cohorts of
patients with well-defined pain states.
Importantly, comparisons between human and rodent cortical
astrocytes have revealed significant differences, which possibly
reflect the higher developmental stage of humans (Oberheim et al.,
2009). Human astrocytes have a threefold larger diameter and
tenfold more primary processes compared to rodents and the
domain of a single astrocyte has been estimated to contain up to
2 million synapses (Oberheim et al., 2006; Witcher et al., 2010).
Whether or how these differences are important for the role
undertaken by astrocytes in nociceptive signalling in preclinical
and clinical settings remain to be determined.
9. Concluding remarks
Evidence from models of inflammatory, neuropathic and
cancer-induced pain demonstrates that the occurrence of reactive
astrocytes is dependent on the specific pain model. Activation of
the astrocytic specific JNK pathway is not observed in all chronic
pain models associated with reactive astrocytes, indicating that
reactive astrocytes can present with different functionalities. Also,
a diversity of stimuli and effective outcomes are associated with
JNK pathway activation, and it is possible that distinct or several
JNK mediated pathways are engaged in the specific models of
chronic pain. Thus, the transition of astrocytes into a reactive state
seems to be mediated via several pathways and it is plausible that
subpopulations and response heterogeneity occurs. Therefore,
reactive astrocytes cannot be considered as a single entity, and
future investigations of the functional properties of astrocytes in
various models of chronic pain will contribute to a better under-
standing of the significance of the reactive states of astrocytes in
chronic pain.
Astrocytes can respond to stimulation by calcium wave propa-
gation mediated by passive diffusion of IP3 through gap junctions
and by release of ATP into the extracellular environment which in
a paracrine fashion recruits adjacent astrocytes. This astrocytic ATP
can facilitate nociceptive signalling through activation of puriner-
gic receptors expressed on neurons, microglia and astrocytes
thereby contributing to chronic pain processing. Thus, inhibition
of the astrocytic released ATP might result in pain relief even
though such a strategy would require inhibition of all sources
leading to astrocytic ATP release, including P2X7 receptors, IP3
mediated ATP release, as well as block of the P2Y mediated ATP
regenerative loop.
Acknowledgement
R.R. Hansen is supported by the Lundbeck Foundation and The
Henry Smith Charity.
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