REVIEWS

GLIA: A NOVEL DRUG DISCOVERY TARGET FOR CLINICAL PAIN

Linda R. Watkins and Steven F. Maier
In many clinical pain syndromes, painful sensations are greatly amplified so that normally innocuous sensations, such as light touch or warmth, are perceived as pain. Presently available drugs are ineffective in controlling such pain in most patients and abolish the pain in only few. Why do they fail? These drugs were developed to target neurons that transmit nociceptive (pain) information. However, glia have recently been recognized as powerful modulators of nociception, and could hold the key to the control of clinical pain and present a new target for drug discovery. This review examines the evidence for glial regulation of nociception and pharmacological approaches that might successfully control glially driven clinical pain syndromes.
PAIN FACILITATION afflicts tens of millions of people in the United States alone. When such pain states occur, hot, cold and hard pressure pains are grossly amplified (HYPERALGESIA). Indeed, warm, cool and touch are perceived as pain (ALLODYNIA) (FIG. 1). Such pain can long outlast the injury or infection that initiated it, causing years of unremitting suffering. Pain facilitation is poorly controlled by presently available drugs, and precious few patients have their pain eliminated by drug treatment1,2. Before proceeding, it is useful to briefly discuss the terminology to be used in this review. The term pain refers to the unpleasant experience associated with actual or perceived tissue damage by a noxious (damaging) stimulus. As an experience, pain includes not only the perception of a sensory stimulus, but also associated cognitive analysis and emotional responses. Quantifying such experiences is arguably impossible in laboratory animals. So in order to study pain in laboratory animals, terminology was created that refers to the lower order quantifiable aspects of pain. The term nociception (noci- meaning pain) is used to describe neural, physiological or behavioural responses to stimuli that humans would typically report as painful. Similarly,nocisponsive neurons are those that encode the presence of noxious stimuli, nociceptors are sensory receptors activated by such stimuli, and so forth. In this review, the use of the term pain will be restricted to situations in which pain experience is reflected by

PAIN FACILITATION

Perceiving pain out of proportion to the initiating stimulus. Includes hyperalgesia and allodynia, below.
HYPERALGESIA

Perceiving stimuli as more painful than they would normally be.

ALLODYNIA

Perceiving normally nonpainful stimuli as painful.

PHENOTYPE SHIFT

As used here, means that a neuron changes what neurotransmitter it produces and releases, creating a functional change in the pain transmission system.

Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309-0345, USA. Correspondence to L.R.W. e-mail: lwatkins@psych. colorado.edu.
doi:10.1038/nrd1251

human report. The terms hyperalgesia and allodynia, when referring to studies of laboratory animals, imply that the animals show enhanced escape responses to applied stimuli. Decades of research have focused on why clinical pain syndromes might occur. Many plastic changes in neurons in the pain pathway have been found that can amplify the transmission of information about noxious stimuli. For example, injured peripheral nerves can exhibit a number of changes: spontaneous activity can occur that signals the presence of noxious stimuli despite the fact that no such stimulus is present; novel receptors for stress hormones can be expressed, such that sympathetic activation subsequently evokes pain in people and amplified escape behaviours in laboratory animals; PHENOTYPE SHIFTS can occur such that neurons that normally transmit information about innocuous stimuli release neurotransmitters that signal the presence of noxious stimuli; or alterations in cation channels can occur, which induce a hyperexcitable state3,4. Such alterations of neuronal function have guided drug discovery researchers in the quest for successful pain control. Despite intensive efforts, clinical pain control has remained a puzzlingly elusive target. An answer to this pain dilemma might lie in a cell type not considered when presently available drugs were developed. These cells are, perhaps surprisingly, not neurons. They are glia; specifically, microglia and
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b
Incoming A-/C fibre 'pain' signals Normal substance P release, EAA release

Quiescent glia AMPA receptor Pain message to brain via PTNs NK-1 receptor NMDA receptor

PTN Pain stimulus

Incoming A-/C fibre 'pain' signals Enhanced substance P release, EAA release

d
Viruses and bacteria

Nonexistent glia AMPA receptor NK-1 receptor Ca2+ NMDA receptor

PTN: NO, PGs, fractalkine Activated glia

Primary afferent: Substance P, EAAs, ATP, fractalkine

cNOS

IL-1, TNF, IL-6, ROS, NO, PGs, EAAs, ATP

NO

L-arginine

PTN

Enhance PTN excitability

Enhance primary afferent substance P and EAA release

Figure 1 | Schematic of pain and pain modulation. a | Classical pain transmission pathway. When a noxious stimulus is encountered (such as stepping on a tack, as shown), peripheral nocisponsive nerves (A- and C fibres) are excited. These axons transmit action potentials to their presynaptic terminals in the spinal cord dorsal horn. Neurotransmitters released here bind to and activate postsynaptic receptors on pain transmission neurons (PTNs). In turn, the axons of PTNs ascend, predominantly contralaterally, to the brain and carry information about the noxious stimulus to higher centres. The region of the sensory presynaptic terminal and post-synaptic region of the PTN are shown in detail in (bd). b | Normal pain. In normal, everyday situations in which pain is experienced, glia are present but quiescent. Information about noxious stimuli arrives from the periphery along A- and C fibres, causing the release of substance P and excitatory amino acids (EAAs) in amounts appropriate to the intensity and duration of the initiating noxious stimulus. Activation of neurokinin-1 (NK-1) receptors by substance P and activation of -amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) receptors by EAAs cause transient depolarization of the PTNs, thereby generating action potentials that are relayed to higher brain areas. N-methyl-D-aspartate (NMDA)-linked channels are inoperative as they are chronically plugged by magnesium ions. c | Pathological pain: classic view. In reponse to intense and/or prolonged barrages of incoming nociceptive information, the PTNs become sensitized and over-respond to subsequent incoming nociceptive signals. The intense and/or prolonged barrage depolarizes the PTNs such that the magnesium ions exit the NMDA-linked channel. The resultant influx of calcium ions activates constitutively expressed nitric oxide synthase (cNOS), causing conversion of L-arginine to nitric oxide (NO). Because it is a gas, NO rapidly diffuses out of the PTNs. This NO acts presynaptically to cause exaggerated release of substance P and EAAs. Postsynaptically, NO causes the PTNs to become hyperexcitable. Glia have not been considered to have a role in creating pain facilitation by this neuronally driven model. d | Pathological pain: new view. Here, glial activation is conceptualized as a driving force for creating and maintaining pathological pain states. The role of glia is superimposed on the NMDANO-driven neuronal changes detailed in c, so only the aspects added by including glia in the model are described here. Glia are activated (shown as hypertrophied relative to b, as this reflects the remarkable anatomical changes that these cells undergo on activation) by three sources: bacteria and viruses which bind specific activation receptors expressed by microglia and astrocytes; substance P, EAAs, fractalkine and ATP released by either A- or C fibre presynaptic terminals (shown) or by brain-to-spinal cord pain enhancement pathways (not shown); and NO, prostaglandins (PGs) and fractalkine released from PTNs. Following activation, microglia and astrocytes cause PTN hyperexcitability and the exaggerated release of substance P and EAAs from presynaptic terminals. These changes are created by the glial release of NO, EAAs, reactive oxygen species (ROS), PGs, pro-inflammatory cytokines (for example, interleukin-1 (IL-1), IL-6, tumour-necrosis factor (TNF)), and nerve growth factor. Modified, with permission, from REF. 12 Elsevier Science Ltd (2001).

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astrocytes within the spinal cord. This review will first examine the evidence that activated glia drive the creation and maintenance of allodynia and hyperalgesia. This literature review will demonstrate that astrocytes and microglia in the spinal cord must now be recognized as active participants in the creation and maintenance of pain facilitation induced by inflammation and damage to peripheral tissues, peripheral nerves, spinal nerves and the spinal cord. On activation, these glia release a variety of neuroexcitatory substances that potentiate pain transmission by neurons. Of these glial products, pro-inflammatory cytokines will be shown to be common spinal mediators of allodynia and hyperalgesia. Given the failure of presently available drugs to provide adequate clinical pain management, this newly recognized role of glia in general, and pro-inflammatory cytokines in particular, is exciting because it predicts novel approaches for effective pain control by targeting glial activation. A number of drugs will be introduced in the course of this discussion that are effective in controlling glially driven exaggerated nociceptive states in laboratory animals. Each of these drugs will be discussed in the final section of this review, with regard to their potential clinical application.
What does it mean that glia are activated?

GLIAL FIBRILLARY ACIDIC PROTEIN

(GFAP). An astrocyte-specific protein. Increases in GFAP are frequently used as a marker of astrocyte activation.
p38 MAP KINASE

An intracellular signalling cascade, activated in response to pro-inflammatory cytokine receptor binding. Activation of this cascade leads to production of pro-inflammatory cytokines.

In the following discussion, frequent reference will be made to activated glia. Activation is a fundamentally different phenomenon in neurons compared with that in glia. The term activation refers to an enhanced ability of a cell to perform a function beyond that present in a basal state. For neurons, activation is unidimensional, as it mainly relates to the production of action potentials. By contrast, activation of glia is multi-dimensional because glia perform numerous functions (see below). So there are many different activational states, with various components expressed with different time-courses and intensities that are dependent on the stimulus that triggers activation. Astrocytes are basally active in carrying out a number of functions, but unless there is an external stimulus they do not become activated. Astrocytes serve several functions in the normal central nervous system (CNS), including the regulation of: extracellular ion and neurotransmitter concentrations; the availability of neurotransmitter/neuromodulator precursors to nearby neurons; and extracellular pH. No evidence is apparent in the literature that these functions are regulated by stimuli that activate astrocytes or by substances released by activated glia. As such, they do not seem likely to be affected by drugs that target these aspects of activated astrocyte function. Astrocyte activation occurs in response to CNS trauma, ischaemia, tumours, neurodegeneration, and the presence of immunogenic components of viruses and bacteria5. Activation is morphologically characterized by hypertrophy and increased production of intermediate filaments (GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP), vimentin and/or nestin)5, and functionally by increased production of a variety of pro-inflammatory substances6.

Notably, functional changes and morphological changes are not time-locked, so functional changes can be detected in the absence of increased intermediate filaments, and vice versa. In contrast to astrocytes, microglia in the normal CNS are quiescent cells with no recognized function7. In this state, they exhibit a ramified morphology, no activation of p38 MITOGEN-ACTIVATED PROTEIN (MAP) KINASE, and little or no expression of the receptors, cellsurface markers or functional activities characteristic of activated microglia7. Microglial activation occurs in response to the same range of stimuli that activate astrocytes. It is a graded phenomenon, characterized by a specific morphology (retracted processes and hypertrophy; amoeboid morphology under strongly pathological circumstances), proliferation, increased expression of one or more cellsurface markers or receptors (such as the complement 3 receptor associated with adhesion, migration and phagocytosis and scavenger receptors associated with phagocytosis), and/or changes in functional activities (migration to areas of damage, phagocytosis, production/release of pro-inflammatory substances)7. Notably, the changes in receptors, cell-surface markers and/or the production of pro-inflammatory substances can occur in the absence of morphological changes, proliferation or phagocytosis7,8. So, as is the the case for astrocyte activation, microglial activation is a multi-dimensional process. The manner in which activation is expressed is dependent on the type and intensity of the inductive stimulus, and different patterns and time-courses of responses can occur.
Spinal cord glia as powerful modulators of pain

Until recently, glia were thought of simply as housekeepers for neurons, regulating the extracellular ionic environment and removing debris. However, in recent years it has become recognized that glia dynamically modulate the function of neurons under both physiological and pathological conditions9. In the discussion to follow, the term glia will be used to refer to both microglia and astrocytes. As will be reviewed below, both cell types are activated in the spinal cord in response to experimental manipulations that induce pain facilitation. Both cell types, on activation, can produce and release a variety of nociceptionenhancing substances. In addition, each stimulates the further activation of the other, perhaps forming a functional unit. The use of the term glia in the present context reflects the fact that most presently available data support the notion that both microglia and astrocytes are involved in allodynia and hyperalgesia, but that these data cannot yet allow us to determine the relative contributions of each. Glia first came to the attention of pain researchers in the early 1990s when Garrison et al. reported that peripheral nerve damage that created exaggerated nociceptive responses (neuropathic pain behaviours) also activated spinal cord glia (FIG. 2). Furthermore, the N-methyl-D-aspartate (NMDA) antagonist MK801, which blocks neuropathy induced allodynia and

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hyperalgesia, also blocked glial activation10,11. These studies established that glial activation, at minimum, closely tracked neuropathy induced allodynia and hyperalgesia as well as their pharmacological resolution. The potential implications of the data collected by Garrison et al. were breathtaking. If glial activation was indeed a causal factor in the development of allodynia and hyperalgesia, rather than simply being correlated with these conditions, it would be a dramatic departure
a Intact side b Nerve-damaged side

c Intact side

d Nerve-damaged side

Figure 2 | A historical look at glial involvement in pain. The first evidence that glia were involved in pain modulation came from the work of Garrison et al. who showed that astrocytes in spinal cord were activated (as reflected by immunohistochemistry for the astrocyte-specific activation marker, glial fibrillary acidic protein) in response to sciatic nerve damage. They examined the effect of chronic constriction injury (CCI), as it is one of the best-validated animal models of partial nerve injury leading to chronic pain. Although previous studies had identified central nervous system glial activation as a rapid response to peripheral nerve injury, the work of Garrison et al. was the first to link such glial activation to a functional outcome, namely, enhanced nociception. In the upper panels (a and b), the spinal cord ipsilateral of sciatic CCI is compared with the spinal cord on the healthy sciatic side. Clearly, these two sides look different. To more clearly see what this difference is due to, the lower panels (c and d) provide a higher-power image of dorsal horn astrocytes. Compared with astrocytes on the healthy spinal cord side (c), astrocytes on the nervedamaged side (d; same magnification as c) are hypertrophied and more darkly stained, which is a sign of astrocyte activation. Modified, with permission, from REF. 10 Elsevier Science Ltd (1991).

from the classical view that exaggerated pain states are created and maintained solely by neurons. In practical terms, if glia were key players, it would open up whole new approaches for clinical pain control, as drugs that alter glial function were likely to be unique from drugs targeting neurons. So, how could one test whether glia are necessary or sufficient for allodynia and hyperalgesia? Neither question proved easy to address, given the limited pharmacological tools available, and the even more limited knowledge of how glia might alter nociception. What became clear, however, was that every model tested that induced allodynia and/or hyperalgesia was associated with the activation of both astrocytes and microglia12 (FIG. 3). So, finding ways to test glial involvement became essential. The question of whether glia are necessary for allodynia and hyperalgesia addresses whether exaggerated nociceptive responses will occur if neurons, but not glia, are present. In practice, this translates to pharmacologically removing glia by disrupting their function. Two drugs have been employed for this purpose: fluorocitrate, which selectively disrupts the Krebs energy cycle of glia by inhibiting the glia-specific enzyme aconitase13,14; and minocycline, which selectively disrupts the activation of microglia without directly affecting neurons or astrocytes15. Both agents have been found to be effective in blocking diverse models of allodynia/hyperalgesia1621. Two intriguing, and interrelated, findings are worth noting. First, fluorocitrate, which disrupts astrocyte as well as microglia function, seems to exert more profound blockade of exaggerated nociceptive states than does minocycline, which targets only microglia. Second, minocycline is far more effective in blocking, than reversing, exaggerated nociceptive responses20,21. The most important role of microglia might be involvement in the initial induction of exaggerated responses to noxious stimuli. Microglial activation leads, in turn, to astrocyte activation that maintains the facilitation of nociception20. Developing an effective strategy for testing whether glial activation is sufficient to induce allodynia/hyperalgesia is dependent on identifying ways to selectively activate these cells. Although studies of glial cultures have identified neurotransmitters that can activate glia9, that information is of little use here for two reasons. First, virtually all data on glial responses to neurotransmitters have been derived from glia isolated from the brain. Given the marked heterogeneity in both receptor expression and response properties of glia isolated from various CNS regions22,23, extrapolating results from brain to spinal cord glia is problematic. Indeed, there are even heterogeneities between glia in nocisponsive layers of the superficial dorsal horn (laminae IIII) versus other spinal regions2426. Little information is available as to what neurotransmitters excite dorsal spinal cord glia to produce and release neuroactive substances27,28. Given that dorsal horn glial activation occurs in response to peripheral injury and inflammation12, it would seem reasonable to predict that neurotransmitters are released in spinal cord in response to peripheral injury, and that inflammation will be found to activate these cells.

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Mechanisms of glial pain enhancement

Figure 3 | Microglia, as well as astrocytes, are activated by manipulations that create enhanced nociception in animal models. Although the earliest studies focused on the activation of astroctyes, microglia are also activated. These photomicrographs provide one example of microglial activation in response to a manipulation (intrathecal HIV-1 glycoprotein 120) that produces robust mechanical allodynia and thermal hyperalgesia. Microglia are stained for expression of complement-3 receptor, which is upregulated when microglia are activated. a | Normal microglia in the dorsal horn of the spinal cord after peri-spinal injection of vehicle (control). b | Same magnification view of dorsal horn microglia after peri-spinal injection of a viral protein (HIV-1 glycoprotein 120), which induces exaggerated pain responses. Modified, with permission, from REF. 12 Elsevier Science Ltd (2001).

IMMUNOCOMPETENT

Functioning similar to immune cells; that is, expressing receptors for bacteria/viruses and releasing pro-inflammatory cytokines and other immune cell products on activation.
NEUROPATHIC PAIN

Allodynia/hyperalgesia caused by inflammation of and/or trauma to peripheral nerves.

IL-6-NEUTRALIZING ANTIBODIES

Antibodies that bind to IL-6 and so prevent IL-6 from binding to its receptor.

However, studying the effects of such neurotransmitters on glia in vivo is confounded by the fact that these substances would also activate spinal neurons. The first approach that has been used to examine whether glial activation is sufficient to create enhanced nociception rests on the fact that both astrocytes and microglia are IMMUNOCOMPETENT cell types. That is, whereas microglia are of haematopoietic (immune) cell lineage29,30 (although this is not without controversy31), astrocytes can also behave, in many regards, like immune cells, despite their neural-tube origins32. Of direct relevance here is that they express receptors for, and are activated by, viruses and bacteria33. So studies have examined the effect of injecting the immunogenic portions of bacteria and viruses over spinal cord. Glial activation and exaggerated pain responses occur as a result16,18,34. Glial activation is causal to the allodynia and hyperalgesia observed in these studies, as the pain changes are blocked by disruption of glial function16,18,34. A second and very recent approach for examining this issue has tested the role of fractalkine (also known as chemokine (C-X3-C motif) ligand-1) in pain facilitation. Fractalkine is a protein that is tethered to the extracellular surface of neurons35. In spinal cord, no cell types other than neurons express fractalkine36,37. In response to strong neuronal activation, fractalkine can be released and diffuse away38. In spinal cord, only microglia express receptors for fractalkine (FIG. 4). So, fractalkine is a putative neuron-to-glia signal. The administration of exogenous fractalkine produces allodynia and hyperalgesia through binding to CX3CR1, the only known receptor for fractalkine36,37. Furthermore, the blockade of endogenous fractalkine attenuates allodynia and hyperalgesia induced in animal models of NEUROPATHIC PAIN, indicating that peripheral-nerve inflammation and injury lead to the release of fractalkine from neurons in dorsal spinal cord36,37. So neuron-to-glia signalling, here evoked by fractalkine activation of microglia, again seems to be sufficient to create pain facilitation.

The mechanisms by which glial activation enhances neuronal transmission of nociceptive information are only partially understood. Ultimately, activated spinal cord glia must lead to hyperalgesia and allodynia by releasing substances that act on neurons in the pain pathway(s). These substances could have a variety of actions: they could direct excitation, sensitization, or potentiation of action potentials; direct the upregulation of neuronal receptors; and promote the induction of the release of other transmitters/modulators that can act on nocisponsive neurons. To date, most evidence has supported a putative role for the glial pro-inflammatory cytokines tumournecrosis factor (TNF), interleukin-1 (IL-1) and IL-6. Pro-inflammatory cytokines are classically known as a family of proteins released by activated immune cells. There is no amino-acid sequence motif or three-dimensional structure that links them; rather, they are grouped on the basis of their biological activities39. TNF, IL-1 and IL-6 came to be classified as pro-inflammatory because they orchestrate the early immune response to infection and injury by communicating with white blood cells, thereby attracting them to the site of infection/injury, and causing them to become activated and respond39. Peripherally, and in the CNS, these proinflammatory cytokines are often sequentially formed in a cascade in which TNF is typically made first, causing the induction of IL-1, which in turn causes the induction of IL-6. It is an important feature that the effects of proinflammatory cytokines synergize (especially TNF and IL-1), such that far more powerful effects are observed when more than one cytokine is present39. TNF and IL-1, in particular, are very potent biological molecules, producing large effects when administered in the CNS in the femptogram to picogram range33. Indeed it has been estimated that as few as four molecules of IL-1 need bind to a cell to induce a physiological response39. Appropriately, given this potency, the production and release of pro-inflammatory cytokines is tightly regulated, and numerous negative-feedback control systems exist (such as the production of antiinflammatory cytokines, decoy receptors and antagonists of receptors for pro-inflammatory cytokines) which can help suppress the production and function of pro-inflammatory cytokines33. Both astrocytes and microglia can release proinflammatory cytokines on activation33 (FIG. 5), and glia and neurons express receptors for them. In the brain, pro-inflammatory cytokines have a wide array of effects on the regulation of core body temperature, sleep, learning and memory, and hypothalamopituitary hormones40. The injection of exogenous pro-inflammatory cytokines over the spinal cord enhances nociception4144, and electrophysiological studies document rapid enhancement of neuronal excitability in response to noxious stimuli following the injection of proinflammatory cytokines to the region43,45. Conversely, the blockade of pro-inflammatory cytokine function, using either an IL-1-receptor antagonist, soluble TNF receptors or anti-IL-6-NEUTRALIZING ANTIBODIES, prevents

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p38 MAP kinase inhibitors were originally referred to as cytokine-suppressive anti-inflammatory drugs, reflecting the importance of this signalling pathway. These compounds inhibit allodynia and hyperalgesia induced by peripheral tissue inflammation, peripheral nerve injury, spinal nerve injury, spinal cord inflammation, and peri-spinal substance P and NMDA administration in animal models18,21,4852. At least some of these compounds cross the bloodbrain barrier51,52, and so are effective systemically as well as after peri-spinal administration. An alternative approach to suppressing cytokines is to use xanthine derivatives, such as propentofylline. Propentofylline controls enhanced nociception induced by spinal nerve transection53. Notably, it is equally effective in reversing and preventing these changes, after both systemic and peri-spinal delivery53. Furthermore, propentofylline decreases both microglial and astrocytic activation in spinal cord53. Other compounds that have been tested in animal models include the disease-modifying anti-rheumatic drug leflunomide, the immunosuppressive drug methotrexate and the immunomodulatory drug thalidomide. Systemic leflunomide was more effective in attenuating enhanced nociception induced by peripheral nerve injury than nociception caused by damage to spinal roots54. Methotrexate, delivered systemically as well as at the site of spinal root injury, both prevented and reversed neuropathic pain behaviours in rats55, an exciting outcome from a clinical perspective. Thalidomide, to date, has only been tested systemically. It has proven effective in delaying enhanced nociceptive responses in rats induced by peripheral nerve damage56. However, its ability to reverse neuropathic pain behaviours in rats has been questioned56. Peripherally, thalidomide attenuated nerve-damage-induced TNF, but had no effect on either IL-1 or IL-6 expression in the damaged nerve57. Given that the focus of these investigators was thalidomide-regulation of peripheral nerve changes, rather than spinal cord glia, they did not assess the effect of systemic thalidomide on central glial activation or pro-inflammatory cytokines. Because thalidomide crosses the bloodbrain barrier58,59, changes in spinal cord glial function would be expected. The newest approach to controlling glially enhanced nociception is upregulation of the expression of a cytokine not yet discussed in this review, IL-10. Rather than being a pro-inflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Like pro-inflammatory cytokines, anti-inflammatory cytokines are a family of proteins that can be released by immune cells and immunocompetent cells, such as glia60,61. Endogenously, anti-inflammatory cytokines serve as negative-feedback regulators that keep potentially pathological activation of immune and immune-like cells under control60,61. Within this family, IL-10 is by far the most powerful member. IL-10 is attractive as a suppressor of glial pathological excitation for a number of reasons: it can inhibit proinflammatory cytokine production at multiple levels, including transcription, translation and release; it can downregulate the expression of receptors for proinflammatory cytokines, so that even if pro-inflammatory

Figure 4 | Neuron-to-glia communication. When pain processing is enhanced by inflammation or damage to peripheral tissues or peripheral nerves, signals must be relayed from sensory nerves to spinal cord glial cells to cause glial activation. There are at least two possible routes of neuron-to-glia communication that could lead to glial activation and consequent enhancement of nociception. First, neurotransmitters that relay information of the presence of peripheral noxious stimuli could bind to and activate glia. Although probable, this has not yet been proven for spinal cord. Second, neurons could release a selective neuron-to-glia signal that binds to and activates glia. This avenue of neuron-to-glia signalling has only very recently begun to be productively explored. One candidate signal is fractalkine, a protein expressed on the extracellular surface of neurons that, on strong neuronal activation, can be released into the extracellular fluid. In spinal cord, only microglia express receptors for fractalkine, making it a putative neuron-to-glia signal. Fractalkine, either injected exogenously or released endogenously in response to peripheral nerve damage, enhances nociception in animal models. The photomicrographs are of astrocyte and microglia mixed cultures. These photomicrographs demonstrate that microglia, but not astrocytes express fractalkine-binding sites. Green fluorescence (a and c) reveals glial fibrillary acidic protein (GFAP), an astrocyte-specific marker. Red fluorescence (b and c) reveals binding of fluorescent fractalkine. The lack of yellow (co-localization of green and red) indicates that astrocytes do not express binding sites for fractalkine. By contrast, all microglia in the field bind this putative neuron-to-glia signal. Panel c shows the mixed glial culture with superimposed fluorescence images. Modified with permission from REF. 133 Springer-Verlag (2003).

PERI-SPINAL INJECTION

Administering a drug into the cerebrospinal fluid surrounding the spinal cord; also called intrathecal.

their effects in brain40. Likewise, PERI-SPINAL INJECTION of antagonists of pro-inflammatory cytokine function prevents and/or reverses allodynia and hyperalgesia in virtually every animal model tested17,19,34,36,46. Such models include inflammation and/or injury to peripheral tissues, peripheral nerves, spinal nerves and spinal cord. The fact that established allodynia and hyperalgesia can be reversed by pro-inflammatory cytokine antagonists supports the conclusion that these glial proteins are involved in the maintenance, as well as the initial induction, of these enhanced nociceptive states. That is an important point when one is attempting to identify drug targets for controlling pre-existing clinical pain syndromes. Recognition of the importance of pro-inflammatory cytokines in the induction and maintenance of allodynia and hyperalgesia has led to the testing of various cytokine-suppressive drugs in animal models. One approach to control pro-inflammatory cytokines is to block intracellular pathways that lead to their production. Another approach is to block intracellular pathways that are activated by the binding of pro-inflammatory cytokines to their receptors. Although multiple intracellular signalling cascades have been implicated in proinflammatory cytokine signalling and production, p38 MAP kinase is crucially involved in both33,47. Indeed,

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TACE

TNF-

Other cells

CXCR4 TNFRI

ERK1/2

Ca2+ Glial cell

Figure 5 | Pro-inflammatory cytokines are constitutively expressed in an inactive precursor form, allowing rapid release. Constitutive expression and rapid release of tumour-necrosis factor (TNF) is illustrated. a | Constitutive extracellular surface expression of TNF on astrocytes. Exposure of living rat astrocytes (glial fibrillary acidic protein-positive, red) to an anti-TNF antibody selectively reveals TNF sequences exposed on the extracellular surface (green) of about 50% of astrocytes. b | Molecular events coupling receptor activation of astrocytes (CX3CR4 shown in example) to TNF release. TNF is released in response to stimulation of various astrocyte receptors. Illustrated is TNF action on a G-protein-coupled receptor (CXCR4) by its endogenous ligand (SDF-1-). The resultant intracellular signalling activates extracellular signal-regulated kinase (ERK1/2), which activates TNF--converting enzyme (TACE). TACE is a specific enzyme required to cleave the extracellular domain of membrane-bound pro-TNF (a 26-kDa protein) to generate the released mature TNF (18-kDa protein) through a process known as ectodomain shedding. Once cleaved, the mature (active) TNF both exerts auto-stimulation of the same cell and diffuses away to exert paracrine actions on surrounding glia and neurons. Adapted with permission from REF. 134 Oxford University Press (2002).

cytokines are released they are less effective due to decreased availability of receptors; it can upregulate endogenous antagonists to pro-inflammatory cytokines, thereby limiting their effectiveness; and evidence to date indicates that neurons in the spinal cord do not express receptors for IL-10, so normal neuronal functions would be unaffected by the presence of IL-10 (REFS 6062). Taken together, this is a powerful profile of effects. Behaviourally, studies of IL-10 in rats demonstrate that it prevents or reverses every enhanced nociceptive state examined to date. These models include pain induced by spinal inflammation, inflammatory neuropathy, traumatic neuropathy, spinal trauma and peri-spinal dynorphin19,36,6365.
Drug discovery outlook

SYNOVIAL TISSUES

Tissues encapsulating joints.

The potential efficacy of a drug is dependent on many factors. What follows is a summary of clinically relevant aspects of the various drugs that target glia and which have successfully controlled enhanced nociceptive states in animal models. No drug presently available for use in humans was developed to target glia. Rather, they were developed to suppress the function of the peripheral immune system. Indeed, their efficacy in suppressing pain in humans and enhanced nociceptive behaviours in rats following systemic administration supports the argument that suppressing pro-inflammatory cytokine production by SYNOVIAL TISSUES, Schwann cells and other immunocompetent cell types in peripheral tissues, peripheral nerves and/or dorsal root ganglia3 can decrease transmission of nociceptive information to the spinal cord. In the present context, the discussion of these compounds will be focused on their potential for also suppressing the pain-enhancing effects of spinal cord glial activation. There is a great need for new drugs to reach clinical trials for controlling the pathological side of spinal cord glial activation.

Disrupting glial activation. The two drugs that have been examined in animal models for their ability to disrupt glial activation, to date, are fluorocitrate and minocycline. Fluorocitrate is a reversible glial poison not appropriate for human use. Although fluorocitrate is a selective glial inhibitor at low doses and short postdrug time intervals 13,14, higher doses and longer post-drug times can indirectly affect neuronal functions. This indirect effect on neurons can result from elevated extracellular concentrations of excitatory amino acids due to the inhibition of glial transport13,14. Seizures have also been reported in response to glia-toxic doses of this compound66. On the other hand, minocycline exhibits selectivity for microglia. It is a tetracycline derivative that has anti-inflammatory effects which are independent of its antimicrobial actions. In rats, it can inhibit microglial activation, p38-MAP-kinase activation, IL-1-convertingenzyme (caspase-1) activation, IL-1 release and the production of nitric oxide28,67. Although the animal literature largely supports the conclusion that minocycline inhibits activation of microglia independent of direct effects on astrocytes and neurons, neuroprotective effects of minocycline on neuronal cultures exposed to toxic levels of nitric oxide have been reported68. Although all of these indices are positive with regards to minocyclines potential for controlling glially driven allodynia and hyperalgesia, concern is raised by the fact that minocycline fails to reverse, or is far less effective at reversing, established enhanced nociceptive states in animal models, relative to agents that inhibit astrocyte as well as microglial activity20,21. These initial studies indicate that microglia might have a more important role in the initial creation of enhanced nociceptive states in animals, whereas astrocytes might become the key glial cell type as allodynia/

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hyperalgesia persists20. If further animal studies support this conclusion, this is an important concept for guiding drug development. It would indicate that minocycline is unlikely to be an effective drug for clinical pain control. Pro-inflammatory cytokine antagonists. A variety of biological modifiers have been developed to target IL-1 and TNF. Anakinra (Kineret; Amgen) is a recombinant human IL-1-receptor antagonist approved for use in rheumatoid arthritis69. Infliximab (Remicade; Centocor) is a chimeric immunoglobulin G1 antibody specific for human TNF-, approved for use in rheumatoid arthritis and Crohns disease70. Etanercept (Enbrel; Amgen/Wyeth) is a human TNF receptor/Fc fusion protein approved for use for the treatment of psoriatic arthritis70. Although anakinra and etanercept have been successfully used in animal studies to both prevent and reverse glially driven allodynia and hyperalgesia19,34,46, peri-spinal administration was required, as this family of compounds does not cross the intact bloodbrain barrier to any significant degree. Animal studies support the idea that disruption of pro-inflammatory cytokine action is an excellent target for drug discovery, but that the potential of these compounds is limited by the need for a chronic indwelling subdural catheter to allow daily peri-spinal administration. Pro-inflammatory cytokine synthesis inhibitors. This family of compounds includes propentofylline (Kronos; Aventis) and thalidomide (Thalomid; Celgene). Other compounds (leflunomide, methotrexate, p38-MAPkinase inhibitors and IL-10) also act as inhibitors of pro-inflammatory cytokine synthesis but, in addition, disrupt pro-inflammatory cytokine signalling. These will therefore be discussed separately later. Propentofylline is an orally active xanthine derivative which crosses the bloodbrain barrier and is prescribed for Alzheimers disease and vascular dementia71,72. It has a complex mechanism of action, but its profile of actions strongly predicts its ability to resolve glially driven allodynia and hyperalgesia. Propentofylline has been reported to reduce ischaemia-induced glial activation and proliferation, lipid peroxidation, and IL-1 and TNF production7375. In addition, it increases the uptake of extracellular glutamate76 and increases extracellular adenosine72, an endogenous anti-inflammatory77 and pain suppressor78. In glial cultures, propentofylline inhibits release of TNF, IL-1 and superoxide; IL-6 and nitric oxide release are unaffected79. Given that it is approved for other uses, crosses the bloodbrain barrier, suppresses glial activation and reverses (as well as blocks) neuropathic pain behaviours in animals53, propentofylline would seem to be an intriguing candidate for clinical trials that target glially enhanced pain. However, Aventis is not pursuing further drug development on this compound due to an unfavourable patent situation, inconsistent outcomes of studies as to its efficacy in Alzheimers and vascular dementia, and problems with oral delivery of the drug71. By contrast, thalidomide ([+]--phthalimidoglutarimide) and potentially safer analogues are being actively developed by Celgene for clinical use80. After being banned from the market in 1963 due to the production of severe birth defects81, thalidomide has been approved for treatment of erythema nodosum leprosum80. Thalidomide is orally active and readily crosses the bloodbrain barrier58,82. It has a number of actions that are consonant with its potential use in controlling glially driven exaggerated pain. In vitro, thalidomide and its analogues can inhibit TNF, IL-1 and IL-6, as well as increase IL-10 production 8386. Systemic administration decreases TNF production by immune cells, either decreases or has no effect on IL-6 (REFS 87,88), and has no effect on IL-1. Thalidomide also inhibits TNF- and IL1-induced NUCLEAR FACTOR-B (NF-B) transcriptional activation, probably as a result of inhibiting the degradation of INHIBITOR OF B (IB)82,89. Last, thalidomide and its analogues can increase the concentration of IL-10, an anti-inflammatory cytokine discussed below57,90. Animal models of neuropathy have documented that systemically administered thalidomide delays the development of allodynia/hyperalgesia, and is correlated with reduced TNF, unaltered IL-1 or IL-6, and increased IL-10 at the site of nerve injury56,57. Given the high bloodbrain barrier permeability of this compound, thalidomide might be capable of inhibiting pro-inflammatory cytokine production by spinal cord glia, but this potential has yet to be investigated. Although thalidomide failed to reverse neuropathy induced enhanced nociception in rats56, it is notable that recent human studies report success in relieving longstanding, intense pain due to complex regional pain syndrome91,92. This pain syndrome is not controlled by drugs developed to target neurons, and recently the hypothesis that spinal cord glia contribute to this enigmatic pain syndrome has been proposed3. So thalidomide is a compound worthy of further consideration. How to reconcile these data with reports of thalidomide-induced neuropathy58,59,80 is being examined. Disrupting pro-inflammatory cytokine signalling and synthesis. This group of compounds includes leflunomide (Arava; Aventis), methotrexate (Rheumatrex; Wyeth-Ayerst), inhibitors of p38 MAP kinase, and IL-10. Leflunomide is an orally active malononitrilamide, a disease-modifying anti-rheumatic drug. It is approved for the treatment of rheumatoid arthritis in humans93. It is metabolized by the liver to form the active metabolite A771726 (REF. 94). Leflunomide inhibits several pain-relevant substances. For example, it decreases rheumatoid arthritis synovial tissue expression of IL-1, TNF, nitric oxide and cyclooxygenase-2 (REFS 9597). Also, it disrupts TNF-induced NF-B activation, cytotoxicity, production of reactive oxygen species and lipid peroxidation98. Although this profile indicates that leflunomide would suppress allodynia and hyperalgesia resulting from glial activation, concern is raised by the fact that leflunomide has broad-ranging immunosuppressive effects. This results from its inhibition of the mitochondrial

NUCLEAR FACTOR-B

(NF-B). A transcription factor that is constitutively expressed. Its activation leads (among other effects) to the production of pro-inflammatory cytokines. Although constitutively expressed, its ability to move to the nucleus to bind to DNA is tonically inhibited by binding of IB.
INHIBITOR OF B

(IB). An inhibitor of NF-B activation. It is binding to NF-B that keeps this transcription factor from being able to move to the nucleus to activate messenger RNA transcription.

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Binding Adenovirus CAR

Receptor-mediated endocytosis

Endosome

Lysosome

H+

Nucleus

Figure 6 | One of the newest approaches to the treatment of pathological pain by the control of glial activation. Very recently, a number of laboratories have begun exploring the possibility that interleukin-10 (IL-10) might prove to be a powerful weapon in the battle to control clinical pain states. IL-10 is an anti-inflammatory cytokine, meaning that it naturally serves the role of a powerful negative feedback signal on pro-inflammatory cytokine expression and function. Although intrathecal administration of exogenous IL-10 protein reverses enhanced nociception, the effects last less than one day. So gene therapy, to induce prolonged production and release of IL-10 protein, is being explored. Early data indicate that IL-10 can powerfully suppress every animal model of chronic pain examined to date. Several versions of gene therapy have been demonstrated to work following intrathecal administration, including adenovirus. This figure illustrates the basics of gene therapy, using adenovirus as the example. Adenovirus virons bind to the coxsackie adenovirus receptor (CAR) and integrins on the plasma membrane, and enter the cell by receptormediated endocytosis. As the endosome acidifies (H+), the capsid is broken down and released from the endosome. Double-stranded viral DNA is released from the degraded capsid and enters the nucleus through the nuclear pore. Modified, with permission, from REF. 131 Macmillan Magazines Ltd (2003).

enzyme dihydro-orotate dehydrogenase, an enzyme required for the synthesis of DNA and RNA, cell proliferation and differentiation, phospholipid synthesis and protein glycosylation99,100. So although leflunomide has shown promise in an animal model of neuropathy54, enthusiasm for using this compound to control clinical pain syndromes is dampened by its severe immunosuppressive effects. In addition, no studies have yet examined whether leflunomide can reverse established exaggerated pain states, beyond inhibiting their initial development54. Additionally, no information is available regarding the potential bloodbrain barrier permeability of the active metabolite A771726, nor is much known about its actions on glia. All that is known at present is

that A771726 inhibits induced release of nitric oxide from rat astrocytes101. If A771726 does indeed access the spinal cord following systemic administration, it might, in part, explain its success in controlling inflammatory pain states such as rheumatoid arthritis, which activate spinal cord glia102 as well as synovial immune cells. In contrast to leflunomide, methotrexate is known to cross the bloodbrain barrier103,104. This makes its systemic administration for controlling spinal cord glial activation potentially feasible. Methotrexate is a derivative of glutamic acid that was first marketed to treat neoplastic diseases105. It is orally active, although variably absorbed. Low-dose methotrexate has been

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used in humans to treat autoimmune diseases, including rheumatoid arthritis 105. Its mechanism of action involves elevating the concentration of extracellular adenosine, a potent endogenous anti-inflammatory106,107. Low-dose methotrexate also decreases production of pro-inflammatory cytokines95,108,109, hydrogen peroxide110 and prostaglandin E2 (REF. 110). In addition, methotrexate can increase IL-10 production111. Furthermore, methotrexate has been proposed to be a competitive inhibitor of IL-1 binding to its receptor112, as methotrexate disrupts IL-1-induced effects113,114. This profile indicates that systemic methotrexate would be able to suppress allodynia and hyperalgesia resulting from spinal cord glial activation. As noted previously, methotrexate both prevented and reversed established enhanced nociceptive responses in rats and reduced some anatomical indices of spinal cord glial activation55. However, there are concerns about the use of this drug as an inhibitor of spinal cord glial activation. For example, the dosage needs to be carefully considered. Methotrexate can activate astrocytes (as reflected by GFAP expression), induce astrocyte proliferation, as well as cause degenerative changes in these cells103,104. Indeed, astrocytes are the primary targets for methotrexate neurotoxicity103,115. So dosage would need to be adjusted to avoid potential glia-activating and glia-toxic effects while maintaining beneficial suppression of glial proinflammatory cytokines. Although glia-toxic side effects are a concern for methotrexate, no such concerns arise for inhibitors of p38 MAP kinase. This group of compounds are being developed for clinical targets including Crohns disease, pancreatitis and rheumatoid arthritis116. These compounds vary in their bioavailability, with some being orally active (for example, SCIO-469 and SCIO-323; Scios) and others requiring injections (for example, CNI-1493; Cytokine Pharmasciences). Some, such as CNI-1493 and NPC037282 (Scios) are active in spinal cord after systemic administration51,52. They vary in their cross-reactivity with other intracellular signalling cascades116,117. Highly selective p38 MAP kinase inhibitors are in development49. This group of drugs is intriguing with respect to their potential for controlling glially driven exaggerated pain, for a number of reasons: pro-inflammatory cytokines activate (that is, phosphorylate) p38 MAP kinase118; p38 MAP kinase activation is one of the major intracellular signalling cascades leading to de novo production of pro-inflammatory cytokines47,119; and (as noted above), at least some of these compounds cross the bloodbrain barrier, making systemic administration of the drugs plausible51,52. In rat spinal cord, microglial p38 MAP kinase activation has been detected immunohistochemically in response to nociception-enhancing peripheral inflammation, peripheral nerve injury, spinal nerve ligation and peri-spinal substance P48,49,120,121. p38 MAP kinase inhibitors do not affect basal nociception. Rather, they block exaggerated nociception induced by tissue inflammation17,48, peripheral nerve inflammation19, spinal nerve ligation50,120, spinal cord inflammation18, and peri-spinal NMDA and substance P48,49. Although CNI-1493 reverses established allodynia induced by sciatic nerve inflammation19, SB203580 (Calbiochem) and SD-282 (Scios) failed to reverse established spinal sensitization induced by spinal nerve ligation50 or peripheral inflammation 48. Whether these different outcomes reflect different nociceptive potencies of the models, different durations of phospho-p38 MAP kinase involvement in pain maintenance across different models, the inhibition of Jun N-terminal kinase as well as p38 MAP kinase by CNI-1493 (REFS 116,117) or other factors is not yet clear. One potential issue for this class of drugs is that p38 MAP kinase is expressed by neurons as well as by glia. Peripheral inflammation, chronic constriction injury and spinal nerve ligation increase activated p38 MAP kinase in small-diameter neurons in the dorsal root ganglia, as well as activating p38 MAP kinase in dorsal horn microglia50,120,121. Activation of p38 MAP kinase in dorsal horn neurons, as well as in microglia, occurs in response to topical capsaicin and peri-spinal NMDA49,51. It will be important to identify the downstream effects of p38 MAP kinase activation in neurons, so as to understand the impact of inhibitors on the pain pathway. The last compound to be discussed is the antiinflammatory cytokine IL-10. The use of IL-10 is an intriguing approach to glially driven allodynia and hyperalgesia, as IL-10 can suppress the production and activity of TNF, IL-1 and IL-6. IL-10 can exert this effect by inhibiting: p38 MAP kinase47,60; NF-B activation, translocation and DNA binding60; pro-inflammatory cytokine messenger RNA transcription, stability and translation47,122124; and pro-inflammatory cytokine release61. In addition, IL-10 stabilizes mRNAs of suppressors of cytokine signalling, thereby increasing the production of a family of proteins that further inhibits pro-inflammatory cytokine production60. IL-10 can also interrupt pro-inflammatory cytokine signalling by downregulating the expresion of receptors for pro-inflammatory cytokines125. Last, it can upregulate endogenous antagonists of pro-inflammatory cytokines, including IL-1-receptor antagonist and soluble TNF receptors126,127. The known effects of IL-10 are restricted to the suppression of pro-inflammatory functions of activated immune and glial cells, leaving non-inflammatory aspects of cellular functions unaffected61. Although some neurons express IL-10 receptors, the only known action of IL-10 on neurons is inhibition of cell death (apoptosis)128. Although speculative, this profile of IL-10 actions indicates that IL-10 might be able to suppress the pathological responses of glia, while not markedly altering basal functions of either glia or neurons. Although there are at present no drugs that specifically target IL-10, Avigen is developing gene therapy methods (FIG. 6) to overexpress this anti-inflammatory cytokine for use in exaggerated pain states129, as well as other applications. IL-10 does not cross the bloodbrain barrier130, so systemic administration is not an option. Constant infusion into the spinal cord

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cerebrospinal fluid (CSF) space through an indwelling catheter would not only be extraordinarily expensive given the short half-life of this protein and the cost of producing recombinant human IL-10, but also raises concerns of infection along the chronic catheter track. Given that IL-10 is an endogenous protein, gene therapy can be employed to drive cells surrounding the CSF space to produce IL-10 for pain control and obviate the need for indwelling spinal catheters. Gene therapy technology is rapidly advancing and new methodologies indicate that prolonged (many months to years) release of gene-therapy-induced protein can be attained after a single injection of the vector into the spinal CSF space131. Importantly, methodologies now exist which allow the gene therapy to be controlled. That is, vectors can be engineered such that IL-10 production can be either activated or deactivated in response to systemically administered drugs that induce or suppress IL-10 mRNA transcription132. So although this IL-10 gene therapy is not yet available for human use, this is a potentially exciting new approach for clinical pain control.

Summary

Astrocytes and microglia in the spinal cord are now recognized as active participants in the creation and maintenance of pain facilitation induced by inflammation and damage to peripheral tissues, peripheral nerves, spinal nerves and spinal cord. On activation, these glia release a variety of neuroexcitatory substances that potentiate pain transmission by neurons. Of these glial products, pro-inflammatory cytokines seem to be common spinal mediators of allodynia and hyperalgesia. Given the failure of presently available drugs to provide adequate clinical pain management, this newly recognized role of glia is exciting as it predicts novel approaches for effective pain control by targeting glial activation. As summarized in this review, several compounds seem worthy of further development and testing in the hope of reaching clinical trials for this application. Even more importantly, perhaps, is the message to pharmaceutical and biotechnology companies that spinal cord glia are key players in hyperalgesia and allodynia, and are prime targets for new drug development aimed at the as-yet-elusive outcome: clinical pain control.

1.

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Acknowledgements
Our laboratory is supported by grants from the National Institute on Drug Abuse, National Institute of Neurological Diseases and Stroke, and National Institute of Mental Health.

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Competing interest statement

The authors declare competing financial interests: see Web version for details.

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Online links
DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ CX3CR1 | fractalkine Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/Omim/ Alzheimers disease | Crohns disease | rheumatoid arthritis Access to this interactive links box is free online.

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