Immune and inflammatory mechanism in neuropathic pain

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Review

Immune and inflammatory mechanisms in neuropathic pain

Gila Moalem, David J. Tracey

School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia

A R T I C L E I N F O A B S T R A C T

Article history:

Accepted 17 November 2005

Available online 4 January 2006

Tissue damage, inflammation or injury of the nervous system may result in chronic

neuropathic pain characterised by increased sensitivity to painful stimuli (hyperalgesia), the

perception of innocuous stimuli as painful (allodynia) and spontaneous pain. Neuropathic

pain has been described in about 1% of the US population, is often severely debilitating and

largely resistant to treatment. Animal models of peripheral neuropathic pain are now

available in which the mechanisms underlying hyperalgesia and allodynia due to nerve

injury or nerve inflammation can be analysed. Recently, it has become clear that

inflammatory and immune mechanisms both in the periphery and the central nervous

system play an important role in neuropathic pain. Infiltrationof inflammatory cells, as well

as activation of resident immune cells in response to nervous system damage, leads to

subsequent production and secretion of various inflammatory mediators. These mediators

promote neuroimmune activation and can sensitise primary afferent neurones and

contribute to pain hypersensitivity. Inflammatory cells such as mast cells, neutrophils,macrophages and T lymphocytes have all been implicated, as have immune-like glial cells

such as microglia and astrocytes. In addition, the immune response plays an important role

in demyelinating neuropathies such as multiple sclerosis (MS), in which pain is a common

symptom, and an animal model of MS-related pain has recently been demonstrated. Here,

we will briefly review some of the milestones in research that have led to an increased

awareness of the contribution of immune and inflammatory systems to neuropathic pain

and then review in more detail the role of immune cells and inflammatory mediators.

2005 Elsevier B.V. All rights reserved.

Keywords:

Neuropathic pain

Nerve injury

Immunity

Inflammation

Abbreviations:

5HT, 5-hydroxytryptamine,

serotonin8R, 15S-diHETE, (8R, 15S)-

dihydroxyeicosa-(5E-9,11,13Z)-

tetraenoic acid

ADP, adenosine diphosphate

AMP, adenosine monophosphate

ATP, adenosine triphosphate

BDNF, brain-derived neurotrophic

factor

CCR2, chemokine (CC motif)

receptor 2

CGRP, calcitonin-gene-related

peptide

COX-1, cyclooxygenase-1COX-2, cyclooxygenase-2

CX3CR1, G-protein-coupled

chemokine receptor for fractalkine

DRG, dorsal root ganglion

GABA, gamma aminobutyric acid

GBS, GuillainBarr syndrome

GDNF, glial-cell-line-derived

neurotrophic factor

B R A I N R E S E A R C H R E V I E W S 5 1 ( 2 0 0 6 ) 2 4 0 2 6 4

Corresponding author. Fax: +61 2 9385 8016.E-mail address: [email protected] (D.J. Tracey).

0165-0173/$ see front matter 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.brainresrev.2005.11.004

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s r e v


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IL-1, interleukin-1

L-NAME, NG-nitro-L-arginine methyl

ester

LTB4, leukotriene B4MCP-1, monocyte chemoattractant

protein-1

MHC, major histocompatibility

complex

MIP-1, macrophage inflammatory

protein-1

MRP-14, cytosolic-calcium-binding

protein of 14 kDa

MS, multiple sclerosis

NGF, nerve growth factor

NO, nitric oxide

NOS, nitric oxide synthase

NSAID, non-steroidal

anti-inflammatory drug

NT-3, Neurotrophin-3

NT-4/5, Neurotrophin-4/5

P2X, ATP-gated ion channels

P2Y, G-protein-coupled ion channels

activated by purine/pyrimidine

nucleotides

PAR-2, protease-activated receptor-2

PGE2, Prostaglandin E2PGI2, Prostaglandin I2TCA, tricyclic antidepressant

TEMPOL, 4-hydroxy-2,2,6,6-

tetramethylpiperidine-1-oxyl

Th1 cells, T helper 1 cells

Th2 cells, T helper 2 cells

TLR4, Toll-like receptor 4

TNF, tumour necrosis factor

TrkA, tyrosine kinase receptor A

TrkB, tyrosine kinase receptor B

TrkC, tyrosine kinase receptor C

TRPV1, transient receptor potential

(receptor) vanilloid 1

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

1.1. Chemical environment of sensory neurones contributes to neuropathic pain . . . . . . . . . . . . . . . . . 2421.2. Inflammatory models of neuropathic pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

1.3. Neuropathic pain in autoimmune diseases of the nervous system . . . . . . . . . . . . . . . . . . . . . . . 243

1.4. Direct and indirect actions of mediators released by immune cells . . . . . . . . . . . . . . . . . . . . . . . 243

2. Immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

2.1. Mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

2.2. Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

2.3. Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

2.4. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

2.5. Glia and Schwann cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

3. Mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

3.1. Bradykinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

3.2. ATP and adenosine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

3.3. Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

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3.4. Eicosanoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.5. Cytokines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

3.5.1. Interleukin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 52

3.5.2. Interleukin-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 52

3.5.3. Tumour necrosis factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

3.5.4. Interleukin-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 53

3.6. Neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.7. Nitric oxide and reactive oxygen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2544. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

1. Introduction

Neuropathic pain is caused by lesion or inflammation of

the nervous system and is relatively common, with an

incidence estimated at 0.6% to 1.5% in the US population

(Warfield and Fausett, 2002). It is often severely debilitating

and largely resistant to treatment (Harden and Cohen,

2003) mainly because the underlying mechanisms are still

poorly understood. Symptoms of neuropathic pain may

include allodynia (pain resulting from a stimulus that is

normally non-painful), hyperalgesia (an excessive response

to painful stimuli) and spontaneous pain. The study of

neuropathic pain can be traced back to Weir Mitchell and

his classic work on nerve injuries from the American Civil

War (Mitchell, 1872). Since that time, a great deal has been

written on neuropathic pain and its possible causes, but it

was only with the development of animals models of pain

due to nerve injury that some real progress was made in

understanding some of the mechanisms involved. For

recent reviews of these mechanisms, see Julius and

Basbaum (2001) and Scholz and Woolf (2002). The first

widely used animal model of neuropathic pain was chronic

constriction injury of the rat sciatic nerve (Bennett and Xie,

1988), in which the sciatic nerve is encircled by four

ligatures of chromic gut. This leads to the cardinal

symptoms of neuropathic painhyperalgesia, allodynia

and apparent spontaneous pain. Other widely used animal

models include partial ligation of the sciatic nerve (Seltzer

et al., 1990) and section of one or more of the spinal

nerves which contribute to the sciatic (Kim and Chung,

1992). Research on these animal models of peripheral

neuropathic pain (the pain syndrome resulting from

lesions of the peripheral nervous system) has made it

clear that a number of mechanisms are involved, including

ectopic excitability of sensory neurones, altered gene

expression of sensory neurones and sensitisation of

neurones in the dorsal horn of the spinal cord (Scholz

and Woolf, 2002; Woolf, 2004). However, there is increasing

evidence that inflammatory and immune mechanisms also

play a role, and we will begin by looking at a brief history

of how this evidence has accumulated. We will then look

in more detail at the evidence for involvement of immune

cells and inflammatory mediators in neuropathic pain.

However, the review does not provide exhaustive coverage

of all immune and inflammatory mechanisms that con-

tribute to pain following nerve injury. For readers who

would like to know more about these and other aspects of

neuropathic pain, several recent reviews are available,

dealing with immune and glial mechanisms (Watkins and

Maier, 2002, 2005; DeLeo et al., 2004; McMahon et al., 2005;

Tsuda et al., 2005), inflammatory aspects (Woolf and

Costigan, 1999; DeLeo and Yezierski, 2001; DeLeo et al.,

2004; Sommer and Kress, 2004) and cytokines (Sommer andKress, 2004).

1.1. Chemical environment of sensory neurones

contributes to neuropathic pain

At the time that the first animal models of pain due to

nerve injury were developed, it was widely believed that

injury or loss of myelinated and unmyelinated sensory

axons in the sciatic nerve was the key factor in producing

symptoms of neuropathic pain. However, some studies in

the early 1990s suggested that other factors might also be

involved. For example, Maves showed that chromic gut

alone placed next to the sciatic nerve induced thermal

hyperalgesia without any loss of myelinated axons (Maves

et al., 1993). Of course, it had long been known that C-

polymodal nociceptors are sensitised or activated by

chemical stimuli, including inflammatory mediators (Wood

and Docherty, 1997), but these effects were thought of as

being responsible for inflammatory pain, and little atten-

tion was given to the idea that immune or inflammatory

cells or their mediators might play a role in neuropathic

pain until the advent of useful animal models. It was also

known that nerve injury resulted in activation of mast cells

(Olsson, 1967) and recruitment of neutrophils and macro-

phages (Perry et al., 1987). However, it was not until 1993

that acute changes in the endoneurial microenvironment

were explicitly linked with the initial development of

hyperalgesia (Frisen et al., 1993; Sommer et al., 1993).

Further work confirmed suggestions that the factors

responsible for neuropathic hyperalgesia included Wallerian

degeneration and macrophage activation (Sommer et al.,

1995). At around the same time, Clatworthy followed up the

observations of Maves et al. and showed that suppression

of the inflammatory response in the injured sciatic nerve

blocked the development of hyperalgesia, while enhancing

the inflammatory response augmented the hyperalgesia

(Clatworthy et al., 1995). Evidence on the involvement of

immune cells and inflammatory mediators in hyperalgesia

was reviewed at this time, and it was suggested that these

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mediators (and cytokines in particular) were a probable link

between the activation or recruitment of immune cells to

the injured nerve and the development of hyperalgesia and

neuropathic pain (Tracey and Walker, 1995; Watkins et al.,

1995). Glial cells supporting neurones in the spinal cord

(Meller et al., 1994) and Schwann cells supporting axons in

the peripheral nerve (Constable et al., 1994; Wagner and

Myers, 1996a) were added to the list of cell types whichmight contribute to neuropathic pain. In fact, glial cells and

Schwann cells may also play an active role in the immune

process by releasing mediators, including cytokines, and

acting as antigen-presenting cells following nerve injury

(Argall et al., 1992; Bergsteinsdottir et al., 1992; Constable et

al., 1994). These ideas were not widely accepted at the

time, but, gradually, evidence accumulated to give them

further support. For example, tumour necrosis factor (TNF)

and interleukins 1 and 6 (IL-1, IL-6) had already been

shown to induce acute or short-term hyperalgesia (Ferreira

et al., 1988; Cunha et al., 1992) but were now implicated

directly in neuropathic pain, involving chronic hyperalgesia

and allodynia (Wagner and Myers, 1996b; DeLeo et al., 1997;

Arruda et al., 1998).

Another example is the contribution of nerve growth

factor (NGF), which shares many attributes with typical

cytokines (Bonini et al., 2003). NGF is best known for its

role in protecting some neuronal types from cell death

during development, but Levine and his colleagues showed

that the N-terminal octapeptide of NGF elicited hyperalge-

sia in the rat when applied to injured tissue (Taiwo et al.,

1991). These experiments were provoked by apparent

similarities between the NGF fragment and bradykininan

inflammatory mediator known to activate or sensitise

nociceptors and to elicit hyperalgesia (Dray and Perkins,

1993; Dray, 1997). By this stage, it was known that the level

of NGF rises substantially in inflamed tissue and in injured

nerve (Heumann et al., 1987; Weskamp and Otten, 1987),

and a causal relationship between tissue levels of NGF and

inflammatory hyperalgesia was confirmed by showing that

anti-NGF antibodies reduced inflammatory hyperalgesia

(Lewin et al., 1994; Woolf et al., 1994). NGF was later

shown to contribute to neuropathic hyperalgesia as well

(Herzberg et al., 1997; Theodosiou et al., 1999). The

underlying mechanism is not clear, but it is agreed that

NGF has a cytokine-like action on inflammatory cells

(Otten, 1991) including mast cells, basophils, neutrophils

and lymphocytes. Furthermore, NGF is produced and

released by leukocytes such as macrophages and T

lymphocytes (Vega et al., 2003). NGF is a potent degranu-

lator of mast cells (Pearce and Thompson, 1986), which

appear to play an important role in inflammatory (Woolf et

al., 1996) as well as neuropathic hyperalgesia (Zuo et al.,

2003). It seems likely that mediators released by activated

mast cells contribute to hyperalgesia following nerve injury

histamine has been strongly implicated (Zuo et al., 2003),

but several other mediators may also be involved.

1.2. Inflammatory models of neuropathic pain

It is now apparent that early animal models of neuropathic

pain suffer from the disadvantage that symptoms such as

hyperalgesia are most likely the combined result of nerve

injury itself (e.g. ectopic firing of injured sensory axons) and

the release of algesic mediators by immune cells activated

near the site of injury. In fact, neuropathic pain may develop

without any injury of sensory axons. This was shown in rats

by induction of neuropathic hyperalgesia by placing segments

of chromic gut next to the sciatic nerve (Maves et al., 1993) or

by lesion of the ventral roots of spinal nerves (Li et al., 2002;Sheth et al., 2002). In both cases, typical signs of neuropathic

pain were elicited without damage to sensory axons, implying

that sensitisation or activation of sensory fibres by inflam-

matory mediators is sufficient to cause neuropathic pain. As a

result of observations like these, inflammatory models of

neuropathic pain have been developed in which carrageenan

or complete Freund's adjuvant (Eliav et al., 1999) or zymosan

(Chacur et al., 2001) is applied around the intact sciatic nerve

of the rat. These models should help us to understand the role

of inflammatory and immune mechanisms in neuropathic

pain.

1.3. Neuropathic pain in autoimmune diseases of thenervous system

Autoimmune diseases of the nervous system including

multiple sclerosis (MS) and GuillainBarr syndrome (GBS)

are among the most common disabling neurologic diseases,

which cause not only demyelination with impaired motor

function, but also abnormal sensory phenomena including

chronic neuropathic pain. MS is an autoimmune demyelinat-

ing disease of the central nervous system that causes

relapsing and chronic neurological impairment. Pain is

experienced by approximately 65% of patients with MS at

some time during the course of their disease (Kerns et al.,

2002). GBS is an acute inflammatory demyelinating neuropa-

thy caused by an autoimmune attack on the peripheral

nervous system and characterised by motor disorders such

as weakness or paralysis and variable sensory disturbances

(Hughes et al., 1999). Pain is a common symptom of GBS

occurring in 7090% of cases (Pentland and Donald, 1994;

Moulin et al., 1997). Immune cells including macrophages and

T cells are believed to play a critical role in the pathogenesis of

both MS and GBS and may contribute to the related pain.

Studying neuropathic pain in these autoimmune syndromes

has been hampered by the lack of proper animal models for

autoimmunity-related pain. However, recent study in mice

has shown that transient demyelination of peripheral affer-

ents without axonal loss results in neuropathic pain behav-

iour (Wallace et al., 2003), and a suitable animal model of MS-

related pain has recently been developed using both active

and passive induction of experimental autoimmune enceph-

alomyelitis (Aicher et al., 2004).

1.4. Direct and indirect actions of mediators released by

immune cells

While there is good evidence that inflammatory mediators

contribute to neuropathic pain, it is not clear just how or

where these mediators act. Do they act directly on receptors

located on the cell membrane of the neurone or do they

activate non-neuronal cells (such as mast cells or Schwann



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cells), which then release a secondary mediator that exerts a

direct action on neuronal receptors? Are the neuronal

receptors for chemical mediators located on the nerve

terminals or cell bodies of sensory neurones, even though

these may be many centimetres from the lesion site? The

answer to the first question is that both direct and indirect

mechanisms are likely to operate. Some mediators (such as

prostaglandin E2) probably sensitise nociceptors directly byacting on receptors on the neuronal cell membrane (Pitchford

and Levine, 1991). While IL-1 may also have a direct action on

nociceptors (Sommer and Kress, 2004), it has additional

indirect actions by eliciting the production of prostaglandins

(Cunha et al., 1992) or even more indirectly via neural

pathways from vagal afferents to the brainstem and then to

the spinal cord (Watkins et al., 1995). Theanswer to thesecond

question is that receptors for inflammatory mediators may be

located not only on the neuronal cell body and peripheral

nerve terminals, but also on the axon itself.

Recent work by Grafe and his colleagues makes it clear

that some of the neuronal receptors are located on the axon

itself (Irnich et al., 2002; Lang et al., 2003; Moalem et al.,

2005). This makes it easier to understand how mediators

released by immune cells clustered around the nerve lesion

can influence the excitability of sensory neurones since

these mediators would be released close to axonal receptors

and would not need to diffuse distally to nerve terminals or

proximally to cell bodies. For example, it was shown that

ATP increased the excitability of unmyelinated C-fibres in an

in vitro preparation of an isolated segment of the sural

nerve, in which no cell bodies or nerve terminals were

present. Tissue levels of ATP are raised in inflamed tissue,

and ATP activates nociceptors causing pain and hyperalgesia

(Sawynok and Liu, 2003); it also plays a role in neuropathic

pain (Barclay et al., 2002; Jarvis et al., 2002). It may well be

that ATP contributes to neuropathic pain by its action on

axonal receptors.

2. Immune cells

Several inflammatory and immune-like glial cells have been

implicated in the pathogenesis of neuropathic pain. They

include mast cells, neutrophils, macrophages and T cells in

the peripheral nervous system (Fig. 1) and microglia (Fig. 2)

and astrocytes in the central nervous system. Table 1

summarises immune cells and the mediators they release

with their most significant actions.

2.1. Mast cells

Mast cells are not only critical effector cells in allergic

disorders but are also important initiators and effectors of

innate immunity (Galli et al., 2005). There is a resident

population of mast cells in the peripheral nerve (Olsson,

1968), and these mast cells are degranulated at the site of a

nerve lesion (Olsson, 1967; Zuo et al., 2003). The granules

contain mediators such as histamine, proteases and cytokines

(Metcalfe et al., 1997; Galli et al., 2005), several of which are

capable of sensitising or activating neurones. For example,

histamine sensitises visceral nociceptors (Koda and Mizu-

mura, 2002). Although it elicits itch in normal skin, histamine

application to the skin of patients with neuropathic pain did

not evoke an itch but instead induced a severe increase in

spontaneous burning pain (Baron et al., 2001). Activation of

Fig. 1 Peripheral nerve injury induces activation of resident immune cells as well as recruitment of inflammatory cells to the

nerve. Injury of a peripheral nerveinitiates an inflammatory cascade in whichmast cells residing in the nerve are the first to be

activated. They release mediators such as histamine (hist) and TNF, which sensitise nociceptors and contribute to the

recruitment of neutrophils and macrophages. Mediators released by neutrophils (including the chemokine MIP-1 and the

cytokine IL-1) assist in recruitment of macrophages. Both neutrophils and macrophages in the nerve produce and secrete

mediators such as TNF and PGE2 that can further sensitise nociceptors. Nerve injury also initiates Schwann cell

de-differentiation and the release of several algesic mediators such as pro-inflammatory cytokines, NGF, PGE2 and ATP. These

initial events promote the recruitment of T cells, which can secrete a variety of cytokines depending on their subtype. This

cocktail of mediators serves as a mechanism of enhanced inflammatory response in the injured nerve and contributes to

neuropathic pain.



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mast cells may also cause secretion of mediators without

overt degranulation (Theoharides and Cochrane, 2004) by

synthesis of lipid mediators such as prostaglandin D2 or by

transcription, translation and secretion of a wide range of

cytokines and chemokines (Mekori and Metcalfe, 2000).

Activation and degranulation of mast cells therefore release

some mediators (such as serotonin in the rat) which can elicit

hyperalgesia by a direct action on the nociceptor (Rueff and

Dray, 1993; Sommer, 2004). Some mast cell mediators exert a

broader range of actions by initiating an inflammatory

cascade (Fig. 1). For example, release of histamine, leuko-

trienes and chemokines contributes to recruitment of leuko-

cytes including neutrophils and macrophages (Gaboury et al.,

1995; Malaviya and Abraham, 2000). In fact, recent work in our

laboratory suggests that degranulation of mast cells plays a

key role in the initiation of neuropathic pain since, in rats

subjected to partial ligation of the sciatic nerve, stabilisation of

mast cells with cromoglycate strongly suppressed the devel-

opment of neuropathic pain and reduced the numbers of

neutrophils and macrophages at the lesion site (Zuo et al.,

2003). Treatment of the nerve-injured rats with H1 and H2

histamine receptor antagonists also alleviated neuropathic

hyperalgesia, although to a lesser extent than stabilisation

with cromoglycate. This suggests that nerve injury induces

degranulation of mast cells and release of histamine, whichacts on histamine receptors on endothelial cells to induce

recruitment of leukocytes such as neutrophils and macro-

phages(Yamaki et al., 1998). Both of these cell types contribute

to neuropathic pain (Liu et al., 2000b; Perkins and Tracey,

2000). The question of how mast cells are activated by nerve

injury is still open, but it could be brought about by increased

levels of adenosine (Sawynok et al., 2000) or bradykinin

(McLean et al., 2000). Other mast cell mediators are probably

also involved in the initiation of neuropathic pain. One

example is tryptase, a trypsin-like serine protease that is

specific to mast cells. One of the most prominent mast cell

mediators, it is associated with the mast cell granules and

released by degranulation. Tryptase acts on the protease-

activated receptor-2 (PAR-2), which is known to be present on

primary sensory neurones and to play a role in inflammation.

Recently, it has been shown to trigger inflammatory hyper-

algesia and nociceptive behaviour in rats (Kawabata et al.,

2001; Vergnolle et al., 2001).

2.2. Neutrophils

Neutrophils (or polymorphonuclear leukocytes) are an essen-

tial part of the innate immune system. Their cytoplasm

contains granules and secretory vesicles that can release a

wide range of microbicidal effector molecules including

bactericidal proteins and cytokines, proteinases and reactive

oxygen species (Witko-Sarsat et al., 2000; Faurschou and

Borregaard, 2003). The first steps in migration towards an

inflammatory focus areadhesionto thesurface of thevascular

endothelium (mediated by selectins) followed by rolling and

transendothelial migration. Neutrophils then migrate towards

the inflammatory site up a gradient of chemoattractants.

These include formyl peptides released by bacteria or dying

cells and chemokines released by immune cells (Witko-Sarsat

et al., 2000). Neutrophils contribute to inflammatory hyper-

algesia (Levine et al., 1984, 1985; Bennett et al., 1998b) by

release of the 15-lipoxygenase product 8R, 15S-diHETE (Levine

et al., 1985; White et al., 1990); their release of cytokines such

as TNF (Witko-Sarsat et al., 2000) may also contribute.

Neutrophils are not observed in normal nerves but are found

in significant numbers at the site of injury in injured

peripheral nerves (Perry et al., 1987; Clatworthy et al., 1995;

Perkins and Tracey, 2000; Zuo et al., 2003). Work in our

laboratory showed that depletion of circulating neutrophils at

the time of nerve injury significantly attenuated the induction

of hyperalgesia. However, depletion of neutrophils at day

8 post-injury did not alleviate hyperalgesia after its normal

induction since by this time neutrophil numbers at the site of

nerve injury have declined to negligible levels(Zuo etal.,2003).

It seems likely that nerve injury induces an inflammatory

cascade initiated by mast cell activation. Activated mast cells

release mediators that promote migration of neutrophils from

Fig. 2 Peripheral nerve injury induces glial activation in the

dorsal horn of the spinal cord. Injury to a peripheral nerve

initiates increased release of neurotransmitters such as

glutamate, substance P and possibly ATP from the central

terminals of primary afferents. These neurotransmitters can

activate both second order neurones and glia. For example,

ATP can activate glial cells through binding to their P2X4receptors, and fractalkine released by second order neurones

can activate glia through binding to their CX3CR1 receptors.

The TLR4 receptor is involved in glial activation, but the

nature of its specific ligand in the spinal cord is unclear.These triggers cause production and release of inflammatory

mediators including pro-inflammatory cytokines (such as

TNF and IL-1), glutamate (Glu), prostaglandins (PGs) and

nitric oxide (NO) from glial cells. These agents are then

capable of further enhancing glial activation and production

of inflammatory mediators that sensitise dorsal horn

neurones, thereby contributing to neuropathic pain.



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Table 1 Immune cells, mediators released and actions

Cell type Mediators Actions

Mast cells Cytokines and

chemokines (Galli

et al., 2005; Mekori

and Metcalfe, 2000)

Recruit leukocytes;

IL-1 facilitates the

release of CGRP from

nociceptors (Fukuoka

et al., 1994) and elicits

hyperalgesia (Ferreira

et al., 1988; Follenfant

et al., 1989; Sung et al.,

2004; Zelenka et al.,

2005); TNF sensitises

C-fibres ( Junger and

Sorkin, 2000; Sorkin

et al., 1997) and DRG

neurones (Schfers et

al., 2003a,b) and

produces hyperalgesia

(Cunha et al., 1992;

Schfers et al., 2003a,b;

Zelenka et al., 2005).

Chemokines produce

excitatory effects on

DRG neurons and

produce allodynia

(Oh et al., 2001)

Histamine

(Mekori and

Metcalfe, 2000)

Sensitises nociceptors

(Koda and Mizumura,

2002), recruits

leukocytes (Gaboury

et al., 1995; Yamaki

et al., 1998); enhances

neuropathic pain in

patients (Baron et al.,

2001)

Leukotriene B4

(Galli et al., 2005;

Mekori and

Metcalfe, 2000)

Recruits neutrophils

(Bennett et al.,

1998a,b; Levine et al.,

1984)

Mast cell protease

(e.g. tryptase)

(Metcalfe et al.,

1997)

Activates PAR-2

receptor (Kawabata

et al., 2001; Vergnolle

et al., 2001)

Nerve growth

factor (Bonini et

al., 2003)


(Bennett et al.,

1998a,b; Bennett,

2001; Koltzenburg et

al., 1999a,b), indirectactions via immune

cells and sympathetic

neurones (Bennett et

al., 1998a,b; Lewin et

al., 1994; Woolf et al.,

1996)

Prostaglandins

(PGD2, PGE2) (Galli

et al., 2005; Mekori

and Metcalfe, 2000)

PGE2 sensitises

nociceptors (Taiwo

and Levine, 1989),

depolarises spinal

neurons (Baba et al.,

2001)

Table 1 (continued)


Serotonin (in

murine rodents)

(Galli et al., 2005)


(Lang et al., 1990;

Rueff and Dray, 1993)

and sensory axons

(Moalem et al., 2005)

Neutrophils 8R, 15S-diHETE

(Levine et al., 1986)


(White et al., 1990)

Cytokines

(Witko-Sarsat et al.,

2000) and chemokines

(Scapini et al., 2000)

See above entry for

cytokines and

chemokines

Defensins (Faurschou

and Borregaard, 2003)

Recruit monocytes,

T-cells (Faurschou and

Borregaard, 2003;

Welling et al., 1998)

Reactive oxygen

species (Witko-Sarsat

et al., 2000)

Sensitise neurons in

PNS and CNS (Aley et

al., 1998; Kim et al.,

2004; Liu et al., 2000a,b;

Meller and Gebhart,

1993; Twining et al.,

2004)

Opioid peptides

(Brack et al., 2004)

Anti-nociceptive (Brack

et al., 2004)

Macrophages Cytokines (Nathan,

1987) and

chemokines

See above entry for

cytokines and

chemokines

Prostaglandins

(PGE2, PGI2)

(Nathan, 1987)

PGE2, PGI2 sensitise

nociceptors (Taiwo and

Levine, 1989), PGE2 acts

on spinal neurons (Baba

et al., 2001)

Reactive oxygen

species (Nathan, 1987)

See above entry for

reactive oxygen species

T lymphocytes Pro-inflammatory

cytokines (Mosmann

et al., 1986; Sad et

al., 1995)

Increase

hyperalgesia and

allodynia (Moalem

et al., 2004)

Anti-inflammatory

cytokines (Mosmann

et al., 1986; Sad et al.,

1995)

Decrease hyperalgesia

and allodynia (Moalem

et al., 2004)

Glia and

Schwann

cells

Cytokines and

chemokines

(Hanisch, 2002)

See above entry for

cytokines and

chemokines

Excitatory amino

acids (Araque et al.,

2001; Watkins et al.,

2001)

Modulate synaptic

transmission and

neuronal excitability

(Araque et al., 2001)



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small blood vessels into inflamed tissue (Gaboury et al., 1995),

and neutrophils in turn release mediators such as chemokines

and defensins which are chemotactic for macrophages (Well-

ing et al., 1998; Scapini et al., 2000) (Fig. 1). Finally, it is worth

noting that neutrophils may also have an anti-inflammatory

and anti-nociceptive role. MRP-14 is a calcium-binding protein

that forms a significant proportion of the cytoplasmic protein

in neutrophils. It de-activates macrophages in vitro and

suppresses inflammatory pain in mice (Giorgi et al., 1998).

Under inflammatory conditions, neutrophils may secrete

opioid peptides, which bind to opioid receptors on peripheral

sensory neurones and mediate anti-nociception (Brack et al.,

2004). The evidence for an anti-nociceptive role for neutro-

phils appears to contradict the pro-nociceptive role outlined

above. However, neuropathic pain and inflammatory pain

differ in some respects, even though they share some basic

mechanisms. In particular, a tertiary peripheral site of opioid

analgesia is initiated by acute inflammation which may notbe

activated in neuropathic pain (Bridges et al., 2001). This

difference may help to explain why opioids are less effective

in relieving neuropathic pain than inflammatory pain (Delle-

mijn, 1999).

2.3. Macrophages

Macrophages phagocytose foreign particles such as microbes

but also have an important role in removing injured or dead

tissue. In the context of this review, they play a vital part in

phagocytosing the dead or dying remnants of injured

Schwann cells and axotomised axons in Wallerian degener-

ation (Brck, 1997). There is a resident population of

macrophages in the peripheral nerve and dorsal root ganglia;

their equivalent in the CNS is the microglia. In peripheral

nerve, the resident macrophages are assisted with clearance

of cellular debris by an influx of haematogenous macro-

phages (Bendszus and Stoll, 2003; Mueller et al., 2003).

Clearance of debris is a prerequisite to regeneration in

peripheral nerves (Perry and Brown, 1992; Correale and Villa,

2004), and Wallerian degeneration is delayed in mice with a

genetic defect which delays the recruitment of macrophages

(Perry and Brown, 1992; Araki et al., 2004). Hyperalgesia is

also delayed in these mice, suggesting that macrophages

contribute to pain resulting from nerve injury (Myers et al.,

1996). This suggestion was confirmed by the demonstration

that depletion of macrophages in nerve-injured rats allevi-

ated neuropathic hyperalgesia (Liu et al., 2000b). However,

another study found only a limited role of macrophages inthe generation of mechanical allodynia following nerve

injury (Rutkowski et al., 2000). Furthermore, in a model of

neuropathic pain, the development of mechanical allodynia

was totally abrogated in mice with a knockout for the

chemokine receptor CCR2, involved in monocyte recruitment

(Abbadie et al., 2003). It is noteworthy that macrophages also

invade the dorsal root ganglia following peripheral nerve

lesion and may contribute to neuropathic pain by release of

excitatory agents that generate ectopic activity in sensory

neurones (Hu and McLachlan, 2002).

What are the signals that recruit macrophages? Essential

roles are played by monocyte chemoattractant protein-1,

MCP-1; macrophage inflammatory protein-1, MIP-1; and

the IL-1 (Perrin et al., 2005); the latter two are known to be

released by neutrophils (Witko-Sarsat et al., 2000). Release of

defensin by neutrophils may also be involved (Welling et al.,

1998). Which are the mediators that are released by macro-

phages to cause hyperalgesia? Macrophages secrete prosta-

glandins, including prostaglandin E2 and I2 (Nathan, 1987),

which sensitise primary afferents directly. Prostaglandin

release by macrophages is strongly implicated in neuropathic

pain since inhibition of cyclooxygenase, an enzyme respon-

sible for prostaglandin synthesis, relieves hyperalgesia in

nerve-injured rats (Syriatowicz et al., 1999), and cyclooxygen-

ase-2 is upregulated in macrophages in the injured nerve (Ma

and Eisenach, 2002, 2003b). Other algesic mediators that are

released by macrophages (Nathan, 1987) and most likely

contribute to neuropathic pain include reactive oxygen

species and the cytokines TNF (Sommer et al., 1998b), IL-1

and IL-6 (Sommer and Kress, 2004) (Fig. 1).

2.4. Lymphocytes

Lymphocytes can be classed as B lymphocytes, responsible

for antibody production, T lymphocytes, which are the

mediators of cellular immunity, and natural killer cells.

Both T cells and natural killer cells are found at the site of

the nerve lesion in rat models of neuropathic pain (Cui et

al., 2000). T cells are also found in increased numbers in

the dorsal root ganglia and spinal cord after lesions of the

peripheral nerve, leading to the suggestion that they may

play a role in generating neuropathic pain (Hu and

McLachlan, 2002; Sweitzer et al., 2002). This suggestion

has now been confirmed by the finding that congenitally

athymic nude rats, which lack mature T cells, develop

significantly less mechanical allodynia and thermal hyper-

algesia when subjected to sciatic nerve injury than their

heterozygous littermates (Moalem et al., 2004). T cells are

rather heterogeneous, they can be divided into CD4+

(helper) and CD8+ (cytotoxic) cells, and each of these

subpopulations can be further divided into type 1 and

type 2 subsets according to their cytokine secretion profile

Table 1 (continued)


Nitric oxide (Minghetti

and Levi, 1998)

Activates guanylyl

cyclase, sensitises

neurons in CNS (Luo

and Cizkova, 2000;

Minghetti and Levi,

1998)

Prostaglandins

(Minghetti and Levi,

1998; Muja and

DeVries, 2004)

See above entries

for prostaglandins

ATP (Anderson et al.,

2004; Liu et al., 2005)

Activates nociceptors

(Hamilton and

McMahon, 2000),

increases sensory

C-fibre excitability

(Irnich et al., 2002;

Moalem et al., 2005)



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(Mosmann et al., 1986; Sad et al., 1995). The functions of T

helper 1 (Th1) and Th2 cells correlate well with their

distinctive cytokines. Th1 cells produce IL-2 and interferon-

gamma and are involved in cell-mediated inflammatory

reactions, while Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10

and IL-13, are involved in antibody and allergic responses

and inhibit synthesis of pro-inflammatory cytokines by Th1

cells (Mosmann and Sad, 1996). Th1 and Th2 cytokines aremutually inhibitory for the functions of the reciprocal

phenotype. Recent work by Moalem et al. has demonstrated

that passive transfer of type 1 T cells, which produce pro-

inflammatory cytokines, into nude rats increased their

sensitivity to noxious stimuli to a level comparable with

that of heterozygous rats. By contrast, passive transfer of

type 2 T cells, which release anti-inflammatory cytokines,

into heterozygous rats reduced their pain sensitivity

(Moalem et al., 2004). These findings indicate that T cells

take part in the course of neuropathic pain (Fig. 1) and

suggest that Th1 and Th2 cells have opposite effects on

neuropathic pain as a result of the distinct sets of

cytokines they release. The role of B cells in neuropathic

pain remains to be elucidated.

2.5. Glia and Schwann cells

Glial cells surround and support neurones in the nervous

system and are 10 to 50 times as numerous as neurones. In

the last few years, it has become clear that glia are by no

means passive bystanders in the nervous system but have

important metabolic and immune functions. In the periph-

eral nervous system, glia are represented by Schwann cells,

while, in the CNS, there are three types of glial cell that can

be distinguished the astrocytes and oligodendrocytes

(macroglia) and the microglia. Microglia form a population

of resident macrophages in the CNS, constituting about 5

10% of glial cells in the CNS. In the resting state, they have a

small soma with fine processes, but, when activated by

stimuli such as trauma, ischemia or inflammation, they take

on an amoeboid shape and function as phagocytes ( Vilhardt,

2005). At this stage, they express the major histocompatibil-

ity complex (MHC), which has a role in presenting antigen to

T lymphocytes, and release mediators that include pro-

inflammatory cytokines (Piehl and Lidman, 2001). Astrocytes

represent the largest cell population in the CNS. They closely

interact with neurones to provide active maintenance of

homeostasis by regulating extracellular ion and neurotrans-

mitter concentrations and extracellular pH in their micro-

environment. The relative contribution of microglia and

astrocytes to neuropathic pain is still under investigation.

However, recent studies suggest that microglia are more

important for the initiation, while astrocytes are more

important for the maintenance of neuropathic pain (Colburn

et al., 1999; Raghavendra et al., 2003). In this review, we will

use the term glia for both microglia and astrocytes in the

CNS. Evidence has been accumulating since the early 1990s

that spinal glia contribute to neuropathic pain (DeLeo and

Colburn, 1999; Watkins et al., 2001; McMahon et al., 2005;

Tsuda et al., 2005). Microglia are activated by peripheral

nerve injury (Colburn et al., 1999; Tanga et al., 2004), and

inhibition of microglial activation in the several models of

neuropathic pain inhibits the development of hyperalgesia

and allodynia (Raghavendra et al., 2003; Ledeboer et al.,

2005). Glia are activated by several mediators including ATP,

bradykinin, substance P and prostaglandins (Hosli and Hosli,

1993; Watkins et al., 2001). ATP is known to activate

nociceptors in the periphery by an action on P2X3 receptors

(Cook et al., 1997), but, in the CNS, it activates P2X4 receptors

that are selectively expressed by activated microglia andappear to be required for neuropathic pain (Tsuda et al.,

2005). Recent evidence suggests that activation of microglia

by fractalkine (Milligan et al., 2004; Verge et al., 2004) and by

the Toll-like receptor 4 (TLR4) (Tanga et al., 2005) is also

involved in the initiation of neuropathic pain (Fig. 2).

Fractalkine is a chemokine expressed on the surface of

spinal neurones, which activates the CX3CR1 receptor on

microglia, while the Toll-like receptors recognise invariant

molecular structures of pathogens and specific ligands

released after nerve injury.

Although we have some idea of how glial cells are

activated, there is little concrete evidence on how activated

glial cells regulate neuronal function to bring about symptoms

of neuropathic pain. Activated glia release a number of

mediators that excite spinal neurones (Watkins et al., 2001),

including prostanoids and nitric oxide (Minghetti and Levi,

1998), cytokines and chemokines (Hanisch, 2002), excitatory

amino acids (Araque et al., 2001) and ATP (Anderson et al.,

2004). However, the role and relative importance of these glial

mediators in neuropathic pain still need to be established.

Interestingly, microglia have been recently implicated in

mirror-image pain, pain arising from sites contralateral to

the site of pathology (Koltzenburg et al., 1999b; Twining et al.,

2004). The mechanisms underlying the expansion of pain to

the contralateral site are not clear. However, one possible

mechanism is through spinal gap junction activation. Recent

study has demonstrated that treatment with a gap junction

decoupler reverses neuropathy-induced mirror-image pain

(Spataro et al., 2004). Thus, strong activation of glia at one site

can lead to gap junctional propagation of calcium waves that

activates distant glia, leading to release of pain-enhancing

substances.

Schwann cells in the peripheral nervous system provide

the myelin sheath in myelinated axons and also envelop

small bundles of unmyelinated axons (Remak bundles). The

primary role of Schwann cells was long thought to be the

provision of an insulating sheath around myelinated axons

(facilitating conduction), maintenance of suitable environ-

ment for peripheral axons, and guiding regenerating axons

back to their targets after injury. In recent years, an immune

function for Schwann cells has received increasing recogni-

tion since they interact with the immune system in T-cell-

mediated immune responses by expressing MHC class II

molecules (Bergsteinsdottir et al., 1992). Schwann cells

release several algesic mediators, including TNF (Murwani

et al., 1996; Wagner and Myers, 1996a; Shamash et al., 2002 ),

IL-1 (Bergsteinsdottir et al., 1991; Shamash et al., 2002), IL-6

(Bolin et al., 1995), NGF (Matsuoka et al., 1991), prostaglandin

E2 (Muja and DeVries, 2004) and ATP (Liu et al., 2005).

Schwann cells also express a number of ion channels and

receptors for mediators including glutamate (Liu and Ben-

nett, 2003), ATP (Grafe et al., 1999; Irnich et al., 2001; Liu et



10/25

al., 2005) and IL-1 (Skundric et al., 1997). Given the close

relationship between Schwann cells and peripheral axons

and the presence of axonal receptors for mediators such as

ATP (Irnich et al., 2001, 2002), it seems very likely that

Schwann cells contribute to neuropathic pain by sensitising

or activating the axons of nociceptors (Fig. 1). However, it

has not yet been possible to obtain direct evidence for this

as selective inhibition or depletion of Schwann cells in anintact animal has not yet been achieved.

3. Mediators

The mediators released by inflammatory and immune cells

may act directly to sensitise or activate neurones (usually

nociceptors in the periphery or dorsal horn neurones in the

spinal cord). Alternatively, they may act on a non-neuronal

cell, which on activation releases another mediator that does

act directly on the neurone. These mediators form a long and

increasing list that includes bradykinin, ATP and adenosine,serotonin, eicosanoids, cytokines, neurotrophins and reactive

oxygen species (Dray, 1995). This list is not exhaustive, and

some inflammatory mediators such as protons and histamine

(see the Mast cells section) are not reviewed here.

3.1. Bradykinin

Bradykinin and kallidin are peptides that are formed from

precursors in the blood and other tissues. In the blood,

bradykinin is formed from high molecular weight kininogen

as part of the clotting cascade, while in other tissues kallidin

(lysyl-bradykinin) is formed from a low molecular weight

kininogen. At least two subtypes of bradykinin receptors (B1

and B2) have been characterised on the basis of in vivo and in

vitro functional studies, using selective agonists and antago-

nists of these receptors (Regoli et al., 1998). Bradykinin

produces pain hypersensitivity by sensitising the peripheral

terminals of nociceptors and by potentiating glutamatergic

synaptic transmission in the spinal cord (Wang et al., 2005).

Bradykinin also sensitises nociceptors by disinhibition of the

TRPV1 receptor (Stucky et al., 1998; Di Marzo et al., 2002). B2

receptors are constitutive and are found on a number of cell

types including sensory neurones and microglia (Dray, 1997).

Increased expression of B1 receptors plays a prominent role in

inflammatory hyperalgesia (Dray and Perkins, 1993; Dray,

1997), and this upregulation is facilitated by cytokines such as

IL-1 (Marceau, 1995). Bradykinin acts mainly on the B2

receptor, while des-Arg9-bradykinin, the major metabolite of

bradykinin, has high affinity for the B1 receptor but little

affinity for the B2 receptor (Dray and Perkins, 1993; Khasar et

al., 1995). Both B1 and B2 receptors have been shown to be

involved in peripheral inflammatory hyperalgesia (Dray and

Perkins, 1993; Dray, 1997). Bradykinin stimulates macrophages

to release TNF and IL-1 (Tiffany and Burch, 1989), as well as

factors that are chemotactic for neutrophils and monocytes

(Sato et al., 1996). Bradykinin also elicits the release of

histamine from mast cells but does so by non-specific binding

tothe cell surface rather than toB1 or B2receptors (Reissmann

et al., 2000). In fact, peptide antagonists of B1 or B2 receptors

may also bind non-specifically to the cell surface and elicit

degranulation (Griesbacher and Rainer, 1999).

Injury of the sciatic nerve in rats led to upregulation of B2

and B1 receptors on lumbar dorsal root ganglia (Eckert et al.,

1999; Levy and Zochodne, 2000), and in rats with a chronic

constriction injury of the sciatic nerve, constant infusion of B2

or B1 receptor antagonists reduced hyperalgesia (Yamaguchi-

Sase et al., 2003). A recent study in nerve-injured mice foundthat B2 receptors were mainly expressed by the cell bodies of

unmyelinated axons and that their expression was drastically

decreased after nerve injury. By contrast, B1 receptors were

newly expressed on the cell bodies of myelinated axons,

suggesting that nerve injury induced a switch in the type of

sensory neurone and the type of bradykinin receptor involved

in nociception (Rashid et al., 2004). Bradykinin and its B2

receptor have recently been implicated in central pain

transmission. This work showed that the B2 receptor is

expressed by dorsal horn neurones and is involved in central

sensitisation evoked by primary afferents (Wang et al., 2005).

Activation of B2 receptors on glial cells in the CNS could play a

role in neuropathic pain, butthere isno evidencefor this asyet.

3.2. ATP and adenosine

Purines such as ATP and adenosine are well known for their

role in energy metabolism but also have widespread effects on

neurones and immune cells. ATP is a classical neurotrans-

mitter but is also released by non-neuronal cells and from

disrupted cells in injured tissue (Cook and McCleskey, 2002).

Adenosine is a neuromodulator that is produced in the

extracellular space by degradation of extracellular ATP and

is also transported from the cytoplasm into the interstitial

space by transport proteins (Linden, 1994). Inosine is produced

by deamination of adenosine. It mayreach millimolar levelsin

ischemic tissue and can degranulate mast cells ( Jin et al.,

1997).

In 1977, Bleehen and Keele reported that ATP, ADP, AMP

andadenosine allproducedpain when applied to a blister base

in man (Bleehen and Keele, 1977). Since then, it has been

found that adenyl compounds and other purines activate

nociceptors and elicit pain by their actions on purinergic

receptors (Burnstock and Wood, 1996; Sawynok, 1998; Hamil-

ton and McMahon, 2000). Receptors for purines have been

classified into adenosine (P1) receptors and nucleotide (P2)

receptors (Ralevic and Burnstock, 1998). P2 receptors can be

subdivided into P2X receptors, which are ATP-gated ion

channels (Chizh and Illes, 2001), and P2Y receptors, which

are G-protein coupled receptors activated by purine or

pyrimidine nucleotides such as ATP or UTP (von Kugelgen

and Wetter, 2000; Burnstock and Knight, 2004).

Initial experiments on the role of ATP in neuropathic pain

implicated P2X3 receptors, although results were apparently

contradictory. Immunocytochemical studies in rats and

humans found that P2X3 receptor expression was significantly

reduced after axotomy (Bradbury et al., 1998; Yiangou et al.,

2000) or partial nerve ligation (Kage et al., 2002), while the

number of P2X3 immunoreactive neurones in the DRG or

trigeminal ganglia was increased after chronic constriction

injury of peripheral nerves (Eriksson et al., 1998; Novakovic et

al., 1999). This apparent discrepancy may be due to the



11/25

different types of nerve injury, leading to downregulation of

P2X3 receptors in injured cells but upregulation in intact cells

(Tsuzuki et al., 2001; Kennedy et al., 2003). Electrophysiological

recordings from DRG neurones supported the idea that

axotomy results in an increase in purinergic sensitivity

(Chen et al., 2001), most likely mediated by P2X receptors

(Zhou et al., 2001). While these results are suggestive, they do

not directly address the issue of neuropathic pain. This wasdone by reducing the level of expression of P2X3 subunits in

DRG cells with intrathecal administration of antisense oligo-

nucleotides. In the partial nerve ligation model of neuropathic

pain, this inhibited the initiation of mechanical hyperalgesia

and reversed established hyperalgesia (Barclay et al., 2002).

This result was confirmed using a novel non-nucleotide

antagonist of P2X3 and P2X2/3 activation. Blocking these

receptors in the chronic constriction model of neuropathic

pain attenuated both thermal and mechanical allodynia

(Jarvis et al., 2002).

P2X4 and P2X7 receptors have also been implicated in

neuropathic pain. P2X4 receptors are upregulated in spinal

microglia following nerve injury, and pharmacological block-

ade or inhibition of P2X4 receptors reversed the accompa-

nying allodynia (Tsuda et al., 2003; Inoue et al., 2004). P2X7receptors are expressed by immune cells, including mast

cells, macrophages and T lymphocytes. They have a key role

in the secretion of IL-1. Recently, it was shown that

disruption of the P2X7 purinoreceptor gene abolishes neuro-

pathic pain and that the receptor is upregulated in human

DRGs and injured nerves from neuropathic pain patients

(Chessell et al., 2005).

P2Y1 and P2Y2 receptors are found in sensory neurones

including nociceptors (Molliver et al., 2002; Xiao et al., 2002;

Stucky et al., 2004) and in Schwann cells (Mayer et al., 1998). In

normal rats, P2Y1 mRNA is found in about 20% of DRG

neurones(large as well as small), butthis percentage increases

to about 70% after section of the sciatic nerve, suggesting a

possible role in neuropathic pain (Xiao et al., 2002). Intrathecal

administration of P2Y receptor agonists appears to have an

inhibitory effect on neuropathic pain, acting via P2Y2 and/or

P2Y4 receptors and P2Y6 receptors (Okada et al., 2002).

Adenosine elicits cutaneous pain and hyperalgesia when

applied peripherally (Bleehen and Keele, 1977; Taiwo and

Levine, 1990a). It also activates axonal receptors on human C-

fibres (Irnich et al., 2002; Lang et al., 2002). However, its role in

the CNS is primarily inhibitory (see Sawynok and Liu, 2003 for

review). Thus, in rats with spinal nerve ligation, spinal

administration of adenosine resulted in a dose-dependent

reduction in tactile allodynia (Lavand'homme and Eisenach,

1999). Inhibition of adenosine kinase increases the availability

of extracellular adenosine in the spinal cord, and spinal

administration of adenosine kinase inhibitors relieves tactile

allodynia or suppresses neuronal responses in nerve-injured

rats (Suzuki et al., 2001; Zhu et al., 2001). These inhibitors may

therefore hold some promise for the treatment of patients

with neuropathic pain.

3.3. Serotonin

Serotonin (5-hydroxytryptamine, 5HT) is a neurotransmitter

synthesised and released by neurones in the CNS. Messenger

RNAs for 5HT1B, 5HT1D, 5HT2A,5HT2C, 5HT3 and 5HT7 receptors

have been found in dorsal root ganglia (Pierce et al., 1996), and

5HT receptors are also found in the cytoplasm of Schwann

cells (Yoder et al., 1997).

There is a large projection of serotonergic neurones from

the raphe nuclei to laminae 1 and 2 of the spinal cord, and

these serotonergic axons contribute to the descending inhibi-

tion of pain (Millan, 2002). While serotonin suppressesnociception at the level of the spinal cord, it enhances it in

the periphery, where serotonin sensitises nociceptors to

thermal stimuli and to bradykinin application (Lang et al.,

1990; Rueff and Dray, 1993). Serotonin also increases the

excitability of sensory C-fibres in isolated segments of

peripheral nerve (Moalem et al., 2005). In the periphery,

serotonin is not released as a neurotransmitter by the axon

terminals of serotonergic neurones but is an inflammatory

mediator released by platelets and murine mast cells (seroto-

nin is not released by human mast cells). When injected into

the rat's paw, it induces a hyperalgesia thought to bedue to an

action on 5HT1A receptors (Taiwo and Levine, 1992; Hong and

Abbott, 1994).

Work in our laboratory suggested that serotonin con-

tributes to established mechanical hyperalgesia in the

partial ligation model of neuropathic pain. Subcutaneous

injection of a 5HT2A receptor blocker or a 5HT3 receptor

blocker alleviated hyperalgesia in a dose-dependent fashion

when injected into the hindpaw of the nerve-injured limb.

Injection of the blockers into the contralateral hindpaw did

not relieve hyperalgesia, suggesting that the effect was

mediated locally rather than systemically (Theodosiou et

al., 1999). Other research on the involvement of serotonin in

neuropathic pain has focussed on its role in the descending

inhibition of pain. Tricyclic antidepressants (TCAs) are

widely used to treat neuropathic pain in patients and inhibit

reuptake of monoamines such as serotonin and noradren-

aline into nerve terminals. This is thought to increase the

efficacy of descending monoaminergic pathways that inhibit

pain. Some TCAs are effective in alleviating neuropathic

pain in animal models, but selective inhibitors of serotonin

reuptake were not effective in animal models or in patients

(Max et al., 1992; Jett et al., 1997). However, fenfluramine,

which not only inhibits serotonin reuptake but also elicits

releases of serotonin from presynaptic terminals, produced a

significant reduction in mechanical allodynia in rats with

tight ligation of the L5/L6 spinal nerves (Wang et al., 1999).

Serotonin reuptake is mediated by the serotonin transporter

(5HTT), and mice deficient for the serotonin transporter did

not develop thermal hyperalgesia following chronic constric-

tion of the sciatic nerve (Vogel et al., 2003). Whether this was

due to altered tissue levels of serotonin or downregulation of

5HT receptors was not clear.

3.4. Eicosanoids

Arachidonic acid is a polyunsaturated fatty acid that is found

in the cell membrane. It is the major precursor of the

eicosanoids, and, once it has been freed from the cell

membrane by phospholipase A2, it is converted by enzymes

in the arachidonate cascade to compounds that include

prostaglandins, thromboxanes and leukotrienes.



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Arachidonic acid is metabolised to prostanoids by the

cyclooxygenase pathway and to leukotrienes by the lipox-

ygenase pathway (Wolfe and Horrocks, 1994; Smith et al.,

2000). It may also be converted to isoprostanes by peroxida-

tion; the isoprostanes are inflammatory mediators that

augment nociception (Evans et al., 2000).

Prostaglandins PGE2 and PGI2 induce hyperalgesia in the

periphery (Taiwo and Levine, 1990b) where they act directlyon the terminals of nociceptors (Taiwo and Levine, 1989)

and probably on macrophages as well (Ma and Eisenach,

2003a). More recently, it has been established that prosta-

glandins also contribute to nociception at the level of the

spinal cord (Malmberg and Yaksh, 1992; Vanegas and Schaible,

2001).

There are two primary isoforms of cyclooxygenase, COX-1

and COX-2. COX-1 is expressed constitutively in most tissues

and has a homeostatic or housekeeping role, while COX-2

expression is usually lowbut canbe induced by factors such as

inflammatory cytokines in cells including mast cells, macro-

phages (Smith et al., 2000) and spinal neurones (Samad et al.,

2002). In fact, COX-1 may also be induced in some conditions

such as spinal cord injury (Schwab et al., 2000; Samad et al.,

2002). Prostaglandins exert their effects by actions on G-

protein-coupled receptors including the EP receptors (EP14 for

PGE2) and the IP receptor for PGI2 or prostacyclin (Hata and

Breyer, 2004).

Work in our laboratory showed that mechanical hyper-

algesia in nerve-injured rats wasalleviated for up to 10 days by

subcutaneous injection of indomethacin (a classic inhibitor of

cyclooxygenase) into the affected hindpaw. Subcutaneous

injection of selective COX-2 inhibitors or an EP1 receptor

blocker relieved thermal as well as mechanical hyperalgesia,

but with a shorter timecourse. We concluded that increased

expression of prostaglandins in the region of the nerve lesion

contributed to neuropathic pain (Syriatowicz et al., 1999). It

was then shown in several animal modelsof neuropathic pain

that the number of COX-2 immunoreactive cells was dramat-

ically increased in the region of the nerve lesion, that most of

these cells were infiltrating macrophages (Ma and Eisenach,

2002, 2003b) and that this was the case in human patients as

well as nerve-injured rats (Durrenberger et al., 2004). In fact,

the increase in COX-2 expression appears to be biphasic, with

an early increase in Schwann cells (1 day after spinal nerve

injury) and an increase in macrophage expression at about 7

14 days (Takahashi et al., 2004). As one might expect,

increased levels of PGE2 are found in the injured nerves

(Schfers et al., 2004). Furthermore, cells immunoreactive for

EP receptors are found in the injured nerve, but not in normal

intact nerves. Once again, many of the immunoreactive cells

prove to be macrophages (Ma and Eisenach, 2003a). These

observations, based on several animal models of sciatic nerve

injury, support the idea that upregulation of COX-2 and EP

receptorsin the injured nerve contributes to neuropathic pain.

However, in the spared nerve injury model (Decosterd and

Woolf, 2000), where degenerating axons distal to the lesion do

not mingle with intact axons, treatment with the COX-2

inhibitor rofecoxib had no effect on the development of

allodynia and hyperalgesia (Broom et al., 2004).

Increased expression of cyclooxygenase after nerve injury

is not restricted to the nerve itself. COX-2 is also upregulated

in the dorsal horn of the spinal cord and thalamus following

peripheral nerve injury (Zhao et al., 2000), and the resulting

allodynia is attenuated by intrathecal injection of ketorolac, a

COX-1 preferring inhibitor (Ma et al., 2002). COX-1 is expressed

constitutively in glial cells and motor neurones of normal rats.

It is also upregulated after ligation of L5L6 spinal nerves so

that the number of cells immunoreactive for COX-1 is

increased in the superficial laminae of the dorsal horn at 4days after spinal nerve ligation and remains high for at least 2

weeks (Zhu and Eisenach, 2003). How do increased levels of

prostaglandins in the spinal cord mediate neuropathic pain?

There is evidence for at least twomechanisms. Thefirst is that

PGE2 depolarises wide dynamic range neurones in the deep

dorsal horn (Baba et al., 2001), and the second is that PGE2 also

blocks glycinergic inhibition of neurones in the superficial

laminae of the dorsal horn (Ahmadi et al., 2002; Harvey et al.,

2004).

Given the evidence for a role of cyclooxygenases in the

development of neuropathic pain and the efficacy of cycloox-

ygenase inhibitors in treating inflammatory pain, it is

surprising that COX inhibitors are relatively ineffective in the

treatment of neuropathic pain in patients (Namaka et al.,

2004). This may be due to the predominant role of prostaglan-

dins in the development rather than maintenance of neuro-

pathic pain or that higher effective concentrations of COX

inhibitors are achieved in experimental animals than in

patients.

There are three mammalian lipoxygenases, which catalyse

the insertion of oxygen at positions 5, 12 and 15 of arachidonic

acid. These are therefore referred to as 5-, 12- and 15-

lipoxygenases. The 5-lipoxygenases form leukotrienes, and

leukotriene B4 (LTB4) produces hyperalgesia by eliciting the

release of a mediator from neutrophils (Levine et al., 1984,

1985), which was identified as the 15-lipoxygenase product

(8R, 15S)-dihydroxyeicosa-(5E-9,11,13Z)-tetraenoic acid (8R,

15S-diHETE) (Levine et al., 1986). This was shown to sensitise

nociceptors in the rat and may contribute to the role of

neutrophils in neuropathic pain (see the Neutrophils section).

Nerve growth factor elicits hyperalgesia when injected into

the rat paw, and inhibition of 5-lipoxygenase inhibits release

of LTB4, accumulation of neutrophils and the associated

hyperalgesia (Amann et al., 1996; Bennett et al., 1998b). It is

likely that NGF produces hyperalgesia by inducing the release

of LTB4 from mast cells, leading in turn to recruitment of

neutrophils (Bennett et al., 1998b), which then release algesic

mediators and chemokines. Neutrophils and nerve growth

factor have both been directly implicated in the genesis of

neuropathic pain (Herzberget al., 1997; Theodosiou et al., 1999;

Perkins and Tracey, 2000), and so it is likely that LTB4 and 8R,

15S-diHETE are also involved.

3.5. Cytokines

Cytokines are small proteins that mediate interactions

between cells over relatively short distances. They are mostly

involved in responses to disease or infection. Many of them

are referred to as interleukins, denoting a mediator released

by one leukocyte and acting on another, but they are

synthesised by most cell types. Several are pro-inflammato-

ry, such as IL-1, IL-6 and TNF, while others such as IL-10 are



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anti-inflammatory. Exogenous administration of these pro-

inflammatory cytokines elicits pain and hyperalgesia (Som-

mer and Kress, 2004), but they do not appear to be involved

in normal pain (Watkins et al., 2001). In fact, these three

pro-inflammatory cytokines induce the production of each

other, and they act synergistically (Watkins et al., 1999). This

leads to the possibility of positive feedback, which could lead

to chronic pain if not appropriately regulated. Other cyto-kines such as IL-4 and IL-10 are anti-inflammatory and

suppress genes that code for IL-1, TNF and chemokines. The

algesic effects of pro-inflammatory cytokines are often

indirect, so that they may not act directly on the nociceptor

but instead induce the expression of agents (such as PGE2)

that themselves sensitise nociceptors. In inflammation and

Wallerian degeneration, cytokine production is organised

into a sequence with TNF playing a leading role (Shamash et

al., 2002; Cunha et al., 2005). This sequence appears to be one

of the mechanisms underlying neuropathic pain (for reviews,

see DeLeo and Colburn, 1999; Sommer, 2001; Sorkin, 2002;

Sommer and Kress, 2004). Chemokines are small chemotactic

cytokines that are important for leukocyte migration and

recruitment to damaged sites. Recently, they have been

shown to contribute directly to nociception by producing

excitatory effects on DRG neurones and inducing allodynia

after injection into the rat's paw (Oh et al., 2001). The

contribution of chemokines to pain has been reviewed

recently (Abbadie, 2005; White et al., 2005).

3.5.1. Interleukin-1

Binding of IL-1 to its receptor IL1-RI on the cell surface

initiates several signalling events, such as translocation of NF-

B into the nucleus (Dinarello, 1999). NF-B then upregulates

transcription of several genes, including COX-2, inducible

nitric oxide synthase, TNF, IL-1 and IL-6 (Pahl, 1999; Tegeder

et al., 2004). IL-1 may act directly as well as indirectly on

nociceptors. Thus, Fukuoka and colleagues found that IL-1

facilitated release of CGRP from nociceptors in an in vitro

nerve-skin preparation. The latency of the effectwas too short

for upregulation of receptors or changes in gene expression,

and the cell bodies were not present, suggesting a direct

sensitisation of nociceptors (Fukuoka et al., 1994; Sommer and

Kress, 2004). IL-1 elicits hyperalgesia when injected periph-

erally into the rat paw (Ferreira et al., 1988; Follenfant et al.,

1989), intraneurally into rat sciatic nerve (Zelenka et al., 2005),

intrathecally in the rat spinal cord (Sung et al., 2004) or

centrally into various regions of the brain (see Bianchi et al.,

1998 for review). In fact, Watkins and her colleagues have

shown that IL-1 may induce an illness hyperalgesia via a

kind of supraspinal loop involving sensory fibres in the

vagus nerve, the nucleus of the solitary tract, nucleus raphe

magnus and projections to the spinal cord (Watkins et al.,

1994; Watkins and Maier, 1999). IL-1 is implicated in

neuropathic pain since IL-1 and IL-1 are both upregulated

in injured peripheral nerve (Gillen et al., 1998; Okamoto et

al., 2001; Shamash et al., 2002). Neuropathic pain is

alleviated in nerve-injured mice by administration of neu-

tralizing antibodies to the IL-1 receptor (Sommer et al., 1999;

Schfers et al., 2001) and in nerve-injured rats by intrathecal

IL-1 receptor antagonist in combination with soluble TNF

receptor (Sweitzer et al., 2001).


IL-6 is synthesised by many cell types, including mast cells,

monocytes, lymphocytes, neurones and glial cells. Once IL-6

has bound to its receptor IL-6R, it initiates two major

intracellular cascadesone involving Janus kinases and

STAT factors, the other using the Ras-dependent MAP kinase

pathway (De Jongh et al., 2003). Injury of the sciatic nerve

induces upregulation of IL-6 and its receptor in the region ofthe lesion, where it appears in macrophages and Schwann

cells (Bolin et al., 1995; Kurek et al., 1996; Ito et al., 1998; Grothe

et al., 2000). Upregulation of IL-6 in these macrophages

appears to be induced by PGE2 (Ma and Quirion, 2005). Sciatic

nerve injury also increases IL-6 levels in the DRG and spinal

cord, particularly in the superficial laminae of the dorsal horn

(Murphy et al., 1995; DeLeo et al., 1996; Lee et al., 2004).

Exogenous IL-6 induces thermal hyperalgesia when injected

into the lateral cerebral ventricles of the rat (Oka et al., 1995)

and increases the heat-evoked release of CGRP from cutane-

ous nociceptors (Opree and Kress, 2000; Obreja et al., 2002).

However, there is little evidence that IL-6 plays a role in

normal nociception. One study did report that intraplantar

injection of IL-6 induced mechanical hyperalgesia in naiverats

(Cunha et al., 1992). However, this finding has not been

reproduced and later work found that intraplantar IL-6 had no

effect on mechanical thresholds (Czlonkowski et al., 1993) or

produced a thermal hypoalgesia (Flatters et al., 2004). In fact,

peripheral IL-6 inhibitedheat responses of nociceptors in an in

vitro preparation.

There is limited evidence that IL-6 contributes to the

development of neuropathic pain. In the in vivo spinal cord,

peripheral IL-6 inhibited the responses of dorsal horn neu-

rones to thermal and mechanical stimuli; after spinal nerve

ligation, peripheral IL-6 released the inhibition of mechanical

responses (Flatters et al., 2004). In IL-6 knockout mice,

nociceptive responses to thermal and mechanical stimuli are

the same as in wild-type mice (Bianchi et al., 1999; Murphy et

al., 1999), but the development of mechanical allodynia after

spinalnervelesion is delayed in IL-6 knockoutsby comparison

with wild-type mice (Ramer et al., 1998). It is not clear whether

thermal hyperalgesia induced by a spinal nerve lesion is

reduced in IL-6 knockouts (Murphy et al., 1999) or not (Ramer

et al., 1998).

3.5.3. Tumour necrosis factor

Tumour necrosis factor (TNF, TNFSF2, formerly TNF) is a

member of a large superfamily of proteins, which have an

unusual trifold symmetry. There is an equally large super-

family of receptors; the receptors activated by TNF are the

constitutively expressed TNFR1 (TNFRSF1A, p55-R) and the

inducible TNFR2 (p75-R) (Locksley et al., 2001). TNFR1 is linked

to pathways for cell death, whereas TNFR2 is not. However,

activation of either receptor results in p38 MAP kinase

signalling (Schfers et al., 2003b), translocation of NFB to

the nucleus and activation of COX-2-dependent prostanoid

release, as described above for IL-1 (Dinarello, 1999). TNF is

constitutively expressed in cutaneous mast cells (Walsh et al.,

1991), but, in injury or inflammation, it may be released by

other cells including neutrophils and macrophages. Intraplan-

tar injection of TNF into the rat's paw induces mechanical

hyperalgesia (Cunha et al., 1992). This can be attributed to



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sensitisation of C-fibres since subcutaneous injection in the

distribution of the sural nerve lowered the thresholds of

cutaneous C-fibres to mechanical stimulation and led to

ongoing activity in some of these fibres ( Junger and Sorkin,

2000). In fact, topical application of TNF to a restricted portion

of the sciatic nerve led to ectopic firing of A- and C-fibres

(Sorkin et al., 1997), and intraneural injection of TNF into rat

sciatic nerve at physiological doses induced thermal hyper-algesia and mechanical allodynia (Zelenka et al., 2005),

suggesting the presence of TNF receptors on the axons. An

alternative possibility is insertion of the TNF trimer into the

cell membrane of the axon (Baldwin et al., 1996), creating a

pore that is permeable to sodium ions and may be voltage-

dependent (Kagan et al., 1992).

Injury of the sciatic nerve leads to upregulation of TNF and

its receptors in the nerve (George et al., 1999; Shubayev and

Myers, 2000; George et al., 2005); this upregulation is found

mainly in Schwann cells and endothelial cells (Wagner and

Myers, 1996a). Nerve injury also leads to increased TNF

expression in the dorsal horn of the spinal cord and in the

locus coeruleus and hippocampus (Ignatowski et al., 1999). An

important role for TNF in neuropathic pain is indicated by

reducing TNF levels in nerve-injured rats or mice (Sommer,

2001). Inhibiting TNF synthesis with thalidomide or treatment

with anti-TNF neutralising antibodies at the time of nerve

injury blocked the development of hyperalgesia and allodynia

in these animal models (Sommer et al., 1998a,b, 2001a). Treat-

ment with etanercept, a recombinant TNF receptor (p75)-Fc

fusion protein that acts as a TNF antagonist, reversed estab-

lished hyperalgesia in mice with a chronic constriction injury

of the sciatic nerve (Sommer et al., 2001b), and endoneurial

injection of neutralising antibodies against TNF receptors

showed that neuropathic hyperalgesia is dependent on the

TNFR1 receptor (Sommer et al., 1998b). Furthermore, applica-

tion of exogenous TNF to the DRG increased the sensitivity of

injured and adjacent uninjured primary sensory neurones and

elicited faster onset of allodynia and spontaneous pain

behaviour after spinal nerve ligation (Schfers et al., 2003a).


IL-10 is generally regarded as anti-inflammatory. Its expres-

sion increases gradually over at least 6 weeks following nerve

injury (Okamoto et al., 2001), and treatment with a single dose

of IL-10 at the site of a chronic constriction injury significantly

reduces hyperalgesia, probably due in part to suppression of

TNF expression and macrophage recruitment (Wagner et al.,

1998). These results are consistent with findings that thalid-

omide (an inhibitor of TNF synthesis) decreases TNF levels,

increases endoneurial IL-10 levels, and alleviates hyperalgesia

in rats with a CCI nerve lesion (Sommer et al., 1998a; George et

al., 2000). Recent work confirms theefficacyof IL-10 in alleviat-

ing neuropathic pain, using virally driven spinal production of

IL-10 in neuropathic pain models (Milligan et al., 2005a,b).

3.6. Neurotrophins

The neurotrophins are dimeric proteins that are essential for

the normal development of the vertebrate nervous system.

This family includes nerve growth factor (NGF), brain-derived

neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5.

Glial-cell-line-derived neurotrophic factor (GDNF) also has

neurotrophic properties but is structurally unrelated to the

dimeric neurotrophin family (Lewin and Barde, 1996).

The neurotrophins act on a trio of tyrosine kinase (Trk)

receptors U TrkA, TrkB and TrkC, which primarily bind NGF,

BDNF and NT-4/5, and NT-3 respectively. There is also a low

affinity or pan-neurotrophin receptor, p75NTR, which acti-

vates a separate set of signalling pathways that interact withthose activated by Trk receptors (Huang and Reichardt, 2003;

Nykjaer et al., 2005). During embryonic development, all

small-diameter sensory neurones express TrkA, the high

affinity receptor for NGF. Most of these neurones will become

nociceptors, with thinly myelinated axons (A fibres) or

unmyelinated axons (C-fibres). After birth, the expression of

TrkA is downregulated so that TrkA is only expressed by those

small sensory neurones which are peptidergic, whereas the

non-peptidergic remainder (IB4 binding neurones) now

expresses receptor components for another neurotrophin,

GDNF (Bennett, 2001).

Neurotrophins are synthesised and released by a several

cell types of immune cells, including mast cells and lympho-

cytes (Moalem et al., 2000; Bonini et al., 2003). BDNF is

constitutively expressed by peptidergic nociceptors (Thomp-

son et al., 1999).

Nerve injury produces marked changes in expression of

neurotrophins and their receptors, primarily in Schwann cells

(Frostick et al., 1998). Schwann cells in the intact peripheral

nerve synthesise NGF, BDNF and GDNF, and this synthesis is

dramatically upregulated by nerve injury (Lindholm et al.,

1987; Meyer et al., 1992; Hammarberg et al., 1996). BDNF

synthesis is also upregulated in sensory neurones following

axotomy (Tonra et al., 1998). In the intactsciatic nerve, mRNAs

for the neurotrophin receptors TrkB and TrkC are expressed,

but TrkA and p75NTR mRNAs are undetectable. Levels of

p75NTR mRNA are markedly increased in Schwann cells after

nerve injury, but TrkA levels remain undetectable (Funakoshi

et al., 1993).

It is now recognised thatsome neurotrophins (in particular,

NGF and BDNF) play a significant role in nociception (Bennett,

2001) so that NGF sensitises nociceptors in the periphery

(Koltzenburg et al., 1999a) while BDNF increases the respon-

siveness of dorsal horn neurones in the spinal cord (Pezet et

al., 2002). Intraplantar or systemic injection of NGF induces

hyperalgesia (Lewin et al., 1994; Andreev et al., 1995; Theodo-

siou et al., 1999). Tissue levels of NGF are increased by

inflammation, and the hyperalgesia resulting from inflamma-

tion is prevented by administration of anti-NGF neutralising

antibodies or a TrkA-IgG fusion molecule (Woolf et al., 1994;

McMahon et al., 1995). Some of the hyperalgesic action of NGF

is exerted directly on sensory neurones, but indirect actions

via mast cells, neutrophils and sympa

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Immune and inflammatory mechanism in neuropathic pain