European Journal of Pharmacology 716 (2013) 169178

Contents lists available at ScienceDirect

European Journal of Pharmacology

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

Review

Peripheral mechanisms of burn injury-associated pain

Helen Laycock, Joao Valente, Carsten Bantel, Istvan Nagy n
Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Imperial College London, Chelsea and Westminster Hospital,
369 Fulham Road, London SW10 9NH, United Kingdom

art ic l e i nf o

a b s t r a c t

Article history:
Received 11 July 2012
Received in revised form
22 January 2013
Accepted 29 January 2013
Available online 13 March 2013

It is estimated that burn injury affects about 1 in 3000 people annually world-wide. Burn injury induces
severe pain, which is difcult to control in the great majority of cases making burn injury-associated pain
a serious clinical challenge. In order to meet this clinical need, novel targets should be identied. Like
pain developing in other peripheral pathologies, burn injury-associated pain is also initiated and
maintained by signalling between the injured tissues and primary sensory bres that supply those
tissues. This signalling is underlain by the formation and accumulation of a mixture of agents at the site
of the injury, some of which could be targets for intervention. However, at present the composition
of this burn injury tissue uid is incompletely established. Here, we summarise our current understanding of the composition of this burn injury tissue uid and explore how already known agents in that
tissue uid may activate nociceptors to initiate and maintain pain associated with burn injury.
& 2013 Elsevier B.V. All rights reserved.

Keywords:
Burn
Inammatory
Nociception
Nociceptor
Primary sensory neuron

Contents
1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pathology of burn injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nociceptive signalling in burn tissues . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Mechanism and molecules involved in the development of
3.2.
Mechanism and molecules involved in the development of
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Pain resulting from burn injuries is one of the most excruciating pain sensations that can be experienced. It is estimated that
1 in 3000 people suffers burn injuries annually world-wide.
According to WHO statistics, almost 11 million required medical
attention due to the severity of their injuries in 2004 (WHO,
2008). Although, Europe has a lower incidence of burn injury than
other areas in the world, annually 0.22.9/100,000 inhabitants
suffer severe burn injury (Brusselaers et al., 2010). Improvements
in resuscitation, wound care, critical illness support and infection
control have improved the outcomes of burn injuries, and
increased survival signicantly (Brusselaers et al., 2005; DeSanti,

Corresponding author. Tel.: 44 2087468897; fax: 44 2033155109.

E-mail addresses: i.nagy@imperial.ac.uk, i.nagy@ic.ac.uk (I. Nagy).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.01.071

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immediate and early pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
pain during the inammatory phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2005). Yet, pain in burn injury patients, both in the acute and
chronic setting, is still a major clinical challenge and an unmet
medical need (Carrougher et al., 2003; Dauber et al. 2002). Poorly
treated pain in burn injury however, can lead to immense suffering, loss of engagement in treatment, development of chronic pain
(Browne et al., 2011) and post traumatic stress disorder (Patterson
et al., 2006). Further, the lack of appropriate pain control may
signicantly hamper successful functional recovery, which can
lead to reduced integration into society (Esselman et al., 2001).
Therefore, providing appropriate pain control following burn
injury is of imperative importance.
The lack of appropriate pain control in burn-injured patients
reects our surprisingly limited understanding of the neuronal
mechanisms involved in burn injury-associated pain. Understanding the signalling between the burned and subsequently inamed
tissues, and the sensory neurons that innervate those tissues,
particularly at a cellular and molecular level therefore, must be

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H. Laycock et al. / European Journal of Pharmacology 716 (2013) 169178

one of the priorities in pain research. In this review, we will give

an account on already known and putative signalling events
occurring between burned tissues and primary sensory neurons,
in the hope that we initiate further studies to elucidate peripheral
mechanisms of burn injury-associated pain which will ultimately
lead us to better pain management in these patients.

2. Pathology of burn injury

The aetiology of burn injury is diverse and it includes contacts
with hot liquids, (scalds), various chemicals (chemical burn), strong
electrical currents (electrical burn), ames or hot objects (contact
burn). All tissues, which have some contact with the external
environment, are susceptible to direct burn damage, including the
skin, gastrointestinal and respiratory tracts. However, the majority
of burn injuries are due to heat impact (Brusselaers et al., 2010) and
affect the skin. Therefore, in this review we will concentrate on
mechanisms involved in the development and maintenance of pain
associated with heat-induced injury of the skin.
Although burn injury has a diverse aetiology, there is some
consistency in the overall pathological process of burns to the skin

and its subsequent impact on pain. The skin is a bilayer organ with
both protective and immunological functions (DeSanti, 2005;
Kao and Garner, 2000). It comprises of the outer epidermal layer,
predominantly of keratinocytes, and the inner dermal layer. The
dermis further divides into the supercial papillary dermis, and
the deeper, reticular dermis. The papillary dermis has greater
capacity to regenerate than the reticular dermis. Therefore, the
depth of the injury to the skin determines healing and management (DeSanti, 2005; Kinsella and Rae, 1997). The depth of injury
has also been used to classify or predict pain following burn injury
(Kinsella and Rae, 1997; see Table 1).
The progression of burn injury involves initial tissue damage,
inammation and then healing and remodelling. Primary tissue
damage occurs predominantly via thermal denaturing of proteins
and loss of the plasma membrane integrity leading to cell death
and the leakage of a series of agents (Evers et al., 2010; Fig. 1).
Following the initial loss of tissues, there is an enormous inammatory response (Allgower et al., 1995; Fig. 1). Locally this involves
a prolonged inux of inammatory cells that release various agents
which, on the one hand, coordinate the action of immuno-competent
cells, while on the other hand, act on sensory neurons inducing
either direct activation or sensitisation (Fig. 1). The action of

Table 1
Correlation between depth of burn injury, pain and healing time.
Table is adapted from Evers 2010.
Depth

Layer of skin involved

Pain

Healing time

Supercial

Epidermis

Moderate to severe pain

37 days

Supercial partial

Supercial papillary dermis

Severe pain
Intact nerve endings in wound
Pain exacerbated by contact with surfaces

13 weeks, long term pigmentation

changes may occur

Deep partial

Deeper reticular dermis

Minimal pain
Nerve endings damaged but not completely destroyed
Endings have ability to transmit noxious stimuli

36 weeks with scars

Deep

Full thickness of skin and into the

subcutaneous fat or deeper

Minimal/pain free state

Complete destruction of nerves

Does not heal by primary intention and

requires skin grafting

Fig. 1. Schematic drawing of event leading to the initiation, development and maintenance of pain following burn injury. The dashed line and solid line boxes identify events
leading to the formation of the early and late burn injury tissue uid.

H. Laycock et al. / European Journal of Pharmacology 716 (2013) 169178

immuno-competent cells is accompanied by localised oedema,

altered perfusion and systemic inammatory response (Fig. 1). The
healing phase involves re-epithelialisation of the wound by the
body itself or by surgical aid. During this period there is nerve
healing, re-growth and sprouting. Lastly the rehabilitation stage is
characterised by wound closure, scar maturation and optimisation
of functional outcome (Summer et al., 2007a).
General characteristics of burn injury-associated pain
While supercial burn injuries are associated with severe pain,
the area of deep burn injuries, due to the loss sensory nerve
endings, are signicantly less painful (Table 1). However burns
involve a combination of depths of injury and certainly with deep
burns there will be more shallow burn areas around the deep burn
area. Therefore, essentially all burn injured patients experience
severe pain in smaller or larger areas of the injury.
Pain from burn injury encompasses a number of different
modalities, including heat and mechanical hyperalgesia and allodynia (Pedersen and Kehlet, 1998). Furthermore, the hyperalgesia
associated with burn injury is both primary, within the damaged
tissue area, as well as secondary, in the adjacent uninjured tissue
(Moiniche et al., 1993; Raja et al., 1984). Regarding its emergence,
burn injury-associated pain is usually classied as background
pain, procedural pain and breakthrough pain which all can occur
in all the three phases of the burn injury. While this classication
is appropriate for describing the emergence of pain, for the
elucidation of the cellular and molecular mechanisms involved
in the development of pain, it is more useful to look at pain
mechanistically at the phase of injury, from the burn to subsequent recovery. This classication encompasses pain arising due to
heat impact and subsequent cell death, pain due to the inammatory response, and nally pain associated with tissue healing
and remodeling (Summer et al., 2007a). In the present review we
will focus on mechanisms involved in the development of pain
during and immediately after the heat impact and during the
inammatory response.
Like the development of pain associated with other peripheral
pathologies, the development of burn-injury associated pain also
involves nociceptive signaling between the injured tissues and
nerve bres innervating those tissues, as well as nociceptive
signaling in the central nervous system. It is believed however,
that the great majority of the characteristics of burn injuryassociated pain are due to signaling between the burn tissues
and primary sensory nerve bres. Below we will discuss signaling occurring between the injured tissues and primary sensory
neurons.

3. Nociceptive signalling in burn tissues

3.1. Mechanism and molecules involved in the development of
immediate and early pain
Heat which can cause tissue damage (above ~43 C) evokes
pain immediately. This pain is due to heat-evoked direct activation
of a sub-class of primary sensory bres in the skin, where the heat
impact has occurred (Fig. 1). Heat-sensitive primary sensory bres
are nociceptors, which are processes of small diameter, polymodal
nociceptive primary sensory neurons. These nociceptive cells are
responsive to high intensity mechanical, thermal and chemical
stimuli (Nagy, 2004). Previously, two ion channels, the transient
receptor potential vanilloid type 1 ion channel (TRPV1; Caterina
et al., 1997; Nagy and Rang, 1999a) and the transient receptor
potential vanilloid type 2 ion channel (TRPV2; Ahluwalia et al.,
2002; Caterina et al., 1999; Nagy and Rang, 1999b) were implicated
in the detection of heat above 43 C and 52 C, respectively, in
nociceptive primary sensory bres/neurons. Recently, a third ion

171

channel, the transient receptor potential melastatin 3 ion channel

(TRPM3; Vriens et al., 2011) was also shown to respond to noxious
heat in primary sensory neurons, though the heat threshold of
TRPM3, unlike of those of TRPV1 and TRPV2, is below the body
temperature. Nevertheless, activation of TRPV1, TRPV2, as well as
TRPM3, results in the development of nocifensive behaviour in
laboratory animals including increased responses to both noxious
and innocuous heat (heat hyperalgesia and allodynia; Caterina
et al., 1997, 2000; Davis et al., 2000; Vriens et al., 2011). In
addition, TRPV1 activation, through spinal cord processes, also
results in the development of mechanical allodynia (LaMotte et al.,
1991). Increased expression of the activated form of the extracellular signal-regulated kinase 1/2 (pERK1/2) in spinal cord
neurons is a marker for spinal nociceptive processing (Ji et al.,
1999). Hence, our recent nding that there are signicantly less
pERK1/2-expressing spinal cord neurons in TRPV1/ than in wild
type mice, 5 min following a scalding type injury, shows that
TRPV1 indeed contributes to the development of pain during the
heat impact (White et al., 2011).
In addition to inducing depolarising currents which can induce
the generation of action potentials, noxious heat, through activating TRPV1 and TRPV2, and possibly also TRPM3, induces the
release of various agents including the neuropeptides substance
P (SP) and calcitonin gene-related peptide (CGRP) from the peripheral terminals of nociceptive primary sensory bres (Helme
et al., 1986; Kessler et al., 1999; Zimmermann et al., 2005; Fig. 1).
These neuropeptides, together with other molecules released
from either heat-shocked cells or degenerating tissues form an
early burn injury tissue uid, a mixture of agents found in the
intercellular space at the area of the burn injury (Fig. 1). At present
very little is known about the composition of this uid however,
many of its molecules may signicantly contribute to the development of pain during and immediately after the heat impact.
Both SP and CGRP are likely to be part of the early burn injury
tissue uid as their levels are elevated in burn injury patients
(Onuoha and Alpar, 2001) and have been identied in animal
models of acute burn injury (Jonsson et al., 1986; Saria, 1984; Papp
and Valtonen, 2006). Both SP and CGRP are important mediators of
neurogenic inammation (Holzer, 1998) during which CGRP
induces vasodilatation, whereas SP induces plasma extravasation
and the release of agents from immuno-competent cells such as
lymphocytes, monocytes/macrophages, eosinophils, neutrophils
and mast cells (Scholzen et al., 1998; Fig. 1). Through this action,
SP and CGRP initiate an inammatory response for the protection
and restoration of tissue integrity and function. However, many of
the mediators released by SP and CGRP during the neurogenic
inammation also activate or sensitise nociceptive primary sensory
neurons, and that activation and/or sensitisation result in further
release of SP and CGRP (Fig. 1).
In addition to acting on vascular smooth muscle and endothelial
cells, and immuno-competent cells, both SP and CGRP also act
directly on nociceptive primary sensory neurons inducing excitation
(Szucs et al., 1999; Natura et al., 2005; Segond von Banchet et al.,
2002; Fig. 1). This SP- and CGRP-induced activation may recruit
additional primary afferents. Further this activation, again, may result
in further release of these neuropeptides (Fig. 1). Thus, SP and CGRP,
both through direct and indirect mechanisms, initiate and maintain
reverberating signalling between burn tissues and nociceptive
primary sensory bres which result in increased nociceptive input
into the central nervous system, hence pain (Fig. 1). Through these
mechanisms therefore SP and CGRP, in areas where sensory bres
survive, seem to contribute to the development of pain arising
immediately as well as later stages after the heat impact.
Noxious temperatures denature proteins and destroy cell
membranes. Hence, the content of degenerating cells is released
into the intercellular space where they also become part of the

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early burn injury tissue uid (Fig. 1). It is believed that ATP should
be a principal algogenic component of the early burn injury tissue
uid because ATP is released from degenerating cells, and its
extracellular concentration during tissue injury is sufcient enough
to activate purinergic P2X3 and P2X2/3 ion channels expressed by
nociceptors (North, 2003). In addition to the initial pain however,
ATP should also be involved in maintaining pain in later stages of
burn injuries because P2X3 receptor expression is up-regulated
following burn injury (Gao et al., 2010, Xu et al., 2009). This upregulation should be use-dependent, hence driven by the presence
of ATP.
Mice lacking P2X2 and/or P2X3 exhibit reduced mechanical and
thermal stimulation-evoked responses as well as reduced inammatory pain-related behaviour (Cockayne et al., 2000, 2005;
Souslova et al., 2000). Hence, P2X2 and P2X3 channels might be
involved in the development of both heat and mechanical hyperalgesia and allodynia in burn injury. Notably, the ATP metabolite
adenosine has been found in burn-induced blister within 12 h of
the injury (Shaked et al., 2007). At present, it is not clear whether
this adenosine is formed from ATP leaked from degenerated
cells or released de novo for example by immuno-competent cells.
Nevertheless, adenosine has been show to exert antinociceptive
rather then nociceptive effects (Sjolund et al., 1999) and its role in
burn pain is unclear.
A recent work has revealed that exposure of skin to noxious
heat (48 C for 10 min) in vitro results in the production of the
lipid metabolites 9- and 13-hydroxyoctadecadienoic acids (9- and
13-HODE) which activate TRPV1 directly (Patwardhan et al., 2010;
Fig. 1). Both of these compounds, when injected into the hind paw
of laboratory animals, produce nocifensive behavioural and heat
hyperalgesia (Patwardhan et al., 2010). At present it is not known
whether, in addition to cells in the skin such as keratinocytes,
other cells such as invading immuno-competent cells or broblast
in deep connective tissue layers also have enzymatic capacity to
synthesise 9- and 13-HODE. Nevertheless, Patwardhan et al. (2010)
ndings indicate that 9- and 13-HODE could be part of the early
burn injury tissue uid, at least in areas where some keratinocytes
survive the impact. In addition to 9- and 13-HODE, other lipid
metabolites, which are produced either in surviving cells or
produced from disrupted cell membranes, are also expected to
be present in the early burn injury tissue uid.
High temperature-induced effects on the integrity of vessels
(Jackson, 1953) and subsequent formation of molecules are also
highly likely to contribute to the early burn injury tissue uid
and the development of pain immediately after the heat impact
(Fig. 1). Damaged vasculature leads to the activation of the
coagulation cascade, which results in the formation of the zone
of coagulation (Jackson, 1953). Thrombin formed during the
coagulation cascade, on the one hand, catalyses the formation of
brin, while on the other hand, could activate protease-activated
receptor (PAR) 1, 3 and 4, which have been shown to be expressed by
sub-populations of nociceptive primary sensory neurons (Russell
et al., 2010; Zhu et al., 2005). Activation of PAR-1 and PAR-4 results
in TRPV1 sensitisation (Vellani et al., 2010), hence thrombin may
contribute to the development of the burning pain immediately after
the burn injury.
The coagulation zone of the burn-injured site is surrounded by
the zone of stasis (Jackson, 1953). The resulting hypoxia in these
two zones, through subsequent acidosis, could also be a major
contributor to pain immediately after, as well as later stages of,
burn injury (Fig. 1). Protons may act on at least four types of ion
channels in nociceptive primary sensory bres (White et al., 2010)
TRPV1 (Caterina et al., 1997), the transient receptor vanilloid type
4 (TRPV4; Suzuki et al., 2003), P2X2/P2X2/3 (King et al., 1997) and
acid-sensing ion channels (ASIC; White et al., 2010). Among the
ASICs, ASIC3 seems to be the most important, responding to

protons in primary sensory neurons (Deval et al., 2008). While

protons activate TRPV1, TRPV4 and ASICs directly, they only
modulate the activity of P2X2/P2X2/3 channels evoked by ATP
(Caterina et al., 1997; King et al., 1997). However, protons also
sensitise the effect of other activators on TRPV1 (Tominaga et al.,
1998). Through these ion channels, protons can produce both heat
hyperalgesia and allodynia (TRPV1, TRPV4; P2X2/P2X2/3,) and
mechanical allodynia (TRPV4, P2X2/P2X2/3; ASICs; Caterina et al.,
2000; Cockayne et al., 2000, 2005; Davis et al., 2000; Grant et al.,
2007; Sluka et al., 2003; Souslova et al., 2000; Todaka et al., 2004).
3.2. Mechanism and molecules involved in the development of pain
during the inammatory phase
At the inammatory stage, the composition of the extracellular
uid should be signicantly changed when compared to the early
burn injury tissue uid because invading immunocompetent cells
produce and release a large number of various agents (Fig. 1).
In addition, molecules released from the degenerating cells come
into contact with cells brought in by the circulation, and it is
expected that many of them are metabolised. Therefore, it is useful
to differentiate this burn injury tissue uid from that emerging in
the early phase, and refer to it as the late burn injury tissue uid.
Similarly to the composition of the early burn injury tissue uid,
we know little about the composition of the late burn injury tissue
uid. Establishing the molecular components of the late burn
injury tissue uid is, however important, as among its components
there must be mediators which maintain pain in the inammatory
phase of the injury.
Often, when investigators try to establish the components of
the late burn injury tissue uid, they assess serum levels and
extrapolate those data to the injury site. However, as pointed out
by Summer et al. (2007b), mediators are released locally and may
undergo metabolism prior to reaching the systemic circulation.
Conversely, agents which are detected in serum may never reach
the site of the burn injury (Summer et al., 2007b). Therefore, it is
more useful to assess the composition of the burn injury tissue
uid locally. Such analysis of late burn injury tissue uid, so far,
has revealed the presence of many agents, including prostaglandin
(PG) F1, PGF2, PGE2, thromboxane B2 (Heggers et al.,1980),
histamine (Horakora and Beavan, 1974), adenosine (Shaked et al.,
2007), interleukin (IL) 1, IL-1, epithelial growth factor (EGF),
basic broblast growth factor (bFGF), transforming growth factor2 (TGF2), TGF, TGF1, IL-6, platelet-derived growth factor
(PDGF) and IL-8 (Ono et al.,1995). In addition to these agents,
others, though not yet shown, are also highly likely to be present.
Nevertheless, looking at the list of so far known agents in the late
burn injury tissue uid, it is clear that the inammatory stage
of burn injury is characterised by a complex interplay between
the component of this tissue uid, the immuno-competent cells
inltrating the wound, and primary sensory bres. Below we will
discuss briey how known and some of the putative agents could
contribute to pain developing in the inammatory phase of burn
injury.
Though bradykinin has not been shown directly in burn injury
tissue uid, previously it has been noted that blister uid taken
from severely burned patients has a bradykinin like effect (SaintBlancard et al., 1966). Further, bradykinin is formed in injured
tissues locally, and by injection it induces a burning pain sensation
(Marceau et al., 1998). Hence, bradykinin should be a major
component of the late burn injury tissue uid. Bradykinin is a
potent activator of primary nociceptive primary sensory bres/
neurons (Oh and Weinreich, 2004) through the G protein-coupled
B1 and B2 receptors (Moreau et al., 2005). Both B1 and B2 receptors
are expressed by nociceptors though B1 expression occurs only in
pathological conditions, for example in inammation (Fox et al.,

H. Laycock et al. / European Journal of Pharmacology 716 (2013) 169178

2003; Ma, 2010; Marceau et al., 1998; Marchand et al., 2005; Prado
et al., 2002; Regoli et al., 2001; Vellani et al., 2004). Bradykinin
exerts its excitatory effects mainly via other channels including
TRPV1 and the transient receptor potential ankyrin type 1 ion
channel (TRPA1; Amaya et al., 2006; Bautista et al., 2006; Cesare
et al., 1999; Kollarik and Undem, 2004; Katanosaka et al., 2008).
Regarding TRPV1 activation, bradykinin can achieve that activation
through at least two indirect mechanisms; through lowering the
heat threshold of TRPV1 so that body temperature activates this
ion channel (Cesare and McNaughton, 1996; Sugiura et al., 2002),
and through inducing the production of the lipid metabolite
12-hydroperoxyeicosatetraenoic acid, which directly activates
TRPV1 (Shin et al., 2002). However, our recent nding that the
number of pERK1/2-expressing neurons in the spinal dorsal horn
in wild type and TRPV1/ mice is not different 30 min, 60 min and
3 h after the burn injury indicates that TRPV1 might not be
involved the maintenance of pain during the inammatory phase
of the injury (White et al., 2011).
While TRPA1 has been identied as a molecule responsive to
noxious cold stimuli, its activation for example by cinnamaldehyde
results in burning pain (Namer et al., 2005). Further, there is
compelling evidence that TRPA1 plays a pivotal role in the development of inammatory thermal hyperalgesia and mechanical
allodynia (Petrus et al., 2007; Tsagareli et al., 2010). Therefore,
bradykinin-evoked TRPA1 activation/sensitisation could contribute
to both thermal and mechanical pain in burn injury. However, at
present, there is no direct evidence whether or not TRPA1 activation
contributes to pain in burn injury.
In 1953 Armstrong demonstrated that serotonin (5-HT) when
applied topically to a blister base caused pain similar to that
caused by the blister uid itself (Armstrong et al., 1953). Indeed,
5-HT has been found in microdyalisates taken from burn injury
sites as well as uninjured sites of burn injured patients (Samuelson
et al., 2008). Mast cells and platelets, which are accumulated and
activated at the site of the burn injury, release 5-HT (KushnirSukhov et al., 2007; White et al., 1994). Hence, it is reasonable to
assume that these cells constitute the source of 5-HT found by
Samuelson et al. (2008). 5-HT induces nociceptor sensitisation
(Dray, 1995; Steen et al., 1996) and mechanical hyperalgesia
(Oliveira et al., 2007), which might be mediated both directly
and indirectly through multiple 5-HT receptors expressed by
primary sensory neurons (Loyd et al., 2011) as well as some
immuno-competent cells (Tambeli et al., 2006). 5-HT directly
activates sensory bres through 5-HT3 receptors (Hicks et al.,
2002). However, Oliveira et al. (2007) have shown that the 5-HTinduced hyperalgesia depends on local release of prostaglandins
and norepinephrine, and that the effect of norepinephrine
depends on 2 adrenergic receptors. In addition, 5-HT2 receptor
activation by serotonin sensitises ASICs (Qui et al., 2012). However,
it should also be noted that activation of 5-HT1B and 5-HT1D
receptors exerts inhibitory actions on primary sensory neurons
(Edvinsson and Petersen, 2007).
Similarly to 5-HT, histamine, when applied to blister base also
induces pain (Armstrong et al., 1953). Further, histamine is also
present in burn tissues (Horakora and Beavan, 1974). More
recently, histamine levels were shown to be increased in an injury
depth-dependent manner (Papp et al., 2005). Histamine is also
likely to be released from activated mast cells and platelets in burn
injured tissues (Mannaioni et al., 1997; Riley, 1953). Histamine
induces Ca2 inux from extracellular uid as well as Ca2
outux from intracellular stores in primary sensory neurons
(Nicolsson et al., 2002). Hence, histamine induces neuronal excitation through activating the H1 receptors (Nicolsson et al., 2002;
Parada et al., 2001; Ting et al., 2007). Recently, Kajihara et al.
(2010) have reported that H1 receptor-mediated excitation in
primary sensory neurons, at least in part, depends on TRPV1.

173

These authors have also shown that histamine potentiates the

excitatory action of protons of on TRPV1. In addition to H1, H4
receptors have also been shown to contribute to the development
of thermal hyperalgesia following acute inammation (Coruzzi
et al., 2007). Hence, histamine may contribute to the development
of burning pain in the inammatory phase of burn injury.
A number of eicosanoids have been found in burn tissue and
blister uid. Burn tissue and inammatory cells, including neutrophils and macrophages, both release arachidonic acid which
forms the basis for the syntheses of prostanoids and thromboxanes via the cyclooxygenase (COX) enzyme system or leukotrienes
via the 5 lipoxygenase enzyme system.
Among prostaglandins, PGE2 might be one of the most important in the development of pain in burn injury. PGE2 injection into
rat paws leads to hyperalgesia (Kuhn and Willis, 1973). This effect
is produced through multiple mechanisms in primary sensory
neurons; PGE2 sensitises the effect of bradykinin (Kumazawa et al.,
1996), sensitises TTX-resistant sodium channels (England et al.,
1996), inhibits voltage-gated potassium currents (England et al.,
1996), sensitises TRPV1 by reducing its heat threshold below the
body temperature (Moriyama et al., 2005) and sensitises the effect
of histamine (Nicolsson et al., 2007). Further, PGE2 increases the
expression of the pro-inammatory cytokine IL-6, of the neuropeptides SP and CGRP, and of the SP-responding neurokinin
1 receptor in primary sensory neurons (Ma, 2010; Segond von
Banchet et al., 2003; St Jacques and Ma, 2011). In addition to PGE2,
other prostaglandins at least through their metabolites, such as
the PGD2 metabolite 15-deoxy-(12,14)-prostaglandin J(2) activate
TRPA1 (Taylor-Clark et al., 2008). Notably, exposure of primary
sensory neurons to bradykinin induces the synthesis and release
of PGE2 from these neurons (Inoue et al., 2006).
Leukotrienes (LT) have been known to be present in the late
burn injury tissue uid for a long time (Denzlinger et al., 1985).
They are released by macrophages and neutrophils once these
immuno-competent cells have inltrated wounds. In addition to
having various affects on non-neuronal cells, LT also act on
primary sensory neurons. Among LT, LTB4 seems to be the most
important agent in contributing to the development of pain in
burn injury. Injection of this agent induces both heat and mechanical hyperalgesia (Bisgaard and Kristensen, 1985; Martin et al.,
1988). The cognate receptor for LTB4, BLT1 is expressed by a major
sub-population of TRPV1 expressing primary sensory neurons
(Andoh and Kuraishi, 2005, Okubo et al., 2010). However, LTB4
can directly activate TRPV1 (Hwang et al., 2000). Hence it is not
surprising that LTB4 induces Ca2 inux into primary sensory
neurons (Andoh and Kuraishi, 2005), hence induces excitation.
In addition to BLT1 and TRPV1, LTB4 may also act on primary
sensory neurons through the peroxisome proliferator-activated
receptor (LoVerme et al., 2006; Narala et al., 2010). However,
through this latter action LTB4 should produce an antinociceptive
effect (LoVerme et al., 2006).
Platelet activating factor (PAF) production is increased by
polymorphonuclear cells isolated from burn patients (Lavaud
et al., 1988). PAF decreases nociceptive thresholds in rats as well
as humans (Dallob et al., 1987; McGivern and Basran, 1984).
Further, intrathecal administration of PAF induces both burn and
mechanical pain which depends on the expression of the PAF
receptor (Tsuda et al., 2007). PAF has a direct excitatory effect on
primary sensory neurons (Tsuda et al., 2007). However, that effect
could be mediated not through its cognate receptor, as the PAF
receptor mRNA has been found only in satellite cells which
surround primary sensory neurons (Hasegawa et al., 2010).
Through the PAF receptor expressed in satellite cells, PAF also
seems to up-regulate the production of tumour necrosis factor
alpha (TNF) and IL-1 in satellite cells (Hasegawa et al., 2010).
Hence, PAF seems to indirectly contribute to the development of

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H. Laycock et al. / European Journal of Pharmacology 716 (2013) 169178

burn injury-associated pain in the inammatory stage. This

indirect algesic effect consists of multiple mechanisms, including
activation of TRPV1 (Marotta et al., 2009).
Interleukin 1 is released by macrophages and neutrophils and
has been demonstrated at low levels in blister uid (Ono et al.,1995).
Il-1 is also produced by primary sensory neurons (Copray et al.,
2001; Hasegawa et al., 2010) and the Il-1 receptor 1 is also
expressed by a sub-population of these neurons (von Banchet
et al., 2011). Il-1 induces heat hyperalgesia and mechanical allodynia
when injected subcutaneously (Saeh-Garabedian et al., 1995; Sachs
et al., 2002). This effect is underlain by Il-1-evoked increase in
responses of primary afferent bres to mechanical as well as thermal
stimuli (Fukuoka et al., 1994). The excitatory effects of Il-1 on
primary sensory neurons are mediated by multiple mechanisms
involving up-regulation of TTX-resistant Na currents (Binshtok
et al., 2008), suppression of voltage-gated K currents (Takeda
et al., 2008) and sensitisation of TRPV1 (Obreja et al., 2002). Recently
von Banchet et al. (2011) have shown that Il-1 application prevents
B2 receptor internalisation, hence Il-1 increases the effect of
bradykinin on primary sensory neurons. In addition to the short
term effects on primary sensory neurons, Il-1 also have long term
effects on other cells; it up-regulates the synthesis and release of the
pivotal inammatory mediator, nerve growth factor (NGF) (SaehGarabedian et al., 1995).
Interleukin 6 has been demonstrated in high levels in blister
uid (Ono et al.,1995). Recently, Il-6 has been shown in increased
levels at burn injury sites in rats, which correlated with ipsilateral
mechanical hyperalgesia (Summer et al. 2007b). Summer et al.
(2007b) also found that the burn injury-induced mechanical
hyperalgesia is attenuated by anti-Il-6 antibodies. These ndings
strongly argue for a pivotal role of Il-6 in the development of burn
injury-associated pain. This view is supported by a recent nding
that PGE2 (Heggers et al.,1980) which itself is present in the late
burn injury tissue uid up-regulates Il-6 expression in primary
sensory neurons (St Jacques and Ma, 2011).
Intraplantar injection of Il-6 causes mechanical pain in rats
(Cunha et al., 1992). Primary sensory neurons may express both Il6 and its receptor as well as its signal transducer glycoprotein,
gp130 (Gadient and Otten, 1996; Obreja et al., 2005). Recently,
Obreja et al. (2002) showed that Il-6 when bound to its soluble
receptor sensitises primary afferents which is underlain by
increased TRPV1-mediated currents and reduced heat threshold
of this ion channel (Obreja et al., 2005). However, as mentioned,
TRPV1 might not be involved in maintaining pain in the inammatory phase of burn injury (White et al., 2011). At present it is
not known what other molecules might be targeted by Il-6
signalling in primary sensory neurons.
Tumour necrosis factor alpha is another proinammatory
cytokine which has been found in blister uid (Onuoha and
Alpar, 2001). Serum levels of TNF are also increased following
burn injury (Onuoha and Alpar, 2001). In naive conditions, TNF is
predominantly synthesised by mast cells (Ackermann and Harvima,
1998). However in injury, TNF is synthesised by a variety of cells
including keratinocytes (Corsini and Galli, 1998), broblasts
(Fujisawa et al., 1997), macrophages and neutrophils (KhanolkarYoung et al.,1995). Schwann cells also synthesise TNF particularly
after peripheral nerve injury, which occurs in burn injury (Wagner
and Myers, 1996). Further, TNF induces the production of other
cytokines, including Il-1, Il-6, and Il-8, and triggers COX which
synthesise prostaglandins (Cunha et al., 1992). TNF should have a
direct action on primary sensory neurons because one of the TNF
cognate receptors, TNFR1, is expressed in a large proportion of
neurons in the dorsal root ganglion. In addition to the direct effect
however, circulating TNF may also have an indirect effect on
primary sensory neurons as another TNF receptor, TNFR2, is
expressed by the satellite cells, and those cells produce various

other inammatory mediators including TNF itself (Hasegawa

et al., 2010) which can act directly on primary sensory neurons.
Local injection of TNF induces thermal hyperalgesia and/or
mechanical allodynia and/or hyperlagesia (Fernandes et al., 2011;
Opree and Kress, 2000; Sommer et al., 1998). The algesic effect
of local TNF injection could be underlain by multiple effects.
Exposure of primary sensory neurons to TNF results in increased
TRPV1-mediated responses (Nicol et al., 1997). This effect can be
blocked by indomethacine, as well as by a COX-2 inhibitor (Nicol
et al., 1997). Hence the sensitising effect of TNF on TRPV1 seems
to be indirect. In addition to TRPV1, TRPA1 also seems to be a
target for TNF-induced signalling because blocking the activity of
TRPA1 at peripheral tissues results in reduced mechanical hyperalgesia (Fernandes et al., 2011). However, P2X3 seems to be
unaffected by TNF (de Oliveira Fusaro et al., 2010).
Nerve growth factor is thought to be a pivotal inammatory
mediator in the development of inammatory heat hyperalgesia
(Huang et al., 2006). Indeed, increased NGF levels were found
following tissue injury including that evoked by burn (Ueda et al.,
2002). Further, anti-NGF treatment alleviates burn-induced hyperalgesia (Summer et al., 2006) indicating that this mediator indeed
plays a key role in the development of pain following burn injury.
NGF is produced by monocytes, eosinophils and mast cells
(Leon et al., 1994). A series of other inammatory mediators, some
of which have been identied in the late burn injury microfuid,
up-regulate NGF synthesis (Abe et al., 2007; Hattori et al., 1993;
Lipnik-Stangelij, 2006; Toyomoto et al., 2004; Woolf et al., 1997).
The great majority of SP- and CGRP-containing primary sensory
neurons responds to NGF because they express the high afnity
NGF cognate receptor tyrosine kinase A (trkA; McMahon et al.,
1994). Local injection of NGF induces heat hyperlagesia and
mechanical allodynia (Lewin et al., 1993; Thompson et al., 1995).
Data, which have emerged in the last decade, have shown that one
of the main targets through which NGF induces heat hyperalgesia
is TRPV1 because NGF signicantly increases TRPV1 responsiveness. This effect is achieved by at least two mechanisms. First, NGF
induces TRPV1 phosphorylation which results in sensitised
responses (Bonnington and McNaughton, 2003; Chuang et al.,
2001). Second, NGF also induces translocation of TRPV1 from the
cytoplasm to cell membrane (Zhang et al., 2005). In addition to
these effects, NGF also up-regulates TRPV1 transcription (Xue
et al., 2007). Recent ndings however indicate that in addition to
TRPV1, other ion channels also constitute targets for NGF-trkA
signalling in primary sensory neurons. TRPA1, ASIC3 and P2X3
expression is controlled and up-regulated by NGF (D'Arco et al.,
2007; Mamet et al., 2003; Obata et al., 2005; Ramer et al., 2001;).
Further, NGF also sensitises TRPA1-, ASIC3- and P2X3-mediated
responses (D'Arco et al., 2007; Malin et al., 2001; Mamet et al.,
2002). Taken together, NGF indeed seems to be a key player in
maintaining burn injury-associated heat and mechanical pain.
Stimulation of peripheral beta adrenergic receptors on primary
nociceptive afferents has been linked to inammatory hyperalgesia. This view is supported by the nding that epinephrine
injection, in a dose-dependent manner, reduces the mechanical
threshold which effect is attenuated by the selective 1 antagonist
atenolol (Dina et al., 2001, Khasar et al., 1999). Variation in
catechol-o-methyltransferase (COMT) genotype had been linked
to prediction of acute pain (Diatchenko et al., 2005). The question
whether or not COMT genotype is related to the severity of burn
injury pain has recently been addressed in burn patients (Orrey
et al., 2012). Using a haplotype based approach to COMT variables,
one of three haplotypes located in the central COMT locus has
previously been associated with low pain sensitivity, increased
COMT activity, and has been deemed to be pain protective. If no
copies of this gene were present in burn injured patients they
were presumed to have a COMT pain vulnerable genotype.

H. Laycock et al. / European Journal of Pharmacology 716 (2013) 169178

Comparing the vulnerable, with the non-vulnerable genotype, a

statistically signicant increase in overall pain, least pain and
awakening pain scores were demonstrated. Expression of the
vulnerable genotype was also a stronger predictor of overall pain
severity than size, type or depth of burn. Based on these data, we
cannot conclude at present that exhibiting the vulnerable genotype is sign for a genetic predisposition for burn injury-associated
pain. However, these data do call for further studies on the role
of pathways involving catecholamines in the development of
pain with peripheral origin including pain associated with burn
injuries.

4. Conclusions
Burn injury is associated with enormous suffering due the
severe pain it induces. Whilst burn injury-associated pain can
be regarded as part of a tissue-protecting/tissue regenerationpromoting mechanism, due to its severity, pain in burn injury
becomes counteractive in every sense rather than benecial.
At present, the most common analgesia used in burn injury pain
in burn units are opioids. However, in the majority of the cases,
opioids do not provide sufcient pain control and may even induce
severe side effects in already highly vulnerable patients. Therefore,
the discovery of novel targets for the development of new
analgesics to improve pain control in burn-injured patients is
important.
Cognate receptors in nociceptors for agents found in burn
injury tissue uid may represent ideal novel targets for drug
development and intervention. However as outlined above, the
composition of these tissue uids is incompletely established at
present. Further, at present it seems unlikely that there is any
leading compound(s) within burn tissue uids, the inactivation
of which alone, would result in a signicant reduction of pain.
Therefore, we should devote more resources and use high
throughput approaches to establish the composition of burn injury
tissue uid both in animal models of burn injury and in burn
injured patients. In addition, investigating the cellular and molecular effects of the components of these burn injury tissue uids
on sensory neurons as well as on tissue regeneration is also
needed, as downstream molecules in nociceptors might provide
better targets than those of individual cognate receptors for
agents alone.
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