Neuro Endocrino PDF

Proceedings of the Nutrition Society (2008), 67, 409–418 doi:10.
1017/S0029665108008690
g The Author 2008
A Meeting of the Nutrition Society, hosted by the Irish Section, was held at the O’Reilly Hall, University College Dublin, Dublin,
Republic of Ireland on 18–20 June 2008
Symposium on ‘The challenge of translating nutrition research into public

health nutrition’
Session 3: Joint Nutrition Society and Irish Nutrition and Dietetic

Institute Symposium on ‘Nutrition and autoimmune disease’
PUFA, inflammatory processes and rheumatoid arthritis
Philip C. Calder
Institute of Human Nutrition, School of Medicine, University of Southampton, IDS Building, MP887, Southampton General
Proceedings of the Nutrition Society
Hospital, Tremona Road, Southampton SO16 6YD, UK
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease manifested by

swollen and painful joints, bone erosion and functional impairment. The joint lesions are
characterised by infiltration of T lymphocytes, macrophages and B lymphocytes into the
synovium and by synovial inflammation involving eicosanoids, cytokines and matrix metallo-
proteinases. In relation to inflammatory processes, the main fatty acids of interest are the n-6
PUFA arachidonic acid, which is the precursor of inflammatory eicosanoids such as PGE2 and
leukotriene B4, and the n-3 PUFA EPA and DHA, which are found in oily fish and fish oils.
Eicosanoids derived from the n-6 PUFA arachidonic acid play a role in RA, and the efficacy of
non-steroidal anti-inflammatory drugs in RA indicates the importance of pro-inflammatory
cyclooxygenase pathway products of arachidonic acid in the pathophysiology of the disease.
EPA and DHA inhibit arachidonic acid metabolism to inflammatory eicosanoids. EPA also
gives rise to eicosanoid mediators that are less inflammatory than those produced from
arachidonic acid and both EPA and DHA give rise to resolvins that are anti-inflammatory and
inflammation resolving. In addition to modifying the lipid mediator profile, n-3 PUFA exert
effects on other aspects of immunity relevant to RA such as antigen presentation, T-cell re-
activity and inflammatory cytokine production. Fish oil has been shown to slow the develop-
ment of arthritis in an animal model and to reduce disease severity. Randomised clinical trials
have demonstrated a range of clinical benefits in patients with RA that include reducing pain,
duration of morning stiffness and use of non-steroidal anti-inflammatory drugs.
Cytokine: Eicosanoid: Fatty acid: Fish oil: Inflammation
Immune system overview commensal gut bacteria. The system has two functional
divisions: the innate (or natural) immune system and the
The immune system is responsible for the host’s response acquired (also termed specific or adaptive) immune system.
to the presence of bacteria, viruses, fungi and parasites; it Both components involve various blood-borne factors and
is also involved in protection against growth of certain cells. Immune cells originate in bone marrow and are
tumours and in the response to injury and trauma. The found circulating in the bloodstream, organised into lym-
immune system acts to distinguish between ‘self’ and ‘non- phoid organs such as the thymus, spleen, lymph nodes
self’, permitting tolerance to self antigens and to non- and gut-associated lymphoid tissue or dispersed in other
threatening environmental agents such as food proteins and locations. The immune response is typified by cellular
Abbreviations: HLA, human leucocyte antigen; IFN, interferon; LT, leukotriene; NSAID, non-steroidal anti-inflammatory drug; RA, rheumatoid arthritis.
Corresponding author: Professor Philip Calder, fax + 44 2380 795255, email pcc@soton.ac.uk
410 P. C. Calder
interactions and by the movement of cells to sites of Synovial fluid from patients with RA contains high
infection or other immune activity. The four key functional levels of pro-inflammatory cytokines including TNFa,
activities of the immune response are: IL-1b, IL-6, IL-8 and granulocyte–macrophage colony-
stimulating factor(5). Synovial cells cultured ex vivo spon-
to act as an exclusion barrier; taneously produce TNFa, IL-1b, IL-6, IL-8 and granulo-
to distinguish self from non-self;
cyte–macrophage colony-stimulating factor for extended
to develop tolerance to, or to eliminate the source of,
periods of time(5). Synovial fluid from patients with RA
non-self antigens;
also contains high levels of anti-inflammatory cytokines
to retain memory of immunological encounters.
such as transforming growth factor b, IL-10, IL-1 receptor
In order to allow effective functioning of the immune antagonist and soluble TNF receptors(5). Thus, the inflamed
system many different cell types, each specialised in a synovial joint contains excessive amounts of both pro- and
limited range of functions, are involved. These cells work anti-inflammatory cytokines, but given the ongoing state of
in a coordinated integrated manner in order to assure a inflammation there must be an imbalance in favour of the
successful immune response. former.
Loss of tolerance can lead to disease and to adverse
patient outcome. For example, autoimmune diseases result
from loss of tolerance to self antigens, allergic diseases Arachidonic acid, eicosanoids and the link with
result from loss of tolerance to normally benign environ- inflammation

mental or food components and inflammatory bowel dis- Eicosanoids are key mediators and regulators of inflam-
eases result from loss of tolerance to commensal gut mation(6,7) and are generated from C20 PUFA. As inflam-
bacteria. The loss of tolerance leads to an immunological matory cells typically contain a high proportion of the n-6
response that is damaging to the host. PUFA arachidonic acid (20 : 4n-6) and low proportions of
other C20 PUFA, arachidonic acid is usually the major
substrate for eicosanoid synthesis. Eicosanoids, which
Rheumatoid arthritis include PG, thromboxanes, leukotrienes (LT) and other
oxidised derivatives, are generated from arachidonic acid
Rheumatoid arthritis (RA) is a chronic inflammatory
by the metabolic processes summarised in Fig. 1. They
autoimmune disease that affects about 1 % of the adult
are involved in modulating the intensity and duration
population and is more common in women than in men(1).
of inflammatory responses(6,7), have cell- and stimulus-
RA is characterised by symmetric polyarthritis(1). Joint
specific sources and frequently have opposing effects.
inflammation is manifested by swelling, pain, functional
Expression of both isoforms of cyclooxygenase is
impairment, morning stiffness, osteoporosis and muscle
increased in the synovium of patients with RA(5,8) and in
wasting. Erosion of bone occurs commonly in the joints of
joint tissues in rat models of arthritis(8). PGE2, LTB4 and
the hands and feet. The joint lesions are characterised by
5-hydroxyeicosatetraenoic acid are found in the synovial
infiltration of activated T lymphocytes, macrophages and
fluid of patients with active RA(9). Infiltrating leucocytes
antibody-secreting B lymphocytes into the synovium (the
such as neutrophils, monocytes and synoviocytes are
tissue lining the joints) and by proliferation of fibroblast-
important sources of eicosanoids in RA(9). PGE2 has a
like synovial cells called synoviocytes(1,2). These cells and
number of pro-inflammatory effects, including increasing
new blood vessels form a tissue termed pannus that leads
vascular permeability, vasodilation, blood flow and local
to progressive destruction of cartilage and bone, which is
pyrexia and potentiation of pain caused by other agents. It
most likely to be a result of cytokine- and eicosanoid-
also promotes the production of some matrix metallo-
mediated induction of destructive enzymes such as matrix
proteinases and stimulates bone resorption. The efficacy of
metalloproteinases. RA is also characterised by signs of
non-steroidal anti-inflammatory drugs (NSAID), which act
systemic inflammation, such as elevated plasma concen-
to inhibit cyclooxygenase activity, in RA indicates the
trations of some cytokines (e.g. IL-6), acute-phase proteins
importance of this pathway in the pathophysiology of the
and rheumatoid factors.
disease. However, although these drugs provide rapid relief
Genetic studies have linked susceptibility to, and sever-
of pain and stiffness by inhibiting joint inflammation, they
ity of, RA to genes in the MHC II locus; in human subjects
do not influence the course of the disease. LTB4 increases
these genes encode the human leucocyte antigen (HLA) II
vascular permeability, enhances local blood flow, is a
proteins involved in antigen presentation. RA is associated
potent chemotactic agent for leucocytes, induces release
with specific alleles of the HLA-DRB1 gene, although
of lysosomal enzymes and enhances release of reactive
other HLA-DR alleles may also play a role(3). As the
oxygen species and inflammatory cytokines such as TNFa,
function of HLA-DR is antigen presentation to T lympho-
IL-1b and IL-6.
cytes, the genetic association indicates a role for T-cells in
RA(4). In total the HLA region contributes 30–50 % of the
genetic component of RA. The second largest genetic risk
Very-long-chain n-3 PUFA and inflammatory processes
for RA lies with a variant in the protein tyrosine phos-
phatase non-receptor 22 gene, which encodes an intracel- Oily fish and fish oils contain the very-long-chain n-3
lular protein tyrosine phosphatase(3). The variant may act PUFA EPA (20 : 5n-3) and DHA (22 : 6n-3). Increased
to reduce the ability to down regulate activated T-cells. consumption of these fatty acids results in their incor-
Recently, novel risk loci have been described(3). poration into immune cell phospholipids(10–13), which
PUFA and rheumatoid arthritis 411
Arachidonic acid in
cell membrane phospholipids
Phospholipase A2
Free arachidonic acid
COX-1 15-LOX 12-LOX 5-LOX

COX-2
PGG2 15-HPETE 12-HPETE 5-HPETE
PGH2 15-HETE 12-HETE LTA4 5-HETE

LTC4 LTB4
PGD2 PGE2 PGI2 TXA2 PGF2α Lipoxin A4
LTD4
PGJ2
LTE4
Fig. 1. Outline of the pathway of eicosanoid synthesis from arachidonic acid. COX, cyclooxygenase; HETE,
hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; TX,
thromboxane.
occurs in a dose–response fashion and is partly at the DHA decreases endotoxin-induced class II molecule up-
expense of arachidonic acid. The changed membrane fatty regulation(32) EPA and DHA treatment has been reported
acid composition is believed to influence immune cell to diminish the up-regulation of HLA-DR and HLA-DP
function and inflammatory processes(14) (Fig. 2). There associated with IFN-g stimulation of human monocytes(33).
have been numerous reviews of the influence of n-3 PUFA It has subsequently been demonstrated that these fatty
on many aspects of immune function in recent years(10–27) acids decrease the ability of human monocytes to present
and the reader is referred to these articles for details antigen(34). Three studies, one in mice(35), one in rats(36)
beyond those provided in the following sections. and one in human subjects(37) have reported effects of
dietary n-3 PUFA on class II expression. Feeding mice fish
oil, which contains EPA and DHA, results in a reduction in
Antigen-presenting cell function MHC II expression on peritoneal cells (mainly B lympho-
There have been several studies of the effects of n-3 PUFA cytes and macrophages)(35). A human supplementation
on MHC II or HLA expression or antigen presentation via study with fish oil has reported decreased expression of
class II molecules(28). These studies have typically found HLA-DR, -DP and -DQ on IFN-g-stimulated blood
that class II expression and antigen presentation via class II monocytes(37), with similar effects to those seen with n-3
molecules are decreased by n-3 PUFA. An in vitro study in PUFA in vitro(33). These studies did not examine antigen
which spleen cells were incubated with EPA has reported presentation activity. However, a study that involved
decreased ability of those cells to present antigen(29); feeding an EPA-rich oil to mice has shown decreased
this study did not report class II expression. Incubating antigen (keyhole limpet (Megathura crenulata) haemo-
murine macrophages with DHA decreases expression of cyanin) presentation by spleen cells to T-cell clones(29).
the class II molecules (termed Ia in mice)(30). Likewise, Perhaps the most thorough study of this type to date is that
incubating mouse macrophages with EPA or DHA in which feeding a fish oil-rich diet to rats was found to
decreases interferon (IFN)-g-induced up-regulation of class result in decreased expression of MHC II on dendritic
II molecules(31) and incubating mouse dendritic cells with cells(36). These cells were found to have a much reduced
412 P. C. Calder
Increased supply of
long-chain n-3 PUFA
Decreased arachidonic acid

and increased long-chain
n-3 PUFA in inflammatory cell
membrane phospholipids
Altered membrane Altered signal

structure and fluidity transduction Altered inflammatory
(gross, rafts, acylation) Altered pattern of pathways gene expression
lipid mediator
synthesis
Promotion of a less-inflammatory phenotype
Fig. 2. Mechanisms by which n-3 PUFA can affect inflammatory cell activity.
capacity to present antigen (keyhole limpet haemocyanin) Inflammatory mediator production

to antigen-sensitised spleen T-cells. The reduction in anti-
gen presentation is probably much greater than could be Eicosanoids. Increased consumption of very-long-chain
explained by the reduction in class II expression, suggest- n-3 PUFA such as EPA and DHA, results in decreased
ing that other interactions between antigen-presenting cells amounts of arachidonic acid present in immune cell mem-
and T lymphocytes are affected by dietary n-3 PUFA. It branes and available for synthesis of eicosanoids(10–13).
was reported that levels of the co-stimulatory molecules Thus, feeding fish oil to laboratory rodents or supple-
CD2, CD11a and CD18 are also decreased on dendritic menting the diet of human subjects with fish oil has
cells from fish oil-fed rats(36). been reported to result in decreased production of a range
of eicosanoids including PGE2, thromboxane B2, LTB4,
5-hydroxyeicosatetraenoic acid and LTE4 by inflammatory
T lymphocyte reactivity
cells(10–13). A recent study has demonstrated the dose–
In vitro studies have demonstrated that EPA and DHA response effect to dietary EPA of PGE2 production by
decrease T-cell proliferation(38–41) and the production of endotoxin-stimulated human mononuclear cells and sug-
helper T-cell 1-type cytokines such as IL-2(38,39,42). Feeding gests that an EPA intake of > 2 g/d is required in order to
studies in rodents and supplementation studies in human be effective(53).
subjects have also shown that fish oil decreases T-cell EPA is also able to act as a substrate for both cyclo-
proliferation(43–48) and production of helper T-cell 1-type oxygenase and 5-lipoxygenase, giving rise to eicosanoids
cytokines such as IL-2(42,45,47,48) and IFN-g (42,48), although with a slightly different structure from those formed from
it is important to note that not all human studies report arachidonic acid. Thus, fish oil supplementation of the
such an effect(11). The reason for these discrepancies in human diet has been shown to result in increased pro-
the literature is not entirely clear, but dose of n-3 PUFA duction of LTB5, LTE5 and 5-hydroxyeicosapentaenoic
used, technical factors and differences among subjects acid by inflammatory cells(10–13). The functional impor-
studied are likely to be contributing factors. tance of this outcome is that the mediators formed from
The mechanism by which long-chain n-3 PUFA affect EPA are frequently less potent than those formed from
T-cell reactivity was initially thought to relate to altered arachidonic acid; for example, LTB5 is less potent as a
patterns of eicosanoid synthesis; however, through the use neutrophil chemotactic agent than LTB4(54,55).
of eicosanoid synthesis inhibitors and pure eicosanoids Resolvins and related compounds: novel EPA- and
in vitro this mechanism has been shown to be unlikely(40). DHA-derived anti-inflammatory mediators. Recent stu-
Studies over the last few years have demonstrated that the dies have identified a novel group of trihydroxyeicosa-
inhibitory effects of n-3 PUFA in general, and of EPA in pentaenoic acid mediators, termed E-series resolvins,
particular, relate to membrane-mediated effects that impact formed from EPA by a series of reactions involving
on the early stages of cell signalling(49–52). cyclooxygenase-2 (acting in the presence of aspirin) and
Antigen-presenting
Self antigen
cell Y
YYY
Self antigen-specific
B IgG = autoantibodies
∗
MHCII
∗
IFN-γ
T-cell
receptor
IL-2
IL-12 Damage to host tissue

Th Th1
IFN-γ
IFN-γ ∗ Inflammation
Th2
Macrophage
Fig. 3. Cellular sites of anti-inflammatory actions of long-chain n-3 PUFA. IFN, interferon; Th, helper T-cell; Y,
IgG. , Sites of action of n-3 PUFA; , inhibits.
5-lipoxygenase. These mediators appear to exert potent shown to have beneficial effects in animal models of
anti-inflammatory actions(56–58). In addition, DHA-derived arthritis. For example, compared with vegetable oil, feed-
trihydroxydocosahexanoic acid mediators termed D-series ing mice fish oil delays the onset (mean 34 d v. 25 d) and
resolvins are produced by a similar series of reactions reduces the incidence (69 % v. 93 %) and severity (mean
and these resolvins are also anti-inflammatory(59,60). peak severity score 6.7 v. 9.8) of type II collagen-induced
Metabolism of DHA via a series of steps, several involving arthritis(72). In another study both EPA and DHA were
5-lipoxygenase, generates a dihydroxydocosatriene termed found to suppress streptococcal cell wall-induced arthritis
neuroprotectin D1, again a potent anti-inflammatory in rats, with EPA being more effective(73).
molecule(61). The identification of these novel EPA- and
DHA-derived mediators is an exciting new area of n-3
fatty acids and inflammatory mediators and the implica- Trials of n-3 PUFA in rheumatoid arthritis
tions to a variety of conditions may be of great impor- Several studies have reported anti-inflammatory effects of
tance(62,63). fish oil in patients with RA, such as decreased LTB4 pro-
Inflammatory cytokines. Cell-culture studies have duction by neutrophils(74–77) and monocytes(76,78), decrea-
demonstrated that EPA and DHA can inhibit the produc- sed PGE2 production by mononuclear cells(79), decreased
tion of IL-1b and TNFa by monocytes(64) and the pro- IL-1 production by monocytes(80), decreased plasma IL-1b
duction of IL-6 and IL-8 by venous endothelial cells(65,66). concentrations(81), decreased serum C-reactive protein
Fish oil feeding decreases ex vivo production of TNFa, concentrations(74,82) and normalisation of the neutrophil
IL-1b and IL-6 by rodent macrophages(67–69). Supple- chemotactic response(83). A number of randomised
mentation of the diet of healthy human volunteers with placebo-controlled double-blind studies of fish oil in RA
fish oil decreases production of TNF or IL-1 or IL-6 have been reported. The characteristics and findings of
by mononuclear cells in some studies(10–13), although a these trials are summarised in Table 1. The dose of long-
number of other studies have shown little effect of n-3 chain n-3 PUFA used in these trials was between 1.6 and
PUFA on production of inflammatory cytokines in human 7.1 g/d and averaged about 3.5 g/d (see Table 1). Almost
subjects(11). The reason for these discrepancies in the all these trials have shown some benefit of fish oil
literature is not entirely clear, but dose of n-3 PUFA used, (Table 1). Such benefits include reduced duration of
technical factors and differences among subjects studied, morning stiffness, reduced number of tender or swollen
including genetic differences(70,71), are likely to be con- joints, reduced joint pain, reduced time to fatigue,
tributing factors. increased grip strength and decreased use of non-steroidal
anti-inflammatory drugs (Table 1). A number of reviews of
these trials have been published(84–90) and each has con-
n-3 PUFA and animal models of rheumatoid arthritis
cluded that there is benefit from fish oil. In an editorial
The effects of n-3 PUFA from fish oil on antigen pre- commentary discussing the use of fish oil in RA it was
sentation, T-cell reactivity and inflammatory lipid and concluded that ‘the findings of benefit from fish oil in
peptide mediator production (Fig. 3) suggest that these rheumatoid arthritis are robust’, ‘dietary fish oil supple-
fatty acids might have a role both in decreasing the risk of ments in rheumatoid arthritis have treatment efficacy’ and
development of RA and in decreasing severity in those ‘dietary fish oil supplements should now be regarded as
patients with the disease. Indeed, dietary fish oil has been part of the standard therapy for rheumatoid arthritis’(91).
414 P. C. Calder
Table 1. Summary of the results of placebo-controlled studies using dietary long-chain n-3 PUFA (in the form of fish oil) in patients with
rheumatoid arthritis
Dose of Duration Clinical outcomes improved with long-chain
Reference EPA + DHA (g/d) (weeks) Placebo n-3 PUFA
Kremer et al.(74) 1.8 + 1.2 12 Paraffin oil No. of tender joints; duration of morning
stiffness
Kremer et al. (75)
2.7 + 1.8 14 Olive oil No. of tender joints; no. of swollen joints;
time to fatigue; physician’s global
assessment
Cleland et al.(76) 3.2 + 2.0 12 Olive oil No. of tender joints; grip strength
van der Tempel et al.(77) 2.0 + 1.3 12 Coconut oil No. of swollen joints; duration of morning
stiffness
Kremer et al.(80) 1.7 + 1.2 24 Olive oil No. of tender joints; no. of swollen joints;
grip strength; physician’s global
assessment
assessment; duration of morning stiffness
Tullekan et al.(78) 2.0 + 1.3 12 Coconut oil No. of swollen joints; joint pain
Skoldstam et al.(95) 1.8 + 1.2 24 Mixed oils No. and severity of tender joints; physician’s
global assessment; use of NSAID
Esperson et al.(81) 2.0 + 1.2 12 Mixed oils No. and severity of tender joints
Nielsen et al.(96) 2.0 + 1.2 12 Vegetable oil No. of tender joints; duration of morning
stiffness
Kjeldsen-Kragh et al.(97) 3.8 + 2.0 16 Maize oil No. and severity of tender joints; duration of
morning stiffness
Lau et al.(98) 1.7 + 1.1 52 Air Use of NSAID
Geusens et al.(99) 1.7 + 0.4 52 Olive oil Physician’s pain assessment; patient’s
global assessment; use of NSAID and/or
disease modifying anti-rheumatic drugs
Kremer et al.(100) 4.6 + 2.5 26–30 Maize oil No. of tender joints; duration of morning
stiffness; physician’s assessment of pain;
physician’s global assessment; patient’s
global assessment
Volker et al.(101) Total 40 mg/kg 15 Mixed oils No. of swollen joints; duration of morning
(approx 2.2–3.0) stiffness; patient’s assessment of pain;
patient’s global assessment; physician’s
global assessment; health assessment by
questionnaire
Adam et al.(102) Approx 2.4 + 1.8 12 Maize oil No. of swollen joints; no. of tender joints;
global assessment; patient’s assessment
of pain
Remans et al.(103) 1.4 + 0.2 ( + 0.5 g-linolenic 16 Liquid supplement None
acid) in a liquid supplement without added PUFA
Berbert et al.(104) Total 3.0 Soyabean oil Duration of morning stiffness; joint pain;
time to onset of fatigue; Ritchie’s articular
index*; grip strength, patient’s global
assessment
Sundrarjun et al.(82) 1.9 + 1.5 24 Not stated None
Galarraga et al.(105) 1.5 + 0.7 36 Air Use of NSAID; patient’s assessment of pain
Approx, approximately; NSAID, non-steroidal anti-inflammatory drugs.

*Based on the summation of a number of quantitative evaluations of the pain experienced by the patient when the joints were subjected to pressures when exerted
over the articular margin or in some instances on movement of the joint(106).
A meta-analysis that included data from nine trials pub- one study that did not use a control for fish oil and one
lished between 1985 and 1992 inclusive and from one study in which transdermal administration of n-3 PUFA by
unpublished trial has concluded that ‘dietary fish oil sup- ultrasound, rather than the oral route, was used(93). This
plementation for three months significantly reduced tender meta-analysis has concluded that fish oil supplementation
joint count (mean difference - 2.9; P = 0.001) and morn- has no effect on ‘patient report of pain, swollen joint count,
ing stiffness (mean difference –25.9 min; P = 0.01)’(92). disease activity or patient’s global assessment’. However,
A more recent meta-analysis of data from trials published this conclusion may be flawed, because of the inappropri-
between 1985 and 2002 included one study of flaxseed oil, ate manner in which studies were combined and because of
Table 2. Summary of the findings of the meta-analysis of Goldberg & Katz(94)

No. of patients
No. of Significance of effect
Outcome studies Control n-3 PUFA of n-3 PUFA: P
Patient-assessed pain 13 247 254 0.03

Physician-assessed pain 3 61 62 0.45
Duration of morning stiffness 8 150 156 0.003
No. of painful and/or tender joints 10 210 215 0.003
Ritchie articular index* 4 68 67 0.40
NSAID consumption 3 79 77 0.01
NSAID, non-steroidal anti-inflammtory drugs.

a poor understanding of the study designs used. For RA. Most of these trials have reported clinical improve-
example, the meta-analysis fails to recognise that patients’ ments (e.g. improved patient assessed pain, decreased
ability to reduce the need for using NSAID or their ability morning stiffness, fewer painful or tender joints, decreased
to be withdrawn from NSAID, as was done in some use of NSAID), and when the trials have been pooled in
designs, must indicate a reduction in pain with n-3 PUFA meta-analyses significant clinical benefit has emerged.
use. This meta-analysis does state that ‘in a qualitative
analysis of seven studies that assessed the effect of n-3
fatty acids on anti-inflammatory drug or corticosteroid Acknowledgements
requirement, six demonstrated reduced requirement for
The author is a consultant to Vifor Pharma, Villars-
these drugs’ and concludes that ‘n-3 fatty acids may reduce
sur-Glane, Switzerland and Danone Research Centre for
requirements for corticosteroids’(93). The effects of long-
Specialised Nutrition, Wageningen, The Netherlands.
chain n-3 PUFA on tender joint count were not assessed by
this meta-analysis, which reiterated the findings of the
earlier meta-analysis that ‘n-3 fatty acids reduce tender References
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Review Article
Special Issue: Eicosanoid Receptors and Inflammation
TheScientificWorldJOURNAL (2007) 7, 1440–1462
ISSN 1537-744X; DOI 10.1100/tsw.2007.188
Endogenous Receptor Agonists: Resolving

Inflammation
Gerard Bannenberg1,*, Makoto Arita2,*, and Charles N. Serhan3

1
Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, CSIC,
Madrid, Spain; 2PRESTO, Japan Science and Technology Agency, Department of
Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo,
Tokyo, Japan; 3Center for Experimental Therapeutics and Reperfusion Injury,
Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and
Women's Hospital, Harvard Medical School and Harvard School of Dental Medicine,
Boston, MA
E-mail: gbannenberg@cnb.uam.es; marita@mol.f.u-tokyo.ac.jp; cnserhan@zeus.bwh.harvard.edu
Received January 22, 2007; Revised June 15, 2007; Accepted July 2, 2007; Published September 1, 2007
Controlled resolution or the physiologic resolution of a well-orchestrated inflammatory

response at the tissue level is essential to return to homeostasis. A comprehensive
understanding of the cellular and molecular events that control the termination of acute
inflammation is needed in molecular terms given the widely held view that aberrant
inflammation underlies many common diseases. This review focuses on recent advances
in the understanding of the role of arachidonic acid and ω-3 polyunsaturated fatty acids
(PUFA)–derived lipid mediators in regulating the resolution of inflammation. Using a
functional lipidomic approach employing LC-MS-MS–based informatics, recent studies,
reviewed herein, uncovered new families of local-acting chemical mediators actively
biosynthesized during the resolution phase from the essential fatty acids
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These new families of
local chemical mediators are generated endogenously in exudates collected during the
resolution phase, and were coined resolvins and protectins because specific members of
these novel chemical families control both the duration and magnitude of inflammation in
animal models of complex diseases. Recent advances on the biosynthesis, receptors,
and actions of these novel anti-inflammatory and proresolving lipid mediators are
reviewed with the aim to bring to attention the important role of specific lipid mediators
as endogenous agonists in inflammation resolution.
KEYWORDS: resolution, inflammation, neutrophil, arachidonic acid (AA), eicosapentaenoic acid

(EPA), docosahexaenoic acid (DHA), omega-3 polyunsaturated fatty acids (ω-3 PUFA),
resolvins, protectins, docosatriene, lipoxin (LX), prostaglandin (PG), human leukocytes, lipid
mediators, apoptosis, chemokines, anti-inflammatory, aspirin
*Corresponding authors. 1440

©2007 with author.
Published by TheScientificWorld; www.thescientificworld.com
Bannenberg et al.: Endogenous Receptor Agonists: Resolving Inflammation TheScientificWorldJOURNAL (2007) 7, 1440–1462
INTRODUCTION
Inflammation
The correct function of body tissues is indispensable for the proper function of the body as a whole. When
tissues are injured through physical damage or are infected by exogenous microbial organisms, local and
systemic responses are activated with the singular objectives to eliminate the offending factors as rapidly
as possible, restore tissue integrity, and retain information about the offending agent in order to facilitate
recognition and elimination on a future encounter. The outcome of these responses is a rapid
physiological reaction of the body towards damage and infection, that is, inflammation. The inflammatory
response effectively operates towards the removal of dead tissue and microbes, yet temporarily allows the
body to deviate from homeostasis in order to recover normal functioning. Five cardinal signs of
inflammation are often denoted to describe this deviation from apparent health at an ultrastructural level:
calor (heat), rubor (redness), tumor (swelling), dolor (pain), and functio laesio (loss of tissue
function)[1]. These signs are reflections of hyperemia (dilation of postcapillary venules causing increased
tissue perfusion and decreased blood flow associated with tissue redness and hotness), an increase in
capillary permeability (causing local fluid accumulation by exudation of serum and consequent rise in
oncotic pressure), and peripheral nervous tissue stimulation by tissue swelling and increased sensitivity
and perception of pain stimuli (causing increased awareness of tissue disturbance). Depending on the
extent of the inflammatory response, a temporary diminution of normal organ function will result.
The inflammatory response is composed of a large number of molecular, cellular, and physiological
changes that allow for the effective delivery of blood-borne leukocytes and lymphocytes to the damaged
or infected tissue. Together with the local synthesis of regulatory mediators, and exudation of serum
factors and acute phase proteins, a seemingly complex and multifactor, yet temporally highly scheduled,
series of events takes place with the purpose to remove microbes and damaged tissue by phagocytosis, the
central effector mechanism of inflammation[2]. The precise composition of components that are
employed, as well as the temporal execution of cellular events during the inflammatory response by each
tissue, depends on the molecular nature of microbial ligands and exposed structural components of
damaged tissue. Specific molecular epitopes that “flag” tissue damage are recognized by recognition
systems, such as complement, collectins, and tissue factor. Likewise, specific microbial epitopes are
detected by the innate immune system via receptors localized on tissue sentinel cells, such as dendritic
cells and macrophages[3]. A binary recognition of conserved microbial molecular patterns in combination
with molecular components of damaged tissue has been proposed to function as an effective means to
discriminate different types of damage, in the presence or absence of infection[4]. Recognition of
microbial antigens also activates specific humoral and cell-mediated immunological responses targeted to
inactivate particular types of microbes, and allows the building of a pool of memory lymphocytes that
permits future recognition of the infectious or tissue damaging agents.
In a typical acute inflammatory response, a remarkably conserved temporal order of events appears to
operate[1] (Fig. 1). After an initial increase in protein exudation, polymorphonuclear leukocytes
accumulate in the inflamed tissue[5,6]. These normally circulating cells migrate to the inflamed tissue in a
series of steps[7]. After first adhering to endothelial cells that line the capillaries perfusing the affected
tissue, neutrophils migrate via transcellular diapedesis across the endothelium into the infected or
damaged tissue[8]. Both positive cues and negative counter-regulatory signals at all steps involved in
cellular migration from blood to tissue determine the magnitude of cellular infiltration and
activation[9,10,11,12]. These activated neutrophils rapidly clear microorganisms and dead tissue by
phagocytosis[13]. After terminating their phagocytotic function, the neutrophils die via apoptosis.
Following the initial accumulation of neutrophils, a delayed infiltration of monocytes follows[1,14].
Monocytes differentiate into macrophages and on their turn remove remaining apoptotic
polymorphonuclear leukocytes by nonphlogistic phagocytosis[15,16]. In this type of phagocytosis,
immunomodulatory and tissue-protective mediators are released, which stop further events that maintain
the evolution of inflammation. Monocyte-derived macrophages also play an important role in regulating
1441
FIGURE 1. Representation of the temporal cellular events in the evolution of inflammation.
1442
the subsequent restoration of tissue. Specific inflammatory cells may be called into action in particular
types of infection; for example, eosinophils are particularly effective in eliminating infections by larger
pathogens, such as helminthes. Lymphocytic inflammatory responses appear to become predominant
during delayed-type hypersensitivity reactions. The preserved temporal order of events suggests that
inflammation evolution is the activation of conserved elements that constitute a molecular program being
executed according to the local needs for clearing the original inciting stimuli.
Resolution of Inflammation
Effective clearance of microbial infections and damaged tissue is normally followed by resolution of
inflammation. This process can be defined at the cellular level as the disappearance of accumulated
polymorphonuclear leukocytes, and at macroscopic level as the reconstruction of tissue architecture and
restoration of tissue function[1,17]. Complete resolution of tissues after bacterial infection will occur
without loss of functional or structural damage and is directly related to the efficiency of microbial
clearance[18,19,20]. Several mechanisms appear to drive the disappearance of inflammatory leukocytes,
and operate in parallel or alternatively in different tissues. Apoptosis of leukocytes is one important route
of elimination[21,22,23]. Cells that have completed their function as phagocytes undergo programmed
cell death in response to local mediators that positively and negatively regulate the rate of
apoptosis[24,25,26]. As polymorphonuclear leukocytes die, they simultaneously function as cytokine
sinks that actively lower the level of proinflammatory cytokines from the inflammatory locus[27,28]. The
apoptotic neutrophils are subsequently phagocytosed by macrophages (efferocytosis) in a so called
nonphlogistic fashion, i.e., in the absence of further generation of proinflammatory mediators and
stimulating the formation of anti-inflammatory mediators, such as transforming growth factor (TGF)-β,
lipoxin (LX) A4, and interleukin (IL)-10[16,29,30]. Another important route of elimination of leukocytes
is egress from the inflamed tissue, as shown for eosinophils in pulmonary inflammation[31].
Macrophages that have completed the elimination of apoptotic neutrophils on their turn disappear from
the resolving inflammatory tissue by either apoptosis or egress via the lymphatic system[32].
Overall, an important conclusion of research into inflammation and resolution over the last 10 years is
that both evolution and resolution are active components of the inflammatory response[24]. Resolution is
not merely the passive dilution of inflammation, but displays the features of activation of specific
molecular events that contribute to removal of inflammatory cells and restoration of tissue integrity. In
view of the early activation of resolution as an integral part of the normal inflammatory response, and the
execution of resolution as discrete molecular and cellular events, one can consider resolution (catabasis)
as an essential characteristic of inflammation. The definition of catabasis is viewed as wider than just the
decline from a state of disease, but rather the return to homeostasis, and emphasizes the cellular and
molecular events involved in that return, which are likely to involve a set of conserved
mechanisms[33,34]. To aid in defining the anti-inflammatory and resolution-stimulating actions of local
mediators and drugs, we have provided a set of indices that define the cellular events during resolution
with quantitative parameters[33]. An important conclusion that has emanated from a close observation of
these measurable indices is that distinct lipid mediators that are generated at precise time points during the
inflammatory response not only inhibit granulocytic inflammation, but also activate and accelerate
resolution. As specific receptors for these anti-inflammatory and resolution-promoting lipid mediators
have been shown to exist, it is now possible to firmly put forward the idea that specific chemical
mediators function as endogenous receptor agonists stimulating the resolution of inflammation[24,35,36].
Defects in the normal development of inflammation are causal factors for and/or associated with
tissue damage that underlies a large number of human diseases. Key roles for inflammation have been
demonstrated in diseases as varied as Alzheimer’s disease and cardiovascular disease[37], as well as
cancer[38], which join the well-known inflammatory disorders, such as rheumatoid arthritis and
periodontal disease[39,40,41]. Uncontrolled acute inflammatory responses, or chronic inflammation with
recurrent episodes of acute inflammation, are associated with tissue damage that can cause discomfort and
severely compromise normal tissue function. Inflammatory responses that are too active or do not resolve
1443
can, consequently, instead of aiding the body to fight infections or restore tissue architecture, become
harmful in themselves. The underlying mechanism of loss of tissue function as a result of inflammation is
often ascribed to the situation where activated leukocytes generate reactive oxygen and nitrogen species,
and release proteases at rates that damage tissue more rapidly than that it can restore[42,43]. For a normal
execution of the inflammatory process, mechanisms must therefore exist that limit the extent of leukocyte
accumulation during inflammation and tone down the magnitude of inflammation evolution. Lipid
mediators derived from polyunsaturated fatty acids (PUFA) arachidonic acid (AA), and the ω-3 PUFA
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), appear to play a particularly important
role as activators of endogenous counter-regulatory, anti-inflammatory, and proresolution mechanisms.
Recent studies have revealed the first molecular details of the mechanisms whereby AA- and ω-3 PUFA–
derived lipid mediators act as endogenous receptor agonists to trigger the active termination of
inflammation via stimulating resolution. In the following sections, a closer look will be taken at the
formation and actions of several resolution-stimulating lipid mediators.
LIPID MEDIATORS AS AGONISTS FOR RESOLUTION
AA-Derived Anti-Inflammatory Lipid Mediators
An important conclusion derived from studies carried out in the 1950s and 1960s that aimed to describe
the chemical structure and formation of the prostaglandins, was that AA is the common endogenous
precursor for the biosynthesis of a large family of bioactive lipid mediators, the eicosanoids[44,45]. AA is
a substrate for both lipoxygenases and cyclooxygenases, fatty acid oxygenases that incorporate one or two
molecules of molecular oxygen into a bis-allylic moiety of PUFA[45,46]. Fatty acid oxygenation by these
specialized oxygenases was shown to be a required first enzymatic step in the formation of a large family
of eicosanoids in animals that includes the prostaglandins, thromboxane, and leukotrienes. The generation
of these lipid mediators requires the posterior modification of the primary oxygenated fatty acids by
specific eicosanoid synthases, and many lipid mediators can be synthesized by a single cell type that
possesses both cyclooxygenase and terminal synthase[47]. Several of the eicosanoids, such as
prostaglandin (PG) E2, PGD2, leukotrienes, and thromboxane, were initially studied with respect to their
role in inflammation and reproduction[48,49]. For example, PGE2 exerts potent phlogistic
(proinflammatory) actions that include vasodilation, potentiation of the vascular permeability activated by
histamine and bradykinin, sensitization of nerve terminals to pain stimuli, and the generation of fever
during infections[50,51]. Leukotrienes are potent chemoattractants for neutrophils and mediators of
vascular permeability[48]. A major focus of research on AA-derived lipid mediators has been, and still is,
the understanding of their proinflammatory actions, as well as therapeutic manipulation of their
biosynthesis. Although one of the prostaglandins, prostacyclin, was identified early as exerting
physiological antagonistic actions to those of thromboxane in the regulation of platelet aggregation and
vascular smooth muscle contraction[52], the potential anti-inflammatory or counter-regulatory actions of
the eicosanoids have been surprisingly poorly explored or disregarded[53].
Lipoxins and Aspirin-Triggered Lipoxins
The insight that different cell types interact, communicate, and act in concert in physiological settings,
such as inflammation, did lead to studies in the beginning of the 1980s with the objective to determine
whether oxygenated AA products formed in one cell type could serve as substrates in a second interacting
cell type[54,55]. The demonstration that a second dioxygenation of 15S-hydroxy-eicosatetraenoic acid
(15S-HETE) occurs in leukocytes, led to the description of a new class of eicosanoids, the lipoxins. Both
LXA4 and LXB4 were isolated, their structures elucidated, and synthesized chemically[56,57]. Later
studies confirmed that transcellular biosynthesis of LXA4 is a primary route of eicosanoid biosynthesis in
interacting cell types, such as neutrophils and epithelial cells, or platelets and neutrophils (Fig.5). LXA4
1444
was subsequently identified to possess potent counter-regulatory actions in inflammation[57]. An

important finding was that neutrophils can store 15S-HETE in an acylated form in specific phospholipids
pools, and which can be rapidly released on cell stimulation and made available for further dioxygenation
to form a second signal, such as LXA4, to regulate the function of the neutrophil[58]. In view of several
identified cellular actions of LXA4 that appeared to be inhibitory rather than stimulatory, LXA4 was
termed to act as a “chalone”, definable as a substance carried though the blood, which has an inhibitory
effect on tissue, or organ that responds to its action. This terminology was further refined when it was
discovered that LXA4 does not indiscriminately inhibit cellular responses, but rather actively stimulates
leukocytes and other cells via specific lipoxin binding sites[60]. The actions of LXA4 more resemble that
of a signal that actively “stops” neutrophil migration and activation in inflammation[61], and, on the other
hand, activates in a non-phlogistic fashion monocyte migration[62]. LXA4 is now known to exert potent
anti-inflammatory actions that are mediated through the activation of a specific G-protein coupled seven-
transmembrane domain-spanning lipoxin receptor[36,63]. In humans, there is one LXA4 receptor named
ALX/FPRL1, while in mice there are at least two receptors for LXA4, ALX1/Fpr-rs1 and ALX2/Fpr-
rs2[36,64,65]. LXA4 can be considered the first example of an endogenous amphiphilic autacoid that
activates receptor-mediated molecular pathways to counter-regulate acute inflammation. The anti-
inflammatory and counter-regulatory actions of LXA4 operate at multiple levels in a variety of resident
and mobile cell types. These include immediate actions on the regulation of signal transduction pathways
activated by proinflammatory and cell growth-stimulatory mediators[66,67,68,69,70]. LXA4 furthermore
regulates gene transcription and transcription factor activity[71,72,73,74]. Stimulation of neutrophils and
eosinophils with LXA4 activates a potent inhibition of chemotaxis, adhesion and migration of these
leukocytes in response to pro-inflammatory mediators[75,76,77,78]. It also potently reduces neutrophil
degranulation, and regulates reactive oxygen and nitrogen species generation by leukocytes[74,79,80].
Actions of LXA4 on other leukocytes include (1) the stimulation of monocyte migration, (2) stimulation
of nonphlogistic phagocytosis by macrophages[81], (3) inhibition of dendritic cell migration and IL-12
formation[82], and (4) inhibition of natural killer cell cytotoxic activity[83]. In experimental animal
models of inflammation, the administration of LXA4 potently (low μg/kg bodyweight) reduces neutrophil
accumulation in inflamed tissues, pointing to the activation of endogenous anti-inflammatory circuits by
LXA4[57,84]. Moreover, transgenic mice with myeloid-selective overexpression of human ALX display a
markedly reduced inflammatory infiltration of leukocytes in peritonitis, which further supports the
counter-regulatory role of ALX[85]. Specific actions of LXA4 have been identified on epithelial cells; in
the gastrointestinal tract LXA4 regulates IL-8 formation and has been found to play an important tissue
protective role[72,86,87]. Moreover, LXA4 acts as an endogenous receptor antagonist at the cysteinyl
leukotriene receptor CysLT1[88]. Importantly, LXA4 modulates vascular, respiratory, and nervous system
physiology[89,90,91,92]. Taken together, a number of actions of LXA4 appear to function in concert to
counter-regulate inflammation and modulate physiology in order to favor resolution of inflammation.
LXB4 shares many of the actions of LXA4, but the study of its actions has not received as detailed
consideration as LXA4. Of interest, LXB4 acts via the activation of a distinct and still unknown receptor.
Specific intracellular proteins such as protein kinase C[93,94] and the nuclear receptor AhR[95] have also
been shown to constitute receptors for lipoxins, but the physiological relevance of these interactions
remains to be investigated in full.
A diastereoisomer of LXA4, 15-epi-LXA4, is formed when AA is oxygenated by cyclooxygenase
(COX)-2 when this enzyme is acetylated by aspirin, followed by a second oxygenation catalyzed by 5-
lipoxygenase. This unique biosynthetic route permits the formation of a bioactive analogue of LXA4,
named aspirin-triggered LXA4 (ATL), which is more resistant to the normal routes of metabolic
degradation of lipoxins in vivo. These routes are oxidation of LXA4 to 15-oxo-LXA4 by 15-hydroxy-
prostaglandin dehydrogenase, reduction to 13,14-dehydro-LXA4 by 15-oxo prostaglandin 13-
reductase/LTB4-12-hydroxy dehydrogenase, and ω-oxidation by specialized cytochrome P450
enzymes[96,97]. 15-Epi-LXA4 displays identical biological activities as native LXA4, and possess
comparable affinity at the ALX receptor[36,98]. 15-Epi-LXA4 has been taken as a lead for introducing
structural modifications with the purpose to incorporate structural elements that further contribute to resist
1445
metabolic degradation. These stable ATL analogs that resist metabolic inactivation have been proven to
be equally efficacious ALX receptor agonists compared with their parent compound, and are often more
potent anti-inflammatory compounds when administered in vivo[36,98]. Initial studies that employed the
stable lipoxin analog 15-R/S-methyl-LXA4 were key to bringing forward the concept of
immunomodulation by lipoxins[99]. Administration of this compound in the mouse air pouch model not
only lowered the levels of proinflammatory mediators MIP-2 and IL-1β, yet also stimulated the formation
of IL-4, a cytokine with potent regulatory actions on inflammatory leukocytes, such as monocytes. The
reorientation by LXA4 of a cytokine-chemokine axis proved to be an essential propensity of lipoxins for
regulating the acute inflammatory response[100]. The aspirin-triggered lipoxin analogs also potently
activate the formation in epithelial cells of the microbicidal and opsonizing protein bactericidal-
permeability inducing protein[101]. Recently, stimulation of nitric oxide formation by the aspirin-
triggered lipoxins has been shown to account for at least part of the anti-inflammatory actions of
aspirin[102].
Further modifications to the chemical structure of LXA4 have been made with the aim to confer
metabolic resistance to β-oxidation, another route of biotransformation of lipid mediators[103]. The
replacement of carbon-3 in the LXA4 structure with an oxygen atom has provided so-called 3-oxa lipoxin
analogs that maintain efficacy as anti-inflammatory compounds after oral, topical, and systemic
administration, and display enhanced pharmacokinetic properties. Selected 3-oxa-15-epi-LXA4 analogs
display increased efficacy compared to analogs that are sensitive to β-oxidation in particular models of
inflammation, such as second-organ lung injury and dermal inflammation[84,104]. Interestingly, in spite
of the development of metabolically stable lipoxin analogs, we have recently described that even after
oral administration of nanogram amounts of native LXA4, systemic anti-inflammatory activity is
maintained[84]. This finding likely reflects the specific and high-affinity binding by lipoxin receptors of
its native ligand, recognition of which only requires localized picomolar to low nanomolar concentration.
Specific lipoxin transport mechanisms, such as the voltage- and sodium-independent LXA4 transporter
that has been described in leukocytes[105], may potentially aid in concentrating and transporting lipoxins
to their sites of action.
Importantly, the actions of LXA4 and ATL are not limited to toning down or counter-regulating
inflammation evolution, but also specifically stimulate resolution at several levels. By activating non-
phlogistic monocytic infiltration, lipoxins facilitate the delivery of the cells that clear dying neutrophils
and regulate tissue restoration[62]. Moreover, LXA4 itself stimulates macrophages in their function as
phagocytes of apoptotic neutrophils[81]. An early decrease in exudates levels of a range of
proinflammatory cytokines and chemokines is activated by ATL, and may be explained by the recent
observation that lipoxins stimulate chemokine sequestration by apoptotic neutrophils[27]. In the murine
air pouch model of inflammation, LXA4 is generated when neutrophils start expressing 15-lipoxygenase
at the onset of resolution. At this stage, a large proportion of neutrophils are apoptotic and now complete
a LXA4 biosynthetic circuit together with 5-lipoxygenase that is already present in these cells[106]. In this
setting, the precise temporal generation of LXA4 specifically turns on resolution of inflammation. PGE2
formation during the inflammatory response is critical for activating the formation of LXA4 ([106], see
below). Cyclooxygenase-derived prostaglandins also initiate the generation of LXA4 in acid-induced
acute lung injury in mice, and this action was found to be essential for the resolution of airway
inflammation[107]. Additionally, macrophages exposed to apoptotic lymphocytes start to produce LXA4,
which may further enhance apoptotic cell removal[29]. At the same time, this process activates the
generation of TGF-β, which can down-regulate a number of proinflammatory circuits[29]. The ATL
stable analog ATLa2 (16-p-fluorophenoxy-15-epi-LXA4-methyl ester) augments TGF-β release prior to
and during the resolution phase of zymosan A–stimulated peritonitis[33].
LXA4 is known to regulate the proliferation of a number of cell types, including a potent inhibitory
action on lung fibroblast proliferation in response to connective-tissue growth factor, and mesangial cell
proliferation stimulated by platelet-derived growth factor, tumor necrosis factor (TNF)-α, or leukotriene
(LT) D4[108,109]. LXA4 furthermore reduces collagen biosynthesis and fibroblast growth induced by
TGF-β[110]. These actions indicate that lipoxins may counteract the fibrotic response, which is one of
1446
several outcomes of tissue remodeling during inflammation. These findings most likely point to distinct
temporal roles of lipid mediators such as promoting leukocyte clearance during the neutrophil-dominated
resolution phase, and regulating tissue restoration after the inflammatory response resolves.
Prostaglandin E2
PGE2 is a ubiquitously formed eicosanoid in mammalian tissues. At least three different enzyme systems
are known that can form PGE2 from PGH2. Both cytosolic PGE2 synthases and microsomal PGE2
synthases are known, and may couple to specific cyclooxygenase-initiated biosynthetic
routes[111,112,113]. The relatively widespread synthesis of PGE2 matches the number of biological
functions that this autacoid displays, and along with the multitude of functions is the presence of at least
four PGE2 (EP) receptors, EP1–4. Although PGE2 is widely viewed as a proinflammatory mediator, a
substantial number of studies indicate that PGE2 also plays distinct roles in down-regulating immune-
inflammatory responses, promotion of tissue repair, and as an activator of resolution[114]. The lung has
long been known as a “privileged” site for such beneficial actions of PGE2[115]. Specific actions of PGE2
in stimulating resolution of inflammation have been documented in allergic edema during infection of rats
with the helminth Angiostrongylus costaricensus[116]. High concentrations of PGE2 are formed during
inflammation and specifically peak at the onset of resolution in the mouse air pouch model[106,117]. This
is an essential event in establishing the complete biosynthetic pathway for LXA4 in a single cell type, the
apoptotic neutrophil. PGE2 activates the transcription of 15-lipoxygenase in exudate neutrophils during
TNF-α–induced inflammation. The generation of LXA4 on its turn was shown to activate the resolution of
inflammation[106]. Such a distinct role for PGE2 in eicosanoid class switching to form LXA4 during
resolution does not appear to be involved in two other models of self-resolving inflammation. In
zymosan-stimulated peritonitis in mouse, PGE2 levels rise during the resolution interval, but is not
accompanied with an increase in LXA4[33]. The LXA4 concentration in the inflammatory exudate peaks
during the proinflammatory phase where it may act to counterbalance LTB4-activated chemoattraction,
migration, and activation of neutrophils[77]. In a second well-studied model of self-resolving
inflammation, carrageenin-induced pleurisy in rat, PGE2 is formed during the evolution of inflammation,
but is not formed at all immediately prior to or during resolution[118]. These apparent incongruous
results are perhaps providing us with the first indications of the existence of distinct types of resolution
responses that are activated in particular settings of inflammation. Inflammation in the air pouch is a
model for a slowly healing wound that is additionally stimulated by TNF-α, whereas zymosan-stimulated
peritonitis and carrageenin-induced pleurisy are models of infection with minimal tissue damage.
Resolving an inflammatory response to a wound vs. a sterile infection most likely also requires a different
set of molecular actors in order to effectively return to homeostasis.
Prostaglandin D2
PGD2 is an important lipid mediator that acts on smooth muscle, inhibits platelet aggregation, influences
pain sensation, and regulates our sleeping behavior[119]. Both the central nervous system and peripheral
organs can synthesize PGD2 via the isomerization of PGH2 catalyzed by lipocalin-type and hematopoietic
PGD synthases. PGD2 has been shown to exert an important function in limiting neutrophil infiltration in
the early phases of experimentally induced colitis in rat[120]. A specific function for PGD2 in
inflammation resolution has been indicated by a number of studies that have shown increased expression
of the hematopoietic PGD synthase during resolution of heart inflammation after endotoxin treatment of
mice[121], increased PGD2 formation during resolution of peritonitis in mice[33], and a reduction of
neutrophilic inflammation by local administration of hematopoietic PGD synthase–expressing fibroblasts
in the murine air pouch model[122]. Furthermore, inhibition of PGD2 formation, by inhibition of COX-2,
impairs resolution of carrageenin-stimulated pleurisy in mice[118]. The role of PGD2 in inflammation has
become a subject of intense study after it was found nonenzymatic breakdown products of PGD2 were
1447
shown to display potent anti-inflammatory actions[49,123,124]. These products are prostaglandins

possessing a cyclopentenone structure and include PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2. Specific
proresolution actions of the cyclopentenones may be of particular importance in T-helper 1 (Th1)–type
inflammatory reactions, where PGD2-derived compounds regulate the intensity of delayed-type
hypersensitivity responses and act as endogenous braking signals for lymphocytes to stimulate
resolution[125]. Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2 also potently stimulate neutrophil apoptosis[126],
their formation is triggered during phagocytosis of apoptotic neutrophils by macrophages[127], and
regulate macrophage activation and proinflammatory gene expression[49]. The actions of the PGD2-
derived cyclopentenones appear to be induced independently from activation of the specific membrane
receptors for PGD2, DP1, or CRTH2/DP2 receptor. Rather, their chemical reactivity permits covalent
adduct formation with reactive sulfhydryl groups, a mechanism whereby these compounds regulate the
activity of intracellular regulatory proteins. In particular, a number of redox-sensitive transcription
factors, and their regulators, are now known to be regulated by cyclopentenone
prostaglandins[128,129,130]. These include PPARγ, Nrf2/Keap1, NF-κB, AP-1, H-Ras, and p53. The
activation of specific transcription factors subsequently triggers the transcription of genes that play
regulatory roles in tissue protection and restoration during resolution[131]. Some points of controversy
with regard to the cyclopentenone PGD2 products continue to provide an interesting front of research.
First of all, careful studies of the biosynthesis of PGD2 products in vivo have indicated that the bioactive
anti-inflammatory PGD2 dehydration products do not appear to be formed in vivo during situations where
PGD2 is formed[132]. Furthermore, the use of supraphysiological concentrations of sulfhydryl-reactive
products may have given experimental results that do not accurately reflect the in vivo situation. In
contrast, mice deficient in hematopoietic PGD2 synthase display defects in resolution of T-cell–dominated
inflammation in a model of delayed-type hypersensitivity. Resolution can be stimulated in these mice by
administration of 15-deoxy-Δ12,14-PGJ2, indicating that PGD2-derived cyclopentenone products do play an
important role in terminating inflammation[125]. It remains a possibility that the reactive prostaglandin
cyclopentenones are not detectable in their free form due to their fast rates of reaction with ubiquitous low
molecular weight and protein sulfhydryl groups. Indeed, intracellular 15-deoxy-Δ12,14-PGJ2 was detected
in activated macrophages by monoclonal antibody specific to 15-deoxy-Δ12,14-PGJ2[133]. Taken together,
the PGD2-derived cyclopentenone products remain of interest, because their high potency and specific
modes of action point to valuable therapeutic targets for stimulation of resolution. Covalent modifications
of regulatory proteins by reactive cyclopentenones also indicate that not all agonists for resolution
obligatorily act through reversible high-affinity interactions with cell-surface G-protein coupled receptors.
Interestingly, the formation of the PGD2 dehydration products was originally found as an albumin-
promoted reaction. The levels of both albumin and PGD2 are both markedly increased in inflammatory
exudates before and during resolution[33]. The conditions for extracellular PGJ2 formation are therefore,
at least theoretically, present, and detailed temporal studies on specific regulatory protein-PGJ2 adducts
may shed new light on the endogenous role of these potent PGD2-derived anti-inflammatory compounds
in resolution. Furthermore, the role of activation of the PGD2 receptors DP1 and CRTH2/DP2 has not been
addressed with respect to their involvement in resolution.
Prostaglandin F2α
The role of PGF2α has been relatively little studied with respect to the resolution of inflammation. PGF2α
can be synthesized by separate reductive routes from either PGH2 and PGE2[134,135]. An 11-epi-PGF2α
epimer, with a different pharmacological profile from PGF2α, is formed by reduction from PGD2. One
recent study has indicated that COX-2–derived PGF2α may contribute to resolution of pleural
inflammation in mice[136]. In this study, an exacerbation of murine pleural inflammation during the
resolution phase after inhibition of COX-2 by administration of a highly-selective cyclooxygenase-2
inhibitor was reversed by administration of the selective FP-receptor agonist fluprostenol. More detailed
studies are required to demonstrate the precise role and contribution of PGF2α as an endogenous agonist
for stimulating resolution.
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ω-3 PUFA-Derived Anti-Inflammatory/Proresolution Lipid Mediators
Studies at the beginning of the 20th century already indicated the essential roles of ω-3 PUFA in
health[137]. Many studies have since addressed the beneficial properties that underlie the protective
action of EPA and DHA, the major ω-3 PUFA in mammalian organisms[138,139,140,141]. A large
randomized trial carried out with >11,000 patients with cardiovascular disease (Gruppo Italiano per lo
Studio della sopravvivenza nell’Infarto miocardico study) showed that patients taking almost 1 g of ω-3
PUFA per day displayed an approximately 45% reduction in sudden death[142,143]. Inspection of the
details of this trial indicated that patients also were taking aspirin. Aspirin therapy was not taken into
account for the beneficial outcomes of this trial. The human lipoxygenases (5-lipoxygenase [LO], 12-LO
and 15-LO) can oxygenate ω-3 PUFA to several monohydroxylated products. The biological importance
of these lipoxygenase-derived products was not known[144,145,146]. Cyclooxygenase can oxygenate
EPA to form PGH3, which can subsequently be transformed to analogs of the natural AA-derived
prostaglandins. However, these prostaglandin-like compounds appear to be far less potent agonists for the
specific prostaglandin receptors. DHA can also be oxygenated in a nonenzymatic fashion to isoprostane-
like compounds (neuroprostanes) in the brain[147], or undergo autooxidation to racemic mixtures of
monohydroxy compounds[148]. Early studies had indicated that cyclooxygenase does not oxygenate
DHA[149]. In summary, several decades of research to understand the molecular basis for the essential
roles of ω-3 PUFA in human health have not provided a clear molecular mechanism to explain their
regulatory and immunoprotective roles.
Resolvins
Recently, we have identified novel oxygenated products derived from EPA and DHA that are generated
via enzymatic pathways. Importantly, these compounds were identified first during the resolution phase
of acute inflammation[150,151,152], namely, in a murine model of inflammation that spontaneously
resolves, the dorsal skin air pouch[53,153]. Exudates were collected during the resolution phase and lipid
mediator profiles were carefully recorded using LC-UV-MS/MS. Exudate cells from resolving air
pouches that had been supplied with EPA and aspirin generated 18R-HEPE, 5-HEPE, and 5,12,18R-
trihydroxy-EPE on stimulation of the cells with calcium ionophore (Fig. 2). Human endothelial cells
expressing cyclooxygenase-2 incubated with aspirin and EPA formed 18R-HEPE. Incubation of 18R-
HEPE with human neutrophils generated 5,12,18R-trihydroxy-EPE, as well as LXA5[150]. 5,12,18R-
Trihydroxy-EPE proved to be a very potent inhibitor of human neutrophil transmigration across
endothelial cells towards chemoattractants, such as LTB4 and fMLP[150]. Isolated cyclooxygenase-2
treated with aspirin oxygenated EPA to form 18R-HEPE as well as 15R-HEPE. Of interest is the finding
that two other commonly used anti-inflammatory drugs, acetaminophen and indomethacin, were found to
also permit the oxygenation of EPA by COX-2 to form 15R- and 18R-HEPE. In summary, these initial
experiments indicated that vascular endothelial cells containing COX-2 acetylated by aspirin convert EPA
to 18R-HEPE, which is oxygenated by 5-LO in human neutrophils to form a 5(6)-epoxide–containing
intermediate that undergoes epoxide hydrolysis and rearrangement to 5,12,18R-triHEPE (Fig. 2). This
compound was named Resolvin E1 (RvE1), a name that reflects the temporal frame in which biosynthesis
occurs (resolution phase), as well as the spatial cellular organization (interaction) necessary for bringing
together the required biosynthetic enzymes. The complete stereochemical assignment was established
using biogenic and synthetic materials and proved to be 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-
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FIGURE 2. Biosynthesis of E-series resolvins. EPA is the endogenous substrate for the formation of E-series resolvins, RvE1 and RvE2. EPA
is first oxygenated by COX-2, acetylated in its catalytic center by aspirin, to form 18R-hydroperoxy-EPE, which is subsequently oxygenated
by neutrophil 5-LO to form 5S-hydroperoxy-18R-HEPE. RvE1 is formed via the formation of an epoxide and subsequent hydrolysis. RvE2 is
formed by reduction of the hydroperoxy group.
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EPA[150,154]. RvE1 displays potent anti-inflammatory actions in vivo; intravenous administration of 100
ng compound reduces exudate cell numbers in the murine air pouch model[33,151,154]. In zymosan A–
stimulated murine peritonitis, local administration of 300 ng RvE1 reduces exudate leukocyte numbers
and activates resolution at an earlier time point, without shortening the resolution interval Ri. RvE1
specifically reduces the exudate levels of a number of proinflammatory cytokines and chemokines (IL-6,
TNF-α, KC, JE, MIP-1α, MIP-2, and RANTES) during resolution[33]. RvE1 reduces pathogen-stimulated
dendritic cell IL-12 generation and migration in the spleen[154]. RvE1 also reduces Porphyromonas
gingivalis–induced oral inflammation and alveolar bone loss during periodontitis[155], as well as has
protective actions in trinitrobenzene-sulfonic acid–induced colitis in mice[156]. RvE1 binds to a G-protein
coupled receptor to down-regulate the activity of NF-κB. This RvE1 receptor is expressed in myeloid,
gastrointestinal, kidney, brain, and cardiovascular tissues[154]. Prior studies identified this receptor to be a
receptor for the serum peptide chemerin, and is hence termed ChemR23[157]. It is also known as
chemokine-like receptor-1. The inhibitory action of RvE1 on dendritic cell migration has been demonstrated
to be mediated through ChemR23 activation[154]. Interestingly, human adherent monocytes display a
marked ChemR23 expression, but neutrophils do not. This observation indicates that tissue
monocytes/macrophages could substantially contribute to the resolution-promoting actions of RvE1. The
ChemR23 receptor is up-regulated by the anti-inflammatory cytokine TGF-β[158]. Given the potent actions
of RvE1 on neutrophils, the existence of a second receptor for RvE1 on human neutrophils is implied.
Just as lipoxins are susceptible to metabolic degradation to regulate tissue concentrations, RvE1 is
transformed by 15-hydroxy-prostaglandin dehydrogenase in the lung to form 18-oxo-RvE1, which is
inactive in vivo. ω-Hydroxylation of RvE1 was found to be a major route of metabolism in human
neutrophils[159]. A stable analog of RvE1, 19-(p-fluorophenoxy)-RvE1, was synthesized to circumvent
these two major routes of metabolic inactivation, and was shown to retain the anti-inflammatory properties
of RvE1[159]. Recently, RvE2 (5S,18-dihydroxy-EPA) (Fig. 2) was identified and proved to be as potent as
RvE1 to block the infiltration of neutrophils in zymosan-stimulated murine peritonitis[160].
Asprin-Triggered D-Series Resolvins
Not only EPA proved to be a substrate for the generation of novel compounds that possess potent anti-
inflammatory, immunoregulatory, and tissue-protective actions[161,162]. DHA was subsequently shown to
be oxygenated by aspirin-acetylated COX-2 as well. Resolving exudates from mice given aspirin and DHA
contained 17R-hydroxy-DHA (17R-HDHA)[151]. The same compound was formed by microvascular
endothelial cells treated with aspirin. Recombinant COX-2 can convert DHA to 13S-hydroxy-DHA, but
when acetylated by aspirin the oxygenation of DHA switches to forming the epimeric 17R-hydroperoxy-
DHA. This aspirin-triggered biosynthetic pathway has been shown to operate at sites of exudate formation
(murine air pouch model) and in brain. 5-LO in human neutrophils can further oxygenate 17R-HDHA to
form 4-hydroperoxy-17R-HDHA and 7-hydroperoxy-17R-HDHA. Both of these two intermediates can
undergo further transformations to the corresponding 4,17R- and 7,17R-dihydroxylated compounds via
reduction of the hydroperoxy group, as well as to trihydroxylated compounds formed via an intermediate
epoxy group, and subsequent spontaneous or epoxide hydrolase–catalyzed hydrolysis (Fig. 3). Potent
inhibitory actions by these novel aspirin-triggered DHA-derived bioactive products (aspirin-triggered D-
series resolvins) were demonstrated on IL-1β secretion by glioma cells, and on neutrophil infiltration in
zymosan A–stimulated peritonitis and the murine air pouch model[151].
Protectins and 17S D-Series Resolvins
Interestingly, neither aspirin nor exogenous DHA was required for the in vivo formation of D-series
resolvins that are hydroxylated at the C17 position as the S-stereoisomers (Fig. 3)[152]. Release of
endogenous DHA can be demonstrated during the inflammatory response[33], most probably via the
stimulated deacylation of DHA from intracellular phospholipid pools[163]. Endogenous DHA can be
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converted via lipoxygenase-initiated mechanisms in vivo to the 17S alcohol-containing series of resolvins
(Fig. 3) and to compounds that possess a docosatriene structure (Fig. 4)[152,161]. One specific DHA-
derived lipid mediator, 10,17S-docosatriene, was termed protectin D1 (PD1). When it is generated
in neural tissue[164], this compound is termed neuroprotectin D1 (NPD1). The complete stereochemistry
FIGURE 3. Biosynthesis of D-series resolvins. DHA is the endogenous substrate for the formation of novel D-series resolvins. These lipid
mediators are formed by oxygenation via the action of a lipoxygenase at the carbon-17 position forming 17S-hydroperoxy-DHA, followed by a
second oxygenation step catalyzed by neutrophil 5-LO. Note that acetylated COX-2 transforms DHA to 17R-hydroperoxy-DHA allowing the
formation of the corresponding 17R D-series resolvins (aspirin-triggered resolvins).
and double bond configuration of PD1 has recently been established. This docosatriene-containing
structure is formed from 17S-hydroperoxy-DHA via an intermediate epoxide that opens via hydrolysis
and subsequent rearrangement to form (in vivo) 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-
hexaenoic acid[164]. PD1 formation has been demonstrated in brain, blood, Th2-skewed lymphocytes,
after ischemia/reperfusion in kidney, and during the resolution phase of zymosan A–stimulated
inflammation[33,152,161,165]. Potent tissue-protective and anti-inflammatory actions are exerted by
PD1. PD1 reduces neutrophil accumulation in murine peritonitis after intravenous administration of 100
ng compound[152]. Neutrophil transmigration across endothelial cells was reduced approximately 50%
by 10 nM PD1, whereas the Δ15-trans isomer of PD1 was inactive. Potent protective actions afforded by
this lipid mediator have been determined in experimental models of stroke[161], and in the protection of
retinal epithelial cells from oxidative stress[161,162]. Furthermore, a markedly reduced formation of PD1
and decreased 15-LO expression has been found in Alzheimer’s disease brain tissue[41]. Both 17S-
hydroxy-DHA and NPD1 have been shown to potently reduce TNF-α-stimulated IL-1β gene transcription
in human glial cells[152].
The potent and stereospecific actions of PD1 strongly suggest that PD1 acts through a yet unidentified
receptor. A further indication to the existence of a specific PD1 receptor is the observation that the
1452
inhibitory actions of PD1 are additive with those of RvE1 in vivo[164]. An explicit role for PD1 in
resolution of inflammation has been exposed[33]; the formation of PD1 is activated specifically during
the resolution phase of murine peritonitis. Local administration of 300 ng PD1 prior to initiation of
zymosan A–stimulated peritonitis reduced maximal neutrophil infiltration (decreased φmax), shortened the
resolution interval (decreased Ri), and activated resolution at an earlier time point (decreased Tmax).
Furthermore, like RvE1, PD1 reduced proinflammatory cytokine levels specifically during the resolution
phase in this model of inflammation[33].
FIGURE 4. Biosynthesis of PD1. 17S-Hydroperoxy-DHA, generated by oxygenation of DHA, can also be transformed to a 16S(17)-epoxy-
docosatriene intermediate that, via hydrolysis, leads to the formation of a 10R,17S-dihydroxy-triene–containing lipid mediator named PD1, as
well as a 16,17S-docosatriene. A second lipoxygenation-catalyzed dioxygenation of 17S-hydroperoxy-DHA leads to the formation of the double-
dioxygenated 10S,17S-dihydroxy geometric isomer of PD1, as well as 7,17S-dihydroxy-DHA. Note that aspirin-acetylated COX-2 can oxygenate
DHA to form 17R-hydroperoxy-DHA allowing the generation of the corresponding 17R-epimeric aspirin-triggered products.
D-series resolvins with protective actions are also formed during inflammation. In response to
bilateral ischemia/reperfusion of mouse kidney injury, small amounts of 17S-DHA and RvD2 (7S,16,17S-
trihydroxy-DHA) were found in kidney tissue and in plasma[166]. On intravenous and subcutaneous
administration of DHA, the formation of these products was markedly increased, and the formation of
RvD1 (7S,8,17S-trihydroxy-DHA) and lower levels of RvD3 (Fig. 3), RvD5, and RvD6[167] were
activated as well. Administration of RvD1, RvD2, and RvD3 prior to and during ischemia afforded
kidney tissue protection, both functionally and morphologically; exerted a marked anti-inflammatory
action; and reduced the subsequent development of interstitial fibrosis[166]. The generation of RvD1 in
brain has been demonstrated after an ischemic insult to murine brain[161].
Taken together, a number of recent studies have now revealed that both EPA and DHA are
endogenous precursors for the generation of a new and large family of oxygenated products that possess
potent protective anti-inflammatory and resolution-stimulating actions. These actions are mediated via the
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activation of specific receptors, of which ChemR23 is the first identified receptor. Further support for the
role of EPA and DHA as endogenous precursors for the synthesis of protective resolvins and protectins
has recently been demonstrated employing transgenic Fat-1 mice that are able to synthesize ω-3 PUFA
endogenously. These mice form resolvins and protectins endogenously and display a reduced
inflammatory response in colitis compared to wild-type mice that are essentially dependent on dietary
sources of EPA and DHA[168,169]. It is important to stress that lipid mediators of the resolvins and
protectin family exert their action at picomolar to nanomolar concentrations[150,151,152,161,162],
whereas EPA- and DHA-derived prostaglandin-like products, which have been characterized in earlier
studies, are far less potent or devoid of biological action[144,149,170]. It is of interest that microbial
organisms also possess the capacity to form oxygenated fatty acids that could feed into endogenous
biosynthetic circuits forming resolvins. For instance, EPA can be converted by microbial cytochrome P450
to form 18R-HEPE, which can subsequently be transformed to RvE1 by neutrophil 5-LO. Similarly, LTB5
can be transformed in vitro to RvE1 by this cytochrome P450 [150,171]. This route of biosynthesis was
useful in the biogenic synthesis of RvE1. Whether this route is used in vivo in certain biological settings
remains of interest. Symbiotic and pathogenic microbes have developed biosynthetic routes in
coevolution with their natural hosts, and have learned to exploit the formation of anti-inflammatory, host-
protective, and immunomodulatory biosynthetic routes for their survival[171,172,173].
SUMMARY AND CONCLUSION
A physiologically normal inflammatory response can be viewed as having defined temporal phases of
inflammation evolution and resolution. A number of studies in the field have now led to the idea that the
inflammatory response comprises a number of programmed events that are executed in a temporally
defined fashion. Several important checkpoints in inflammation can be assigned that activate the next
stage of the response. It is important to note that different components may activate the employment of
subsets of molecular components, or possibly in a different temporal order, depending on the original
molecular stimulus that initiated inflammation. For example, we have seen that in acute inflammation
initiated by administration of TNF-α in the mouse air pouch, resolution on inflammation turns on when
apoptotic neutrophils start expressing 15-LO, which in concert with already present 5-LO completes the
formation of a LXA4 biosynthetic circuit[106]. In zymosan A–initiated murine peritonitis, LXA4 appears
to play a role during the evolution of inflammation, possible as a counteracting signal to regulate the
magnitude of neutrophil infiltration[33]. Yet, in this model, the proresolving docosatriene PD1 is
specifically formed during resolution. Together with the resolution-promoting and accelerating actions of
resolvins and protectins, these findings point to central roles for endogenous lipid mediators in the
activation of resolution. Delineating the different “programs” of inflammation is an important and
interesting aim for future investigations, as therapeutic intervention in inflammation may need to beget
precision in order to target specific components of the inflammatory response.
Inflammation underlies and accompanies a majority of the diseases that afflict human beings. Yet,
treatment of inflammatory disease is generally limited to the use of compounds that antagonize the
evolution of inflammation. It still remains a major challenge to treat chronic or recurrent inflammatory
diseases. It is only recently that we have started to consider that the stimulation of inflammation
resolution may constitute a viable therapeutic approach for treatment of inflammatory disease. We have
learned that in experimental models of inflammation, resolution of inflammation is not merely the
termination of inflammation, but displays discrete molecular and cellular changes that point to the active
progression of inflammation to the healthy state. If the view is taken that resolution is the execution of a
program that forms an essential part of the acute inflammatory response, than it follows that the
temporally defined generation of pro- and anti-inflammatory and proresolution lipid mediators is an
integral part of this program. Our growing appreciation that these mediators exert their biological actions
1454
FIGURE 5. Generation and actions of anti-inflammatory/proresolution lipid mediators during acute inflammation and resolution. The formation
of anti-inflammatory and pro-resolution lipid mediators is characterized by heterotypic cellular interactions that bring together the required fatty
acid oxygenases permitting transcellular biosynthesis. (A) Biosynthesis of LXA4 by transformation of AA via oxygenation by neutrophil 5-LO
and platelet 12-LO. (B) Biosynthesis of LXA4 by transformation of AA via sequential oxygenation by epithelial cell 15-LO and neutrophil 5-LO.
(C) Formation of aspirin-triggered E- and D-series resolvins from the ω-3 PUFA EPA and DHA. (D) Formation of protectins and D-series
resolvins from DHA via sequential oxygenation steps involving neutrophil lipoxygenases and other inflammatory cells lipoxygenases. (E)
Formation of LXA4 by apoptotic neutrophils expressing both 5- and 15-LO[106]. (F) Formation of LXA4 by macrophages that are exposed to
apoptotic lymphocytes[29]. The formed LXA4 contributes to the nonphlogistic nature of apoptotic cell removal by phagocytosis, and LXA4 itself
can stimulate the phagocytosis of apoptotic neutrophils[81].
1455
via the activation of specific receptors indicates that agonist-receptor interactions are critical regulatory
events in the progression of the normal inflammatory response. Possibly, defects in the generation of the
discussed proresolution lipid mediators, their agonist-receptor interactions, and subsequent signaling
actions, underlie inflammatory disease. This puts forward the very interesting prospect for active
modulation of life-threatening acute inflammation or chronic inflammatory disease through activation of
specific endogenous resolution circuits.
ACKNOWLEDGMENTS
Work reviewed here in the CNS laboratory was supported in part by the National Institutes of Health
USA grant nos. GM38765 and P50-DE016191 (CNS). Gerard Bannenberg was supported by a
Postdoctoral Fellowship from the Arthritis Foundation, and is a current Ramón y Cajal fellow supported
by the Spanish Ministry of Education and Science, and the Centro Nacional de Biotecnología, Consejo
Superior de Investigaciones Científicas, Madrid, Spain. Makoto Arita was supported by a Postdoctoral
Fellowship for Research Abroad from the Japanese Society for the Promotion of Science and Uehara
Memorial Foundation, and is a current PRESTO researcher funded by the Japanese Science and
Technology Agency.
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This article should be cited as follows:

Bannenberg, G., Arita, M., and Serhan, C.N. (2007) Endogenous receptor agonists: resolving inflammation.
TheScientificWorldJOURNAL 7, 1440–1462. DOI 10.1100/tsw.2007.188.
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Proceedings of the Nutrition Society (2008), 67, 409–418 doi:10.1017/S0029665108008690
g The Author 2008
A Meeting of the Nutrition Society, hosted by the Irish Section, was held at the O’Reilly Hall, University College Dublin, Dublin,
Republic of Ireland on 18–20 June 2008
Symposium on ‘The challenge of translating nutrition research into public

health nutrition’
Session 3: Joint Nutrition Society and Irish Nutrition and Dietetic

Institute Symposium on ‘Nutrition and autoimmune disease’
PUFA, inflammatory processes and rheumatoid arthritis
Philip C. Calder
Institute of Human Nutrition, School of Medicine, University of Southampton, IDS Building, MP887, Southampton General
Hospital, Tremona Road, Southampton SO16 6YD, UK
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease manifested by

swollen and painful joints, bone erosion and functional impairment. The joint lesions are
characterised by infiltration of T lymphocytes, macrophages and B lymphocytes into the
synovium and by synovial inflammation involving eicosanoids, cytokines and matrix metallo-
proteinases. In relation to inflammatory processes, the main fatty acids of interest are the n-6
PUFA arachidonic acid, which is the precursor of inflammatory eicosanoids such as PGE2 and
leukotriene B4, and the n-3 PUFA EPA and DHA, which are found in oily fish and fish oils.
Eicosanoids derived from the n-6 PUFA arachidonic acid play a role in RA, and the efficacy of
non-steroidal anti-inflammatory drugs in RA indicates the importance of pro-inflammatory
cyclooxygenase pathway products of arachidonic acid in the pathophysiology of the disease.
EPA and DHA inhibit arachidonic acid metabolism to inflammatory eicosanoids. EPA also
gives rise to eicosanoid mediators that are less inflammatory than those produced from
arachidonic acid and both EPA and DHA give rise to resolvins that are anti-inflammatory and
inflammation resolving. In addition to modifying the lipid mediator profile, n-3 PUFA exert
effects on other aspects of immunity relevant to RA such as antigen presentation, T-cell re-
activity and inflammatory cytokine production. Fish oil has been shown to slow the develop-
ment of arthritis in an animal model and to reduce disease severity. Randomised clinical trials
have demonstrated a range of clinical benefits in patients with RA that include reducing pain,
duration of morning stiffness and use of non-steroidal anti-inflammatory drugs.
Cytokine: Eicosanoid: Fatty acid: Fish oil: Inflammation
Immune system overview commensal gut bacteria. The system has two functional
divisions: the innate (or natural) immune system and the
The immune system is responsible for the host’s response acquired (also termed specific or adaptive) immune system.
to the presence of bacteria, viruses, fungi and parasites; it Both components involve various blood-borne factors and
is also involved in protection against growth of certain cells. Immune cells originate in bone marrow and are
tumours and in the response to injury and trauma. The found circulating in the bloodstream, organised into lym-
immune system acts to distinguish between ‘self’ and ‘non- phoid organs such as the thymus, spleen, lymph nodes
self’, permitting tolerance to self antigens and to non- and gut-associated lymphoid tissue or dispersed in other
threatening environmental agents such as food proteins and locations. The immune response is typified by cellular
Abbreviations: HLA, human leucocyte antigen; IFN, interferon; LT, leukotriene; NSAID, non-steroidal anti-inflammatory drug; RA, rheumatoid arthritis.
Corresponding author: Professor Philip Calder, fax + 44 2380 795255, email pcc@soton.ac.uk
410 P. C. Calder
interactions and by the movement of cells to sites of Synovial fluid from patients with RA contains high
infection or other immune activity. The four key functional levels of pro-inflammatory cytokines including TNFa,
activities of the immune response are: IL-1b, IL-6, IL-8 and granulocyte–macrophage colony-
stimulating factor(5). Synovial cells cultured ex vivo spon-
to act as an exclusion barrier; taneously produce TNFa, IL-1b, IL-6, IL-8 and granulo-
to distinguish self from non-self;
cyte–macrophage colony-stimulating factor for extended
to develop tolerance to, or to eliminate the source of,
periods of time(5). Synovial fluid from patients with RA
non-self antigens;
also contains high levels of anti-inflammatory cytokines
to retain memory of immunological encounters.
such as transforming growth factor b, IL-10, IL-1 receptor
In order to allow effective functioning of the immune antagonist and soluble TNF receptors(5). Thus, the inflamed
system many different cell types, each specialised in a synovial joint contains excessive amounts of both pro- and
limited range of functions, are involved. These cells work anti-inflammatory cytokines, but given the ongoing state of
in a coordinated integrated manner in order to assure a inflammation there must be an imbalance in favour of the
successful immune response. former.
Loss of tolerance can lead to disease and to adverse
patient outcome. For example, autoimmune diseases result
from loss of tolerance to self antigens, allergic diseases Arachidonic acid, eicosanoids and the link with
result from loss of tolerance to normally benign environ- inflammation

mental or food components and inflammatory bowel dis- Eicosanoids are key mediators and regulators of inflam-
eases result from loss of tolerance to commensal gut mation(6,7) and are generated from C20 PUFA. As inflam-
bacteria. The loss of tolerance leads to an immunological matory cells typically contain a high proportion of the n-6
response that is damaging to the host. PUFA arachidonic acid (20 : 4n-6) and low proportions of
other C20 PUFA, arachidonic acid is usually the major
substrate for eicosanoid synthesis. Eicosanoids, which
Rheumatoid arthritis include PG, thromboxanes, leukotrienes (LT) and other
oxidised derivatives, are generated from arachidonic acid
Rheumatoid arthritis (RA) is a chronic inflammatory
by the metabolic processes summarised in Fig. 1. They
autoimmune disease that affects about 1 % of the adult
are involved in modulating the intensity and duration
population and is more common in women than in men(1).
of inflammatory responses(6,7), have cell- and stimulus-
RA is characterised by symmetric polyarthritis(1). Joint
specific sources and frequently have opposing effects.
inflammation is manifested by swelling, pain, functional
Expression of both isoforms of cyclooxygenase is
impairment, morning stiffness, osteoporosis and muscle
increased in the synovium of patients with RA(5,8) and in
wasting. Erosion of bone occurs commonly in the joints of
joint tissues in rat models of arthritis(8). PGE2, LTB4 and
the hands and feet. The joint lesions are characterised by
5-hydroxyeicosatetraenoic acid are found in the synovial
infiltration of activated T lymphocytes, macrophages and
fluid of patients with active RA(9). Infiltrating leucocytes
antibody-secreting B lymphocytes into the synovium (the
such as neutrophils, monocytes and synoviocytes are
tissue lining the joints) and by proliferation of fibroblast-
important sources of eicosanoids in RA(9). PGE2 has a
like synovial cells called synoviocytes(1,2). These cells and
number of pro-inflammatory effects, including increasing
new blood vessels form a tissue termed pannus that leads
vascular permeability, vasodilation, blood flow and local
to progressive destruction of cartilage and bone, which is
pyrexia and potentiation of pain caused by other agents. It
most likely to be a result of cytokine- and eicosanoid-
also promotes the production of some matrix metallo-
mediated induction of destructive enzymes such as matrix
proteinases and stimulates bone resorption. The efficacy of
metalloproteinases. RA is also characterised by signs of
non-steroidal anti-inflammatory drugs (NSAID), which act
systemic inflammation, such as elevated plasma concen-
to inhibit cyclooxygenase activity, in RA indicates the
trations of some cytokines (e.g. IL-6), acute-phase proteins
importance of this pathway in the pathophysiology of the
and rheumatoid factors.
disease. However, although these drugs provide rapid relief
Genetic studies have linked susceptibility to, and sever-
of pain and stiffness by inhibiting joint inflammation, they
ity of, RA to genes in the MHC II locus; in human subjects
do not influence the course of the disease. LTB4 increases
these genes encode the human leucocyte antigen (HLA) II
vascular permeability, enhances local blood flow, is a
proteins involved in antigen presentation. RA is associated
potent chemotactic agent for leucocytes, induces release
with specific alleles of the HLA-DRB1 gene, although
of lysosomal enzymes and enhances release of reactive
other HLA-DR alleles may also play a role(3). As the
oxygen species and inflammatory cytokines such as TNFa,
function of HLA-DR is antigen presentation to T lympho-
IL-1b and IL-6.
cytes, the genetic association indicates a role for T-cells in
RA(4). In total the HLA region contributes 30–50 % of the
genetic component of RA. The second largest genetic risk
Very-long-chain n-3 PUFA and inflammatory processes
for RA lies with a variant in the protein tyrosine phos-
phatase non-receptor 22 gene, which encodes an intracel- Oily fish and fish oils contain the very-long-chain n-3
lular protein tyrosine phosphatase(3). The variant may act PUFA EPA (20 : 5n-3) and DHA (22 : 6n-3). Increased
to reduce the ability to down regulate activated T-cells. consumption of these fatty acids results in their incor-
Recently, novel risk loci have been described(3). poration into immune cell phospholipids(10–13), which
Arachidonic acid in
cell membrane phospholipids
Phospholipase A2
Free arachidonic acid
COX-1 15-LOX 12-LOX 5-LOX

COX-2
PGG2 15-HPETE 12-HPETE 5-HPETE
PGH2 15-HETE 12-HETE LTA4 5-HETE

LTC4 LTB4
PGD2 PGE2 PGI2 TXA2 PGF2α Lipoxin A4
LTD4
PGJ2
LTE4
Fig. 1. Outline of the pathway of eicosanoid synthesis from arachidonic acid. COX, cyclooxygenase; HETE,
hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; TX,
thromboxane.
occurs in a dose–response fashion and is partly at the DHA decreases endotoxin-induced class II molecule up-
expense of arachidonic acid. The changed membrane fatty regulation(32) EPA and DHA treatment has been reported
acid composition is believed to influence immune cell to diminish the up-regulation of HLA-DR and HLA-DP
function and inflammatory processes(14) (Fig. 2). There associated with IFN-g stimulation of human monocytes(33).
have been numerous reviews of the influence of n-3 PUFA It has subsequently been demonstrated that these fatty
on many aspects of immune function in recent years(10–27) acids decrease the ability of human monocytes to present
and the reader is referred to these articles for details antigen(34). Three studies, one in mice(35), one in rats(36)
beyond those provided in the following sections. and one in human subjects(37) have reported effects of
dietary n-3 PUFA on class II expression. Feeding mice fish
oil, which contains EPA and DHA, results in a reduction in
Antigen-presenting cell function MHC II expression on peritoneal cells (mainly B lympho-
There have been several studies of the effects of n-3 PUFA cytes and macrophages)(35). A human supplementation
on MHC II or HLA expression or antigen presentation via study with fish oil has reported decreased expression of
class II molecules(28). These studies have typically found HLA-DR, -DP and -DQ on IFN-g-stimulated blood
that class II expression and antigen presentation via class II monocytes(37), with similar effects to those seen with n-3
molecules are decreased by n-3 PUFA. An in vitro study in PUFA in vitro(33). These studies did not examine antigen
which spleen cells were incubated with EPA has reported presentation activity. However, a study that involved
decreased ability of those cells to present antigen(29); feeding an EPA-rich oil to mice has shown decreased
this study did not report class II expression. Incubating antigen (keyhole limpet (Megathura crenulata) haemo-
murine macrophages with DHA decreases expression of cyanin) presentation by spleen cells to T-cell clones(29).
the class II molecules (termed Ia in mice)(30). Likewise, Perhaps the most thorough study of this type to date is that
incubating mouse macrophages with EPA or DHA in which feeding a fish oil-rich diet to rats was found to
decreases interferon (IFN)-g-induced up-regulation of class result in decreased expression of MHC II on dendritic
II molecules(31) and incubating mouse dendritic cells with cells(36). These cells were found to have a much reduced
412 P. C. Calder
Increased supply of
long-chain n-3 PUFA
Decreased arachidonic acid

and increased long-chain
n-3 PUFA in inflammatory cell
membrane phospholipids
Altered membrane Altered signal

structure and fluidity transduction Altered inflammatory
(gross, rafts, acylation) Altered pattern of pathways gene expression
lipid mediator
synthesis
Promotion of a less-inflammatory phenotype
Fig. 2. Mechanisms by which n-3 PUFA can affect inflammatory cell activity.
capacity to present antigen (keyhole limpet haemocyanin) Inflammatory mediator production

to antigen-sensitised spleen T-cells. The reduction in anti-
gen presentation is probably much greater than could be Eicosanoids. Increased consumption of very-long-chain
explained by the reduction in class II expression, suggest- n-3 PUFA such as EPA and DHA, results in decreased
ing that other interactions between antigen-presenting cells amounts of arachidonic acid present in immune cell mem-
and T lymphocytes are affected by dietary n-3 PUFA. It branes and available for synthesis of eicosanoids(10–13).
was reported that levels of the co-stimulatory molecules Thus, feeding fish oil to laboratory rodents or supple-
CD2, CD11a and CD18 are also decreased on dendritic menting the diet of human subjects with fish oil has
cells from fish oil-fed rats(36). been reported to result in decreased production of a range
of eicosanoids including PGE2, thromboxane B2, LTB4,
5-hydroxyeicosatetraenoic acid and LTE4 by inflammatory
T lymphocyte reactivity
cells(10–13). A recent study has demonstrated the dose–
In vitro studies have demonstrated that EPA and DHA response effect to dietary EPA of PGE2 production by
decrease T-cell proliferation(38–41) and the production of endotoxin-stimulated human mononuclear cells and sug-
helper T-cell 1-type cytokines such as IL-2(38,39,42). Feeding gests that an EPA intake of > 2 g/d is required in order to
studies in rodents and supplementation studies in human be effective(53).
subjects have also shown that fish oil decreases T-cell EPA is also able to act as a substrate for both cyclo-
proliferation(43–48) and production of helper T-cell 1-type oxygenase and 5-lipoxygenase, giving rise to eicosanoids
cytokines such as IL-2(42,45,47,48) and IFN-g (42,48), although with a slightly different structure from those formed from
it is important to note that not all human studies report arachidonic acid. Thus, fish oil supplementation of the
such an effect(11). The reason for these discrepancies in human diet has been shown to result in increased pro-
the literature is not entirely clear, but dose of n-3 PUFA duction of LTB5, LTE5 and 5-hydroxyeicosapentaenoic
used, technical factors and differences among subjects acid by inflammatory cells(10–13). The functional impor-
studied are likely to be contributing factors. tance of this outcome is that the mediators formed from
The mechanism by which long-chain n-3 PUFA affect EPA are frequently less potent than those formed from
T-cell reactivity was initially thought to relate to altered arachidonic acid; for example, LTB5 is less potent as a
patterns of eicosanoid synthesis; however, through the use neutrophil chemotactic agent than LTB4(54,55).
of eicosanoid synthesis inhibitors and pure eicosanoids Resolvins and related compounds: novel EPA- and
in vitro this mechanism has been shown to be unlikely(40). DHA-derived anti-inflammatory mediators. Recent stu-
Studies over the last few years have demonstrated that the dies have identified a novel group of trihydroxyeicosa-
inhibitory effects of n-3 PUFA in general, and of EPA in pentaenoic acid mediators, termed E-series resolvins,
particular, relate to membrane-mediated effects that impact formed from EPA by a series of reactions involving
on the early stages of cell signalling(49–52). cyclooxygenase-2 (acting in the presence of aspirin) and
Antigen-presenting
Self antigen
cell Y
YYY
Self antigen-specific
B IgG = autoantibodies
∗
MHCII
∗
IFN-γ
T-cell
receptor
IL-2
IL-12 Damage to host tissue

Th Th1
IFN-γ
IFN-γ ∗ Inflammation
Th2
Macrophage
Fig. 3. Cellular sites of anti-inflammatory actions of long-chain n-3 PUFA. IFN, interferon; Th, helper T-cell; Y,
IgG. , Sites of action of n-3 PUFA; , inhibits.
5-lipoxygenase. These mediators appear to exert potent shown to have beneficial effects in animal models of
anti-inflammatory actions(56–58). In addition, DHA-derived arthritis. For example, compared with vegetable oil, feed-
trihydroxydocosahexanoic acid mediators termed D-series ing mice fish oil delays the onset (mean 34 d v. 25 d) and
resolvins are produced by a similar series of reactions reduces the incidence (69 % v. 93 %) and severity (mean
and these resolvins are also anti-inflammatory(59,60). peak severity score 6.7 v. 9.8) of type II collagen-induced
Metabolism of DHA via a series of steps, several involving arthritis(72). In another study both EPA and DHA were
5-lipoxygenase, generates a dihydroxydocosatriene termed found to suppress streptococcal cell wall-induced arthritis
neuroprotectin D1, again a potent anti-inflammatory in rats, with EPA being more effective(73).
molecule(61). The identification of these novel EPA- and
DHA-derived mediators is an exciting new area of n-3
fatty acids and inflammatory mediators and the implica- Trials of n-3 PUFA in rheumatoid arthritis
tions to a variety of conditions may be of great impor- Several studies have reported anti-inflammatory effects of
tance(62,63). fish oil in patients with RA, such as decreased LTB4 pro-
Inflammatory cytokines. Cell-culture studies have duction by neutrophils(74–77) and monocytes(76,78), decrea-
demonstrated that EPA and DHA can inhibit the produc- sed PGE2 production by mononuclear cells(79), decreased
tion of IL-1b and TNFa by monocytes(64) and the pro- IL-1 production by monocytes(80), decreased plasma IL-1b
duction of IL-6 and IL-8 by venous endothelial cells(65,66). concentrations(81), decreased serum C-reactive protein
Fish oil feeding decreases ex vivo production of TNFa, concentrations(74,82) and normalisation of the neutrophil
IL-1b and IL-6 by rodent macrophages(67–69). Supple- chemotactic response(83). A number of randomised
mentation of the diet of healthy human volunteers with placebo-controlled double-blind studies of fish oil in RA
fish oil decreases production of TNF or IL-1 or IL-6 have been reported. The characteristics and findings of
by mononuclear cells in some studies(10–13), although a these trials are summarised in Table 1. The dose of long-
number of other studies have shown little effect of n-3 chain n-3 PUFA used in these trials was between 1.6 and
PUFA on production of inflammatory cytokines in human 7.1 g/d and averaged about 3.5 g/d (see Table 1). Almost
subjects(11). The reason for these discrepancies in the all these trials have shown some benefit of fish oil
literature is not entirely clear, but dose of n-3 PUFA used, (Table 1). Such benefits include reduced duration of
technical factors and differences among subjects studied, morning stiffness, reduced number of tender or swollen
including genetic differences(70,71), are likely to be con- joints, reduced joint pain, reduced time to fatigue,
tributing factors. increased grip strength and decreased use of non-steroidal
anti-inflammatory drugs (Table 1). A number of reviews of
these trials have been published(84–90) and each has con-
n-3 PUFA and animal models of rheumatoid arthritis
cluded that there is benefit from fish oil. In an editorial
The effects of n-3 PUFA from fish oil on antigen pre- commentary discussing the use of fish oil in RA it was
sentation, T-cell reactivity and inflammatory lipid and concluded that ‘the findings of benefit from fish oil in
peptide mediator production (Fig. 3) suggest that these rheumatoid arthritis are robust’, ‘dietary fish oil supple-
fatty acids might have a role both in decreasing the risk of ments in rheumatoid arthritis have treatment efficacy’ and
development of RA and in decreasing severity in those ‘dietary fish oil supplements should now be regarded as
patients with the disease. Indeed, dietary fish oil has been part of the standard therapy for rheumatoid arthritis’(91).
414 P. C. Calder
Table 1. Summary of the results of placebo-controlled studies using dietary long-chain n-3 PUFA (in the form of fish oil) in patients with
rheumatoid arthritis
Dose of Duration Clinical outcomes improved with long-chain
Reference EPA + DHA (g/d) (weeks) Placebo n-3 PUFA
Kremer et al.(74) 1.8 + 1.2 12 Paraffin oil No. of tender joints; duration of morning
stiffness
Kremer et al. (75)
2.7 + 1.8 14 Olive oil No. of tender joints; no. of swollen joints;
time to fatigue; physician’s global
assessment
Cleland et al.(76) 3.2 + 2.0 12 Olive oil No. of tender joints; grip strength
van der Tempel et al.(77) 2.0 + 1.3 12 Coconut oil No. of swollen joints; duration of morning
stiffness
assessment
assessment; duration of morning stiffness
Tullekan et al.(78) 2.0 + 1.3 12 Coconut oil No. of swollen joints; joint pain
Skoldstam et al.(95) 1.8 + 1.2 24 Mixed oils No. and severity of tender joints; physician’s
global assessment; use of NSAID
Esperson et al.(81) 2.0 + 1.2 12 Mixed oils No. and severity of tender joints
Nielsen et al.(96) 2.0 + 1.2 12 Vegetable oil No. of tender joints; duration of morning
stiffness
Kjeldsen-Kragh et al.(97) 3.8 + 2.0 16 Maize oil No. and severity of tender joints; duration of
morning stiffness
Lau et al.(98) 1.7 + 1.1 52 Air Use of NSAID
Geusens et al.(99) 1.7 + 0.4 52 Olive oil Physician’s pain assessment; patient’s
global assessment; use of NSAID and/or
disease modifying anti-rheumatic drugs
Kremer et al.(100) 4.6 + 2.5 26–30 Maize oil No. of tender joints; duration of morning
stiffness; physician’s assessment of pain;
physician’s global assessment; patient’s
global assessment
Volker et al.(101) Total 40 mg/kg 15 Mixed oils No. of swollen joints; duration of morning
(approx 2.2–3.0) stiffness; patient’s assessment of pain;
global assessment; health assessment by
questionnaire
Adam et al.(102) Approx 2.4 + 1.8 12 Maize oil No. of swollen joints; no. of tender joints;
global assessment; patient’s assessment
of pain
Remans et al.(103) 1.4 + 0.2 ( + 0.5 g-linolenic 16 Liquid supplement None
acid) in a liquid supplement without added PUFA
Berbert et al.(104) Total 3.0 Soyabean oil Duration of morning stiffness; joint pain;
time to onset of fatigue; Ritchie’s articular
index*; grip strength, patient’s global
assessment
Sundrarjun et al.(82) 1.9 + 1.5 24 Not stated None
Galarraga et al.(105) 1.5 + 0.7 36 Air Use of NSAID; patient’s assessment of pain
Approx, approximately; NSAID, non-steroidal anti-inflammatory drugs.

A meta-analysis that included data from nine trials pub- one study that did not use a control for fish oil and one
lished between 1985 and 1992 inclusive and from one study in which transdermal administration of n-3 PUFA by
unpublished trial has concluded that ‘dietary fish oil sup- ultrasound, rather than the oral route, was used(93). This
plementation for three months significantly reduced tender meta-analysis has concluded that fish oil supplementation
joint count (mean difference - 2.9; P = 0.001) and morn- has no effect on ‘patient report of pain, swollen joint count,
ing stiffness (mean difference –25.9 min; P = 0.01)’(92). disease activity or patient’s global assessment’. However,
A more recent meta-analysis of data from trials published this conclusion may be flawed, because of the inappropri-
between 1985 and 2002 included one study of flaxseed oil, ate manner in which studies were combined and because of
Table 2. Summary of the findings of the meta-analysis of Goldberg & Katz(94)

No. of patients
No. of Significance of effect
Outcome studies Control n-3 PUFA of n-3 PUFA: P
Patient-assessed pain 13 247 254 0.03

Physician-assessed pain 3 61 62 0.45
Duration of morning stiffness 8 150 156 0.003
No. of painful and/or tender joints 10 210 215 0.003
Ritchie articular index* 4 68 67 0.40
NSAID consumption 3 79 77 0.01
NSAID, non-steroidal anti-inflammtory drugs.

a poor understanding of the study designs used. For RA. Most of these trials have reported clinical improve-
example, the meta-analysis fails to recognise that patients’ ments (e.g. improved patient assessed pain, decreased
ability to reduce the need for using NSAID or their ability morning stiffness, fewer painful or tender joints, decreased
to be withdrawn from NSAID, as was done in some use of NSAID), and when the trials have been pooled in
designs, must indicate a reduction in pain with n-3 PUFA meta-analyses significant clinical benefit has emerged.
use. This meta-analysis does state that ‘in a qualitative
analysis of seven studies that assessed the effect of n-3
fatty acids on anti-inflammatory drug or corticosteroid Acknowledgements
requirement, six demonstrated reduced requirement for
The author is a consultant to Vifor Pharma, Villars-
these drugs’ and concludes that ‘n-3 fatty acids may reduce
sur-Glane, Switzerland and Danone Research Centre for
requirements for corticosteroids’(93). The effects of long-
Specialised Nutrition, Wageningen, The Netherlands.
chain n-3 PUFA on tender joint count were not assessed by
this meta-analysis, which reiterated the findings of the
earlier meta-analysis that ‘n-3 fatty acids reduce tender References
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Neuron. 2006 October 5; 52(1): 77–92.
Mechanisms of Neuropathic Pain
James N. Campbell1,* and Richard A. Meyer1

1 Johns Hopkins University School of Medicine Baltimore, Maryland 21287
Abstract
Neuropathic pain refers to pain that originates from pathology of the nervous system. Diabetes,
infection (herpes zoster),nerve compression, nerve trauma, “channelopathies,” and autoimmune
disease are examples of diseases that maycause neuropathic pain. The development ofbothanimal
models and newer pharmacological strategies has led to an explosion of interest in the underlying
mechanisms. Neuropathic pain reflects both peripheral and central sensitization mechanisms.
Abnormal signals arise not only from injured axons but also from the intact nociceptors that share
the innervation territory of the injured nerve. This review focuses on how both human studies and
animal models are helping to elucidate the mechanisms underlying these surprisingly common
disorders. The rapid gain in knowledge about abnormal signaling promises breakthroughs in the
treatment of these often debilitating disorders.
Chronic pain has been estimated to affect one-sixth of the population. The phrase “neuropathic
pain” came into common use only in the last decade and increasingly has been appreciated as
a frequent source of chronic pain, perhaps trailing only osteoarthritis as a cause. Neuropathic
pain results from pathology in the nervous system. Consider the following clinical presentation:
A 47-year-old woman presented for consultation for complaints of pain on the right chest wall.
The patient underwent a mastectomy on the right side as a treatment for cancer 5 years
previously. As the surgical pain faded, the patient noted increasing chest-wall pain that
extended well beyond the surgical borders. Clothing lightly touching the skin increased the
pain. Reconstructive surgery was deferred because of concerns about the ongoing pain. In
addition to the ongoing burning pain, the patient also complained of sudden “pain attacks” one
to several times a day. These attacks lasted seconds to minutes and were described as
debilitating. Examination revealed a well-healed surgical scar. Light stroking of the skin
provoked significant pain in an area from the clavicle down to the T8 dermatome. Despite the
pain to light tactile stimuli, the patient also had areas of decreased sensibility as demonstrated
by the inability to detect a fine probe applied to the skin.
The above case represents a classical presentation of a patient with neuropathic pain. The
notable features that point to neuropathic processes are as follows:
• Widespread pain not otherwise explainable
• Evidence of sensory deficit
• Burning pain
• Pain to light stroking of the skin
• Attacks of pain without seeming provocation
The liability for pain appears to vary from person to person, from nerve to nerve, between
males and females, and even with age. What appears to be the same lesion may induce no pain
*Correspondence: jcampbel@jhmi.edu.
Campbell and Meyer Page 2
in one person but severe pain in another. In addition to ongoing pain (i.e., stimulus-independent
pain), patients may have heightened pain to stimuli applied to their skin. This enhanced
stimulus-dependent pain is called hyperalgesia. In some patients, lightly stoking the skin may
evoke pain. This pain to light touch is often called allodynia (Treede et al., 1992).
In this review, we will focus on the mechanisms of neuropathic pain. Lesions of the CNS (e.g.,
spinal cord) and peripheral nerves may lead to pain, but the majority of experimental studies
have focused on consequences of lesions of peripheral nerves, and therefore, we will focus on
this area in this review.
Anatomical Considerations
Neuropathic pain is distinguished from other pain conditions where the pain generator begins
with disease of nonneural tissues. These nonneuropathic pain entities are said to be nociceptive
and include conditions such as osteoarthitis and inflammatory pain. By definition, neuropathic
pain originates from a lesion of the nervous system. Innumerable diseases may be the culprits.
Examples include autoimmune disease (e.g., multiple sclerosis), metabolic diseases (e.g.,
diabetic neuropathy), infection (e.g., shingles and the sequel, postherpetic neuralgia), vascular
disease (stroke), trauma, and cancer. A rule without apparent exception is that the lesion leading
to pain must directly involve the nociceptive pathways (Boivie et al., 1989). Accordingly, for
example, lesions of the medial lemniscal system (e.g., dorsal columns) do not induce pain
(Cook and Browder, 1965).
Whereas evidence supports the hypothesis that pain-generating lesions of the nervous system
must involve the nociceptive pathways, the converse clearly does not hold up. Namely, not all
lesions of nociceptive pathways induce pain. A lesion of the peripheral nerve may induce pain,
but simply severing dorsal roots seems to have little chance of creating lasting pain (Li et al.,
2000). For example, dorsal roots are cut to treat spasticity and sometimes to remove tumors.
The authors know of no case of neuropathic pain arising from these types of lesions in man.
Here, we do not take up the issue of CNS lesions, but it is nevertheless worth noting that though
spinal cord lesions carry a substantial risk of inducing pain, some evidence suggests that the
lesion must include the gray matter. Vireck has found that simple cordotomy where only the
white matter is lesioned does not induce abnormal pain behavior in a primate model, whereas
lesions that include the gray matter may (Vierck and Light, 1999). Lesions of the brainstem
and thalamus carry a risk of causing pain if the nociceptive pathways are involved (Boivie,
2006). In nearly all of these cases, there is the paradoxical juxtaposition of ongoing pain and
a sensory deficit to noxious stimulation (even when hyperalgesia is present). Lesions confined
to the cortex appear not to be associated with abnormal pain (but it is also unclear whether
lesions confined to the cerebral cortex induce deficits of pain sensibility [Head and Holmes,
1911]).
Nervous System Diseases Associated with Pain

As noted above, a rich variety of diseases of the nervous system are associated with pain (Table
1) (Scadding and Koltzenburg, 2006). In the introduction, a case of traumatic painful
neuropathy was presented (Campbell, 2001). The offending lesion may involve a simple
axotomy (cutting of the nerve) or merely nerve entrapment. Ongoing pain may be the only
manifestation, but in many cases, hyperalgesia with pain to light-stroking stimuli (allodynia)
may be manifest. In some cases, there is striking cooling hyperalgesia, where mild lowering
of skin temperature exacerbates the pain. Weir Mitchell described a striking variant of
neuropathic pain that he termed causalgia (Lau and Chung, 2004). He noted this condition in
patients who had missile lesions of an extremity. Patients presented with striking edema,
profound autonomic features (abnormal sweating and either hot or cold skin), and devastating
pain. A similar presentation can occur without a clear nerve injury, and presently, this is referred

to as “complex regional pain syndrome (CRPS), type 1.” Where a clear nerve lesion is manifest,
the condition is referred to as CRPS, type 2 (Stanton-Hicks et al., 1995).
One of the classic examples of neuropathic pain is Tic douloureux. Without treatment, this is
a debilitating disorder that involves attacks of severe pain in the facial area (also referred to as
trigeminal neuralgia). Often there is little or no pain between attacks. The lightening-like
attacks are referred to one of the dermatomes (V1, V2, or V3). Light touching of the skin in a
so-called trigger zone suffices to evoke an attack. The disease appears to be associated with
mechanical distortion at the entry zone of the nerve root to the brainstem. Demyelination may
be seen at the compression site. Nerve compression from an aberrant blood vessel is one of the
more common causes (Elias and Burchiel, 2002).
Another classical neuropathic pain condition is painful diabetic neuropathy (Dyck et al.,
2000). Diabetes often causes a length-dependent neuropathy (meaning that the longest axons
in the peripheral nerve are most vulnerable). Patients report bilateral burning pain in the toes
and feet. Quantitative sensory testing reveals decreased pain sensibility (with or without
decreased touch sensibility).
Postherpetic neuralgia is a complication of shingles and is an example of how an infection can

lead to pain. Shingles results from an activation of the herpes zoster virus that takes up residence
in the dorsal root ganglion after a chickenpox infection. The shingles eruption consists of
blisters that follow the dermatome(s) of one or more spinal nerves. The blisters heal in time,
but the pain may continue. Allodynia is a particularly prominent feature of postherpetic
neuralgia. This allodynia may be present even with loss of C-fiber innervation of the epidermis.
The clinical manifestations vary with the type of disease (Table 1). These variations suggest
different mechanisms. These differences in mechanism may also be reflected in responses to
therapy. Tic douloureux responds nicely to treatment with the anticonvulsant carbamazepine.
Responses to carbamazepine for other conditions are typically disappointing. Painful diabetic
neuropathy leads to ongoing pain, but allodynia is distinctly unusual, and cooling may relieve
pain. In contrast, allodynia is prominent in traumatic neuropathy and cooling often causes
severe pain.
Animal Models of Neuropathic Pain

Whereas the distinction between nociceptive and neuropathic pain has utility, in the actual
clinical setting, the mechanisms are often intertwined (e.g., back pain with radiculopathy).
Fortunately, however, this need not be the case with regard to animal models. A great advance
in the study of neuropathic pain emanated from the discovery that placement of loose chromic
ligatures on the sciatic nerve in rat brought about behavior that appeared analogous to human
neuropathic pain conditions (Bennett and Xie, 1988). The rats engaged in protective behavior
and had lowered thresholds to heat, cooling, and mechanical stimuli.
Subsequent work indicated that an idiosyncratic immune-mediated response to the chromic

suture played a major role in the development of the model (Maves et al., 1993). The nerve
swelled, leading to nerve compression and axotomy. But can an axotomy by itself induce pain?
The answer is unequivocally yes. Chung and colleagues (Kim and Chung, 1992) devised a now
frequently used model in rat whereby one or more spinal nerves that innervates the foot is cut
(SNL model in Figure 1A). A substantial innervation of the foot remains, reflecting input from
the adjacent spinal nerves. This remaining innervation allows tests for hyperalgesia to be
undertaken. A simple axotomy provides a foundation by which to study mechanism in diseases
where axotomy is part of the disease process. Some of the common traumatic nerve injury
models are illustrated in Figure 1A. These injuries lead to hyperalgesia (Figures 1B and 1C).

Trauma has not been the only model used in studies of neuropathic pain. One of the leading
causes of pain in humans is diabetic neuropathy. Injection of streptozotocin leads to an animal
model of diabetes and is associated with the development of neuropathy, similar to what is
seen in humans. Hyperalgesia can be measured in rodent models, thus providing a means to
study treatments and mechanisms of pain in this neuropathy model (Ahlgren and Levine,
1993). One of the dose-limiting problems in chemotherapy is neuropathy, and these
neuropathies are often associated with pain. Animal models of paclitaxel and vincristine-
induced painful neuropathies have been developed (Flatters and Bennett, 2004).
Though hyperalgesia is relatively easy to demonstrate in animal models, the measurement of

ongoing pain is more problematic. This issue is important because ongoing pain without clear
hyperalgesia appears to be a common occurrence in humans (Basbaum et al., 2006). Substantial
denervation of a limb in rat often leads to autotomy behavior. Some have advocated that
autotomy is an indication of ongoing pain, the presumed rationale being that the animal desires
to eliminate the painful body part (Devor, 1991). An alternative explanation is that the animal
chews an anesthetic part for reasons unrelated to pain. Some have argued that spontaneous foot
lifting may provide a behavioral measure of spontaneous pain (Djouhri et al., 2006). Alternative
measures of ongoing pain involve the use of cellular markers of increased neuronal activity.
Increased expression of the immediate early gene protein, c-Fos, in the dorsal horn (and perhaps
other more rostral sites) is an example (Bullitt, 1990). Small-animal functional magnetic
resonance imaging (fMRI) and/or PET imaging may in the future offer alternative measures.
Animal models of neuropathic pain in some cases have been inconsistent with human models.
Allodynia in painful diabetic neuropathy in humans is infrequent yet appears to be robust in
rat models. NK1 antagonists appeared to have promise for treatments of pain based on animal
models yet to date have not proven useful in patients. Vierck (2006) argues that “reflex”
measures of pain in animal neuropathic models are intrinsically flawed and are neither sensitive
nor specific predictors of drug efficacy in man. For example, he points out that the paw-
withdrawal threshold method tests motor neuron response rather than simply providing a
measure of pain. Moreover, he indicates that rostral signaling pathways may be ignored when
one merely measures the paw-withdrawal threshold. His solution is to utilize operant models.
However, operant measures may be flawed as well in that they introduce other complexities
such as motivational factors that may be extraneous to the question of how much pain is felt.
Furthermore, the simple paw-withdrawal technique has in fact demonstrated some consistency
with human pain studies. In a recent pharmacological study that used a paw-withdrawal method
in rodents with the SNL model (LaBuda and Little, 2005), hyperalgesia was reversed with
gabapentin, amitriptyline, and fluoxetine, but not indomethacin, treatment. This correlates with
the observation that the first three drugs are useful in treatment of neuropathic pain in humans
but that nonsteroidal drugs are in general not useful.
A careful comparison of operant and reflex models in terms of their predictive capacity has
yet to be performed. For drugs with a predominantly supraspinal action, operant measures may
be more appropriate, whereas simple paw-withdrawal thresholds may be sufficient for drugs
that work at peripheral or dorsal horn targets. Though the particulars of how pain is assessed
remain an issue, a greater concern may relate to substantial inherent differences in the biology
of nociception in rodent and man. Regardless of this, new drugs for treatment of neuropathic
pain have been developed, and their utility in animal models played an influential role in
moving these treatments into clinical trials.
Secondary Hyperalgesia and Central Sensitization

A starting place in understanding neuropathic pain is to consider what happens with injury to
nonneural tissues. Skin injury produces ongoing pain and two types of hyperalgesia: primary

and secondary (Meyer et al., 2006). Primary hyperalgesia occurs at the site of tissue injury and
is mediated in part by sensitization of primary afferent nociceptors. This is reflected by
increased responses to heat stimuli, for example. Secondary hyperalgesia occurs in the
uninjured tissue surrounding the site of injury and is thought to be due to sensitization in the
central nervous system. Secondary hyperalgesia is characterized by hyperalgesia to
mechanical, but not heat, stimuli. This mechanical hyperalgesia is comparable to the
hyperalgesia seen in patients with neuropathic pain. Two types of mechanical hyperalgesia are
observed: pain to light-stroking stimuli (i.e., allodynia) and enhanced pain to punctate stimuli.
Two psychophysical studies in human volunteers provide strong evidence that secondary
hyperalgesia is due to sensitization in the central nervous system. In both studies, intradermal
injection of capsaicin, the algesic substance in hot peppers, was used to produce a large zone
of secondary hyperalgesia. In the first study, the nerve supplying the area to be injected was
anesthetized with a local anesthetic (LaMotte et al., 1991). Injection of capsaicin still produced
a large flare, consistent with the concept that the flare reflects a peripherally mediated release
of vasoactive peptides subsequent to antidromic spread of action potentials in the nociceptive
terminals. In the opposite control arm, capsaicin injection at the same time produced intense
pain and a large zone of secondary hyperalgesia. When the anesthesia wore off, no zone of
mechanical hyperalgesia was found in the test arm, but hyperalgesia persisted in the control
arm. This experiment provides evidence that the barrage of nociceptor activity associated with
the capsaicin injection leads to an altered central processing of input from mechanosensitive
afferents.
In the second study, a fine electrode was placed into the superficial peroneal nerve (Torebjörk
et al., 1992). Electrical intraneural microstimulation produced a non-painful tactile percept that
was referred to a small zone on the top of the foot. Capsaicin was injected adjacent to this area
so that the zone of secondary hyperalgesia overlapped the area of referred sensation.
Microstimulation after the injection produced a painful percept. Because this stimulation
bypasses any peripheral sensitization processes at the cutaneous receptor, this experiment also
provides evidence for altered central processing of mechanoreceptor input. Additional studies
have shown that the tactile pain arises from central sensitization to the inputs of Aβ fibers,
whereas the punctate hyperalgesia is due to a central sensitization to the inputs of capsaicin-
insensitive Aδ nociceptors (Magerl et al., 2001).
The tactile fibers have known convergence onto dorsal horn cells that in addition receive inputs
from nociceptive primary afferents. The inputs from the nociceptors in the injury zone are
presumed to sensitize these so-called wide-dynamic range neurons and thereby enhance the
synaptic efficacy of the tactile fibers. Nociceptive-specific neurons (dorsal horn neurons in
lamina I) may be sensitized in similar fashion.
Central sensitization to the inputs of tactile afferents in patients has been demonstrated in
patients with neuropathic pain. A blood pressure cuff inflated in the proximal extremity leads
to an orderly loss of sensation beginning first with loss of tactile function. When this was done
in patients, the loss of tactile function coincided with loss of allodynia (Campbell et al.,
1988). It was also noted that the detection of pain with tactile stimuli was fast and indicated
that primary afferents transmitting the stimulus were likely in the A-β range.
Where Do the Abnormal Signals in Neuropathic Pain Originate?

As shown in Figure 2, multiple sites along the neural axis are altered after nerve injury.
Abnormalities occur in the injured and uninjured afferents supplying the affected region.
Central sensitization, similar to that discussed above following tissue injury, is observed. In
addition, changes in descending control systems have been reported. Finally, an immune
response, both peripherally and centrally, is observed.

A Role for Primary Afferents

The importance of primary afferent inputs in neuropathic pain is strongly suggested by several
pharmacological studies. For example, systemic administration of AM1241, a selective CB2

cannabinoid receptor agonist, results in reversal of signs of mechanical and thermal
hyperalgesia following an SNL lesion (Ibrahim et al., 2003). Because CB2 is not expressed in
the central nervous system, the effects are likely due to a peripheral mechanism.
Systemic administration of artemin dose-dependently reversed the behavioral signs of

neuropathic pain in rats with an SNL injury (Gardell et al., 2003). Artemin is a member of the
glial-derived neurotrophic factor (GDNF) family that signals through a common tyrosine
kinase receptor (RET) and GFRα3. GFRα3 is an accessory protein that is predominantly
expressed in small-diameter, unmyelinated neurons (Orozco et al., 2001). The artemin-induced
changes in neuropathic pain behavior are therefore most likely due to its action on nociceptive
primary afferents.
Antisense oligodeoxynucleotides (ODNs) directed against Nav 1.8 reversed signs of

mechanical hyperalgesia (Porreca et al., 1999). Nav 1.8 is a tetrodotoxin-resistant sodium
channel that is primarily expressed in small-diameter primary afferent neurons. This treatment
was effective even when applied 6–14 days after nerve injury, demonstrating that ongoing
peripheral neuronal input is critically involved in the maintenance of neuropathic pain.
The Injured Afferent Hypothesis

The above evidence makes a clear case for the role of primary afferents, and one might assume
that the culprit is the injured afferent itself. Indeed, much evidence favors this hypothesis. When
nerve is injured a neuroma forms. The spontaneous activity and ectopic sensitivity to
mechanical, thermal, and chemical stimuli that originate from the traumatic neuroma have been
well documented (Blumberg and Jänig, 1984; Devor, 2006a). Clinical evidence has suggested
that local-anesthetic blockade of an injured nerve in patients relieves pain, though no study,
surprisingly, has really addressed this issue in a well-designed blinded fashion (Arner et al.,
1990; Burchiel et al., 1993; Gracely et al., 1992; Koltzenburg et al., 1994). Additional support
for the role of injured afferents comes from experiments in the rat L5 SNL model in which
anesthetics (Sukhotinsky et al., 2004) or tetrodotoxin (TTX) (Lyu et al., 2000) directed at the
L5 ganglia reversed the neuropathic behavior.
Regardless of the reasonableness of the so-called injured afferent hypothesis, several lines of
evidence suggest that there is more to the story.
Surprisingly, several authors report that the L5 SNL model in rat leads to spontaneous activity
in A fiber, but not C fiber, afferents as recorded in the L5 dorsal root (Boucher et al., 2000;
Liu et al., 2000). This spontaneous activity in myelinated fibers appears to be produced mainly
in afferents that (preinjury) serve muscle and joint, but not skin (Michaelis et al., 2000; Proske
et al., 1995). These findings produce a dilemma because activity in C fibers is thought to be
necessary to evoke the central sensitization mechanisms that account for the hyperalgesia (Ji
and Woolf, 2001). One possible explanation is that A fiber-spontaneous activity can initiate
central sensitization after nerve injury, if the afferent undergoes a phenotypic switch. A de
novo expression of neuropeptides normally only expressed in nociceptive afferents could
occur. For example, substance P immunoreactivity increases in large- and medium-size DRG
neurons after axotomy (Noguchi et al., 1994). However, studies investigating substance P
release from myelinated afferents following nerve injury are inconclusive (Allen et al., 1999;
Malcangio et al., 2000). Another explanation is that the spontaneous activity occurs in “A-β”
nociceptors, the existence of which has been documented in rodent and primate (Djouhri and
Lawson, 2004; Treede et al., 1998).

Other behavioral data suggest that signals originating from the injured spinal nerve are not
essential for hyperalgesia to occur. An L5 dorsal rhizotomy immediately before or after an L5
spinal nerve ligation did not prevent or reverse neuropathic behavior (Eschenfelder et al.,
2000; Li et al., 2000). The counter to this observation is that dorsal rhizotomy by itself may
produce signs of mechanical hyperalgesia (Colburn et al., 1999; Eschenfelder et al., 2000).
More importantly, neuropathic pain behavior is observed after an L5 ganglionectomy, where
the injured afferents are removed altogether (Sheth et al., 2002). The prevailing message seems
to be that hyperalgesia can develop in the absence of neural activity from the injured nerve.
The Intact Nociceptor Hypothesis

According to this hypothesis, the intact nociceptors that survive injury and that innervate the
region affected by the transected nerve fibers sensitize and have spontaneous activity. These
changes in the intact nociceptors may induce ongoing pain and may account for certain aspects
of hyperalgesia.
Following peripheral nerve lesions in primate and rodent models, spontaneous activity
developed in uninjured, unmyelinated nociceptive afferents that shared the same innervation
terrritory of the transected fibers (Ali et al., 1999; Djouhri et al., 2006; Wu et al., 2001).
Although the average discharge frequency was low (seven action potentials/5 min), the
incidence of spontaneously active fibers was high (50%). Low rates of spontaneous activity
may therefore assume importance if this phenomenon is occurring in large numbers of C fibers,
in particular given the high convergence of C fiber input in the CNS. Consistent with this
hypothesis is the observation that low-frequency electrical stimulation in C fibers can lead to
hyperalgesia in humans (Klede et al., 2003) and behavioral signs of hyperalgesia in rats (Wu
et al., 2002). The development of spontaneous activity has also been observed in uninjured,
myelinated afferents in the L4 dorsal root (Boucher et al., 2000), and this activity originated
within the dorsal root ganglion.
Sensitization has also been reported in the intact nociceptors. Following peripheral or spinal
nerve lesion in rat or rabbit, uninjured afferents develop adrenergic sensitivity (Sato and Perl,
1991) and an increased sensitivity to tumor necrosis factor-α (TNFα) (Schäfers et al., 2003).
One study has suggested that sensitization to mechanical and heat stimuli occurs in the
uninjured, unmyelinated afferents as well (Shim et al., 2005). Increased responsiveness to heat
stimuli could be due to the observed increases in expression of mRNA and protein for the
transient receptor potential receptor V1 (TRPV1) in the DRG of uninjured afferents (Fukuoka
et al., 2000; Hudson et al., 2001). In addition, the incidence of cold-responsive neurons in the
L4 DRG is increased after an L5 SNL (Djouhri et al., 2004), and there is an increase in
expression of the putative cold-sensitive channel TRPA1 (Katsura et al., 2006). These
observations may account for the occurrence of cooling hyperalgesia seen in patients after
nerve trauma.
One compelling line of evidence in favor of the intact nociceptor hypothesis stems from
experiments where local treatments are applied to the partly denervated skin. Local treatments
can only affect the intact fibers. In patients, local treatment with capsaicin may significantly
relieve ongoing pain. This is of interest because the effects of capsaicin are specific to
nociceptors. Experiments with animal models point to similar conclusions. Mibefradil, a T-
type calcium-channel blocker, reversed mechanical and thermal hyperalgesia (Dogrul et al.,
2003), and local application of a glutamate receptor antagonist reversed signs of thermal
hyperalgesia (Dogrul et al., 2000). In both studies, contralateral injections were without effects.
Intraplantar injection of morphine on the ipsilateral, but not contralateral, side significantly
reversed mechanical allodynia (Pertovaara and Wei, 2001).

The intact L4 dorsal root ganglion has been studied extensively to determine changes in
phenotype after injury to the adjacent L5 spinal nerve (L5 SNL). In addition to increased
expression of TRPV1 and TRPA1, mRNA for calcitonin gene-related peptide (CGRP) is
upregulated (Fukuoka et al., 1998). Furthermore, the number of uninjured DRG neurons
expressing brain-derived neurotrophic factor (BDNF) is increased (Fukuoka et al., 2001). In
the SNI model, there is an upregulation of P2X3 mRNA in uninjured neurons (Tsuzuki et al.,
2001).
Sympathetically Maintained Pain and the Intact Nociceptor Hypothesis

CRPS is a striking disorder manifest by severe pain typically in an extremity. Patients typically
have edema, hyperalgesia, and may even have a motor disability that is difficult to explain
from a purely electrophysiological perspective. In certain of these patients, selective anesthetic
blockade of the sympathetic nervous system leads to dramatic relief of pain (sympathetically
maintained pain, or SMP). Blockade of α -adrenergic receptor function with intravenous
infusion of the antagonist phentolamine also leads to pain relief (Raja et al., 1991). In patients
with SMP, in whom a sympathetic ganglion block was done to relieve pain and block release
of norepinephrine, injection of physiological concentrations of norepinephrine evoked
substantial pain (Figure 3A).
In a primate model of neuropathic pain, an L6 SNL leads to hyperalgesia just as it does in

rodent models (Carlton et al., 1994). About one-half of the afferents to the top of the foot are
lost, and the other half reach the foot from the adjoining and putatively normal spinal nerves
(Ali et al., 1999). More than 60% of the intact nociceptors had spontaneous activity (normally
infrequently seen), and more than 50% had sensitivity to the select α -adrenergic agonist
phenylephrine (Figure 3B). These studies indicate that the mechanisms of SMP relate to an
adrenergic sensitization of nociceptors (Figure 3C). Moreover, this chemical sensitization
occurs in the intact nociceptors that survive a proximal nerve injury. Thus, SMP is a specific
example of the importance of the intact nociceptors in the pathogenesis of a particular
neuropathic pain disorder.
The Role of Growth Factors

Trophic factors in the target tissue have an ongoing influence on sensory and motor fibers.
Nerve injury induces changes in growth-factor expression (Griffin, 2006). The change in
expression occurs in the tissue deprived of innervation (e.g., the skin), the Schwann cells
affected by Wallerian degeneration (denervated Schwann cells), the DRG, and the dorsal horn.
Of note, in the SNL model, the L5 root is lesioned, but the L4 root shares the innervation
territory, the same Schwann cells, and has convergent input to dorsal horn cells served by the
L5 afferents. Nerve injury leads to increased levels of NGF in the skin supplied by L4. The
binding of this NGF to the Trk-A receptors on the L4 fibers leads to transport of NGF back to
the L4 DRG. Evidence suggests that this increased level of NGF in the L4 DRG affects factors
such as BDNF. Indeed, the changes in gene expression in the intact L4 DRG and the lesioned
L5 DRG are quite different.
Griffin has introduced the terms “undertrophed” and “overtrophed” to describe a putative
mechanism by which growth factors may dually affect both the injured and uninjured afferents
(Griffin, 2006). The injured axons by losing connectivity with the peripheral tissues lose trophic
influences. The increased expression of trophic factors in the denervated tissues on the other
hand “overtroph” the remaining (intact) fibers. NGF might be one of the villains in the case of
overtrophing, whereas GDNF might be the missing growth factor, absence of which generates
the pain-signaling mechanisms in the injured L5 spinal nerve (Boucher and McMahon,
2001).

Transduction, Channel Function, and Ectopic Discharge

Primary afferents transduce different forms of energy into generator potentials. If of sufficient
magnitude, these generator potentials lead to action potentials. The action potentials propagate
along the axon to the CNS. Pathological pain may result from disorders involved in any of
these steps.
There are two ways nociceptors may be sensitized. One is for the transduction channel to
sensitize (i.e., a bigger generator potential to a given natural stimulus). The other mechanism
regards a decrease in threshold of the sodium channels responsible for spike initiation. It is
logical therefore to ask whether primary afferent sensitization may account for features of
neuropathic pain. NGF regulates TRPV1 expression, and thus, over-trophing mechanisms
could link growth factors to transduction-channel function (Cortright and Szallasi, 2004). A
role for TRPV4, a channel that may be involved in transduction of mechanical stimuli, was
suggested in a neuropathic pain model produced by administration of taxol (Alessandri-Haber
et al., 2004). Abnormal transduction, at least in part, likely underlies the pathophysiology of
the “intact nociceptor.” Sensitization of the “intact” nociceptor in the SNL model may be
responsible for the hyperalgesia (Ringkamp and Meyer, 2006). Another mechanism relates to
the acquisition of transduction capacity for stimuli that ordinarily do not activate nociceptors.
This has been clearly demonstrated in the case of SMP, where intact nociceptors acquire
sensitivity to norepinephrine. Cooling hyperalgesia is prominent in neuropathic pain.
Abnormal expression of transduction channels such as TRPA1, or TRPM8 in nociceptors,
could account for this phenomenon.
Transduction can be displaced to a point of nerve injury as well. This misplaced transduction
could account for the so-called “Tinel sign,” a useful clinical finding, wherein patients
experience paresthesias (tingling sensation) or dysthesthesia (an unpleasant or frankly painful
sensation) when the area over the nerve injury is tapped. The Tinel sign may be present at a
point of entrapment where continuity of the axon has not necessarily been interrupted. The
presumed mechanism is that a disturbance of fast axonal transport (which serves as delivery
mechanism for newly synthesized transduction proteins) leads to ectopic expression of the
transduction proteins. Axotomy leads to neuroma formation, where budding regenerative nerve
sprouts devoid of growth guidance from denervated Schwann cells form an entangled mass.
Many neuromas are “painful,” but for unclear reasons (genetic factors, location?) many are
not. Areas of nerve injury may be tethered to adjacent moving structures (e.g., tendons), such
that otherwise normal movements evoke an increase in pain presumably by activating
nociceptive afferents through activation of mechanoreceptive transduction channels.
Nociceptor sensitization could also relate to changes in voltage-gated sodium channels,

particularly in the region of spike initiation (Devor, 2006b). For example, if the threshold for
sodium-channel activation decreases, the response to a given generator potential will be

enhanced.
One reason for interest in sodium-channel function centers on clinical evidence that drugs that
affect sodium-channel function may be very effective in relieving neuropathic pain. For
example, the anticonvulsant carbamazepine, which works by stabilizing sodium channels in
an inactive state, is considered the treatment of choice for trigeminal neuralgia. The effects are
consistent enough that clinicians are trained to question the diagnosis if the patient does not
have at least some response to the drug. As a class, each of the anticonvulsants that work
through a sodium-channel mechanism (lamotrigine, phenytoin, in addition to carbamazepine)
have some level of efficacy in neuropathic pain, though side effects often preclude usefulness.
Another class of drugs that is effective in neuropathic pain is the local anesthetics (Tremont-
Lukats et al., 2006). At doses that have no effect on normal sensibility, local anesthetics may
favorably affect neuropathic pain.

There are multiple types of voltage-gated sodium channels that are expressed in the dorsal root
ganglion and may have a role in neuropathic pain. The channels that have received greatest
interest are Nav 1.3, 1.7, 1.8, and 1.9. These channels are found in small DRG cells and therefore
are likely involved in action potential generation and conduction in nociceptors. Channels
Nav 1.3 and 1.7 are TTX sensitive, whereas Nav 1.8 and 1.9 are TTX insensitive. Some animal
data suggest that TTX given systemically may relieve hyperalgesia without adversely affecting
normal functions (Marcil et al., 2006). Of the channels tested, only the TTX-sensitive channel
Nav 1.3 is upregulated in the DRG of the injured axons (Black et al., 1999). This channel has
kinetic properties that could favor repetitive spiking.
Whereas prior efforts have concentrated on the α -subunit expression, other complexities affect
sodium-channel function. Expression of the β subunits could be part of this complexity. β2
subunits regulate channel gating, assembly, and cell-surface expression of TTX-sensitive
channels. Expression of these subunits is increased in injured sensory afferents (Pertin et al.,
2005), and in β2 null mice, mechanical hyperalgesia is reduced in a spared nerve injury model.
β2 null mice display marked reduction in TTX-sensitive sodium current in small DRG neurons
(Lopez-Santiago et al., 2006).
That channel function may strongly affect nociception has been further emphasized by the
discovery of the genetic underpinnings of a striking neuropathic pain disorder known as
erythromelalgia. Here, patients present with profound heat hyperalgesia and ongoing burning
pain typically affecting the feet. The feet may also be intensely red, indicating profound
vasodilation. Point mutations in the TTX-sensitive channel Nav1.7 account for the disorder
(Figure 4). These mutations lead to a hypo-excitability in sympathetic neurons (accounting for
the increased perfusion in the feet) and a hyperexcitability in small-sensory neurons
(accounting for the pain and hyperalgesia) (Rush et al., 2006). Release of vasoactive peptides
in the skin from the tonically active nociceptors may also contribute to the vasodilatation.
The Role of Central Sensitization

Central sensitization refers to the augmented response of central signaling neurons. Though
thalamic and cortical levels may be involved, most attention has focused on the dorsal horn
and in this review we will concentrate on this area.
When it occurs as a result of inflammation, central sensitization is strongly dependent on

ongoing input from nociceptors. Thus, if nociceptive input is blocked in the injury zone,
secondary hyperalgesia abates promptly (LaMotte et al., 1991). This same dependence on
peripheral input probably also applies to central sensitization associated with neuropathic pain,
but this issue requires further clarification. This distinction is of great importance clinically. If
mechanisms of pain are completely centralized, it might be argued that attention to peripheral
mechanisms is unlikely to be therapeutically beneficial.
Central sensitization involves homosynaptic and heterosynaptic mechanisms (Magerl et al.,

1998; Woolf et al., 1988). Homosynaptic sensitization means that the conditioning stimulus
and the test stimulus involve the same input. In dorsal horn nociceptive neurons, this is evident
in a phenomenon referred to as “windup,” where continual low-frequency stimulation of C
fiber afferents leads to an increasing response in the dorsal horn cell. Windup lasts tens of
seconds and represents a short-term form of sensitization.
Herterosynaptic sensitization means that the conditioning stimulus and the test stimulus involve
different sets of afferents. Heterosynaptic sensitization accounts for allodynia. Here,
nociceptive inputs alter synaptic efficacy such that A-β mechanoreceptors acquire the capacity
to evoke responses. Electrophysiological evidence for heterosynaptic sensitization has been

shown in primate studies of dorsal horn cells where the response to light stroking of the skin
was enhanced after capsaicin injection (Simone et al., 1991).
Ample evidence indicates that heterosynaptic and homosynaptic sensitization occur in

neuropathic pain conditions (Campbell et al., 1988; Ji et al., 2003). Figure 5 provides a model
for how this may occur in the L5 SNL model of neuropathic pain. Injured myelinated fibers
from the L5 root develop spontaneous activity. Some of this activity could be in A-β
mechanoreceptors (that have acquired the capacity to release substance P) or in A-δ
nociceptors. The input of these fibers onto cells in the L5 segment could lead to homosynaptic
sensitization and enhanced ongoing activity. The enhanced activity in L5 spinal thalamic tract
cells could play a role in ongoing pain sensation. The injured L5 afferents could also project
to the adjacent L4 segment where they could produce heterosynaptic sensitization to input from
the L4 segment. This could account for the hyperalgesia to mechanical and heat stimuli that is
signaled by activity in the intact L4 afferents.
Uninjured nociceptors in L4 root can develop spontaneous activity after an L5 SNL. This
spontaneous activity can lead to homosynaptic sensitization such that stimulus-evoked activity
in these nociceptors results in an augmented response of the dorsal horn cells. The spontaneous
activity in uninjured L4 nociceptors could also produce heterosynaptic sensitization such that
the response of L4 spinal thalamic cells to L4 mechanoreceptor input is enhanced. A brush
stimulus applied to the foot activates the A-β fibers in the L4 root and acquires the capacity to
drive activity in the L4 dorsal horn cell, consistent with heterosynaptic sensitization.
Homosynaptic and heterosynaptic sensitization involve many mechanisms, but fundamentally

these mechanisms come down to two processes: (1) increased release of excitatory
neurotransmitter (e.g., glutamate, substance P) and/or (2) enhanced synaptic efficacy. These
changes in turn may relate to several cellular mechanisms (Basbaum, 1999). These mechanisms
may be considered in five categories: (1) presynaptic changes, (2) postsynaptic changes, (3)
interneuron changes, (4) changes in descending modulation, and (5) immune/microglial
mechanisms (discussed in the following section). Evidence suggests a role for each of these
mechanisms in neuropathic pain. Long-term potentiation (LTP), a phenomenon that has
received intensive study in the hippocampus, appears to apply to the dorsal horn to some degree.
However, given the abnormal ongoing input of primary afferents documented after nerve
injury, the acute central-sensitizing effects of primary afferents may have an ongoing role.
Thus, both short-term sensitization and LTP mechanisms likely apply. A role for long-term
depression (LTD) in inhibitory cells is also considered below.
Presynaptic Mechanisms
Release of glutamate is inhibited presynaptically by several metabotropic G protein-coupled
receptors, including μ-opioid receptors, GABA-b, and adenosine receptors. Downregulation

of μ-opioid receptors has been documented both pre- and postsynaptically after nerve injury
(Kohno et al., 2005) and could lead to an enhanced release of glutamate.
Neurotransmitter release is triggered by voltage-gated calcium channel activity on the central

terminals of primary afferents. Nerve injury leads to an upregulation of the α -2-δ subunit of
these channels in the DRG and spinal cord (Li et al., 2004). This could be associated with
increased calcium entry and augmented release of glutamate. The anticonvulsant drugs
gabapentin and pregabalin are effective in neuropathic pain presumably because they bind to
and block this subunit (Bian et al., 2006; Freynhagen et al., 2005). Although these drugs reverse
hyperalgesia in neuropathic models, there is no apparent effect on normal pain sensibility
(Suzuki et al., 2005). This argues for a special role of the α -2 subunit in neuropathic pain.

There is also evidence to suggest that under conditions of nerve injury, A-β fibers undergo a
phenotypic switch, such that they begin to express substance P (Noguchi et al., 1994). As a
consequence, stimulation of these fibers leads to substance P release, providing an additional
potential mechanism for sensitization of dorsal horn neurons.
Postsynaptic Mechanisms
There is excellent documentation of the role of postsynaptic mechanisms in central
sensitization. Slow depolarization induced by substance P and other peptides leads to an
opening of the NMDA glutamate-gated channel, which in turn leads to calcium entry
(Dougherty et al., 1993; Duggan, 1995; Yoshimura and Yonehara, 2006). Calcium may gain
entry through AMPA receptors that do not contain the GluR2 subunit, as well, and this
mechanism may play a role in dorsal horn LTP (Gu et al., 1996). A conditional knockdown of
one NMDA subunit, NR1, abolished hyperalgesia to formalin but had no effect on normal pain
sensibility (South et al., 2003). Evidence exists for a multitude of intracellular events linked
to postsynaptic sensitization that result from calcium entry. Protein kinases A and C and other
transcriptional factors likely play important roles in these events (Kawasaki et al., 2004).
Another possible mechanism of postsynaptic sensitization involves an increased trafficking of

AMPA receptors to the cell surface. The expression of AMPA receptors is increased in the
superficial dorsal horn after an SNL lesion (Harris et al., 1996). In addition, upregulation of
the mRNAs for both AMPA and NMDA receptors has been demonstrated in diabetes
(Tomiyama et al., 2005).
We have suggested that allodynia stems from sensitization to the inputs of tactile afferents in
the dorsal horn. However, certain evidence suggests that the “Aβ sensitization” phenomenon
may actually arise from alternative pathways. A lesion of the ipsilateral dorsal column
abolished mechanical hyperalgesia but, interestingly, not heat hyperalgesia (Ossipov et al.,
2002; Sun et al., 2001). These data suggest that dorsal horn trafficking may account for heat
hyperalgesia but that other centers, such as the thalamus, may actually account for the allodynia.
Role of Disinhibition Mechanisms

Inhibitory neurons play an important role in governing the sensitivity of dorsal horn neurons
(Gu et al., 1996). Decreased expression of inhibitory receptors (on primary afferent terminals
and postsynaptic neurons) has been noted following nerve injury, and sensitization could result
from this disinhibition mechanism (Kohno et al., 2005). Several other mechanisms involving
inhibitory interneurons have been suggested. We have discussed LTP. The reciprocal
phenomenon is LTD. Salter has suggested that LTD may be evoked by activation of NMDA
receptors present on GABAergic cells due to differential expression of the subunits in the
NMDA receptor (Salter, 2006). LTD in inhibitory cells may lead to central sensitization
(Randic et al., 1993). To what extent this mechanism applies to the dorsal horn is unclear.
Nerve injury also induces a downregulation of the potassium-chloride transporter KCC2. This
trans-synaptic effect on superficial dorsal horn cells (presumed projection neurons of the
spinothalamic tract), leads to a less-negative equilibrium potential for chloride, such that
opening the chloride channel with GABA induces depolarization sufficient to induce
excitation. Consistent with this finding, knockdown of the expression of KCC2 leads to
hyperalgesia (Coull et al., 2003).
Further evidence indicates that nerve injury induces a selective apoptosis of inhibitory
GABAergic inter-neurons (Moore et al., 2002). Apoptosis of inhibitory GABAergic neurons
argues for a hard-wire change in circuitry (Scholz et al., 2005). This is a critical therapeutic
issue. If central sensitization evolves such that nociceptive signaling becomes independent of

inputs from primary afferents, then treating the peripheral nerve may no longer be
therapeutically useful. Moreover, the relative importance of peripheral and central mechanisms
may well depend on the underlying disease. Some diseases may involve primary afferents as
well as the CNS. An example is postherpetic neuralgia where damage may extend from the
primary afferents to the dorsal horn.
Descending Modulatory Mechanisms

Descending modulatory pathways also appear to influence dorsal horn sensitization
mechanisms in neuropathic pain. Cortical, thalamic, and periaqueductal inputs converge on
the rostroventromedial medulla (RVM). This center gives rise to both inhibitory and excitatory
inputs to the dorsal horn via an ipsilateral pathway in the dorsolateral funiculus (DLF). Many
of the cells in the RVM express μ-opioid receptors. Selective ablation of these cells either
before or after establishment of the SNL model can eliminate hyperalgesia (Porreca et al.,
2001). Lidocaine microinjection (Burgess et al., 2002) and hemisection experiments (Bian et
al., 1998) have yielded similar results.
Supraspinal Mechanisms
Recent fMRI studies reveal changes in the processing of cutaneous stimuli at supraspinal
centers in conditions of hyperalgesia and allodynia. Experimental studies of secondary
hyperalgesia allow the comparison of fMRI images before and after the development of
hyperalgesia. In these studies, mechanical stimulation of the skin produced enhanced pain in
the zone of secondary hyperalgesia and led to enhanced activation in areas of the brain
associated with pain signaling (Baron et al., 1999; Maihofner et al., 2004). Whether these
changes in supraspinal processing are passive or whether they reflect additional plasticity
mechanisms is difficult to know. Functional imaging studies in patients with neuropathic pain
reveal that light stroking that produces pain recruits a complex cortical network, including
nociceptive, motor, and cognitive areas (Maihofner et al., 2006; Schweinhardt et al., 2006).
Functional imaging may lead to new insights into the mechanisms of neuropathic pain as well
as to objective measures of altered sensations (Borsook et al., 2004).
The Immune System and Neuropathic Pain

Clearly the immune system is critical to the pain associated with inflammatory pain. The
immune system appears to play a critical role in neuropathic pain as well (Marchand et al.,
2005). Nude rats that lack mature T cells had diminished hyperalgesia (Moalem et al., 2004).
Passive transfer of type 1, but not type 2, T cells led to similar levels of hyperalgesia as those
seen in rats with intact T cell function. In addition, cytokines, II-1, II-6, and TNFα all have
demonstrated roles in neuropathic pain (Manning, 2006). Several variables are worth noting
in approaching the question of immune mechanisms.

• One must distinguish the immune mechanisms associated with a particular disease
from the immune mechanisms that are particular to the issue of neuropathic pain.
• Immune mechanisms need to be considered separately in terms of what happens in
the peripheral nervous system versus those that are important in the central nervous
system.
• Finally, immune mechanisms may play a role in initiating neuropathic pain,
maintaining neuropathic pain acutely, and/or play a role in chronic neuropathic pain.
Peripheral Immune Mechanisms

Nociceptors have a rich variety of immune receptors, and evidence exists for roles of the
interleukins (IL-1β and IL-6), TNFα, bradykinin, prostanoids, and others (Opree and Kress,

2000). In the CCI model of neuropathic pain, a “neuritis” develops, likely reflecting the
inflammatory response to the suture material (Kleinschnitz et al., 2004). In this model, immune
mechanisms might be a particular issue simply because of the foreign suture material. A more
interesting question is what happens with simple surgical axotomy (e.g., the SNL model).
Nerve damage evokes a cascade of immune responses. Nerve damage leads to macrophage
infiltration, T cell activation, and increased expression of proinflammatory cytokines. With
axotomy, Schwann cells are in a sense denervated. These denervated Schwann cells (as well
as cells in the partially denervated target tissue) may communicate with intact fibers that pass
within the same nerve. This provides a mechanism by which to explain changes in the intact
nociceptor (see above). Schwann cell denervation recruits macrophages via secretion of
leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 (MCP-1) (Sugiura
et al., 2000). MCP-1 serves as a ligand for CCR2 (Tofaris et al., 2002). CCR2 knockout mice
do not develop mechanical hyperalgesia after a partial nerve ligation injury (Abbadie et al.,
2003). Cytokine IL-1β leads to increased expression of nerve growth factor (NGF), and NGF
may sensitize nociceptors (Kanaan et al., 1998). Knockout of the IL-1 receptor type I and
genetically mediated overexpression of an IL-1 receptor antagonist decreased hyperalgesia.
That the effect was peripheral was supported by the observation that spontaneous activity
recorded from the dorsal root fibers was reduced (Wolf et al., 2006).
TNFα has received much attention as a culprit in neuropathic pain. Endoneurial administration
induces hyperalgesia (Wagner and Myers, 1996). Preemptive treatment with the TNF-
sequestering drug etanercept decreases hyperalgesia but has no effect once hyperalgesia is
established (Sommer et al., 2001). TNFα can initiate activity in nociceptors (Sorkin et al.,
1997). Inhibition of TNFα blocks phosphorylation of the MAP kinase p38 in DRG and
hyperalgesia but again only when given preemptively (Schäfers et al., 2003). Another cytokine
that has received attention is IL-6. A role in the CCI model has been suggested (Okamoto et
al., 2001), but other peripheral actions are unclear at present.
Central Immune Mechanisms

Somewhat surprising is the mounting evidence that central immune mechanisms play a role in
neuropathic pain (Figure 6) (Watkins and Maier, 2005). As with peripheral mechanisms, much
evidence supports the view that initiation, but not maintenance, of neuropathic pain behaviors
is associated with immune mechanisms (Xu et al., 2006). Microglia serve as the macrophages
of the CNS. Nerve injury distal to the dorsal root ganglia leads to microglial activation.
However, activation after dorsal rhizotomy is much less, in keeping with the observation that
dorsal rhizotomy leads to less-impressive hyperalgesia compared to spinal nerve lesions
involving the identical root (Willis et al., 2006). Interthecal delivery of the antibiotic
minocycline, an inhibitor of microglial activation, attenuates neuropathic pain (Raghavendra
et al., 2003).
The mechanisms are far from clear. Nociceptor activity leads to rapid activation of the MAP
kinase ERK in microglia and initially not in other central cells (Tsuda et al., 2005). Inhibition
of this activation attenuates pain behavior. The mechanisms for this activation might be multi-
factorial. Evidence exists for a role of the ATP receptors P2X4 and/or P2X7, both of which
are expressed on microglia. The chemokine fractalkine may also be involved, as blockade of
its receptor, CX3CR1, attenuates hyperalgesia in neuropathic pain models (Milligan et al.,
2005). The activated microglia produce the toxic cytokines IL-1, IL-6, and TNFα as well as
nitric oxide, excitatory amino acids, ATP, and prostaglandins (Inoue, 2006).
The toll-like receptor-4 (TLR4) is a molecule involved in the innate immune response and is
mainly expressed on microglia. Spinal mRNA for TLR4 is increased after L5 SNL. A knockout
or point mutation in TLR4, as well as downregulation through intrathecal delivery of anti-sense

ODN, has a significant effect on neuropathic pain behavior, but only when given in the early
stages (Tanga et al., 2005).
Conclusion
We began this review by presenting a patient with chest-wall pain following a mastectomy.
The simultaneous finding of decreased sensation and ongoing pain was suggested to be a
paradox. Animal models, as well as studies in humans, however, have taken us a long way
toward understanding this case of neuropathic pain. Ongoing pain likely represents
spontaneous discharge in afferents. The injured afferents are an obvious source of abnormal
input.
Sodium-channel dysfunction likely plays an important role in leading to ectopic generation of

action potentials. The patient had episodes of abrupt “pain attacks.” These attacks may
correspond to bursts of spontaneous activity in the injured afferents related to sodium-channel
dysfunction. Somewhat surprisingly, however, the intact afferents that share the innervation
territory of the injured afferents also discharge spontaneously. Studies of their cell bodies in
the DRG reveal striking phenotype changes. Trophic factors released from the partly
denervated skin working through their receptors on the peripheral terminals may account for
these abnormalities. The abnormalities seen in these “intact” nociceptors likely account for the
fact that patients such as this may respond to therapies applied at the level of the skin. Central
changes also play a role in neuropathic pain. Many of the mechanisms probably are the same
as those observed with inflammatory pain. The patient had pain and hyperalgesia well outside
the region of nerve injury. This is precisely the case in inflammatory pain, where secondary
hyperalgesia extends well beyond the injury site. The patient had substantial pain when the
skin was lightly stroked (allodynia). Multiple lines of evidence indicate that this allodynia is
due to central sensitization, such that tactile afferents acquire synaptic efficacy, which enables
them to trigger activity in central pain signaling neurons. Prevailing work has focused on the
dorsal horn as the site for this sensitization, but more rostral pathways may be involved as well.
Though not easily understood as yet, peripheral nerve injury induces some striking transynaptic
effects. One is apoptosis that appears to preferentially affect GABA inhibitory cells. Several
lines of evidence indicate that immune mechanisms are involved both peripherally and
centrally. Activation of microglial cells occurs in the dorsal horn, and this activation may play
a vital role in initiating central sensitization. The role of this activation in ongoing neuropathic
pain is less clear. The sensation of pain begins with a simple thesis: nociceptors encode
information about noxious stimuli and propagate these messages to the CNS and pain is felt.
In the case of neuropathic pain, however, we see that a rich biology is at play. Studies are
quickly uncovering the mechanisms, and no longer can we consider the presence of this
problem quite so mysterious.
Acknowledgements
We greatly appreciated the suggestions from Drs. Jasenka Borzan, Michael Caterina, and Matthias Ringkamp on earlier
drafts of this manuscript and the technical assistance of Cheryl Carmona. This research was supported by the National
Institutes of Health (NS-014447 and NS-041269) and the Blaustein Pain Treatment Center.
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Figure 1. Animal Models of Neuropathic Pain

(A) Four different nerve injury models are shown. In the spinal nerve ligation (SNL) model,
one or more spinal nerves going to the foot are ligated and cut (Kim and Chung, 1992). In the
partial sciatic ligation (PSL) model, a portion of the sciatic nerve is tightly ligated (Seltzer et
al., 1990). The chronic constriction injury (CCI) model involves placement of four loose
chromic-gut ligatures on the sciatic nerve. An immune response to the sutures leads to nerve
swelling and nerve constriction. In the spared nerve injury (SNI) model, the common peroneal
and tibial nerves are cut, sparing the sural nerve (Decosterd and Woolf, 2000). In each model,
only a portion of the afferents going to the foot are lesioned.
(B and C) Each of these nerve injury models leads to hyperalgesia, which is manifest by
enhanced responses to mechanical, heat, and/or cooling stimuli. (B) To test for mechanical
hyperalgesia, Von Frey monofilaments with different bending forces are applied to the plantar
surface of the foot. The threshold force for paw withdrawal decreases dramatically after the
nerve injury (adapted with permission [Li et al., 2000])
(C) To test for heat hyperalgesia, a radiant heat source is focused onto the plantar surface of
the foot, and the reaction time for paw withdrawal is measured. The difference in reaction time
between the ipsilateral and contralateral foot is calculated. After the SNL, the withdrawal of
the ipsilateral foot is faster than the contralateral foot (negative latency difference), indicating
the presence of heat hyperalgesia (adapted from Kim and Chung [1992] reprinted from Pain,
pp. 355–363, copyright 1992, with permission from the International Association for the Study
of Pain).
Data are presented as mean ± SEM.

Figure 2. A Spinal Nerve Injury Leads to Alterations at Many Sites along the Neural Axis for Pain
Eight different sites of pathophysiological changes are shown. (1) Spontaneous neural activity
and ectopic sensitivity to mechanical stimuli develops at the site of nerve injury. (2) The
expression of different molecules in the dorsal root ganglion of the injured nerve is up- or
downregulated, reflecting the loss of trophic support from the periphery. Spontaneous neural
activity develops in the dorsal root ganglia. (3) The distal part of the injured nerve undergoes
Wallerian degeneration, exposing the surviving nerve fibers from uninjured portions of the
nerve to a milieu of cytokines and growth factors. (4) Partial denervation of the peripheral
tissues leads to an excess of trophic factors from the partly denervated tissue that can lead to
sensitization of primary afferent nociceptors. (5) The expression of different molecules in the
dorsal root ganglion of the uninjured nerve is up- or downregulated, reflecting the enhanced
trophic support from the periphery. (6) Sensitization of the postsynaptic dorsal horn cell
develops, leading to an augmentation of the response to cutaneous stimuli. (7) Activated
microglial cells contribute to the development of this dorsal horn sensitization. (8) Changes in
descending modulation of dorsal horn neurons also may contribute to the enhanced
responsiveness of dorsal horn neurons.

Figure 3. A Model for Sympathetically Maintained Pain

(A) After acutely relieving pain by performing a sympathetic block, norepinephrine in
physiological concentrations was injected intra-dermally in a blinded fashion into the
previously hyperalgesic area. The norepinephrine injections into the affected, but not the
unaffected, extremity produced pain. Norepinephrine does not induce pain in control subjects.
These data suggest that the nociceptors are sensitized to catechols in patients with SMP
(adapted with permission [Ali et al., 2000]). ACUC, area under curve.
(B) In primates, normal nociceptors do not respond to catechol administration. However, in a
monkey-spinal nerve ligation model, nociceptors developed a response to the α 1 adrenergic
agonist phenylephrine administered topically to their receptive field (adapted and used with

permission [Ali et al., 1999]). AP, action potentials. (C) A model to account for SMP. After a
partial nerve lesion, some afferent fibers still remain in the skin. Factors released in the skin
induce the sympathetic efferents to sprout into more superficial areas of the skin (Yen et al.,
2006). These factors also lead the nociceptors to express α 1 adrenergic receptors. Now, the
release of norepinephrine from the sympathetic terminals leads to activation of the nociceptive
terminals, which accounts for the coupling of sympathetic activity with nociceptor responses.

Figure 4. A Genetic Basis for Erythromelalgia

(A) Schematic of the Nav 1.7 voltage-gated sodium channel that is found exclusively in sensory
and sympathetic neurons. A number of point mutations have been identified in this sodium
channel in families of patients with erythromelalgia (Waxman and Dib-Hajj, 2005). The two
mutations shown here produced single amino acid substitutions at the sites indicated.
(B) Whole-cell-patch-clamp recordings in transfected HEK293 cells revealed that these point
mutations lead to an augmentation of the response of the channel to a slow depolarizing ramp
(100 mV in 500 ms). These mutations would be expected to increase the excitability of the
peripheral sensory neuron. (Adapted with permission [Cummins et al., 2004]. Copyright 2004
by the Society for Neuroscience.).

Figure 5. Central Sensitization Mechanisms Involved in the Spinal Nerve Ligation Model
Spontaneous activity from the injured afferents (L5) and intact nociceptors (L4) may sensitize
central pain-signaling neurons. The spontaneous activity in the L5 fibers is restricted to
myelinated afferents. Nociceptive C fibers from L4 spontaneously discharge and may
themselves be sensitized. The enhanced discharge of the primary afferents leads to augmented
response of dorsal horn cells to nociceptor input and increased synaptic efficacy of inputs from
mechanoreceptors (mechanism for allodynia). Alterations in descending modulation and
inhibitory interneuron function also likely play a role.

Figure 6.
Microglial Cells in the CNS Are Activated following Peripheral Nerve Injury and Release
Cytokines that Alter the Responses of Dorsal Horn Cells

Table 1
Examples of Neuropathic Pain Conditions
Disease Pathology Symptoms Hyperalgesia Special Features

Cooling Heat Mechanical
Traumatic neuropathy Axotomy distal to Ongoing pain ++ + ++ Great variety of

DRG presentations
Tic douloureux Compression (often Lightening- ? ? +++ Mechanical
vascular) of like attacks of stimuli evoke
trigeminal nerve near pain attacks of pain
brainstem
Painful diabetic neuropathy Length- Burning pain − + +/− Neuropathy
dependent neuropathy in the feet sometimes
affects small
fibers exclusively
Postherpetic neuralgia Results from damage Burning pain +/− +/− ++ Pain follows the
by herpes zoster distribution of the
infection of peripheral affected
nerve dermatomes

ARTICLE IN PRESS
Medical Hypotheses (2006) x, xxx–xxx
http://intl.elsevierhealth.com/journals/mehy
Chronic pain: A non-use disease

L. Pruimboom *, A.C. van Dam
Faculty of Human Sciences, University of Gerona, C/Francesc Masia 65 Salt, Spain
Received 20 July 2006; accepted 1 August 2006
Summary One of the major problems in modern medicine is to find remedies for the group of people with chronic
pain syndromes. Low back pain is one of the most frequent syndromes and perhaps the most invalidating of all of them.
Chronic pain seems to develop through several pathways affecting the spinal cord and the brain: (1) neuro-anatomical
reorganisation, (2) neuro-physiological changes, and (3) activation of glia cells (immune reaction in the central nervous
system). Although all of these pathways seem to provide a (partial) plausible explanation for chronic pain, treatments
influencing these pathways often fail to alleviate chronic pain patients. This could be because of the probability that
chronic pain develops by all three mechanisms of disease. A treatment influencing just one of these mechanisms can
only be partially successful. Other factors that seem to contribute to the development of chronic pain are
psychosocial. Fear, attention and anxiety are part of the chronic pain syndrome being cause or consequence. The three
pathways and the psycho-emotional factors constitute a psycho-neuro-immunological substrate for chronic pain
syndromes; a substrate which resembles the substrate for phantom pain and functional invalidity after stroke. Both
phantom pain and functional invalidity are considered non-use syndromes. The similarity of the substrate of both these
two neurological disorders and chronic pain makes it reasonable to consider chronic pain a non-use disease (the
hypothesis).
To test this hypothesis, we developed a ‘‘paradoxal pain therapy’’. A therapy which combines the constraint induced
movement therapy and strategies to dissociate pain from conditioning factors like fear, anxiety and attention. The aim
of the therapy is to establish a behaviour perpendicular on the pathological pain-behaviour. Clinically, the treatment
seems promising, although we just have preliminary results. Further clinical and laboratory studies are needed to
measure eventual changes at neuro-anatomical and neuro-psychological level using modern neuro-imaging instruments
(PET, SPECT, fMRI). Randomised clinical trials should be carried out to test our hypothesis for all-day use in clinical
practice. The hypothesis: chronic pain is a non-use disease produced by psycho-emotional factors like fear, attention
and anxiety. Optimal treatment should be based on physiological use, and dissociation of pain and the mentioned
psycho-emotional factors. Paradoxal pain therapy could serve these treatment conditions.
c 2006 Published by Elsevier Ltd.
Introduction
Finding remedies for patients with chronic pain is

* Corresponding author. Present address: Faculty of Human
one of the most difficult and at the same time
Sciences, University of Gerona, C/Los Helechos 5a, 35500
Arrecife, Las Palmas, Spain. Tel.: +34 928844959.
one of the most intruding objectives of modern sci-
E-mail address: leo.pruimboom@fudgif.udg.es entific investigation. The last decade has brought a
(L. Pruimboom). lot of new insides in the mechanisms of disease for

0306-9877/$ - see front matter c 2006 Published by Elsevier Ltd.
doi:10.1016/j.mehy.2006.08.036
Please cite this article in press as: Pruimboom L, van Dam AC, Chronic pain: A non-use disease, Medical Hypotheses
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
2 Pruimboom and van Dam
people with chronic pain syndrome. Neurological lated to changes of the homeostasis, ranging from
imaging techniques like positron evoked tomogra- tissue damage to hypoxia and acidosis. The intero-
phy (PET) and functional MRI (fMRI) have made it ceptive afferents enter the spinal cord via lamina
possible to visualise changes at neurological, histo- I neurons (in the so called heat-pinch-cold neurons),
logical and immunological level. Resuming the re- cross to the other side of the spinal cord and ascend
sults of the most revealing work of the last as a spinothalamic tract through homeostatic cen-
decade [1–3] it seems obvious that mechanisms tres in the brain stem and thalamus, ending up in
of disease for developing chronic pain include: the insular cortex and gyrus cingularis. Their theory
is that chronic pain is caused by central sensitisat-
1. Neuro-anatomical reorganisation in the spinal ion of the so called ‘‘neuromatrix of homeostasis’’
cord and the brain. and involves neuro-anatomical and neuro-physio-
2. Neuro-physiological changes in the spinal cord logical pathways. For an excellent review of la-
and the brain. belled line interoceptive afferents and pain, I
3. Activation of glia cells (immune reaction in the would like to refer to [5].
central nervous system). Wieseler-Frank et al. [6,7] describe chronic pain
as a process of activation of glia cells in the spinal
Craig et al. [2,4] have developed a reasonable cord. Glia cells (microglia and astrocytes) normally
amount of evidence that pain is an efferent homeo- exhibit minor (astrocytes) or no activity (microglia)
static feeling produced as a reaction on labelled in physiological circumstances. Several factors,
line interoceptive afferent information. Interocep- like long term use of morphine, virus, trauma and
tive afferents transport all kind of information re- ischemia, can activate glia cells (Fig. 1). Once glia
Figure 1 Glia-cell activation as a direct cause for enhanced activity of excitatory neurons in the spinal cord,
provoking a pain sensation. Interoceptive afferents can be activated by a great deal of peripheral homeostatic
imbalances. All of them can activate glia cells in lamina I of the spinal cord. Glia cell activation can continue with
absence of the initial activator.
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
Chronic pain: A non-use disease 3
cells are activated they produce pro-inflammatory behaviour characteristics are anxiety and continu-
cytokines like interleukin 6 (IL6), interleukin 1 ous focusing on the pain. Fear, anxiety and chronic
(IL1) and tumor necrosis factor alpha (TNFa). These focusing can be considered ‘‘homeostatic feeling’’.
substances are able to activate excitatory neurons All these homeostatic feeling activate the so called
in the spinal cord responsible for afferent pain interoceptive cortex (insular cortex) and the gyrus
(interoceptive) transmission. Chronic activation of cingularis [4,5]. When fear, anxiety, pain and pain-
glia cells can produce long term changes in neuro- focusing activate the interoceptive cortex at the
nal activity in spinal cord and brain neurons respon- same time, in the end they will be neurologically
sible for pain transmission; changes which could be connected:
responsible for the development of chronic pain
‘‘When two neurons fire together they wire
syndromes.
together and if they don’t they won’t’’
Results of clinical trials using antagonistic drugs
of pro-inflammatory cytokines (glia-modulator) The described process of classical and operant
show that these kind of treatments are promising conditioning will lead to the typical neuro-psycho-
for people with chronic pain syndromes [8–11]. logical substrate for chronic pain patients:
Current treatment of patients with chronic pain
syndrome is based on the neuro-immunological There is no pain without fear [21];
pathways of developing chronic pain. Tricyclic There is no pain without anxiety [21];
antidepressants [12,13], gabapentin and morphine There is no pain without attention [22] and;
[14] are the most frequently used drugs combined There is no pain without gain [21].
with anti-inflammatory drugs (NSAID), glia-modula-
tor drugs and cognitive psychotherapy. Effective- This pain-behaviour reduces the use of the af-
ness of these treatments has been proven, fected area. Reduced or non-use of a part of the
although there still is a group of non-reacting pa- body results in a neuro-anatomical reorganisation
tients, and secondary effects of pain-drugs are in various parts of the brain [15,23]; a situation sim-
considerable. ilar to patients with phantom pain. Patients with
For these reasons we have developed a new phantom pain are successfully treated with con-
therapy for patients with chronic pain based on straint induced movement [(CIMT,15,23)]. A therapy
the similarity of the scientific substrate of both based on limitation of the non-affected side of the
phantom pain and chronic pain [15,16]. Phantom body and massive use (6 h a day) of a functional pros-
pain is considered a non-use syndrome. If chronic thesis adapted to the amputated extremity.
pain patients present a similar substrate as pa- ‘‘Paradoxal pain therapy’’ is based on a combi-
tients with non-use syndromes, than chronic pain nation of CIMT and dissociation-techniques of neu-
could also be considered a non-use syndrome. If ronal connections.
so then:
First Chronic pain patients should hardly use The hypothesis

there affected part of the body;
Second Constraint induced use of the affected Chronic pain is a non-use disease provoked by psy-
part of their body should change the pain cho-emotional factors like fear, attention and anx-
intensity and function and; iety [25]. Treatment should be based on 1.
Third Deep learning and massive training of the physiological use, and 2. on dissociation of pain
affected part of the body should provide and the mentioned psycho-emotional factors.
a re-reorganisation of neuro-anatomical ‘‘Paradoxal pain therapy’’ could serve these treat-
structure in the brain and spinal cord. ment conditions.
Neuro-physiological changes should
include neurotransmitters and glia-cell
activation. Paradoxal pain therapy
The theory 1. Deep learning therapy.

2. Dissociation-techniques Inform the environ-
People with chronic pain tend to ‘‘fly’’ from their ment to ‘‘forget’’ the pain of the affected
affected part of the body. Fear for pain and more patient. Pain-associated behaviour of the
damage seems to be the mayor reason for this part patient is not rewarded anymore (loss of gain
of the typical pain-behaviour [17–20]. Other pain- and attention).
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
3. Constraint induced use of affected area (from Hence, these conditions for constraint
fearful non-use to relaxed massive use). induced movement training in patients
4. Illusion technique as an early pain-distraction with chronic pain vary compared with the
strategy. CIMT for patients with phantom pain or
stroke. The adaptation is based on the fact
Ad. 1. Patients are informed about the influence that the affected part of the body is still
of emotions (fear, anxiety) and attention there and tissue overload has to be
on their pain syndrome. Further informa- avoided. Studies of Moseley [24] made it
tion is given about the pathways that clear that overload can be prevented if
lead to the neuro-immunological changes the patient chooses intensity (load, the
in the spinal cord and brain [24]. It is number of repetitions, pause between
important to state that the patient’s the repetitions and the context in which
problem is not a psychological but a neu- the task is executed). The CIMT period
rological problem. The profound explana- duration in chronic pain patients varies
tion (deep learning) activates from 8 days to 14 days. After one month
motivational areas in the brain; areas a new training program starts (if neces-
which belong to the so called central sary) adapted to the actual functions of
reward circuit [25]. Several parts of the the patient. Task difficulty depends on
central reward circuit are able to pro- function and not on pain. Task may not
duce a serial of pain-inhibiting neuro- provoke fear or anxiety.
transmitters like endorphins, dopamine Restoration of function is probably the
and GABA. Activation of these areas by missing link treating patients with chronic
deep learning enhances pain-killing pro- pain. Current cognitive/behavioural- and
cesses and diminishes fear and anxiety medical therapies to often focus on pain
by rational understanding of the pain pro- killing alone [26]. The CIMT recovers nor-
cess. Deep learning explanation has to be mal function, offering physiological infor-
profound and understandable for every mation to the brain areas responsible for
individual and includes neuro-physiologi- interoceptive/homeostatic analysis.
cal, neuro-anatomical and psycho-emo- These areas can produce the homeostatic
tional pathways related with chronic feeling ‘‘pain’’ but would not do so if the
pain. information, which enters these areas is
Ad. 2. Patient and the direct environment are completely normal.
informed about the influence of attention Ad. 4. Patients with chronic pain often (almost
related to pain and pain-behaviour. Pain- always) start a new day focusing on their
behaviour should be avoided (for pain. ‘‘How will I feel today? Will I be in
instance, hand supporting the low back agony? This means that although they feel
in patients with chronic low back pain) no pain at that moment, their brain (and
by the patient and ignored by his/her spinal cord) is already occupied with pain
environment. and fear for pain. To prevent this focusing
Ad. 3. Patient trains her/his affected part of the moment (with a huge impact on pain-con-
body using a massive training program of ditioning processes) at the beginning of
difficult motor tasks, constraining other the day, patients have to carry out a so
parts of the body. The therapist can help called ‘‘illusion therapy’’. This therapy
to execute the tasks if necessary. Before (therapy conditions see frame) has proven
starting the program the therapist valo- its effects in laboratory investigations of
rises the actual functions of the patient. patients with pain in our clinic. Effects
After inventorying the different tasks, seem to be mediated by changes in endor-
training begins. The amount of repetitions phin and dopamine production in the
depends on resistance declared by the reward circuit of the brain. Both natural
patient. This means that the patient and neurotransmitters are able to regulate
the therapist choose respectively, the neurological activation in pain transmis-
intensity of training and the task. A train- sion neurons [27]. Another goal is to dis-
ing session lasts not more than 60– tract patient’s attention to the pain,
90 min. The constraint part of the body being distraction-therapy one of the most
has to be constraint for at least 50% of promising treatments for chronic pain
wakeful daytime. patients [28–30].
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
[3] Taub E et al. A placebo-controlled trial of constraint-

Illusion therapy induced movement therapy for upper extremity after
stroke. Stroke 2006;37(4):1045–9.
When you wake up in the morning you plan on [4] Craig AD. Pain mechanisms: labeled lines versus convergence
in central processing. Annu Rev Neurosci 2003;26:1–30.
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I go to the cinema). Planning includes time (at neurons exhibit responses that correspond with burning
eight p.m.) and capability of execution. At pain. J Neurophysiol 2004;92(4):2604–9.
last you have to carry out your plan. [6] Wieseler-Frank J, Maier SF, Watkins LR. Central proinflam-
Frequency 5 times a week matory cytokines and pain enhancement. Neurosignals
2005;14(4):166–74.
The same wish can’t be repeated more than 5 [7] Wieseler-Frank J, Maier SF, Watkins LR. Immune-to-brain
times a month (to prevent adaptation). communication dynamically modulates pain: physiological
and pathological consequences. Brain Behav Immun
2005;19(2):104–11;
Cami J, Farre M. Drug addiction. New Engl J Med
Discussion and conclusion 2003;349(10):975–86.
[8] Raghavendra V et al. Anti-hyperalgesic and morphine-
sparing actions of propentofylline following peripheral
Scientific research in the last decades has nerve injury in rats: mechanistic implications of spinal glia
brought a lot of new insides in the treatment and proinflammatory cytokines. Pain 2003;104(3):655–64.
of patients suffering chronic pain syndromes. Cur- [9] Raghavendra V, Tanga FY, DeLeo JA. Attenuation of
rent treatments include morphine, tricyclic anti- morphine tolerance, withdrawal-induced hyperalgesia,
and associated spinal inflammatory immune responses by
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cognitive psychotherapy with acceptable results. 327–34.
However side-effects are considerable and re- [10] Dorazil-Dudzik M et al. The effects of local pentoxifylline
bound effects (more pain after a period of treat- and propentofylline treatment on formalin-induced pain
ment) are frequent. A further problem is that and tumor necrosis factor-alpha messenger RNA levels in
the inflamed tissue of the rat paw. Anesth Analg
normal function is not recuperated. The men- 2004;98(6):1566–73. Table of contents.
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edge about neurological, immunological and insula to painful heat studied with high resolution func-
physiological processes related to chronic pain tional imaging. Neuroimage 2005;27(1):201–9.
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chronic pain present changes on neurological, [13] Max MB. Treatment of post-herpetic neuralgia: antidepres-
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of their pain. These changes seem to be pro- for neuropathic pain. New Engl J Med 2005;352(13):1324–34.
[15] Mark VW, Taub E. Constraint-induced movement therapy
duced by chronic activity of interoceptive affer- for chronic stroke hemiparesis and other disabilities. Restor
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ultimate consequence is a lack of use of the af- ment therapy: a new family of techniques with broad
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Rehabil Res Dev 1999;36(3):237–51.
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include massive use of the affected part of the in patients with subacute low back pain. Pain 2006.
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(2006), doi:10.1016/j.mehy.2006.08.036
Review Wound Healing
Nutritional Support
for Wound Healing
Douglas MacKay, ND,
and Alan L. Miller, ND
Abstract
Introduction
Healing of wounds, whether from accidental
Wound healing involves a complex series
injury or surgical intervention, involves the
of interactions between different cell types,
activity of an intricate network of blood cells,
cytokine mediators, and the extracellular matrix.
tissue types, cytokines, and growth factors.
The phases of normal wound healing include he-
This results in increased cellular activity, which
mostasis, inflammation, proliferation, and remod-
causes an intensified metabolic demand for
eling. Each phase of wound healing is distinct,
nutrients. Nutritional deficiencies can impede
although the wound healing process is continu-
wound healing, and several nutritional factors
ous, with each phase overlapping the next. Be-
required for wound repair may improve healing
cause successful wound healing requires adequate
time and wound outcome. Vitamin A is required
blood and nutrients to be supplied to the site of
for epithelial and bone formation, cellular
damage, the overall health and nutritional status
differentiation, and immune function. Vitamin
of the patient influences the outcome of the dam-
C is necessary for collagen formation, proper
aged tissue. Some wound care experts advocate a
immune function, and as a tissue antioxidant.
holistic approach for wound patients that consid-
Vitamin E is the major lipid-soluble antioxidant
ers coexisting physical and psychological factors,
in the skin; however, the effect of vitamin E on
including nutritional status and disease states such
surgical wounds is inconclusive. Bromelain
as diabetes, cancer, and arthritis. Keast and Orsted1
reduces edema, bruising, pain, and healing
wittily state, “Best practice requires the assess-
time following trauma and surgical procedures.
ment of the whole patient, not just the hole in the
Glucosamine appears to be the rate-limiting
patient. All possible contributing factors must be
substrate for hyaluronic acid production in the
explored.”
wound. Adequate dietary protein is absolutely
Wound repair must occur in a physiologic
essential for proper wound healing, and tissue
environment conducive to tissue repair and regen-
levels of the amino acids arginine and
eration. However, several clinically significant
glutamine may influence wound repair and
factors are known to impede wound healing, in-
immune function. The botanical medicines
cluding hypoxia, infection, tumors, metabolic dis-
Centella asiatica and Aloe vera have been used
orders such as diabetes mellitus, the presence of
for decades, both topically and internally, to
debris and necrotic tissue, certain medications, and
enhance wound repair, and scientific studies
are now beginning to validate efficacy and
explore mechanisms of action for these Douglas J. MacKay, ND – Technical Advisor, Thorne
botanicals. To promote wound healing in the Research, Inc; Senior Editor, Alternative Medicine Review;
private practice, Sandpoint, ID.
shortest time possible, with minimal pain, Correspondence address: Thorne Research, PO Box 25,
discomfort, and scarring to the patient, it is Dover, ID 83825 E-mail: duffy@thorne.com
important to explore nutritional and botanical Alan L. Miller, ND – Technical Advisor, Thorne Research,
influences on wound outcome. Inc; Senior Editor, Alternative Medicine Review.
Correspondence address: Thorne Research, PO Box 25,
(Altern Med Rev 2003;8(4):359-377) Dover, ID 83825 E-mail: alanm@thorne.com
Alternative Medicine Review ◆ Volume 8, Number 4 ◆ 2003 Page 359

Wound Healing Review
a diet deficient in protein, vitamins, or minerals. tissue. The neutrophils engulf debris and
In addition, increased metabolic demands are microorganisms, providing the first line of defense
made by the inflammation and cellular activity in against infection. Neutrophil migration ceases
the healing wound, which may require increased after the first few days post-injury if the wound is
protein or amino acids, vitamins, and minerals.2 not contaminated. If this acute inflammatory phase
The objective in wound management is persists, due to wound hypoxia, infection,
to heal the wound in the shortest time possible, nutritional deficiencies, medication use, or other
with minimal pain, discomfort, and scarring to the factors related to the patient’s immune response,
patient. At the site of wound closure a flexible and it can interfere with the late inflammatory phase.3
fine scar with high tensile strength is desired. In the late inflammatory phase, monocytes
Understanding the healing process and nutritional converted in the tissue to macrophages, which di-
influences on wound outcome is critical to suc- gest and kill bacterial pathogens, scavenge tissue
cessful management of wound patients. Research- debris and destroy remaining neutrophils. Mac-
ers who have explored the complex dynamics of rophages begin the transition from wound inflam-
tissue repair have identified several nutritional mation to wound repair by secreting a variety of
cofactors involved in tissue regeneration, includ- chemotactic and growth factors that stimulate cell
ing vitamins A, C, and E, zinc, arginine, glutamine, migration, proliferation, and formation of the tis-
and glucosamine. Botanical extracts from Aloe sue matrix.
vera, Centella asiatica, and the enzyme brome- The subsequent proliferative phase is
lain from pineapple have also been shown to im- dominated by the formation of granulation tissue
prove healing time and wound outcome. Eclectic and epithelialization. Its duration is dependent on
therapies, including topical application of honey, the size of the wound. Chemotactic and growth
sugar, sugar paste, or Calendula succus to open factors released from platelets and macrophages
wounds, and comfrey poultices and hydrotherapy stimulate the migration and activation of wound
to closed wounds are still in use today. Although fibroblasts that produce a variety of substances
anecdotal reports support the efficacy of these essential to wound repair, including glycosami-
eclectic therapies, scientific evidence is lacking. noglycans (mainly hyaluronic acid, chondroitin-
4-sulfate, dermatan sulfate, and heparan sulfate)
The Four Phases of Wound Healing and collagen.2 These form an amorphous, gel-like
Tissue injury initiates a response that first connective tissue matrix necessary for cell migra-
clears the wound of devitalized tissue and foreign tion.
material, setting the stage for subsequent tissue New capillary growth must accompany
healing and regeneration. The initial vascular re- the advancing fibroblasts into the wound to pro-
sponse involves a brief and transient period of vide metabolic needs. Collagen synthesis and
vasoconstriction and hemostasis. A 5-10 minute cross-linkage is responsible for vascular integrity
period of intense vasoconstriction is followed by and strength of new capillary beds. Improper
active vasodilation accompanied by an increase cross-linkage of collagen fibers has been respon-
in capillary permeability. Platelets aggregated sible for nonspecific post-operative bleeding in
within a fibrin clot secrete a variety of growth fac- patients with normal coagulation parameters.4
tors and cytokines that set the stage for an orderly Early in the proliferation phase fibroblast activity
series of events leading to tissue repair. is limited to cellular replication and migration.
The second phase of wound healing, the Around the third day after wounding the growing
inflammatory phase, presents itself as erythema, mass of fibroblast cells begin to synthesize and
swelling, and warmth, and is often associated with secrete measurable amounts of collagen. Collagen
pain. The inflammatory response increases levels rise continually for approximately three
vascular permeability, resulting in migration of weeks. The amount of collagen secreted during
neutrophils and monocytes into the surrounding this period determines the tensile strength of the
wound.
Page 360 Alternative Medicine Review ◆ Volume 8, Number 4 ◆ 2003

Figure 1. Nutrient Impacts on the Phases of Wound Healing
Wounding
Calendula succus – topical antimicrobial
Hemostasis
Drugs, herbs, vitamins, amino acids, or minerals that effect blood-clotting

mechanisms should be avoided prior to surgery.
Inflammatory Phase
Vitamin A – enhances early inflammatory phase
Bromelain and adequate protein intake – prevent prolonging inflammatory phase
Vitamin C – enhances neutrophil migration and lymphocyte transformation
Proliferative Phase
Vitamin C – necessary for collagen synthesis
Centella asiatica – promotes type-1 collagen synthesis
Glucosamine – enhances hyaluronic acid production
Vitamin A – promotes epithelial cell differentiation
Zinc – required for DNA synthesis, cell division, and protein synthesis
Calendula succus and Aloe vera – support formation of granulation tissue
Remodeling
Protein deficiency – inhibits wound remodeling
The final phase of wound healing is Figure 1 summarizes the phases of wound
wound remodeling, including a reorganization of healing and nutrients that impact the various
new collagen fibers, forming a more organized lat- phases.
tice structure that progressively continues to in-
crease wound tensile strength. The remodeling
process continues up to two years, achieving 40-
70 percent of the strength of undamaged tissue at
four weeks.2

Vitamins and Minerals Essential to with fractures, tendon damage, or vitamin A defi-
ciency may also benefit from perioperative vita-
Wound Healing min A supplementation. Additional research is
Vitamin A necessary to establish the effectiveness of univer-
Vitamin A is required for epithelial and sal perioperative vitamin A supplementation in
bone tissue development, cellular differentiation, healthy individuals.
and immune system function. Substantial evidence Concern among some practitioners re-
supports the use of vitamin A as a perioperative garding the potential toxicity of higher doses of
nutritional supplement.5 In addition to facilitating vitamin A has led to uneasiness about using it
normal physiological wound repair, Ehrlich and perioperatively. The vast majority of toxicity cases
Hunt have shown vitamin A reverses the cortico- have occurred at daily vitamin A dosages of
steroid-induced inhibition of cutaneous and fas- 50,000-100,000 IU in adults over a period of
cial wound healing.6-8 Vitamin A has also corrected weeks to years.15 Short-term supplementation of
non-steroid induced, post-operative immune de- 25,000 IU daily appears to be safe for most non-
pression9 and improved survival in surgically-in- pregnant adults. Caution must be exercised in
duced abdominal sepsis.10 Levenson et al suggest supplementing vitamin A in patients for whom the
vitamin A benefits the wound by enhancing the anti-inflammatory effect of steroids is essential,
early inflammatory phase, including increasing the such as in rheumatoid arthritis or organ transplants,
number of monocytes and macrophages at the as well as in pregnant women and women of child-
wound site, modulating collagenase activity, sup- bearing age.5
porting epithelial cell differentiation, and improv-
ing localization and stimulation of the immune Vitamin C
response.10,11 Ascorbic acid is an essential cofactor for
Animal studies show vitamin A may in- the synthesis of collagen, proteoglycans, and other
crease both collagen cross-linkage and wound- organic components of the intracellular matrix of
breaking strength. Greenwald et al inflicted sur- tissues such as bones, skin, capillary walls, and
gical flexor profundus damage and immediate re- other connective tissues. Ascorbic acid deficiency
pair on adult chickens. They found chickens that causes abnormal collagen fibers and alterations
ate a diet supplemented with vitamin A (150,000 of the intracellular matrix that manifests as cuta-
IU/kg chicken chow) demonstrated wound-break- neous lesions, poor adhesion of endothelium cells,
ing strength more than double that of controls fed and decreased tensile strength of fibrous tissue.16
standard chicken chow.12 In addition, rats with Clinical manifestations of ascorbic acid deficiency
dorsal skin incisions and concurrent comminuted include bleeding gums, poor immunity, easy bruis-
femoral fractures exhibited delayed cutaneous ing and bleeding, and slow healing of wounds and
healing. Supplemental vitamin A enhanced wound fractures.17 Ascorbic acid is necessary for the hy-
healing in these animals, demonstrated by in- droxylation of proline and lysine residues in
creased breaking strength of the dorsal skin inci- procollagen, which is necessary for its release and
sions in rats fed supplemental vitamin A compared subsequent conversion to collagen. Hydroxypro-
to the non-supplemented group. The authors be- line also stabilizes the collagen triple-helix struc-
lieve the improved wound healing is a result of an ture.18 In addition to collagen production, ascor-
increased rate of collagen cross-linkage.13 bic acid enhances neutrophil function,19 increases
Levenson and Demetrio recommend vi- angiogenesis,20 and functions as a powerful anti-
tamin A supplementation of 25,000 IU daily be- oxidant.21
fore and after elective surgery.14 Research supports Although ascorbic acid is required for
perioperative vitamin A supplementation in pa- reparation of damaged tissue, researchers have
tients known to be immune depleted or steroid demonstrated the benefit of vitamin C only in vi-
treated. Surgical patients with sepsis and those tamin C-deficient individuals using low doses of

ascorbic acid.22 In a study by Hodges et al, four Events leading to wounds, including trauma and
subjects (ages 33-44) were depleted of vitamin C surgery, are perceived as physiological stressors
for 99 days to induce scurvy. On day 100, a 5-cm that have also been correlated with a decrease in
incision was made in the left thigh of each subject plasma ascorbic acid.34,35 Thus, the acute stress
and they began the oral administration of 4, 8, 16, experienced by trauma or surgery patients may
or 32 mg ascorbic acid daily. Healing was mea- unmask marginal vitamin C deficiencies, leading
sured by histological and electron microscope to deficiency symptoms.
technique. It was shown that 4 mg daily of vita- Cutaneous healing wounds have been
min C was just as effective as 32 mg daily for found to have lower ascorbic acid content than
wound healing in these vitamin C-deficient sub- intact tissue. Levels of vitamin C were compared
jects.22 The efficacy of using vitamin C to improve to normal skin in two-, four-, seven-, and 14-day-
wound healing in non-deficient individuals re- old wounds in animals. Vitamin C levels decreased
mains uncertain. It should be noted, however, that approximately 60 percent post-wound and had not
even the highest dose in this study (32 mg) is be- exhibited full recovery by day 14.36 In addition,
low the RDA for vitamin C. Higher doses and low levels of antioxidants, including ascorbic acid,
larger differences between doses might have accompanied by elevated levels of markers of free
yielded more significant differences. radical damage have been detected in elderly rat
Humans lack the ability to store vitamin cutaneous wounds exhibiting delayed healing.
C, and certain populations are more likely to be Eighteen-month-old wounded male rats were com-
deficient in ascorbic acid, including the elderly, pared to 3-4 month-old rats pre-wound and seven
alcoholics, drug abusers, and under-nourished in- days post-wound. Normal skin of aged and young
dividuals.23 Subclinical vitamin C deficiency is rats showed no difference in ascorbic acid con-
being recognized increasingly in the general popu- tent; however, a 59-percent decrease in ascorbic
lation. Published cases show that restricted eating acid content was observed in wound tissues of
patterns, prolonged hospitalization, severe ill- aged animals compared to its content in young
nesses, and poor dietary intake in both children adult wounds.37 Rasik and Shukla propose the de-
and adults cause deficiency with significant clini- lay in wound healing of older rats is at least par-
cal consequences.4,24-26 In one study 12 patients tially a result of increased free radical damage.37
with post-surgical diffuse hemorrhage, each ex- The programmed sequences of the cellu-
hibiting normal coagulation parameters, were lar and molecular processes occurring during
found to have low plasma ascorbic acid levels. wound repair are also dependent on immune func-
Each patient received 250-1,000 mg oral vitamin tion. Infection resulting from impaired immunity
C daily. Within 24 hours of vitamin C administra- is one of the most commonly encountered and
tion there was no further evidence of bleeding or clinically significant impediments to wound heal-
need for subsequent blood transfusions in any pa- ing. 3 In addition, cellular immunity and
tient. The authors concluded vitamin C deficiency dysregulation of cytokines can impair wound heal-
should be included in the differential diagnosis for ing.38 Ascorbic acid has been shown to improve
nonspecific bleeding in surgical patients.4 immune function in humans.39-42 Human volun-
In mammals, ascorbic acid is necessary teers who ingested 2-3 g ascorbate daily for sev-
for a normal response to physiological stressors, eral weeks exhibited enhanced neutrophil motil-
with the need for ascorbic acid increasing during ity to chemotactic stimulus and stimulation of lym-
times of injury or stress.27 Studies have shown the phocyte transformation.43 Neutrophil motility and
physiological stress of intense exercise generates lymphocyte transformation were also stimulated
excess reactive oxygen species (ROS), increasing by 1 g intravenous ascorbic acid in six healthy
the demand on the antioxidant defense system.28- volunteers. Alterations in these activities were re-
30
A similar elevation of ROS has been noted within lated to serum ascorbic acid levels.
wounds; therefore, substances that increase tissue
antioxidants are thought to benefit healing.31-33

The combined effect of ascorbic acid on Perioperative zinc supplementation is re-

collagen synthesis, antioxidant status, and commended for zinc-depleted patients.23 Data is
immunomodulation make it an appropriate supple- lacking to show zinc supplementation improves
ment for wound repair protocols. Research pro- healing in non-deficient individuals; however, zinc
vides evidence for the use of low doses of vitamin deficiency in humans is widespread, and injured
C in vitamin C-deficient individuals, but many and stressed individuals are more prone to devel-
practitioners believe larger doses of ascorbic acid oping deficiencies. Ehrlich et al suggest zinc is
in non-deficient individuals are indicated for op- lost in significant amounts after surgery because
timal wound repair. Levenson and Demetriou re- of fistulas, stress, and diarrhea.50 Zinc deficien-
commend supplementing 1-2 g ascorbic acid daily cies have also been identified in individuals with
from wound onset until healing is complete.14 Such deep partial- or full-thickness burns and chronic
doses may be justified due to the lack of adverse venous leg ulceration.51,52
effects at these levels44 combined with the poten- Further research is needed on the efficacy
tial for deficiency in certain individuals. In addi- of zinc supplements for wound healing. Justifica-
tion, the transient increase in metabolic require- tion for perioperative zinc supplementation in-
ments for vitamin C resulting from the physiologic cludes the absence of adverse effects at moderate
stress of trauma or surgery and the metabolic re- doses (15-30 mg daily) and evidence that zinc
quirement of vitamin C for collagen synthesis are deficiency impairs wound healing. Zinc supple-
indications for higher doses of vitamin C in non- mentation of 15-30 mg daily is recommended
deficient individuals. perioperatively to prevent unmasking of marginal
deficiencies. Higher levels of zinc supplementa-
Zinc tion may be necessary in patients with malnutri-
Approximately 300 enzymes require zinc tion, malabsorption, chronic diarrhea, or other risk
for their activities. Zinc is an essential trace min- factors of zinc deficiency.
eral for DNA synthesis, cell division, and protein
synthesis,45 all necessary processes for tissue re- Vitamin E
generation and repair. Zinc deficiency has been Vitamin E is popular among consumers
associated with poor wound healing and decreased for skin care and to prevent scar formation. It func-
breaking strength of animal wounds,46 which can tions as the major lipophilic antioxidant, prevent-
result from decreased protein and collagen syn- ing peroxidation of lipids and resulting in more
thesis during healing found in zinc-deficient ani- stable cell membranes. The antioxidant-membrane
mals.47 Senapati and Thompson found zinc levels stabilizing effect of vitamin E also includes stabi-
were 50-percent higher in muscle and skin from lization of the lysomal membrane, a function
abdominal wounds of rats during wound healing, shared by glucocorticoids.53 Systemic vitamin E
but mild deficiency reduced this accumulation.48 and glucocorticoids inhibit the inflammatory re-
Zinc demands are thought to be the high- sponse and collagen synthesis, thereby possibly
est from time of wounding throughout the early impeding the healing process. The effect of vita-
inflammatory phase. Sequential changes in zinc min E on wound healing is complex; it may have
concentrations were studied in the incisional alternate effects in different types of wounds and
wound model in the rat. Zinc levels increased from in the presence of other nutrients, as well as dif-
wounding and peaked on the fifth day – at a time ferent functions for water soluble versus lipid
of high inflammation, granulation tissue forma- soluble preparations of vitamin E.
tion, and epidermal cell proliferation.49 Zinc con- Animal studies of vitamin E supplemen-
centrations returned to normal by the seventh day, tation on surgical wounds show conflicting results.
when inflammation had regressed. It has been sug- Greenwald et al showed flexor tendon repair in
gested that increased local demand for zinc result- chickens treated with vitamin E had breaking
ing from surgery and wounding exposes otherwise strength less than half that of controls measured
marginal zinc deficiencies in humans.48

after days 7 and 45 from surgical repair.12 Another time critical to interpretation of this study. It was
animal study showed impaired collagen synthesis also noted that breakdown products and contami-
in rats treated with vitamin E after wounding.54 nants could account for the inflammatory response
The researchers cite the glucocorticoid-like effect encountered.59 In a second, larger blinded study,
of vitamin E as the cause of the negative results. the effects of topical steroids, vitamin E, or the
However, these effects are mitigated by vitamin base cream carrier for these substances on scar
A, as vitamin A is a lysomal destabilizer that re- outcome of 159 post-operative patients were
verses several of the deleterious effects of gluco- evaluated. Both topical steroids and topical vita-
corticoids.8 min E failed to impact scar thickness, range of
Paradoxical results found by Galeano et motion, or ultimate cosmetic appearance.60
al showed a hydrophilic vitamin E preparation The available data on vitamin E and
positively impacted delayed wound healing in dia- wound healing could lead to several possible con-
betic mice. Increased breaking strength and col- clusions: (1) systemic vitamin E may have a nega-
lagen content of the wound was found in treated tive impact on surgical wounds due to its lysoso-
animals. These authors speculate inhibition of lipid mal-stabilizing properties; (2) vitamin A may miti-
peroxidation accounted for the positive results.55 gate these negative effects; and (3) hydrophilic and
In addition, prophylactic administration of vita- hydrophobic preparations of vitamin E may have
min E has been shown to increase breaking different actions related to wounds. The benefit
strength and normalize healing of wounds exposed of topical vitamin E on surgical wound healing
to preoperative irradiation56 and to decrease the and scar formation remains inconclusive and, al-
development of intraperitoneal adhesions in ani- though anecdotal reports support topical use of
mals.57 vitamin E for scar therapy, research shows it may
Since the discovery of vitamin E as the have a negative effect on scarring and wound out-
major lipid-soluble antioxidant in skin, it has been come.
used topically for a wide variety of skin lesions.
Anecdotal reports claim topical vitamin E is valu- Other Dietary Supplements and
able for speeding wound healing and improving
cosmetic outcome of burns and other wounds, in-
Wound Healing
cluding surgical scars. Such claims are disputed Bromelain
by two human clinical trials. In a double-blind Bromelain is a general name given to a
study of 15 patients with surgically-induced family of proteolytic enzymes derived from
wounds, emollient lotion and emollient lotion Ananas comosus, the pineapple plant. Through-
mixed with vitamin E were applied to healing out the 1960s and 1970s a series of studies found
wounds. The wounds were randomly divided into the effects of orally administered bromelain in-
two parts and the different topical applications clude the reduction of edema, bruising, pain, and
were applied to the same half of each wound twice healing time following trauma and surgical pro-
daily. Physicians and patients independently evalu- cedures.61-64 More recently, researchers from the
ated the scars for cosmetic appearance on weeks Czech Republic found that patients with long bone
1, 4, and 12. In 90 percent of cases, topical vita- fractures administered a proteolytic enzyme com-
min E either had no effect, or actually worsened bination containing 90 mg bromelain per tablet
the cosmetic appearance of scars.58 In addition, had less post-operative swelling compared to pa-
33 percent of the patients studied developed con- tients given placebo.65 Fractures were treated by
tact dermatitis to topical vitamin E. A response to surgically inserting rods through the long axis of
this study, published in Dermatologic Surgery, the fractured bone (intramedullary fixation) or by
pointed out that d-alpha tocopherol is an extremely constructing an external framework of pins and
unstable compound, rendering details of its source, rods going through the skin and muscle to con-
formulation, storage condition, and stability over nect to the fractured bone (external fixators). The
treatment group was given three 90-mg tablets

three times daily for three days after surgery and, Tassman et al noted that, while post-sur-
subsequently, two tablets three times daily for two gical oral bromelain administration was effective
weeks. On the fourteenth post-operative day the in reducing pain, swelling, and healing time, a
limb volume of the treatment group was reduced protocol using pre- and post-surgical bromelain
by 17 percent compared with nine percent in the is recommended.63 Studies have shown bromelain
control group. The total number of analgesics con- prevents aggregation of blood platelets in patients
sumed by the treatment group was also signifi- with high platelet aggregation values, which has
cantly reduced in comparison to the control led to recommendations by physicians and sur-
group.65 geons to avoid oral bromelain prior to any surgi-
Studies by Tassman et al show bromelain cal procedure. In one human trial, bromelain was
reduced swelling, bruising, pain, and healing time administered orally to 20 volunteers with a his-
in patients following dental surgeries.63,66 In a tory of heart attack or stroke, or with high platelet
double-blind study of dental surgery patients, bro- aggregation values. Bromelain decreased platelet
melain was found to decrease swelling to 3.8 days, aggregation in 17 of the subjects and normalized
compared with seven days in patients given pla- values in eight of the nine subjects who previously
cebo. In addition, duration of pain was reduced to had high aggregation values.72 Contrary to this,
five days in the treatment group, compared to eight other human studies have shown oral bromelain
days in the placebo group.63 to be free of any significant effects on clotting
In an uncontrolled trial, bromelain was parameters.73,74 In one study, 47 patients with vari-
reported to positively influence swelling, pain at ous disorders leading to edema and inflammation
rest and during movement, and tenderness in pa- found no significant effects of oral bromelain (40
tients with blunt injuries to the musculoskeletal mg four times daily for one week) on bleeding,
system.67 Although bromelain has been shown to coagulation, and prothrombin time.
reduce post-operative and trauma-related pain, this It is noteworthy that the studies pertain-
is probably related to its anti-inflammatory action ing to bromelain and platelet aggregation are over
rather than a direct analgesic effect.68 30 years old. The potential benefit of pre- and post-
Aside from its documented anti-inflam- surgical oral bromelain on hematoma resorption,
matory activity, bromelain is of interest to surgeons pain, inflammation, and healing time justifies the
because of its ability to increase resorption rate of need for concise, well-designed clinical trials
hematomas. Bromelain’s influence on hematoma evaluating different doses of bromelain on clot-
resorption was demonstrated using artificially in- ting parameters. Until further data is available re-
duced hematomas in humans. Hematomas in the garding bromelain’s action on platelets, oral bro-
treatment group resolved significantly faster than melain administration should be withheld or used
controls when oral bromelain was given at the time with caution before surgery.
of hematoma induction and for seven days there-
after.69 Glucosamine
Seltzer investigated two different doses of Hyaluronic acid is an important part of
bromelain in patients undergoing rhinoplasty. the extracellular matrix and one of the main gly-
Fifty-three patients were randomized to receive cosaminoglycans secreted during tissue repair.
either one of two doses of bromelain or placebo. Production of hyaluronic acid by fibroblasts dur-
In patients receiving placebo, swelling and ecchy- ing the proliferative stage of wound healing stimu-
mosis persisted for seven days, compared to two lates the migration and mitosis of fibroblasts and
days in both bromelain groups.70 However, a ran- epithelial cells. Glucosamine appears to be the
domized trial of 154 facial plastic surgery patients rate-limiting substrate for hyaluronic acid synthe-
receiving either 400 mg bromelain daily or pla- sis.75 In vitro studies suggest the mechanism of
cebo for one day before and four days after sur- glucosamine on repair processes involves stimu-
gery found no statistically significant differences lation of the synthesis of glycosaminoglycans and
in edema between the two groups.71

Table 1. Perioperative Nutritional Protocol
Nutrient Dose Action
Vitamin A** 25,000 IU daily Enhances early inflammatory

phase of wound healing;
supports epithelial cell
differentiation;
improves localization and
stimulation of immune response.
Vitamin C** 1-2 g daily Synthesis of collagen,

proteoglycans, and other
organic components of the
intracellular matrix;
tissue antioxidant;
supports immune response.
Zinc** 15-30 mg daily Required for DNA synthesis, cell

division, and protein synthesis.
Glucosamine** 1,500 mg daily Enhances hyaluronic acid

production in the wound.
Protein** Minimum of 0.8 g/kg body Prevents delayed healing and

weight daily surgical complications.
Bromelain (use 500-1,000 mg daily Reduces edema, bruising, pain,

post-surgery only) and healing time.
** Use from two weeks prior to surgery until healing is complete
collagen.76 Animal studies have shown the con- well as the first few days after surgery or trauma
tent of glycosaminoglycans within the site of par- might enhance hyaluronic acid production in the
tially ruptured muscles increased maximally five wound, promoting swifter healing and possibly
days after trauma and decreased thereafter.77 This fewer complications related to scarring.
suggests the timing of glucosamine supplementa-
tion may determine its therapeutic impact on Protein and Wound Healing
wounds. Adequate protein intake is essential for
Clinical trials using glucosamine for proper wound healing. Protein depletion appears
perioperative support are lacking. However, the to delay wound healing by prolonging the inflam-
administration of oral glucosamine both before as matory phase; by inhibiting fibroplasia, collagen

and proteoglycan synthesis, and neoangiogenesis stress and have the greatest impact on protein re-
(proliferation phase); and by inhibiting wound quirements, increasing protein need 75-100 per-
remodeling.78,79 cent.86
Experimental protein depletion in animals Table 1 summarizes nutrients recom-
caused a decrease in the tensile strength of wounds. mended for perioperative nutritional support.
Rats fed a diet deficient in protein exhibited de-
creased wound integrity and strength versus con- Amino Acids in Wound Healing
trol animals.80 In a study of 108 human patients It is well accepted that sufficient protein
with experimental wounds, individuals with either is necessary for wound healing. This appears to
low serum protein or serum albumin had signifi- be due to the increased overall protein need for
cantly weaker wounds than those with normal pro- tissue regeneration and repair. Researchers have
tein values.81 investigated the effects of specific amino acids on
Protein calorie malnutrition increases the healing process and determined that arginine
morbidity and mortality in the surgical/trauma and glutamine appear to be necessary for proper
patient. Many studies have found hospitalized wound healing.
patients in a state of malnutrition at admission.
Thus, it is important to increase protein intake to
optimize healing and immune function, and to
Arginine
prevent post-surgical complications in these indi- Arginine is a non-essential amino acid that
viduals.82-84 plays a key role in protein and amino acid synthe-
Protein supplementation of elderly pa- sis. It is acquired from the diet and derived en-
tients with liquid protein formulas significantly dogenously from citrulline in a reaction catalyzed
enhanced healing of pressure ulcers. The change by the enzyme arginine synthetase. Adequate tis-
in ulcer area was significantly correlated with the sue arginine appears to be essential for efficient
amount of protein in the diet.85 wound repair and immune function.87
The surgical or trauma patient exists in a Arginine (17 g/day) was given to 30 eld-
state of metabolic stress, with the severity of the erly patients (>65 years of age) who sustained an
stress depending on the severity of the wounded experimental surgical injury. Supplemented pa-
state. An injured patient requires more protein than tients demonstrated significantly greater hydroxy-
a non-injured patient because of the increased proline (a sign of collagen deposition) and pro-
metabolic activity of wound healing, acute-phase tein accumulation at the wound site, compared to
protein production in response to stress, and amino non-supplemented controls. Lymphocyte re-
acid mobilization from muscle used for hepatic sponse, signifying greater immune activity, was
gluconeogenesis. elevated in the supplemented group, as was insu-
In a non-injured state, adults require ap- lin-like growth factor-1, which is a control mol-
proximately 0.8 g dietary protein/kg body wt/day. ecule for wound repair.88 Other studies have found
Elderly patients have a higher protein requirement similar results.89,90
(1-1.2 g/kg body wt/ day) due to a decreased abil-
ity to synthesize proteins. The surgical/trauma Glutamine
patient can require significantly more protein. Glutamine is used by inflammatory cells
Minor surgery may not significantly increase the within the wound for proliferation and as a source
protein requirement; however, if the patient is al- of energy.91,92 Fibroblasts use glutamine for these
ready protein malnourished, wound healing will same purposes, as well as for protein and nucleic
be adversely affected unless dietary protein intake acid synthesis. Because optimal functioning of
is increased. Major surgery can increase protein these cells is paramount to the healing process,
requirements 10 percent, while a patient with glutamine is a necessary component of the pro-
multiple traumas may need 75-percent more process of tissue repair. Glutamine is a non-essential
tein. Burn wounds cause tremendous metabolic amino acid that can become a “conditionally es-

sential” amino acid in

certain circumstances,
including tissue injury.93 Table 2. Post-surgery or Trauma Protocol
Glutamine is released
from skeletal muscle fol-
lowing injury or surgery, Nutrient Dose
which can cause a rela-
tive deficiency of Bromelain 500-1,000 mg daily
glutamine in skeletal
muscle and the gut, as Vitamin A 25,000 IU daily
intestinal uptake is fre-
quently diminished as
well.
Vitamin C 1-2 g daily
Studies utilizing
oral glutamine pre- and Zinc 15-30 mg daily
post-surgery, and in burn
patients, have shown Protein Minimum 0.8 g/kg body weight daily
mixed results. Oral feed-
ing of glutamine in sur- Glucosamine 1,500 mg daily
gery patients did not af-
fect plasma glutamine or
nitrogen turnover. Intra- Recommended from wounding until healing is complete.
venous glutamine in sur-
gery patients as an ala-
nine-glutamine dipeptide Botanical Medicines in Wound
showed consistently better post-operative results,
as seen by significantly decreased length of hos- Healing
92
pital stays (average of four days or less). A sig- Centella asiatica and Aloe vera
nificantly smaller incidence of pneumonia, bacte- Centella asiatica and Aloe vera have been
remia, and sepsis was noted in patients with mul- used for decades as folk remedies for burns,
tiple trauma given enteral glutamine feedings.94 wounds, and scars. Improved wound healing has
Whether glutamine supplementation will enhance been reported from topical or internal application
wound healing in less severely injured individu- of these two botanical medicines. Continued use
als is not known. of these plants as healing agents has led to scien-
A mixture of arginine (14 g/day), tific investigation of their efficacy as wound heal-
glutamine (14 g/day), and beta-hydroxy-beta- ing agents.
methylbutyrate (HMB) (3 g/day) was given to 18 Centella asiatica (gotu kola) has been
elderly (>70 years) individuals who then under- documented to aid wound healing in several sci-
went experimental implantation of sterile entific studies.96-99 One of the primary mechanisms
polytetrafluoroethylene tubes that could later be of action of Centella appears to be the stimulation
excised and studied for fibroblastic migration and of type-1 collagen production.100 Animal studies
collagen deposition. Supplementation with this have consistently shown topical application of
mixture resulted in significantly greater wound Centella asiatica to a sutured wound significantly
collagen deposition than in 17 controls not supple- increased the breaking strength of the
mented.95 wound.96,99,101,102 Asiaticoside, a saponin extracted
Table 2 summarizes nutrients recom- from Centella asiatica, is thought to be one of its
mended for post-surgery or trauma care.

active constituents. Shukla et al showed a 0.2-per- Eclectic Wound Therapies

cent asiaticoside solution applied topically twice Humans have always been faced with the
daily for seven days to punch wounds in guinea dilemma of how to treat wounds. Many diverse
pigs resulted in 56-percent increase in hydroxypro- and interesting approaches to wound management
line, 57-percent increase in tensile strength, in- have been applied throughout medical history.
creased collagen content, and better epithelializa- Thirty years ago physicians believed pus in a
tion compared to controls. Using the same punch wound was laudable and anxiously awaited its
wound model the researchers demonstrated an oral arrival;108 surgeons today attempt every conceiv-
dose of 1 mg/kg for seven days produced a 28- able means to prevent its presence. Although sci-
percent reduction in wound area and a significant entific validation is absent, some wound-care
increase of tensile strength and hydroxyproline therapies applied by eclectic physicians are still
content of the wound.102 considered valuable and effective therapies today.
Topical treatment with Aloe vera has been Honey and sugar or sugar paste have been
shown to improve healing in frostbite and electri- used to treat wounds for decades. Both are con-
cal injury in animals.103,104 In addition, Aloe vera sidered to be antimicrobial and have been associ-
has improved the healing of wounds in both nor- ated with scarless healing in some cavity
mal and diabetic rats.105,106 Topical application and wounds.109 Hyaluronic acid consists of disaccha-
oral administration of Aloe vera to rats with heal- ride chains made from modifications of the
ing dermal wounds increased the collagen con- monosaccharide glucose. One possible mechanism
tent of the granulation tissue as well as the degree in scar prevention is that glucose in honey or de-
of cross-linkage. Collagen increased 93 percent rived from sugar may be converted into hyaluronic
with topical treatment and 67 percent with oral acid at the wound surface, forming an extracellu-
treatment compared to controls. The increase was lar matrix that promotes wound healing.109 Fetal
attributed to increased stimulation by Aloe vera wounds heal without scar formation and the ex-
of collagen synthesis or increased proliferation of tracellular matrix of fetal wounds is rich with hy-
fibroblast synthesis of collagen, or both.107 In a aluronic acid and lacks excessive collagen.109 The
similar study, the effects of oral and topical Aloe glucose in honey or derived from sugar may fa-
vera on full thickness dermal wounds in rats ex- cilitate a balance between hyaluronic acid and
hibited an increase in glycosaminoglycan compo- collagen, similar to that found in fetal wounds.
nents of the extracellular matrix and, in particu- Preparations of fresh juice from Calen-
lar, hyaluronic acid and dermatan sulphate lev- dula officinalis preserved in alcohol, known as
els.107 Calendula succus, are used topically to promote
Aloe vera and Centella asiatica have been wound healing. Naturopathic doctors utilize Cal-
widely used for a host of curative purposes in- endula succus to cleanse wounds after minor sur-
cluding facilitating wound repair. In spite of their gical procedures and throughout the healing pro-
wide use as folk remedies the biochemical basis cess. External Calendula succus is listed in The
for their action or influence on tissue repair is just Complete German Commission E Monographs for
beginning to be understood. Human clinical trials promoting wound healing. Topical application is
are needed to determine safety and benefits of thought to have anti-inflammatory and granulatory
perioperative oral administration of these botani- action.110
cals. Topical application of both Aloe vera and Knitbone and bruisewort are common
Centella asiatica extracts to healing wounds or names for Symphytum officinalis (comfrey) that
surgical scars appears to be safe and facilitates give clues to its traditional uses. The active ingre-
improved wound repair. dient in comfrey is thought to be allantoin, which
is reported to promote cell division and the growth
of connective tissue, bone, and cartilage. Com-
frey poultices are applied externally on intact skin

for bruises, sprains, and

fractures. Medical litera-
ture regarding comfrey is Table 3. Topical Wound Care
limited to its potential
liver toxicity when taken
internally. However, Topical preparation Action
many anecdotal reports
claim comfrey is ex- Aloe vera Increases collagen content and degree of
tremely effective at procollagen cross-linkage within the wound.
moting swift healing in
bruises, sprains, and frac- Centella asiatica Stimulates type-1 collagen production.
tures. External applica-
tion to intact skin does Honey or sugar paste Glucose converted into hyaluronic acid at
not appear to have the the wound surface forming an extracellular
same toxicity concerns as matrix that promotes wound healing; also
internal consumption. considered antimicrobial.
Table 3 summa-
rizes botanicals and other Calendula succus Anti-inflammatory and promotes
topical treatments for granulation.
wound healing.
Adequate tissue Symphytum officinale Promotes cell division and the growth of
perfusion, blood flow, bone, cartilage, and other connective
and oxygen levels are re- tissues; applied topically to closed wounds.
quired for wound heal-
ing. Tissue perfusion de-
livers oxygen and nutri-
ents to regenerating tissue. The synthesis of fibro- influences the biochemical processes necessary for
blasts and the enzymatic hydroxylation of proline the phases of normal healing to occur. Undernour-
and lysine residues on the forming collagen chains ished or malnourished individuals heal less effi-
are dependent, in part, on the availability of oxy- ciently and are at greater risk for complications
111
gen. Hydrotherapy utilizes external hot and cold during and after surgery. Part of treating the whole
applications of water to manipulate the quantity patient and not just the “hole in the patient” is
of blood flow through a given tissue. Adequate appreciating the complex interactions and the nu-
blood flow brings oxygen, nutrients, and red and trients involved in the wound-healing process. The
white blood cells to target tissues. This basic physi- relationship between malnutrition and poor wound
ological manipulation of blood flow can support healing is well documented,112-114 while the impact
the wound healing process. Hydrotherapy is an of optimal levels of dietary and supplemental nu-
inexpensive and powerful adjunct to wound care; trient intakes for wound healing is relatively un-
however, there are some limitations to applying known.
hydrotherapy to open wounds, burns, and in pa- Promotion of good nutrition is recom-
tients with peripheral neuropathies. mended, particularly in populations at risk for
marginal and frank nutritional deficiencies, includ-
ing the elderly,115 severely injured,116 smokers,117,118
Discussion patients with maldigestion or poor assimilation,3
Wound healing proceeds quickly and ef- and hospitalized patients119 before elective surgery.
ficiently in a physiologic environment conducive Evidence supporting supplementation of nutrients
to tissue regeneration and repair. Nutritional sta- known to benefit the healing process in healthy
tus of patients at the time of trauma or surgery

individuals is lacking. Several journal reviews cite 2. Stadelmann WK, Digenis AG, Tobin GR.
a high prevalence of complementary and alterna- Physiology and healing dynamics of chronic
cutaneous wounds. Am J Surg 1998;176:26S-
tive medicine (CAM) use by surgical patients.120-
126
38S.
The authors of these articles caution against
3. Stadelmann WK, Digenis AG, Tobin GR.
the use of CAM therapies because of potential Impediments to wound healing. Am J Surg
adverse reactions, the most common being poten- 1998;176:39S-47S.
tial vitamin, mineral, herb, or amino acid interac- 4. Blee TH, Cogbill TH, Lambert PJ. Hemor-
tions with platelet aggregation or anesthetics or rhage associated with vitamin C deficiency in
other pharmaceuticals given perioperatively.124-126 surgical patients. Surgery 2002;131:408-412.
The potential benefit of nutrients is seldom dis- 5. Petry JJ. Surgically significant nutritional
cussed. supplements. Plast Reconstr Surg
Evidence exists that vitamins A and C, 1996;97:233-240.
zinc, arginine, glutamine, glucosamine, bromelain, 6. Ehrlich HP, Hunt TK. Effects of cortisone and
Aloe vera, and Centella asiatica may be benefi- vitamin A on wound healing. Ann Surg
1968;167:324-328.
cial to wounded or surgical patients; however,
7. Hunt TK, Ehrlich HP, Garcia JA, Dunphy JE.
many patients will be advised to avoid them. More
Effect of vitamin A on reversing the inhibitory
extensive, well-defined, blinded clinical trials to effect of cortisone on healing of open wounds
evaluate the safety, efficacy, and drug interactions in animals and man. Ann Surg 1969;170:633-
of these potential beneficial substances are needed. 641.
From the current available data it would 8. Ehrlich HP, Tarver H, Hunt TK. Effects of
appear that an adequate protein supply, as well as vitamin A and glucocorticoids upon inflamma-
supplementation of 25,000 IU vitamin A, 1-2 g tion and collagen synthesis. Ann Surg
1973;177:222-227.
vitamin C, 15-30 mg zinc, 3-15 g arginine, 3-15 g
glutamine, and 1,500 mg glucosamine per day 9. Cohen BE, Gill G, Cullen PR, Morris PJ.
Reversal of postoperative immunosuppression
prior to and after surgery would benefit adult pain man by vitamin A. Surg Gynecol Obstet
tients. Wounded patients could also benefit from 1979;149:658-662.
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complete. Post-operative topical application of of vitamin A and beta carotene on intra-
Aloe vera and Centella asiatica extracts may fa- abdominal sepsis. Arch Surg 1984;119:161-
cilitate the creation of a flexible, fine scar with 165.
high tensile strength at the wound site. In addi- 11. Levenson SM, Gruber CA, Rettura G, et al.
tion, 750-1,000 mg bromelain post-operatively Supplemental vitamin A prevents the acute
radiation-induced defect in wound healing.
may reduce edema, bruising, pain, and healing
Ann Surg 1984;200:494-512.
12. Greenwald DP, Sharzer LA, Padawer J, et al.
Several eclectic wound therapies have survived Zone II flexor tendon repair: effects of
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Scientific research is needed to validate safety and 1990;49:98-102.
efficacy of these eclectic therapies. 13. Seifter E, Crowley LV, Rettura G, et al.
Influence of vitamin A on wound healing in
rats with femoral fracture. Ann Surg
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PERSPECTIVES
Background
OPINION
The view represented in most textbooks
regards pain and temperature as distinct, dis-
criminative, cutaneous sensations that are
How do you feel? Interoception: the relayed to somatosensory areas of the cortex
by the thalamic ventrobasal complex8,9 (FIG. 1).
sense of the physiological condition Visceral sensations are assigned to a system
that relays vagal, glossopharyngeal, facial and
of the body spinal afferent activity by way of the brain-
stem parabrachial nucleus to the same ventro-
basal complex, but then to the insular cortex
A. D. Craig — a fact that is used to explain why such
sensations are less distinct10,11.
As humans, we perceive feelings from our taste) and interoceptive (visceral) modalities, However, these concepts could not explain
bodies that relate our state of well-being, and he categorized temperature and pain as several important discrepancies. First, the
our energy and stress levels, our mood aspects of touch4. involvement of the somatosensory cortex is
and disposition. How do we have these Recent findings on the functional anatomy challenged by the fact that pain and tempera-
feelings? What neural processes do they of the lamina I spinothalamocortical system ture sensations remain after cortical lesions of
represent? Recent functional anatomical indicate that interoception should be rede- the postcentral gyrus. For this reason, Head
work has detailed an afferent neural fined as the sense of the physiological condi- and Holmes12 assigned awareness of pain sen-
system in primates and in humans that tion of the entire body, not just the viscera5–7. sation to the posterolateral thalamus and pre-
represents all aspects of the physiological This system is a homeostatic afferent pathway scribed a modulatory role for the cortex.
condition of the physical body. This system that conveys signals from small-diameter pri- Second, the association of pain with touch
constitutes a representation of ‘the mary afferents that represent the physiological implies the existence of convergent, modifi-
material me’, and might provide a status of all tissues of the body. It projects first able pain-and-touch cells (considered to be
foundation for subjective feelings, emotion to autonomic and homeostatic centres in the the so-called ‘wide dynamic range’ cells of the
and self-awareness. spinal cord and brainstem, thereby providing deep dorsal horn9,13,14), despite the obvious
the long-missing afferent complement of the inability of such modality-ambiguous cells to
The feelings we perceive from our bodies efferent autonomic nervous system. Together engender the distinct sensations elicited by dif-
include temperature, pain, itch, tickle, sen- with afferent activity that is relayed by the ferent stimuli (for example, sharp versus burn-
sual touch, muscular and visceral sensations, nucleus of the solitary tract (NTS), it generates ing pain). Third, although there is a clear need
vasomotor flush, hunger, thirst, AIR HUNGER a direct thalamocortical representation of the for an afferent pathway to elicit efferent auto-
and others related to the body’s state. Early state of the body in primates that is crucial for nomic adjustments to somatic events that pro-
German physiologists regarded such general temperature, pain, itch and other somatic feel- duce feelings from the body (for example, for
bodily feelings as Gemeingefühl, or ‘common ings. This anatomical organization shows that the ongoing homeostatic process of thermo-
sensation’, and distinguished them from the these feelings are highly resolved, sensory regulation15), the concept of a ‘sympathetic
five senses1. Sir Charles Sherrington concep- aspects of ongoing homeostasis that represent afferent’ system has often been dismissed10,11.
tualized a sense of ‘the material me’ by con- the physiological condition of the body itself The prevailing views also stumbled on the
sidering that all of these feelings are related — a distinct shift from the concept that pain fundamental fact, recognized long ago16, that,
and form a foundation for the sense of one’s and temperature are aspects of touch. in the central nervous system, pain, tempera-
physical self 2. William James presented simi- Converging evidence from functional imaging ture and itch sensations are distinguished
lar natural philosophical arguments and studies substantiate this interoceptive cortical from touch by their association with the
regarded the feelings from our bodies as the image and indicate that its re-representation spinothalamic projection, which ascends in
basis for self-awareness and emotion3. in the anterior insular cortex of the non- the contralateral spinal cord, rather than with
However, Sherrington later codified the dominant (right) hemisphere, possibly the dorsal column/medial lemniscal system in
senses into teloreceptive (vision and hear- uniquely in humans, constitutes a basis for the the ipsilateral spinal cord. Because of this
ing), proprioceptive (limb position), extero- subjective evaluation of one’s condition, that arrangement, spinal hemisection produces
ceptive (touch), chemoreceptive (smell and is,‘how you feel’. contralateral loss of pain and temperature
NATURE REVIEWS | NEUROSCIENCE VOLUME 3 | AUGUST 2002 | 6 5 5
© 2002 Nature Publishing Group

PERSPECTIVES
a Conventional pain pathway b Conventional visceroceptive pathway c Lamina I spinothalamocortical pathway

Somatosensory Gustatory cortex
cortex in anterior insula
and frontal
operculum
Ventroposterior Ventroposterior Ventromedial

lateral nucleus medial nucleus nucleus
(parvocellular part) (posterior part)
Parabrachial
nucleus
Gustatory
Solitary part
nuclear
Reticular complex General visceral
formation part
Homeostatic
VII control
regions
IX
Anterolateral X
system
Lateral
spinothalamic
tract
Figure 1 | Pain, visceroceptive and spinothalamocortical pathways. Comparison of organizational charts of a | the conventional view of pain pathways, b | the
conventional view of visceroceptive pathways and c | the lamina I spinothalamocortical pathway.
sensations, and ipsilateral loss of fine-touch is their inherent association with emotion its homeostatic role. Accordingly, this feeling is
perception (the Brown–Sequard syndrome). (but see the discussion below on sensual directly dependent on the body’s needs19,20. For
Phylogenetic arguments that defined so-called touch). These feelings all have not only a sen- example, compare the pleasant feeling of cool
‘palaeo-’ and ‘neo-’ spinothalamic systems sory, but also an affective, motivational aspect. water when your body is overheated with the
attempted to explain how pain and tempera- The emotional aspect of thermal sensation, in gnawing discomfort generated by the very
ture were part of the touch system at the thala- particular, is easily under-appreciated; we typi- same cool stimulus when you are ‘chilled to the
mocortical level, although separate at the cally project temperatures onto the object we bone’. Thermoregulation is a primal evolution-
spinal level17,18. However, the fundamental are touching or onto our environment, as ary requirement for all animals, particularly
commonality of pain, temperature and other Aristotle did. However, temperature is ulti- homeothermic mammals, and the affective
bodily sensations as interoceptive, rather than mately a physiological condition of the tissue aspects of such feelings correspond to the
exteroceptive, remained unrecognized. itself (and likewise for all bodily feelings), and motivations that are essential for behavioural
A key feature that distinguishes pain, tem- it generates an inseparable affect (pleasantness thermoregulation and homeostasis — that is,
perature and other bodily feelings from touch or unpleasantness) — a feeling — that signals survival21,22. Thermoreception is therefore
656 | AUGUST 2002 | VOLUME 3 www.nature.com/reviews/neuro

PERSPECTIVES
representative, because all of the feelings from horn is genetically coordinated to coincide project to the main homeostatic integration
the body are directly related to homeostatic with the arrival of lamina I neurons. The sites in the brainstem. The latter include
needs and associated with behavioural moti- lamina I neurons, which arise from the prog- regions that also receive vagal and glossopha-
vations that are crucial for the maintenance of enitors of autonomic interneurons in the lat- ryngeal afferent activity (by way of the solitary
body integrity, and their neural representation eral horn, migrate to their superficial dorsal nucleus) and are heavily interconnected with
reflects this homeostatic primacy. position during a ventromedial rotation of the hypothalamus and amygdala — that is, the
The ascending neural activity that repre- the entire dorsal horn, which occurs simulta- ventrolateral medulla, catecholaminergic cell
sents temperature and other physiological neously with the arrival of the small-diameter groups A1, A2 and A5–A7, the parabrachial
conditions of the body is conveyed by the afferents23. A common transcription factor nucleus and the periaqueductal grey. Lamina I
lamina I spinothalamocortical pathway (FIG. 1). (DRG11) is activated in the B cells and lam- terminations in the medulla coincide precisely
The physiological and anatomical characteris- ina I neurons at this time24. This rotation with the developmental border between the
tics of this system will first be reviewed, and places the small-diameter afferent fibres that
then the functional imaging and clinical enter by the lateral division of the dorsal root
Limbic sensory and motor cortex
evidence that documents the primary intero- in contact with lamina I neurons. This rota-
ceptive cortex will be summarized. Finally, the tion concurrently results in the characteristic
cortical re-representations of interoceptive recurrent trajectory of the large-diameter
state that provide the basis for subjective fibres, which do not connect with lamina I Hypothalamus
awareness of the feelings from the body neurons25. This course of development
will be described and related to concepts of supports the view that the small-diameter
self-awareness and consciousness. afferents and lamina I constitute a cohesive
homeostatic afferent system. Parabrachial PAG
Mesencephalic
The functional anatomy of lamina I The Aδ- and C-type primary afferent fibres
Lamina I, the most superficial layer of the that are relayed by lamina I relate homeostatic
spinal (and trigeminal) dorsal horn, is the information — that is, much more than sim- A1 + A2 RVLM + VMM
Medullary
only neural region that receives monosynap- ply ‘pain and temperature’ sensations — from
tic input from small-diameter (Aδ and C) all tissues. These fibres convey slow activity that
primary afferent fibres, which innervate is sensitive to changes in a wide variety of phys- Lamina I ANS
essentially all tissues of the body6. The adja- iological conditions — not only temperature Spinal
cent lamina II (substantia gelatinosa) was and mechanical stress, but also local metabo-
once described as the pain-processing region lism (acidic pH, hypoxia, hypercapnia, hypo-
Fine afferents Fine efferents
of the dorsal horn, but it contains only local glycaemia, hypo-osmolarity and lactic acid),
interneurons in cat and monkey, and it cell rupture (ATP and glutamate), cutaneous
essentially receives C-fibres only from skin. parasite penetration (histamine), mast cell acti- Figure 2 | Hierarchical organization of neural
(In rodents, lamina II is less distinct from vation (serotonin, bradykinin and eicosa- homeostasis involving the sympathetic
lamina I.) Lamina I is the source of output noids), and immune and hormonal activity nervous system. Small-diameter afferent fibres
from the superficial dorsal horn, contribut- (cytokines and somatostatin)6,7,26–30. This per- that report the physiological condition of all tissues
ing half of the spinal input to the brainstem spective emphasizes that the category ‘nocicep- of the body terminate in lamina I of the spinal and
and thalamus. It is usually associated with tors’, although heuristically of enormous value, trigeminal dorsal horns. The ascending projections
of lamina I neurons provide the bases for somato-
‘pain and temperature’ and, indeed, its pro- is actually a theoretical simplification, because autonomic reflex arcs at the spinal, medullary and
jections contain LABELLED LINES that specifically the empirical mechanical, thermal and poly- mesencephalic levels. At the spinal level, lamina I
subserve these sensations. However, the evi- modal thresholds of small-diameter afferents projects strongly to the sympathetic regions in the
dence indicates that these are simply particu- extend broadly across the ‘pain’ threshold in all intermediomedial and intermediolateral cell
lar aspects of interoception and that lamina I tissues, as expressed clearly by many investiga- columns of the thoracolumbar cord, where the
neurons fundamentally represent many tors31–34. Microneurographic data indicate sympathetic preganglionic cells of the autonomic
nervous system (ANS) originate. In the medulla,
aspects of the physiological condition of the that only the summated activation of ‘C-noci-
lamina I neurons project to the A1 and A2
tissues of the body. ceptors’ causes a conscious perception of ‘pain’ catecholaminergic cell groups and to other sites
The association of small-diameter affer- in humans35, and C-fibres often have slow (including the rostral ventrolateral medulla, RVLM),
ents and lamina I neurons begins with their ongoing discharge without provocation, which are interconnected with the pre-
development, which is temporally coordi- which is not perceived34,36,37 and is likely to be sympathetic neurons that project to spinal levels.
nated and distinguished from the large- related to current tissue metabolic status. In The A2 cell group (that is, the nucleus of the
solitary tract) also receives direct parasympathetic
diameter exteroceptive and proprioceptive addition, many cutaneous C-fibres are selec-
(vagal and glossopharyngeal) afferent input (not
afferents that project to the deep dorsal horn; tively and exquisitely sensitive to slow, weak shown), and A1 projects to the hypothalamus.
this developmental differentiation seems to mechanical stimuli that evoke sensual (‘lim- In the pons and mesencephalon, lamina I neurons
reflect a simple physiological distinction bic’) touch38, as are neurons in lamina I and in project to the parabrachial nucleus, the main
between the inside and outside of the body. the inner substantia gelatinosa39. brainstem homeostatic integration site, and to the
Whereas the large-diameter fibres originate The ascending projections of lamina I periaqueductal grey (PAG), the main homeostatic
from the first wave of large (A) dorsal root neurons, which have been detailed in cat and brainstem motor site. The hypothalamus (the main
autonomic control centre) and the limbic cortical
ganglion cells, the small-diameter afferents monkey, unambiguously indicate their role in regions project to all of these sites in the
originate from small (B) cells and enter the homeostasis6,40,41 (FIGS 1 and 2). First, they pro- brainstem, and to lamina I and the sympathetic
spinal cord in a second wave, subsequent to ject strongly to the sympathetic cell columns preganglionic regions in the spinal cord. VMM,
the larger fibres. Their arrival in the dorsal of the thoracolumbar spinal cord. Next, they ventromedial medulla.

PERSPECTIVES
Box 1 | The thermal-grill illusion of pain several distinct, modality-selective classes that
receive input from particular subsets of small-
Thunberg’s (1896) thermal grill evokes a diameter fibres. These classes of neurons serve
burning, ice-like pain sensation with interlaced as labelled lines, because they differ morpho-
innocuous warm (40 °C) and cool (20 °C) bars. logically, physiologically and biochemically,
A similar, albeit stronger, sensation is elicited and their activity corresponds to distinct sen-
by pouring warm water on feet that are numb sations47,48. Primarily on the basis of cutaneous
with cold (an unmistakable signal of stimulation (and the heuristic concept of
thermoregulatory distress), and only burning
nociception), we now recognize two classes of
pain is evoked by a cool stimulus (<24 °C)
neurons that signal sharp pain (FIRST PAIN) and
when A-fibre conduction (and normal cool
burning pain (SECOND PAIN) that selectively
sensation) is blocked by pressure on a
peripheral nerve. These demonstrations reveal
receive inputs from Aδ-nociceptors and poly-
two basic phenomena: the increasing modal C-nociceptors, respectively49,50. In addi-
activation of the polymodal C-nociceptive tion, there are two types of thermoreceptive
sensory channel by cold below a threshold of Warm Cold lamina I cell that respond selectively to cooling
~24 °C (the normal thermoneutral ambient or to warming, distinct types of chemorecep-
temperature), and the central inhibition of Burning tive cell that respond selectively to histamine
that activity by innocuous thermosensory pain or to noxious chemicals, and other classes that
activity (‘cold inhibits pain’). respond selectively to muscle or joint afferents
The burning pain sensation caused by HPC or to mechanical ‘slow brush’ (tickle)39,51–54.
polymodal C-nociceptors (HPC), which are A particularly convincing demonstration of
sensitive to noxious heat and pinch, as well as to cold, is normally masked centrally by the the selectivity inherent in this pathway is pro-
activity of the specific cutaneous Aδ-fibre thermoreceptors that are responsible for the sensation vided by the histamine-responsive cells that
of cooling. When the activity of cooling receptors is reduced, the polymodal nociceptive activity we recently identified, which constitute a
that is evoked by cooling is disinhibited centrally and causes a burning sensation at cool (<24 °C) specific pathway for the sensation of itch55.
temperatures that is normally felt only at noxious cold (<15 °C) temperatures. Further types remain to be documented more
The thermal-grill illusion can be explained physiologically by an unmasking of the cold- clearly. For example, although most viscero-
evoked activity of polymodal nociceptive lamina I spinothalamic neurons (which are selectively ceptive lamina I cells reported so far have con-
activated by polymodal C-nociceptors) due to the reduction of the normal cold-evoked activity vergent cutaneous input54,56,57, such cells might
of thermoreceptive lamina I spinothalamic neurons (which are selectively activated by Aδ-type
have been sensitized by the requisite surgical
cooling thermoreceptors) by spatial summation of the simultaneous warm stimuli in the
preparation and repeated visceral search stim-
thermoreceptive but not the nociceptive neurons 62. The grill effectively produces a relative
ulation, and distinct types of visceroceptive
balance of polymodal and thermosensory activity similar to that caused by a noxious cold
stimulus of ~10 °C; the equivalence of these sensations has been verified psychophysically.
cell probably exist that even distinguish renal
Reduction of activity in the thermoreceptive sensory channel disinhibits or unmasks the artery from renal vein occlusion by respond-
polymodal nociceptive channel at thalamocortical levels, producing a burning pain sensation. ing selectively to renal osmoreceptors or
Functional imaging has confirmed that the thermal grill produces a pattern of activity in the mechanoreceptors58, similar to the selectively
cortex that is identical to the activation produced by noxious cold63. The cortical activation metaboreceptive muscle-responsive lamina I
unmasked by the grill is in the anterior cingulate cortex (ACC), indicating that ACC activation is neurons that we recently identified (see
selectively associated with the perception of thermal pain — that is, the affect that signals below)59. In stark contrast to these neurons,
thermoregulatory distress. So, the thermosensory activity in the lamina I spinothalamocortical the cells in the deep dorsal horn provide a
pathway to interoceptive cortex unmasks activity in the medial lamina I pathway, which activates modality-ambiguous integration of all affer-
the ACC and is associated with burning pain. This indicates that the medial thalamic lamina I ent inputs, including mechanoreceptive,
spinothalamic tract projection to the mediodorsal thalamic nucleus (ventral caudal part, MDvc) proprioceptive and nociceptive activity9,60,61.
might be the crucial site for the inhibition of thermal pain by cold; this suggestion is supported by The thermoreceptive lamina I cells that are
preliminary physiological recordings from the MDvc in our laboratory. sensitive to cooling respond predominantly to
Aδ-fibre input and have ongoing discharge at
normal skin temperature that is inhibited by
alar and basal plates that defines the classic example, respiration is linearly modulated by warming. They uniquely evince a linearly
‘general visceral afferent’ integration region42, graded changes in innocuous thermosensory increasing response to skin temperatures
and throughout the brainstem they co-localize activity45. Completing this anatomical circuitry, below a neutral skin temperature (~34 °C)
with the catecholaminergic cell columns that lamina I receives descending modulation that corresponds directly with human psycho-
identify homeostatic regions. These spinal and directly from brainstem pre-autonomic physics48. Their activity reaches a plateau at the
bulbar lamina I projections substantialize (pro- sources (A5, A7 and raphe nuclei). Most strik- cold temperatures that we perceive as noxious
vide the anatomical substrate for) the somato- ingly, the only spinal regions that receive (<15 °C); at these temperatures, the polymodal
autonomic reflexes that are activated by small- descending controls directly from the hypo- nociceptive cells that signal burning (second)
diameter afferents (including visceroceptive thalamic paraventricular nucleus — the main pain are increasingly active. Analysis of the
activity) and are crucial for cardiorespiratory diencephalic autonomic control centre — are thermal-grill illusion of pain (BOX 1) indicates
and other homeostatic functions43,44. the autonomic motor nuclei and lamina I46. that the perception of thermal distress and
Importantly, these reflex pathways are not sim- Lamina I neurons relate many aspects of cold pain depends on the comparison of these
ply emergency mechanisms; rather, they pro- the ongoing physiological status of the tissues two sensory channels in the forebrain62,63, as
vide continuous homeostatic feedback. For of the body. In cat and monkey, they comprise well as on core temperature20. Notably, the

PERSPECTIVES
polymodal nociceptive channel responds to dense clusters of large, glutamatergic lamina I Right orbitofrontal
cold with accelerating activity below ~24 °C — boutons that are organized topographically
corresponding to our increasing thermoregu- (in the rostrocaudal direction) and associated
latory distress below a thermoneutral ambient with TRIADIC ARRANGEMENTS of GABA (γ-amino-
Left anterior insula Right anterior insula
temperature — and, when Aδ-type thermo- butyric acid)-containing presynaptic den-
receptive afferent activity is reduced by a nerve drites and proximal relay cell dendrites in
block or by the thermal grill, the activity in cytoarchitectonically distinguishable cell
Anterior insula
this channel elicits a burning-pain sensation nests70,72. The VMpo is contiguous rostrally
at such temperatures48,62,64. This emphasizes with the basal part of the ventromedial
that pain is not a binary (yes or no) modality, nucleus, or VMb (which, for historical reasons,
and compels the conceptual shift of viewing is denoted by some as the parvicellular part of Mid/posterior dorsal insula
the role of polymodal C-nociceptors and the the ventroposterior medial nucleus, or VPMpc,
lamina I neurons that convey their activity as even though it does not project to the somato-
a homeostatic afferent pathway rather than sensory cortex as the remainder of the VPM VMpo VMb
simply a ‘nociceptive’ pathway. does) and, in primates, VMb receives a direct
To directly address the role of lamina I in (bilateral) input from the nucleus of the
homeostasis, we recently examined the solitary tract, which conveys vagal and glosso-
Parabrachial
responses of lamina I neurons to muscle pharyngeal (general visceral and gustatory)
contraction. We identified a class of lamina I afferent activity73. Therefore, these two thala-
spinobulbar neurons that respond selectively mic nuclei together represent all homeostatic
during and after muscle contraction. These afferent inflow (both sympathetic and para- Lamina I NTS
neurons provide a substrate for the EXERCISE sympathetic) from the body, and they form a
59
PRESSOR REFLEX . Some of the muscle Aδ- and separate, rostrocaudally organized column
C-fibres that are represented by these lamina I that is orthogonal to the mediolateral align-
cells are sensitive to contraction; others are ment of the exteroceptive and proprioceptive Fine Fine
sympathetic parasympathetic
sensitive to lactic acid and other metabolites representations in the ventroposterior nuclei, afferents afferents
released during muscular exercise, and can be to which they are connected at the representa-
viewed as metaboreceptors (or ERGORECEPTORS) tion of the mouth. The direct lamina I path-
that continuously drive a variety of regional way to VMpo and the direct pathway from
and whole-body homeostatic adjustments to the nucleus of the solitary tract to VMb are Figure 3 | The organizational chart for
muscular work36,65–67. It is important to rec- distinguishable only in primates; essentially, interoception. Small-diameter afferents that
innervate tissues in parallel with sympathetic
ognize that large increases in such activity homeostatic afferent input in sub-primates
efferents (‘sympathetic afferents’) provide input to
can cause the familiar aching or burning sen- reaches the forebrain after integration in the lamina I; small-diameter afferents that innervate
sation from muscles, and synchronous acti- brainstem parabrachial nucleus10 and by direct tissues in parallel with parasympathetic efferents
vation causes a painful cramping sensation68, spinohypothalamic projections (in rats)74. (‘parasympathetic afferents’) provide input to the
but the activity elicited continuously by mus- Nociceptive and thermoreceptive neurons nucleus of the solitary tract (NTS). In mammals,
cle contraction normally produces homeo- with properties similar to lamina I neurons such activity is integrated in the parabrachial
nucleus, which projects to insular cortex by way of
static adjustments without the perception of have been identified in VMpo in macaque and
the ventromedial thalamic nucleus (VMb, also
a behaviourally motivating signal. This class owl monkeys70,75, and such neurons have also known as VPMpc). In primates, however, a direct
of neurons clearly confirms the role of lam- been recorded in the region of VMpo in awake projection from lamina I to the ventromedial
ina I in ongoing homeostasis, and substanti- humans76,77. These different modalities are nucleus (VMpo), and a direct projection from the
ates the concept that it relates the current segregated within VMpo. Significantly, micro- NTS to the VMb, provide a rostrocaudally
physiological condition of all the tissues of stimulation in this region of the thalamus in contiguous column that represents all
contralateral homeostatic afferent input and
the body. awake humans elicits discrete pain or cooling
projects topographically to the mid/posterior
or visceral sensations77–80. Anatomical tracing dorsal insula. In humans, this cortical image is
Lamina I spinothalamocortical pathway in monkeys indicates that VMpo and VMb re-represented in the anterior insula on the same
Anterolateral cordotomy in humans interrupts project in a rostrocaudally topographic fash- side of the brain. The parasympathetic activity is
contralateral temperature, pain, itch, sensual ion to a cytoarchitectonically distinct field in then re-represented in the left (dominant)
touch and visceral sensations, if made at the the fundus of the superior limiting sulcus at hemisphere, whereas the sympathetic activity is
location of ascending lamina I axons in the the dorsal margin of insular cortex, with the re-represented in the right (non-dominant)
hemisphere. These re-representations provide the
lateral spinothalamic tract 6,69. In primates, VMb projection field extending rostrally from
foundation for a subjective evaluation of
lamina I spinothalamic neurons project to a the VMpo projection to include the previously interoceptive state, which is forwarded to the
dedicated thalamocortical relay nucleus in recognized gustatory cortex6,81,82. A smaller, orbitofrontal cortex, where hedonic valence is
the posterolateral thalamus — the posterior ancillary VMpo projection terminates in area represented in mammals.
part of the ventromedial nucleus, or VMpo70 3a in the fundus of the central sulcus. These
(FIGS 1 and 3). The VMpo is small in the anatomical and functional findings directly
macaque monkey thalamus, and it is only pri- contradict the conventional view of pain pro- Functional imaging with PET (positron
mordially represented in sub-primates, but it cessing and the view of some investigators that emission tomography), SPECT (single-
is proportionately very large in the human nociceptive activity simply passes through the photon emission computed tomography) and
thalamus71. It has appropriate anatomical posterior thalamus to VPM and VPL and fMRI (functional magnetic resonance imag-
characteristics for a thalamic relay nucleus: thence to the somatosensory cortex9. ing) verify that the anatomically identified

PERSPECTIVES
a Graded cooling b C-fibre touch c Thermal pain d Chronic pain
e Dynamic exercise f Respiration/isometric exercise g Graded itch h Cold allodynia
Figure 4 | Activation of the interoceptive cortex in the dorsal posterior insula by various modalities. a | Graded cooling (reproduced, with permission, from
REF. 5 © 2000 Macmillan Magazines Ltd). b | Sensual (limbic) touch evoked by slow brushing in a polyneuropathy patient with only C-mechanoreceptor activation
(reproduced, with permission, from REF. 101 © 2002 Macmillan Magazines Ltd). c | Thermal pain (reproduced, with permission, from REF. 91 © 2002 Elsevier Science).
d | Chronic pain (reproduced, with permission, from REF. 92 © 2001 International Association for the Study of Pain). e | Dynamic exercise on a bicycle (reproduced,
with permission, from REF. 96 © 1999 The American Physiological Society). f | Forced respiration and isometric exercise with a hand grip (reproduced, with
permission, from REF. 95 © 1999 John Wiley & Sons). g | Graded itch elicited by cutaneous histamine injection (reproduced, with permission, from REF. 94 © 2001
International Association for the Study of Pain). h | Cold allodynia (reproduced, with permission, from REF. 93 © 2000 International Association for the Study of Pain).
VMpo/VMb projection field in the dorsal cortical field. So, tactile stimulation of the (hypertonic saline injection), hunger and air
insular cortex is activated by temperature, hand activates a portion of S2 close to the lip hunger95,97–99. Consistent with the anatomical
pain and numerous interoceptive modalities of the lateral sulcus, more than 16 mm lateral tracing studies, gustatory stimuli activate the
that cause feelings from the body, consistent to the site in the dorsal posterior insula that is dorsal insular cortex more rostrally100.
with the functional anatomical data on activated by cooling the hand. Activation of this interoceptive cortex was
lamina I (FIG. 4). This site is distinct from the This same region in the dorsal posterior also observed using light, slow brushing in a
parietal somatosensory cortices, supporting insula (albeit slightly more rostrally) shows patient with polyneuropathy who has only
the idea of a neural distinction of intero- (graded) activity in every imaging study that peripheral nerve C-fibre conduction, consis-
ception from exteroception. Our PET study uses noxious heat applied to the hand87–91. It tent with the essential role of lamina I and
using graded innocuous cooling of the hand shows ongoing activity in chronic pain the lateral spinothalamic tract for sensual
showed correlated activity only at this site in patients92, as well as evoked activity in (limbic) touch101,102.
the fundus of the superior limiting sulcus of NEUROPATHIC PAIN patients, in which pain can be So, the evidence indicates that, in primates,
the posterior insula5. This site coincides with caused by normally innocuous stimuli93. (In the dorsal insular cortex contains a sensory
the location of cortical lesions that produce anaesthetized monkeys, noxious cold stimuli representation of the small-diameter afferent
thermanaesthesia and hypalgesia83,84, and it produce fMRI activation at precisely the sites activity that relates to the physiological condi-
verifies that the terminus of the lamina I identified in our anatomical tracing studies6.) tion of the entire body. This cortical region
spinothalamocortical pathway is the same in Furthermore, this same dorsal posterior insu- seems to constitute a primary interoceptive
humans as in monkeys. In human atlases, this lar site shows graded activity that is correlated image of homeostatic afferents. Embedded in
site has been included with the parietal oper- with itch sensation in humans94. This intero- the interoceptive cortex are the cortical repre-
culum as the second somatosensory region ceptive site is activated by both isometric and sentations of several highly resolved, distinct
(S2), on the basis of comparative anatomical dynamic exercise95,96, like the lamina I neurons sensations, including temperature, pain, itch,
tracing work that was carried out before the that respond selectively during and after mus- muscular and visceral sensations, sensual
recognition of VMpo85. However, this site is cle contraction59. It is activated by a range of touch and other feelings from the body. The
distinct from S2. The S2 has a topography modalities associated with visceral sensations, importance of the interoceptive cortex for
that is aligned from lateral to medial (face to such as the Valsalva manoeuvre, manipula- physical well-being is underscored by its clear
foot 86), which is orthogonal to the rostro- tions of blood pressure (maximal inspiration; delimitation in the monkey by in situ hybridiza-
caudal topography in the interoceptive VMpo lactate or cholecystokinin injection), thirst tion labelling for the mRNA of receptors for

PERSPECTIVES
corticotropin-releasing factor, which is Box 2 | Central pain syndrome

thought by many to be a definitive indicator of
an association with homeostatic stress103. Damage to the brain can cause the
Notably, the absence of this direct intero- central pain syndrome, in which Area 3a Area 24c
ceptive representation in sub-primates implies ongoing, intractable pain is referred to

that they cannot experience feelings from the contralateral deep and cutaneous
body in the same way that humans do. In tissues, where there is always a loss of
humans, lesions of the dorsal insula interrupt thermal (cool and warm) sensation
and often a paradoxical loss of acute Dorsal
these feelings83,84, disrupt homeostatic process- posterior
pain (pinprick and heat)
ing104 and cause permanent loss of discrimina- insula
sensation6,158. Central pain is
tive thermal sensation. By contrast, in cats
characteristically described as
that have been trained on a high-resolution burning and hyperpathic
thermosensory discrimination task, lesions of (augmenting), and is often Ventromedial Mediodorsal
the rudimentary thalamocortical lamina I accompanied by mechanical or nucleus nucleus
relay cause only a weak, transient behavioural thermal (cold) allodynia — that is, (posterior part) (ventral
caudal
deficit, whereas a spinal lesion of the ascend- pain is evoked by normally innocuous part)
ing lamina I axons causes permanent disrup- stimuli. It apparently results from
tion105. These findings indicate that behaviour damage to the lamina I
in sub-primates reflects motivational signals spinothalamocortical pathway at any
from homeostatic integration centres in the level, including interoceptive cortex,
brainstem and hypothalamus, and they reveal but it is not produced by lesions of the
that homeostatic integration in the brain- exteroceptive lemniscal pathways,
–
stem parabrachial nucleus is sufficient to the parietal somatosensory cortices Periaqueductal
or the medial thalamus. It occurs in grey
motivate thermoregulatory behaviour. Indeed,
~5% of stroke patients and 25–40% –
in humans, a lesion of the lamina I spino- Parabrachial
thalamocortical pathway that eliminates ther- of patients with multiple sclerosis nucleus
mal (and evoked pain) sensation can result in or spinal cord injury. Tricyclic
the central pain syndrome (BOX 2), which antidepressants are efficacious for
probably reflects a similar motivational signal about half of patients with central
pain, but opiates are usually
to forebrain behavioural mechanisms by
ineffective. Head and Holmes12 first
brainstem homeostatic drives that are released
postulated that this is a release
from descending insular cortical modula-
phenomenon that results from loss of
tion5,6,106. The occurrence of central pain after an inhibitory effect of discriminative
lesions that disrupt evoked pain sensibility was pain processing on the emotional
a key argument made against the existence of aspect of pain, and others suggested
a specific neural representation of pain13; how- release of phylogenetically old
ever, the available evidence indicates that cen- brainstem inputs or hyperactive
tral pain can be regarded as thermoregulatory bursting in somatosensory pathways.
distress, consistent with the recognition that A recent proposal, the thermosensory-
pain is both an aspect of interoception and a inhibition hypothesis, is based on the Lateral
behavioural drive caused by a physiological spinothalamic
anatomy of the lamina I projection tract
imbalance that homeostatic systems alone system. This hypothesis posits that
cannot rectify. central pain results from the loss of
So, like other feelings from the body, pain descending controls from interoceptive cortex on brainstem homeostatic sites that drive
normally consists of both a sensation and a thermoregulatory behaviour by way of the medial thalamus and the anterior cingulate cortex
motivation. The cortical region associated (see figure; minus symbols denote inhibition). The disinhibition proposed by this hypothesis is
with such motivation seems to be the anterior conceptually similar to the unmasking shown by the thermal-grill illusion (BOX 1). This proposal
cingulate cortex (ACC). The insula has long views central pain as a thermoregulatory dysfunction, and it emphasizes the concept that pain is
been regarded as limbic sensory cortex, not only a feeling, but also a behavioural drive that signals a homeostatic imbalance.
because of its association with visceral sensa-
tion by Penfield’s stimulation studies in studies of pain89,90,114. The ACC is activated the parabrachial nucleus and other brainstem
humans, whereas the ACC can be regarded as selectively during the thermal-grill illusion of homeostatic sites seems likely116. Like the lam-
limbic motor cortex, because of its association pain, and therefore signifies the urgency of ina I pathway to VMpo, this direct medial
with autonomic and emotional control107,108. thermal distress63; similarly, the unpleasant- pathway to the ACC does not exist in sub-
Both are strongly interconnected with the ness of thermal pain has been directly corre- primates, in which a phylogenetically earlier
amygdala, hypothalamus, orbitofrontal cortex lated with ACC activation using hypnotic lamina I projection is relayed by the thalamic
and brainstem homeostatic regions109,110. manipulation115. In primates, the underlying submedial nucleus to the orbitofrontal cortex6.
Many recent functional imaging studies have pathway seems to be an ancillary lamina I Although the underlying anatomy differs,
documented the role of the ACC in behav- spinothalamocortical route through the ven- data from studies in rats support a primordial
ioural drive and volition111–113. A particular tral caudal portion of the medial dorsal role of the ACC in behavioural motivation
portion of the ACC is activated in virtually all nucleus6, where convergence with input from by homeostatic distress, probably due to

PERSPECTIVES
a Anger b Subjective coolness c Graded disgust in faces d Trustworthiness in faces
in
mb
e Sexual arousal
ac
S2 S2
Figure 5 | Activation of the right (non-dominant) anterior insular cortex associated with different subjective feelings. a | Recall-induced anger, in which
activation is also visible in the right orbitofrontal, anterior cingulate (ac) and interoceptive (S2) cortices (reproduced from REF. 123 © 2000 Macmillan Magazines Ltd).
in, insular cortex; mb, mammillary bodies. b | Regression analysis of subjective ratings of the intensity of cooling the hand (reproduced from REF. 5 © 2000
Macmillan Magazines Ltd). c | Activation after exposure to graded disgust in computer-generated faces (reproduced from Nature (REF. 124) © 1997 Macmillan
Magazines Ltd). d | Activation elicited by subjective ratings of trustworthiness in faces (reproduced from REF. 129 © 2002 Macmillan Magazines Ltd). e | Activation
elicited by sexual arousal (reproduced, with permission, from REF. 128 © 1999 Kluwer Academic Publishers).
parabrachial and other brainstem inputs117,118. direct evidence that the underlying anatomi- anger, anticipatory anxiety and pain, panic,
So, activation of the ACC is associated with cal pathway involves first the contralateral disgust, sexual arousal, trustworthiness and
motivation, and activation of the insula is anterior insula, which must contain an ini- even subjective responses (positive or nega-
associated with feeling, which together form tial re-representation of interoceptive cortex tive) to music121–127 (FIG. 5). For example, anger
an emotion. on the same side, and then, by way of a cal- was associated with activation in the right
losal pathway, a lateralized, second-order anterior insula and the orbitofrontal cortex, as
Subjective awareness of feelings re-representation on the right side that is well as in the interoceptive dorsal posterior
How do we perceive the feelings from our subsequently forwarded to orbitofrontal insular region123 (albeit labelled ‘S2’).
bodies? We examined this question in our cortex. Recent fMRI data indicate precisely Activation correlated with the implicit level of
PET study of innocuous thermal sensibility5, the same progression of insular activity for the disgust elicited by computer-generated facial
in which human volunteers provided a sub- subjective appreciation of pain91. Sequential expressions was shown in the anterior insular
jective magnitude rating of the perceived cortical processing occurs in all sensory sys- and orbitofrontal cortices on the right side124,
intensity of each stimulus (irrespective of tems, and it seems reasonable to conclude even though this emotion is usually regarded
emotional valence). Their ratings were not that evolutionary development of such a as a derivative of gustation. Sexual arousal
perfectly correlated with stimulus tempera- progression for interoception would be elicited by erotic films (measured by testos-
ture, and regression analysis of these per- advantageous for survival by differentiating terone release and intumescence) was
cepts showed strongly correlated activity in and refining the representation of internal strongly correlated with right anterior insular
the right (ipsilateral, non-dominant) anterior feelings. Analogous re-representation and activation128. Subjective assessment of the
insula (FIG. 5) and the orbitofrontal cortex, lateralization of gustatory processing in the trustworthiness of faces also activated right
which differed significantly from the linear left anterior insular and orbitofrontal cor- anterior insula129, and such activation has
representation of stimulus temperature in tices in humans has been proposed on the been explicitly interpreted as representing the
the interoceptive cortex. This revealed that basis of clinical findings119, and corroborated subjective feeling (or ‘gut reaction’) elicited by
subjective evaluation of the thermal stimuli by functional imaging120. each face130.
— the feeling elicited in each person by the These observations match a wide variety These convergent findings are consistent
graded coolness — depended on post- of functional imaging studies that have asso- with the concept that the re-representation of
processing in those cortical regions. Close ciated activation of right anterior insular and the cortical image of the interoceptive condi-
inspection of the correlation maps (see orbitofrontal cortices with subjective emotion of the body serves as a limbic sensory
figure 2 in REF. 5, levels 20–24 mm) provided tion, such as recall-generated sadness or substrate for subjective feelings and emotions.

PERSPECTIVES
This fits the idea that the evaluation of the thermal stimulus, or any other bodily feeling, than a basic sensory awareness of the environ-
state of the body in response to a pertinent according to the body’s homeostatic needs. In ment. It is required for extended autobio-
stimulus serves as the basis for emotional feel- fact, the orbitofrontal cortex guides feeding graphical and cognitive continuity, because it
ings — the James–Lange theory of emotion3. behaviour and behavioural valence in the rat engenders the fundamental image of the
In particular, these data are consistent with as well; it is not a phylogenetically recent physical self as a feeling (sentient) entity.
the neurological hypothesis, based on analy- development. Anatomical data in the
ses of clinical cases and imaging data, that the macaque monkey indicate that this region Conclusions and future directions
right (non-dominant) anterior insula is inte- receives convergent input from several sen- The identification of an entire neural system
gral for the generation of the mental image of sory systems, and it seems to receive a direct that can be cogently conceptualized as a rep-
one’s physical state, which underlies basic projection from gustatory and interoceptive resentation of the physiological condition of
emotional states and is required for the moti- insular cortex133. In other words, whereas the the material body has several fundamental
vation to make rational decisions that affect macaque monkey has an anatomically visible implications. It provides a rational explana-
survival and quality of life — the essence of interoceptive thalamocortical pathway, it has tion for the long-recognized association of
the ‘somatic marker’ hypothesis of conscious- direct interoceptive cortical projections to pain, temperature, itch and other feelings
ness131 (see also REFS 99,132) (BOX 3). Indeed, orbitofrontal cortex, and therefore might not from the body, separate from the lemniscal
Damasio explicitly suggested that a re-map- have the intervening re-representations of system that represents exteroceptive touch
ping of the representation of the state of the the subjective state of the body in the right and proprioception. It incorporates specific
body (interoceptive sensation) could provide anterior insular cortex that are clearly present labelled lines for several physical conditions
an ‘as if ’ circuit that would allow the brain to in humans. that generate distinct feelings, and it substan-
judge and predict the effects of emotionally These functional anatomical considera- tiates their common integration in the hier-
relevant stimuli on the body, which is what tions indicate clearly that primates differ from archical homeostatic network. It provides the
these functional anatomical data seem to sub-primates in the encephalization of a long-missing peripheral and central afferent
indicate. So, a refined image of the state of the direct cortical image of the physiological con- complement to the efferent autonomic ner-
body seems to provide the basis for awareness dition of the body. Furthermore, they strongly vous system. These findings reveal a direct
of the physical self across time. suggest that humans differ from monkeys not cortical image of the state of the body that
Nonetheless, it is equally important to rec- only in the relative size of the interoceptive differentiates primates from sub-primates
ognize that the ACC (as well as subcortical thalamocortical pathway, but also in the neuroanatomically. The size and multiple
structures, such as the amygdala and ventral development of sequential re-representations re-representations of this interoceptive image
striatum133–136) is co-activated with the ante- of the physiological state of the body in the seem to differentiate humans from sub-
rior insula in virtually all imaging studies of right anterior insula. The interoceptive re- human primates. Finally, these findings
emotion111,112,121,123,127,137, consistent with the representation that is lateralized in the right signify the cortical representation of feelings
view that an emotion is both a feeling and a anterior insula of humans corresponds with from the body as the likely basis for human
motivation. Notably, this provides the active the ability to perceive the self as a physical and awareness of the physical self as a feeling
agent that is missing from the somatic- separate entity — that is, self-awareness. The entity. This association provides a funda-
marker hypothesis, in which Damasio conjec- functional imaging data strongly support the mental framework for the involvement of
tured that the subjective ‘I’ is generated only integral role of the right anterior insula in these feelings with emotion, mood, motiva-
as an illusory by-product of the re-mapping. the feelings we perceive that are the basis of tion and consciousness. These concepts
For example, a recent imaging study of our perceptions of our selves, and therefore emerge directly from the functional anatomy
placebo analgesia revealed concomitant acti- of consciousness. As Damasio131 has elegantly of the lamina I spinothalamocortical system,
vation of both the ACC and the right anterior described, this level of consciousness is more rather than from preconceived ideas.
insula138; according to the present interpreta-
tion, this reflects the active modulation by the
behaviourally motivating agent (limbic motor Box 3 | The ‘somatic marker’ hypothesis of consciousness
cortex, the ACC) of the feeling represented by On the basis of neurological analyses of patients with forebrain lesions, Antonio Damasio123,131
the image of the expected state of the body (in has advanced the ‘somatic marker’ hypothesis of consciousness. He proposes that the subjective
the right anterior insula). This interpretation process of feeling emotions requires the participation of brain regions that are involved in the
is consistent both with the interoceptive lam- mapping and/or regulation of our continuously changing internal states — that is, in
ina I spinothalamocortical projections and homeostasis. These feelings help to guide behavioural decisions that affect survival and quality
with the overall anatomical organization of of life by producing a ‘perceptual landscape’ that represents the emotional significance of a
primate frontal cortex into medial (motor) particular stimulus that is being experienced, or of a projected future action by means of a
and orbital (sensory) networks139,140. further ‘as-if-body loop’ mechanism. The feelings are grounded in the body itself, based on
The orbitofrontal cortex, which correlated multi-tiered and evolutionarily developed neural mechanisms that control the body’s state.
most strongly with subjective thermal per- These feelings distinguish between inner-world representations and outer-world representations,
ception5, is associated with the discrimina- and allow the brain to build a meta-representational model of the relationship between outer
tion of positive and negative rewards (pleas- and inner entities159. So, the representational image of the body’s state provides a neural basis for
antness/unpleasantness, or hedonic valence, distinguishing self from non-self, and re-representations of this image enable the behavioural
corresponding to approach/avoidance encod- neural agent to project the effects of possible actions onto the state of the body, as well as the
ing) in relation to the body’s needs136,141,142. For resultant changes in such feeling states due to interactions with other (external) agents. This
hypothesis posits that degrees of conscious awareness are related to successive upgrades in the
example, this region engenders stimulus-spe-
self-representational maps159. The anatomical features of Damasio’s hypothesis include a central
cific satiety to food143, so it would probably
role for the anterior insular cortex in the representation of such feeling states.
also differentiate the affect associated with a

PERSPECTIVES
Glossary In contrast to the many discriminable sen- stress — including a direct cortical image of
sations from the body, the subjective apprecia- physical well-being — provides a sound epis-
AIR HUNGER
Hypercapnia with mechanically restricted ventilation.
tion of visceral sensation is more diffuse, less temological foundation for integrated
well localized, and usually below perceptive approaches to the treatment of pain, meta-
ERGORECEPTION thresholds. This was one of the main reasons bolic, eating and psychosomatic disorders. For
Afferent activity relating tissue energy and for the long-standing mis-categorization of example, this provides an easy formulation for
metabolic needs.
pain and temperature as exteroceptive rather somatization under emotional stress. Similarly,
EXERCISE PRESSOR REFLEX
than interoceptive.Although it would be highly these considerations imply that mysterious
Increased blood pressure and heart rate caused by inefficient for gastrointestinal processing to pain syndromes, such as fibromyalgia (deep
activity in small-diameter afferents from muscle. require constant behavioural supervision, this aches and pains), could be related to homeo-
perceptual difference remains to be explained static dysfunction (for example, salt or water
FIRST PAIN
Sharp, pricking pain associated with rapidly
adequately11. Notably, many observations indi- balance or cardiovascular function), rather
conducting Aδ-fibres. cate that there is opponent processing between than to tissue damage, and this possibility
parasympathetic and sympathetic afferents deserves vigorous study. Consideration of these
LABELLED LINES that parallels their efferent opponency. findings led directly to the recent proposal that
Anatomically and physiologically distinct neurons that
For example, there are obligatory mutual the central pain syndrome is a thermo-
are specifically associated with particular sensations.
inhibitory interactions between spinal and regulatory disorder5,106. The recognition that
NEUROPATHIC PAIN vagal small-diameter afferent activities in the sensual touch is incorporated into the intero-
Intractable pain associated with damage to the medulla that are essential for cardiorespiratory ceptive system has strong implications for the
peripheral or central nervous system. control149,150. Similarly, vagal afferent activation neurobiological and health effects of con-
SECOND PAIN
inhibits both pain sensation151 and spinal specific contact — visitors to zoos will remem-
Dull, burning pain associated with slowly conducting visceroceptive processing152.Vagal stimulation ber that monkeys, chimpanzees and bonobos
C-fibres. can reportedly reduce stress and depression normally spend an enormous amount of
clinically. Similarly, opposing effects on auto- time grooming and cuddling each other, and
TRIADIC ARRANGEMENT
nomic function have been elicited by stimula- readers will remember the classic studies by
Ultrastructural contacts between an afferent terminal,
a relay cell dendrite and a GABA-containing tion of human insular cortex on both sides153, H. Harlow showing the importance of con-
presynaptic dendrite that is characteristic of high- and corresponding cortical lateralization has specific contact for emotional development.
fidelity transmission in sensory relay nuclei. been observed with micturition154 and gusta- Last, the observation that the neuroanatomical
tion120. Such a basic organization would be substrate for subjective emotion in humans is
It is important to recognize that this neural parsimonious with many considerations and based on an abstracted meta-representation
sensory system is part of an entire network could explain the perceptual differences, but of the physiological state of the body, consis-
involved in homeostasis; that is, in the auto- this certainly needs further study, particularly tent with the conjectures of James3 and
nomic, hormonal and behavioural neural because of the potential clinical significance155. Damasio131, provides a basis for the volitional
mechanisms that maintain optimal physiologi- The association of the re-representation of modulation of feelings, emotion and efferent
cal conditions in the body and that respond in the interoceptive pathway with self-awareness activity affecting the state of the body that is
an integrated and ongoing fashion to all inte- implies the existence of neuroanatomically unique to humans138, and clearly emphasizes
rior and exterior environmental challenges, verifiable correlates of conscious behaviour. the role of the body’s health in human
ranging from exercise, dehydration or altitude To this end, we are now comparing the size consciousness and interaction.
to injury, sepsis or social interactions. The and cytoarchitectonic differentiation of the A. D. (Bud) Craig is at the Atkinson Pain Research
organization of this network is focused at the thalamic relay VMpo in different primate Laboratory, Division of Neurosurgery,
spinal level on cardiovascular and direct end- species. Preliminary observations are sup- Barrow Neurological Institute, Phoenix,
organ control11,144, at the brainstem level on portive; VMpo in the pygmy chimpanzee, Arizona 85013, USA.
e-mail: bcraig@chw.edu
integrated control of fluid, electrolyte, energy, which can recognize itself in a mirror156, is
immune and cardiorespiratory balances44,145, clearly similar to that in the human, albeit doi:10.1038/nrn894
at the forebrain level in sub-primates on considerably smaller. By contrast, VMpo in 1. Weber, E. H. Handwörterbuch des Physiologie mit
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PERSPECTIVES
Background
OPINION
The view represented in most textbooks
regards pain and temperature as distinct, dis-
criminative, cutaneous sensations that are
How do you feel? Interoception: the relayed to somatosensory areas of the cortex
by the thalamic ventrobasal complex8,9 (FIG. 1).
sense of the physiological condition Visceral sensations are assigned to a system
that relays vagal, glossopharyngeal, facial and
of the body spinal afferent activity by way of the brain-
stem parabrachial nucleus to the same ventro-
basal complex, but then to the insular cortex
A. D. Craig — a fact that is used to explain why such
sensations are less distinct10,11.
As humans, we perceive feelings from our taste) and interoceptive (visceral) modalities, However, these concepts could not explain
bodies that relate our state of well-being, and he categorized temperature and pain as several important discrepancies. First, the
our energy and stress levels, our mood aspects of touch4. involvement of the somatosensory cortex is
and disposition. How do we have these Recent findings on the functional anatomy challenged by the fact that pain and tempera-
feelings? What neural processes do they of the lamina I spinothalamocortical system ture sensations remain after cortical lesions of
represent? Recent functional anatomical indicate that interoception should be rede- the postcentral gyrus. For this reason, Head
work has detailed an afferent neural fined as the sense of the physiological condi- and Holmes12 assigned awareness of pain sen-
system in primates and in humans that tion of the entire body, not just the viscera5–7. sation to the posterolateral thalamus and pre-
represents all aspects of the physiological This system is a homeostatic afferent pathway scribed a modulatory role for the cortex.
condition of the physical body. This system that conveys signals from small-diameter pri- Second, the association of pain with touch
constitutes a representation of ‘the mary afferents that represent the physiological implies the existence of convergent, modifi-
material me’, and might provide a status of all tissues of the body. It projects first able pain-and-touch cells (considered to be
foundation for subjective feelings, emotion to autonomic and homeostatic centres in the the so-called ‘wide dynamic range’ cells of the
and self-awareness. spinal cord and brainstem, thereby providing deep dorsal horn9,13,14), despite the obvious
the long-missing afferent complement of the inability of such modality-ambiguous cells to
The feelings we perceive from our bodies efferent autonomic nervous system. Together engender the distinct sensations elicited by dif-
include temperature, pain, itch, tickle, sen- with afferent activity that is relayed by the ferent stimuli (for example, sharp versus burn-
sual touch, muscular and visceral sensations, nucleus of the solitary tract (NTS), it generates ing pain). Third, although there is a clear need
vasomotor flush, hunger, thirst, AIR HUNGER a direct thalamocortical representation of the for an afferent pathway to elicit efferent auto-
and others related to the body’s state. Early state of the body in primates that is crucial for nomic adjustments to somatic events that pro-
German physiologists regarded such general temperature, pain, itch and other somatic feel- duce feelings from the body (for example, for
bodily feelings as Gemeingefühl, or ‘common ings. This anatomical organization shows that the ongoing homeostatic process of thermo-
sensation’, and distinguished them from the these feelings are highly resolved, sensory regulation15), the concept of a ‘sympathetic
five senses1. Sir Charles Sherrington concep- aspects of ongoing homeostasis that represent afferent’ system has often been dismissed10,11.
tualized a sense of ‘the material me’ by con- the physiological condition of the body itself The prevailing views also stumbled on the
sidering that all of these feelings are related — a distinct shift from the concept that pain fundamental fact, recognized long ago16, that,
and form a foundation for the sense of one’s and temperature are aspects of touch. in the central nervous system, pain, tempera-
physical self 2. William James presented simi- Converging evidence from functional imaging ture and itch sensations are distinguished
lar natural philosophical arguments and studies substantiate this interoceptive cortical from touch by their association with the
regarded the feelings from our bodies as the image and indicate that its re-representation spinothalamic projection, which ascends in
basis for self-awareness and emotion3. in the anterior insular cortex of the non- the contralateral spinal cord, rather than with
However, Sherrington later codified the dominant (right) hemisphere, possibly the dorsal column/medial lemniscal system in
senses into teloreceptive (vision and hear- uniquely in humans, constitutes a basis for the the ipsilateral spinal cord. Because of this
ing), proprioceptive (limb position), extero- subjective evaluation of one’s condition, that arrangement, spinal hemisection produces
ceptive (touch), chemoreceptive (smell and is,‘how you feel’. contralateral loss of pain and temperature

PERSPECTIVES
a Conventional pain pathway b Conventional visceroceptive pathway c Lamina I spinothalamocortical pathway

Somatosensory Gustatory cortex
cortex in anterior insula
and frontal
operculum
Ventroposterior Ventroposterior Ventromedial

lateral nucleus medial nucleus nucleus
(parvocellular part) (posterior part)
Parabrachial
nucleus
Gustatory
Solitary part
nuclear
Reticular complex General visceral
formation part
Homeostatic
VII control
regions
IX
Anterolateral X
system
Lateral
spinothalamic
tract
Figure 1 | Pain, visceroceptive and spinothalamocortical pathways. Comparison of organizational charts of a | the conventional view of pain pathways, b | the
conventional view of visceroceptive pathways and c | the lamina I spinothalamocortical pathway.
sensations, and ipsilateral loss of fine-touch is their inherent association with emotion its homeostatic role. Accordingly, this feeling is
perception (the Brown–Sequard syndrome). (but see the discussion below on sensual directly dependent on the body’s needs19,20. For
Phylogenetic arguments that defined so-called touch). These feelings all have not only a sen- example, compare the pleasant feeling of cool
‘palaeo-’ and ‘neo-’ spinothalamic systems sory, but also an affective, motivational aspect. water when your body is overheated with the
attempted to explain how pain and tempera- The emotional aspect of thermal sensation, in gnawing discomfort generated by the very
ture were part of the touch system at the thala- particular, is easily under-appreciated; we typi- same cool stimulus when you are ‘chilled to the
mocortical level, although separate at the cally project temperatures onto the object we bone’. Thermoregulation is a primal evolution-
spinal level17,18. However, the fundamental are touching or onto our environment, as ary requirement for all animals, particularly
commonality of pain, temperature and other Aristotle did. However, temperature is ulti- homeothermic mammals, and the affective
bodily sensations as interoceptive, rather than mately a physiological condition of the tissue aspects of such feelings correspond to the
exteroceptive, remained unrecognized. itself (and likewise for all bodily feelings), and motivations that are essential for behavioural
A key feature that distinguishes pain, tem- it generates an inseparable affect (pleasantness thermoregulation and homeostasis — that is,
perature and other bodily feelings from touch or unpleasantness) — a feeling — that signals survival21,22. Thermoreception is therefore

PERSPECTIVES
representative, because all of the feelings from horn is genetically coordinated to coincide project to the main homeostatic integration
the body are directly related to homeostatic with the arrival of lamina I neurons. The sites in the brainstem. The latter include
needs and associated with behavioural moti- lamina I neurons, which arise from the prog- regions that also receive vagal and glossopha-
vations that are crucial for the maintenance of enitors of autonomic interneurons in the lat- ryngeal afferent activity (by way of the solitary
body integrity, and their neural representation eral horn, migrate to their superficial dorsal nucleus) and are heavily interconnected with
reflects this homeostatic primacy. position during a ventromedial rotation of the hypothalamus and amygdala — that is, the
The ascending neural activity that repre- the entire dorsal horn, which occurs simulta- ventrolateral medulla, catecholaminergic cell
sents temperature and other physiological neously with the arrival of the small-diameter groups A1, A2 and A5–A7, the parabrachial
conditions of the body is conveyed by the afferents23. A common transcription factor nucleus and the periaqueductal grey. Lamina I
lamina I spinothalamocortical pathway (FIG. 1). (DRG11) is activated in the B cells and lam- terminations in the medulla coincide precisely
The physiological and anatomical characteris- ina I neurons at this time24. This rotation with the developmental border between the
tics of this system will first be reviewed, and places the small-diameter afferent fibres that
then the functional imaging and clinical enter by the lateral division of the dorsal root
Limbic sensory and motor cortex
evidence that documents the primary intero- in contact with lamina I neurons. This rota-
ceptive cortex will be summarized. Finally, the tion concurrently results in the characteristic
cortical re-representations of interoceptive recurrent trajectory of the large-diameter
state that provide the basis for subjective fibres, which do not connect with lamina I Hypothalamus
awareness of the feelings from the body neurons25. This course of development
will be described and related to concepts of supports the view that the small-diameter
self-awareness and consciousness. afferents and lamina I constitute a cohesive
homeostatic afferent system. Parabrachial PAG
Mesencephalic
The functional anatomy of lamina I The Aδ- and C-type primary afferent fibres
Lamina I, the most superficial layer of the that are relayed by lamina I relate homeostatic
spinal (and trigeminal) dorsal horn, is the information — that is, much more than sim- A1 + A2 RVLM + VMM
Medullary
only neural region that receives monosynap- ply ‘pain and temperature’ sensations — from
tic input from small-diameter (Aδ and C) all tissues. These fibres convey slow activity that
primary afferent fibres, which innervate is sensitive to changes in a wide variety of phys- Lamina I ANS
essentially all tissues of the body6. The adja- iological conditions — not only temperature Spinal
cent lamina II (substantia gelatinosa) was and mechanical stress, but also local metabo-
once described as the pain-processing region lism (acidic pH, hypoxia, hypercapnia, hypo-
Fine afferents Fine efferents
of the dorsal horn, but it contains only local glycaemia, hypo-osmolarity and lactic acid),
interneurons in cat and monkey, and it cell rupture (ATP and glutamate), cutaneous
essentially receives C-fibres only from skin. parasite penetration (histamine), mast cell acti- Figure 2 | Hierarchical organization of neural
(In rodents, lamina II is less distinct from vation (serotonin, bradykinin and eicosa- homeostasis involving the sympathetic
lamina I.) Lamina I is the source of output noids), and immune and hormonal activity nervous system. Small-diameter afferent fibres
from the superficial dorsal horn, contribut- (cytokines and somatostatin)6,7,26–30. This per- that report the physiological condition of all tissues
ing half of the spinal input to the brainstem spective emphasizes that the category ‘nocicep- of the body terminate in lamina I of the spinal and
and thalamus. It is usually associated with tors’, although heuristically of enormous value, trigeminal dorsal horns. The ascending projections
of lamina I neurons provide the bases for somato-
‘pain and temperature’ and, indeed, its pro- is actually a theoretical simplification, because autonomic reflex arcs at the spinal, medullary and
jections contain LABELLED LINES that specifically the empirical mechanical, thermal and poly- mesencephalic levels. At the spinal level, lamina I
subserve these sensations. However, the evi- modal thresholds of small-diameter afferents projects strongly to the sympathetic regions in the
dence indicates that these are simply particu- extend broadly across the ‘pain’ threshold in all intermediomedial and intermediolateral cell
lar aspects of interoception and that lamina I tissues, as expressed clearly by many investiga- columns of the thoracolumbar cord, where the
neurons fundamentally represent many tors31–34. Microneurographic data indicate sympathetic preganglionic cells of the autonomic
nervous system (ANS) originate. In the medulla,
aspects of the physiological condition of the that only the summated activation of ‘C-noci-
lamina I neurons project to the A1 and A2
tissues of the body. ceptors’ causes a conscious perception of ‘pain’ catecholaminergic cell groups and to other sites
The association of small-diameter affer- in humans35, and C-fibres often have slow (including the rostral ventrolateral medulla, RVLM),
ents and lamina I neurons begins with their ongoing discharge without provocation, which are interconnected with the pre-
development, which is temporally coordi- which is not perceived34,36,37 and is likely to be sympathetic neurons that project to spinal levels.
nated and distinguished from the large- related to current tissue metabolic status. In The A2 cell group (that is, the nucleus of the
solitary tract) also receives direct parasympathetic
diameter exteroceptive and proprioceptive addition, many cutaneous C-fibres are selec-
(vagal and glossopharyngeal) afferent input (not
afferents that project to the deep dorsal horn; tively and exquisitely sensitive to slow, weak shown), and A1 projects to the hypothalamus.
this developmental differentiation seems to mechanical stimuli that evoke sensual (‘lim- In the pons and mesencephalon, lamina I neurons
reflect a simple physiological distinction bic’) touch38, as are neurons in lamina I and in project to the parabrachial nucleus, the main
between the inside and outside of the body. the inner substantia gelatinosa39. brainstem homeostatic integration site, and to the
Whereas the large-diameter fibres originate The ascending projections of lamina I periaqueductal grey (PAG), the main homeostatic
from the first wave of large (A) dorsal root neurons, which have been detailed in cat and brainstem motor site. The hypothalamus (the main
autonomic control centre) and the limbic cortical
ganglion cells, the small-diameter afferents monkey, unambiguously indicate their role in regions project to all of these sites in the
originate from small (B) cells and enter the homeostasis6,40,41 (FIGS 1 and 2). First, they pro- brainstem, and to lamina I and the sympathetic
spinal cord in a second wave, subsequent to ject strongly to the sympathetic cell columns preganglionic regions in the spinal cord. VMM,
the larger fibres. Their arrival in the dorsal of the thoracolumbar spinal cord. Next, they ventromedial medulla.

PERSPECTIVES
Box 1 | The thermal-grill illusion of pain several distinct, modality-selective classes that
receive input from particular subsets of small-
Thunberg’s (1896) thermal grill evokes a diameter fibres. These classes of neurons serve
burning, ice-like pain sensation with interlaced as labelled lines, because they differ morpho-
innocuous warm (40 °C) and cool (20 °C) bars. logically, physiologically and biochemically,
A similar, albeit stronger, sensation is elicited and their activity corresponds to distinct sen-
by pouring warm water on feet that are numb sations47,48. Primarily on the basis of cutaneous
with cold (an unmistakable signal of stimulation (and the heuristic concept of
thermoregulatory distress), and only burning
nociception), we now recognize two classes of
pain is evoked by a cool stimulus (<24 °C)
neurons that signal sharp pain (FIRST PAIN) and
when A-fibre conduction (and normal cool
burning pain (SECOND PAIN) that selectively
sensation) is blocked by pressure on a
peripheral nerve. These demonstrations reveal
receive inputs from Aδ-nociceptors and poly-
two basic phenomena: the increasing modal C-nociceptors, respectively49,50. In addi-
activation of the polymodal C-nociceptive tion, there are two types of thermoreceptive
sensory channel by cold below a threshold of Warm Cold lamina I cell that respond selectively to cooling
~24 °C (the normal thermoneutral ambient or to warming, distinct types of chemorecep-
temperature), and the central inhibition of Burning tive cell that respond selectively to histamine
that activity by innocuous thermosensory pain or to noxious chemicals, and other classes that
activity (‘cold inhibits pain’). respond selectively to muscle or joint afferents
The burning pain sensation caused by HPC or to mechanical ‘slow brush’ (tickle)39,51–54.
polymodal C-nociceptors (HPC), which are A particularly convincing demonstration of
sensitive to noxious heat and pinch, as well as to cold, is normally masked centrally by the the selectivity inherent in this pathway is pro-
activity of the specific cutaneous Aδ-fibre thermoreceptors that are responsible for the sensation vided by the histamine-responsive cells that
of cooling. When the activity of cooling receptors is reduced, the polymodal nociceptive activity we recently identified, which constitute a
that is evoked by cooling is disinhibited centrally and causes a burning sensation at cool (<24 °C) specific pathway for the sensation of itch55.
temperatures that is normally felt only at noxious cold (<15 °C) temperatures. Further types remain to be documented more
The thermal-grill illusion can be explained physiologically by an unmasking of the cold- clearly. For example, although most viscero-
evoked activity of polymodal nociceptive lamina I spinothalamic neurons (which are selectively ceptive lamina I cells reported so far have con-
activated by polymodal C-nociceptors) due to the reduction of the normal cold-evoked activity vergent cutaneous input54,56,57, such cells might
of thermoreceptive lamina I spinothalamic neurons (which are selectively activated by Aδ-type
have been sensitized by the requisite surgical
cooling thermoreceptors) by spatial summation of the simultaneous warm stimuli in the
preparation and repeated visceral search stim-
thermoreceptive but not the nociceptive neurons 62. The grill effectively produces a relative
ulation, and distinct types of visceroceptive
balance of polymodal and thermosensory activity similar to that caused by a noxious cold
stimulus of ~10 °C; the equivalence of these sensations has been verified psychophysically.
cell probably exist that even distinguish renal
Reduction of activity in the thermoreceptive sensory channel disinhibits or unmasks the artery from renal vein occlusion by respond-
polymodal nociceptive channel at thalamocortical levels, producing a burning pain sensation. ing selectively to renal osmoreceptors or
Functional imaging has confirmed that the thermal grill produces a pattern of activity in the mechanoreceptors58, similar to the selectively
cortex that is identical to the activation produced by noxious cold63. The cortical activation metaboreceptive muscle-responsive lamina I
unmasked by the grill is in the anterior cingulate cortex (ACC), indicating that ACC activation is neurons that we recently identified (see
selectively associated with the perception of thermal pain — that is, the affect that signals below)59. In stark contrast to these neurons,
thermoregulatory distress. So, the thermosensory activity in the lamina I spinothalamocortical the cells in the deep dorsal horn provide a
pathway to interoceptive cortex unmasks activity in the medial lamina I pathway, which activates modality-ambiguous integration of all affer-
the ACC and is associated with burning pain. This indicates that the medial thalamic lamina I ent inputs, including mechanoreceptive,
spinothalamic tract projection to the mediodorsal thalamic nucleus (ventral caudal part, MDvc) proprioceptive and nociceptive activity9,60,61.
might be the crucial site for the inhibition of thermal pain by cold; this suggestion is supported by The thermoreceptive lamina I cells that are
preliminary physiological recordings from the MDvc in our laboratory. sensitive to cooling respond predominantly to
Aδ-fibre input and have ongoing discharge at
normal skin temperature that is inhibited by
alar and basal plates that defines the classic example, respiration is linearly modulated by warming. They uniquely evince a linearly
‘general visceral afferent’ integration region42, graded changes in innocuous thermosensory increasing response to skin temperatures
and throughout the brainstem they co-localize activity45. Completing this anatomical circuitry, below a neutral skin temperature (~34 °C)
with the catecholaminergic cell columns that lamina I receives descending modulation that corresponds directly with human psycho-
identify homeostatic regions. These spinal and directly from brainstem pre-autonomic physics48. Their activity reaches a plateau at the
bulbar lamina I projections substantialize (pro- sources (A5, A7 and raphe nuclei). Most strik- cold temperatures that we perceive as noxious
vide the anatomical substrate for) the somato- ingly, the only spinal regions that receive (<15 °C); at these temperatures, the polymodal
autonomic reflexes that are activated by small- descending controls directly from the hypo- nociceptive cells that signal burning (second)
diameter afferents (including visceroceptive thalamic paraventricular nucleus — the main pain are increasingly active. Analysis of the
activity) and are crucial for cardiorespiratory diencephalic autonomic control centre — are thermal-grill illusion of pain (BOX 1) indicates
and other homeostatic functions43,44. the autonomic motor nuclei and lamina I46. that the perception of thermal distress and
Importantly, these reflex pathways are not sim- Lamina I neurons relate many aspects of cold pain depends on the comparison of these
ply emergency mechanisms; rather, they pro- the ongoing physiological status of the tissues two sensory channels in the forebrain62,63, as
vide continuous homeostatic feedback. For of the body. In cat and monkey, they comprise well as on core temperature20. Notably, the

PERSPECTIVES
polymodal nociceptive channel responds to dense clusters of large, glutamatergic lamina I Right orbitofrontal
cold with accelerating activity below ~24 °C — boutons that are organized topographically
corresponding to our increasing thermoregu- (in the rostrocaudal direction) and associated
latory distress below a thermoneutral ambient with TRIADIC ARRANGEMENTS of GABA (γ-amino-
Left anterior insula Right anterior insula
temperature — and, when Aδ-type thermo- butyric acid)-containing presynaptic den-
receptive afferent activity is reduced by a nerve drites and proximal relay cell dendrites in
block or by the thermal grill, the activity in cytoarchitectonically distinguishable cell
Anterior insula
this channel elicits a burning-pain sensation nests70,72. The VMpo is contiguous rostrally
at such temperatures48,62,64. This emphasizes with the basal part of the ventromedial
that pain is not a binary (yes or no) modality, nucleus, or VMb (which, for historical reasons,
and compels the conceptual shift of viewing is denoted by some as the parvicellular part of Mid/posterior dorsal insula
the role of polymodal C-nociceptors and the the ventroposterior medial nucleus, or VPMpc,
lamina I neurons that convey their activity as even though it does not project to the somato-
a homeostatic afferent pathway rather than sensory cortex as the remainder of the VPM VMpo VMb
simply a ‘nociceptive’ pathway. does) and, in primates, VMb receives a direct
To directly address the role of lamina I in (bilateral) input from the nucleus of the
homeostasis, we recently examined the solitary tract, which conveys vagal and glosso-
Parabrachial
responses of lamina I neurons to muscle pharyngeal (general visceral and gustatory)
contraction. We identified a class of lamina I afferent activity73. Therefore, these two thala-
spinobulbar neurons that respond selectively mic nuclei together represent all homeostatic
during and after muscle contraction. These afferent inflow (both sympathetic and para- Lamina I NTS
neurons provide a substrate for the EXERCISE sympathetic) from the body, and they form a
59
PRESSOR REFLEX . Some of the muscle Aδ- and separate, rostrocaudally organized column
C-fibres that are represented by these lamina I that is orthogonal to the mediolateral align-
cells are sensitive to contraction; others are ment of the exteroceptive and proprioceptive Fine Fine
sympathetic parasympathetic
sensitive to lactic acid and other metabolites representations in the ventroposterior nuclei, afferents afferents
released during muscular exercise, and can be to which they are connected at the representa-
viewed as metaboreceptors (or ERGORECEPTORS) tion of the mouth. The direct lamina I path-
that continuously drive a variety of regional way to VMpo and the direct pathway from
and whole-body homeostatic adjustments to the nucleus of the solitary tract to VMb are Figure 3 | The organizational chart for
muscular work36,65–67. It is important to rec- distinguishable only in primates; essentially, interoception. Small-diameter afferents that
innervate tissues in parallel with sympathetic
ognize that large increases in such activity homeostatic afferent input in sub-primates
efferents (‘sympathetic afferents’) provide input to
can cause the familiar aching or burning sen- reaches the forebrain after integration in the lamina I; small-diameter afferents that innervate
sation from muscles, and synchronous acti- brainstem parabrachial nucleus10 and by direct tissues in parallel with parasympathetic efferents
vation causes a painful cramping sensation68, spinohypothalamic projections (in rats)74. (‘parasympathetic afferents’) provide input to the
but the activity elicited continuously by mus- Nociceptive and thermoreceptive neurons nucleus of the solitary tract (NTS). In mammals,
cle contraction normally produces homeo- with properties similar to lamina I neurons such activity is integrated in the parabrachial
nucleus, which projects to insular cortex by way of
static adjustments without the perception of have been identified in VMpo in macaque and
the ventromedial thalamic nucleus (VMb, also
a behaviourally motivating signal. This class owl monkeys70,75, and such neurons have also known as VPMpc). In primates, however, a direct
of neurons clearly confirms the role of lam- been recorded in the region of VMpo in awake projection from lamina I to the ventromedial
ina I in ongoing homeostasis, and substanti- humans76,77. These different modalities are nucleus (VMpo), and a direct projection from the
ates the concept that it relates the current segregated within VMpo. Significantly, micro- NTS to the VMb, provide a rostrocaudally
physiological condition of all the tissues of stimulation in this region of the thalamus in contiguous column that represents all
contralateral homeostatic afferent input and
the body. awake humans elicits discrete pain or cooling
projects topographically to the mid/posterior
or visceral sensations77–80. Anatomical tracing dorsal insula. In humans, this cortical image is
Lamina I spinothalamocortical pathway in monkeys indicates that VMpo and VMb re-represented in the anterior insula on the same
Anterolateral cordotomy in humans interrupts project in a rostrocaudally topographic fash- side of the brain. The parasympathetic activity is
contralateral temperature, pain, itch, sensual ion to a cytoarchitectonically distinct field in then re-represented in the left (dominant)
touch and visceral sensations, if made at the the fundus of the superior limiting sulcus at hemisphere, whereas the sympathetic activity is
location of ascending lamina I axons in the the dorsal margin of insular cortex, with the re-represented in the right (non-dominant)
hemisphere. These re-representations provide the
lateral spinothalamic tract 6,69. In primates, VMb projection field extending rostrally from
foundation for a subjective evaluation of
lamina I spinothalamic neurons project to a the VMpo projection to include the previously interoceptive state, which is forwarded to the
dedicated thalamocortical relay nucleus in recognized gustatory cortex6,81,82. A smaller, orbitofrontal cortex, where hedonic valence is
the posterolateral thalamus — the posterior ancillary VMpo projection terminates in area represented in mammals.
part of the ventromedial nucleus, or VMpo70 3a in the fundus of the central sulcus. These
(FIGS 1 and 3). The VMpo is small in the anatomical and functional findings directly
macaque monkey thalamus, and it is only pri- contradict the conventional view of pain pro- Functional imaging with PET (positron
mordially represented in sub-primates, but it cessing and the view of some investigators that emission tomography), SPECT (single-
is proportionately very large in the human nociceptive activity simply passes through the photon emission computed tomography) and
thalamus71. It has appropriate anatomical posterior thalamus to VPM and VPL and fMRI (functional magnetic resonance imag-
characteristics for a thalamic relay nucleus: thence to the somatosensory cortex9. ing) verify that the anatomically identified

PERSPECTIVES
a Graded cooling b C-fibre touch c Thermal pain d Chronic pain
e Dynamic exercise f Respiration/isometric exercise g Graded itch h Cold allodynia
Figure 4 | Activation of the interoceptive cortex in the dorsal posterior insula by various modalities. a | Graded cooling (reproduced, with permission, from
REF. 5 © 2000 Macmillan Magazines Ltd). b | Sensual (limbic) touch evoked by slow brushing in a polyneuropathy patient with only C-mechanoreceptor activation
(reproduced, with permission, from REF. 101 © 2002 Macmillan Magazines Ltd). c | Thermal pain (reproduced, with permission, from REF. 91 © 2002 Elsevier Science).
d | Chronic pain (reproduced, with permission, from REF. 92 © 2001 International Association for the Study of Pain). e | Dynamic exercise on a bicycle (reproduced,
with permission, from REF. 96 © 1999 The American Physiological Society). f | Forced respiration and isometric exercise with a hand grip (reproduced, with
permission, from REF. 95 © 1999 John Wiley & Sons). g | Graded itch elicited by cutaneous histamine injection (reproduced, with permission, from REF. 94 © 2001
International Association for the Study of Pain). h | Cold allodynia (reproduced, with permission, from REF. 93 © 2000 International Association for the Study of Pain).
VMpo/VMb projection field in the dorsal cortical field. So, tactile stimulation of the (hypertonic saline injection), hunger and air
insular cortex is activated by temperature, hand activates a portion of S2 close to the lip hunger95,97–99. Consistent with the anatomical
pain and numerous interoceptive modalities of the lateral sulcus, more than 16 mm lateral tracing studies, gustatory stimuli activate the
that cause feelings from the body, consistent to the site in the dorsal posterior insula that is dorsal insular cortex more rostrally100.
with the functional anatomical data on activated by cooling the hand. Activation of this interoceptive cortex was
lamina I (FIG. 4). This site is distinct from the This same region in the dorsal posterior also observed using light, slow brushing in a
parietal somatosensory cortices, supporting insula (albeit slightly more rostrally) shows patient with polyneuropathy who has only
the idea of a neural distinction of intero- (graded) activity in every imaging study that peripheral nerve C-fibre conduction, consis-
ception from exteroception. Our PET study uses noxious heat applied to the hand87–91. It tent with the essential role of lamina I and
using graded innocuous cooling of the hand shows ongoing activity in chronic pain the lateral spinothalamic tract for sensual
showed correlated activity only at this site in patients92, as well as evoked activity in (limbic) touch101,102.
the fundus of the superior limiting sulcus of NEUROPATHIC PAIN patients, in which pain can be So, the evidence indicates that, in primates,
the posterior insula5. This site coincides with caused by normally innocuous stimuli93. (In the dorsal insular cortex contains a sensory
the location of cortical lesions that produce anaesthetized monkeys, noxious cold stimuli representation of the small-diameter afferent
thermanaesthesia and hypalgesia83,84, and it produce fMRI activation at precisely the sites activity that relates to the physiological condi-
verifies that the terminus of the lamina I identified in our anatomical tracing studies6.) tion of the entire body. This cortical region
spinothalamocortical pathway is the same in Furthermore, this same dorsal posterior insu- seems to constitute a primary interoceptive
humans as in monkeys. In human atlases, this lar site shows graded activity that is correlated image of homeostatic afferents. Embedded in
site has been included with the parietal oper- with itch sensation in humans94. This intero- the interoceptive cortex are the cortical repre-
culum as the second somatosensory region ceptive site is activated by both isometric and sentations of several highly resolved, distinct
(S2), on the basis of comparative anatomical dynamic exercise95,96, like the lamina I neurons sensations, including temperature, pain, itch,
tracing work that was carried out before the that respond selectively during and after mus- muscular and visceral sensations, sensual
recognition of VMpo85. However, this site is cle contraction59. It is activated by a range of touch and other feelings from the body. The
distinct from S2. The S2 has a topography modalities associated with visceral sensations, importance of the interoceptive cortex for
that is aligned from lateral to medial (face to such as the Valsalva manoeuvre, manipula- physical well-being is underscored by its clear
foot 86), which is orthogonal to the rostro- tions of blood pressure (maximal inspiration; delimitation in the monkey by in situ hybridiza-
caudal topography in the interoceptive VMpo lactate or cholecystokinin injection), thirst tion labelling for the mRNA of receptors for

PERSPECTIVES
corticotropin-releasing factor, which is Box 2 | Central pain syndrome

thought by many to be a definitive indicator of
an association with homeostatic stress103. Damage to the brain can cause the
Notably, the absence of this direct intero- central pain syndrome, in which Area 3a Area 24c
ceptive representation in sub-primates implies ongoing, intractable pain is referred to

that they cannot experience feelings from the contralateral deep and cutaneous
body in the same way that humans do. In tissues, where there is always a loss of
humans, lesions of the dorsal insula interrupt thermal (cool and warm) sensation
and often a paradoxical loss of acute Dorsal
these feelings83,84, disrupt homeostatic process- posterior
pain (pinprick and heat)
ing104 and cause permanent loss of discrimina- insula
sensation6,158. Central pain is
tive thermal sensation. By contrast, in cats
characteristically described as
that have been trained on a high-resolution burning and hyperpathic
thermosensory discrimination task, lesions of (augmenting), and is often Ventromedial Mediodorsal
the rudimentary thalamocortical lamina I accompanied by mechanical or nucleus nucleus
relay cause only a weak, transient behavioural thermal (cold) allodynia — that is, (posterior part) (ventral
caudal
deficit, whereas a spinal lesion of the ascend- pain is evoked by normally innocuous part)
ing lamina I axons causes permanent disrup- stimuli. It apparently results from
tion105. These findings indicate that behaviour damage to the lamina I
in sub-primates reflects motivational signals spinothalamocortical pathway at any
from homeostatic integration centres in the level, including interoceptive cortex,
brainstem and hypothalamus, and they reveal but it is not produced by lesions of the
that homeostatic integration in the brain- exteroceptive lemniscal pathways,
–
stem parabrachial nucleus is sufficient to the parietal somatosensory cortices Periaqueductal
or the medial thalamus. It occurs in grey
motivate thermoregulatory behaviour. Indeed,
~5% of stroke patients and 25–40% –
in humans, a lesion of the lamina I spino- Parabrachial
thalamocortical pathway that eliminates ther- of patients with multiple sclerosis nucleus
mal (and evoked pain) sensation can result in or spinal cord injury. Tricyclic
the central pain syndrome (BOX 2), which antidepressants are efficacious for
probably reflects a similar motivational signal about half of patients with central
pain, but opiates are usually
to forebrain behavioural mechanisms by
ineffective. Head and Holmes12 first
brainstem homeostatic drives that are released
postulated that this is a release
from descending insular cortical modula-
phenomenon that results from loss of
tion5,6,106. The occurrence of central pain after an inhibitory effect of discriminative
lesions that disrupt evoked pain sensibility was pain processing on the emotional
a key argument made against the existence of aspect of pain, and others suggested
a specific neural representation of pain13; how- release of phylogenetically old
ever, the available evidence indicates that cen- brainstem inputs or hyperactive
tral pain can be regarded as thermoregulatory bursting in somatosensory pathways.
distress, consistent with the recognition that A recent proposal, the thermosensory-
pain is both an aspect of interoception and a inhibition hypothesis, is based on the Lateral
behavioural drive caused by a physiological spinothalamic
anatomy of the lamina I projection tract
imbalance that homeostatic systems alone system. This hypothesis posits that
cannot rectify. central pain results from the loss of
So, like other feelings from the body, pain descending controls from interoceptive cortex on brainstem homeostatic sites that drive
normally consists of both a sensation and a thermoregulatory behaviour by way of the medial thalamus and the anterior cingulate cortex
motivation. The cortical region associated (see figure; minus symbols denote inhibition). The disinhibition proposed by this hypothesis is
with such motivation seems to be the anterior conceptually similar to the unmasking shown by the thermal-grill illusion (BOX 1). This proposal
cingulate cortex (ACC). The insula has long views central pain as a thermoregulatory dysfunction, and it emphasizes the concept that pain is
been regarded as limbic sensory cortex, not only a feeling, but also a behavioural drive that signals a homeostatic imbalance.
because of its association with visceral sensa-
tion by Penfield’s stimulation studies in studies of pain89,90,114. The ACC is activated the parabrachial nucleus and other brainstem
humans, whereas the ACC can be regarded as selectively during the thermal-grill illusion of homeostatic sites seems likely116. Like the lam-
limbic motor cortex, because of its association pain, and therefore signifies the urgency of ina I pathway to VMpo, this direct medial
with autonomic and emotional control107,108. thermal distress63; similarly, the unpleasant- pathway to the ACC does not exist in sub-
Both are strongly interconnected with the ness of thermal pain has been directly corre- primates, in which a phylogenetically earlier
amygdala, hypothalamus, orbitofrontal cortex lated with ACC activation using hypnotic lamina I projection is relayed by the thalamic
and brainstem homeostatic regions109,110. manipulation115. In primates, the underlying submedial nucleus to the orbitofrontal cortex6.
Many recent functional imaging studies have pathway seems to be an ancillary lamina I Although the underlying anatomy differs,
documented the role of the ACC in behav- spinothalamocortical route through the ven- data from studies in rats support a primordial
ioural drive and volition111–113. A particular tral caudal portion of the medial dorsal role of the ACC in behavioural motivation
portion of the ACC is activated in virtually all nucleus6, where convergence with input from by homeostatic distress, probably due to

PERSPECTIVES
a Anger b Subjective coolness c Graded disgust in faces d Trustworthiness in faces
in
mb
e Sexual arousal
ac
S2 S2
Figure 5 | Activation of the right (non-dominant) anterior insular cortex associated with different subjective feelings. a | Recall-induced anger, in which
activation is also visible in the right orbitofrontal, anterior cingulate (ac) and interoceptive (S2) cortices (reproduced from REF. 123 © 2000 Macmillan Magazines Ltd).
in, insular cortex; mb, mammillary bodies. b | Regression analysis of subjective ratings of the intensity of cooling the hand (reproduced from REF. 5 © 2000
Macmillan Magazines Ltd). c | Activation after exposure to graded disgust in computer-generated faces (reproduced from Nature (REF. 124) © 1997 Macmillan
Magazines Ltd). d | Activation elicited by subjective ratings of trustworthiness in faces (reproduced from REF. 129 © 2002 Macmillan Magazines Ltd). e | Activation
elicited by sexual arousal (reproduced, with permission, from REF. 128 © 1999 Kluwer Academic Publishers).
parabrachial and other brainstem inputs117,118. direct evidence that the underlying anatomi- anger, anticipatory anxiety and pain, panic,
So, activation of the ACC is associated with cal pathway involves first the contralateral disgust, sexual arousal, trustworthiness and
motivation, and activation of the insula is anterior insula, which must contain an ini- even subjective responses (positive or nega-
associated with feeling, which together form tial re-representation of interoceptive cortex tive) to music121–127 (FIG. 5). For example, anger
an emotion. on the same side, and then, by way of a cal- was associated with activation in the right
losal pathway, a lateralized, second-order anterior insula and the orbitofrontal cortex, as
Subjective awareness of feelings re-representation on the right side that is well as in the interoceptive dorsal posterior
How do we perceive the feelings from our subsequently forwarded to orbitofrontal insular region123 (albeit labelled ‘S2’).
bodies? We examined this question in our cortex. Recent fMRI data indicate precisely Activation correlated with the implicit level of
PET study of innocuous thermal sensibility5, the same progression of insular activity for the disgust elicited by computer-generated facial
in which human volunteers provided a sub- subjective appreciation of pain91. Sequential expressions was shown in the anterior insular
jective magnitude rating of the perceived cortical processing occurs in all sensory sys- and orbitofrontal cortices on the right side124,
intensity of each stimulus (irrespective of tems, and it seems reasonable to conclude even though this emotion is usually regarded
emotional valence). Their ratings were not that evolutionary development of such a as a derivative of gustation. Sexual arousal
perfectly correlated with stimulus tempera- progression for interoception would be elicited by erotic films (measured by testos-
ture, and regression analysis of these per- advantageous for survival by differentiating terone release and intumescence) was
cepts showed strongly correlated activity in and refining the representation of internal strongly correlated with right anterior insular
the right (ipsilateral, non-dominant) anterior feelings. Analogous re-representation and activation128. Subjective assessment of the
insula (FIG. 5) and the orbitofrontal cortex, lateralization of gustatory processing in the trustworthiness of faces also activated right
which differed significantly from the linear left anterior insular and orbitofrontal cor- anterior insula129, and such activation has
representation of stimulus temperature in tices in humans has been proposed on the been explicitly interpreted as representing the
the interoceptive cortex. This revealed that basis of clinical findings119, and corroborated subjective feeling (or ‘gut reaction’) elicited by
subjective evaluation of the thermal stimuli by functional imaging120. each face130.
— the feeling elicited in each person by the These observations match a wide variety These convergent findings are consistent
graded coolness — depended on post- of functional imaging studies that have asso- with the concept that the re-representation of
processing in those cortical regions. Close ciated activation of right anterior insular and the cortical image of the interoceptive condi-
inspection of the correlation maps (see orbitofrontal cortices with subjective emotion of the body serves as a limbic sensory
figure 2 in REF. 5, levels 20–24 mm) provided tion, such as recall-generated sadness or substrate for subjective feelings and emotions.

PERSPECTIVES
This fits the idea that the evaluation of the thermal stimulus, or any other bodily feeling, than a basic sensory awareness of the environ-
state of the body in response to a pertinent according to the body’s homeostatic needs. In ment. It is required for extended autobio-
stimulus serves as the basis for emotional feel- fact, the orbitofrontal cortex guides feeding graphical and cognitive continuity, because it
ings — the James–Lange theory of emotion3. behaviour and behavioural valence in the rat engenders the fundamental image of the
In particular, these data are consistent with as well; it is not a phylogenetically recent physical self as a feeling (sentient) entity.
the neurological hypothesis, based on analy- development. Anatomical data in the
ses of clinical cases and imaging data, that the macaque monkey indicate that this region Conclusions and future directions
right (non-dominant) anterior insula is inte- receives convergent input from several sen- The identification of an entire neural system
gral for the generation of the mental image of sory systems, and it seems to receive a direct that can be cogently conceptualized as a rep-
one’s physical state, which underlies basic projection from gustatory and interoceptive resentation of the physiological condition of
emotional states and is required for the moti- insular cortex133. In other words, whereas the the material body has several fundamental
vation to make rational decisions that affect macaque monkey has an anatomically visible implications. It provides a rational explana-
survival and quality of life — the essence of interoceptive thalamocortical pathway, it has tion for the long-recognized association of
the ‘somatic marker’ hypothesis of conscious- direct interoceptive cortical projections to pain, temperature, itch and other feelings
ness131 (see also REFS 99,132) (BOX 3). Indeed, orbitofrontal cortex, and therefore might not from the body, separate from the lemniscal
Damasio explicitly suggested that a re-map- have the intervening re-representations of system that represents exteroceptive touch
ping of the representation of the state of the the subjective state of the body in the right and proprioception. It incorporates specific
body (interoceptive sensation) could provide anterior insular cortex that are clearly present labelled lines for several physical conditions
an ‘as if ’ circuit that would allow the brain to in humans. that generate distinct feelings, and it substan-
judge and predict the effects of emotionally These functional anatomical considera- tiates their common integration in the hier-
relevant stimuli on the body, which is what tions indicate clearly that primates differ from archical homeostatic network. It provides the
these functional anatomical data seem to sub-primates in the encephalization of a long-missing peripheral and central afferent
indicate. So, a refined image of the state of the direct cortical image of the physiological con- complement to the efferent autonomic ner-
body seems to provide the basis for awareness dition of the body. Furthermore, they strongly vous system. These findings reveal a direct
of the physical self across time. suggest that humans differ from monkeys not cortical image of the state of the body that
Nonetheless, it is equally important to rec- only in the relative size of the interoceptive differentiates primates from sub-primates
ognize that the ACC (as well as subcortical thalamocortical pathway, but also in the neuroanatomically. The size and multiple
structures, such as the amygdala and ventral development of sequential re-representations re-representations of this interoceptive image
striatum133–136) is co-activated with the ante- of the physiological state of the body in the seem to differentiate humans from sub-
rior insula in virtually all imaging studies of right anterior insula. The interoceptive re- human primates. Finally, these findings
emotion111,112,121,123,127,137, consistent with the representation that is lateralized in the right signify the cortical representation of feelings
view that an emotion is both a feeling and a anterior insula of humans corresponds with from the body as the likely basis for human
motivation. Notably, this provides the active the ability to perceive the self as a physical and awareness of the physical self as a feeling
agent that is missing from the somatic- separate entity — that is, self-awareness. The entity. This association provides a funda-
marker hypothesis, in which Damasio conjec- functional imaging data strongly support the mental framework for the involvement of
tured that the subjective ‘I’ is generated only integral role of the right anterior insula in these feelings with emotion, mood, motiva-
as an illusory by-product of the re-mapping. the feelings we perceive that are the basis of tion and consciousness. These concepts
For example, a recent imaging study of our perceptions of our selves, and therefore emerge directly from the functional anatomy
placebo analgesia revealed concomitant acti- of consciousness. As Damasio131 has elegantly of the lamina I spinothalamocortical system,
vation of both the ACC and the right anterior described, this level of consciousness is more rather than from preconceived ideas.
insula138; according to the present interpreta-
tion, this reflects the active modulation by the
behaviourally motivating agent (limbic motor Box 3 | The ‘somatic marker’ hypothesis of consciousness
cortex, the ACC) of the feeling represented by On the basis of neurological analyses of patients with forebrain lesions, Antonio Damasio123,131
the image of the expected state of the body (in has advanced the ‘somatic marker’ hypothesis of consciousness. He proposes that the subjective
the right anterior insula). This interpretation process of feeling emotions requires the participation of brain regions that are involved in the
is consistent both with the interoceptive lam- mapping and/or regulation of our continuously changing internal states — that is, in
ina I spinothalamocortical projections and homeostasis. These feelings help to guide behavioural decisions that affect survival and quality
with the overall anatomical organization of of life by producing a ‘perceptual landscape’ that represents the emotional significance of a
primate frontal cortex into medial (motor) particular stimulus that is being experienced, or of a projected future action by means of a
and orbital (sensory) networks139,140. further ‘as-if-body loop’ mechanism. The feelings are grounded in the body itself, based on
The orbitofrontal cortex, which correlated multi-tiered and evolutionarily developed neural mechanisms that control the body’s state.
most strongly with subjective thermal per- These feelings distinguish between inner-world representations and outer-world representations,
ception5, is associated with the discrimina- and allow the brain to build a meta-representational model of the relationship between outer
tion of positive and negative rewards (pleas- and inner entities159. So, the representational image of the body’s state provides a neural basis for
antness/unpleasantness, or hedonic valence, distinguishing self from non-self, and re-representations of this image enable the behavioural
corresponding to approach/avoidance encod- neural agent to project the effects of possible actions onto the state of the body, as well as the
ing) in relation to the body’s needs136,141,142. For resultant changes in such feeling states due to interactions with other (external) agents. This
hypothesis posits that degrees of conscious awareness are related to successive upgrades in the
example, this region engenders stimulus-spe-
self-representational maps159. The anatomical features of Damasio’s hypothesis include a central
cific satiety to food143, so it would probably
role for the anterior insular cortex in the representation of such feeling states.
also differentiate the affect associated with a

PERSPECTIVES
Glossary In contrast to the many discriminable sen- stress — including a direct cortical image of
sations from the body, the subjective apprecia- physical well-being — provides a sound epis-
AIR HUNGER
Hypercapnia with mechanically restricted ventilation.
tion of visceral sensation is more diffuse, less temological foundation for integrated
well localized, and usually below perceptive approaches to the treatment of pain, meta-
ERGORECEPTION thresholds. This was one of the main reasons bolic, eating and psychosomatic disorders. For
Afferent activity relating tissue energy and for the long-standing mis-categorization of example, this provides an easy formulation for
metabolic needs.
pain and temperature as exteroceptive rather somatization under emotional stress. Similarly,
EXERCISE PRESSOR REFLEX
than interoceptive.Although it would be highly these considerations imply that mysterious
Increased blood pressure and heart rate caused by inefficient for gastrointestinal processing to pain syndromes, such as fibromyalgia (deep
activity in small-diameter afferents from muscle. require constant behavioural supervision, this aches and pains), could be related to homeo-
perceptual difference remains to be explained static dysfunction (for example, salt or water
FIRST PAIN
Sharp, pricking pain associated with rapidly
adequately11. Notably, many observations indi- balance or cardiovascular function), rather
conducting Aδ-fibres. cate that there is opponent processing between than to tissue damage, and this possibility
parasympathetic and sympathetic afferents deserves vigorous study. Consideration of these
LABELLED LINES that parallels their efferent opponency. findings led directly to the recent proposal that
Anatomically and physiologically distinct neurons that
For example, there are obligatory mutual the central pain syndrome is a thermo-
are specifically associated with particular sensations.
inhibitory interactions between spinal and regulatory disorder5,106. The recognition that
NEUROPATHIC PAIN vagal small-diameter afferent activities in the sensual touch is incorporated into the intero-
Intractable pain associated with damage to the medulla that are essential for cardiorespiratory ceptive system has strong implications for the
peripheral or central nervous system. control149,150. Similarly, vagal afferent activation neurobiological and health effects of con-
SECOND PAIN
inhibits both pain sensation151 and spinal specific contact — visitors to zoos will remem-
Dull, burning pain associated with slowly conducting visceroceptive processing152.Vagal stimulation ber that monkeys, chimpanzees and bonobos
C-fibres. can reportedly reduce stress and depression normally spend an enormous amount of
clinically. Similarly, opposing effects on auto- time grooming and cuddling each other, and
TRIADIC ARRANGEMENT
nomic function have been elicited by stimula- readers will remember the classic studies by
Ultrastructural contacts between an afferent terminal,
a relay cell dendrite and a GABA-containing tion of human insular cortex on both sides153, H. Harlow showing the importance of con-
presynaptic dendrite that is characteristic of high- and corresponding cortical lateralization has specific contact for emotional development.
fidelity transmission in sensory relay nuclei. been observed with micturition154 and gusta- Last, the observation that the neuroanatomical
tion120. Such a basic organization would be substrate for subjective emotion in humans is
It is important to recognize that this neural parsimonious with many considerations and based on an abstracted meta-representation
sensory system is part of an entire network could explain the perceptual differences, but of the physiological state of the body, consis-
involved in homeostasis; that is, in the auto- this certainly needs further study, particularly tent with the conjectures of James3 and
nomic, hormonal and behavioural neural because of the potential clinical significance155. Damasio131, provides a basis for the volitional
mechanisms that maintain optimal physiologi- The association of the re-representation of modulation of feelings, emotion and efferent
cal conditions in the body and that respond in the interoceptive pathway with self-awareness activity affecting the state of the body that is
an integrated and ongoing fashion to all inte- implies the existence of neuroanatomically unique to humans138, and clearly emphasizes
rior and exterior environmental challenges, verifiable correlates of conscious behaviour. the role of the body’s health in human
ranging from exercise, dehydration or altitude To this end, we are now comparing the size consciousness and interaction.
to injury, sepsis or social interactions. The and cytoarchitectonic differentiation of the A. D. (Bud) Craig is at the Atkinson Pain Research
organization of this network is focused at the thalamic relay VMpo in different primate Laboratory, Division of Neurosurgery,
spinal level on cardiovascular and direct end- species. Preliminary observations are sup- Barrow Neurological Institute, Phoenix,
organ control11,144, at the brainstem level on portive; VMpo in the pygmy chimpanzee, Arizona 85013, USA.
e-mail: bcraig@chw.edu
integrated control of fluid, electrolyte, energy, which can recognize itself in a mirror156, is
immune and cardiorespiratory balances44,145, clearly similar to that in the human, albeit doi:10.1038/nrn894
at the forebrain level in sub-primates on considerably smaller. By contrast, VMpo in 1. Weber, E. H. Handwörterbuch des Physiologie mit
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[Frontiers in Bioscience 14, 5291-5338, June 1, 2009]
Neurobiology of depression, fibromyalgia and neuropathic pain
Vladimir Maletic1 Charles L. Raison2

1
University of South Carolina, School of Medicine, Department of Neuropsychiatry and Behavioral Sciences, Columbia, SC,
2
Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Atlanta, GA
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Epidemiology and Comorbidity of Major Depression, Fibromyalgia and Neuropathic Pain
4. Clinical Relevance of Neurobiological Understanding of MDD, FM and NeP
5. Genetics of MDD, FM and NeP
5.1. Genetics of MDD
5.1.1. Genes Involved in Regulation of Serotoninergic Function
5.1.2. The Role of Genes Regulating Neurotrophic Factors
5.1.3. The Role of Genetic Epistasis in MDD
5.1.4. Polymorphism of Genes Regulating MAO
5.1.5. COMT Gene Polymorphism
5.1.6 Polymorphism of Genes Involved in Regulating Inflammatory Response
5.2 Genetics of Nociception and FM
6.Functional and Structural Alterations in Central Nervous System Circuitry Involved in Regulation of Emotion and Pain
6.1. Structural and Functional Brain Changes in Depression
6.1.1. Abnormalities in Prefrontal Cortex (PFC)
6.1.2. Abnormalities in Anterior Cingulate Cortex (ACC)
6.1.3. Hippocampal Changes
6.1.4. Amygdala Alterations
6.1.5. Insular Abnormalities
6.1.6. Abnormalities in Subcortical Areas
6.1.7. Abnormalities in Connections Between Brain Regions in MDD
6.2. The Role of Peripheral and Central Nervous System in FM and Chronic Pain
6.2.1. Abnormalities in Peripheral Nerves and Spinal Cord Pathways
6.2.2. The Role of Supraspinal Structures in Pain
6.2.3. Descending Pain Modulation in Chronic Pain Disorders
6.2.4. Structural CNS Changes in FM and NeP
6.3. Implications of CNS Overlap of Depression and Pain
7. Neuroendocrine, Autonomic and Immune Dysregulation in MDD, FM and NeP
7.1. Abnormalities of HPA axis in Depression and Pain
7.2. Autonomic Nervous System Abnormalities in Depression and Pain
7.3. Inflammatory Signaling in Mood and Pain Disorders
8. Neurotransmitters Implicated in MDD, FM and NeP
8.1.1. Glutamate and GABA in MDD
8.1.2. Monoamine Neurotransmitters in MDD
8.1.3. Endogenous Opioids and Other Peptide Neurotransmitters in MDD
8.2.1. Glutamate and GABA in Pain Disorders
8.2.2. Monoaminergic Neurotransmitters in Pain Disorders
8.2.4. Endogenous Opioids in Chronic Pain
8.2.5. Contribution of SP to Conditions of Chronic Pain
9. Cellular, Subcellular and Neurotrophic Changes in MDD, FM and NeP
9.1. An Overview of Glial Architecture and Function
9.2. Contributions of Glial Pathology to MDD
9.3. Neuron-Glia Interactions in Chronic Pain
10.Conclusion
11. Acknowledgements
12. References
1. ABSTRACT from the fact that these disorders share multiple biological
and environmental underpinnings. This perspective
This article synthesizes recent data suggesting suggests that these biologically complex conditions result
that the high rates of comorbidity observed between major from similar genetic vulnerabilities interacting with
depression, fibromyalgia and neuropathic pain likely result environmental adversity. Shared genetic determinants
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include poorly functional alleles regulating monoaminergic, Given these findings, it should perhaps not be
glutamatergic, neurotrophic, opioid and inflammatory surprising that recent developments in fields as diverse as
cytokine signaling. Chief among environmental risk factors social neuroscience and psychoimmunology point to the
are psychosocial stress and illness, both of which promote, fact that pain and depression co-exist symptomatically
in vulnerable individuals, relative resistance to because they are driven by largely overlapping
glucocorticoids, increased sympathetic/decreased pathophysiological processes in the brain and body. This
parasympathetic activity and increased production and review will attempt to review current scientific
release of proinflamnmatory mediators. Dysregulation of understandings of these shared processes and to
stress/inflammatory pathways promotes alterations in brain demonstrate how recent advances in our knowledge
circuitry that modulates mood, pain and the stress response. regarding the epidemiology, phenomenology and etiology
Over time, these functional changes likely promote of FM, chronic pain and major depression (MDD) provide
disruptions in neurotrophic support and disturbances of important clues for how the clinician may best approach
glia-neuronal communication. These changes, in turn, have these challenging clinical issues. Indeed, we believe that
been associated with the related processes of central gaining a better understanding of the shared
sensitization in pain disorders and “kindling” in depression, neurobiological bases of MDD, FM and neuropathic pain
both of which may account for the progressive and self- (NeP) will provide the clinician with important tools for
perpetuating nature of these disorders, especially when improving clinical decision making, and—by extension—
inadequately treated. patient outcomes.
2. INTRODUCTION 3. EPIDEMIOLOGY AND COMORBIDITY OF

MAJOR DEPRESSION, FIBROMYALGIA AND
Nowhere are the limitations of current psychiatric NEUROPATHIC PAIN
diagnostic schemas more apparent than at the interface of
Major Depressive Disorder (MDD) and chronic pain. Although 10-12% of the population worldwide
Indeed, one might emerge from a search through the DSM- and in the US endorse chronic widespread pain, only about
IV-TR with the impression that depression and pain had 2% (3.4% of women and 0.5% of men) of individuals meet
little in common. Pain is not listed as a symptom of any the American College of Rheumatology (ACR) criteria for
mood disorder, and depressive and anxiety complaints are Fibromyalgia (FM). FM is characterized by chronic
strikingly marginalized in the list of symptoms required to widespread pain (tenderness in at least 11 of 18 pre-defined
meet criteria for a chronic pain disorder. Unfortunately, points), lasting at least three months, typically accompanied
several decades of research demonstrate that this officially by fatigue and sleep disturbance (9, 10). While no single
sanctioned segregation of mood and pain maps poorly onto etiology has been identified for the condition, a unifying
both clinical and neurobiological reality. hypothesis that continues to receive increasing scientific
support suggests that FM is a consequence of sensitization
In reality, comorbidity between depression and of the central nervous system (9).
pain appears to be more the rule than the exception, with a
30-60% co-occurrence rate reported in a recent review (1). Neuropathic pain (NeP) is typically a
Similarly high rates of comorbidity have been repeatedly consequence of a direct nerve injury or damage to neural
observed between fibromyalgia (FM) and major tissue, resulting in abnormal sensory processing (11).
depression, leading to many years of debate between Common features of NeP include hyperalgesia (augmented
researchers as to whether the conditions are most sensitivity to painful stimuli), allodynia (abnormal pain
parsimoniously considered as separate illnesses with high response to non-noxious stimuli) and paresthesias (11, 12).
comorbidity or as differential symptom presentations of a Shared clinical features, consistent with a role for central
single underlying condition (2). Moreover, overwhelming sensitization, have prompted authors such as Martinez-
evidence suggests that chronic pain and depression do more Lavin and others to suggest that FM and NeP share an
than co-occur—they also promote the development of each overlapping pathophysiology (12, 13). Estimates suggest
other, such that chronic pain is strong predictor of that various forms of NeP (diabetic neuropathy,
subsequently developing major depression, and vice versa. postherpetic neuralgia, trigeminal neuralgia, spinal cord
When comorbid, pain and depression also significantly injury, radiculopathy, etc.) afflict up to 26 million people
complicate the treatment of each condition. For example, worldwide, including approximately 1.5% of the US
once treatment of depression is initiated, pain is a major population (11, 12). Consistent with these estimates, a large
obstacle to achieving remission (3, 4) and a significant risk survey of 6000 adults in the United Kingdom reported an
factor for relapse (5). In a three-year longitudinal study by 8% prevalence of chronic pain, most of which was of
Geerlings et al. the presence of painful symptoms neuropathic origin. As with FM, women were significantly
substantially reduced the chance of recovery in a group of more likely than men to be affected (14).
elderly depressed patients (n=327). Significantly more
patients with MDD alone attained recovery (47%), Many patients suffering with either FM or NeP
compared with only 9% of patients with MDD combined (or both) will also meet diagnostic criteria for major
with painful symptoms (6). Finally, a higher level of pain depressive disorder (MDD). Moreover, patients with FM
may delay the onset of remission in the treatment of MDD are at significantly increased risk of subsequently
(7), thereby decreasing the chances for an optimal outcome developing MDD and related psychiatric conditions. For
(8). example, Arnold et al reported that patients with FM were
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4.3 times more likely than healthy control subjects to precipitated or aggravated by stress (19, 28, 30, 33-36). In
develop MDD at some point in their lives and 4.7 times addition to peripheral and central sensitization, FM and
more likely to develop an anxiety disorder (15). NeP are also characterized by altered limbic and cortical
Surprisingly, Weir et al. found that although women were function and structure (28, 37, 38). The circuitry involved
much more likely to develop fibromyalgia in the first place, in modulating pain (typically altered in FM and NeP) has
once FM had developed the risk of males and females common elements with the circuits regulating stress
subsequently developing MDD was comparable: with a risk response and mood (36, 39-43). Remarkably, fMRI studies
ratio of 2.91 (95% CI 2.15-3.94) vs. 2.85 (95% CI 2.38-3.42) have demonstrated that brain areas (i.e. dorsal anterior
(16). Overall depression and anxiety are among the most cingulate) central to experience of negative affect in response
common comorbidities of FM, with prevalence rates ranging to physical pain also mediate distress in response to the “pain”
in studies from 20-80% and 13-63.8%, respectively (17). of social exclusion (44). These findings strongly suggest that
emotional and physical pain co-occur so often because they
The high comorbidity between depression and share the same central nervous system pathways (45).
pain is not restricted to subjects with FM, but is also Consistent with this, similar functional and structural changes
relevant for patients suffering with NeP. For example, in an in amygdala and hippocampus have been described in MDD,
Austrian study that surveyed 7707 individuals (18), the FM and NeP (46-51). Dysfunction of these limbic formations
prevalence of NeP was 3.3%, with two-thirds of the sample is believed to contribute to perturbations in neuroendocrine,
suffering from pain for longer than a year. Depression was autonomic and immune functioning that may further contribute
reported in 34% of the sample, anxiety in 25% (18). The to the generation and/or worsening of mood and pain
severity of pain tended to be enduring and associated with symptoms (28, 37, 42, 52). In this regard, increasing data
significant impairment of functioning. demonstrate that excessive sympathetic activation,
combined with elevated proinflammatory cytokine
4. CLINICAL RELEVANCE OF A production and release, likely plays a role in the etiology of
NEUROBIOLOGICAL UNDERSTANDING OF MDD, MDD, FM and NeP (19, 42, 53). Finally, at the cellular level
FM and NeP all three conditions are associated with disturbed neuron-glia
relationship, glutamatergic dysregulation and alterations in
Relationships between MDD, FM and NeP are intracellular signaling cascades and neurotrophic trafficking
complex and intricate. Based on clinical and (Figure 1) (54-61).
neurobiological similarities, some researches have even
made a claim that FM and NeP may be variations of the One clear clinical implication of the
same condition (13, 19). However, other researchers have neurobiological links between depression and pain is that
pointed out that, unlike FM which is characterized each set of symptoms worsens the other and/or makes the
exclusively by altered neural function, NeP has a other more likely to develop. A second implication arises
discernable neuro-pathologic substrate, such as a lesion of from the many empirical studies demonstrating that the
the peripheral nerve or CNS (e.g. central pain due to brain core symptoms of pain disorders (i.e. pain, chronic fatigue,
tumor) (20). Diagnostic quandaries aside, it is increasingly sleep disturbance and cognitive complaints) significantly
apparent that, like MDD, conditions characterized by complicate the treatment of MDD because these symptoms
chronic pain share a common a progressive course that is tend to be especially non-responsive to conventional
reflected in cognitive alterations, as well as structural treatments (4, 62-64). Conversely, both depression and
changes within the brain itself (21-27). anxiety are extremely common in FM and NeP (17, 18) and
may intensify the experience of pain (43, 65). In summary,
As noted above, a preponderance of current available evidence suggests that MDD and FM/NeP
scientific evidence suggests that NeP and FM are mutually amplify each other, thus contributing significantly
characterized by central sensitization (28, 29). Relevant to to treatment resistance in both depressive and pain
its relationship with pain, MDD is widely considered to disorders. Consistent with this, timely treatment of MDD
represent a “kindling” phenomenon. In the context of may optimize the chance of remission and decrease the
depression, kindling implies that each episode of chances of enduring structural changes (8, 21, 66-68). Full
depression makes subsequent depressive episodes more and sustained remission may also decrease the chance of
likely and less dependent upon an external impetus such as future recurrences (69). In this regard, the comorbidity of
stress or sickness (30). Robert Post—who first proposed MDD and pain may hinder an early and appropriate
kindling as a construct for understanding the tendency of diagnosis of MDD (70, 71), thereby delaying treatment and
mood disorders to worsen over time—has recently subsequent benefits of remission. Consistent with this,
suggested that kindling and sensitization may have similar resolution of painful symptoms doubled the remission rate
neurobiological underpinnings, such as neuroplastic in a recent study of depressed patients: 36.2% of the
changes and alterations in gene expression (31). In this subjects who had >50% reduction of pain on a visual
vein, some authors have gone so far as to posit “neuro- analogue scale (VAS) attained remission vs. only 17.8% of
sensitization” as a common etiology for chronic pain, the individuals who had <50% reduction on the VAS (3).
depression and anxiety disorders (i.e. PTSD) (32).
5. GENETICS OF MDD, FM AND NEP
Consistent with the predictions of a sensitization
model for pain and mood disorders, FM, NeP and MDD Given the high degree of overlap between MDD,
have many common features. All 3 conditions are either FM and NeP in terms of symptom profiles, disease course
5293
Figure 1. An integrated view of the shared neurobiological underpinnings of major depression (MDD), fibromyalgia (FM) and
neuropathic pain (NeP) .
and underlying neurobiology, it should perhaps not come as 5.1. Genetics of MDD
a surprise that these conditions also appear to share a Dozens of different genes have been implicated
number of genetic determinants. Interestingly, many of in the development of MDD. Among the many genes that
these shared genes contribute to the structure and/or have been identified as conferring vulnerability to the
functioning of pathways in the brain and body that evolved disorder, studies have most consistently supported a role
to respond to danger signals from the internal and external for polymorphisms in genes that regulate the serotonin
environment, whether these signals arise from transporter promoter locus (5HTTPR), the serotonin
environmental threat (i.e. stress), tissue damage (i.e. pain) 5HT2A receptor, catechol-O-methyl transferase (COMT),
or infection (i.e. inflammation). One result of the monoamine oxidase (MAO) and brain-derived
tremendous complexity and interdependency of these neurotrophic factor (BDNF). More recently, genes
systems is that MDD, FM and NeP are clearly genetically regulating the synthesis and/or activity of CRF and
heterogeneous conditions to which multiple polymorphic glutamate receptors have also been implicated (75). An
genes contribute to various degrees and in various exciting development in the field of psychiatric genetics is
combinations. On the other hand, because CNS and the recognition that genes are more likely to code for
peripheral danger pathways tend to respond in a “endophenotypic traits” that increase the risk of
coordinated and highly stereotyped manner to a variety of psychiatric morbidity, than they are to code directly for
environmental threats (72), it should also come as no any specific psychiatric disorder (74). For mood and pain
surprise that alterations in genes linked to very different disorders, these endophenotypic factors center primarily
physiological elements in this circuitry (i.e. neurotransmitters on circuitry essential for affective function, cognition and
and inflammatory cytokines) are capable of producing similar threat appraisal, as well as for stress and immune system
abnormalities in emotional state and pain perception, reflecting activity.
what is often referred to as a “final common pathway”
phenomenon. Nonetheless, even this level of analysis fails to 5.1.1. Genes involved in regulation of serotonergic
do justice to the full complexity of genetic contributions to function
MDD, FM and NeP, as demonstrated by epistatic interactions As noted above, “depression genes” frequently
in which two “bad” genes—rather than additively contributing do not contribute directly to the development of the
to disease development—actually cancel each other out (73). disorder, but rather confer vulnerability to developing
Finally, even the notion that genes specifically exist for MDD, depression in response to environmental threat. For
FM and NeP reifies these states of emotional distress and example, a study by Caspi et al. demonstrated that the
physical pain in a way that does violence to emerging scientific inconsistently observed association between depression
data demonstrating that risk factor genes do not cause these and the short allele of 5HTTPR was likely explained by
conditions, but rather predispose the brain and body to the fact that the allele increased the likelihood of
physiologically respond to internal and external environmental developing both depression and suicidal ideation in
conditions in ways that lead to symptom production (74). individuals exposed to life stressors, but not in individuals
5294
spared environmental adversity (76). This finding has allele of the 5HTTPR and the “met” variant of the val66met
been replicated several times. For example, Kendler et al BDNF was recently reported by Pezawas and colleagues. In
observed identical interactions between the 5HTTPR short a volumetric MRI study of 111 healthy, non-depressed,
variant (“s” allele), stressful life events and the risk of individuals, researchers found that—contrary to
developing MDD. In addition they described a gender expectations—the met BDNF allele (which increases
effect, such that women with the short 5HTTPR allele depression risk) provided protection to circuitry linking
were especially likely to develop depression in response to amygdala and rostral ACC (rACC). As noted above, this
stress (odds ratio of 7 compared to males with the long circuitry, which is involved in emotional modulation and
allele) (77). stress responses, tends to function sub-optimally and be
reduced in volume in 5HTTPR “s” carriers. However, these
The “s” allele of the 5HTTLPR gene has been abnormalities are significantly muted in 5HTTPR “s”
associated with a number of alterations in brain function carriers who also carry the met BDNF allele, because this
and morphology, including compromised functional and allele made amygdala-rACC circuitry relatively insensitive
structural integrity of amygdala-ACC circuitry involved in to 5HT signaling, thereby protecting these individuals from
regulation of emotional, behavioral and endocrine loss of rACC gray matter volume (73). This finding may
response to stress, as well as in processing the experience have significant clinical implications, given that rACC also
of pain (40, 41, 78-81). The short allele has also been plays a role in pain signaling and predicts treatment
linked to reduced anterior cingulate cortex (ACC), response to antidepressants (91).
amygdala and hippocampal volumes (78). These structural
changes, in turn, may result in aberrant connectivity Similarly, studies have reported that 5HTT,
between ACC and amygdala which is believed to interfere COMT, and MAOA polymorphisms and gender have a
with emotional homeostasis and effective modulation of convergent effect on the HPA-axis response to
the stress response (78, 79, 82). Gotlib et al. have psychological stress and endocrine challenges (92).
proposed relationships between the “s” 5HTT allele, Individuals with less functional 5HTT, COMT and MAOA
responses to stress, HPA reactivity and susceptibility to alleles had blunted baseline ACTH and cortisol responses
MDD, given data that girls homozygous for the “s” allele to DEX/CRH challenge, reflective of potential
reacted to stress with prolonged elevation of cortisol, susceptibility towards MDD. This study also implies that
compared to ones with “l” allele, indicating greater monoamines play an important role in neuroendocrine
reactivity to stress and possible increased susceptibility homeostasis and maintenance of health (92).
towards depression (83).
5.1.4. Polymorphism of genes regulating MAO
Of course, genes such as the “s” 5HTTPR allele Polymorphisms in the MAOA gene have been
don’t exist in isolation but interact in myriad ways with shown to interact with maltreatment with adverse impact
other genes—a process known as epistasis. For example, on children’s mental health. Depressed MAO-H (higher
the combination of the 5HTTPR “s” allele with a activity) carriers, tend to have compromised amygdala-
functionally less competent allele of the 5HT1A receptor prefrontal connectivity and greater illness severity (93).
gene has been associated with exaggerated amygdala
reactivity in medicated MDD patients (84). 5.1.5. COMT gene polymorphisms
The COMT Val108/158 Met genotype has a
5.1.2. The role of genes regulating neurotrophic factors significant impact on hippocampal and prefrontal gray
The activity of genes known to modulate matter volume (94). Beyond structural differences, Met
neurotrophic factors, cellular resilience, neuroplasticity and 158 COMT allele has been associated with increased
neurogenesis may be compromised in MDD patients (85, limbic reactivity to unpleasant stimuli and altered
86). For example, the val66met allele confers reduced connectivity between amygdala and PFC (95).
BDNF functioning and has been associated with structural
brain changes common in MDD, including reduced gray 5.1.6. Polymorphisms in genes involved in regulating
matter volume in dorsolateral prefrontal cortex (DLPFC), the inflammatory response
lateral-orbital prefrontal cortex (LOPFC) and hippocampus Polymorphisms in inflammation-related genes
(87, 88). Dwivedi et al reported altered genetic expression have been reported to confer susceptibility to major
of BDNF in hippocampus and PFC of depressed suicide depression and to effect antidepressant response (96, 97).
subjects, further supporting the relevance of neurotrophic Inflammatory reactions are an integral component of the
factors in MDD (89). stress response. Interestingly, proinflammatory cytokines
have a profound impact on mood, HPA axis regulation,
5.1.3. The Role of genetic epistasis in MDD monoamine signaling, as well as regulation of neurotrophic
Epistasis has been reported with the 5HTTPR factors and pain modulation (42, 53).
gene and a variant of the gene for BDNF known as
val66met. In a seminal study, Kaufman et al. reported that In summary, genes regulating monoamine
the “s” allele of 5HTTPR and the “met” variant of the receptors, transporters and enzymes involved in their
val66met BDNF allele increase the risk of developing metabolism, all seem to contribute to vulnerability towards
depression in an additive fashion in the context of MDD (98). These genes combined with ones regulating
environmental adversity early in life (90). On the other corticosteroid, neurotrophic and inflammatory signaling
hand, a very different epistatic interaction between the “s” influence structural integrity and functional connectivity in
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areas involved in generating adaptation to stress (86, 99, a risk for FM development, genetically-linked alterations
100). Consistent with this, a recent review suggested that of 5HTTLPR density in FM patients may be associated
genes associated with depression may act in unison by with symptom severity (108).
accelerating sensitization to stress (101).
Other genes implicated in mood and anxiety
5.2. Genetics of nociception and FM disorders that may also contribute to chronic pain states,
Significant data suggest that FM is genetically include COMT, as well as the 5HT2A and D4 dopamine
related to a wide range of conditions subsumed under the receptors. Zubieta et al reported an association between a
rubric of “affective spectrum disorders” (ASD), including common val158met polymorphism of COMT (catechol-
depressive and anxiety disorders, premenstrual dysphoric O-methyl-transferase, an enzyme involved in
disorder, attention deficit hyperactivity disorder (ADHD), catecholamine metabolism) and sensory and affective pain
FM, irritable bowel syndrome (IBS) as well as migraine ratings (116). Met homozygous carriers showed a
and cataplexy conditions. For example, a recent family diminished mu-opioid receptor response to pain, and a
study concluded that patients with FM were twice as stronger subjective experience of pain when compared to
likely to have at least one of these other conditions, heterozygous subjects. Other studies have found that this
compared with individuals without FM (OR 2.0, 95% CI COMT polymorphism may play a role in the stress
1.2-3.2, p = 0.004) (102). A recent study has refined our response, the trait of novelty seeking, cognition, MDD,
understanding of genetic links between mood and schizophrenia, anxiety disorders and ADHD (92, 117,
pain/somatic disorders by suggesting intriguing patterns of 118). The 5HT2A receptor gene is also a candidate for
genetic overlap and environmental specificity for these involvement in FM, given a recent study reporting that
conditions. Specifically, in a large twin study of the carriers of the T/T allele, while under-represented in the
relationship between two psychiatric disorders (MDD and FM population, had significantly higher pain scores than
generalized anxiety disorder [GAD]) and somatic FM carriers of the C/C allele or healthy controls (119).
syndromes such as FM, chronic fatigue, IBS and recurrent Interestingly, a separate study noted that the same T/T
headache, multivariate analyses suggested the influence of allele, in the presence of high maternal nurturance, was
two factors: one, most likely genetic, shared between associated with lower depressive symptoms than the C/C
somatic disorders, MDD and GAD and a second one, genotype, consistent with the notion that “vulnerability”
more specific to somatic conditions, that was more genes may have been maintained in the human gene pool
environmentally based. Thus, these conditions are a because they are “opportunity” genes given the proper
product of interaction of genes, some shared, some more environmental exposure (120). Finally, some reports have
specific to individual conditions, and environmental established a connection between a gene coding for the
factors (103). D4 dopamine receptor and a vulnerability towards FM
(119, 121). Other studies link the D4 receptor gene to a
It is not surprising that genetic studies of personality trait called “novelty seeking” and to ADHD
nociception are in their infancy given the genetic (118, 119).
heterogeneity of pain disorders, as well as the complexity
of relevant gene/environment interactions (104). In conclusion, genetic studies of depression, pain
Nonetheless, a number of genes known to modulate and FM have noted alterations in genes regulating (likely in
human nociception have emerged as potential risk factors a convergent manner) monoamine and inflammatory
for impaired pain processing, including genes coding for signaling (92, 115, 119). It is tempting to speculate that
opioid receptors, transient receptor potential cation shared genetic vulnerabilities towards depression and pain
channels (TRPV1), fatty acid amino hydrolase (FAAH) states may be reflected in dysregulation of circuitry
and GTP cyclohydrolase 1 (104, 105). Genes implicated involved in modulating stress responses, pain and
in mood disorders have also been identified as risk factors emotional states.
for FM and related pain states. These genes include
5HTTPR, the 5HT2A receptor, COMT and the dopamine 6. FUNCTIONAL AND STRUCTURAL
D4 receptor, as well the proinflammatory cytokines IL-1 ALTERATIONS IN CENTRAL NERVOUS SYSTEM
and IL-6 (105-110). Although not found by all studies CIRCUITRY INVOLVED IN REGULATION OF
(107), the association between FM and the “s” allele of EMOTION AND PAIN
5HTTLPR is particularly interesting given its association
with a wide range of conditions that are either risk factors Because emotional reality appears to be actively
for—or frequent concomitants to—FM, including anxiety, construed, rather than just passively experienced (122), a
neuroticism (best conceptualized as a tendency towards person’s world literally changes with changes in brain
excessive emotional reactivity to stressful stimuli), MDD, functioning. In the case of depression, these changes warp
Bipolar Disorder, Psychosis and even ADHD (111-114). reality into a frightening or empty realm that calls forth a
Given the significant role played by stress in the initiation depth of emotional pain that often defies verbal
of chronic pain and fatigue states, it is interesting that the description (123).This emotional pain frequently
“s” 5HTTLPR allele has also been shown to affect stress engenders an equally disabling experience of physical
responses and to be a risk factor for the development of pain, even when no peripheral source for such pain can be
depression and fatigue in response to chronic identified. Conversely, anyone who has long struggled
inflammatory exposure during interferon-alpha treatment with chronic physical pain knows that the dark emergence
for hepatitis C (115). In addition to potentially conferring of emotional despair commonly compounds the physical
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ache with a sense of affective terror. Given the numerous structures (134, 137) (Figure 2). Decreased activity in
links between depression and pain conditions such as FM DLPFC in MDD may contribute to the compromised
or NeP outlined thus far, it should come as no surprise that working memory, impaired sustained attention and
the different disorders also resemble each other in terms executive dysfunction seen in the disorder (137). In
of abnormalities in CNS structure and function. It is to addition to functional abnormalities in MDD, a recent
these abnormalities that we now turn. three-year prospective study reported significantly greater
DLPFC gray matter decline in un-remitted MDD patients
6.1. Structural and functional brain changes in compared to control subjects and to subjects with MDD
depression who attained remission (21).
6.1.1. Abnormalities in prefrontal cortex (PFC)
Prefrontal cortical (PFC) abnormalities are a 6.1.2. Abnormalities in anterior cingulate cortex
common finding in MDD. However, because different (ACC)
subregions of the PFC have profoundly different The anterior cingulate cortex (ACC) is probably
functional roles, a more detailed description of these areas the brain area most often implicated in the
and their activities is required to gain a sense of how this pathophysiology of MDD, with most studies emphasizing
brain region contributes to MDD. In this discussion we the subgenual ACC (sgACC) as being especially relevant.
will focus on three subregions most often implicated in The sgACC has a role in assessing the salience of emotional
MDD: ventromedial prefrontal cortex (VMPFC), lateral and motivational information and making necessary
orbital prefrontal cortex (LOPFC) and dorsolateral adjustments in behavior. It is also involved in modulation of
prefrontal cortex (DLPFC). sympathetic and neuroendocrine responses. Functional
imaging studies suggest increased metabolism in this area in
VMPFC tends to demonstrate increased activity depressed patients (when corrected for reduced volume)
in MDD (26, 124). VMPFC has rich reciprocal (138). Structural studies have noted significantly decreased
connections with limbic formations and the hypothalamus volume of sgACC in MDD subjects. For example, Drevets et
(125-127). VMPFC not only serves as a major recipient of al. noted that sgACC had 48% lesser volume in individuals
limbic projections, but also modulates amygdala and with familial depression than in healthy controls (130). These
hippocampal activity through complex feedback alterations likely contribute to disturbances of motivation,
connections (126, 128). VMPFC also plays a key role in limbic regulation (especially amygdala), and neuroendocrine
regulating appetitive drives and pain responses (129). function, all of which are commonly seen in patients with
Increased VMPFC activity in MDD patients has been MDD (48, 80, 138). Although more anterior areas of the
associated with melancholy ruminations and intensity of ACC have been a primary focus of research, more dorsal
negative affect (130, 131). These observations suggest areas of the ACC have received increasing attention for
that this prefrontal subregion, together with anterior their potential role in MDD. For example, in a study of
cingulate cortex (ACC) and limbic areas, is a component patients with MDD, Chen and colleagues reported that
of an integrated network involved in processing reduced gray matter volume of dorsal ACC and DLPFC were
emotionally relevant information for the purpose of correlated with increased symptom severity in MDD patients
guiding behavior and orchestrating adaptive autonomic (91). This report resonates with the work of Matthews et al.
and endocrine responses (130). Given the key regulatory who noted reciprocal connectivity between supragenual ACC
role of VMPFC, it is intriguing that several authors have and amygdala in unmedicated depressed patients (48). In
found significant VMPFC volume reduction (up to 32%) summary, recent research lends credence to the hypothesis
in MDD patients compared to healthy controls (132, 133). that these “executive network” areas have a role in “top-
down” modulation of limbic areas and that when this
Lateral orbital prefrontal cortex (LOPFC) seems modulation fails, depression is likely to ensue (134, 139).
to be involved in suppressing maladaptive and Finally, several lines of evidence suggest that ACC
perseverative emotional responses. LOPFC may also have abnormalities are associated with treatment response.
a major role in volitional regulation of emotion and Mayberg et al. has noted that activity in sgACC normalizes in
cognitive reappraisal (122, 134). LOPFC activity is patients who respond to either an active antidepressant or
enhanced in depression (26), most likely in a placebo (137), and diminished function and volume of pre-
compensatory role to modulate excessive limbic activity. genual ACC has also been associated with a delayed
Lacerda et al have found a significant reduction in LOPFC antidepressant treatment response (91).
gray matter volume of MDD patients compared with
healthy subjects (133). Medial dorsal PFC (MDPFC), 6.1.3. Hippocampal changes
associated with automatic emotional and attentional The hippocampus is a key limbic area located at
control and self-appraisal (134, 135), also suffers a greater the “crossroads” of circuitry that regulates the stress
gray matter decline over time in MDD patients compared response by providing inhibitory feedback to the HPA
to healthy controls (20). axis, in addition to its role in mood modulation and
memory formation (68, 125). However, its central
Dorsolateral prefrontal cortex (DLPFC) is a location may render the hippocampus vulnerable to
primary component of an executive function network in functional dysregulation that accompanies extreme
the brain that also includes dorsal ACC (dACC) and parts stress and mood disorders (125). Reflecting this
of the parietal cortex (130, 314, 136). DLPFC tends to vulnerability, alterations in hippocampal volume are
have a “top-down” regulatory influence over limbic among the most common imaging findings in
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Figure 2. Cortical and subcortical areas involved in voluntary and automatic regulation of mood and the stress response.
Reprinted with permission from (134).
MDD patients (68). Recent data strongly suggest that investigators found a significant association between
physiological abnormalities, inherent to depression, may hippocampal increases in N-acetylaspartate (NAA) (a
actually lead to reductions in hippocampal volume. marker of neuronal density, function and myelination) and
Indeed, Frodl et al found a significant decline over a choline compounds (Cho) (a marker of membrane
three-year period in hippocampal gray matter volume in integrity and metabolism) and treatment response. These
MDD patients compared to healthy controls (21). authors also reported reduced glutamine (Gln) in
Successful treatment appeared to have a protective effect, depressed subjects. Glutamate (Glu), a major excitatory
given that remitted patients had a significantly greater neurotransmitter is metabolized into Gln, after being taken
hippocampal density than non-remitted ones (21). up by astrocytes. Gln has major significance for
Consistent with this, one post hoc analysis (68) has found maintenance of Glu/GABA balance in neuron-glia
a significant increase in hippocampal volume (21%, signaling. This is the first study providing evidence that
p=0.004) after 6-7 months of antidepressant treatment in a successful treatment with two different antidepressants, a
small subset of patients with atypical depression. predominantly noradrenergic and a predominantly
Similarly, Sheline et al found an inverse relationship serotonergic one, may lead to functional and structural
between the days of untreated depression and restoration of hippocampal neurons and astroglia (142).
hippocampal volume (140), and Colla and colleagues
noted a negative correlation between duration of 6.1.4. Amygdala alterations
depression and hippocampal volume, corrected for age As with other relevant brain regions,
and intracranial volume (141). The potential importance neuroimaging studies suggest that functional
of restoration of hippocampal structure and function for abnormalities of the amygdala may contribute to
successful treatment of depression was further highlighted depressive symptom development and that
by a study conducted by Block et al (142). Using pathophysiological processes inherent to depression may,
magnetic resonance spectroscopy (MRS), these in turn, damage this extremely important brain structure.
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Functional neuroimaging studies have, for the most part, 6.1.7. Abnormalities in connections between brain
found increased activity in the amygdala of depressed regions in MDD
individuals (143). Because the amygdala plays an Several studies have found altered connectivity
important role in rapidly assessing and assigning between limbic and paralimbic prefrontal areas in MDD
emotional value to surprising and ambiguous stimuli, it is (144, 157-159). Combined with studies that have found
not surprising that patients with MDD respond to angry white matter abnormalities in patients with MDD (158),
and fearful faces with increased amygdala activity, even these studies point to a compromise in the integrity of
when these faces are presented below the level of fronto-subcortical and prefrontal-limbic circuits in the
conscious awareness (143-145). It is intriguing to disorder (48, 157, 159). Additional involvement of fronto-
consider that this type of amygdala over-activity may cerebellar-thalamic circuitry is likely (151). Therefore,
translate into the increased anxiety and emotional cumulative evidence suggests that disruption of circuitry
misattribution, both of which are commonly observed that provides cognitive-emotional integration and control
in patients with MDD (146, 147). Because the of stress responses may be responsible for complex
amygdala and other limbic structures have significant manifestations of MDD. In summary, structural and
bidirectional connections with the hypothalamus, it is functional studies support an organic basis for the
also not surprising that sympathetic and emotional, cognitive and neuroendocrine symptomatology
neuroendocrine dysregulation are a frequent of MDD (128, 138, 158). Unfortunately, current research
concomitant of mood disorders (125, 128). Structural indicates that cognitive impairments in MDD tend to be
neuroimaging studies of the amygdala suggest that this persistent, non-specific and progressive, with a greater
brain region is negatively impacted by physiological number of episodes being associated with increasing
processes inherent to depression. The same prospective cognitive burden (160, 161).
study that recently reported an association between
ongoing depression and loss of brain volume in DLPFC 6.2. The role of the peripheral and central nervous
and hippocampus also noted a decline in amygdala system in FM and chronic pain
gray matter (esp. left) over time in MDD patients, 6.2.1. Abnormalities in peripheral nerves and spinal
relative to control subjects (21). cord pathways
Pain pathways implicated in NeP and FM have
6.1.5. Insular abnormalities peripheral and central components—in this section we
Insular cortex has emerged in recent years as trace how pain signals originate in the periphery and reach
another limbic structure of tremendous relevance to the the brain
regulation of both affect and pain. Insula has rich
connections with other limbic (e.g., amygdala and Pain signals are detected by peripheral
hippocampus) and paralimbic cortical areas (ACC and nociceptive nerve endings and conveyed to neurons
LOPFC). Because of this, it is widely considered to be located in dorsal root ganglia (DRG). From the DRG pain
a primary target of thalamo-cortical projections. As information is conducted by lightly myelinated A-delta
such, the insula plays an essential role in sensory- and un-myelinated slow C-fibers to secondary sensory
affective integration that creates a bodily sense of self. neurons localized in the dorsal column of the spinal cord.
The anterior insula is also involved in modulating the Aside from functional alterations in nerve membranes and
influence of sensory and emotional distractors (148- endocellular signaling (discussed elsewhere), peripheral
150). Consistent with these roles and interconnections, sensitization may occur as a result of alterations in
altered activation of insula in MDD subjects has been synaptic connectivity resulting from sprouting of
described in several studies (26, 150, 151). In general, sympathetic axons within DRG (which may further
MDD patients appear to have decreased activity of augment pain transmission), ectopic discharges and
insula, which tends to improve with antidepressant ephaptic (direct electrical transfer of signal)
treatment (26, 150). Neuroimaging studies, utilizing communication. Central sensitization in NeP may in part
labeling of 5HT2 receptors, have also found evidence be mediated by collateral sprouting (whereby non-
of altered sertononergic transmission in insula in MDD nociceptive A-beta fibers form new connections with
subjects (148). nociceptive neurons in the dorsal horn), as well as damage
to inhibitory GABA inter-neurons (29, 162, 163).
6.1.6. Abnormalities in sub-cortical areas
Functional and structural changes have been Damage to these initial components of pain
noted in basal ganglia and thalamus of patients with mood processing circuitry (e.g. nerve injury) is the primary
disorders (152). Functional studies have noted increased cause of altered pain signaling in NeP. On the other hand,
thalamic activity in depressed individuals. Thalamic there are fewer data to support an important role for
hyper-metabolism is associated with increased activity in abnormalities in peripheral or spinal cord pain signaling in
sgACC and decreased metabolic activity in dACC (153), FM. Nonetheless, some evidence does indicate potential
both of which have been observed in MDD. Some, but not peripheral contributions to the disorder. For example,
all, studies have found alterations in cerebellar function in Salemi et al. have performed skin biopsies in 53 FM
patients with MDD (26, 152). This is of particular interest patients and found mononuclear and fibroblast-like cells
because the vermis of the cerebellum has been implicated adjacent to nociceptive neuronal fibers that stained
in generating automatic emotional responses—including positive for inflammatory cytokines, suggesting a role for
empathy—to facial expressions (154-156). neurogenic inflammation in the etiology of the FM (164).
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Other authors have found evidence of changes in Schwann fMRI studies like these provide objective
cell morphology in the skin (165) and altered blood flow evidence of altered cerebral processing of painful stimuli
in the muscles of FM patients (166, 167). in FM patients. More recently, abnormalities in some of
the same brain regions have also been observed using
6.2.2. The role of supraspinal structures in pain magnetic resonance spectroscopy (MRS). For example,
After synaptic processing in the dorsal horn of Petrou et al. noted altered choline compounds (markers of
the spinal cord, pain signals are propagated via membrane metabolism and myelination) in DLPFC of FM
spinothalamic (paleo-spinothalamic) and patients compared to healthy controls (173) Alterations in
spinoparabrachial (neo-spinothalamic) tracts to higher choline significantly correlated with subjective pain
CNS pain centers. Spinothalamic signals are relayed intensity. Another MRS study found evidence of increased
through thalamus to somatosensory cortices I and II (SI glutamatergic activity in the insula of FM patients that
and SII) and associated areas, including insula, ACC correlated with measures of clinical pain (174). In an
and posterior cingulate cortex (PCC). ACC, in turn, has earlier study, the same group described decreased mu-
close bidirectional connections with amygdala and opiate receptor binding potential in the cingulate cortex,
hippocampus. Spinoparabrachial fibers convey amygdala and nucleus accumbens of FM individuals
information to the parabrachial nucleus in the compared to controls, a finding that may explain why
brainstem and then on to amygdala, hippocampus and opiate medications are often ineffectual in reducing FM
hypothalamus. Ascending pain signals and information pain (50).
from supraspinal pain circuitry are integrated in the
mesencephalic periaqueductal gray area (PAG), which Temporal summation of “second pain” (TSSP) is
also has a pivotal role in regulating descending pain one of the landmark clinical features of FM. TSSP results
pathways (163, 168). DLPFC and LOPFC appear to from repetitive stimulation of C-fibers and is believed to
initiate the descending pain modulatory sequence, reflect a summation mechanisms in dorsal horn neurons
explaining how attention and anticipation may (i.e., “windup”), therefore it is a basic substrate for
influence the intensity of pain. These prefrontal areas, widespread central sensitization in FM (175). Consistent
richly innervated by dopamine fibers, can trigger opioid with this, Staud et al found that TSSP correlated with
release in PAG, substantially reducing the intensity of elevated activity in S1, S2, thalamus, insula and ACC in
experienced pain (descending pain modulatory system FM subjects relative to healthy controls (176). It appears
will be discussed in more detail, later in the text (38, 169). that enhanced pain sensitivity in the context of FM may
arise more from altered activity of the entire “pain
Imaging studies have consistently identified matrix”, rather than from individual components.
several brain areas as having a major role in pain
processing, including primary and secondary 6.2.3. Descending pain modulation in chronic pain
somatosensory cortices (S1 and S2), thalamus, insula, disorders
ACC and PFC. Together these brain areas are commonly Imaging studies have consistently suggested that
referred to as the “pain matrix” (170) and many studies both FM and NeP are characterized by dysregulation of
indicate that function is disrupted in this matrix in the pain processing circuitry involving ACC and insula. These
context of chronic pain states, including FM and NeP. For two formations appear to have a significant role in
example, Bailiki et al. utilized fMRI to study chronic back regulating descending pain modulatory pathways that
pain patients. These authors reported an association include PAG and rostro-ventral medulla (RVM) (177-
between the intensity of spontaneous pain and activation 179). Insular cortex appears to exert regulatory control
of medial PFC (mPFC) (49), an area known to have a over descending inhibitory tracts that project from the
role in automatic emotional regulation (134). On the RVM to the dorsal horn of the spinal cord via the
other hand, in this population, duration of pain was dorsolateral funiculi (DLF). ACC has a principal role in
most strongly associated with increased activity in the modulating descending pain facilitatory pathways, which
insula (49). A second fMRI study noted a greater project from RVM via the ventrolateral funiculi (VLF) to
activation in DLPFC and ACC in response to non- the dorsal columns. NE, 5HT and Ach are the principal
painful stimuli in patients with FM relative to control neurotransmitters in inhibitory descending pathways
subjects, a finding likely to reflect alterations in (177). Primary neurotransmitters in facilitatory
processes central to the cognitive and emotional descending pain pathways include glutamate, 5HT,
aspects of pain, such as attention and anticipation neurotensin, cholecystokinins and BDNF (177, 178, 180).
(171). In response to an equivalent pressure stimulus, Dysregulation of descending pain pathways, as a result of
patients with FM have been shown to demonstrate inadequate activity in the inhibitory pathway, excessive
increased activity in several areas of the CNS pain activity in the facilitatory pathway or both, has been
matrix when compared to normal control subjects, proposed as an important etiological factor for the
including S2, insula, posterior cingulate cortex (PCC), secondary hyperalgesia/ allodynia often observed in FM
ACC, superior temporal gyrus and inferior parietal and NeP (178, 180, 181).
lobule (172). Moreover, mild pressure applied to
subjects with FM elicited subjective pain and cerebral 6.2.4 Structural brain changes in FM and NeP
responses similar to the responses seen in normal In addition to functional differences, several
subjects when twice as much pressure was applied studies have found significant structural changes in the
(172). brains of FM patients. Kuchinad et al. reported
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significantly reduced gray matter density in the cingulate in ways that cause depression and pain conditions to
cortex, insula, mPFC and the para-hippocampal lobe of worsen each other in a feed-forward circuit. For example,
FM patients when compared to a control group (24). As multiple lines of evidence (i.e. electrophysiological,
with depression, the physiological changes that biochemical, and imaging) are indicative of compromised
accompany FM may themselves damage brain structures function and structure of the amygdala in chronic pain
over time, given that in this study duration of illness (43). Depressive and anxious feelings, as well as chronic
correlated with greater gray matter changes, such that stress, may enhance amygdala responsivity to pain (43).
each year of disease had an impact equivalent to 9.5 times Rainville et al. have demonstrated that negative emotions,
the loss due to normal aging. Changes in these areas a defining feature of MDD, enhance the perception of pain
appear to contribute to the compromised pain regulation, and associated autonomic responses to painful stimuli
emotional modulation, stress responsivity and cognitive (65). The very similar functional and morphological
functioning, often described in FM patients (24). For alterations of DLPFC in MDD and chronic pain (21, 22,
example, Luerding et al. reported that neurocognitive 26), may conspire to disrupt the “top-down” regulation of
deficits in FM patients correlated with reduced gray depression and pain, eventually impacting the function of
matter volume in DLPFC and ACC (areas typically descending pain pathways (while at the same time
associated with executive function), additionally pain compromising cognition, and therefore, coping abilities)
scores were noted to be negatively correlated with gray (182).
matter volume in mPFC (25). A recent MRS study noted
lower NAA levels in hippocampus of FM patients Ventral striatum/ nucleus accumbens (N.Acc.) is
compared to controls. NAA levels are commonly a critical component of both stress/pain response and
considered to be a marker of neuronal and axonal integrity reward/analgesic systems (183-185). N.Acc. receives
and function. Hippocampal alterations may be associated cognitive information from the PFC, emotionally relevant
with impaired pain regulation, cognition, sleep and signals from amygdala, and contextual information from
neuroendocrine function in FM (47). hippocampus (183). It is richly innervated with
dopaminergic and opiate fibers (183, 184, 186). Feedback
Fewer neuroimaging studies have been conducted fibers from N.Acc have a key role in modulating VTA
in neuropathic pain than in either depression or FM. dopaminergic output, and therefore play an important role
Nonetheless, fMRI studies of neuropathic pain have in maintaining the balance between meso-cortical and
implicated brain areas known to be functionally abnormal meso-limbic dopaminergic activity (183). Neuroimaging
in FM and chronic non-neuropathic pain, including PFC, evidence suggests that N.Acc/VTA regulation may be
thalamus, insula and ACC (38, 41). Apkarian et al. disrupted in MDD and chronic pain states (187, 188).
utilized volumetric MRI to assess gray matter changes in a Excessive activation of amygdala/sgACC/vmPFC in
group of chronic back pain (CBP) sufferers, the majority chronic stress, pain and depression may interrupt VTA
of who had neuropathic pain (22). The investigators found dopaminergic transmission leading to impaired reward
significantly reduced gray matter volume in DLPFC and perception, cognitive dysfunction, decreased interest in
thalamus of CBP patients when compared to controls. novelty, compromised pain regulation and possibly,
Moreover, decreased gray matter density in DLPFC was vulnerability towards substance abuse (184-188).
correlated with pain intensity, duration and negative
affective characteristics in this population. Disturbingly, In summary, MDD, pain and chronic stress
the magnitude of gray matter reduction in CBP patients synergistically activate limbic circuitry, mutually
was equivalent to 10-20 years of normal aging. Wiech et amplifying the distress signal, while simultaneously
al. have reviewed the evidence that establishes a degrading the regulatory influence of prefrontal cortical
supporting role for DLPFC and dorsal ACC in volitional structures.
pain control via activation of descending pain modulatory
pathways (182). Considering the well-established role of Depressed patients activate DLPFC and VLPFC
DLPFC in top-down regulation of limbic and paralimbic in response to pain in a significantly greater manner than
prefrontal areas, it is conceivable that morphological healthy controls, which may be interpreted as a
changes in DLPFC may contribute to the compromised compensatory attempt to suppress negative emotions
emotional and pain modulation apparent in NeP patients (189). Consistent with this view, Graff- Guerrero et al.
(22). Prefrontal structures, through processes of attention, reported decreased cerebral blood flow (CBF) in areas
expectation and reappraisal, also appear to play an associated with emotional response to pain, such as
important role in the cognitive modulation of pain (182). amygdala, hippocampus, insula, ACC and PCC after
antidepressant treatment. Changes in CBF coincided with
6.3. Implications of the CNS overlap of depression and increased experimental pain threshold and tolerance (190).
pain Depressed patients may also have an impaired ability to
A striking feature of neuroimaging studies, modulate pain due to heightened emotional reactivity. For
reviewed thus far, is the significant overlap in brain areas example, Strigo at al. noted increased activation of insula,
that are functionally and/or structurally abnormal in dACC, and amygdala in MDD subjects anticipating pain
MDD, FM and NeP. These brain areas include DLPFC, compared to controls (191). Depressed patients also had
mPFC, LOPFC, insula, ACC, amygdala, hippocampus decreased activation of rACC and PAG, implicating
and thalamus. As would be expected from this overlap, compromised functioning in descending pain modulatory
symptoms of pain and depression also ramify each other pathways. Greater activation of amygdala in this study
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was associated with increased levels of perceived nervous system and inflammatory response system in
helplessness, indicating a possible bidirectional major depression, FM, NeP and other conditions of
relationship between depression and pain (191). Changes chronic pain and distress.
in ACC function and structure have been noted in MDD,
FM and NeP (21, 24, 41, 91). Some authors have 7.1. Abnormalities of the HPA axis in depression and
proposed that altered ACC function may result in pain
increased activity of facilitatory descending pathways 7.1.1. HPA abnormalities in depression
(179, 192). Similarly, changes in insula in MDD and FM Hyperactivity of the HPA axis (with resultant
may negatively impact inhibitory descending pathways hypercortisolism) is one of the most replicated
(24, 26, 177, 181). physiological abnormalities seen in patients with major
depression (193). Many studies suggest that this
In summary, when viewed as a continuum MDD, hyperactivity commences with overproduction of
FM and NeP overlap significantly in terms of associated corticotropin releasing hormone (CRH) (the primary
brain changes. When viewed as separate diagnostic secretagogue for the HPA axis), which is released from
conditions they clearly exist in complicated bidirectional the paraventricular nucleus (PVN) of the hypothalamus,
relationships with each other, such that depression may whenever higher brain areas (or signals arising in the
give rise to altered pain processing and alterations in pain body) indicate potential threat to the organism (194).
processing may promote affective states conducive to the Findings indicative of CRH over production in MDD
development of depression. Shared patterns of include 1.) elevated cerebrospinal fluid (CSF)
dysregulation in circuitry involved in modulating emotion, concentrations of CRH; 2.) increased CRH mRNA and
pain and the stress response, especially as manifested by protein in the PVN (postmortem samples); 3.) blunted
excessive activation of amygdala and compromised ACTH response to CRH challenge (likely reflecting
hippocampal function and structure in MDD and FM, may ACTH receptor downregulation secondary to increased
also have neuroendocrine, autonomic and immune CRH), and 4.) downregulation of CRH receptors in the
repercussions, to which we turn next. frontal cortex of suicide victims (many of whom were
presumably depressed) (195-200). Reflecting the fact that
7. NEUROENDOCRINE, AUTONOMIC AND CRH drives production of adrenocorticotropin (ACTH)
IMMUNE DYSREGULATION IN MDD, FM AND and the subsequent release of glucocorticoids (i.e. cortisol
NEP in primates, corticosterone in rodents) from the adrenal
glands, MDD is also frequently associated with
A central thesis of this article is that disorders of hypercortisolemia. Evidence for this in MDD includes 1)
mood and pain share so many commonalities in symptoms increased number of cortisol and ACTH secretory pulses
and disease course precisely because these conditions are across the circadian cycle resulting in elevated
characterized by similar patterns of dysregulation in the concentrations of cortisol in the peripheral circulation,
activity and structure of brain regions and circuits that urine and CNS; 2) an exaggerated cortisol response to
play a primary role in sensing, evaluating and responding adrenocorticotropin (ACTH); and 3) enlargement of
to danger, whether the danger be from the external (e.g. a pituitary and adrenal glands, suggestive of hypertrophy
predator or social threat) or internal (e.g. infection, tissue secondary to HPA overdrive.(201-205)
damage) environment. This perspective provides clarity to
the otherwise rather remarkable finding that disorders as The notion that MDD is characterized by
diagnostically diverse as major depression, post-traumatic insufficient glucocorticoid signaling seems at first blush to
stress disorder, FM and chronic fatigue syndrome share be directly contradicted by the multiple findings indicative
similar abnormalities in neuroendocrine, autonomic and of increased—rather than decreased—HPA axis activity in
immune function. At the most basic level, we would the disorder. However, the signaling strength of any
suggest that these brain-body commonalities reflect the hormonal system depends not just on hormone levels, but
fact that many conditions currently parsed into separate also on how effectively the signal “gets through”, which is
DSM diagnoses can be understood as states of CNS and also dependent upon receptor sensitivity, as well as
peripheral danger system hyperactivity. Moreover, even ancillary factors such as binding proteins (206). And in the
the patterns of hyperactivity observed across these context of MDD, many years of data demonstrate that
multifarious conditions are strikingly stereotyped and glucocorticoid receptors (GR) have reduced activity,
have as their primary features insufficient glucocorticoid leading to a relative failure in cortisol signaling, even in the
signaling, increased sympathetic and/or reduced context of increased cortisol levels (207, 208). Indeed, a
parasympathetic tone and activation of innate immune strong association exists between increased cortisol blood
inflammatory pathways (42). Increasing evidence suggests levels and decreased GR sensitivity in MDD (209),
that these patterns of stress—immune system derangement suggesting that rather than being indicative of excessive
not only purvey a heightened risk for the development of glucocorticoid tone, the hypercortisolism of depression is
medical illness, but may also provide physiological more often a reflection of relative GR resistance and hence
feedback to the brain that promotes the development of inadequate glucocorticoid signaling.
both depression and chronic pain. In this section we will
attempt to enlarge upon the role of danger system In addition to being a primary mediator of the
dysregulation in depression and pain by describing and stress response, cortisol is also the body’s primary anti-
comparing abnormalities in the HPA axis, autonomic stress and anti-inflammatory molecule, so one would
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expect that insufficient glucocorticoid signaling would lead reduced sensitivity is highlighted by findings that high
to an inability of neuroendocrine stress pathways to levels of cortisol following the DST (i.e. glucocorticoid
terminate activity and to a release of inflammatory resistance) strongly predicted the future development of
processes from regulatory control (206). Both CWP over a 15-month period in a group of 267 at-risk
abnormalities are seen in MDD and strongly argue for the individuals based on a somatasizing personality style (226).
fact that glucocorticoid signaling is insufficient in the Also suggesting that reduced glucocorticoid sensitivity may
disorder. Rates of impaired glucocorticoid responsiveness contribute to symptom development are findings from a
in the HPA axis (as a result of the axis being insensitive to trial of a multidisciplinary intervention in FM. The
cortisol-mediated inhibitory feedback control) vary from investigators found that symptomatic improvement over a
approximately 25 to 80% (201), depending on depressive 3-week period was associated with increased mRNA
symptomatology (highest rates are found for melancholic, expression for the active form of the glucocorticoid
or endogenous, subtypes) (210), age (older subjects are receptor (which would be expected to enhance
more likely to exhibit glucocorticoid resistance) and the glucocorticoid signaling) (227).
technique used for assessment (the DEX-CRH stimulation
test is more sensitive than the traditional dexamethasone To resolve whether the end products of the HPA
suppression test [DST]).(201, 211) Of note, both the DST axis (i.e. glucocorticoids) contribute to depression, fatigue
and the DEX-CRH test have been shown to powerfully and pain primarily through overactivity or under-activity
predict clinical response and relapse (212-215). And in the (i.e. insufficient signaling), several lines of evidence can be
case of the DEX-CRH test, there is evidence that impaired marshaled based on known effects of glucocorticoids on a
glucocorticoid responsiveness represents a genetically- variety of relevant physiological and behavioral endpoints.
based risk factor for the development of depression (216). Because glucocorticoids exert profound anti-inflammatory
Complimenting in vivo findings, in vitro studies have effects in the brain and body, one would expect evidence
demonstrated that peripheral immune cells from patients for reduced inflammatory activity in MDD, FM and CFS if
with major depression exhibit decreased sensitivity to the these conditions were states of excessive glucocorticoid
well-known immunosuppressive effects of glucocorticoids activity. As will be reviewed in some detail below, the
(207). Whereas normal subjects show a marked inhibition opposite appears to be the case as evidence for increased
of in vitro natural killer (NK) cell activity, lymphocyte inflammation in these conditions mounts. Similarly, if
proliferation or cytokine production following increased glucocorticoid signaling contributes to symptom
glucocorticoid (usually DEX) exposure, patients with major development in these disorders, one would expect that
depression, especially DST non-suppressors, consistently adding exogenous corticosteroids would worsen symptoms.
show an attenuated inhibitory response (217-220). In fact, quite the opposite appears to be the case, given
studies showing that hydrocortisone, although not widely
7.1.2. HPA abnormalities in FM and chronic pain used due to risk for adverse events, demonstrates
Although not entirely consistent, most studies antidepressant efficacy and improves symptoms in patients
report that conditions of chronic pain, fatigue and other with CFS and PTSD, both of which are frequently
somatic symptoms (i.e. FM, chronic fatigue syndrome comorbid with FM (228-231). If cortisol routinely
[CFS]) are characterized not by the hypercortisolism of contributed to the high rate of FM development that occurs
depression but by decreased cortisol production and after motor vehicle accidents, one would expect high levels
release, both at baseline and in response to a variety of of the hormone to predict subsequent symptom
stressors (206, 221). Interestingly, depressive conditions development, but in fact the opposite appears to be the
characterized by atypical symptoms (i.e. increased sleep, case, with several studies showing that reduced cortisol
increase appetite and profound lethargy) also appear to be responses to motor vehicle accidents strongly predicts
associated with reduced HPA axis activity (blunted subsequent PTSD, which is, in turn, a significant risk factor
cerebrospinal CRH) (222), perhaps reflecting the fact that for the development of FM-type symptoms. Consistent with
pain and exhaustion feature prominently in this type of a protective effect for cortisol, patients who receive stress
depressive presentation. As with MDD, the pressing doses of hydrocortisone as part of their treatment while in a
question revolves around whether decreased cortisol medical intensive care unit (ICU) have reduced rates of
production and release leads to glucocorticoid insufficiency ICU-related PTSD symptoms (232), and subjects
in conditions of chronic pain and fatigue or whether administered cortisol or prednisone prior to exposure to
changes in glucocorticoid receptor activity compensate (or standardized laboratory stressors report less distress and
even over-compensate) for the reduced levels of hormone. less fatigue (233, 234). Finally, the administration of
The relevance of this question is highlighted by the fact that hydrocortisone to aging veterans with PTSD was shown to
the idea of glucocorticoid receptor supersensitivity in FM, enhance both episodic and working memory (235).
CFS (and PTSD) has gained significant currency in recent
years based on studies showing increased sensitivity to Less controversial than whether glucocorticoid
feedback inhibition of both the HPA axis and the immune signaling is excessive or impaired in the context of pain and
system in these frequently comorbid conditions (223, 224). depression is the consistent finding that the diurnal rhythm
However, although not as widely considered, a significant of the HPA axis is flattened in MDD, anxiety and
number of studies support the opposite conclusion—that conditions associated with fatigue and pain, such as FM
(as with MDD) FM, chronic widespread pain (CWP) and and CFS.(236-238) In healthy individuals, cortisol
CFS are conditions of reduced glucocorticoid sensitivity production peaks around wake time and then falls thereafter
(225). The potential pathophysiological relevance of this (excepting “blips” of increased cortisol in response to
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meals) until the early morning hours when it commences its As with MDD, disturbances in
climb toward wake-time (239). In a variety of conditions SNS/parasympathetic balance have been frequently
linked to stress, pain and emotional misery, this pattern is reported in FM and highly comorbid conditions such as
disrupted, such that cortisol levels are lower around PTSD and CFS and may contribute to disease pathology
wakening, show more disorganized variability during the by perpetuating neuroimmune dysregulation and
day and do not fall sufficiently in the evening. Such facilitating neurogenic inflammation (42, 263). In one of
flattening is an ominous development indeed, given that it the rare studies in men with FM, Cohen et al. conducted
predicts the following: subsequent development of chronic power spectral analysis of HRV and found that orthostatic
widespread pain in medically healthy individuals (226), intolerance in FM patients may result from sympathetic
development of fatigue and depression in patients receiving hyperactivity and concomitant reduced parasympathetic
interferon (IFN)-alpha (240) and increased mortality in tone (264). Several other authors have identified
patients with cancer (241). In animal models, flattening of sympathetic overactivity as a component of the stress
the cortisol rhythm impairs the production and release of response that both precipitates and perpetuates FM and
BDNF and impairs neurogenesis in brain regions in which NeP symptoms such as fatigue, sleep disturbance, anxiety,
such neurogenic processes are tightly linked to depression, vasomotor instability and gastrointestinal
antidepressant response (242, 243). Behavioral complaints (19, 265). In an intricately designed,
interventions that improve pain symptoms in FM patients controlled, study, Torpy et al. observed excessive
also steepen the cortisol slope.(227). sympathetic and HPA activity in FM patients after an IL-6
injection, supporting the notion that FM may, at least in
7.2. Autonomic nervous system abnormalities in part, be disorder of the stress system dysregulation (266).
depression and pain A number of studies report increased heart rate in FM
Given the known role of glucocorticoids in (267). HRV is also reduced and shows abnormalities in
modulating autonomic nervous system (ANS) activity circadian patterning (268-270), although the association
(206), if the hypercortisolism of depression and the between reduced HRV and FM may be stronger in women
hypocortisolism of FM, CWP and CFS were really than men (271). One study noted that FM patients show
functionally different, one would expect to find these enhanced SNS and reduced parasympathetic activity when
disorders reliably associated with very different types of recumbent, but fail to adequately activate SNS activity or
ANS abnormalities. In fact, the opposite is the case. withdraw vagal tone upon standing (272). This lack of
Although inconsistencies in the data exist, the majority of reactivity has also been documented in response to
available evidence suggests that depressive and pain psychological stressors (224). Of note, biofeedback
disorders are characterized by a common ANS signature training that enhanced HRV (i.e. reduced SNS activity and
comprised of increased SNS signaling and diminished increased vagal tone) significantly improved pain and
parasympathetic (or cholinergic) tone. other symptoms in patients with FM (262), suggesting
that—as with MDD—ANS abnormalities may actually
Evidence supporting increased SNS/decreased contribute to symptom development and might be an
parasympathetic tone in MDD includes increased heart effective target for intervention in FM. Similar patterns of
rate at rest and in response to stress, increased blood ANS disturbance have been reported in conditions that are
pressure, increased systemic vascular resistance, increased highly comorbid with FM, such as CFS and PTSD (236,
whole body sympathetic activity based on measures of 273, 274).
postganglionic norepinephrine (NE) release and NE
clearance, reduced overall heart rate variability (HRV), 7.3. Inflammatory signaling in mood and pain disorders
reduced high frequency HRV (a measure of Interestingly, abnormalities in HPA axis and
parasympathetic tone), impaired autonomic ANS functioning frequently observed in both mood and
information flow (AIF) on 24-h ambulatory pain disorders have in common a proclivity for activating
electrocardiograms, impaired baroreflex, higher the same peripheral innate immune inflammatory
ventricular repolarization time and increased incidence pathways that are activated in response to pathogen
of multiple firing within a sympathetic burst based on invasion and/or tissue trauma (206). Several studies link
single-unit muscle sympathetic nerve analysis (244- glucocorticoid resistance with increased inflammation
254), Conversely, although not entirely consistent, data (220, 275). An even larger literature demonstrates a tie
suggest that various forms of treatment, including between SNS activation and/or parasympathetic
repeated transcranial magnetic stimulation, ECT, withdrawal and enhanced inflammatory tone (248, 275-
antidepressants and psychotherapy correct 283). Not surprisingly, therefore, a rapidly increasing
sympathetic/parasympathetic imbalances by literature indicates that mood and pain disorders are
attenuating SNS activity and/or increasing vagal tone associated with increased inflammation.
(255-260). Epidemiologic studies link intake of omega-
3 fatty acid with a reduced depression risk and When compared to non-depressed individuals, as
improved heart rate variability (261). Interestingly, a a group patients with major depression exhibit all of the
recent study suggests that applying biofeedback to cardinal features of inflammation, including elevations in
directly induce these ANS changes improves inflammatory cytokines and their soluble receptors in
depressive symptoms, strongly suggesting that ANS peripheral blood and cerebrospinal fluid (CSF), as well as
activity may be as much a cause of depressive elevations in blood concentrations of acute phase proteins,
symptomatology as a result (262). chemokines, adhesion molecules and inflammatory
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mediators such as prostaglandins (284-304). Associations have also recently been reported in FM and chronic pelvic
between depression and increased proinflammatory pain (330, 331). Although microglial activation in the
cytokines and/or c-reactive protein (CRP) have been CNS of humans with chronic pain has not been directly
apparent across the adult life span, whether comparisons demonstrated, it is intriguing that several studies find
are made between clinically depressed patients and elevations in proinflammatory cytokines in the
matched controls (284, 285, 288) or whether they derive cerebrospinal fluid of patients with chronic pain (332,
from large population-based studies (289, 291, 305-310). 333). In further support of the important role of immune
Recent investigations have reported positive correlations factors in etiopathogenesis of disorders characterized by
between levels of various inflammatory mediators and chronic pain and fatigue, researchers at the Centers for
depressive symptom severity (284, 285, 304, 305, 309, Disease Control and Prevention have recently reported
311-315); however, the association between immune that subjects with CFS or with CFS symptoms such as
activation and depression appears to be robust enough to fatigue and pain, had significantly higher levels of plasma
be detectable in the context of mild depressive symptoms CRP than did well controls (334). This study utilized a
that do not meet criteria for major depression (314). large population-based sample and found correlations with
Indeed, even single depression-related symptoms—such unwellness symptoms such as pain, fatigue and sleep
as fatigue, insomnia, fear and anger/hostility—appear to disturbance even after adjustment for multiple potential
increase the likelihood of inflammatory activation in confounding factors such as age, sex, race, body mass
otherwise healthy individuals (314, 316-321). index and depressive symptom status. In this study,
Inflammation has generally been assessed at a single time increased CRP was associated with increased physical, but
point; however, a recent study found that IL-6 production not emotional symptoms, highlighting the especially close
was abnormal across the entire circadian cycle in patients link between somatic symptoms and inflammatory
with major depression (284). Although most studies have activity.
focused on cytokine production/release, emerging data
indicate that depression may also be associated with In summary, altered neuroimmune,
activation of “downstream” inflammatory second neuroendocrine and autonomic regulation may interact to
messenger signaling pathways, such as the NF-kappa-B perpetuate states of pain and depression (53, 335-337).
pathway (208). Although recent research has focused Peripheral mediators of an aberrant stress response
primarily on cytokines which mediate the innate immune (cortisol, NE, proinflammatory cytokines) may not only
response, including IL-1, tumor necrosis factor (TNF)- be responsible for many of the clinical symptoms of
alpha, and IL-6, findings of increased markers of T cell MDD, FM and NeP, but may also have a role in
activation (e.g. soluble IL-2 receptor) in depressed perpetuating disturbed homeostasis in cortico-limbic
patients raises the specter that both acquired (e.g. T and B circuitry, therefore maintaining a vicious cycle (42, 53,
cell) and innate (e.g. macrophage) immune responses may 335-337) (Figure 3). In this sense MDD, FM and NeP
participate in inflammation in depression (322). may be seen as composed not just of psychosomatic
components (i.e. brain driving bodily dysregulation), but
Perturbation of neuroimmune control has also also of somato-psychic components (i.e. bodily processes
repeatedly been cited as a shared feature of conditions promoting CNS dysregulation). On a deeper level, one
such as FM, NeP and CFS that are characterized by pain, may even question the utility of such causal dichotomies
fatigue, sleep disturbances and cognitive complaints (53, between mind/brain and body.
164, 323, 324). Although results have not always been
consistent, several authors have reported elevations of 8. NEUROTRANSMITTERS IMPLICATED IN
TNF-alpha, IL-8 and IL-6 in FM (324-326). The MDD, FM AND NEP
inflammatory cytokine IL-8 is released by the pain
promoting peptide substance P, modulates HPA axis 8.1.1. Glutamate and GABA in MDD
activity and is involved in the induction of Multiple lines of evidence implicate aberrant
sympathetically mediated pain (324-327). Consistent with glutamate (Glu) and GABA transmission in the
these effects, elevation of IL-8 appears to be one of the etiopathogenesis of MDD, NeP and FM. As the principal
more consistent findings in FM (325, 326, 328). excitatory neurotransmitter in the circuitry linking limbic
Moreover, several studies found associations between and cortical areas, Glu is virtually ubiquitous in the brain.
blood levels of IL-8 and subjective pain ratings in FM Cortical glutamatergic projections have a principal role in
(325, 328). In addition to elevations in proinflammatory modulating activity in the source neuronal regions for
activity, data suggest that chronic pain conditions may norepinephrine (locus ceruleus [LC]), serotonin (nuclei
also be characterized by reductions in anti-inflammatory raphe [NR]) and dopamine (substantia nigra [SN] and
activity. For example, chronic widespread pain has been ventral tegmental area [VTA]) (338, 339). Glutamatergic
shown to be associated with reduced gene expression and neurotransmission relies on several classes of receptors.
blood protein levels of the anti-inflammatory cytokines Ionotropic receptors include N-methyl-D-aspartate
IL-4 and IL-10 (329). Elevations in inflammatory activity (NMDA-R), alpha-amino-3-hydroxyl-5 methyl-4-
may be a widespread phenomenon in FM, given a recent isoxazole-propionate (AMPA-R) and kainite receptors
study that reported TNF-alpha, IL-1 and IL-6 in the skin (KR). In order for NMDA-R to be activated, neural
of a majority of FM patients but in none of the controls membranes must also express AMPA-R. NMDA –R
(164). Elevations in peripheral levels of the chemokine “uncoupled” from AMPA-R are also referred to as “silent
MCP-1, which is known to activate microglia in the CNS, NMDA” receptors (340). Increased neural activity leads to
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Figure 3. Mood disorders, fibromyalgia (FM) and neuropathic pain (NeP) may have shared systemic consequences.
Compromised homeostatic function of prefrontal cortical-limbic circuitry in MDD, FM and NeP appears to disrupt autonomic,
neuroendocrine and neuroimmune regulation. Stress, pain and depression lead to excessive and untimely release of corticotropin-
releasing hormone (CRH), adenocorticotropic hormone (ACTH) and glucocorticoids. Sympathetic over-activity, combined with
diminished parasympathetic tone, contributes to immune activation and release of proinflammatory cytokines (e.g. TNF-alpha,
IL-1, IL-6) from macrophages and other immune cells. Inflammatory cytokines further interfere with monoaminergic and
neurotrophic signaling. They may also down-regulate central glucocorticoid receptor sensitivity, leading to further disruption of
feedback control of the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. In depression and pain states,
disturbances of serotonin (5HT), norepinephrine (NE) and dopamine (DA) transmission may impair regulatory feedback loops
that turn off the stress response, with a resultant compromise in the function of descending pain modulatory pathways. Elevated
mediators of the inflammatory response, combined with excessive sympathetic tone may further impact dorsal column processing
of pain signals by contributing to activation of microglia and astroglia. Activated microglia exchange signals with astrocytes and
nociceptive neurons, amplifying pain-related transmission of glutamate (Glu), substance P (SP), adenosine triphosphate (ATP),
brain-derived neurotrophic factor (BDNF), pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-alpha, nitrogen oxide (NO) and
prostaglandins (PGs). PVN= Paraventricular nucleus of hypothalamus; IL = interleukin; TNF-alpha = Tumor necrosis factor-
alpha; Ach=acetyl choline
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[Frontiers in Bioscience 14, 5291-5290, January 1, 2009]
repeated activation of synaptic NMDA-R, promoting clinical studies noted that antidepressants and ECT
synthesis of brain derived neurotrophic factor (BDNF). improve GABA deficits (350, 351).
Binding of BDNF to tyrosine kinase-B (TrkB) receptors
facilitates synaptic delivery of AMPA-R, thereby mediating 8.1.2. Monoamine Neurotransmitters in MDD
neural plasticity and long-term potentiation (LTP), which Despite decades of elaboration, the “monoamine
are key mechanisms for translating experience into hypothesis” of depression remains beset with
enduring modification of synaptic transmission (340, 341). controversies. Prompted by the evident treatment efficacy
Additionally, BDNF modulates dendritic arborization and of medications that modulate 5HT, dopamine (DA) and
synaptic spine density and morphology, contributing to a norepinephrine (NE), it has long been held that
micro- feed-forward circuit that allows the brain to “re- insufficient monoamine signaling may play a cardinal role
wire” itself in response to increased neural activity (341). in the etiopathogenesis of MDD (352, 353). In support of
Conversely, Glu binding to extra-synaptic NMDA-Rs, has this hypothesis, researches have noted alterations of NE
an opposite effect and suppresses BDNF synthesis (338). and 5HT receptor density in cortical and limbic
Glu also binds to three different classes of pre-synaptic and formations of depressed patients (353). In a PET imaging
post-synaptic metabotropic receptors (mGlu-Rs), which study comparing dopamine type-1 receptor (D1) binding
have a role in modulating neurotransmitter release and potential (BP) in MDD patients vs. healthy subjects,
post-synaptic activation of AMPA-Rs and NMDA-Rs(342). researchers found significantly reduced D1 BP in the
depressed group. This reduction in D1 BP correlated
Early studies that examined serum and plasma negatively with illness duration and anhedonia ratings
glutamate levels in MDD subjects had equivocal findings (354). We have already provided evidence implicating
(338). More recently, MRS studies have noted decreased genes that code for 5HTT, COMT, MAO and monoamine
Glx (a measure combining glutamate, homocarnosine, receptors in perpetuating vulnerability towards depression.
GABA and glutamine spectroscopic signatures) in ACC Meyer et al. have reported elevated levels of MAO-A in
and amygdala of depressed adults, children and elderly the brains of depressed patients. Since MAO-A is a
patients. In one of these studies, this alteration normalized primary monoamine-lowering enzyme in the nerve cells,
with successful ECT treatment (see (338) and (343) for a its excessive activity may contribute to a disturbance of
review). A significantly higher Glu/GABA ratio was monoamine signaling in MDD (355). Changes in platelet
reported in the occipital cortex of depressed individuals 5HT and NE receptor density have also been noted in
compared to controls. Additional reports indicate changes depressed and suicidal patients (353). Patho-histological
in the NMDA receptor glycine binding site in depressed studies have reported a decrease in the number of NE
suicide victims and NMDA subunits in LC of depressed neurons in LC of depressed patients whose death was
subjects (338). Altered function of glutamatergic fibers unrelated to suicide when compared to matched controls
originating from vmPFC and projecting to sympathetic (356). The same authors also noted a decrease in the number
and parasympathetic brainstem centers has been described of 5HT neurons in dorsal nuclei raphe of depressed patients
in MDD patients (344). This abnormality may provide a compared to healthy controls (356). Neuromelanine-sensitive
link between emotional dysregulation and excessive stress MRI imaging has revealed an attenuated signal in LC of
reactivity, repeatedly noted in depressed individuals (344). depressed patients (357). On the other hand, research
Open label studies of lamotrigine and rizulole (both assessing NE and 5HT metabolites in the CSF has yielded
inhibitors of glutamate release), as well as a controlled inconsistent results. Levels of 5-hydroxyindoleacetic acid (5-
randomized trial of ketamine (an NMDA antagonist) in HIAA) have been reported to be lower in depressed patients
treatment resistant depressed patients provide preliminary and especially low in victims of violent suicide (353).
evidence of efficacy of glutamatergic modulators in the However, because MDD is a biologically heterogeneous
treatment of MDD (345). syndrome, it is not surprising that there are differing reports
on the levels of monoamine biomarkers. For example, 3-
Several studies have reported that MDD is methoxy-4-hydroxyphenylglycol (MHPG), a major NE
associated with reduced plasma GABA levels (see (346) metabolite, was found to be elevated in plasma of agitated
for a review), as well as decreased GABA neurons in and anxious depressed patients, while depressed individuals
lateral orbital PFC (LOPFC) and DLPFC (347). This is a with psychomotor retardation were found to have lower
particularly interesting finding, given the previously MHPG and homovanillic acid (HVA) (a primary dopamine
described role of DLPFC and LOPFC in cognition, metabolite) levels than healthy controls. Patients with
voluntary regulation of emotion and suppression of psychotic depression had elevated plasma levels of HVA.
maladaptive affect. Some preliminary data suggest that (358). Another study, using catheters placed in the internal
altered cortical GABA and Glu concentrations may not jugular vein noted reduced NE and DA release in the brains
only differentiate MDD patients from controls but also of patients suffering from refractory depression relative to
differentiate depressive subtypes from each other healthy volunteers (359). In contrast, using a similar
(melancholy and atypical) (348). A recent MRS study technique, brain 5HT turnover was found to be elevated in
found reductions in GABA levels in dmPFC, DLPFC and depressed patients when compared to healthy subjects (360).
ACC in MDD. It should be noted that these are the same
brain areas in which patho-histological studies established Monoamine transporters also seem to be
alterations in glial density in MDD patients compared to influenced by the disease process. For example, the
healthy controls (349). Finally, several preclinical and dopamine transporter (DAT) has a 15% lower binding
potential in depressed patients compared to controls (361),
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and 5HT transporter (5-HTT) binding potential (which example, dopaminergic input tends to up-regulate
can be considered as a marker of 5-HTT density) tends to serotonergic and down-regulate noradrenergic activity.
be elevated during a depressive episode, especially a more Elevated serotonergic activity has a mostly inhibitory
severe one (362). Pharmacologic strategies that deplete effect on NE and DA, while increases in NE tend to
monoamines suggest that the therapeutic effect of suppress DA but can modulate 5HT transmission in either
antidepressants may be selectively reversed by depleting direction (368, 369). Complex cross talk between
the monoamine affected by that particular antidepressant dopaminergic and noradrenergic systems appears to take
(363). place in VTA, LC and dorsal hippocampus (370).
Extracellular DA in the prefrontal cortex originates not
However, a converging body of evidence brings only from dopamine but also from NE terminals (371).
into question simplistic interpretations of the “monoamine Furthermore, not only do monoamines influence each
theory”. If monoamines are the primary abnormality in other via complex interactions but GABA and Glu tend to
MDD, it is hard to understand why in the STAR*D trial have a bidirectional regulatory influence on monoamines
50% of the patients failed to respond to the first line SSRI (351, 372-375). In summary, extant evidence supports the
treatment, 65% did not achieve remission and more than a view of a complex dysregulation of interrelated
half of those who did still had two or more residual neurotransmitter systems and distributed brain networks
symptoms (364). While in vivo preclinical evidence involved in regulation of mood, cognition and the stress
describes prompt LC activation following SSRI response, rather than a simple deficit of monoamine
application (365), clinical studies have shown that it may signaling (122, 376).
take as long as five to eight weeks until optimal
antidepressant response is attained (366). Mayberg et al. 8.1.3. Endogenous Opioids and other peptide
evaluated SSRI responses utilizing PET imaging. These neurotransmitters in MDD
investigators found no difference between responders and The role of the peptide neurotransmitters galanin
non-responders following a week of antidepressant and substance-P (SP) and endogenous opiates in the
treatment. Rather, the pattern of activity associated with pathophysiology of MDD has been a focus of recent
depression was reversed only after six weeks of treatment, research (377-379). Galanin coexists in serotonergic DNR
suggesting an adaptation to chronic antidepressant neurons and noradrenergic LC neurons. Galanin receptors
administration as the basis of therapeutic effect (137). are located in PFC, amygdala, hippocampus,
Wong et al. measured CSF NE and plasma cortisol levels hypothalamus and the brainstem nuclei LC and DNR
in a group of patients suffering from melancholic MDD. (379). Galanin receptor modulators are showing early
Samples obtained around the clock showed that depressed promise in preclinical and clinical studies as
patients had significantly higher levels of NE and cortisol antidepressant, anxiolytic and neurogenesis-promoting
than healthy subjects. NE and cortisol levels were strongly agents (379).
correlated to each other (200). A variety of antidepressant
medications and ECT have been reported to suppress the A recent review emphasized the pivotal role of
activity of tyrosine hydroxylase (TH), a key enzyme SP in communicating peripheral inflammation and stress
regulating NE synthesis (367), while remarkably response to the brain. SP appears to occupy a key position
consistent evidence shows reductions in CSF levels of in bidirectional communication between the brain and the
MHPG (the main NE metabolite) in depressed patients body (377). Additionally, SP fibers and the main
taking antidepressants (331). Imaging studies have neurokinin-1 receptor (NK-1), are well represented in the
uncovered insufficient activity and reduced volume of prefrontal cortical and limbic areas, involved in the
vmPFC in depressed individuals (26, 124, 132, 137, 344). regulation of mood, anxiety and stress response (380).
Because vmPFC plays a key role in regulating amygdala Recent studies have demonstrated that emotional stress
activity and sympathetic/parasympathetic balance, it is not results in SP efflux in amygdala and septal areas (380). A
surprising that depressed patients tend to exhibit excessive preclinical study indicates that stress-induced release of
sympathetic activation in response to stressful stimuli SP in LC, facilitates neurotransmission via NE projections
(221, 344). A recent preclinical study demonstrated a to mPFC, thus relaying the stress signal to this area (380).
significant reduction in LC electrophysiological activity SP also modulates the firing rate of serotonergic DNR
following 14 days of treatment with a diverse group of neurons and dopaminergic VTA neurons, as has been
antidepressants, including desipramine (a predominantly demonstrated in studies utilizing systemic administration
noradrenergic TCA), paroxetine and citalopram (SSRIs) of NK-1 receptor antagonists (381).
and mirtazepine, an alpha-2 antagonist (331).
Kennedy et al. utilized PET imaging to assess
Barton et al. recently reported elevated 5HT endogenous opioid transmission in female MDD patients.
turnover in unmedicated MDD patients. Marked reduction These authors reported a reduction in mu-opioid binding
in brain 5HT turnover, accompanied by clinical in ACC, ventral basal ganglia, hypothalamus and
improvement, ensued after twelve weeks of SSRI amygdala of MDD patients compared to healthy subjects
treatment (360), suggesting a relatively insufficient 5HT (378). Supporting data in depressed patients, demonstrate
transmission in MDD. perturbation of opioid transmission in the aforementioned
areas involved in emotional and neuroendocrine
Strong evidence suggests that monoamines also regulation.
regulate each other through complex interactions. For
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8.2.1. Glutamate and GABA in pain disorders GABA has an important modulatory role in pain
Glutamate is the principal neurotransmitter in transmission, from interneurons in dorsal column to
both ascending and descending pathways involved in complex cerebral circuitry involved in pain processing and
inhibiting and facilitating pain transmission in the CNS, regulation of descending pain pathways (168, 395).
including the dorsal column, thalamo-cortical and cortico- Alterations in peripheral benzodiazepine receptors (PBR)
limbic circuitry involved in pain processing (174, 177, on platelets (396) and monocytes (291) have been
180, 382, 386). Excessive Glu transmission leads to reported in FM patients relative to healthy controls. For
conjoined activation of AMPA and NMDA receptors all example, upregulation of PBRs, a proxy indicator of
along the neuroaxis involved in pain perception and GABA-ergic function, correlated with severity of FM
modulation, which tends to promote increased synthesis of symptoms (396). Insular dysfunction associated with
neurotrophic factors (177, 180, 382). Such an GABA, DA and opiate neurotransmission has been
enhancement of neurotrophic signaling has been documented in both clinical and preclinical models of FM
associated with enduring neuroplastic changes, which (397). Moreover, extensive preclinical studies support a
are—in turn—the substrate for long-term potentiation role for GABA in the mediation of neuropathic pain (395).
(LTP) and “central sensitization” (177, 180, 382). Similarly, dysfunction of dorsal horn GABA interneurons
Additionally, activation of metabotropic Glu receptors due to injury may play a key role in NeP pathogenesis
(mGluR) has been implicated in both peripheral and (163, 398). Directly supporting a role for GABA in the
central pain sensitization (387). development of chronic pain, preclinical models suggest
that early GABA administration to spinal cord may
Based on concomitant increases in Glu, nerve prevent subsequent development of neuropathic pain
growth factor (NGF) and BDNF in the CSF of FM (395). Consistent with these data, pharmacologic
patients, investigators have recently hypothesized that manipulation of GABA-A receptors successfully
increased Glu neurotransmission may facilitate NGF and prevented NeP and alleviated pain in a preclinical model
BDNF synthesis in pain relevant pathways, which in turn involving sciatic nerve crush injury (399). On the other
may promote the expression of NMDA receptors in neural hand, inadequate GABA-opiate system regulation in PAG
membranes, effectively closing a positive feed-forward may result in compromised function of the descending
circuit that enhances pain signaling (388). Temporal pain pathway in models of NeP (162, 395).
summation of second pain (TSSP) and “windup”, also
believed to reflect a summation process characteristic of 8.2.3. Monoaminergic neurotransmitters in pain
dorsal horn neurons, are cardinal manifestations of central disorders
sensitization in FM. Repeated release of Glu and SP from Monoaminergic nuclei in mesencephalon and
C-fiber terminals, followed by excessive NMDA brainstem have rich projections that innervate cortical,
activation, appears to be the neural substrate for these limbic and thalamic areas involved in regulating mood,
phenomena (175). For example, a study that measured stress responses and pain processing (see previous text).
the concentration of excitatory amino acids in CSF of Additionally, serotonergic and noradrenergic brainstem
FM patients reported a relationship between elevated nuclei provide innervation for descending pain-
Glu and examination-based measures of pain intensity modulating tracts (180),(177), (400). Several authors have
in FM patients, including the tender point index (TPI) reported decreased CSF levels of the monoamine
(389). Harris et al, using fMRI imaging to study the metabolites (5-HIAA, MHPG and HVA) in FM patients
effect of a non-pharmacologic treatment in a group of relative to healthy controls (401, 402). Serotonergic
FM patients demonstrated a relationship between abnormalities in FM are of particular importance given the
changes in Glu levels in insula and improvements in role played by 5HT in regulating SP transmission, another
multiple pain domains (174). Thus, alterations in Glu mediator heavily implicated in pain regulation (402).
transmission at both cortical and dorsal column levels Serum concentrations of 5HT were found to be lower,
may play an important role in the etiology of FM. while 5-HT receptor density on circulating platelets was
Consistent with this, alterations in spinal glutamatergic higher, in FM patients compared to controls (401, 403). In
signaling and subsequent NMDA activation, as well as vivo microanalytical studies have reported elevated levels
excessive limbic NMDA mediated transmission, has of 5-HT, NE, SP, TNF-alpha and IL-1 in skeletal muscle
also been documented in preclinical models of of myofascial pain sufferers (404). FM patients were also
neuropathic pain (11, 390-392). noted to have lower plasma levels of tryptophan, which is
the metabolic substrate for 5HT (405). Although not all
Elevated glutamatergic transmission in NeP may, the studies support serotonergic involvement in FM,
in part, be attributable to upregulation of the alpha-2-delta alterations in 5HT signaling may contribute to the HPA
subunit of voltage-gated calcium channels. A group of axis and sleep dysregulation commonly observed in
authors reported a 17-fold increase in alpha-2-delta studies of FM (9. 405). In the context of NeP, 5-HT
subunit in dorsal root ganglia of animals subjected to an appears to have a dual role, participating in
experimental model of neuropathic pain (393). Such antinociceptive descending modulation (400, 406) on the
increased expression of alpha-2-delta subunits may one hand, and contributing to signaling in facilitatory
produce an excessive influx of calcium into the nerve descending fibers, on the other. Moreover, together with
cells, resulting in augmented release of excitatory amino NE, 5HT participates in sensitizing peripheral C-fiber
acids and SP, with a resultant amplification of pain (394). axons in preclinical models of NeP (407).
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We have previously described elevated receptor availability in amygdala of FM patients compared

sympathetic tone in FM and NeP, and recent studies have to controls (50). Reduced mu-opioid receptor availability
confirmed that sympathetic manipulation enhances pain correlated with affective pain ratings, possibly explaining
sensitivity in a subgroup of heat sensitive NeP patients the reduced efficacy of exogenous opiates in FM patients
(408). Decreased CSF concentrations of MHPG and HVA and elevated CSF and plasma levels of endogenous
(primary metabolites of NE and DA) in FM patients may opioids in this population (50, 405). Preliminary evidence
be related to compromised neurotransmission in inhibitory provides an intriguing view of the role that glial cells may
descending pathways (401, 402). A recent study utilizing play in the modulation of pain. In preclinical models,
PET imaging assessed dopaminergic function in FM morphine promoted release of Glu and inflammatory
patients (409). Unlike healthy controls, FM patients were mediators from astrocytes, leading to paradoxical increase
not able to mount a release of DA in basal ganglia in in pain transmission. Nonetheless, the role of this
response to painful stimulation. While the amount of mechanism in the clinical setting remains to be
released DA correlated with pain intensity in normal determined (415).
controls, no such relationship was noted in FM patients
(262). Because DA has a role in motivation and cognition, Preclinical models of NeP suggest an intriguing
it is intriguing to speculate that it may mediate at least dissociation between supraspinal and spinal opioid
some psychiatric symptoms that are common in the nociceptive systems. In an animal model of NeP, an
context of FM (39). Finally, monoamines may play a intrathecal injection of morphine had a significantly
synergistic role in modulating NeP, given a recent reduced analgesic effect, whereas supraspinal
preclinical study that found that combined 5-HT and NE administration had a potent antinociceptive effect (386).
uptake inhibition conferred greater analgesic benefits than Additionally, pain-facilitating neurons that originate from
uptake inhibition of a single monoamine. Interestingly the rostral ventromedial medulla (RVM) and are typically
addition of DA uptake inhibition further ameliorated NeP modulated by the opioid system seem to be sensitized
pain in this animal model (410). after nerve injury in preclinical models of NeP. This
sensitization likely contributes to allodynia and
8.2.4. Endogenous opioids in chronic pain hyperalgesia in NeP (416).
The dopaminergic system is closely linked to the
opioidergic system, which is probably the best established 8.2.5. Contributions of SP to conditions of chronic pain
antinociceptive pathway in the CNS (38). Opioid release Much like opioid and monoamine projections, the
in cortical and limbic areas involved in emotional substance-P (SP) system is present in several cortical and
processing and the stress response appears to regulate limbic regions involved in the stress response and the
pain-associated cognition, effectively attenuating emotive regulation of emotion and pain (380, 417). In preclinical
aspects of pain and sadness (411, 412). Sustained pain models, both chronic pain and chronic stress have been
reflexively activates the endogenous opioid system in associated with down-regulation of SP NK-1 receptors
ACC, PFC, insula, thalamus and hypothalamus (413). and BDNF synthesis in hippocampus, suggesting
Cognitive modulation of pain, mediated by ACC and similarities in mechanisms underlying chronic pain and
DLPFC activity, is conveyed by mu-opioid receptors depression (417). Activation of nociceptive fibers leads to
(414). The coupling of rACC and PAG opioid analgesic a concomitant release of glutamate and SP in dorsal horn
systems may have a crucial role in placebo anesthesia and synapses (9, 418). NK-1 receptors are expressed by
alteration of pain perception by change in expectation lamina-I dorsal horn neurons which project to brainstem
(413). Subcortical opioid circuits are more involved in and thalamus, modulating descending inhibitory and
modulating the sensory component of pain. Moreover, facilitatory pathways (418). It is no surprise that altered
activation of the endogenous opioid system in conditions SP transmission and subsequent dorsal horn neuron
of stress and danger may modulate affective and sensory sensitization has been implicated in the
components of pain independently (411). The etiopathogenesis of both FM and NeP (9, 419, 420). SP
hyperalgesic effect of psychological distress appears to be release seems to be promoted by elevations of
suppressed by endogenous opioid release, particularly in proinflammatory mediators and reduced by
women (411). Activation of descending pain inhibitory administration of dexamethasone in preclinical models
tracts leads to opioid release in the dorsal horn of the of NeP (421). C-fibers are also capable of retrograde
spinal column, where a hyperpolarizing analgesic effect is release of SP into the injured tissue, which then
mediated by mu- , delta- and kappa- opioid receptors contributes to “neurogenic inflammation” mediated by
(400). Glu acting through NMDA receptors, and SP acting proinflammatory substances, especially IL-8 (168,
through NK-1 activation, both modulate endogenous 326). This reiterative loop may amplify and perpetuate
opioids, which in turn have an inhibitory influence on NE chronic pain. Several authors have reported elevations
release (400). of SP in CSF of FM patients relative to healthy
controls, often associated with a reduction of
Evidence suggests altered opioid signaling in FM monoamine metabolites and endogenous opiates (401,
and NeP (12, 405). A recent PET imaging study has 422-425). However, elevated levels of SP in the CSF do
revealed decreased availability of mu-opioid receptors in not appear to be specific to FM. Given its association with
nucleus accumbens, dACC and amygdala of FM patients other painful conditions, it may be better considered as a
relative to healthy controls (50). A significant relationship biological marker of chronic pain than as a marker of any
was also detected between depression and mu-opioid particular diagnostic condition (37).
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Glu and SP have a synergistic pro-nociceptive Moreover, at times of peak neuronal activity glia cells
effect. Proinflammatory cytokines enhance the release of release lactate in response to increased energy needs.
both transmitters, and are in turn, themselves stimulated
by SP. IL-8, whose release is facilitated by SP, promotes Recent research suggests that in addition to
sympathetic pain (326, 400). Pro-nociceptive effects of SP myelinating axons, Schwann cells and oligodendroglia are
are opposed by endogenous opiates and galanin, a peptide instrumental in the long term maintenance of axonal
over-expressed in sensory neurons following peripheral functional integrity, with this role being independent of
nerve damage that is also known to promote a sustained myelin formation (431). Neurotrophic factors, including
inhibitory effect on dorsal horn synapses (400). BDNF that are generated and released by oligodendroglia
have a significant role in axonal resilience and recovery
Only selected neurotransmitters and mediators of (432). Loss of glial support appears to promote axonal
pain have been addressed in this article. A more degeneration and local inflammation (431).
exhaustive review of substances involved in pain Oligodendroglia appear to have a dominant role in human
modulation is beyond the scope of this review. In white matter Glu clearance. An altered ability to remove
summary, there is a remarkable similarity and overlap in Glu, likely mediated by TNF-alpha, may be the
how multiple neurotransmitter systems, utilizing underpinning of Glu excitotoxicity (433).
glutamate, GABA, monoamines, endogenous opioids and
SP, through complex supraspinal and spinal interactions, Astrocytes and oligodendrocytes most likely have
modulate the stress response, emotions and pain. a common glial precursor, with local microenvironments
during development determining the eventual fate of the
9. CELLULAR, SUBCELLULAR AND cell (434). On the other hand, microglia are the only cell
NEUROTROPHIC CHANGES IN MDD, FM AND population in the brain of mesodermal origin (435).
NeP Microglia have a complex role in the CNS. They act as
resident macrophages, constantly screening the CNS
9.1. An overview of glial architecture and function environment. Depending on circumstances, microglia
Accumulating evidence suggests that MDD may perform a variety of functions, including the apparently
be associated with significant CNS cellular pathology, opposite functions of neuroprotection and perpetuation of
especially in glia cells. The human nervous system has cellular damage, as well as the support of
approximately 100 billion neurons and one trillion glia oligodendrogliagenesis (436). Microglia may be
cells (426). Traditionally, glia cells have been cast as a conceived as the principal CNS recipients of peripheral
passive supportive matrix for neurons (426). However, in stress signals . Under usual circumstances, microglia have
dramatic contrast to prior assumptions, contemporary a protective role; however, in pathologic conditions these
research has established astroglia and oligodendroglia as cells amplify and perpetuate stress signals, contributing to
full-fledged neuronal partners in neurotransmission (54). oligodendroglial and neuronal damage (437, 438). Like
Indeed, brain architecture is defined by astrocytes. Each astrocytes and oligodendroglia, microglia are endowed
protoplasmic astrocyte occupies its own territory, with a wide variety of receptors, including those for
covering all the neural elements within its domain (427). chemokines, cytokines, GABA, glutamate, Ach, NE,
Each human astrocyte contacts and encapsulates dopamine, SP and opioids. They are capable of
approximately two million synapses (427). In addition to synthesizing and releasing prostaglandins,
managing the content of the synaptic cleft, astroglia may proinflammatory cytokines, oxygen- and nitrogen-reactive
have a role in synchronizing the activity of all neurons species (ROS and NRS), neurotrophic factors, Glu and
within their domains (428). Brain connectivity is quinolinic acid (439).
effectively shaped by astroglia through regulation of
synaptogenesis, synaptic strength and plasticity (427). 9.2. Contributions of glial pathology to MDD
Almost all classes of serotonin, norepinephrine, Glial cell pathology has been reported in the
dopamine, cholinergic, GABA, glutamate, neurotrophin sgACC, DLPFC, orbitofrontal cortex and the amygdala of
and cytokine receptors are expressed on glial cell unmedicated MDD patients (54, 440, 441)) It appears that
membranes (54, 55, 428). Additionally, astroglial both astroglia and oligodendroglia may be affected.
membranes express monoamine (5HTT, NAT, DAT) and Uranova et al. describe a prominent 19% reduction in
glutamate transporters. Evidence suggests that oligodendroglia in the DLPFC (BA 9) of MDD patients
neurotrophic factors, such as brain-derived and glia- (441). Having in mind the crucial role that DLPFC plays in
derived neurotrophic factors (BDNF and GDNF), as well executive function and “top-down” limbic regulation, the
as cytokines (i.e. TNF-alpha) are synthesized within glia implications of this finding are striking, because it may
cells (54, 55). However, unlike neurons, which release provide a neurobiological substrate for both the emotional
neurotransmitters in response to action potentials, glia dysregulation and cognitive dysfunction commonly
cells discharge the content of Glu vesicles in response to observed in MDD. Hamidi et al. have described reductions
graded increases in cytoplasmic Ca++. Astroglia also have in oligodendroglia density in amygdala of MDD patients
a role in regulating neuronal energy supply. Cerebral (442). Genetic factors may contribute to these
perfusion is significantly influenced by astroglia. On one oligodendroglia abnormalities, given a recent
end astroglial extensions “sense” synaptic activity, on the transcriptional profiling study of MDD subjects that has
other end their distal processes, or “feet”, modulate identified aberrant expression of 17 genes related to
vascular tone and capillary permeability (429, 430). oligodendroglia function (443). Rajkowsa et al. have noted
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a significant reduction of glia density in DLPFC and OPFC amplifies cytokine signals from the periphery (55, 429,
of MDD subjects (444). Further supporting glial 437, 448, 452, 453), Multiple effects are likely to result
involvement in mood disorders (445), Stockmeier et al. from this feed-forward proinflammatory, pro-oxidant
observed a significant decrease in glial density in the activity. For example, oligodendroglia are likely to suffer
dentate gyrus of the hippocampus in MDD subjects (446). oxidative damage due to overexposure to reactive oxygen
Additionally, age dependant decrements in the astroglial and nitrogen species (ROS and RNS). TNF-alpha released
markers were reported in a group of MDD subjects (447). by microglia and astrocytes in the context of diminished
neurotrophic support has a direct toxic impact on
A study using immunohistochemistry assessed oligodendroglia, consequently contributing to
microglia density in DLPFC, ACC, mediodorsal thalamus demyelination. Because oligodendroglia also play a role in
and hippocampus of depressed patients. The authors Glu uptake, their damage may add to excessive
suggest that significant microgliosis (i.e. increased number accumulation of this neurotoxic transmitter (455),
of microglia) in depressed patients who committed suicide (433),(456).
relative to healthy controls, might be a marker of pre-
suicidal stress. Proinflammatory mediators released from Proinflammatory cytokines (e.g. IL-1, IL-6, TNF-
microglia may have altered 5HT and NE transmission and alpha) and prostaglandins (e.g. PGE-2) synergistically
contributed to suicidality (448). induce indoleamine 2,3- dioxygenase (IDO), an enzyme
that converts tryptophan to kynurenine, thereby
In contradistinction to widespread glial diminishing its availability for 5-HT and melatonin
abnormalities, neuronal changes appear to be subtler and synthesis (56, 457). Proinflammatory cytokines, such as
more discrete in MDD. For example, some authors have IL-1and TNF-alpha, may also compromise serotonergic
noted decreased pyramidal somal size in hippocampus neurotransmission by increasing reuptake of the
(446), ACC (449), DLPFC (BA 9) and OPFC (BA 47) in neurotransmitter from the synaptic cleft (90). For example,
postmortem studies of MDD patients (440). The mRNA expression for these cytokines was associated with
distribution of this cellular pathology overlaps remarkably elevated 5HT transporter activity in MDD patients (458).
with findings from structural and functional imaging
studies. Si et al. investigated the influence of age and MDD In addition to reducing serotonin availability,
on packing density of glial fibrillary acedic protein IDO may contribute to depression directly as a result of the
(GFAP)-positive astrocytes in DLPFC. Individuals afflicted production of kynurenine and its downstream metabolites.
with MDD had a much steeper correlation between For example, the downstream metabolite quinolinic acid
reduction of GFAP levels and age relative to healthy (QUIN) is a potent NMDA agonist and stimulator of Glu
controls, suggesting a synergy between the aging process release. While the complete enzymatic pathway for QUIN
and disease state in reducing glia density in DLPFC (286). production is only present in microglia, activation of IDO
in astrocytes may aid in the conversion of tryptophan into
Microglia-Astroglia-Oligodendroglia-Neuron kynurenic acid (KA), a compound known to down-regulate
“units” may be conceptualized as neural microsystems that dopaminergic transmission and NMDA activity (56, 459-
interface with peripheral macrosystems, including 461). Whether KA is more likely to protect against
autonomic, immune and endocrine signals, providing an depression via its inhibitory effects on NMDA transmission
ongoing integration of these peripheral regulatory systems or to promote depression via its ability to down-regulate
with cerebral activity. We have previously described dopamine signaling is an important, and unanswered,
circumstances leading to neuroendocrine, autonomic and question.
neuroimmune dysregulation in MDD, with concomitant
elevations of proinflammatory cytokines, catecholamines Astrocytes have recently been found to be a
and circulating corticosteroids (sections 7.1-7.3). Peripheral source of GABA in the CNS and may be important
mediators of the inflammatory response propagate their modulators of GABA activity in hippocampus (462). In the
influence on the brain via several pathways, including 1.) context of MDD, a shift in the microglia/astroglia balance,
afferent neural fibers (i.e. vagal nerve); 2.) stimulation of favoring microglial activity, may lead to excessive Glu
immune cells in circumventricular organs (e.g. area transmission relative to GABA output (56, 459, 462); a
postrema, eminentia mediana, pineal gland), leading to dominance of T-helper-1 (Th-1) signaling (mostly
release of proinflammatory cytokines; and 3.) induction of proinflammatory) over Th-2 activity (predominantly
blood-brain barrier (BBB) cells (ependymal, endothelial associated with release of the anti-inflammatory cytokines
and choroid plexus cells), which respond by releasing IL-1, IL-4 and IL-10); and inadequate neurotrophic support for
IL-6, TNF-alpha, prostaglandins, NO and MCP-1. Limited oligodendroglia, contributing to demyelination (456, 459).
evidence suggests that transport of proinflammatory
cytokines across BBB may also be possible. (450),(451). Astrocytes contain serine racemase, an enzyme
responsible for the conversion of L-serine to D-serine
Microglia are the chief recipients of (463). Compromised astrocytic function in MDD may
inflammatory signals conveyed from the periphery. therefore lead to altered D-serine release. D-serine is an
Activated microglia respond by releasing additional endogenous ligand of the glycine receptor and therefore a
amounts of IL-1, IL-6, TNF-alpha, PGs, NO and H2O2, co-modulator of NMDA function and synaptic plasticity
which in turn induce astroglia to release more of these (463). Astroglial coverage of the synapse determines the
inflammatory mediators. This positive feedback loop extent of D-serine available to synaptic NMDA receptors.
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If astrocytic processes retract from the synapse, the amount of Multiple signaling pathways have a convergent
D-serine is reduced, resulting in long term depression of influence on neurotrophic factor synthesis. We have already
synaptic function (430). Conversely, excessive astrocytic suggested that altered microglia-astroglia-neuron
release of D-serine and consequent NMDA over-stimulation communication may lead to increased NO and prostaglandin
may be toxic to neurons (429). In support of this view, a recent production which may, in turn, have a negative impact on
study has reported that elevation of plasma L-serine and Glu in neurotrophic signaling. Preclinical data also demonstrate that a
depressed patients was directly associated with symptom loss of diurnal rhythm of corticosterone secretion impairs
severity, possibly hinting at astrocytic insufficiency of BDNF function (242). Altered astroglia function in MDD may
converting L-serine to D-serine (464) (Figure 4). have significant ramifications on neurotrophic signaling.
Stimulated astroglia release Glu almost exclusively into the
The disturbance in sympathetic/parasympathetic extrasynaptic space where it binds to extrasynaptic NMDA
balance associated with MDD may have significant receptors (430). Unlike activation of synaptic NMDA
repercussions on glia-neuron communication. Parasympathetic receptors, which promote BDNF release, activation of
activity has a stabilizing effect on microglia activity, while extrasynaptic NMDA receptors powerfully suppress BDNF
sympathetic input can play a dual role, either inhibiting or synthesis through a CREB-dependent mechanism (478). Glia-
activating microglia (439). In stressful situations it appears that derived neurotrophic factor (GDNF) is an important regulator
sympathetic discharge induces microglial release of of neuronal health and cognitive function. A recent study
proinflammatory cytokines (465). Consistent with this, a recent reported decreased serum levels of GDNF in depressed
study that reported increased nuclear factor- kappa B (NF- patients compared to healthy controls. Moreover, eight weeks
kappa B) activity in human volunteers after stress also of antidepressant treatment was associated with a significant
observed that NF-kappa B responses to stress in animals was elevation of serum GDNF in depressed patients (479). Juric et
obviated by alpha-1 receptor blockade (277). NF-kappa B al. demonstrated a significant increase in BDNF in astroglial
plays a pivotal role in catecholamine-induced synthesis of cultures treated with NE, DA and 5HT, establishing an
proinflammatory cytokines (466). Proinflammatory cytokines important relationship between monoaminergic neuronal
and glucocorticoids have opposing effects on NF-kappa-B activity and astroglial neurotrophic support (480).
regulation (208). Under usual circumstances glucocorticoids
have an immunosuppressant effect. MDD and sustained stress In summary, disturbed neuron-glia relationships
are accompanied by insufficiency of glucocorticoid signaling in MDD may result in diminished neurotrophic support
and glucocorticoid receptor (GR) resistance (42. 206). (BDNF and GDNF), altered energy supply and oxidative
Glucocorticoid resistance, in turn, may be associated with a regulation, massive releases of glutamate accompanied by
“permissive state”, leading to an augmented inflammatory compromised uptake, accumulation of ROS and RNS, all of
response (42, 206). However, in the context of aberrant stress which may jointly contribute to neurotoxicity (56, 452,
responses, glucocorticoids have also been reported to increase 481).
NMDA activation in microglia and neurons (467, 468).
Regardless, it appears that stress-MDD-HPA-sympathetic- 9.3. Neuron-Glia Interactions in chronic pain
inflammatory dysregulation may be a self-perpetuated As with MDD, alterations in neuron-glia
vicious cycle with the potential to negatively impact relationships may be of fundamental importance in the
glia-neuron signaling. etiopathogenesis of chronic pain conditions (482-484). In
the case of NeP, preclinical models are the primary source
Proinflammatory cytokines have been reported to of information about the cascade of interactions between
induce nitric oxide synthase (NOS), a pivotal enzyme spinal glia cells and neurons, and the role of these
regulating nitrogen oxide (NO) synthesis. NOS induction interactions in the development of NeP-associated
following exposure to inflammatory cytokines has been symptoms, such as allodynia and hyperalgesia. Microglia
reported in microglial, astrocytic and neuronal preparations have been reported to respond to a range of pathologic
(469-471). NMDA signaling is also associated with increased conditions conducive to development of NeP, such as
release of NO. A recent postmortem study reported elevated ischemia, infection and mechanical damage. These
NOS in hippocampal neurons of depressed patients (471). responses include morphological transformation,
Additionally, Xiong et al. reported a suppressant effect of NO proliferation and migration to the site of the injury (398). In
on BDNF synthesis (472). pathologic circumstances, a repetitive barrage of synaptic
firing by nociceptive A-delta and C-fibers induces
Neural injury caused by proinflammatory cytokines increased responsiveness of the dorsal horn pain-projecting
may in part be attributable to induction of cyclooxygenase neurons, a phenomenon also known as “central
(COX) and ensuing prostaglandin (PG) synthesis and release sensitization” (482). In this context, an intricate co-release
(473). Direct PG injection into rodent dorsal hippocampus was pattern of glutamate, SP, calcitonin gene-related peptide
sufficient to impair memory and reduce post-conditioning (CGRP), fractalkine, BDNF and ATP ensues (163, 330,
BDNF levels (473). Other consequences of COX-2 activation 482, 484). BDNF signaling, mediated by TrkB receptors,
include NMDA and QUIN mediated neurodegeneration (474, perpetuates the release of glutamate, CGRP and SP, and
475) and induction of NOS with ensuing accumulation of NO stimulates noradrenergic fiber sprouting following nerve
(476). Finally, preclinical studies suggest that age-related injury, thus adding to the development of pathologic pain
increases in hippocampal IL-1, TNF-alpha and PGE-2 can be (485, 486). Both astrocytes and microglia release D-serine
prevented by a selective COX-2 inhibitor (477).
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Figure 4. Microglia are the primary recipients of peripheral inflammatory signals as they reach the brain. Activated microglia
initiate an inflammatory cascade by releasing cytokines, chemokines, prostaglandins and reactive nitrogen and oxygen species
(RNS and ROS, respectively). Bi-directional exchanges between microglia and astroglia amplify inflammatory signals within the
central nervous system (CNS). Cytokines including interleukin (IL)-1, IL-6, tumor necrosis (TNF)-alpha and interferon (IFN)-
gamma induce indoleamine 2,3 dioxygenase (IDO), the enzyme responsible for degrading tryptophan, the primary precursor of
serotonin (5-HT), into kynurenine, which is eventually metabolized into quinolinic acid (QUIN), a potent NMDA agonist and
stimulator of glutamate (Glu) release. Multiple astrocytic functions are compromised due to the excessive exposure to cytokines,
prostaglandins, QUIN and RNS/ROS, ultimately leading to downregulation of glutamate transporters, impaired glutamate
reuptake, excessive glutamate release and compromised synthesis and release of neurotrophic factors. Oligodendroglia suffer
damage due to toxic overexposure to cytokines such as TNF-alpha, and diminished neurotrophic support, both of which promote
apoptosis and demyelination. Copious amounts of glutamate are released from astrocytes in the vicinity of extrasynaptic NMDA
receptors, whose activation leads to inhibition of BDNF synthesis. Excessive NMDA activation, caused by QUIN and D-serine,
is compounded by diminished glutamate reuptake by astrocytes and oligodendroglia. NMDA-mediated excitotoxicity, combined
with a consequent decline in neurotrophic support, and an increase in oxidative stress, synergistically disrupts neural plasticity
and induces apoptosis.
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in response to peripheral inflammation, further up- gap-junction propagation of calcium waves that will
regulating NMDA mediated transmission (487). Glutamate activate glia on the contra-lateral side, leading to a release
mediated NMDA receptor activation combined with of pain mediators in an otherwise unaffected area (323).
voltage-gated Ca 2+ currents (VGCCs) cumulatively alter New data suggest that glia may also interfere with opioid
intracellular signaling (482). The subsequent activation of analgesia in pathologic pain states (415). In response to
mitogen-activated protein kinase-1 (MAPK-1) pathways morphine, glia cells release neuroexcitatory substances that
(e.g. extracellular signal-regulated kinase [ERK], p38, c- oppose its analgesic effect (415). It appears that microglial
Jun N-terminal kinase [JNK]) and NF-kappa B pathways in activation is necessary for the initiation of pathological
spinal glia and neurons plays an important role in pain, while astroglia have a major role in propagation and
development of NeP (482-484). Neuronal ERK further maintenance of pain states (493, 494). Consistent with this
sensitizes AMPA and NMDA receptors, thus augmenting view, administration of minocycline (an inhibitor of
excitatory neurotransmission (482). Enhanced purinergic microglial activation) prevented the development of
transmission, mediated by ATP binding to P2X3 receptors, neuropathic pain, but failed to suppress acute pain or
neurokinin-1 (NK-1) receptor activation by SP, glutamate already entrenched pathologic pain (495, 496).
binding to mGlu receptors and BDNF release cumulatively Accumulating preclinical evidence suggests that disturbed
augment nociceptive transmission (482). Synaptic neuron-glia relationships in the context of NeP may extend
glutamate transporters, including glutamate transporter-1 to the supraspinal regions (497-499). For example, a sciatic
(GLT-1) and glutamate-aspartate transporter (GLAST) nerve ligation model of chronic pain produced a significant
(both principal regulators of synaptic glutamate) become astrocyte activation in the cingulate gyrus (497). Similarly,
dysregulated following prolonged and excessive exposure a recent report indicated that spinal cord injury induced
to glutamate (482). Under usual circumstances, GABA microglial activation and ensuing release of
released by inhibitory neurons modulates glutamatergic proinflammatory cytokines in remote locations, such as
transmission in the dorsal horn synapse (488). Following “below-level” spinal cord (i.e. below the level of injury)
peripheral nerve injury, ATP stimulates BDNF release via and the thalamus (500). Pain induced by peripheral
P2X4 receptors in microglia (488). Interestingly, microglia inflammation was associated with elevated BDNF in PAG
are not the only source of BDNF, as oligodendroglia and and subsequent NMDA-mediated descending pain
astrocytes have also been found to release neurotrophins facilitation, via RVM (rostral ventromedial medulla),
following spinal cord injury (489). BDNF signaling suggesting a potential role for the supraspinal
through Trk-B receptors reduces the levels of anion inflammation-neurotrophic factor-glutamate interactions in
transporter KCC2, ultimately resulting in increased persistent pain (498). In a separate study, the same group of
intracellular Cl – concentrations (488). In these pathological authors reported elevations of IL-1 and TNF-alpha in
circumstances, GABA activation paradoxically leads to Cl – RVM, resulting in excessive NMDA activation and
efflux, further depolarizing the dorsal horn neuron (488). allodynia following chronic constriction injury, a
Thus, BDNF has a role in perpetuating “central preclinical model of neuropathic pain. Injection of the
sensitization” by both facilitating excitatory transmission microglial and astroglial inhibitors abolished hyperalgesia
and by interfering with inhibitory regulation (488, 490). and allodynia, providing further proof of supraspinal
astrocyte and microglia involvement in perpetuating the
Activation of MAPK and NF-kappa-B in descending pain facilitation (499).
microglia and astrocytes ultimately leads to increased
synthesis and eventual release of proinflammatory factors, Evidence supporting the neuron-glia pathology in
such as IL-1, IL-6, IL-18, TNF-alpha, PGE-2 and NO (482- the genesis of FM is modest at best. Kim et al. conducted
484). Recent research suggests that the dorsal root ganglia an electron microscopic assessment of the skin biopsy
(DRG) nociceptive neurons also express IL-1, possibly samples from fibromyalgia patients and compared them
contributing to the inflammatory response seen in with healthy controls (165). In 9/13 of FM patients
pathologic pain states (491). Release of proinflammatory unmyelinated Schwann cells were “ballooned”, whereas
mediators leads to a self-perpetuating activation of glia none of the control samples displayed this type of
cells and excessive stimulation of dorsal horn neurons, pathology. Also, in most of the FM patients, axons were
producing sensory abnormalities typically associated with localized in the periphery of the unmyelinated Schwann
nerve injury (483, 484). Additionally, proinflammatory cell sheets (165). This preliminary finding suggests that
mediators may directly impact axons or become transported altered glia morphology and function may play a role in
in a retrograde fashion to the cell bodies in dorsal root FM. Despite the paucity of hard evidence, some authors
ganglia, where they alter gene expression in the primary have posited that dysregulated neuron-glia relationships
nociceptive neurons, producing “neurogenic inflammation” may play a role in FM, based on similarities in biochemical
and contributing to “peripheral sensitization” (163, 168, markers between NeP, other chronic pain states and FM
492). (60, 420). In summary, multiple lines of evidence implicate
disturbances in neuron-glia relationships with concomitant
Altered neuron-glia interactions have recently disruption in the regulation of glutamatergic, neurotrophic
been implicated in the genesis of “mirror pain”, whereby and inflammatory signaling as principal mediators of
pain is propagated to the side contra-lateral to the site of chronic pain states, including NeP, and perhaps FM. Some
pathology (323). Hypothetically, robust glia activation on authors have even characterized chronic pain as a
one side, due to pathological pain processes, can trigger “gliopathy” (501).
5315
10. CONCLUSION all associated with altered sympathetic/parasympathetic

balance, neuroendocrine disturbances, characterized by
Multiple lines of evidence suggest that the co- insufficient HPA regulation and altered immune function.
occurrence of depression and pain is more the rule than the In turn, these peripheral responses signal back to the neural
exception. For example, recent studies demonstrate that 30- structures to further drive the CNS danger pathway
60% of depressed patients also suffer from a painful activation, leading to a maladaptive feedforward circuit,
condition (1), and at least an equal percentage of people which increasingly appears to be implicated in the
with chronic pain also meet criteria for a mood disorder production and maintenance of symptoms.
diagnosis. (502-506). Of course, pain is one of many
syndromes/symptoms that co-occur regularly with MDD. Microglia seem to be the principal recipients of
For example, there is also a considerable overlap between bodily distress/pain signals. Differences in the micro-
MDD and anxiety disorders (507, 508). Some authors have environments in the dorsal column of the spinal cord and
gone so far as to suggest that they are dual manifestations brain areas involved in processing of pain in MDD, NeP
of the same underlying patho-physiological condition (354, and FM may be reflected in the different patterns of
509). Manchikanti et al. even reported a “dose-response” interaction between microglia, astroglia and neurons, in
relationship amongst pain, and depression and anxiety. In these respective conditions. Excessive firing of nociceptive
this study, 14% of the control group experienced GAD and neurons in the context of NeP and FM tends to be
4% experienced MDD. Among the patients who associated with increased release of substance-P, IL-8
experienced chronic pain involving one bodily region, the (which promotes sympathetic pain), IL-18, fractalkine and
prevalence of GAD and MDD was 30% and 20%, CGRP, in addition to chemokines produced by macrophage
respectively. If patients experienced pain in two or more and mononuclear cells (324, 482-484, 492), none of which
regions, the prevalence of GAD and MDD rose to 54% and are part of the usual spinal cord cellular milieu in MDD.
32% (504). Given this overlap, it is no surprise that the Nonetheless, excessive excitatory glutamatergic
presence of pain is a major predictor of depression and transmission and compromised GABA mediated inhibition
anxiety. appear to be common features of depression and pain
disorders. Dysregulation in monoamine, substance P,
It is becoming increasingly clear that galanin and opiate signaling also characterizes both pain
relationships between MDD, anxiety and pain run even syndromes and MDD. On the other hand, depression and
deeper than the surface similarities shared by these pain disorders have been reported to have different
conditions. For example, although not completely patterns of abnormality in the production of
consistent, studies point to a shared genetic underpinning neurotrophic factors. MDD is characterized by the
for these disorders, especially in relation to genes involved reduction of the serum BDNF levels, while serum
in the regulation of monoaminergic and peptide BDNF levels tend to be increased in FM. . Nonetheless,
transmission, inflammatory response, diurnal rhythm and pain, stress and depression have a similar, if not
neurotrophic signaling. All of these are important synergistic, impact on neurotrophic signaling in
modulators of pain, emotional tone and the stress response. hippocampus, with all three conditions being associated
Stress, in turn, is a major precipitant, perpetuant and an with reduced BDNF synthesis. This finding is of
aggravating factor in all three conditions. Consistent with particular interest, given that hippocampus represents a
this, brain circuitry involved in the regulation of mood and veritable “intersection” of pathways involved in
the stress response overlaps to a significant degree with emotional regulation, memory and coordination of the
components of the “pain matrix”, involved in emotional stress response.
and cognitive aspects of pain processing.
MDD, NeP and FM are all associated with
From an evolutionary perspective, it is apparent neuroplastic changes in the CNS. In pathologic pain states,
that both negative emotions and physical pain have a facilitation of pain signaling, presumably based on
tremendous survival value, given that both provide a clear neuroplastic changes in pain pathways, is often designated
signal that current conditions are a threat to an organism’s as the “central sensitization”. Similarly, the recurrent and
goals and/or survival. Thus, it is not surprising, that, both most likely progressive nature of MDD is often ascribed to
peripheral and CNS depression and pain pathways overlap “kindling”, which—like central sensitization—reflects
significantly, nor that these pathways are built into the warp neuroplastic changes. Given this, MDD, NeP and FM may
and woof of the mammalian stress and immune response all be characterized by adaptive processes gone awry as a
systems. While important differences exist in the result of complex interactions between genetic
processing of physical pain, emotional pain and stress vulnerabilities and environmental factors. In this scenario,
responses at the level of pain sensory areas (e.g. thalamus, persistent aberrant processing of emotional, painful and
SI and SII), striking similarities are apparent in the stressful signals eventually becomes “ossified”, presumably
involvement of limbic and paralimbic prefrontal cortical due to ensuing neuroplastic changes. In some regards,
areas (amygdala, hippocampus, insula, ACC, vmPFC), as depression, FM and NeP share dysfunctional
well as more “cognitive” and integrative brain areas, such psychosomatic and somatopsychic communication. It
as rACC, dACC dmPFC and DLPFC. Moreover, pain and should be no surprise, then, that overlapping pathology
depressed mood appear to have an overlapping capacity to gives rise to similar phenomenological manifestations. If
engage autonomic, neuroendocrine and neuroimmune we assume that shared biological underpinnings give rise to
components of the stress response. MDD, FM and NeP are the clinical symptoms of MDD, NeP and FM, it is clear that
5316
a full understanding of this “synergy” has critical treatment 12. Offenbaecher M and M. Ackenheil: Current trends in
implications. neuropathic pain treatments with special reference to
fibromyalgia. CNS Spectr, 10(4), 285-97 (2005)
11. ACKNOWLEDGEMENTS
13. Martinez-Lavin M, S. Lopez, M. Medina and A. Nava:
The authors would like to thank Eli Lilly and Use of the leeds assessment of neuropathic symptoms and
Company, Mathew Iype and Christopher Wikoff of signs questionnaire in patients with fibromyalgia. Semin
Indegene, for financial and technical assistance in Arthritis Rheum, 32(6), 407-11 (2003)
preparation of illustrations. We would also like to
acknowledge Eli Lilly and Company for assistance with 14. Torrance N, B. H. Smith, M. I. Bennett and A. J. Lee:
publication costs. The epidemiology of chronic pain of predominantly
neuropathic origin. Results from a general population
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Key Words: Neurobiology, Depression, Neuropathic Pain,

Fibromyalgia, Hypothalamic-Pituitary-Adrenal Axis,
Inflammation, Glia, Neurotrophic Factors, Review
Send correspondence to: Vladimir Maletic, 38 Parkway

Commons Way, Greer, SC 29650, Tel: 864-848-4448, Fax:
864-848-4428, E-mail: vmaletic@bellsouth.net
http://www.bioscience.org/current/vol14.htm
5338
PAIN
Pain Supplement 6 ( 1999) S 121 -S 126
From the gate to the neuromatrix

Ronald Melzack
Department of Psychology. McGill University, 1205 Dr. Penjkld Avenue. Montreal, Quebec H3A IBI, Canada
Abstract
The gate control theory’s most important contribution to understanding pain was its emphasis on central neural mechanisms. The theory
forced the medical and biological sciences to accept the brain as an active system that filters, selects and modulates inputs. The dorsal horns,
too, were not merely passive transmission stations but sites at which dynamic activities (inhibition, excitation and modulation) occurred. The
great challenge ahead of us is to understand brain function. I have therefore proposed that the brain possesses a neural network - the body-
self neuromatrix - which integrates multiple inputs to produce the output pattern that evokes pain. The body-self neuromatrix comprises a
widely distributed neural network that includes parallel somatosensory, limbic and thalamocortical components that subserve the sensory-
discriminative, affective-motivational and evaluative-cognitive dimensions of pain experience. The synaptic architecture of the neuromatrix
is determined by genetic and sensory influences. The ‘neurosignature’ output of the neuromatrix - patterns of nerve impulses of varying
temporal and spatial dimensions - is produced by neural programs genetically build into the neuromatrix and determines the particular
qualities and other properties of the pain experience and behavior. Multiple inputs that act on the neuromatrix programs and contribute to the
output neurosignature include, (1) sensory inputs (cutaneous, visceral and other somatic receptors); (2) visual and other sensory inputs that
influence the cognitive interpretation of the situation; (3) phasic and tonic cognitive and emotional inputs from other areas of the brain; (4)
intrinsic neural inhibitory modulation inherent in all brain function; (5) the activity of the body’s stress-regulation systems, including
cytokines as well as the endocrine, autonomic, immune and opioid systems. We have traveled a long way from the psychophysical concept
that seeks a simple one-to-one relationship between injury and pain. We now have a theoretical framework in which a genetically determined
template for the body-self is modulated by the powerful stress system and the cognitive functions of the brain, in addition to the traditional
sensory inputs. 6 1999 International Association for the Study of Pain. Published by Elsevier Science B.V.
Keywords; Gate control theory: Neuromatrix; Neurosignature
1. Introduction apparatus that resembled the catapult in the comic-strip

‘Hager the Horrible’ and flung cans of food against a cement
In 1959, Patrick Wall had already achieved a reputation surface. Happily for me, Pat convinced them that they could
as a brilliant young scientist who had done important dispense with some of their space to allow me to continue
research on spinal cord physiology. As a result, when I my research on the effects of early sensory deprivation on
arrived at the Massachusetts Institute of Technology in the perception of pain. Thus began a life-long friendship that
September 1959 as an Assistant Professor of Psychology, has been one of the highlights of my life.
I was eager to meet Pat, who was a Professor in M.I.T.‘s During periodic visits to Pat’s lab, where he was invari-
prestigious Department of Biology. We met more quickly ably doing an experiment on the spinal cord, he and I talked
than I expected because I was appalled to discover on the often about our interests in somesthesis and the particularly
day of my arrival that research with animals could not be challenging problem of pain. W.K. Livingston visited me
done in my building and I would have to go elsewhere to do from time to time, and he was delighted to join Pat and me
it. A colleague suggested that I call Pat Wall, who might be during one of our discussions on alternatives to specificity
able to help me. theory. This topic - the need for a new theory - was
Pat detected the desperation in my voice and invited me foremost in our minds, and after a year or so, Pat and I
over. After a warm, friendly conversation, Pat said that decided to write a paper together.
some research space might be available for me. Members When we began our discussions that led to the gate
of the Department of Food Technology in Pat’s building had control theory of pain, we were convinced that (1) brain
funds from a U.S. Space Agency to discover what might processes had to be integrated into the theory, including
happen to cans of food that make a hard landing on the feedforward and feedback transmission; and (2) the new
moon’s surface. To find out, these investigators used an hypothetical spinal cord mechanism would need sufficient
0304-3959/99/$20.00 0 1999 International Association for the Study of Pain. Published by Elsevier Science B.V
PII: s0304-3959(99)00 145- 1
explanatory power to challenge spinal-cord physiologists
and entice them away from the concept of specificity.
How the theory actually came into being involves an
amusing sequence of events. My early research in psychol-
ogy and physiology led me to speculate that the brain exerts
a powerful, continuous descending inhibitory control over
the input that is transmitted through the dorsal horns
(Melzack et al.. 1958). But this notion of modulation of
input by the brain does not constitute a conceptual model
of pain. It could be part of one, but more was needed. In
19.59. Pat was examining the different nerve impulse
patterns evoked in dorsal horn cells by various stimuli and
the way in which vibration modulated the pattern evoked by
noxious stimulation. In I96 1 1 published an article in Scien-
tific American which reviewed the psychology and physiol-
ogy of pain as it was understood at the time. It emphasized
4
patterning, modulation in the dorsal horns, multiple ascend-
ing pathways and the multidimensional qualities of pain
L
I GATE CONTROL SYSTEM
experience. But it was not a cohesive, succinct theory. In

1962. Pat and I (Melzack and Wall, 1962) proposed a
general theory of somesthesis in the form of eight proposi- INPUT ACTION -)
SYSTEM
tions. The paper, published in Brain. evoked some interest
but had little impact. We then toyed with the idea of using
S
this general ‘theory’ as the basis for a theory that dealt
exclusively with pain but we made no headway and put
the project aside.
Then, things unexpectedly and suddenly started to fall Fig. I. The evolution of the gate control theory. (A) show Noordenbos’
into place. It began in the fall of 1962. when I first stumbled model in which large. fast-conductin g fibers Inhibit small. multisynaptic.
onto William Noordenbos’ 1959 book on pain (Noordenbos, slowly conducting fibers. Noordenbos ( 1959) wy\ of the circle that repre-
sents the dorsal horns: ‘In this circle which includes the wbstantia gelati-
1959). That brilliant little book led me to have a ‘flash of
nosa of Rolandi and its immediate adjacent parts. the multitiher pattern of
insight’. afferent impulses is modified... The nature of this inhibitory interaction will
Fig. IA shows Noordenbos’ concept of pain. He did not not be further discussed...’ (B) An early development that led to the pate
fill in the circle in the dorsal horns to show how large fibers control theory in which the large fiber system is shown to actiwtc psycho-
inhibit small ones. He just said that they did, and showed a lopical variables (such as meanin g and past experience) that then project
down to the dorsal horns and modulate the input. (C) Show:, a further
picture of the substantia gelatinosa to illustrate the complex-
development toward the gate control theory which comprises a theoretical
ity of dorsal horn anatomy. He then went on to explain presynaptic inhibitton exerted by the sutxtantin gelatinosa. The idea\
temporal and spatial summation, referred pain, and other gradually evolved into the model of the gate control theory shown ;Ltthe
properties of pain after nerve injury. However, Noordenbos’ bottom.
story stops at the thalamus - the T at the top. My idea was
to put a cortex on Bill’s thalamus, show the dorsal column by mail or during my many visits to Boston where we
projection as a rapid, precise feedforward system to activate consumed large amounts of duty-free whiskey and talked
psychological processes, with a feedback to the circle to late into the night at Pat’s home.
modulate the input (Fig. IB). Here, at last, was the begin- When the gate control theory of pain was published in
ning of a conceptual model in which brain processes can 1965, we were astonished by the reception. The theory
select, filter and modulate pain signals. generated vigorous (sometimes vicious) debate as well as
When I discussed all this with Pat, he began to have ideas a great deal of research to disprove or support the theory.
too. He soon developed a concept, based on his research on The search for specific pain fibers and spinal-cells by our
the substantia gelatinosa, for a hypothetical mechanism to opponents now became almost frantic. It was not until the
put in the circle. A few weeks later he gave me his picture mid-1970’s that the gate control theory was presented in
(Fig. 1C). It may seem an easy step from our two pictures to almost every major textbook in the biological and medical
the final gate model, but it was not. We invented and sciences. At the same time there was an explosion in
rejected a variety of names for the theory and the compo- research on the physiology and pharmacology of the dorsal
nents of the model. It took countless drafts, changes and horns and the descending control systems. The theory’s
compromises to produce the final paper (Melzack and emphasis on the modulation of inputs in the spinal dorsal
Wall. 1965). I moved to McGill University in 1963, so horns and the dynamic role of the brain in pain processes
that most of the paper was written by exchanging drafts had a clinical as well as a scientific impact. Psychological
R. Mel:nck / Pair? Sup~~lmtcwt 6 (1999) S121-SIX s123
factors, which were previously dismissed as ‘reactions to and is identified as the ‘self’, distinct from other people and
pain’ were now seen to be an integral part of pain processing the surrounding world. The experience of a unity of such
and new avenues for pain control were opened. Similarly, diverse feelings, including the self as the point of orientation
cutting nerves and pathways was gradually replaced by a in the surrounding environment. is produced by central
host of methods to modulate the input. Physical therapists neural processes and cannot derive from the peripheral
and other healthcare professionals who use a multitude of nervous system or spinal cord. Fourth, the brain processes
sensory modulation techniques were brought into the that underlie the body-self are, to an important extent which
picture, and TENS became an important modality for the can no longer be ignored, ‘built-in’ by genetic specification,
treatment of chronic and acute pain. although this built-in substrate must, of course, be modified
A major force in this exciting epoch was John Bonica, by experience. These conclusions provide the basis of a new
who had been trying valiantly to convince his medical conceptual model.
colleagues that pain is a syndrome in its own right that How can we explain our experience of the body? I
merits special attention, research and funding. The arrival propose that a genetically built-in matrix of neurons for
of the gate control theory encouraged John to pursue his the whole body produces characteristic nerve-impulse
cause even more vigorously. At the same time, he promoted patterns for the body and the myriad somatosensory quali-
the gate control theory as a focus for new medical ties we feel. 1 have termed the network, whose spatial distri-
approaches. Out of all this ferment of theory, research and bution and synaptic links are initially determined
clinical advances, John brought together a host of scientists genetically and are later sculpted by sensory inputs, a
and clinicians and formed the International Association for ‘neuromatrix’. Thalamocortical and limbic loops that
the Study of Pain (Bonica. 1974). At the same time, the comprise the neuromatrix diverge to permit parallel proces-
journal Pain was created, with Pat as its founding Editor. sing in different components of the neuromatrix and
He has done, and continues to do, a brilliant job of it. The converge to permit interactions between the output products
journal helped establish the held of pain as a major specialty of processing. The cyclical processing and synthesis of
in the health sciences and professions. nerve impulses in the neuromatrix imposes a characteristic
What was the gate control theory’s most important contri- output pattern or ‘neurosignature’.
bution to biological and medical science? I believe it was Loeser and I (Melzack and Loeser, 1978) have presented
the emphasis on CNS mechanisms. Never again, after 1965, a model. consistent with the gate control theory of pain,
could anyone try to explain pain exclusively in terms of which proposes that synaptic areas along the transmission
peripheral factors. The theory forced the medical and biolo- routes of the major sensory projection systems - from the
gical sciences to accept the brain as an active system that dorsal horns to the somatosensory projection areas in the
filters. selects and modulates inputs. The dorsal horns, too, thalamus and cortex - may become pattern generating
were not merely passive transmission stations but sites at mechanisms. Their activity is capable of producing patterns
which dynamic activities - inhibition, excitation and of nerve impulses which exceed a critical firing level per
modulation - occurred. This then was the revolution: we unit time (or have a particular pattern, or both) and project to
highlighted the central nervous system as an essential other areas that subserve pain experience and the localiza-
component in pain processes. tion of pain at specific sites.
This concept is consistent with the fact that injury may
produce high firing levels that signal pain as well as with the
2. The neuromatrix observation that loss of input to central structures by deaf-
ferentation after amputation, root section or cord transection
Where do we go from here? I believe the great challenge also produce high firing levels and abnormal bursting
ahead of us is to understand brain function. My analysis of patterns that may provide the necessary conditions for
phantom limb phenomena (Melzack, 1989; Melzack et al.. pain. Thus. any input to the hyperactive central cells -
1997) has led to four conclusions which point to a new from nearby injured tissues, from visceral sensory nerve,
conceptual nervous system. First. because the phantom from small afferents in the sympathetic chain and from
limb (or other body part) feels so real. it is reasonable to higher psychoneuronal processes - can trigger abnormal.
conclude that the body we normally feel is subserved by the prolonged firing and produce severe, persistent pains in
same neural processes in the brain. These brain processes discrete areas of the denervated limbs or other body parts.
are normally activated and modulated by inputs from the
body but they can act in the absence of any inputs. Second,
all the qualities we normally feel from the body, including 3. Pain and stress
pain, are also felt in the absence of inputs from the body.
From this we may conclude that the origins of the patterns We are so accustomed to considering pain as a purely
that underlie the qualities of experience lie in neural sensory phenomenon that we have ignored the obvious
networks in the brain: stimuli may trigger the patterns but fact that injury does not merely produce pain: it also disrupt
do not produce them. Third, the body is perceived as a unity the brain’s homeostatic regulation systems, thereby produ-
S124 R. Melzack / Pain Supplement 6 (1999) S1214126
cing ‘stress’ and initiating complex programs to reinstate released within minutes, their initial function may be simply
homeostasis. By recognizing the role of the stress system to inhibit or modulate the release of cortisol. Experiments
in pain processes, we discover that the scope of the puzzle of with animals suggest that their analgesic effects may not
pain is vastly expanded and new pieces of the puzzle appear until as long as 30 min after injury.
provide valuable clues in our quest to understand chronic Cortisol, together with noradrenergic activation, sets the
pain (Melzack, 1998, 1999). stage for response to life-threatening emergency. If the
Hans Selye, who founded the field of stress research, dealt output of cortisol is prolonged, excessive or of abnormal
with stress in the biological sense of physical injury, infec- patterning, it may produce destruction of muscle, bone
tion and pathology, and also recognized the importance of and neural tissue and produce the conditions for many
psychological stresses. In recent years, the latter sense of the kinds of chronic pain.
word has come to dominate the field. However, it is impor- Cortisol is an essential hormone for survival after injury
tant for the purpose of understanding pain to keep in mind because it is responsible for producing and maintaining high
that stress is a biological system that is activated by physical levels of glucose for rapid response after injury, threat or
injury, infection or any threat to biological homeostasis as other emergency. However, cortisol is potentially a highly
well as by psychological threat and insult of the body-self. destructive substance because, to ensure a high level of
Both are correct and important. glucose, it breaks down the protein in muscle and inhibits
The disruption of homeostasis by injury activates the ongoing replacement of calcium in bone. Sustained
programs of neural, hormonal and behavioral activity cortisol release, therefore, can produce myopathy, weak-
aimed at a return to homeostasis. The particular programs ness, fatigue and decalcification of bone. It can also accel-
that are activated are selected from a genetically determined erate neural degeneration of the hippocampus during aging.
repertoire of programs and are influenced by the extent and Furthermore, it suppresses the immune system.
severity of the injury. A major clue to the relationships between injury, stress
When injury occurs, sensory information rapidly alerts and pain is that many autoimmune diseases, such as rheu-
the brain and begins the complex sequence of events to matoid arthritis and scleroderma, are also pain syndromes.
reinstate homeostasis. Cytokines are released within Furthermore, more women than men suffer from autoim-
seconds after injury. These substances, such as gamma- mune diseases as well as chronic pain syndromes. Among
interferon, interleukins 1 and 6, and tumour necrosis factor, the 5% of adults who suffer from an autoimmune disease.
enter the bloodstream in l-4 min and travel to the brain. The two out of three are women. Pain diseases also show a sex
cytokines, therefore, are able to activate fibers that send difference, as Berkley (1997) has argued, with the majority
messages to the brain and, concurrently, to breach the prevalent in women, and a smaller number prevalent in men.
blood-brain barrier at specific sites and have an immediate Of particular importance is the concurrent change in sex
effect on hypothalamic cells. The cytokines together with ratios with changes in sex hormone output as a function of
evaluative information from the brain rapidly begin a age. Estrogen increases the release of peripheral cytokines.
sequence of activities aimed at the release and utilization such as gamma-interferon, which in turn produce increased
of glucose for necessary actions, such as removal of debris, cortisol. This may explain why more females than males
the repair of tissues and (sometimes) fever to destroy suffer from most kinds of chronic pain as well as painful
bacteria and other foreign substances. At sufficient severity autoimmune diseases such as multiple sclerosis and lupus.
of injury, the noradrenergic system is activated. Adrenalin is I propose that some forms of chronic pain may occur as a
released into the blood stream and the powerful locus coer- result of the cumulative destructive effect of cortisol on
uleus/norepinephrine (LUNE) system in the brainstem muscle, bone and neural tissue. Furthermore, loss of fibers
projects information upward throughout the brain and in the hippocampus due to aging reduces a natural brake on
downward through the descending efferent sympathetic cortisol release which is normally exerted by the hippocam-
nervous system. Thus the whole sympathetic system is acti- pus. As a result, cortisol is released in larger amounts,
vated to produce readiness of the heart, blood vessels and producing a greater loss of hippocampal fibers and a cascad-
other viscera for complex programs to reinstate homeostasis ing deleterious effect. This is found in aging primates and
(Chrousos and Gold, 1992; Sapolsky, 1992). presumably also occurs in humans. It could explain the
At the same time, the perception of injury activates the increase of chronic pain problems among older people.
hypothalamic-pituitary-adrenal (HPA) system, in which Cortisol output by itself may not be sufficient to cause any
corticotropin-releasing hormone (CRH) produced in the of these problems, but rather it provides the conditions so
hypothalamus enters the local blood stream which carries that other contributing factors may, all together, produce
the hormone to the pituitary, causing the release of adreno- them. Sex-related hormones, genetic predispositions,
corticotropic hormone (ACTH) and other substances. The psychological stresses derived from social competition
ACTH then activates the adrenal cortex to release cortisol, and the hassles of every day life may act together to influ-
which must inevitably play a powerful role in determining ence cortisol release, its amount and pattern, and the effects
chronic pain. Cortisol also acts on the immune system and of the target organs.
the endogeneous opioid system. Although these opioids are These speculations are supported by strong evidence.
R. Melzack / Pain Supplement 6 (1999) S121S126 S125
Chrousos and his colleagues (Chrousos and Gold, 1992) by multiple influences. These influences range from the
have documented the effects of dysregulation of the cortisol existing synaptic architecture of the neuromatrix - which
system: effects on muscle and bone, to which they attribute is determined by genetic and sensory factors - to influ-
fibromyalgia, rheumatoid arthritis and chronic fatigue ences from within the body and from other areas in the
syndrome. They propose that they are caused by hypocorti- brain. Genetic influences on synaptic architecture may
solism, which could be due to depletion of cortisol as a determine, or predispose toward, the development of
result of prolonged stress. Indeed, Sapolsky (1992) attri- chronic pain syndromes. Fig. 2 summarizes the factors
butes myopathy, bone decalcification, fatigue and acceler- that contribute to the output pattern from the neuromatrix
ated neural degeneration during aging to prolonged that produce the sensory, affective and cognitive dimensions
exposure to stress. of pain experience and behavior.
Clearly, consideration of the relationship between stress- We have traveled a long way from the psychophysical
system effects and chronic pain leads directly to examina- concept that seeks a simple one-to-one relationship between
tion of the effects of suppression of the immune system and injury and pain. We now have a theoretical framework in
the development of autoimmune effects. The fact that which a genetically determined template for the body-self is
several autoimmune diseases, such as Crohn’s disease, modulated by the powerful stress system and the cognitive
multiple sclerosis, rheumatoid arthritis, scleroderma and functions of the brain, in addition to the traditional sensory
lupus, are also classified as chronic pain syndromes suggests inputs.
that the study of these syndromes in relation to stress effects The neuromatrix theory of pain - which places genetic
and chronic pain could be fruitful. Immune suppression, contributions and the neural-hormonal mechanisms of stress
which involves prolonging the presence of dead tissue, on a level of equal importance with the neural mechanisms
invading bacteria and viruses, could produce a greater of sensory transmission - has important implications for
output of cytokines, with a consequent increase in cortisol research and therapy. An immediate recommendation is that
and its destructive effects. Furthermore, prolonged immune interdisciplinary pain clinics should expand to include
suppression may diminish gradually and give way to a specialists in endocrinology and immunology. Such a colla-
rebound, excessive immune response. The immune boration may lead to insights and new research strategies
system’s attack on its own body’s tissues may produce auto-
immune diseases that are also chronic pain syndromes.
Thorough investigation may provide valuable clues for
understanding at least some of the terrible chronic pain
syndromes that now perplex us and are beyond our control.
4. The multiple determinants of pain

ViSUSl, luditory nnd
The neuromatrix theory of pain proposes that the neuro- other sensory input
signature for pain experience is determined by the synaptic Tonic somstic input
(trigger points.
architecture of the neuromatrix, which is produced by
genetic and sensory influences. The neurosignature pattern
is also modulated by sensory inputs and by cognitive events,
such as psychological stress. It may also occur because
stressors, physical as well as psychological, act on stress- Fig. 2. The body-self nemomatrix. The body-self neuromatrix, which
regulation systems, which may produce lesions of muscle, comprises a widely distributed neural network that includes somatosensory,
limbic. and thalamocortical components, is schematically depicted as a
bone, and nerve tissue, thereby contributing to the neuro-
circle containing smaller parallel networks that contribute to the sensory-
signature patterns that give rise to chronic pain. In short, the discriminative (S), affective-motivational (A), and evaluative-cognitive (E)
neuromatrix, as a result of homeostasis-regulation patterns dimensions of pain experience. The synaptic architecture of the neuroma-
that have failed, produces the destructive conditions that trix is determined by genetic and sensory influences. The ‘neurosignature’
may give rise to many of the chronic pains that so far output of the neuromatrix-patterns of nerve impulses of varying temporal
and spatial dimensions-is produced by neural programs genetically built
have been resistant to treatments developed primarily to
into the neuromatrix and determines the particular qualities and other prop-
manage pains that are triggered by sensory inputs. The stress erties of the pain experience and behavior. Multiple inputs that act on the
regulation system, with its complex, delicately balanced neuromatrix programs and contribute to the output neurosignature include
interactions, is an integral part of the multiple contributions (1) sensory inputs from somatic receptors (phasic cutaneous, visceral and
that give rise to chronic pain. tonic somatic inputs); (2) visual and other sensory inputs that influence the
cognitive interpretation of the situation; (3) phasic and tonic cognitive and
The neuromatrix theory guides us away from the Carte-
emotional inputs from other areas of the brain; (4) intrinsic neural inhibitory
sian concept of pain as a sensation produced by injury, modulation inherent in all brain function; and (5) the activity of the body’s
inflammation, or other tissue pathology and toward the stress-regulation systems, including cytokines as well as the endocrine.
concept of pain as a multidimensional experience produced autonomic. immune and opioid systems.
S126 R. Melzack/ Pain Supplement 6 (1999) S12/-S126
that may reveal the underlying mechanisms of chronic pain Melzack R, Wall PD. On the nature of cutaneous sensory mechanisms.
and give rise to new therapies to relieve the tragedy of Brain 1962;85:331-356.
Melzack R, Wall PD. Pain mechanisms: a new theory. Science
unrelenting suffering.
1965;150:971-979.
Melzack R, Stotler WA, Livingston WK. Effects of discrete brainstem
lesions in cats on perception of noxious stimulation. J Neurophysiol
Acknowledgements 1958;21:353-367.
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This paper was supported by grant #A7891 from the congenital limb deficiency or amputation in early childhood. Brain
Natural Sciences and Engineering Research Council of 1997:120:1603-1620.
Melzack R. Pain and stress: clues toward understanding chronic pain. In:
Canada.
Sabourin M. Craik F, Robert M, editors. Advances in psychological
science, Vol. 2. Biological and Cognitive Aspects. Hove: Psychology
Press, 1998. pp. 63-85.
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editors. Psychosocial factors in pain, New York: Guilford Press. 1999.
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on Pain New York: Raven Press, 1974. ial Lecture). Can Psycho1 1989;30:1-16.
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Developmental Cognitive Neuroscience 11 (2015) 18–30
Contents lists available at ScienceDirect
Developmental Cognitive Neuroscience

journal homepage: http://www.elsevier.com/locate/dcn
Broken or maladaptive? Altered trajectories in

neuroinflammation and behavior after early life adversity
Prabarna Ganguly, Heather C. Brenhouse ∗
Northeastern University, Psychology Department, 125 Nightingale Hall, 360 Huntington Avenue, Boston, MA 02115, USA
a r t i c l e i n f o a b s t r a c t
Article history: Exposure to adversity and stress early in development yields vulnerability to mental ill-
Received 1 May 2014 nesses throughout the lifespan. Growing evidence suggests that this vulnerability has
Received in revised form 23 June 2014 mechanistic origins involving aberrant development of both neurocircuitry and neuro-
Accepted 2 July 2014
immune activity. Here we review the current understanding of when and how stress
Available online 11 July 2014
exposure initiates neuroinflammatory events that interact with brain development. We
first review how early life adversity has been associated with various psychopatholo-
Keywords:
gies, and how neuroinflammation plays a role in these pathologies. We then summarize
Early life stress
data and resultant hypotheses describing how early life adversity may particularly alter
Microglia
Development neuro-immune development with psychiatric consequences. Finally, we review how sex
Immunology differences contribute to individualistic vulnerabilities across the lifespan. We submit the
Sex importance of understanding how stress during early development might cause outright
Vulnerability neural or glial damage, as well as experience-dependent plasticity that may insufficiently
prepare an individual for sex-specific or life-stage specific challenges.
© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction exposure to threats has been repeatedly shown to yield a

myriad of deviations in brain circuitry, stress-responsivity,
Early life experiences—both positive and negative—can cognitive function, and general health (Anda et al., 2008;
have profound effects on brain development in mammals. Dube et al., 2009; Brown et al., 2010). In this review, we
Rearing environments that are enriched with good parental will discuss the current progress in understanding inter-
care, suitable protection, and engaging sensory stimulation vening variables that underlie vulnerability, resilience, and
offer resilience to insults later in life such as psycholog- behavior after ELA, with a focus on the evolving knowledge
ical stressors (Francis et al., 2002) or even pathological of neuroimmune influences. We will present findings from
infection (Johnson et al., 2014). In contrast, early life adver- both human and animal research, since a comprehensive
sity (ELA) such as parental deprivation, neglect, abuse, or and clinically relevant view will only come from a synthe-
sis of both realms. Models of ELA vary widely across studies,
and each provides a distinct characteristic of exposure and
Abbreviations: ADHD, attention deficit hyperactivity disorder; COX-2, effects. A full comparison of all models is beyond the scope
cyclooxygenase-2; ELA, early life adversity; HPA, hypothalamic-pituitary of this review; therefore we will present different models
axis; IFN, interferon; IL, interleukin; PFC, prefrontal cortex; PTSD, post- throughout and highlight the implications of differences
traumatic stress disorder; SHP, stress hyporesponsive period; SHRP,
when possible.
spontaneously hypertensive rats; TNF, tumor necrosis factor.
∗ Corresponding author. Tel.: +1 617 373 6856. The idea of modeling the correct kind of ELA is
E-mail addresses: prabarna@gmail.com (P. Ganguly), irrelevant, since there is no single type of exposure, and
h.brenhouse@neu.edu (H.C. Brenhouse). the remarkable plasticity exhibited by the brain is largely
http://dx.doi.org/10.1016/j.dcn.2014.07.001
1878-9293/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/3.0/).
P. Ganguly, H.C. Brenhouse / Developmental Cognitive Neuroscience 11 (2015) 18–30 19
experience-dependent. For example, growing evidence ELA-attributable depression. Therefore, the life-long con-
from human studies strongly suggests that gray mat- sequences of ELA could be viewed very differently as either
ter volume, cortical thickness, and white matter integrity a result of, or a response to, these stressful experiences. This
are differentially altered across brain areas depending conceptual distinction is worthy of attention as we attempt
on the type of ELA exposure (Tomoda et al., 2009). to understand the what’s and why’s of vulnerability to men-
Animal work has revealed that stressful experiences in tal illness after ELA.
general can have functionally relevant effects on den-
dritic arbor, spine, and synapse number in many brain 2. Behavioral effects of early life stress across the
regions, including the hippocampus, amygdala, and the lifespan
prefrontal cortex (PFC), with effects on cognition, emo-
tional regulation and neuroendocrine function (McEwen ELA causes children to experience their environment
and Gianaros, 2011). These effects can occur through exci- as threatening, perceiving themselves as having no value
totoxicity (Moghaddam, 1993), oxidative stress (Madrigal and regarding the future as being not trustworthy (Dube
et al., 2001; Manikandan et al., 2006; Spiers et al., 2013), et al., 2003). A history of ELA consequentially increases
and inflammation (Munhoz et al., 2010). When presented the risk of developing a psychiatric disorder in adulthood
early in life, these processes can prevent typical devel- (Rojo-Moreno et al., 1999; Ritchie et al., 2009; Wright et al.,
opmental patterns of innervation and receptor activity, 2009; Carr et al., 2013). Models of plasticity such as the
and cause unhealthy sensitization of the immune response allostatic load and reactive scope models have been useful
(Hennessy et al., 2011). For example, stress-induced activ- to understand the mechanisms underlying psychopathol-
ity of the immune and neuroendocrine systems (McEwen ogy after ELA (Howell and Sanchez, 2011). In these models,
and Magarinos, 1997; Goshen and Yirmiya, 2009; Sorrells the pathological consequences of ELA have been attributed
et al., 2009) reportedly causes neuronal damage in areas to a dysfunction in homeostasis of neural, endocrine, or
such as the hippocampus (Schneider et al., 1998; Avital immune functions. It has also been proposed that the
et al., 2003; Ross et al., 2003; Frank et al., 2012), striatum effects of ELA on allostatic load can contribute to diathesis
(Relton and Rothwell, 1992) and PFC (de Pablos et al., 2006). for stress-mediating disorders later in life (Grassi-Oliveira
At the same time, altered neurotransmission (Gunn et al., et al., 2008; Rogosch et al., 2011; Danese and McEwen,
2013), synaptogenesis (Aisa et al., 2009; Jutapakdeegul 2012).
et al., 2010), and immune responsivity are consequences Notably, ELA-exposed individuals have an earlier age of
of ELA that could be interpreted as adaptations to the envi- onset for several disorders such as depression and sub-
ronment in preparation for future challenges (Tottenham stance abuse (Andersen and Teicher, 2008; Scott et al.,
and Sheridan, 2009). Indeed, ELA represents stressors that 2012) compared to the general population. These individ-
impact the brain during a time of rapid development uals also have a greater risk of self-harm and have poorer
and, importantly, during a time preceding the tumultuous response to treatment in comparison to non-maltreated
period of adolescence. Here, we will explore the young but people with same psychopathologies (Nemeroff et al.,
growing landscape of how neural and immune develop- 2003). These findings are indicative of the major differences
mental trajectories that drive behavior intersect (or fail to between individuals affected by ELA versus later stressors,
intersect) with environmental demands over the lifespan. and show a need to understand the underlying biology and
ELA impacts the immune system at the time of expo- behavior caused by ELA over a lifespan.
sure (Hennessy et al., 2010, 2011), and can also alter the
normal developmental trajectory of certain immunologi- 3. Neuroinflammation and psychopathology
cal processes (e.g., Coe et al., 1989). One consequence of
these early alterations is a heightened immune response The immune system has been implicated in vulnerabil-
to stressors later in life (see Tables 1 and 2). The ity to psychopathologies over the lifespan. For example,
adaptive advantage of heightened immune function in many clinical studies have provided evidence for the
response to stress can be seen from an evolutionary influence of immunological activation during the prena-
perspective, since a psychological stressor would typi- tal or early postnatal period on behavioral, psychological
cally occur alongside a threat to an animal’s physical and neurological consequences such as schizophrenia and
well-being (e.g., injury, predator). Therefore, a sensitized Parkinson’s disease (Brown et al., 2004; Bilbo and Schwarz,
immune response to future stressors could better prepare 2009; Kohman and Rhodes, 2013). This research has shed
an animal for future threatening environments. In one light on how the interactive influence of the hypothala-
well-characterized example, a behavioral consequence of mic pituitary axis (HPA), sympathetic nervous system, and
heightened inflammation is the phenomenon of sickness immune system can contribute to the effects of ELA.
behavior. The lethargy, social avoidance, and anhedonia The well-orchestrated mammalian immune system has
associated with being exposed to an immunostimulant two major kinds of immune responses: innate and adap-
(e.g., a pathogen) can be viewed as a part of the organism’s tive. Both are responsible for detecting and regulating
effort to recruit all of its resources for fighting against the foreign threats, and inflammation resulting from both
invading pathogen and overcoming the disease (Hartung has been associated with psychopathology (Miuller and
et al., 1988). Sickness behavior purportedly shares phen- Schwarz, 2007; McNally et al., 2008; Raison and Miller,
omenology and immunological physiology with major 2011). The innate immune system is the first line of the
depressive disorder (Maes et al., 2012). In this very sim- host defense, and involves a rapid response of patrolling
ple sequence, we begin to see a role of immunity in cells such as macrophages and microglia. The adaptive
20
Table 1
Rodent studies of ELA inflammatory effects. Papers were identified using search terms “early life stress” or “maternal separation” or “neonatal stress” with “inflammation” or “inflammatory”, excluding transgenic
models or sensitive lines (to eliminate the complication of interactive effects). White cells: infancy; shaded cells: adolescence; gray cells: adulthood.
P. Ganguly, H.C. Brenhouse / Developmental Cognitive Neuroscience 11 (2015) 18–30

a
Fifteen-minute maternal separation is typically referred to as handling, which can induce increased maternal behavior and have opposite effects from ELA; however in this paradigm, a highly stress-reactive
mouse strain was used which displays heightened corticosterone release in response to this type of separation.
b
In this paradigm, pups were also separated from bedding and were not thermoregulated, thus this was presented as ELA.
Table 2
Human studies of ELA inflammatory effects. Papers were identified using search terms “early life stress” or “childhood adversity” or “childhood maltreatment” or “childhood trauma” with “inflammation” or
“inflammatory”. Anti-inflammatory modulators are in italics. Light gray cells: childhood; shaded cells: adolescence; gray cells: adulthood.
P. Ganguly, H.C. Brenhouse / Developmental Cognitive Neuroscience 11 (2015) 18–30

21
22 P. Ganguly, H.C. Brenhouse / Developmental Cognitive Neuroscience 11 (2015) 18–30
system is uniquely associated with the production of various danger signals posed by neurons and/or astrocytes
immune memory responses that are protective and also (Davalos et al., 2005) and have local protective effects via
can be easily activated upon later encounters with specific regulated release of cytokines and phagocytosis of cellu-
pathogens (Chaplin, 2006). When these responses are inap- lar debris. Under conditions of serious injury, microglia
propriately provoked, harmful inflammation is induced become reactive, characterized by heightened release of
from pro-inflammatory cytokines such as IL-1␤, IL-2, IL- inflammatory mediators. When reactive, microglia can be
6 and TNF-␣ secreted by activated microglia, macrophages neurotoxic and damage otherwise healthy neurons (Banati
and lymphocytes (Miuller and Schwarz, 2007; Haroon et al., and Graeber, 1994). For example, activated microglia
2012). Inflammatory activity is countered by other subsets release nitric oxide (NO) (Chao et al., 1992). While low
of astrocytes, T lymphocytes, macrophages and monocytes levels of NO function as signaling molecules, high lev-
that secrete anti-inflammatory cytokines including IL-10, els of this free radical can cause neuronal cytotoxicity
IL-5 and IL-4 (Muller et al., 2000; Raison et al., 2010; Haroon (Uttara et al., 2009). Several studies have shown that excess
et al., 2012). Innate and adaptive immunity both play crit- production of free radicals such as NO contributed sig-
ical roles in early development and aging (Schwarz and nificantly to neuronal loss in schizophrenia, Parkinson’s
Bilbo, 2011), but very few studies have looked at normative and Alzheimer’s disease (Mahadik and Mukherjee, 1996;
lifespan development of the immune system (Siegrist and Christen, 2000; Beal, 2003), likely through excitotoxic-
Aspinall, 2009), which may impact pathogenesis of mental ity and apoptosis (Kehrer, 2000). These mechanisms may
illnesses. partially explain the etiology of psychopathologies that
Pro-inflammatory immune activation has been linked emerge after ELA. It is noteworthy that different insults
to psychiatric disorders such as depression, schizophrenia may summate upon microglia and potentiate each other to
and obsessive–compulsive disorder (Jones and Thomsen, worsen the outcome of the response (Luo and Chen, 2012).
2013; Valkanova et al., 2013). For example, a meta-analysis It is therefore plausible that ELA acts to sensitize microglia
controlling for antipsychotics revealed consistently ele- toward a lower threshold for a reactive state, leading
vated levels of several immune molecules released to increased inflammatory cytokine levels and altered
from macrophages, such as tumor necrosis factor (TNF)- neurotransmission. Alternatively, activated microglia can
␣, interferon (IFN)-␥ and interleukin (IL)-12 in cases express anti-inflammatory cytokines and play a role in tis-
with schizophrenia (Miller et al., 2011). Cell cultures sue protection and repair.
obtained from individuals with schizophrenia also pro- Neuroinflammatory influences on neurotransmission
duced higher levels of circulating IL-8 and IL-1␤, further and morphology also rely largely on glial activity. Activated
implicating immunity in schizophrenia pathology. In microglia may contribute to neuroplastic changes through
obsessive–compulsive disorder studies, polymorphisms in synaptic remodeling, excitatory transmission, and phago-
the TNF-␣ gene have been described (Cappi et al., 2012), cytosis of newborn neurons and cellular debris (reviewed
as well as both increases and decreases in plasma TNF-␣ by Kovacs, 2012). Monoamine transmission is also affected
cytokine levels (Monteleone et al., 1998; Denys et al., 2004; by neuroinflammation. In activated microglia, tryptophan
Konuk et al., 2007). It has been proposed that cytokine gene is metabolized by indoleamine to the NMDA agonist
polymorphisms can have variable effects between individ- quinolinic acid instead of serotonin (Steiner et al., 2011).
uals (Cappi et al., 2012). In another meta-analysis, raised Microglial activity has therefore been directly linked to
levels of the pro-inflammatory proteins IL-6 and C-reactive depressive symptoms through a reduction in serotonin
protein were significantly associated with the later devel- production (Muller, 2014). Importantly, neural-immune
opment of depressive symptoms in prospective studies. In interactions begin during fetal development (Schwarz and
order to understand how these changes in the immune Bilbo, 2011), and a growing landscape of data supports an
response can lead to susceptibility to a number of psy- active role for these interactions on the programming of
chopathologies, we review the two major ways in which lifelong function.
neuroinflammation can affect the brain: neuronal damage
and altered neurotransmission.
A neuroinflammatory response typically occurs when 4. The neuroinflammatory link between ELA and
signals involving infection or irritation are evoked in the behavioral changes
brain, or through vagal afferents, transport of cytokines
into the brain through the blood–brain-barrier (BBB), Pioneering work describing how ELA can alter immuno-
or infiltration of cytokines where the BBB is absent logical development was conducted in the 1980s in
(Schoderboeck et al., 2009; Northrop and Yamamoto, 2012; primates (Laudenslager et al., 1982, 1990; Coe et al., 1987,
reviewed by Schwarz and Bilbo, 2011). Microglia are res- 1988, 1989). Since then, the growing appreciation for an
ident immune cells in the brain and are known to play immunological contribution to mental illness has led to
a major mediating role in neuroinflammation. Although a resurgence of interest in neuroimmune interactions.
the mechanism by which neuroinflammation causes eti- Tables 1 and 2 display studies over the past 15 years directly
ology of behavioral and psychological disorders is not fully investigating the effect of ELA on later immune function
understood, the damaging effects of microglial and astro- in rodents (Table 1) and humans (Table 2). Despite stress
cyte activation on neuron function and circuitry are being paradigms, species/strain differences, and age differences,
elucidated. Under normal circumstances, resting microglia a pattern emerges suggesting ELA leads to increased pro-
continuously survey the brain parenchyma (Nimmerjahn inflammatory responsivity in early adolescence or later.
et al., 2005). Microglia become activated in response to This heightened response is likely a consequence of the
early response to ELA during and immediately following Direct effects of early life infection, as elegantly pre-
exposure. sented by Bilbo and colleagues, further highlight the critical
role of the immune system in brain development with
particular influence from microglia. For example, healthy
4.1. Immediate immune effects of ELA levels of microglial activity are necessary for phagocy-
tosing apoptotic neurons and unneeded synapses during
Measurements taken during or immediately after ELA, development (Schafer et al., 2012; reviewed by Bilbo and
which understandably have largely been studied in non- Frank, 2013). Infection or otherwise exacerbated inflam-
humans (Table 1 and (Laudenslager et al., 1982, 1990; mation in neonate rats can sensitize microglia to become
Coe et al., 1987, 1989), often reveal a suppression of over-activated upon future infections in adulthood, caus-
inflammatory responses in stressed infants. Many of the ing impaired cognitive flexibility (Bilbo and Frank, 2013;
studies showing immune suppression targeted the adap- Williamson and Bilbo, 2014). This is one example of how
tive immune responses of lymphocyte proliferation, while early life programming is affected by experience. Notably,
other studies that measured circulating cytokines or neonatal infection leads to transient neuroinflammation in
macrophage proliferation (Coe et al., 1988) reported mixed the brain (Lieblein-Boff et al., 2013), which differs from the
results including an increased response in preweanlings apparent suppression of neuroimmune activity during ELA.
and infants. While the peripheral response to ELA may It is unclear—and an important topic of investigation—how
not be an overall activation or suppression, early immune early suppression of microglial activity translates to the
programming through ELA appears to sensitize later pro- heightened neuroinflammatory state observed later in life
inflammatory processes and lead to greater vulnerability after ELA. However, we will generalize here to acknowl-
to depression and anxiety in adulthood (Hennessy et al., edge that altered neuronal-glial programming could incite
2010). neuronal circuitry to compensate for an unpredictable
In the brain, glial proliferation, glial activity and direct environment.
neuronal response to cytokines are crucial for healthy
brain development (Eyo and Dailey, 2013). The importance 4.2. Delayed and prolonged immune effects of ELA
of immune activity during early development is gleamed
from the peak of cytokine concentrations, cytokine recep- As mentioned above, ELA has been shown in the short-
tor densities, and glial activity shown in rodent and ex vivo term to suppress some types of inflammatory activity
models during this period (Giulian et al., 1988; Gadient and in the brain and periphery; however ELA often leads to
Otten, 1994). Glia-mediated mechanisms control synap- exacerbated pro-inflammatory activity later in life (see
togenesis (Chamak et al., 1995; Christopherson et al., Tables 1 and 2). Given the important role of the immune
2005), apoptosis (Frade and Barde, 1998), synaptic prun- system in normal brain development, it is not surprising
ing (Stevens et al., 2007; Garay and McAllister, 2010), and that an altered trajectory of inflammatory responses will
myelination (Pang et al., 2013). The impact of ELA on devel- lead to atypical brain development after ELA. However, the
opmental processes therefore likely involves aberrant glial immune-related effects of ELA are not yet fully understood.
activity. It will be important to determine whether different types
ELA from maternal separation has been shown to or time-courses of ELA can differentially affect immunity
decrease microglial number in midbrain areas (Chocyk depending on the presence of a secondary insult, the devel-
et al., 2011), decrease cytokine expression (Dimatelis et al., opmental stage of assessment, or the sex of the individual.
2012), and decrease acute-phase proteins like lipopolysac- ELA has been observed to produce developmentally
charide binding protein (Wei et al., 2012) in the rodent distinct psychiatric outcomes. In humans, externalizing
brain. Taken together, it appears that suppression of glial disorders such as aggression and cognitive dysfunction are
activity during early development is the earliest neuroim- apparent during childhood and early-adolescence (Egeland
mune response to ELA. Behavioral consequences have also et al., 2002; reviewed by Gunnar and Fisher, 2006), while
been noted early on, as this suppressed neuroinflammation disorders such as depression, schizophrenia, and drug
co-occurred with anhedonic and withdrawal behaviors addiction are often not observed until adolescence or
(Hennessy et al., 2010) and altered fear learning (Callaghan early adulthood (Lewis and Levitt, 2002; Andersen and
and Richardson, 2011) in rodents. However, guinea pigs Teicher, 2009; Teicher et al., 2009). The display of cognitive
that are separated from their mother exhibit a charac- deficits and aggression during childhood compared to the
teristic behavior that resembles sickness behavior, and later presence of depressive, addictive, or psychotic symp-
is reportedly blocked with anti-inflammatory treatment toms raises the question whether inflammatory activity
(Hennessy et al., 2007, 2011; Perkeybile et al., 2009), sug- after ELA prospectively affects distinct circuits in the brain
gesting that pro-inflammatory processes play a role in at different developmental stages. Inflammatory cytokine
these early responses to ELA as well. In mature animals, receptors are dense in several areas controlling cogni-
glucocorticoid exposure has been shown to activate neu- tive function such as the hippocampus (Tancredi et al.,
roinflammatory processes, leading to a sensitization of 2000; Vereker et al., 2000; Curran and O’Connor, 2003;
microglia to a pro-inflammatory state (Frank et al., 2012). Butler et al., 2004) and PFC (del Rey et al., 2013), and pro-
However, during early postnatal life, microglia display an inflammatory interventions have been shown to directly
immature phenotype (Schwarz and Bilbo, 2012) and sensi- impair cognitive processing in people (Harrison et al.,
tization of immature microglia to psychological stress has 2009). A direct relationship between aggression and proin-
not been directly investigated. flammatory processes in humans has also been reported
(Coccaro et al., 2014). It is therefore likely that a sensi- shown in several species to induce a dysbalance between
tized inflammatory response yields impaired cognition and inhibitory and excitatory signaling in the PFC (Bock et al.,
externalizing behaviors after ELA. 2014). Both depression and schizophrenia in humans have
Depression and schizophrenia, which manifest in been attributed to both dysfunctional glutamate signaling
adolescence or later, are also purportedly linked to pro- and related inflammatory mechanisms (Myint et al., 2012;
inflammatory processes, however this link is not fully Muller, 2014). Furthermore, microglia contribute to inflam-
understood. While ELA in humans has been associated matory mechanisms through glutamate release and may be
with elevated inflammatory C-reactive protein and IL-6 in overactivated in a sensitized state after ELA.
late childhood (age 10) (Slopen et al., 2013), inflamma- Rodent investigations of behavior and neuroanatomy
tory markers during late childhood and adolescence do not and functional connectivity studies in humans have shed
consistently predict later episodes of depression (Copeland some light onto how corticolimbic development after ELA
et al., 2012; Slopen et al., 2013). That said, Miller and might yield age-dependent vulnerability to neuroimmune
Cole (Miller and Cole, 2012) recently reported that female activity. These studies have revealed that PFC connections
adolescent women exposed to ELA expressed higher lev- with subcortical limbic structures such as the amygdala are
els of IL-6 that forecasted depression 6 months later. In immature during childhood and become adult-like during
patients with schizophrenia, 40% of individuals displayed adolescence (Cunningham et al., 2002; Gee et al., 2013b).
increased microglial activation and inflammatory cytokine However, development of other corticolimbic circuits,
mRNA expression in the dorsolateral PFC, with the greatest such as that of the PFC-nucleus accumbens projections in
effect in those who had been most recently diagnosed, sug- rodents, have been reported to display transient changes
gesting inflammation might be an early causative factor. It during adolescence that are different from both juveniles
is also likely that the subset of patients with schizophrenia and adults (Brenhouse et al., 2008). The interplay of altered
who also displayed an inflammatory profile represented glial programming in early-developing structures with
a unique population that had undergone ELA, since the later maturation of interconnected regions is largely
patients with childhood trauma—but not those without unknown. However, given the complexity of these inter-
such experience—reportedly have heightened inflamma- secting trajectories it is not surprising that lifelong effects
tory markers than healthy controls (Dennison et al., 2012). of ELA are mediated by the time of exposure as well as the
However, these inflammatory changes do not reveal a time of assessment.
mechanism for the delayed manifestation of illnesses like ELA from childhood neglect has also been associated
depression and schizophrenia after ELA. It is possible that with later alterations in reward processing in humans
the late and protracted development of the prefrontal cor- (Mueller et al., 2012). Reactivity of the nucleus accumbens
tex (PFC)—which is not fully mature until early adulthood (Goff et al., 2013) and other basal ganglia regions (Mehta
(Giedd et al., 1999)—delays the full impact of a sensitized et al., 2010) in response to emotion or reward is reduced
immune response after ELA on affective control. in ELA-exposed teenagers. These effects on reward cir-
In a rodent model, we have observed that mater- cuitries are likely due to altered trajectories of connectivity
nal separation ELA leads to a loss of PFC interneurons with cortical regions, such as the premature maturation
(Brenhouse and Andersen, 2011a), which is a purported of cortical-amgydala functional connectivity seen after
mechanism of schizophrenia that is linked to inflammatory maternal deprivation in rodents (Gee et al., 2013a). We
and excitotoxic damage (Behrens and Sejnowski, 2009). know of no direct investigations into neuroimmune effects
We have also observed that rats exposed to maternal sep- from ELA on reward-related regions. However, recent
aration display deficits in PFC-mediated behaviors such as observations in rats raised with enhanced maternal care
learned helplessness (Leussis et al., 2012), social interaction through an early handling paradigm reveal early glial pro-
(Holland et al., 2014), and working memory (Brenhouse gramming in the nucleus accumbens (Schwarz et al., 2011).
and Andersen, 2011b) in adolescence, with increased Specifically, early short-term handling protected rats from
peripheral levels of the inflammatory cytokines IL-␤ and later morphine-induced microglial activation within the
IL-6 (Wieck et al., 2013). It is possible that development nucleus accumbens, through an anti-inflammatory mech-
of the neuroinflammatory response partially underlies the anism. Microglial activation in reward-related areas has
delayed effects of ELA, since maternal separation in rats been associated with heightened drug-associated con-
was shown to yield neuroinflammatory changes in the PFC ditioning (Schwarz et al., 2011; Zhang et al., 2012),
that correlated with interneuron deficits and manifested drug-seeking, and drug-induced neuroplasticity (Kovacs,
in adolescence, but not before (Brenhouse and Andersen, 2012) in rodents. Astrocyte activity in the nucleus accum-
2011a). Specifically, the neuroinflammatory mediator bens was also shown to mediate drug reward in mice
cyclooxygenase-2 (COX-2) was upregulated in the PFC (Narita et al., 2006). Moreover, changes in innate immune
in adolescent males, and PFC interneuron loss was pre- gene expression have been proposed to directly con-
vented with pre-adolescent COX-2 inhibition (Brenhouse tribute to the development of addiction through cortical
and Andersen, 2011a). ELA-exposed males did not display hyper-excitability (Crews and Vetreno, 2011), which is
this increase of COX-2 in juvenility. Since these changes exacerbated during adolescent development (Brenhouse
co-occurred with altered glutamatergic NMDA receptor et al., 2008). Whether ELA shifts cortical excitatory
expression (Wieck et al., 2013), we hypothesize that aber- signaling earlier is not known, however related studies
rant glutamatergic innervation from subcortical regions examining prefrontal-amygdala connectivity in previously
during adolescence could play a role in increased neuroin- institutionalized children suggest that other glutamatergic
flammatory activity within the PFC. Indeed, ELA has been connections with the PFC do indeed develop earlier than in
controls (Tottenham, 2012, 2013). Inflammatory processes 5.2. Sex differences in glial programming and
thereby likely interact with developing connectivity to con- neuroinflammation
tribute to the association of ELA with earlier manifestation
of addictive behaviors, e.g. the 2- to 4-fold increase in the Few studies have directly investigated the sex-specific
likelihood of illicit drug use by age 14 (Dube et al., 2003). effects of ELA on immune programming. As Table 2 illus-
As reviewed by Tottenham and Sheridan (2009), once trates, human studies of inflammatory changes after ELA
environmental exposure occurs, it modifies the architec- often include both sexes, but typically do not assess sex
ture of the circuit in such a way that certain patterns of differences (likely due to limited participants of each
future activity are preferred (Knudsen, 2004). We acknowl- sex). Animal studies of ELA have traditionally focused on
edge here that ELA occurs at a time when the brain is male models, however a small number of reports have
learning how to adapt to its lifelong environment. As these emerged that suggest sexually dimorphic developmen-
modifications develop, the brain encounters developmental changes after ELA. For example, we recently reported
tal time-points that require particular function. Mental that maternally separated female rats displayed earlier
illness or dysfunction may arise if the trajectory of the changes in PFC inhibitory interneurons, with concur-
brain has left it unprepared for transient changes during rent social interaction deficits after maternal separation;
early through late adolescence. This concept was presented male deficits were seen as well, only later in adoles-
in Greenough’s “experience-expectant” model (Greenough cence (Holland et al., 2014). Male deficits were correlated
et al., 1987), whereby the brain expects and “waits” to inter- with increases in COX-2 in the PFC, yet ELA-exposed
act with the environment by shaping itself based on early females did not display COX-2 effects (Holland et al.,
experiences during critical periods. Here we have reviewed 2014). We also observed in preliminary studies that mater-
evidence that neuroimmune development plays an impor- nally separated males, but not females, displayed higher
tant role in such programming after ELA. circulating levels of pro-inflammatory cytokines during
pre-adolescence that predict later cognitive dysfunction
(unpublished observations). Additionally, Chocyk et al.
5. Neuroimmune mechanisms underlying sex
(2011) reported decreased glial cells (type unspecified) in
differences
the basal ganglia of maternally separated juvenile males,
but not females.
Measuring developmental and life-long effects of ELA
Interestingly, microglial colonization of the brain occurs
exposure produces a moving baseline from which to
much earlier in males than in females in several regions
gauge anatomical and functional differences. Therefore the
including the parietal cortex, hippocampus, and amygdala
addition of sex as a factor in developmental studies is
(Schwarz et al., 2012). It has been proposed that sex dif-
challenging. While some animal studies report stronger
ferences in the colonization and function of glia within the
effects of maternal separation ELA in females on measures
normal developing brain may contribute to distinct win-
such as HPA axis responsivity (Desbonnet et al., 2008),
dows of vulnerability between males and females (Schwarz
others report a stronger effect in males (Kunzler et al.,
and Bilbo, 2012). One might hypothesize that early stress
2013) on measures such as catecholamine fiber density.
exposure during a period when microglia have colonized
When accounting for sexually dimorphic moderators like
these regions in males but not females could lead to prefer-
HPA responsivity (Klein and Corwin, 2002), neuroimmune
ential sensitization of the neuroimmune response in males,
development (Schwarz and Bilbo, 2012), and rates of cir-
however this has not been determined.
cuitry maturation (Brenhouse and Andersen, 2011b), it
Further supporting evidence of sexually-dimorphic
becomes clear that the idea of one sex being resilient or
glial programming is gleamed from work by Bilbo and
vulnerable to ELA is oversimplified.
colleagues, who reported that males were more sensi-
tive than females to the long-term effects of early-life
5.1. Sex differences in HPA responsivity and stress coping immune challenges (Bilbo et al., 2012; Schwarz and Bilbo,
2012). Therefore, males may be more vulnerable than
Sex differences in the effects of ELA on HPA responsive- females to the inflammatory consequences of ELA. By ado-
ness have been reported. For example, ELA was found to lescence in many species glial activation is greater in
yield higher cortisol levels in females only, both imme- typically-developing females, and peripheral inflamma-
diately after maternal separation ELA in infant monkeys tory responses are reportedly more robust in females than
(Sanchez et al., 2005) and in adolescence after ELA in males (reviewed by Klein, 2000; Schwarz and Bilbo, 2012).
humans (Burghy et al., 2012). Furthermore, only in females Whether or not ELA or subsequent challenges can impact
was increased HPA activity during childhood shown to this dimorphism is not known. In contrast to the above evi-
predict lower functional connectivity between the amyg- dence suggesting that males are preferentially affected by
dala and PFC and internalizing symptoms (Burghy et al., ELA via glial mechanisms, a separate study reported that
2012). These data have been interpreted as a greater sen- in prenatally-stressed mice, sex-specific hippocampal glial
sitivity to the neuroendocrine effects of ELA in females decreases were found only in juvenile female but not male
(Desbonnet et al., 2008). However, it is interesting to offspring (Behan et al., 2011). As seen in Tables 1 and 2, and
note that in rats, stress exposure in adolescence yielded as discussed in the previous section, other studies inves-
increased microglial activation and neuroinflammation in tigating adult females have indeed shown inflammatory
males, but not females (Pyter et al., 2013), suggesting a sex changes after ELA [e.g., Miller and Cole, 2012]. These incon-
difference in inflammatory response to glucocorticoids. sistencies are likely due to the timing, duration, and type
Fig. 1. Hypothetical schematic showing neuroimmune and neuroendocrine influences over development after ELA. Microglial activity is aberrantly sup-
pressed during ELA (illustrated as freezing). Early neuroimmune dysfunction leads to a heightened neuroimmune response later in life (illustrated as
heat), which impacts developing corticolimbic circuitries. Concurrently, HPA activity is aberrantly overactivated during ELA (which is more pronounced in
females). This leads to a hypersensitivity of the HPA axis later in life which impacts both immune activity and corticolimbic function—themselves devel-
opmentally dynamic. Grayed arrows depict immature connections that can be programmed through immune and neuroendocrine influences; bold arrows
depict maturing connections that can cause latent behavioral changes after ELA.
of ELA assessed. Indeed, sexual dimorphism has also been 6. Future directions and conclusions
observed in circuitry changes after ELA, which can lead to
differential vulnerability to neuroinflammation over devel- Here we have reviewed the converging evidence that
opment. For example, ELA in humans has been shown ELA can deleteriously suppress normal inflammatory
to decrease amygdala-hippocampus functional connectiv- and neuroinflammatory processes during early develop-
ity in adolescent females, but not males (Herringa et al., ment, which may lead to a sensitized immune response
2013). Amygdala development is in particular flux during and heightened neuroinflammation later in life. Fig. 1
adolescence, completing a shift from positive to negative illustrates a simplified hypothetical schematic of how
functional connectivity with the PFC (Gee et al., 2013b). development of the ELA-exposed brain is influenced
Therefore a female-specific effect in the adolescent amyg- by neuroimmune and neuroendocrine actions. Both age
dala is noteworthy when considering potential impacts of and sex of an individual can influence the impact of
ELA and subsequent stress exposure. neuroinflammation, since males and females display dif-
Taken together, we present the perspective that males ferent time-courses of glial development, proliferation, and
and females might be impacted by ELA differently, with colonization. Since neuroinflammation involves aberrant
females more vulnerable to early neuroendocrine-induced glutamate signaling, altered monoamine synthesis, and
changes in corticolimbic circuitry, and males more vulnera- synaptogenesis, immune sensitization directly influences
ble to later neuroinflammation, possibly through microglial circuitry development, which itself is altered after ELA
sensitization. These ideas are entirely understudied and through separate neuroendocrine mechanisms. Concur-
currently speculative, however they highlight the impor- rently, neuroinflammation causes oxidative damage and
tance of parsing mechanistic changes based on sex and excitotoxicity that can directly impair normal develop-
development, and of questioning why these sexually ment, which also will have discrete impacts on behavior
dimorphic responses would exist. in separate sexes and ages.
Revisiting the philosophy that brain development aims Bilbo, S.D., Frank, A., 2013. Beach award: programming of neuroendocrine
to meet the demands of each stage-specific environment, function by early-life experience: a critical role for the immune sys-
tem. Horm. Behav. 63, 684–691.
we see here how ELA derails the typical trajectory. On a Bilbo, S.D., Schwarz, J.M., 2009. Early-life programming of later-life brain
psychosocial level, children that are exposed to threaten- and behavior: a critical role for the immune system. Front. Behav.
ing or negligent environments are met with two forces on Neurosci. 3, 14.
Bilbo, S.D., Smith, S.H., Schwarz, J.M., 2012. A lifespan approach to neu-
their development: First, the stress coping mechanisms in roinflammatory and cognitive disorders: a critical role for glia. J.
place during early development are over-activated, lead- Neuroimmune Pharmacol.: Off. J. Soc. NeuroImmune Pharmacol. 7,
ing to long-term changes that may be evolutionarily ideal 24–41.
Bock, J., Rether, K., Groger, N., Xie, L., Braun, K., 2014. Perinatal program-
for survival in similarly threatening later life environments,
ming of emotional brain circuits: an integrative view from systems to
but are not ideal for typical adolescent and adult chal- molecules. Front. Neurosci. 8, 11.
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encountered with day-to-day challenges (i.e., a need for adolescence in males and females: a cross-species understanding of
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Brenhouse, H.C., Sonntag, K.C., Andersen, S.L., 2008. Transient D1
severely ELA-exposed brain are—at best—wired for a very dopamine receptor expression on prefrontal cortex projection neu-
different type of challenge or—at worst—mis-wired and rons: relationship to enhanced motivational salience of drug cues in
dysfunctional. adolescence. J. Neurosci. 28, 2375–2382.
Brown, A.S., Begg, M.D., Gravenstein, S., Schaefer, C.A., Wyatt, R.J., Bresna-
Given the remarkable plasticity of the brain we expect han, M., Babulas, V.P., Susser, E.S., 2004. Serologic evidence of prenatal
that many of the deleterious effects of ELA can be treated influenza in the etiology of schizophrenia. Arch. Gen. Psychiatry 61,
with interventions that account for gender and target 774–780.
Brown, D.W., Anda, R.F., Felitti, V.J., Edwards, V.J., Malarcher, A.M., Croft,
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geted prevention—not just treatment, should be a high with the risk of lung cancer: a prospective cohort study. BMC Public
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Burghy, C.A., Stodola, D.E., Ruttle, P.L., Molloy, E.K., Armstrong, J.M., Oler,
able population with neurodevelopmental deficits that can
J.A., Fox, M.E., Hayes, A.S., Kalin, N.H., Essex, M.J., Davidson, R.J., Birn,
particularly benefit from interventions aimed at the mech- R.M., 2012. Developmental pathways to amygdala-prefrontal func-
anisms we review here, possibly during a sex-dependent tion and internalizing symptoms in adolescence. Nat. Neurosci. 15,
1736–1741.
critical period.
Butler, M.P., O’Connor, J.J., Moynagh, P.N., 2004. Dissection of tumor-
necrosis factor-alpha inhibition of long-term potentiation (LTP)
reveals a p38 mitogen-activated protein kinase-dependent mecha-
Acknowledgements nism which maps to early-but not late-phase LTP. Neuroscience 124,
319–326.
We would like to thank Freedom Holland for his sig- Callaghan, B.L., Richardson, R., 2011. Maternal separation results in early
emergence of adult-like fear and extinction learning in infant rats.
nificant artistic contribution to Figure 1 and for his help
Behav. Neurosci. 125, 20–28.
proofreading this manuscript. We would also like to thank Cappi, C., Muniz, R.K., Sampaio, A.S., Cordeiro, Q., Brentani, H., Palacios,
Bradley Schlaggar and Beatriz Luna for the generous invi- S.A., Marques, A.H., Vallada, H., Miguel, E.C., Guilherme, L., Hounie,
A.G., 2012. Association study between functional polymorphisms in
tation to contribute to this Special Edition.
the TNF-alpha gene and obsessive–compulsive disorder. Arq. Neurop-
siquiatr. 70, 87–90.
Carr, C.P., Martins, C.M., Stingel, A.M., Lemgruber, V.B., Juruena, M.F., 2013.
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Vol. 51 No. 3/2004
563–585
QUARTERLY
Review
HIF-1: the knowns and unknowns of hypoxia sensing.
Anna Zagórska½ and Józef Dulak½
Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University,

Kraków, Poland
Received: 28 February, 2004; revised: 29 June, 2004; accepted: 09 July, 2004
Key words: hypoxia inducible factor-1, angiogenesis, heme oxygenase, reactive oxygen species, prolyl and
asparaginyl hydroxylases, nitric oxide, carbon monoxide
Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that functions as a

master regulator of cellular and systemic oxygen homeostasis. It consists of two con-
stitutively produced subunits: HIF-1a and HIF-1b. Under normoxic conditions
HIF-1a undergoes hydroxylation at specific prolyl residues which leads to an imme-
diate ubiquitination and subsequent proteasomal degradation of the a subunit. Addi-
.
The work was in part supported by the grants 3 P04 049 22 and P04B 013 21 awarded to J. D. by the
State Committee for Scientific Research (KBN, Poland).
½
Corresponding author: Józef Dulak, Department of Cell Biochemistry, Faculty of Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; phone: (48 12) 664 6375; fax: (48 12)
664 6902; e-mail: jdulak@mol.uj.edu.pl or Anna Zagórska; e-mail: azagorska@op.pl
Abbreviations: AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear
translocator; bHLH, basic helix-loop-helix domain; BMAL, brain muscle ARNT-like protein; CBP,
cAMP-responsive element-binding protein; DMOG, N-dimethyl-oxalylglycine; EPO, erythropoietin;
FIH-1, factor inhibiting HIF-1; Flt-1, fms-like tyrosine kinase-1; FRAP, FKBP-rapamycin associated pro-
tein; HAS, HIF-1 ancillary sequence; HBS, HIF-1 binding site; HIF-1, hypoxia-inducible factor 1; HO-1,
heme oxygenase-1; HRE, hypoxia response element; HUVEC, human umbilical vein endothelial cells;
IPAS, inhibitory PAS protein; MAPK, mitogen-activated protein kinase; MDM2, murine double minute
2; NLS, nuclear localization signal; NOS2, inducible nitric oxide synthase; NPAS2, neuronal PAS do-
main protein 2; ODD, oxygen-dependent degradation domain; PAS, Per-AHR-ARNT-Sim homology do-
main; PHD, PH domain-containing protein; PI3K, posphatidylinositol-3 kinase; PKC, protein kinase C;
pVHL, von Hippel-Lindau tumor suppressor protein; ROS, reactive oxygen species; SNP, sodium
nitroprusside; SOD, superoxide dismutase; TAD, transactivation domain; VEGF, vascular endothelial
growth factor; VSMC, vascular smooth muscle cells.
564 A. Zagórska and J. Dulak 2004
tionally, hydroxylation of an asparaginyl residue blocks the transcriptional activity

of HIF-1 due to inhibition of its interaction with co-activators. In contrast, under
hypoxic conditions, abolition of prolyl hydroxylation results in HIF-1a stabilization,
whereas the lack of asparaginyl hydroxylation allows the transcriptional activity. Ad-
ditionally, the transcriptional activity may be modulated by phosphorylation or re-
dox modification of HIF-1. Despite its name, HIF-1 is induced not only in response to
reduced oxygen availability but also by other stimulants, such as nitric oxide, various
growth factors, or direct inhibitors of prolyl and asparaginyl hydroxylases. There-
fore, it seems to be a crucial transcription factor elicited by a wide range of stresses
such as impaired oxygenation, inflammation, energy deprivation, or intensive prolif-
eration. However, the mechanisms of normoxic activation, as well as of oxygen sens-
ing, are not yet fully known. Further understanding of the processes that control
HIF-1 activity will be crucial for the development of new diagnostic and therapeutic
strategies.
Oxygen homeostasis is strictly controlled in Although the importance of HIF-1 in the in-
order to maintain intracellular levels of oxy- duction of the complex response to hypoxia is
gen within tight limits dictated by the require- widely appreciated, the molecular mecha-
ment for O2 in many metabolic processes on nisms of HIF-1 activation remain unclear.
the one hand, and its high toxicity on the This review aims to summarize current
other. The control occurs at the level of an en- knowledge on HIF-1 regulation under hypoxic
tire organism as well as at the level of a single and normoxic conditions.
cell. It involves numerous mechanisms of reg-
ulation and adaptation to changes in O2 ten-
sion. At the cellular level, decreased O2 ten- HIF-1 TARGET GENES
sion (hypoxia) leads to the activation of alter-
native metabolic pathways that do not require The consensus DNA sequence for HIF-1 bin-
molecular oxygen. The switch from aerobic ding, 5¢-(A/G)CGTG-3¢, is common for many
metabolism to anaerobic glycolysis is medi- genes upregulated by low oxygen tension
ated by the induction of glycolytic enzymes (Semenza et al., 1996). The list of known
and expression of glucose transporters. Addi- genes activated by HIF-1 grows continuously.
tionally, the expression of various stress pro- It includes genes whose protein products act
teins responsible for cell death or survival is both to maintain O2 homeostasis and to adapt
upregulated. Further adaptations which oc- to changes in the oxygen concentration (Ta-
cur at the tissue and systemic levels lead to ble 1). The first group comprises of genes in-
the increase in O2 delivery. They include involved in the development and functioning of
duction of erythropoiesis (red blood cell pro- the vascular system. They either promote
duction), angiogenesis (new vessel forma- angiogenesis (such as VEGF and its receptor
tion), and hyperventilation. Among the vari- Flt-1) or modulate vascular tone (such as in-
ous upregulated proteins there is erythropoie- ducible nitric oxide synthase, heme oxy-
tin (EPO), the main growth factor inducing genase-1, endothelin 1, adrenomedullin, and
maturation of erythrocytes, and vascular en- a1B-adrenergic receptor). The second group is
dothelial growth factor (VEGF), the major represented by genes whose protein products
mediator of angiogenesis and vascular perme- induce erythropoiesis. Besides erythropoie-
ability. The hypoxia-dependent regulation of tin, HIF-1 upregulates ceruloplasmin, trans-
these and other proteins (Table 1) occurs at ferrin, and transferrin receptor which facili-
the transcriptional level and is mediated by tate the supply of iron to the erythroid tis-
hypoxia-inducible transcription factor sues. The third group consists of genes whose
(HIF-1). products are involved in energy metabolism.
Vol. 51 HIF-1: the knowns and unknowns of hypoxia sensing 565
Their cooperation leads to increased glucose HIF-1 STRUCTURE

uptake and a switch to glycolysis as the main
source of energy. The fourth group comprises
HIF-1 is a heterodimer of two bHLH-PAS
genes whose products are responsible for cell
proteins
proliferation and viability. Besides these four
main sets of HIF-1-induced genes, there are Hypoxia-inducible factor-1 is a heterodimer
others also important in response to various composed of the a and b subunits. Both of
stresses (all references in Table 1). them contain two characteristic domains: the
Table 1. Genes upregulated by HIF-1.

basic helix-loop-helix (bHLH) domain and the and PAS-B (aa 228–298) repeats (Wang et al.,
PAS (Per-AHR-ARNT-Sim) domain. The PAS 1995a). Two transactivation domains, N-ter-
domain was termed as an acronym for the minal and C-terminal TADs (also termed
first known members of the family: Dro- NAD and CAD), are localized in the C-termi-
sophila period (Per) and single-minded (Sim) nal half of HIF-1a (aa 531–575 and 786–826,
proteins and mammalian aryl hydrocarbon respectively; Pugh et al., 1997). Moreover, the
receptor (AHR) and aryl hydrocarbon recep- C-terminal part contains a domain responsi-
tor nuclear translocator (ARNT) proteins ble for degradation of HIF-1a under normoxic
(Wang et al., 1995a). It contains two internal conditions (Huang et al., 1998). This oxy-
homology units, the A and B repeats, and is gen-dependent degradation domain (ODD at
involved in interaction between proteins. aa 401–603) contains two PEST-like motifs:
PAS-proteins have been found in many spe- sequences rich in proline (P), glutamic acid
cies, including prokaryotes, which indicates (E), serine (S), and threonine (T) (aa 499–518
their high evolutionary stability (Wang et al., and 581–600) common for many proteins
1995a). with a short half-life (less than 2 h)
The bHLH domain, which is common for a (Rechsteiner & Rogers, 1996). The HIF-1a
large number of transcription factors, is re- half-life under normoxic conditions is less
quired for both protein dimerization and than 10 min and the protein is hardly detect-
DNA binding. In the case of HIF-1, the high- able (Chun et al., 2002). Additionally, HIF-1a
est efficiency of heterodimerization is ob- contains two nuclear localization signals:
tained only if the bHLH and PAS domains are N-NLS (aa 17–74) and C-NLS (aa 718–721).
intact (Jiang et al., 1996a). Moreover, the effi- The C-terminal NLS is crucial in the nuclear
ciency of dimerization and DNA binding of in import of HIF-1a, whereas the N-terminal one
vitro-translated HIF-1a and HIF-1b is weaker seems to be less important (Kallio et al.,
than those isolated from a cell, suggesting 1998). Furthermore, alternative splice vari-
that one or both subunits may undergo ants of HIF-1a and b have been observed
posttranslational modifications (Jiang et al., (Wang et al., 1995a; Gothie et al., 2000) and
1996a). both subunits contain multiple consensus
sites for protein phosphorylation (Wang et
HIF-1a al., 1995b).
HIF-1a is an 826-amino acid (120-kDa) pro- HIF-1b

tein (Fig. 1). In the N-terminal part it contains
the basic domain (aa 17–30), the he- HIF-1b was previously identified as aryl hy-
lix-loop-helix domain (aa 31–71), and the PAS drocarbon nuclear receptor translocator
domain (aa 85–298) with PAS-A (aa 85–158) (ARNT), which heterodimerizes with aryl hy-
Figure 1. Structure of HIF-1a.

Numbers indicate the first and last amino-acid residues of each domain. The main domains are: basic helix-loop-he-
lix domain (bHLH), Per-AHR-ARNT-Sim homology domains (PAS-A and PAS-B), oxygen-dependent degradation
domain (ODD), and transactivation domains (N-TAD and C-TAD). Also position of PEST-like motifs and nuclear lo-
calization signals (NLS) is shown. Further description in the text.
drocarbon receptor (AHR) to form the func- OXYGEN-DEPENDENT HIF-1

tional dioxin receptor. It has two isoforms REGULATION
(774- and 789-aa that constitutes 92 or 94
kDa, respectively) that differ by the presence
of the sequence encoded by a 45-bp alterna-
tive exon (Wang et al., 1995a). Different levels of HIF-1 regulation
Additional HIFs
The regulation of HIF-1 activity concerns
Two other members of the HIF-1a family mostly the a subunit and occurs at multiple
have been identified: HIF-2a, also known as levels such as protein stabilization, post-
endothelial PAS protein (EPAS1), HIF-like translational modifications, nuclear translo-
factor (HLF), HIF-related factor (HRF), or a cation, dimerization, transcriptional activa-
member of PAS domain family 2 (MOP2); and tion, and interaction with other proteins.
HIF-3a. Both of them heterodimerize with Moreover, changes in mRNA expression
one of the members of the ARNT family: (Wang et al., 1995a; Wiener et al., 1996; Yu et
ARNT (HIF-1b), ARNT2, or ARNT3 (BMAL/ al., 1998) and alternative splicing (Gothie et
MOP3) (all references in Semenza, 2000b; al., 2000) of both subunits have been ob-
Wenger, 2002). The structure, regulation and served. Under normoxic conditions, however,
function of all HIFs seem to be similar. How- when HIF-1a and HIF-1b are constitutively
ever, the expression of HIF-2a, HIF-3a, transcribed and translated, the abrogation of
ARNT2, and ARNT3 is tissue specific, which HIF-1 activity results mainly from constitu-
suggests that they may play more specialized tive HIF-1a degradation (Fig. 2).
roles (Semenza, 2000b).
Figure 2. Regulation of HIF-1a protein stability.

Under normoxic conditions HIF-1-prolyl hydroxylases (PHDs) hydroxylate Pro 402 and 564 (P) within the ODD do-
main. After prolyl hydroxylation von Hippel-Lindau tumor suppressor proteins (pVHLs) bind to the ODD domain
and recruit other proteins of E3 ubiquitin ligase complex. HIF-1a is subsequently ubiquitinated and degraded by
the 26S proteasome. Under hypoxic conditions HIF-1-prolyl hydroxylases are inactive which prevents binding of
pVHL. Therefore, HIF-1a escapes ubiquitination and proteasomal degradation, and can be transported to the nu-
cleus where, after dimerization with HIF-1b, it stimulates target genes transcription.
HIF-1a degradation HIF-1 prolyl 4-hydroxylases, members of the

Fe(II)- and 2-oxoglutarate-dependent dioxy-
Ubiquitination genase superfamily. Their activity, however,
is distinct from the activity of procollagen
Under normoxic conditions HIF-1a is sub- prolyl hydroxylases, which belong to the same
jected to ubiquitination and degradation by superfamily (Bruick & McKnight, 2001;
the 26S proteasome proteolysis (Kallio et al., Jaakkola et al., 2001). The prolyl hydroxylase
1999). In HIF-1a ubiquitination the major responsible for HIF-1a hydroxylation was
role is played by the von Hippel-Lindau tumor first identified as a product of egl-9 gene after
suppressor protein (pVHL), whose b-domain searching Caenorhabditis elegans genome da-
interacts directly with the ODD domain of tabase (Epstein et al., 2001). Egl-9 mutant
HIF-1a (Bonicalzi et al., 2001). The inactiva- worms, similarly to vhl-1 mutants, constitu-
tion of pVHL is associated with the von tively expressed HIF-1a. Later three EGL-9
Hippel-Lindau disease, which is a hereditary homologues in mammals were identified and
cancer syndrome characterized by the devel- designated PHD (PH domain containing pro-
opment of highly vascularized tumors with tein) 1, 2, and 3. They are also termed HPHs
constitutive HIF-1 expression (Ivan et al., (HIF-1 prolyl hydroxylases) 3, 2, and 1, re-
2001). pVHL acts as the substrate recognition spectively. They differ in intracellular local-
component of the E3 ubiquitin ligase protein ization: PHD1 was detected exclusively in the
complex and after binding to HIF-1a it re- nucleus, PHD2 — in the cytoplasm, whereas
cruits elongins B and C, cullin 2, and Rbx1 PHD3 in both nucleus and cytoplasm (Metzen
(reviewed in Semenza, 2001). et al., 2003a). Additionally, the existence of a
The interaction between pVHL and HIF-1a fourth PHD has been described (Oehme et al.,
is oxygen-dependent: pVHL associates with 2002). Each of them hydroxylates Pro-564 in
ODD under normoxic but not under hypoxic HIF-1a, whereas only PHD1 and PHD2
conditions, thus the degradation does not oc- hydroxylate Pro-402. Molecular oxygen is a
cur during hypoxia (Maxwell et al., 1999). The substrate of these enzymes, while carbon di-
interaction of pVHL with HIF-1 depends on a oxide and succinate are by-products. During
posttranslational modification of HIF-1a, hypoxia, or in case of a lack of Fe(II) or
which was identified as an oxygen- and 2-oxoglutarate, prolyl hydroxylases cease to
iron-dependent prolyl hydroxylation (Ivan et function, thus HIF-1a does not undergo deg-
al., 2001). The binding of pVHL to the ODD radation (reviewed in Semenza, 2001).
domain, and therefore the ubiquitination of
its N- and C-terminal part (aa 390–417 and
HIF-1 activation
549–582, respectively), must be preceded by
the hydroxylation of proline residues (Pro To obtain full transcriptional activity HIF-1
402 in the N-terminal part and Pro 564 in the must bind to the DNA target sequence and re-
C-terminal part of ODD, Fig. 2). These resi- cruit transcriptional cofactors. This phase
dues are embedded in the conserved amino also undergoes hypoxia-dependent regula-
acid motif, LXXLAP, in which Leu 562 tion. The role of HIF-1a transactivation do-
strongly facilitates the hydroxylation of Pro mains (TADs) is to recruit the transcriptional
564 (Ivan et al., 2001). coactivator complexes to the promoters of
HIF-1 target genes. The central integrating
HIF-1 prolyl hydroxylases coactivator p300/CBP interacts through its
CH1 (cysteine-histidine-rich) domain with
The hydroxylation of the proline residues in HIF-1a TADs (Gu et al., 2001) and recruits
the ODD domain of HIF-1a is catalyzed by the accessory coactivators like histone
acetylotransferases SRC-1, TIF-2, and redox main by preventing its interaction with
factor Ref-1 (Ema et al., 1999). p300/CBP. The asparaginyl hydroxylase that
The molecular mechanism of activation of modifies HIF-1a was recently identified as
the TAD domains was recently discovered. the previously known factor inhibiting HIF-1
Under normoxic conditions the highly con- (FIH-1) (Lando et al., 2002b). The final evi-
served asparagine residue within the C-TAD dence that both residues, Pro within the ODD
domain (Asn 803) is hydroxylated (Lando et and Asn within the C-TAD, are necessary for
al., 2002a) which results in the silencing of the full activation of HIF-1 was provided by
transactivation domains (Fig. 3). The enzyme an experiment in which either critical Pro, or
Figure 3. The role of prolyl and asparaginyl hydroxylation in the stabilization and activation of HIF-1a.
Under normoxic conditions specific prolyl (P) residues within the oxygen-dependent degradation domain (ODD)
and asparaginyl (N) residues within the COOH-terminal transactivation domain (C-TAD) are hydroxylated by re-
spective hydroxylases. The hydroxylation of prolines enables binding of pVHLs and thus constitutes a signal for
proteasomal degradation. The hydroxylation of asparaginyl (N) residues blocks binding of transcriptional
coactivator (p300/CBP) and subsequently inhibits transcriptional activity of HIF-1. In contrast, under hypoxic con-
ditions HIF-1a escapes proteasomal degradation because of abolition of prolyl hydroxylation and may interact with
p300/CBP due to inhibition of asparaginyl hydroxylation.
that hydroxylates the Asn residues is also a Asn, or both were replaced by Ala (Lando et
member of the Fe(II)- and 2-oxoglutarate-deal., 2002a). Replacement of Pro within the
pendant superfamily of dioxygenases, thus it ODD domain resulted in a stable protein even
is blocked by the inhibitors of 2-oxogluta- under normoxia which, however, exhibited
rate-dependent dioxygenases such as dime- low transcriptional activity at normoxia and
thyloxalylglycine (DMOG) and Fe(II) chela- high transcriptional activity at hypoxia. Re-
tors. Treatment of cells with DMOG or iron placement of the critical Asn within the
chelators as well as replacing the critical Asn C-TAD domain does not influence protein ac-
residue with Ala result in activation of C-TAD tivity in comparison to wild-types proteins,
even at normoxia (Lando et al., 2002a). There- because, despite the active C-TAD domain,
fore, p300/CBP interacts with C-TAD only the protein is unstable at normoxia. Finally,
when the Asn is nonhydroxylated which en- the double amino acid substitution resulted in
ables the assembly of transcriptional co- nearly full activity of the protein at normoxia
activator complex, whereas the hydroxylation (Lando et al., 2002a).
of Asn during normoxia silences C-TAD do-
Other levels of HIF-1 regulation HRE may be located within either promoter
or enhancer regions (5¢-flanking, 3¢-flanking,
Nuclear localization of HIF-1a or intervening) of target genes (Fig. 4, refer-
ences in Table 1). Generally, HBS is the mini-
The dimerization of the HIF-1 a and b sub- mal sequence necessary for HIF-1 binding.
units occurs in the nucleus and is necessary However, the structure of HRE, methylation
for the DNA binding and subsequent activa- of the cytosine residue within HBS, or pres-
tion of transcription (Kallio et al., 1997). In ence of additional transcription factors may
contrast to HIF-1b which is present in the nu- influence HIF-1-induced response. Addition-
cleus regardless of oxygen levels, nuclear ally, in the majority of hypoxia-induced genes
translocation of HIF-1a is correlated with HRE contains HIF-1 ancillary sequence
HIF-1 activity. Consequently, it was sug- (HAS), which is located 8–9 nt down- or up-
gested that this translocation could be stream of HBS and is necessary for HIF-1-me-
Figure 4. The localization of hypoxia response element (HRE) in various hypoxia-induced genes.
HRE (black rectangle) may be located within either 5¢-flanking (VEGF and transferrin) or 3¢-flanking (EPO)
enhancer regions, or within the promoter (NOSII) of target genes.
upregulated by hypoxia (Kallio et al., 1998). diated transcription activation (Kimura et al.,
However, when HIF-1a is overexpressed the 2001). HAS is an imperfect inverted repeat of
translocation occurs even under normoxic HBS, thus the secondary structure of HRE
conditions. Therefore, this process seems to seems be crucial for its activatory function
be hypoxia-independent and the nuclear frac- (Fig. 5). It was also shown that HAS recruits
tion of HIF-1a may simply reflect the overall protein complexes distinct from HIF-1
level of this protein in the cell (Hofer et al., (Kimura et al., 2001).
2001). Furthermore, efficient gene activation fre-
quently requires recruiting of more than one
HIF-1 DNA binding HIF-1 or binding of additional transcriptional
factors, which are not hypoxia-dependent.
After the stabilization of the a subunit, nu- Two or three adjacent HBSs were found in
clear translocation, and dimerization, HIF-1 some genes encoding glycolytic enzymes, glu-
binds to its consensus binding site (HBS, cose transporter 1, and transferrin. There is
HIF-1 binding site) within the hypoxia re- also a binding site for the ATF-1/CREB-1 fac-
sponse element (HRE) (Semenza et al., 1996). tor (activating transcription factor-1/cAMP-
The core sequence of HBS is (A/G)CGTG. response element-binding protein-1) in the
HRE of lactate dehydrogenase A gene, for conditions (Frick et al., 2003) or in response
AP-1 (activator protein-1) binding factor in to hypoxia (unpublished data). In contrast, in
VEGF gene, and for the HNF-4 (orphan recep- human microvascular endothelial cells
tor hepatic nuclear factor-4) in the erythropoi- (Józkowicz et al., 2004) and smooth muscle
etin gene (references in Wenger, 2002). All cells (Dulak et al., 2002) hypoxia potently
Figure 5. Human hypoxia response element (HRE) in VEGF, EPO, glucose transporter-1 (GLUT-1), and
lactate dehydrogenase A (LDHA) genes (Kimura et al., 2001).
HRE, besides the HIF-1 binding site (HBS), contains the HIF-1 ancillary sequence (HAS), which is located 8–9 nt
down- or upstream of HBS and constitutes an imperfect inverted repeat of HBS.
these transcription factors modulate HIF-1 modulates VEGF production. These data
response. Such a requirement for additional suggest that the response to hypoxia is cell
transcription factors may (i) amplify hypoxic type specific.
response in particular conditions, (ii) vary re-
sponses of distinct tissues to the hypoxia, and
Molecular mechanism of oxygen sensing
(iii) enable diverse induction of distinct target
genes. Although the mechanisms of HIF-1a stabili-
Another possible level of regulation may be zation and activation are already known, the
the CpG methylation of the cytosine residue signaling pathways that lead to the inhibition
within HBS. Although, in the majority of of prolyl and asparaginyl hydroxylases re-
HIF-1 target genes it remains unmethylated main unclear. The requirement of molecular
as it is located in the methylation-free CpG is- oxygen as a substrate for these enzymes could
lands, the HBS of the erythropoietin gene explain the loss of HIF-1 degradation under
might undergo methylation, which inhibits hypoxic conditions. This assumption, how-
HIF-1 binding (Wenger et al., 1998). To pre- ever, appears to be oversimplified. It has been
vent methylation, HBS is at normoxia occu- shown that collagen prolyl hydroxylases are
pied by other DNA binding factors (reviewed active in hypoxic cells and that maximal
in Wenger, 2002). It might also be hypothe- HIF-1a stabilization occurs under 0.5% O2
sized that methylation of HBS may constitute concentration rather than under anoxia,
a mechanism of cell-specific regulation of which would not be possible if prolyl
hypoxia-induced gene expression. Interest- hydroxylases were direct oxygen sensors
ingly, human macrovascular endothelial cells (Jiang et al., 1996b). Moreover, earlier stud-
do not release VEGF either under normoxic ies provided evidence that other signaling
pathways, such as ROS-, protein phos- b-containing NADPH oxidase that could re-
phorylation-, and nitrosylation-dependent spond to oxygen levels (references in Michiels
pathways are involved in oxygen sensing. et al., 2002), and (iv) induction of HIF-1 target
Therefore, many models of intracellular genes in the presence of exogenous catalase
oxygen sensing have been considered. or antioxidants (Salceda & Caro, 1997). How-
ever, some observations were inconsistent
A hemoprotein as an oxygen sensor with this hypothesis. A nonspecific inhibitor
of NADPH oxidases (diphenylene iodonium,
The initial hypothesis suggested that the DPI) blocked HIF-1 activation in response to
role of oxygen sensor is mediated by an un- hypoxia (Gleadle et al., 1995) and HIF-1 activ-
known protein containing heme as a pros- ity was sustained in cells deficient in a sub-
thetic group. The putative role of heme, unit of NADPH oxidase (Archer et al., 1999).
bound either to HIF-1 PAS domains or to a Moreover, some experiments indicated that
distinct sensor hemoprotein, was suggested the production of ROS was increased rather
because of the fact that HIF-1 activation is in- than decreased during hypoxia (Chandel et
duced not only by the lack of molecular oxy- al., 1998).
gen but also by Fe(II) chelators, such as
desferrioxamine (Wang et al., 1993), and also
by some transient metals that could replace
Fe(II) in heme (Huang et al., 1997). However,
currently these observations seem to confirm
the iron-dependence of prolyl and asparaginyl
hydroxylases rather than hemoproteins
acting as the oxygen sensor.
ROS-dependent signaling pathways Figure 6. Model I of ROS-dependent hypoxia sig-

nal transduction.
Two opposing models postulate signaliza- According to this model hypoxia results in decreased
tion by changes in the cellular levels of reac- reactive oxygen species (ROS) generation, which leads
tive oxygen species (ROS) (reviewed in to HIF-1a stabilization. The same effect is observed in
the presence of antioxidants and catalase (Cat).
Semenza, 2000c; Michiels et al., 2002). The
first of them assumes that ROS are continu-
ously produced by an unknown NADPH The second model concerning ROS-depend-
oxidase that reduces O2 to superoxide anion ent pathway is diametrically opposed to the
(O2·–) which is subsequently converted to hy- previous one as it assumes that hypoxia re-
drogen peroxide (H2O2) by superoxide sults in an increased generation of ROS by
dismutase (SOD). According to this model the mitochondria, which constitutes the signal to
reduction of molecular oxygen concentration HIF-1a stabilization (Fig. 7). According to
would be followed by the reduction of ROS this model under hypoxic conditions the con-
levels, and therefore a decrease in ROS gener- sumption of oxygen at cytochrome c oxidase
ation would be a direct or indirect signal for (mitochondrial complex IV) is lower and elec-
HIF-1 activation (Fig. 6). The experiments trons accumulate at preceding complexes.
confirming this hypothesis showed: (i) de- Such an accumulation leads to increased gen-
creased ROS production under hypoxic condi- eration of ROS at complex III. Compatibly
tions, (ii) suppression of HIF-1 target genes with this theory inhibitors of complexes I and
by exogenous H2O2 in hypoxia, (iii) the pres- III blocked the induction of HIF-1 activity at
ence of a non-mitochondrial cytochrome hypoxia (due to inhibition of ROS produc-
tion), whereas inhibitors of complex IV were chloride or desferrioxamine instead of

able to induce HIF-1-dependent transcription hypoxia (Chandel et al., 1998). The explana-
at normoxia (due to induction of ROS generation of the contradictory results of Chandel et
tion) (Chandel et al., 1998). Another evidence al. (1998), and Vaux et al. (2001) may lie in
that mitochondrial complex III acts as an oxy- different mechanisms of oxygen sensing in
gen sensor was shown in r0 Hep3B cells, lack- the case of anoxia and hypoxia. Prolyl and
ing the functional respiratory chain, in which asparaginyl hydroxylases, which use molecu-
the induction of HIF-1 did not occur under lar oxygen as a substrate, act as direct oxygen
hypoxic conditions (Chandel et al., 1998). sensors in the total absence of oxygen
Moreover, the substrate of complex II (Schroedl et al., 2002). Similarly, des-
(succinate) restored hypoxic response in cells ferrioxamine and cobalt chloride are direct in-
Figure 7. Model II of ROS-dependent hypoxia signal transduction.

According to this model hypoxia results in an increased generation of reactive oxygen species (ROS) due to attenu-
ation of cytochrom c oxidase (mitochondrial complex IV) activity. The generation of ROS is mediated by complex
III at which, in the absence of functional cytochrome c oxidase (due to hypoxia or in the presence of complex IV in-
hibitors), electrons are accumulated. High levels of ROS induce HIF-1a stabilization, whereas low levels lead to
HIF-1a degradation. Low levels of ROS are maintained either when the electron transport chain is fully functional
(normoxia) or when electrons do not reach complex III (in the presence of inhibitors of complex I and II, or III).
with a defect in complex I (Agani et al., 2000). hibitors of the hydroxylases. Thus, at anoxia
Consistent with this theory are also our re- or after treatment with desferrioxamine or
sults showing that overexpression of SOD, cobalt chloride, stabilization of HIF-1a occurs
leading to increased production of H2O2, in- independently of ROS generation and the
duces HIF-1-dependent VEGF expression presence of functional electron transport
(Grzenkowicz-Wydra et al., 2004). chain is not necessary. This hypothesis ex-
However, these findings were not confirmed plains why inhibitors of complex I prevent
in other experiments on r0 cell lines cultured hypoxia-induced but not desferrioxamine- or
under severe hypoxic conditions (0.1% O2) anoxia-induced accumulation of HIF-1a
(Vaux et al., 2001) or after exposure to cobalt (Schroedl et al., 2002). In contrast, in the
hypoxia-mediated HIF-1a stabilization addi- way able to upregulate HIF-1 activity involves
tional intracellular signaling, including the receptor tyrosine kinase®PI3K (pospha-
phosphorylation- and ROS-dependent path- tidylinositol-3 kinase)® prolyl-4-hydroxylases
ways, is required (Schroedl et al., 2002). The (protein kinase B)®FRAP (FKBP-rapamycin
preservation of hypoxia-induced stabilization associated protein) pathway. Some studies
of HIF-1a in cells with a defect in electron showed that inhibition of the PI3K or AKT
transport chain (Vaux et al., 2001) may have kinases impairs HIF-1-dependent gene ex-
resulted either from the presence of marginal pression (references in Wenger, 2002). More-
complex I and III activity, sufficient to gener- over, loss of PTEN (phosphatase and tensin
ate ROS, or from anoxic rather than hypoxic homolog deleted on chromosome ten) activ-
conditions (0.1% O2) used in the experiments. ity, which is a tumor suppressor protein and a
It is possible that signals induced by hypoxia, negative regulator of PI3K, results in in-
as well as signals from some cytokines, creased HIF-1a expression (Zundel et al.,
vasoactive hormones, and nitric oxide (see be- 2000). Finally, FRAP can stimulate HIF-1a
low) lead to the inhibition of proline expression even under normoxic conditions
hydroxylation (Fig. 8). (Zhong et al., 2000). Additionally, the involve-
Figure 8. Prolyl and asparginyl hydroxylases function as direct (anoxia) or indirect (hypoxia) oxygen
sensors.
Phosphorylation cascades ment of ROS-dependent p38 MAP kinase

pathway has been suggested because inhibi-
Initial studies indicated that the activation tion of HIF-1 activity by p38 kinase blockers
of HIF-1 involves protein phosphorylation was observed (Gorlach et al., 2001).
(Wang et al., 1995b). This process might be
mediated by several different protein kinase Other signaling pathways
pathways. First, the p42/p44 (Erk2/Erk1)
mitogen-activated protein kinases (MAPKs) Other suggested signaling pathways con-
are able to phosphorylate HIF-1a (Richard et cern redox-dependent regulation and protein
al., 1999). This modification, however, en- N-nitrosylation. For example, it was shown
hances HIF-1 activity but is not involved in that reduction of cysteine within C-TAD en-
HIF-1a stabilization. Thus, this is rather the hances HIF-1 trans-activation by enabling
way in which growth factors modify HIF-1 the interaction with CBP. This reduction is
function as many of them act via the receptor provided by the system: thioredoxin/the re-
tyrosine kinase®Ras/Raf®MEK (mitogen-ac- dox factor Ref-1 (Ema et al., 1999). Co-ex-
tivated kinase kinase)®MAPK pathway (re- pression of Ref-1 and thioredoxin enhanced
viewed in Wenger, 2002). The second path- the transactivation by C-TAD, but not by
N-TAD, in a hypoxia-dependent manner. Ad- Interaction between HIF-1 and p53

ditionally, upon hypoxic conditions, thio-
redoxin was found to be translocated to the There are many similarities between the two
nucleus where it can interact with Ref-1 transcription factors: HIF-1a and the tumor
(Ema et al., 1999). suppressor p53. Under hypoxic conditions
both proteins accumulate and gain their
transcriptional activity which requires re-
HIF-1 negative regulation
cruitment of p300 as a co-activator. More-
Several negative feedback regulatory path- over, under normoxic conditions p53, simi-
ways that could limit the response to hypoxia larly to HIF-1a, is ubiquitinated by E3 ligase
have been proposed (reviewed in Wenger, (MDM2 for p53) and subsequently degraded
2002). The downregulation of HIF-1a might by 26S proteasome (Giaccia et al., 1998). It
occur on the level of transcription, transla- was observed that the loss of p53 activity re-
tion, protein stabilization, and/or protein ac- sults in an increased accumulation of HIF-1a
tivation. Responsible for the latter could be under hypoxic conditions (Ravi et al., 2000).
Cited2 (also named p35srj), a member of the This relationship was explained by the obser-
CBP/p300-interacting transactivators with a vation that p53 binds to HIF-1a and recruits
glutamic acid and aspartic acid-rich tail. the MDM2 ubiquitin ligase which preferably
Cited2 competes with HIF-1 in binding to the targets HIF-1a for degradation. Thus, in the
cysteine-histidine-rich (CH1) region of p300 case of prolonged anoxic conditions, p53 can
and CBP (Leung et al., 1999). Notably, Cited2 act as a negative regulator of HIF-1 in two
expression is induced by HIF-1 (Bhattacharya ways. First, it inhibits HIF-1 activity by com-
et al., 1999). petitive binding of p300 (Schmid et al., 2003),
Recently it was shown that the down- and second, the interaction between p53 and
regulation occurs also at the level of HIF-1a HIF-1a results in HIF-1a degradation (Ravi et
stabilization as HIF-1 induces synthesis of al., 2000). Such cooperation explains the
prolyl hydroxylases which enable rapid angiogenic switch that occurs during tumori-
HIF-1a hydroxylation and degradation dur- genesis in case of p53 mutation.
ing reoxygenation (D’Angelo et al., 2003).
Also some anti-inflammatory factors could HIF-1 natural inhibitors
act through the inhibition of the HIF-1 path-
way. We recently showed that under hypoxic The natural inhibitors of HIF-1 identified so
conditions prostaglandin-J2 attenuates VEGF far are mainly splice variants of the a subunit
expression by inhibition of HIF-1 activity and thus are able to act as HIF antagonists.
(Józkowicz et al., 2004). Furthermore, heme The first of them, inhibitory PAS protein
oxygenase-1, which is a stress inducible en- (IPAS) is a HIF-a without the transactivation
zyme that degrades heme to carbon monox- domain. High levels of IPAS were indicated in
ide, iron ions, and biliverdin, could enhance the corneal epithelium of the eye where
activity of prolyl and asparaginyl hydroxy- HIF-dependent angiogenesis is significantly
lases by an increased release of iron. impaired (Makino et al., 2001). Other poten-
Biliverdin and its derivate, bilirubin, scaven- tial HIF-1 antagonists are: the zinc-inducible
gers of peroxyl radicals, could also modulate isoform lacking exon 12 (HIF-1aZ) and the
HIF-1a stability due to the attenuation of re- dominant-negative HIF-1a isoform lacking
active oxygen species generation. On the exons 11 and 12. In addition, an antisense
other hand, it was observed that HO-1 over- RNA specific for the 3' untranslated region of
expression enhances VEGF synthesis (re- HIF-1a was identified (references in Wenger,
viewed in Dulak et al., 2004). 2002).
NON-HYPOXIC ACTIVATION OF HIF-1 are responsible for this hypoxia-independent

AND MODULATORS OF HIF-1 induction of HIF-1 in VSCM. The first leads to
ACTIVITY an increase in HIF-1 gene transcription,
whereas the second results in an increase in
Interestingly, an increasing number of stud- translation of HIF-1 mRNA. The central role
ies indicate that HIF-1a stabilization might in both pathways belongs to the diacyl-
occur in a hypoxia-independent manner. The glycerol-sensitive protein kinase C (PKC). It
inducers of HIF-1 activity that act independ- was also shown that the increase in HIF-1
ently of O2 levels may be divided into several translation by angiotensin II is mediated by
groups. The first group consist of direct inhib- ROS-dependent activation of the phospha-
itors of prolyl and asparaginyl hydroxylases tidylinositol-3 kinase (PI3K) pathway (Page et
such as iron chelators, iron replacing mole- al., 2002). Together these two pathways in-
cules, and analogs of 2-oxoglutarate. The sec- crease HIF-1a in VSMC to levels that surpass
ond group is represented by various hor- hypoxic induction.
mones and growth factors. Also hallmarks of Also some growth factors such as insulin, in-
inflammation such as proinflammatory sulin-like growth factor 1 and 2, epidermal
cytokines, nitric oxide, increased tempera- growth factor, fibroblast growth factor 2,
ture, or mechanical stress, may induce HIF-1 platelet-derived growth factor, transforming
activity. growth factor b1, and inflammatory cytokines
such as interleukin-1, or tumor necrosis fac-
tor a, (all references in Wenger, 2002) can
Direct inhibitors of prolyl hydroxylases
evoke HIF-1 activation under normoxic condi-
Transition metal ions, such as cobalt and tions. Most of them act through the tyrosine
nickel, induce HIF-1a stabilization under kinase receptor®PI3K®AKT®FRAP path-
normoxic conditions. Initially this effect was way. This growth factor-dependent HIF-1 acti-
thought to confirm the hypothesis of a heme vation is important in tumorigenesis as the
protein acting as a putative oxygen sensor overexpression of some growth factors may
(Huang et al., 1997). Recently, however, it cause HIF-1-mediated induction of intra-
was suggested that transition metals could in- tumoral angiogenesis.
hibit prolyl hydroxylases by substituting the
ferrous ion coordinated by PHDs (Epstein et
Nitric oxide
al., 2001). Another well-known activator of
HIF-1, desferrioxamine (Wang et al., 1993), HIF-1 induces nitric oxide production
could also inactivate PHDs by removal of iron through the enhancement of inducible nitric
from their catalytic domains. Furthermore, oxide synthase transcription (Table 1). Con-
compounds competing with 2-oxoglutarate versely, NO affects the accumulation and ac-
such as N-dimethyl-oxalylglycine (DMOG), a tivity of HIF-1. Initial studies focusing on the
2-oxoglutarate analog, are also able to inhibit NO influence on HIF-1 activity showed that
PHDs activity (Jaakkola et al., 2001). under hypoxic conditions, or after treatment
with CoCl2, NO inhibits HIF-1a stabilization
and transcriptional activation (Liu et al.,
Vasoactive hormones and cytokines
1998; Sogawa et al., 1998; Huang et al., 1999).
The induction of VEGF expression in vascu- On the other hand, it was indicated that un-
lar smooth muscle cells (VSMC) by vasoactive der normoxic conditions diverse NO donors
hormones like angiotensin II and thrombin is (with the exception of sodium nitroprusside,
mediated through the activation of HIF-1 SNP) or endogenously produced NO (by in-
(Richard et al., 2000). Two separate pathways ducible or endothelial nitric oxide synthase,
NOS2 and NOS3, respectively) causes HIF-1a cently, however, it was reported that NO do-
stabilization and activation of its target genes nor, NOC18, induces HIF-1a synthesis at
(Kimura et al., 2000; Dulak et al., 2000; normoxia, whereas neither hydroxylation nor
Sandau et al., 2001a; 2001b; Józkowicz et al., stabilization of HIF-1a is influenced. Instead,
2001; Dulak & Józkowicz, 2003b). Normoxic it was shown that this effect depends on the
NO-induced upregulation of HIF-1 occurred PI3 and MAP kinases pathways (Kasuno et
through the PI3K/Akt pathway (Sandau et al., 2004).
al., 2001b; Brüne et al., 2001; Natarajan et al.,
2003) and was independent of soluble NO and ROS
guanylyl cyclase (sGC) activity (Brüne et al.,
2001). The type of the NO donor and its con- On the other hand, NO as a free radical re-
centration used in experiments seem to be acts rapidly with ROS, particularly in a diffu-
crucial for the results obtained. The inhibi- sion-controlled fashion with superoxide anion
tory effect of SNP may be evoked by toxic to create peroxynitrate, one of the most reac-
by-products of its decomposition such as tive compounds in nature. If hypoxia acti-
cyanides and iron ions which probably sur- vates HIF-1 through the increase in mitochon-
pass the effect of NO (Dulak et al., 2000; drial ROS generation, NO would inhibit HIF-1
Józkowicz et al., 2001). activation by attenuation of ROS levels. To
confirm this hypothesis it was shown that NO
NO and PHDs blocks HIF-1a stabilization in the presence of
superoxide anion donors or H2O2 (Sandau et
Recently it was reported that the NO-medial., 2001b). Thus ROS and NO might be able
ated activation of HIF-1 at normoxia is caused to induce HIF-1 activity but, when present to-
by attenuation of prolyl hydroxylation gether, they may react to form compounds
(Metzen et al., 2003b) and, therefore, PHDs such as peroxynitrite that lacks this property
might constitute direct or indirect targets of and might cause HIF-1a degradation.
NO. An interaction between NO and iron
could constitute a way of affecting prolyl
Carbon monoxide
hydroxylation as NO directly binds to the fer-
rous ion in heme or non-heme iron-containing Initially it was reported that carbon monox-
proteins. Prolyl hydroxylases contain Fe(II) ide (CO) inhibits hypoxia-induced HIF-1a ex-
in their catalytic domains, thus NO could pression, HIF-1 DNA binding, and HIF-1
compete with molecular oxygen for the cata- transcriptional activity (Huang et al., 1999).
lytic site and subsequently inhibit enzyme Such a correlation would be consistent with
function (Metzen et al., 2003b). This hypothe- the hypothesis of a heme protein acting as an
sis, however, does not explain why NO inhib- oxygen sensor. However the concentrations
its hypoxia-induced HIF-1 accumulation. of CO used in those experiments were very
Helpful in searching for the answer could be high (up to 80%) and therefore non-physiologi-
the fact that NO down-regulates HIF-1a in cal. On the contrary, it is possible that CO, as
hypoxia by activation of PHDs in cytoplasmic an inhibitor of mitochondrial complex IV,
extracts (Wang et al., 2002), although this ef- could lead to an increase in ROS generation
fect has not been confirmed by in vitro pro- and, therefore, induce HIF-1a accumulation.
tein interaction assays (Metzen et al., 2003b). We have shown that low (1% or lower) concen-
Therefore, activation of PHDs by NO might trations of CO strongly induce VEGF expres-
be caused by cytoplasmic components such as sion in vascular smooth muscle cells (Dulak et
protein phosphorylation cascades or/and al., 2002) and in endothelial cells (Józkowicz
ROS-dependent signaling pathways. Re- et al., 2003; reviewed in Dulak & Józkowicz,
2003a). Another interesting hypothesis was in development, physiology, and patho-

proposed after the discovery that mammalian physiology. The central role in the regulation
neuronal PAS domain protein 2 (NPAS2), a of HIF-1 activity belongs to prolyl and aspar-
bHLH-PAS transcription factor involved in aginyl hydroxylases, which function coopera-
regulation of circardian cycle, is regulated not tively as direct or indirect oxygen sensors.
only by the NADPH/NADP ratio but also by The question is why HIF-1 activity is con-
carbon monoxide (Dioum et al., 2002). Incor- trolled by two independent processes. The an-
poration of CO into hemes which are bound to swer might be that multiple levels of the regu-
the PAS domains of NPAS2 inhibits lation enable graded responses to subtle
NPAS2-BMAL1 heterodimerization and, sub- changes in O2 concentration and ensure tight
sequently, its transcriptional activity. The control of the hypoxic response pathway
NPAS2 binding sequence, CACGTG (Rutter (Bruick & McKnight, 2002).
et al., 2001), may be recognized by HIF-1, thus The analysis of HIF-1 expression pattern
reciprocal relationship between these two and the modulation of HIF-1 activity may
transcription factors is possible and requires bring new diagnostic and therapeutic ap-
further studies. proaches in many diseases such as cancer and
vascular disorders (reviewed in Semenza
2000a). It is possible that inhibition of HIF-1
Mechanical and thermal stress
could suppress intratumoral angiogenesis
It has been reported that HIF-1a is present and thus attenuate cancer growth. Addition-
in the nuclei of cardiac myocytes in ally, it was observed that Hif1a+/– mice had
non-hypoxic myocardium (Kim et al., 2002). significantly weaker responses to chronic
This upregulation of HIF-1a occurs as a result hypoxia such as reduced polycythemia, right
of a wall stretch and is mediated by the ventricular hypertrophy, and pulmonary hy-
PI3K®AKT®FRAP pathway. A similar effect pertension (Yu et al., 1999). Thus in case of
was observed in aortic VSMC where hyper- chronic lung disease local downregulation of
tension induced HIF-1a accumulation HIF-1a could be useful in treating or prevent-
(Kuwahara et al., 2002). Therefore, HIF-1 ing these disorders. In contrast, in the case of
seems to be responsible not only for the adap- myocardial ischemia or other ischemic disor-
tation to hypoxia but also for the adaptation ders induction of HIF-1 expression could pre-
to mechanical stress. vent ischemic tissues from permanent dam-
It was also indicated that HIF-1a accumula- ages. HIF-1 upregulation in pro-angiogenic
tion might be evoked by exposure to in- therapies could facilitate the formation of
creased temperature. This effect seems to be fully matured vascularity.
mediated by direct protein stabilization that Therefore, the understanding of the molecu-
involves the heat shock protein HSP90 and is lar mechanisms of HIF-1 activation will be
hypoxia-independent (Katschinski et al., crucial for the development of new drugs act-
2002). ing either as agonists or as antagonists of the
hypoxia response pathway. Possible targets
of future therapies could be not only HIF-1a
CONCLUSIONS itself but also the proteins mediating its stabi-
lization such as prolyl and asparaginyl
Undoubtedly, HIF-1 plays the leading role in hydroxylases.
the induction of complex response to various
stresses and thus contributes to the mainte-
nance of cellular and systemic homeostasis. We are grateful to Dr. Alicja Józkowicz and
This function gives HIF-1 a major importance Prof. Aleksander Koj for useful comments.
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Activation of Spinobulbar Lamina I Neurons by Static
Muscle Contraction
L. B. Wilson, D. Andrew and A. D. Craig
J Neurophysiol 87:1641-1645, 2002.
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J Neurophysiol
RAPID COMMUNICATION 87: 1641–1645, 2002; 10.1152/jn.00609.2001.
Activation of Spinobulbar Lamina I Neurons by Static Muscle

Contraction
L. B. WILSON,1 D. ANDREW,2 AND A. D. CRAIG2

1
Department of Physiology, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2Atkinson Pain
Research Laboratory, Division of Neurosurgery, Barrow Neurological Institute, Phoenix, Arizona 85013
Received 24 July 2001; accepted in final form 14 November 2001
Wilson, L. B., D. Andrew, and A. D. Craig. Activation of spino- afferents terminate in the dorsal horn of the spinal cord, par-
bulbar lamina I neurons by static muscle contraction. J Neurophysiol ticularly within lamina I (Craig and Mense 1983). Spinobulbar
87: 1641–1645, 2002; 10.1152/jn.00609.2001. Spinal lamina I neu- lamina I neurons have never been recorded. Spinothalamic
rons are selectively activated by small-diameter somatic afferents, and lamina I neurons excited by group III muscle afferents and
they project to brain stem sites that are critical for homeostatic control.
Because small-diameter afferent activity evoked by contraction of
noxious muscle stimulation were identified previously (Craig
skeletal muscle reflexly elicits exercise-related cardiorespiratory acti- and Kniffki 1985), but activation of lamina I neurons by

vation, we tested whether spinobulbar lamina I cells respond to muscle contraction has never been demonstrated; this requires
muscle contraction. Spinobulbar lamina I neurons were identified in maintaining microelectrode recordings from these small spinal
chloralose-anesthetized cats by antidromic activation from the ipsilat- neurons during hindlimb muscle contractions strong enough to
eral caudal ventrolateral medulla. Static contractions of the ipsilateral elicit blood pressure changes in an unparalyzed animal under
triceps surae muscle were evoked by tibial nerve stimulation using anesthesia. The purpose of this study was to determine whether
parameters that avoid afferent activation, and arterial blood pressure excitation of identified spinobulbar lamina I neurons by static
responses were recorded. Recordings were maintained from 13 of 17 contraction of skeletal muscle can be demonstrated.
L7 lamina I spinobulbar neurons during static muscle contraction, and
5 of these neurons were excited. Three were selectively activated only
by muscle afferents and did not have a cutaneous receptive field. METHODS
Spinobulbar lamina I neurons activated by muscle contraction provide
an ascending link for the reflex cardiorespiratory adjustments that Five adult cats (3.5– 4.2 kg) were premedicated with ketamine (25
accompany muscular work. This study provides an important first step mg/kg im) and anesthetized with ␣-chloralose (80 mg/kg) and urethan
in elucidating an ascending afferent pathway for somato-autonomic (100 mg/kg) administered via a cannula in the cephalic vein. Cannulae
reflexes. were inserted into the carotid artery (to record blood pressure) and
into the trachea (for artificial respiration). The animals were paralyzed
during surgery with pancuronium (400 ␮g/h) and ventilated; end-tidal
CO2 was maintained near 4.0%. Body temperature was monitored
INTRODUCTION
using a rectal thermistor and maintained at 37.5°C.
Lamina I, the most superficial layer of the spinal or trigem- A laminectomy was performed to expose the lumbosacral spinal
inal dorsal horn, receives modality-selective A␦- and C-fiber cord, and a pool filled with warm (38°C) Tyrode’s solution was
afferent input from somatic and visceral tissues (Craig 2000). constructed. The medulla was exposed by enlarging the foramen
magnum. A bipolar electrode (Rhodes NE- or NEX-100) was inserted
Anatomic findings indicate that lamina I neurons are the major into the left caudal ventrolateral medulla (CVLM) to antidromically
source of spinobulbar projections (Krout and Craig 1996) and activate spinobulbar neurons. The left calcaneal bone was cut, and the
project to specific brain stem sites involved in homeostatic Achilles tendon was connected to a transducer (Grass model FT10) to
regulation (Craig 1995). Thus lamina I may provide an impor- measure the tension developed during contraction of the triceps surae
tant link for the somato-autonomic reflex responses evoked by muscle. The patellar tendon was secured to a post to ensure an
somatic and visceral small-diameter afferent input (Sato and isometric contraction. The popliteal fossa was exposed, and the tibial
Schmidt 1973). nerve was placed on platinum wire electrodes (LeDoux and Wilson
The brain stem plays a pivotal role in homeostatic responses, 2001). A pool made around the exposed nerves and muscles was filled
including cardiovascular and respiratory responses to muscle with warm mineral oil. The preparation is shown diagrammatically in
work (Iwamoto et al. 1985). Static contraction of skeletal Fig. 1. Once the preparation was complete, the paralytic was allowed
to wear off.
muscle activates small-diameter afferents that evoke a reflex Glass-insulated tungsten microelectrodes were used to record ex-
increase in sympathetic nerve activity and cardiovascular func- tracellularly from single lamina I neurons, identified by ongoing unit
tion (Kaufman and Forster 1996; Mitchell and Schmidt 1983; activity or by antidromic search stimuli (see Craig et al. 2001).
Wilson and Hand 1997). Activation of spinobulbar dorsal horn Bipolar or monopolar electrical stimuli were delivered from the elec-
neurons is required for this somato-autonomic reflex, the “ex- trode in the CVLM to determine whether a neuron projected to the
ercise pressor reflex” (Wilson 2001). Small-diameter muscle brain stem by antidromic activation. Two stimulus pulses of 400 ␮A
Address for reprint requests: A. D. Craig, Atkinson Pain Research Labora- The costs of publication of this article were defrayed in part by the payment
tory, Div. of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas of page charges. The article must therefore be hereby marked ‘‘advertisement’’
Rd., Phoenix, AZ 85013 (E-mail: bcraig@chw.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
www.jn.org 0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society 1641
1642 L. B. WILSON, D. ANDREW, AND A. D. CRAIG
and 1-ms duration were delivered at 100 Hz; stronger stimuli that average central conduction velocity of the spinobulbar lamina
evoked movement were avoided unless pancuronium could be admin- I neurons was 11.3 ⫾ 6.2 (SD) m/s (n ⫽ 17; range, 2.2–20.8),
istered. Every unit that followed two pulses at 100 Hz also followed which is significantly faster than spinothalamic lamina I neu-
a train of six antidromic stimuli at 250 Hz when tested (Fig. 2A); rons (mean, 4.7 ⫾ 2.2 m/s, n ⫽ 186, P ⬍ 10 ⫺7, unpaired t-test)
collision was also observed between naturally and antidromically
evoked impulses (Fig. 2B). Each unit was characterized using me-
(Andrew and Craig 2001). Unitary recordings from 13 spino-
chanical and thermal stimulation applied over the entire hindlimb bulbar lamina I neurons and 4 other lamina I neurons were
(Craig et al. 2001), and each cell was tested for excitation by muscle successfully maintained during muscle contraction.
contraction. Muscle contraction was evoked by stimulation of the Figure 2 shows the response to muscle contraction of an
tibial nerve with a 30-Hz train of 25-␮s pulses at 1.3 times motor antidromically identified and histologically confirmed lamina I
threshold for 10 –30 s. These stimulus parameters excite motor axons spinobulbar neuron. This unit began to respond ⬃15 s after the
but do not excite A or C afferent fibers (Degtyarenko and Kaufman onset of static contraction of the triceps surae muscle (Fig. 2E);
2000; Kaufman and Forster 1996). Only moderate contractions that its activity was maintained ⬎1 min following the contraction.
evoked moderate blood pressure changes were evoked because of the It did not respond to cutaneous mechanical or thermal innoc-
need to maintain stable single-unit microelectrode recordings. uous or noxious stimulation or to blood pressure changes these
Electrolytic lesions (⫹20 –50 ␮A, 10 s) were made at spinal re-
stimuli evoked or to tapping or moving the hindlimb, but it did
cording sites and at stimulation sites in the medulla. The tissue blocks
were fixed in formalin, and the lesions were recovered in 50-␮m respond to strong squeezing and stretching of the triceps surae
frozen sections that were stained with thionin (Fig. 2, C and D). muscle. It received time-locked (monosynaptic) input from
group III (A␦) tibial afferent fibers (conduction velocity, 18.5
RESULTS
m/s) but not from group IV (C) fibers (determined during
paralysis with paired 1-mA, 1-ms pulses at 120 Hz).
Recordings were obtained from 17 lamina I neurons identi- The responses of lamina I neurons to static muscle contrac-

fied as spinobulbar neurons by antidromic activation from the tion varied; 9 of the 17 neurons tested were excited, 2 were
CVLM and 5 lamina I cells with unidentified projections. The inhibited and 6 were unaffected. Of the 13 spinobulbar lamina
I neurons tested, 5 were excited by muscle contraction. Of
these, 3 had no detectable cutaneous receptive field, whereas 1
was a polymodal nociceptive neuron (HPC) (Craig et al. 2001)
that responded to noxious heat and pinch and noxious cold on
the hindpaw, and one was a wide-dynamic-range (WDR) neu-
ron that responded to low- and high-threshold stimulation of
the foot. Of the four lamina I neurons with unidentified pro-
jections that were excited by contraction, one received input
only from muscle afferents and three had HPC cutaneous
receptive properties. Most (4/6) of the neurons that were un-
affected by contraction were nociceptive-specific (NS) cells,
all of which projected to the CVLM. (Two had receptive fields
on the leg near the muscle exposure, indicating that muscle-
responsive cells without cutaneous fields did not result from
surgical damage to hindlimb cutaneous afferents.) The mean
central conduction velocity of the spinobulbar neurons excited
by contraction (9.7 m/s; range, 2.2–19.3, SD 7.4) was not
different from that of the other spinobulbar neurons (mean
10.9; range, 4.1–16.9, SD 6.0; P ⬎ 0.7, unpaired t-test).
Various responses of spinobulbar and other lamina I neurons
to muscle contraction are shown in Figs. 2 and 3. These
neurons responded during the contraction (Fig. 3A), after the
contraction (Fig. 3B) or both (Figs. 2E and 3C), and their
responses paralleled the tension developed in the muscle (Fig.
3, A and C), the centrally evoked cardiovascular reflex (Fig.
3A), or both (Fig. 3A). Interestingly, rhythmic muscle contrac-
tion (5 Hz), which is a potent stimulus for group IV (C)
afferents (Adreani et al. 1997), was the most effective stimulus
for activation of the cell shown in Fig. 3C.
DISCUSSION
FIG. 1. Diagram of the preparation. The activity of single lamina I neurons These are the first records of lamina I responses to static
was recorded extracellularly with microelectrodes (R). Electrical stimuli (S) contraction of skeletal muscle as well as the first recordings
were applied to the caudal ventrolateral medulla (CVLM) to test for neuronal from identified spinobulbar lamina I neurons. These technical
projections to the brain stem and to the axons of motoneurons in the tibial
nerve to evoke a static contraction of the triceps surae muscle. A tension advances notwithstanding, the most striking and novel result is
transducer (T) attached to the free end of the muscle was used to measure the the documentation of spinobulbar lamina I neurons that re-
force developed during a contraction. sponded to muscle contraction, particularly those which re-
J Neurophysiol • VOL 87 • MARCH 2002 • www.jn.org

LAMINA I NEURONS AND EXERCISE 1643
FIG. 2. Example of the properties of a lamina I spinobulbar

neuron and its response to static contraction. A: a pair of traces
showing reproducible 1-for-1 following of a train of 6 anti-
dromic stimuli (180 ␮A, 1 ms; ●) delivered at 250-Hz from an
electrode in the ipsilateral CVLM. B: collision of the 1st
impulse of a train of 3 antidromic action potentials (200 Hz, ●)
with a preceding spontaneously occurring impulse (*). 1, the
time at which the 1st antidromic response would have oc-
curred. C: reconstruction of the recording site in the L6 seg-
ment of the spinal cord showing an electrolytic lesion local-
ized to lamina I. D: diagram of the stimulating site in the
medulla: Cu, cuneate nucleus; EC, external cuneate nucleus;
G, gracile nucleus; LR, lateral reticular formation; IO, inferior
olive; V, trigeminal nucleus, X, vagal nucleus, XII, hypoglos-
sal nucleus. E: response of the same neuron to static contrac-
tion of the triceps surae muscle. The traces are, from the top

downward: arterial blood pressure; tension in the triceps surae,
the neural record and a histogram of the unit’s activity (1-s
bins). The thickening of the neural recording is due to stimulus
artifacts during muscle stimulation (1.3⫻ motor threshold, 25
95 s, 30 Hz for 30 s). This neuron received input only from
group III muscle afferents; its central conduction velocity was
2.2 m/s; and, it had no cutaneous receptive field.
sponded selectively and had no cutaneous receptive field. Such solitary tract, the retrotrapezoidal region, and other homeo-
neurons provide an ascending pathway for the cardiovascular static sites in the brain stem (Craig 1995; Westlund and Craig
and respiratory responses that are reflexly elicited by muscular 1996). The neurons we antidromically activated from the ipsi-
work. This evidence directly supports the proposal that modal- lateral CVLM could have projected to any of these sites (and
ity-selective lamina I neurons are an afferent link for homeo- are therefore termed spinobulbar rather than spinomedullary),
static responses to changes in the physiologic condition of the and the anatomic data indicate that they probably did not
body, consistent with a fundamental role in homeostasis and project any further rostrally (Craig 1995). Therefore a role for
interoception, of which pain, temperature, itch and muscular these neurons in somato-autonomic reflexes (Li et al. 2001;
sensations are particular aspects (Andrew and Craig 2001; Stornetta et al. 1989) and homeostatic integration is highly
Craig 1996, 2000; Craig et al. 2001; Sherrington 1900). likely.
Anatomic evidence indicates that lamina I receives dense A recent study reported contraction-evoked activation of
input from small-diameter muscle afferents and also that lam- neurons in the deep dorsal horn (Degtyarenko and Kaufman
ina I provides the predominant spinal input to specific homeo- 2000) that was reduced by stimulation of the mesencephalic
static brain stem sites bilaterally (Craig 1995, 2000; Craig and locomotor region, similar to the effects of motor commands on
Mense 1983; Krout and Craig 1996). We identified these afferent-induced cardiovascular responses. In contrast to mo-
spinobulbar lamina I projection neurons by using antidromic dality-selective lamina I neurons, deep dorsal horn neurons are
activation from the ipsilateral CVLM because lamina I axons modality-ambiguous and integrate nearly all somatic afferent
that ascend to the mesencephalon or thalamus are almost all inflow (Carstens 1997). Further, the axonal projections of such
contralateral (Craig 2000). The lamina I neurons we recorded cells were not identified, and thus their role in brain stem
that had unidentified projections (i.e., not antidromically acti- somato-autonomic integration is unclear.
vated from the ipsilateral CVLM nor in some cases from the The present findings show that spinobulbar lamina I neurons
contralateral thalamus) could also have included contralateral have appropriate characteristics to engage homeostatic re-
spinobulbar cells. Lamina I spinobulbar axons terminate in the sponses to muscle exercise. Exercise demands rapid cardiovas-
caudal and rostral ventrolateral medulla, the nucleus of the cular and respiratory adjustments. Exercise-evoked cardiores-

indirectly excited by metabolites released from the contracting

muscle (metabo-receptive; Kaufman and Forster 1996; Kauf-
man et al. 1983; Kniffki et al. 1981; McCloskey and Mitchell
1972). The central neural correlates of both mechanisms were
observed in the present study. Some lamina I neurons were
excited immediately following the onset of contraction and
their activity paralleled muscle tension, similar to mechano-
sensitive small-diameter muscle afferents. Other neurons were
excited late during a contraction or after the contraction, sim-
ilar to metabo-receptive muscle afferents.
In addition, most (3/5) of the spinobulbar lamina I neurons
in this initial sample that responded to muscle contraction had
no cutaneous input, and conversely, the spinobulbar cells that
responded selectively to cutaneous nociceptors (NS cells) did
not respond to contraction. This observation supports the pos-
sibility that the muscle afferents that initiate reflex cardiores-
piratory changes produced by muscular work are “ergorecep-
tors” that are distinct from nociceptors (see Kniffki et al. 1981;
Mitchell and Schmidt 1983), an idea that has received recent
experimental support (LeDoux and Wilson 2001). This obser-
vation is also consistent with the general modality-selectivity

of lamina I neurons (Craig 2000). On the other hand, muscle-
responsive lamina I cells that did have cutaneous input were
nearly all HPC cells, consistent with the possibility that the
activity in such polymodal nociceptive cells generally repre-
sents increased regional metabolic needs (Craig 1996). Further
investigations can address these issues more fully.
Our observations demonstrate that spinobulbar lamina I neu-
rons associated with homeostasis can be investigated. Detailed
analyses of this unexplored pathway are needed. Only lamina
I neurons that project to the mesencephalon (Hylden et al.
1986) or thalamus (Craig et al. 2001) have been characterized
before. Lamina I receives dense afferent input not only from
skin and muscle but also from the viscera and other tissues of
the body, and it receives descending controls from homeostatic
brain stem sites and from the hypothalamus (Craig 2000). Thus
lamina I spinobulbar neurons could be involved in many so-
mato-autonomic and homeostatic reflexes. Significantly, recent
retrograde labeling results indicate that contralateral spinotha-
lamic and ipsilateral spinobulbar lamina I neurons are com-
pletely separate populations (Andrew and Craig 2000). Our
present physiologic observations support this finding, because
spinobulbar lamina I neurons have characteristics not observed
in spinothalamic lamina I neurons (i.e., contraction-responsive,
FIG. 3. Examples of the variety of responses of lamina I neurons that were wide-dynamic-range, faster conduction velocities), and be-
excited by muscle contraction. The traces show from the top downward: the cause spinobulbar lamina I neurons are not antidromically
tension developed in the triceps surae muscle, the discharge of the single activated from the contralateral thalamus (0 of 10 tested)
neuron histogrammed in 1 s bins and either mean blood pressure (A) or the neural
recording (B and C). A: response from an HPC lamina I neuron with unidentified
(Andrew and Craig, unpublished observations).
projections that was excited by contraction that paralleled the tension devel- To conclude, we demonstrated that spinobulbar lamina I
oped in the muscle and the centrally-evoked cardiovascular reflex. B: response neurons can be identified that are activated by muscle contrac-
of another HPC lamina I neuron with unidentified projections that was only tion; such neurons provide an ascending pathway for the au-
excited after the contraction. C: response of a spinobulbar lamina I neuron tomatic cardiorespiratory adjustments to muscular work, that
(central CV 2.2 m/s) that was excited during and after the contraction; this
neuron had no identifiable cutaneous receptive field but responded to stretching is, the exercise pressor reflex. This study provides the founda-
the triceps muscle and also to rhythmic contractions at 5 Hz. tion for investigations of spinobulbar mechanisms that mediate
homeostatic adjustments elicited by small-diameter afferent
piratory responses are mediated in part by central motor com- activity from all tissues of the body. It remains to be estab-
mands and in part by the “exercise pressor reflex,” which lished how other types of somatic and visceral afferent activity
originates in small-diameter group III and IV (A␦ and C) are represented in this unexplored pathway.
afferents from active muscles (Iwamoto et al. 1985; Mitchell
and Schmidt 1983; Wilson and Hand 1997). These receptors We thank M. Tatum and S. Jordan for excellent technical assistance.
are either directly excited by contraction (mechanosensitive) or This work was supported by National Institute of Neurological Disorders

and Stroke Grant NS-25616 (A. D. Craig) and by the American Heart Asso- KAUFMAN MP AND FORSTER HV. Reflexes controlling circulatory, ventilatory
ciation–Southeast Affiliate (L. B. Wilson). and airway responses to exercise. In: Handbook of Physiology. Exercise:
Regulation and Integration of Multiple Systems, edited by Rowell LB and
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insight review articles
Molecular mechanisms of nociception

David Julius* & Allan I. Basbaum†
*Department of Cellular and Molecular Pharmacology, and †Departments of Anatomy and Physiology and W. M. Keck Foundation Center for
Integrative Neuroscience, University of California San Francisco, San Francisco, California 94143, USA (e-mail: julius@socrates.ucsf.edu)
The sensation of pain alerts us to real or impending injury and triggers appropriate protective responses.
Unfortunately, pain often outlives its usefulness as a warning system and instead becomes chronic
and debilitating. This transition to a chronic phase involves changes within the spinal cord and brain,
but there is also remarkable modulation where pain messages are initiated — at the level of the primary
sensory neuron. Efforts to determine how these neurons detect pain-producing stimuli of a thermal,
mechanical or chemical nature have revealed new signalling mechanisms and brought us closer to
understanding the molecular events that facilitate transitions from acute to persistent pain.
J
ust as beauty is not inherent in a visual image, root ganglia (DRG), respectively, and can be categorized into
pain is a complex experience that involves not three main groups based on anatomical and functional crite-
only the transduction of noxious environmental ria (Fig. 1a). Cell bodies with the largest diameters give rise to
stimuli, but also cognitive and emotional myelinated, rapidly conducting A primary sensory fibres.
processing by the brain. Progress has been made Most, but not all4, A fibres detect innocuous stimuli applied
in identifying cortical loci that process pain messages, but to skin, muscle and joints and thus do not contribute to pain.
far greater advances have been made in understanding the Indeed, stimulation of large fibres can reduce pain, as occurs
molecular mechanisms whereby primary sensory neurons when you activate them by rubbing your hand. By contrast,
detect pain-producing stimuli, a process referred to as small- and medium-diameter cell bodies give rise to most of
nociception. These insights have arisen predominantly the nociceptors, including unmyelinated, slowly conducting
from the analysis of sensory systems in mammals, as well C fibres and thinly myelinated, more rapidly conducting A
as from studies of invertebrates. Of course, invertebrate fibres, respectively. It has long been assumed that A and C
organisms do not experience pain per se, but they do have nociceptors mediate ‘first’ and ‘second’ pain, respectively,
transduction mechanisms that enable them to detect and namely the rapid, acute, sharp pain and the delayed, more
avoid potentially harmful stimuli in their environment. diffuse, dull pain evoked by noxious stimuli5 (Fig. 1b).
These signalling pathways can be regarded as the There are two main classes of A nociceptor6; both
evolutionary precursors of nociceptive processing in respond to intense mechanical stimuli, but can be
vertebrates, and genetic studies have facilitated the distinguished by their differential responsiveness to intense
identification and functional characterization of molecules heat or how they are affected by tissue injury. Most C-fibre
and signalling pathways that contribute to the detection of nociceptors are also polymodal, responding to noxious
noxious stimuli in animals. Indeed, many of the receptors thermal and mechanical stimuli6. Others are mechanically
and ion channels we refer to here are related to molecules insensitive, but respond to noxious heat. Importantly, most
highlighted in the accompanying reviews on C-fibre nociceptors also respond to noxious chemical stimuli,
mechanosensation, vision or olfaction in flies or worms. such as acid or capsaicin, the pungent ingredient in hot chilli
peppers. Finally, the natural stimulus of some nociceptors is
The primary afferent nociceptor difficult to identify. These so-called ‘silent’ or ‘sleeping’ noci-
Nearly a century ago, Sherrington proposed the existence of ceptors are responsive only when sensitized by tissue injury7.
the nociceptor, a primary sensory neuron that is activated by These nociceptor profiles derive largely from analysis of
stimuli capable of causing tissue damage1. According to this fibres that innervate skin. But very different features charac-
model, nociceptors have characteristic thresholds or sensi- terize nociceptors in other tissues6. For example, although
tivities that distinguish them from other sensory nerve corneal afferents can be activated by capsaicin and sensitized
fibres. Electrophysiological studies have, in fact, shown the by inflammatory mediators, pain is normally produced by
existence of primary sensory neurons that can be excited by innocuous tactile stimulation. In teeth, almost any stimulus
noxious heat, intense pressure or irritant chemicals, but not produces pain. Visceral pain is unique in that there are no
by innocuous stimuli such as warming or light touch2. In this first (fast) and second (slow) components; instead, pain is
respect, acute pain can be regarded as a sensory modality often poorly localized, deep and dull8. Tissue damage is also
much like vision or olfaction, where stimuli of a certain not required for visceral pain to occur; it can result from
quality or intensity are detected by cells with appropriately excessive distension (for example, of the colon). And the pain
tuned receptive properties. of ischaemia may have unique features that reflect innerva-
tion of vasculature by distinct subsets of acid-sensitive
Many types of nociceptors for many types of pain primary sensory nociceptors. These features illustrate the
Pain is unique among sensory modalities in that electrophysi- difficulty of defining a nociceptor based only on activation
ological recordings of single primary sensory fibres have been threshold or on whether its activation evokes pain.
made in awake humans, allowing simultaneous measure-
ment of psychophysical responses when regions of the head The neurochemistry of nociceptors
and body are stimulated3. Fibres that innervate regions of the Glutamate is the predominant excitatory neurotransmitter in
head and body arise from cell bodies in trigeminal and dorsal all nociceptors. Histochemical studies of adult DRG, however,
NATURE | VOL 413 | 13 SEPTEMBER 2001 | www.nature.com 203
© 2001 Macmillan Magazines Ltd
a b
Primary afferent axons Aα β
Thermal threshold
Aα and Aβ fibres
Voltage
Myelinated
Large diameter None
Proprioception, light touch
Aδ C
Aδ Fibre
Lightly myelinated Time
Medium diameter ~ 53 °C Type I
Nociception
(mechanical, thermal, chemical) ~ 43 °C Type II First pain
C fibre Second pain

Unmyelinated
Small diameter
Innocuous temperature, itch ~ 43 °C
Nociception
(mechanical, thermal, chemical)
Figure 1 Different nociceptors detect different types of pain. a, Peripheral nerves action potential recording from a peripheral nerve. Most nociceptors are either A or C
include small-diameter (A) and medium- to large-diameter (A,) myelinated afferent fibres, and their different conduction velocities (6–25 and ~1.0 m s–1, respectively)
fibres, as well as small-diameter unmyelinated afferent fibres (C). b, The fact that account for the first (fast) and second (slow) pain responses to injury. Panel b adapted
conduction velocity is directly related to fibre diameter is highlighted in the compound from ref. 75.
reveal two broad classes of unmyelinated C fibre. The so-called pep- spinal cord ‘pain’ transmission neurons (central sensitization), or
tidergic population contains the peptide neurotransmitter substance lowering of nociceptor activation thresholds (peripheral sensitiza-
P, and expresses TrkA, the high-affinity tyrosine kinase receptor for tion). With central sensitization, pain can be produced by activity in
nerve growth factor (NGF)9. The second population does not express non-nociceptive primary sensory fibres. Peripheral sensitization is
substance P or TrkA, but can be labelled selectively with the -D-galac- produced when nociceptor terminals become exposed to products of
tosyl-binding lectin IB4, and expresses P2X3 receptors, a specific tissue damage and inflammation, referred to collectively as the
subtype of ATP-gated ion channel. This categorization is a first approx- ‘inflammatory soup’ (Fig. 3). Such products include extracellular
imation at best — as additional molecular markers become available, protons, arachidonic acid and other lipid metabolites, serotonin,
new subsets are likely to be recognized. It is, however, unclear whether bradykinin, nucleotides and NGF, all of which interact with receptors
these neurochemically distinct groups represent different functional or ion channels on sensory nerve endings. Because nociceptors can
classes of nociceptor. Moreover, because expression of neurotransmit- release peptides and neurotransmitters (for example, substance P,
ters, their receptors and other signalling molecules are dramatically calcitonin-gene-related peptide and ATP) from their peripheral
altered after tissue or nerve injury10,11, both the significance and the terminals when activated by noxious stimuli, they are able to facilitate
complexity of nociceptor neurochemistry are increased. production of the inflammatory soup by promoting the release of
factors from neighbouring non-neuronal cells and vascular tissue, a
Diversity of nociceptor signalling phenomenon known as neurogenic inflammation5.
All sensory systems must convert environmental stimuli into electro- In contrast to vision, olfaction or taste, sensory nerve endings that
chemical signals. In the case of vision or olfaction, primary sensory detect painful stimuli are not localized to a particular anatomical
neurons need only detect one type of stimulus (light or chemical odor- structure, but are instead dispersed over the body, innervating skin,
ants) and use redundant and convergent biochemical mechanisms to muscle, joints and internal organs. Although this has made the bio-
accomplish this goal (Fig. 2a). In this regard, nociception is unique chemical analysis of nociceptive pathways particularly challenging,
because individual primary sensory neurons of the ‘pain pathway’ have the combined application of electrophysiological, pharmacological
the remarkable ability to detect a wide range of stimulus modalities, and genetic methods are generating significant progress in
including those of a physical and chemical nature. Compared with understanding the molecular basis of nociceptor signalling. In some
sensory neurons of other systems, nociceptors must therefore be respects, the field can be compared to the state of cellular immunolo-
equipped with a diverse repertoire of transduction devices (Fig. 2b). At gy some 25 years ago, when one of the main goals was to define
the same time, markedly different stimuli of a chemical (capsaicin and biochemical markers for specific subsets of lymphocytes. The goal for
acid) or physical (heat) variety can excite nociceptors by activating a nociceptor research is to elucidate signalling functions for key
single receptor, enabling the cell to integrate information and respond cell-surface markers and to assign physiological roles to molecularly
to complex changes in the physiological environment. defined sub-populations of sensory neurons.
Primary afferent nociceptors are also unique in the extent to
which their receptive properties can be modulated. Thus, nocicep- Detectors of noxious stimuli
tors not only signal acute pain, but also contribute to persistent and Response to heat
pathological pain conditions (allodynia) that occur in the setting of Remarkably, many functional characteristics of nociceptors are
injury, wherein pain is produced by innocuous stimuli5,12. Allodynia retained when sensory ganglia are dissociated and placed into cul-
can result from two different conditions: increased responsiveness of ture13. Thus, ~45% of small- to medium-diameter neurons exhibit
204 NATURE | VOL 413 | 13 SEPTEMBER 2001 | www.nature.com
Figure 2 Polymodal nociceptors use a greater diversity of a

signal-transduction mechanisms to detect physiological
Light Odorants
stimuli than do primary sensory neurons in other systems.
a, In mammals, light or odorants are detected by a
convergent signalling pathway in which G-protein-coupled
receptors modulate the production of cyclic nucleotide
second messengers, which then alter sensory neuron
excitability by regulating the activity of a single type of
cation channel. b, In contrast, nociceptors use different cGMP cAMP
signal-transduction mechanisms to detect physical and
chemical stimuli. Recent studies suggest that TRP-
b
channel family members (VR1 and VRL-1) detect noxious
heat, and that ENaC/DEG-channel family detect
mechanical stimuli. Molecular transducers for noxious
cold remain enigmatic. Noxious chemicals, such as ?
capsaicin or acid (that is, extracellular protons) may be
detected through a common transducer (VR1), illustrating
aspects of redundancy in nociception. At the same time, a A
N A N
single type of stimulus can interact with multiple detectors,
C C C
as shown by the ability of extracellular protons to activate A N
not only VR1, but also ASICs, which are also members of
the ENaC/DEG-channel family.
heat-evoked membrane currents with a ‘moderate’ threshold of and C fibres, but exhibited markedly reduced pain behaviour at
~45 C, whereas another 5–10% of cells respond with a ‘high’ higher temperatures (>50 C). In other words, VR1–/– animals recog-
threshold of ~52 C and are insensitive to capsaicin14,15. The former nize a noxious heat stimulus, but discriminate poorly among stimuli
corresponds presumably to C and type II A nociceptors, and the of different noxious intensities. Perhaps the threshold for a behav-
latter to type I A nociceptors. What is the molecular basis by which ioural pain response is determined by the thermal threshold of a
specific thermal thresholds are established in these nociceptor sub- small number of nociceptors, whereas discrimination among
types? The answer came from a well-proven path of success in the pain suprathreshold temperatures requires information from a larger
field, namely identifying the molecular targets through which natural cohort of nociceptors that adequately encode stimulus intensity. In
products (for example, morphine from the opium poppy or aspirin some respects, the phenotype of the VR1–/– mouse illustrates the
from willow bark) produce or modulate our sensation of pain. futility of a long-lasting debate as to whether pain is generated by
In the case of moderate thermal nociception by C and type II A activity in specific nociceptors that are connected to the spinal cord
afferents, a transducer was revealed with the cloning and functional through a dedicated pathway, or whether it is the pattern of activity
characterization of the vanilloid receptor VR1 (Fig. 2b), which is across afferents that determines the pain response. Both models are
activated by capsaicin and other vanilloid compounds16. VR1 is a likely to be relevant, depending on the magnitude of the stimulus and
non-selective plasma-membrane cation channel possessing a very steep the context in which it is measured. Moreover, because most nocicep-
temperature dependence (Q1020.6) and a thermal activation thresh- tors are polymodal, our ability to distinguish pain sensations
old of ~43 C, characteristics that are shared with native heat-evoked resulting from heat, cold or pressure must involve decoding of
currents in sensory neurons17,18. The strong correlation between nociceptive signals within the central nervous system.
moderate heat and capsaicin sensitivity, and the similarity of the Another striking and significant phenotype of the VR1–/– mouse is
non-selective cationic currents and pharmacology underlying these its failure to develop increased sensitivity to heat in the context of
responses14,15 support the hypothesis that heat and capsaicin activate a tissue injury20,21. This deficit suggests that VR1 is modulated by one or
common transducer. Heat-evoked single-channel currents are more components of the inflammatory soup, which act on the
observed in membrane patches excised from sensory neurons or VR1- nociceptor to increase its sensitivity to heat.
expressing cells, indicating that VR1 is an intrinsically heat-sensitive What mediates high-threshold heat responses in type I A affer-
channel that functions as a molecular thermometer at the cell surface17. ents? One candidate transducer is the vanilloid receptor-like
The high correlation that exists between heat and capsaicin (VRL-1) channel (Fig. 2b) that shares ~50% sequence identity with
sensitivity in sensory neurons at the whole-cell level is less pronounced VR1 (ref. 22). Functional analysis of VRL-1 in transfected
at the single-channel level19. Although this observation could be mammalian cells and frog oocytes showed that this channel is not
explained by different functional states of the channel, it has raised responsive to vanilloid compounds, but can be activated by noxious
some controversy regarding the link between the activities of native thermal stimuli with a threshold of ~52 C. VRL-1 is most
and cloned channels. But studies of mice lacking functional VR1 chan- prominently expressed by a subset of medium- to large-diameter
nels20,21 clearly show that cultured DRG neurons from VR1-null mice myelinated neurons within the DRG. But because VRL-1 transcripts
are severely deficient in moderate heat-evoked responses, whereas are also expressed outside the sensory nervous system, this channel
high-threshold heat responses persist. VR1–/– mice also have signifi- probably serves multiple physiological functions22,23.
cantly fewer (>threefold) heat-sensitive C fibres, and total heat-evoked VR1 and VRL-1 belong to the larger family of transient receptor
C-fibre output may be decreased by ~85% compared with wild-type potential (TRP) channels, whose core transmembrane structure
mice. Thus, although VR1 is not the only detector of moderate-thresh- resembles that of voltage-gated potassium or cyclic nucleotide-gated
old heat stimuli, it accounts for the majority of such responses and channels (reviewed in ref. 24). The prototypical TRP channel was
must contribute significantly to thermal coding in normal animals. discovered in the Drosophila phototransduction pathway, where it is
Somewhat paradoxically, VR1–/– mice showed normal behaviour- activated downstream of phospholipase C (PLC)-coupled rhodopsin
al responses at temperatures near the threshold for activation of VR1 (see review in this issue by Hardie and Raghu, pages 186–193). Some
mammalian TRP channels are also activated by G-protein-coupled One way to detect pressure or tissue deformation is through acti-
or tyrosine kinase receptors that stimulate PLC, but as in the fly, the vation of a mechanically gated protein. Another mechanism might
underlying gating mechanism remains enigmatic. Recent studies involve a ‘mechanochemical’ process whereby stretch evokes the
indicate that PLC-mediated hydrolysis of the membrane phospho- release of a diffusible chemical messenger that then excites nearby
lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and primary sensory nerve terminals. Extracellular ATP is of particular
the consequent production of lipid second messengers constitute interest because large- and small-diameter sensory neurons express
important steps in TRP channel activation25 (see below). G-protein-coupled ATP receptors or ATP-gated ion channels (P2Y
and P2X receptors, respectively), and because extracellular ATP
Detection of noxious cold excites primary sensory neurons33. Using frog oocytes as a model
The definition of cold sensitivity is less stringent compared with heat system, Nakamura and Strittmatter34 showed that mechanical stimu-
sensitivity, in large part because thermal thresholds for activation of lation can release ATP from the cell, promoting autocrine activation
cold-sensitive fibres may not be as distinct or precipitous as they are of P2Yreceptors on the cell’s surface. In vivo, mechanical force might
with heat, and because the threshold for cold-evoked pain is not as therefore promote ATP release from one or more cell types in the
precise. Whereas noxious heat (47 C) activates ~50% of C fibres that periphery, where it could activate purinergic receptors (for example,
innervate the hindpaw of a rodent, noxious cold (4 C) may excite P2X3) on nearby nociceptor terminals.
only 10–15% of fibres within the same cutaneous receptive field20. In fact, P2X3-deficient mice show reduced urination stemming
However, a significantly greater proportion of C and A fibres are from a failure to empty a full bladder35. Filling and stretching of the
classified as cold-sensitive when the range of stimulus intensities urinary bladder promotes the release of ATP from epithelial cells that
extends below 0 C (ref. 26). Few, if any, cold-sensitive (4 C) afferents lie beneath the smooth muscle layer. Once released, ATP may excite
are heat sensitive, and VR1–/– mice show a normal prevalence of sensory nerve endings embedded within the bladder wall to initiate
cold-responsive fibres in the hindpaw20, indicating that noxious heat the voiding reflex. The deficit observed in P2X3–/– mice provides
and cold are detected by distinct mechanisms. It is not known compelling evidence that ATP transduces signals of mechanical
whether noxious cold depolarizes nerve fibres by inhibiting a tissue distension into depolarization of primary sensory neurons, in
(Na+K+)ATPase or background potassium current27, or by pro- this case through direct activation of an ion channel.
moting calcium and/or sodium influx28,29.
Chemical transducers to make the pain worse
Pressure and mechanical stress As described above, injury heightens our pain experience by increas-
Nociceptors can be activated by mechanical stress resulting from ing the sensitivity of nociceptors to both thermal and mechanical
direct pressure, tissue deformation or changes in osmolarity, stimuli. This phenomenon results, in part, from the production and
enabling the detection of touch, deep pressure, distension of a viscer- release of chemical mediators from the primary sensory terminal and
al organ, destruction of bone, or swelling. Functional, mechanically from non-neural cells (for example, fibroblasts, mast cells,
gated channels have yet to be identified at the molecular level in neutrophils and platelets) in the environment36 (Fig. 3). Some com-
eukaryotes, although model genetic organisms such as bacteria, ponents of the inflammatory soup (for example, protons, ATP,
worms and flies have provided important leads for identifying serotonin or lipids) can alter neuronal excitability directly by inter-
mechanosensory transducers in mammals (see review in this issue by acting with ion channels on the nociceptor surface, whereas others
Gillespie and Walker, pages 194–202). One such ion channel of the (for example, bradykinin and NGF) bind to metabotropic receptors
degenerin (DEG/ENaC) family, called MDEG (alternatively, brain and mediate their effects through second-messenger signalling
sodium channel 1 (BNC1) or acid-sensing ion channel 2 (ASIC2); cascades11. Considerable progress has been made in understanding
Fig. 2b)30, has attracted particular interest in the pain field because its the biochemical basis of such modulatory mechanisms.
messenger RNA is expressed in primary sensory neurons31. Respons-
es of primary sensory fibres from BNC1-deficient mice to a range of Extracellular protons and tissue acidosis
mechanical stimuli identified a specific class of rapidly adapting Local tissue acidosis is a hallmark physiological response to injury,
mechanoreceptors that showed reduced sensitivity to hair move- and the degree of associated pain or discomfort is well correlated with
ment32. Other types of afferents, including C fibres, showed normal the magnitude of acidifica-
responses in these mutant mice, indicating that BNC1 is involved in tion37. Application of acid Representative
Stimulus
some aspects of innocuous mechanical (touch) sensation, but not in (pH 5) to the skin produces receptor
the detection of noxious mechanical stimuli. sustained discharges in a third NGF TrkA
Bradykinin BK2
Serotonin 5-HT3
Mast cell or ATP P2X3
Figure 3 The molecular complexity of the primary afferent nociceptor is illustrated H+ ASIC3/VR1
neutrophil
by its response to inflammatory mediators released at the site of tissue injury. Lipids PGE2/CB1/VR1
Some of the main components of the ‘inflammatory soup’ are shown, including Heat VR1/VRL-1
Substance Pressure
peptides (bradykinin), lipids (prostaglandins), neurotransmitters (serotonin (5-HT) P DEG/ENaC ?
and ATP) and neurotrophins (NGF). The acidic nature of the inflammatory soup is
Histamine DRG cell body
also indicated. Each of these factors sensitize (lower the threshold) or excite the Bradykinin NGF
terminals of the nociceptor by interacting with cell-surface receptors expressed by Tissue 5-HT
these neurons. Examples of these factors and representative molecular targets injury
Prostaglandin
are indicated in the box. Activation of the nociceptor not only transmits afferent ATP H+
messages to the spinal cord dorsal horn (and from there to the brain), but also CGRP
Substance P
initiates the process of neurogenic inflammation. This is an efferent function of the
nociceptor whereby release of neurotransmitters, notably substance P and Blood
calcitonin gene related peptide (CGRP), from the peripheral terminal induces vessel
vasodilation and plasma extravasation (leakage of proteins and fluid from
postcapillary venules), as well as activation of many non-neuronal cells, including Spinal cord
mast cells and neutrophils. These cells in turn contribute additional elements to
the inflammatory soup. Figure adapted from refs 75,76.
206 © 2001 Macmillan Magazines Ltd NATURE | VOL 413 | 13 SEPTEMBER 2001 | www.nature.com
Figure 4 When nociceptors are exposed to Ca2+

Na+
products of injury and inflammation, their BK
VR1
excitability is altered by a variety of intracellular TrkA H+
signalling pathways. The figure highlights the
vanilloid receptor (VR1) and tetrodotoxin-resistant AEA
(TTX-R) voltage-gated sodium channels (Nav1.8
and 1.9) as downstream targets of modulation. PLC-γ
(+)
Responses of VR1 to heat can be potentiated by PLC-β Na+
Gq
direct interaction of the channel with extracellular PKC-ε TTX-R
(+)
protons (H+) or lipid metabolites, such as PKA (Nav1.8
anandamide (AEA). VR1 activity can also be and 1.9)
(-) AC (+)
heightened by agents such as NGF or bradykinin, Gi Gs
which bind to their own cell-surface receptors (TrkA
and BK, respectively) to stimulate phospholipase C
(PLC- or PLC-) signalling pathways. This, in
turn, leads to hydrolysis of plasma membrane lipids AEA PGE2
and the subsequent stimulation of protein kinase C
isoforms, such as PKC-
. Both of these actions CB (or opiate) PG
have been proposed to potentiate VR1 function.
Prostaglandins (PGE2) and other inflammatory products that activate adenylyl cyclase (AC) through Gs-coupled receptors also enhance nociceptor excitability. This occurs, in part, by
a cyclic AMP-dependent protein kinase (PKA)-dependent phosphorylation of Nav1.8 and/or Nav1.9. By activating Gi-coupled receptors, opiates and cannabinoids can counteract
these increases in excitability of the nociceptor, and produce a peripherally mediated analgesia.
or more of polymodal nociceptors that innervate the receptive field20. do indeed show a marked reduction in sustained proton (pH 5)-
At the cellular level, protons depolarize sensory neurons by directly evoked membrane currents. But proton-evoked responses, particu-
activating a non-selective cationic current38,39. In many DRG larly those of a transient nature, are not eliminated completely in
neurons, this response consists of a transient, rapidly inactivating VR1–/– mice and may be mediated by members of the ASIC channel
current that is carried predominantly by Na+ ions, followed by a family.
sustained non-selective cationic current. Responses of a sustained Lazdunski and colleagues44 described a family of two-transmem-
nature have been proposed to underlie persistent pain associated brane-domain proteins that are related to the putative mechanosen-
with tissue acidosis37, but this may not occur in all physiological sory DEG/ENaC channels. These novel cation-channel subunits
settings. For example, during cardiac ischaemia, DRG neurons that were named ASICs because of their ability to be gated by reductions
innervate the epicardium show very large, but transient, response to in extracellular pH when expressed in heterologous systems (Fig. 2b).
extracellular protons40. A number of proton-sensitive channels are Including splice variants, there are at least five ASIC subtypes (1a, 1b,
found on sensory neurons and thus an important goal has been to 2a, 2b and 3) in rats, each having a unique profile of pH sensitivity,
determine which, if any, of these molecules contributes to proton activation and desensitization rates, ionic permeability and tissue
sensitivity of nociceptors in vivo. Two main candidates have emerged distribution. Most subtypes are expressed in DRG, with ASIC1b and
— VR1 and a family of ASICs. ASIC3 (known also as ASIC- and DRASIC, respectively) showing
The similarity between native proton (pH 5)-evoked and exclusive or preferential expression within sensory ganglia. Can
capsaicin-evoked currents in dissociated DRG neurons41 is well ASIC channels account for any aspect of transient or sustained pro-
established, and low extracellular pH can augment responses of ton-evoked currents observed in DRG neurons? Heterologous
cultured DRG neurons to capsaicin42. These observations suggested expression of most ASIC subunits produces currents consisting of a
that protons and vanilloid compounds interact with the same ion- single transient phase, or one having a sustained component that is
channel complex on the nociceptor (perhaps providing a cellular Na+-selective or observed only at non-physiological proton
rationale for the culinary appeal of ‘hot’ and sour soup). Analysis of concentrations (pH 5). However, co-expression of ASIC3 with
the cloned vanilloid receptor in heterologous expression systems has ASIC2b (a splice variant of MDEG, also called MDEG2) generates a
substantiated these observations. Protons have two main effects on more native-like current containing a sustained component with
VR1 function17. First, VR1 can be activated at room temperature non-selective cation permeability30. McCleskey and colleagues40 have
when the extracellular pH drops below 6, producing currents that provided strong evidence that ASIC3/2b heteromeric channels,
resemble the sustained component of proton-evoked responses rather than VR1, underlie the unusually large and mostly transient
observed in sensory neurons. Second, protons potentiate responses proton-evoked currents observed in the relatively small subpopula-
to capsaicin or heat, and do so over a concentration range (pH 6–8) tion (2–3%) of DRG afferents that innervate the heart. This makes
that matches the extent of local acidosis associated with various good physiological sense because occlusion of a cardiac artery
forms of tissue injury. These changes in VR1 activity would be produces modest acidosis (to just below pH 7), but this is sufficient to
expected to increase nociceptor excitability, even at normal body activate cardiac nociceptors or ASIC3/2b.
temperature. Structure–function studies have now identified several By contrast, genetic studies indicate that MDEG (BNC1) gene
negatively charged residues within putative extracellular loops of products (ASIC2a and 2b) do not contribute to acid sensitivity in
VR1 that are important for mediating these effects18,43, supporting most non-cardiac nociceptors32. Thus, BNC1–/– mice showed no
the idea that protons interact directly with the vanilloid receptor to obvious decrement in pH 5-evoked responses among large- or
allosterically modulate channel function. small-diameter DRG neurons. Moreover, acid produced normal
Although proton-evoked changes in VR1 thermal sensitivity sustained discharges in cutaneous C fibres from these animals. Gene
closely resemble those exhibited by nociceptors during inflamma- knockouts of other members of this family will be needed to clarify
tion, does VR1 actually contribute to pH sensitivity of nociceptors in the contribution of ASICs to proton detection and nociceptor
vivo? DRG neurons or sensory nerve fibres from VR1-deficient mice sensitization at various physiological sites.
Peptides and growth factors pro-algesic agents that activate PLC. TRP channels in the fly eye exist
Tissue damage promotes the release or production of bioactive in a complex with other components of the phototransduction
peptides from non-neural cells and plasma proteins at the site of machinery (see review by Hardie and Raghu), an arrangement that
injury45. Principal among these is the nonapeptide bradykinin, influences sensitivity and kinetics of the signalling process57,58.
which, when applied to primary sensory nerve terminals or cultured Interestingly, VR1 forms a signalling complex with TrkA and PLC-
sensory neurons, produces immediate membrane depolarization as in heterologous systems50. Whether a similar signalling scaffold exists
well as sensitization to other noxious, or even innocuous stimuli46. in nociceptors remains to be determined, but phylogenetic compar-
Bradykinin activates G-protein-coupled (BK2) receptors on these isons suggest that this might well be the case.
cells to stimulate PLC-catalysed hydrolysis of PtdIns(4,5)P2,
consequently releasing Ca2+ from intracellular stores and activating Sensitization and activation by lipids
protein kinase C (PKC) (Fig. 4). Treatment of cultured DRG neurons The efficacy of non-steroidal anti-inflammatory agents, such as
with bradykinin augments heat-evoked currents in these cells47, an aspirin, is attributed generally to blockade of cyclooxygenase (COX)
effect that may be mediated through direct or indirect modification enzymes that convert arachidonic acid, a lipid messenger, into pro-
of VR1 by the
-isoform of PKC48 (Fig. 4). PKC-
-knockout mice inflammatory prostanoid products, notably prostaglandin E2
exhibit reduced thermal and mechanical hypersensitivity after (PGE2). Most studies indicate that PGE2 contributes to peripheral
treatment with adrenaline or acetic acid49, but effects of bradykinin sensitization by binding to G-protein-coupled receptors that
have not been reported. In any event, molecular validation of this increase levels of cyclic AMP within nociceptors45. However, it now
pathway will require biochemical proof that VR1 is phosphorylated seems likely that cyclooxygenase products are also present in the
in response to BK2-receptor activation and that mutation of one or spinal cord, where they could interact with receptors on the central
more phosphate-accepting residues abrogates channel modulation. terminals of nociceptors59. This idea has aroused great interest
Bradykinin might also heighten VR1 sensitivity through a PKC- because it argues that COX inhibitors may exert their pain-relieving
independent process50. In this mechanism, channel modulation effects by modulating nociception at both peripheral and central
occurs as a direct consequence of PLC-mediated PtdIns(4,5)P2 sites60.
hydrolysis, in effect releasing VR1 from PtdIns(4,5)P2-mediated Recent studies have provided important information on a likely
inhibition. Precedence for such a regulatory mechanism comes from molecular target through which PGE2 sensitizes primary sensory
studies of cyclic nucleotide-gated channels51 and G-protein-gated fibres. Nociceptors express a specific subclass of voltage-gated
inwardly rectifying potassium channels52,53. Moreover, as mentioned sodium channel that is resistant to blockade by tetrodotoxin61. These
above, several members of the TRP channel family are activated TTX-R Na+ channels are believed to contribute significantly to
downstream of PLC-coupled receptors, and recent evidence from action-potential firing rate and duration in small-diameter sensory
both vertebrate and invertebrate systems suggests that PtdIns(4,5)P2 neurons. Electrophysiological studies suggest that PGE2 increases
hydrolysis also is important in modulating the activity of these excitability of DRG neurons, in part by shifting the voltage depen-
channels24,25. Exogenously applied lipids, such as anandamide, dence of TTX-R Na+-channel activation in the hyperpolarizing
arachidonate or diacylglycerol, have been shown to activate VR1 (see direction. This reduces the extent of membrane depolarization
below) or other TRP channels (see review by Hardie and Raghu, needed to initiate an action potential and favours repetitive spiking.
pages 186–193), raising the possibility that these hydrophobic Pharmacological and biochemical studies indicate that PGE2-depen-
agonists exert their effects by displacing PtdIns(4,5)P2 or other dent modulation of the TTX-R Na+ current involves phosphoryla-
membrane lipids from an inhibitory site on the channel complex. tion of the channel protein by cAMP-dependent protein kinase A62–64
NGF is best known as a survival factor for embryonic neurons and (Fig. 4). It is not known which of the two known TTX-R Na+-channel
is essential for the development of all primary sensory nociceptors. subtypes within DRG (Nav1.8 or Nav1.9)65–67 are targeted by lipids,
But in the adult, NGF serves a very different function, being released and under what conditions this occurs. Behavioural analysis of
by mast cells, fibroblasts and other cell types at sites of injury and Nav1.8-deficient mice revealed modest deficits in acute sensation to
inflammation, where it acts on primary sensory nerve terminals to noxious stimuli68. These animals also showed a delayed onset of
promote thermal hypersensitivity54. Consistent with this action, inflammatory thermal hypersensitivity, but the maximal
Mendell and colleagues55 showed that exposure of cultured DRG response was equivalent to that of wild-type animals. Such observa-
neurons to NGF for several minutes produces acute sensitization of tions support the involvement of Nav1.8 in tissue injury-evoked
capsaicin-evoked responses. Although NGF elicits long-term hypersensitivity, but also suggest that there is redundancy among
changes in gene expression, its ability to promote short-term voltage-gated Na+ channel subtypes in nociceptor function.
nociceptor sensitization suggests that post-translational mecha- Capsaicin is a hydrophobic molecule that bears structural
nisms also are involved. similarity to several lipid second messengers (Fig. 5), leading many to
NGF binds to TrkA tyrosine kinase receptors on peptidergic sen- suggest that lipid metabolites might serve as endogenous vanilloid-
sory neurons to activate mitogen-activated protein (MAP) kinase receptor agonists. The hunt for such ligands has been further
and PLC- signalling pathways56. When frog oocytes expressing TrkA advanced by the realization that the vanilloid receptor belongs to the
and VR1 are treated with NGF for several minutes, proton-, vanil- TRP channel family, some members of which can be activated by
loid- or heat-evoked responses are potentiated, recapitulating the polyunsaturated fatty acids or other lipid metabolites (see review by
sensitization that occurs in cultured DRG neurons or primary senso- Hardie and Raghu, pages 186–193). Indeed, we and others have
ry fibres. A TrkA mutant that specifically disrupts coupling of the shown that the endogenous cannabinoid-receptor agonist anan-
receptor to PLC- abrogates NGF enhancement of VR1, consistent damide (arachidonylethanolamide), or lipoxygenase products of
with the potentiation mechanism outlined for bradykinin50. The arachidonic-acid metabolism (for example, 12- and 15-(S)-
importance of the MAP-kinase pathway in the regulation of gene hydroperoxyeicosatetraenoic acid), activate native or cloned vanil-
expression is well appreciated, but the physiological significance of loid receptors when applied to whole cells or excised membrane
PLC activation to neurotrophin action has been enigmatic. These patches containing VR1 channels69–71. These lipid messengers are
findings now suggest that PLC signalling contributes to NGF-medi- admittedly weak as VR1 agonists (EC50 ~ 1–10 M at 25 C, pH 7.6),
ated thermal hypersensitivity and possibly to other forms of at least when compared to capsaicin, and this has fuelled some debate
short-term, post-translational nociceptor modulation (Fig. 4). as to whether they should be considered ‘endovanilloids’72. But the
VR1–/– mice do not develop thermal hypersensitivity in response to physiological setting in which these molecules are likely to act as VR1
NGF or bradykinin treatment50, indicating that this channel is a agonists is one involving inflammation, and thus the relevant
probable downstream target for modulation by these and other question should be whether they act synergistically with other
nociceptor. Functional presynaptic purinergic receptors have also
Capsaicin been described and the source of ATP can be identified73. By contrast,
O presynaptic vanilloid receptors will never be exposed to noxious
H 3CO
N
thermal temperatures or to the magnitude of pH changes that
H regulate VR1 gating at the peripheral terminal of the nociceptor, and
HO thus other ligands must be considered. Lipid mediators, such as
Olvanil
anandamide, may be relevant. There is evidence for presynaptic reg-
O ulation of neurotransmitter release through an action of anandamide
H3CO
N at both CB1 cannabinoid and vanilloid receptors in the dorsal horn74.
H Similarly, the function of central ASICs is unclear, nor is it known
HO whether novel endogenous ligands for these channels exist.
AM404
Future directions
HO
O One of the main challenges is to understand how both the specific
physiological properties of nociceptors and the circuits that they
N
H engage in the central nervous system determine pain perception and
resultant behaviour. Molecular markers make it possible to identify
Anandamide and manipulate the activity of subsets of nociceptors, so facilitating
O the mapping of spinal cord and brainstem circuits that are engaged by
HO
N
specific nociceptor populations. It is important to understand the
H function of the many neurotransmitters, receptors and transducers
that are expressed by nociceptors and the significance of their tran-
15-HPETE O
scriptional and post-translational regulation in the setting of injury.
Although opioids and non-steroidal anti-inflammatory agents are
HO analgesic drugs of choice for the treatment of pain, their utility is
often limited by unacceptable side-effects due to actions at identical
HOO receptors outside of the pain pathway. Because many of the channels
and receptors described in this review seem to be unique to the
Figure 5 An alignment of natural and synthetic vanilloid receptor agonists illustrates nociceptor (for example, TTX-R Na+ channels, P2X3 and VR1), they
their structural similarity. Olvanil is a synthetic, non-pungent capsaicin analogue that represent promising targets for the development of new and highly
activates VR1 with relatively slow kinetics. Anandamide is an endogenous lipid selective local anaesthetics and analgesics for treating a wide variety
metabolite (similar in structure to arachidonic acid) that was initially discovered as a of persistent pain conditions. ■
ligand for cannabinoid receptors. AM404 is a synthetic drug that blocks cellular re- 1. Sherrington, C. S. The Integrative Action of the Nervous System (Scribner, New York, 1906).
uptake of anandamide. Both compounds activate native and cloned vanilloid 2. Burgess, P. R. & Perl, E. R. Myelinated afferent fibres responding specifically to noxious stimulation of
receptors in vitro with relatively slow kinetics, similar to olvanil. 15-HPETE and other the skin. J. Physiol. 190, 541–562 (1967).
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Mini-Review
The regulatory role of nitric oxide in

proinflammatory cytokine expression during
the induction and resolution of inflammation
Yoshiro Kobayashi1
Division of Molecular Medicine, Department of Biomolecular Science, Faculty of Science, Toho University, Miyama,
Funabashi, Japan
RECEIVED MARCH 16, 2010; REVISED AUGUST 17, 2010; ACCEPTED AUGUST 17, 2010. DOI: 10.1189/jlb.0310149
ABSTRACT cells [1]. The production of chemokines and cytokines is also

Upon inflammation, neutrophils and subsequently subject to negative or positive regulation by a variety of prod-
monocytes infiltrate into the involved site. Neutrophils ucts, such as NO and PGs. Consequently, the regulation of
perform functions such as bacterial killing or tissue de- neutrophil infiltration has been investigated intensively with
struction and then undergo apoptosis, whereas mono- the hope of developing therapeutics for inflammatory diseases.
cytes differentiate into macrophages at the site. Mac- After infiltration, neutrophils then undergo apoptosis,
rophages and other phagocytes finally clear apoptotic whereas monocytes differentiate into macrophages. The mac-
neutrophils, leading to resolution of the inflammation.
rophages phagocytose apoptotic neutrophils, contributing to
One of the key steps during inflammation is leukocyte
resolution of the inflammation. Therefore, much interest has
infiltration, which is controlled chiefly by chemokines for
neutrophils and monocytes. The production of these
centered on the mechanism by which the macrophage re-
chemokines is regulated positively or negatively by sponse is regulated upon clearance of apoptotic neutrophils.
iNOS-derived NO. Although the mechanisms underlying Apoptotic cells inhibit the inflammatory responses of phago-
such dual effects of NO remain unknown, the level of cytes, such as production of proinflammatory cytokines. Al-
NO and duration of NO exposure appear to be deter- though this has been considered to be an active process medi-
mining factors. The clearance of apoptotic neutrophils ated by the production of TGF-␤, IL-10, PGE2, and NO [2, 3],
without causing further proinflammatory responses, on its significance in vivo has not been fully elucidated, except
the other hand, is another key event during inflamma- perhaps for the case of NO [4].
tion. The production of proinflammatory cytokines ap-
NO is produced by three different forms of NOS, namely
pears to be actively suppressed by TGF-␤ and NO,
nNOS (or NOS1), eNOS (or NOS3), and iNOS (or NOS2).
which are produced by phagocytes upon interaction
with apoptotic cells. Overall, NO plays a critical role The former two are constitutive forms and exist as preformed
during inflammation and therefore, remains a potential proteins that produce a small amount of NO upon the eleva-
target for developing therapeutics for inflammatory tion of intracellular calcium concentration, whereas the third
diseases. J. Leukoc. Biol. 88: 1157–1162; 2010. can produce a large amount of NO via de novo synthesis of
NOS protein. Because of the huge difference in the amount of
NO produced, it has been generally assumed that nNOS and
eNOS are critical for a normal physiology, whereas iNOS is
INTRODUCTION
associated with injury. In support of this assumption, iNOS-KO
The hallmark of inflammation is neutrophil and subsequent mice were less susceptible to DSS-induced intestinal injury
monocyte infiltration into the involved site. Their infiltration is than nNOS-KO, eNOS-KO, or WT mice [5].
chiefly governed by various chemokines, such as MIP-2, KC, Compared with nNOS and eNOS, iNOS is expressed in
and MCP-1 in mouse. These chemokines are produced by a many cell types, including macrophages, neutrophils, DCs, en-
variety of cells, including macrophages, neutrophils, endothe- dothelial cells, and epithelial cells [6], raising the possibility
lial cells, and epithelial cells in response to exogenous stimuli, that the effect of iNOS-derived NO may depend on the cell
such as bacteria and fungi, as well as endogenous stimuli, such type. For instance, iNOS-derived NO plays a physiological role
as damage-associated molecular patterns released from dead in cardiac myocytes from endotoxemic mice and is essential
for the cell-shortening response to ␤-adrenergic stimulation,
whereas iNOS-derived NO plays a detrimental role in neutro-
Abbreviations: DSS⫽dextran sodium sulfate, eNOS⫽endothelial NOS,
IRAK⫽IL-1R-associated kinase, KC⫽keratinocyte-derived chemokine, 1. Correspondence: Division of Molecular Medicine, Department of Biomo-
KO⫽knockout, nNOS⫽neuronal NOS, SNAP⫽S-nitroso-N-acetyl-D, L-peni- lecular Science, Faculty of Science, Toho University, 2-2-1, Miyama, Fun-
cillamine abashi 274-8510, Japan. E-mail: yoshiro@biomol.sci.toho-u.ac.jp
0741-5400/10/0088-1157 © Society for Leukocyte Biology Volume 88, December 2010 Journal of Leukocyte Biology 1157
phils and causes a decrease in cell shortening of cardiac myo- cals [13]. It should be noted, however, that another study
cytes [7]. about DSS-induced colitis found that bone marrow-derived,
Whether human macrophages express, produce, or use NO CX3CR1-expressing macrophages were crucial in DSS-induced
in their host defense functions remains a matter of debate, but colitis and that they were a major source of iNOS [14]. The
readers should refer to a recent review [8]. According to this cause for such contradiction is not known but may relate to
review, human macrophages also express iNOS in vivo, espe- the different strains used (C57BL/6 in ref. [12] vs. BALB/c in
cially in disease states and chronic inflammatory processes, ref. [14]).
and it is likely that this NO is functionally relevant to host de- Upon airway administration of LPS, an inflammatory re-
fense. sponse is induced in the lung as a result of activation of
In this review, therefore, I will focus on the role of NO in phagocytic cells, which produce a variety of chemokines. When
cytokine expression during the induction and resolution of inflammatory injury in the lungs was induced by intratracheal
inflammation. instillation of LPS, the injury and MPO activity were increased
substantially in iNOS-KO mice [15]. The MIP-2 and KC levels
in BAL fluids of iNOS-KO mice, however, were similar to those
of WT mice. In contrast, the MCP-1 levels were enhanced in
iNOS-KO mice as compared with WT mice. Moreover, admin-
THE ROLE OF NO IN istration of anti-MCP-1 antibodies to iNOS-KO mice reduced
PROINFLAMMATORY CYTOKINE neutrophil infiltration to the level in WT mice. Consistent with
EXPRESSION DURING THE INDUCTION the increase in injury in iNOS-KO mice, in vitro stimulation of
OF INFLAMMATION
microvascular endothelial cells with LPS and IFN-␥ elevated
The role of NO in inflammation has been studied by using the production of MCP-1 in cells from iNOS-KO mice when
iNOS-KO mice and NOS inhibitors in various types of inflam- compared with endothelial cells from WT mice. Peritoneal
mation models, such as DSS-induced colitis and LPS-induced macrophages from iNOS-KO mice also showed increased pro-
lung injury. The role of iNOS-derived NO in cytokine expres- duction of MCP-1 after stimulation with LPS and IFN-␥. There-
sion appears to differ among the models. fore it is conceivable that when a large amount of NO is pro-
As noted in the introduction, DSS-induced injury in the co- duced in the lungs upon intratracheal instillation of LPS, it
lon was reduced substantially in iNOS-KO mice as compared down-regulates MCP-1 production, neutrophil infiltration into
with WT mice [5], indicating that iNOS-derived NO is harmful lungs, and lung injury. Although MCP-1 is chemotactic for
in this model. As certain broad-spectrum antibiotics prevent monocytes, stimulated neutrophils change their chemokine
colonic injury [9], it has been assumed that DSS-induced epi- receptor expression pattern and become responsive to certain
thelial injury may cause translocation of normal colonic flora CC chemokines [16].
and subsequent leukocyte recruitment to the colon. Among In another study about zymosan-induced peritoneal inflam-
leukocytes, neutrophils are effector cells [5], and CXCR2, a mation, neutrophil infiltration at 1 h was enhanced in
receptor for neutrophil chemokines CXCL1/KC, CXCL2/3/ iNOS-KO mice in comparison with WT mice but suppressed at
MIP-2, and CXCL5/LIX, appears to be crucial in this model 2– 4 h in KO mice [17]. A notable finding was the parallel be-
[10]. Indeed one of the CXCR2 ligands, KC, was detected in tween the MIP-2 and KC levels and neutrophil infiltration in
the gut mucosa in this model [11]. To delineate the role of zymosan-induced peritoneal inflammation. This suggests that
iNOS in bone marrow-derived cells as well as non-bone mar- NO exerts a biphasic, regulatory effect on MIP-2 and KC,
row-derived ones, irradiated WT or iNOS-KO mice were trans- namely, a suppressive effect at 1 h versus an enhancing effect
planted with bone marrow-derived cells from WT or iNOS-KO at 2– 4 h. Knowledge of the NO level at each time-point and
mice, respectively [12]. Chimeric-irradiated mice with bone the producer cell type would facilitate understanding of the
marrow-derived cells of iNOS-KO mice were markedly more biphasic effect of NO.
resistant to DSS-induced injury, and this resistance was lost In addition to the above examples, there have been numer-
when irradiated iNOS-KO mice were given bone marrow-de- ous conflicting papers about the role of NO in the regulation
rived cells of WT mice. In this study, neutrophils and epithe- of cytokine expression (Table 1). In some cases, NO sup-
lial cells were the main source of iNOS in the diseased colon, presses the expression of cytokines, such as MCP-1, IL-8,
whereas macrophages exhibited minimal iNOS expression, as MIP-2, and CCL1 (I-309), whereas in other cases, it augments
evidenced by immunohistochemistry. Therefore, iNOS in neu- or induces the expression of cytokines, such as IL-8, MIP-1␣,
trophils appears to cause injury in the colon. Moreover, a par- MIP-2, and IFN-␤. One explanation for such contradictory ef-
allel was found between MPO activity and histology in this fects of NO on cytokine production may be that NO regulates
study, and MPO activity was lower in the colon of iNOS-KO NF-␬B in a biphasic manner. In a murine monocyte/macro-
mice than in that of WT ones, suggesting that iNOS-derived phage cell line, NO activated NF-␬B at early time-points after
NO enhances neutrophil infiltration. This study found a signif- LPS administration or with lower levels of NO, whereas it in-
icant decrease in the MIP-1␤ and TNF-␣ mRNA levels in hibited NF-␬B activation at later time-points or with higher
iNOS-KO mice, but it is possible that the levels of neutrophil doses [28]. We obtained similar data for murine resident peri-
chemokines such as KC are also reduced in intestinal epithe- toneal macrophages after coculture with apoptotic cells [3].
lial cells of iNOS-KO mice, as various NO donors significantly When a small amount of NO was produced by macrophages
induced IL-8 mRNA in lung epithelial cells via hydroxyl radi- after coculture with late apoptotic cells (secondary necrotic
1158 Journal of Leukocyte Biology Volume 88, December 2010 www.jleukbio.org

Kobayashi Role of NO in cytokine production
TABLE 1. Regulation of Cytokine Expression by NO
Cells Stimulants Products Effects of NO donors (⫹ or –)a Ref.
endothelial cells LPS ⫹ IFN-␥ MCP-1, MIP-2, KC –b [15]

PM LPS ⫹ IFN-␥ MCP-1b –b [15]
smooth muscle cells Oxidized LDL or LPS MCP-1 – [18]
endothelial cell line TNF-␣ IL-8 – [19]
HUVEC glycoxidized LDL MCP-1 – [20]
endothelial cells shear stress MCP-1 – [21]
endothelial cells cytokine MCP-1 – [22]
PM early apoptotic cells MIP-2 –c [3]
mast cell line IFN-␥, PMA IL-8, CCL1 –c [23]
PM late apoptotic cells MIP-2 ⫹c [3]
lung epithelial cells none IL-8 ⫹ [13]
monocytes LPS MIP-1␣d ⫹c [24]
lung fibroblasts hydroxylamine MIP-2, MCP-1 ⫹ [25]
glomerular mesangial cells IL-1␤ MIP-2 ⫹ [26]
RAW264.7 cells LPS IFN-␤ ⫹ [27]
PM, Resident peritoneal macrophages; RAW264.7, mouse macrophage cell line. a⫹ Denotes enhancement; – denotes suppression; bMIP-2 and
KC levels were not changed; cEffects of endogenously produced NO; dMCP-1 levels were not changed.
cells), the NOS inhibitor, N(G)-nitro-l-arginine methyl ester, idly cleared by macrophages, thereby contributing to resolu-
and the NO scavenger, 2-(4-carboxyphenyl)-4, 4, 5, 5-tetra- tion of the inflammation. This implies that early apoptotic
methylimidazoline-1-oxyl-3-oxide, each inhibited MIP-2 produc- cells inhibit the inflammatory responses of phagocytes, such as
tion, but when a large amount of NO was produced by macro- the production of proinflammatory cytokines. This has been
phages after coculture with early apoptotic cells, the NOS in- considered as an active process mediated by the production of
hibitor and the NO scavenger each augmented MIP-2 produc- TGF-␤, IL-10, and PGE2, as macrophages cocultured with early
tion through NF-␬B activation [3]. The other explanation may apoptotic cells produce these anti-inflammatory mediators that
be that the effect of NO on cytokine production depends on suppress the production of proinflammatory mediators in vitro
the type of responder cell, the cytokine, and the stimulant. For [2, 29].
instance, NO suppresses MCP-1 but not MIP-2 production by The coculture of phagocytes with early apoptotic cells, how-
peritoneal macrophages in response to LPS and IFN-␥ [18],
ever, yielded conflicting results concerning NO production
whereas NO augments IFN-␤ production by the RAW264.7
and/or iNOS expression in phagocytes (Table 2). Unless oth-
macrophage cell line in response to LPS [27].
erwise stated, early apoptotic cells were used in the studies be-
low.
Henson’s group [30] reported that apoptotic cells sup-
pressed the NO production and iNOS induction of macro-
REGULATION OF NO AND phages through TGF-␤. They stimulated macrophages with
PROINFLAMMATORY CYTOKINE LPS and IFN-␥ in the presence of apoptotic Jurkat cells and
PRODUCTION BY APOPTOTIC CELLS found that as well as producing arginase I, the macrophages
Apoptotic cells are rapidly cleared by phagocytes, such as mac- produced TGF-␤, which in turn, suppressed iNOS and TNF-␣
rophages and DCs, without causing inflammatory responses. induction and NO production. They used a mouse macro-
Likewise, upon inflammation, apoptotic neutrophils are rap- phage cell line (RAW264 cells) as well as thioglycollate broth-
TABLE 2. Induction or Suppression of iNOS by Apoptotic Cells
Apoptotic cells/phagocytes Stimulants iNOS expression Ref.
Jurkat/mo TG M␾, RAW264 LPS/IFN-␥ suppression [30]

lymphocytes/mo PM Coxiella burnetti suppressiona [31]
mo splenocytes/mo DC IFN-␥ induction [32]
CTLL-2, thymocytes, HL-60/mo PM none induction [3]
thymocytes/mo TM, mo PM none induction [4]
Jurkat/mo epithelial cells (HC-11) none induction (mRNA) [33]
Jurkat/RAW264.7 IFN-␥ no change [34]
a
Indirect evidence (see text). mo, Mouse; TG M␾, thioglycollate broth macrophage; DC, GM-CSF- and IL-4-induced mouse bone marrow-de-
rived DCs; TM, thymic macrophages obtained after whole body X-irradiation.
www.jleukbio.org Volume 88, December 2010 Journal of Leukocyte Biology 1159

elicited macrophages, the latter including inflammatory mono- than WT mice [4]. Thymic macrophages isolated from KO
cytes and monocyte-derived macrophages. They did not pro- mice also produced more MIP-2 and KC than those from WT
vide data concerning iNOS induction in macrophages by mice in coculture with apoptotic thymocytes.
apoptotic cells alone. Microarray analysis showed that when epithelial cells phago-
C. burnetti, the agent of Q fever in humans and coxiellosis in cytosed apoptotic cells, they induced TGF-␤2, VEGF-A, and
other mammals, resides in macrophages and induces NO, iNOS mRNA in coculture with apoptotic Jurkat cells [33].
which then suppresses the formation of large phagolysosome- Whether the same is true for macrophages, however, has not
like replicative vacuoles. Consistent with the findings of Hen- been reported except for secretion of VEGF.
son’s group [30], phagocytosis of apoptotic cells by macro- There has, however, only been one report that exposure of
phages reduced the NO production induced by the bacteria, IFN-␥-stimulated RAW264.7 cells to apoptotic cells suppressed
which is dependent on iNOS [31]. As the bacteria induce NO production but not iNOS expression [34]. Instead of sup-
apoptosis in human monocytes, reduction of NO production pression of iNOS, arginase II was increased in these cells,
by apoptotic cells may represent a counterattack by the bacte- which perhaps reduced the level of arginine. In this case, a
ria. lipid factor from apoptotic cells was found to be responsible
Contrary to the results in the above two papers, when my- for modulation of NO production.
eloid DCs were used as phagocytes, preincubation of DCs with Very recently, it was reported that a NO donor, SNAP, acti-
apoptotic cells augmented the response to IFN-␥ that leads to vates latent TGF-␤1 via activation of soluble guanylate cyclase
the production of NO through iNOS, and NO suppressed the and generation of cGMP in mouse RAW 264.7 cells [37]. The
proliferative response of T cells [32]. Although IL-10 was de- findings potentially link the suppressive activity of NO with
tected in the culture, neither anti-IL-10 antibodies nor anti- TGF-␤.
TGF-␤ antibodies restored the proliferative response of T cells. Although apoptotic human peripheral blood neutrophils
In the absence of IFN-␥, however, apoptotic cells failed to in- have been principally used to study the responses of human
duce NO production in DCs. In view of the findings reported macrophages following an encounter with apoptotic cells [38,
in ref. [32], it is intriguing that IFN-␥R-deficient mice were 39], it should be borne in mind that human peripheral blood
hypersensitive to the anti-CD3-induced cytokine release syn- neutrophils are at a resting stage, whereas infiltrating neutro-
drome and thymocyte apoptosis, in which much less NO was phils are activated. In contrast to resting neutrophils, activated
produced [35], as in this case, IFN-␥ seems to induce NO pro- neutrophils potentially secrete granular proteins, which may
duction presumably via iNOS, thereby suppressing cytokine modulate cytokine production [40]. The microenvironment in
production, thymocyte apoptosis, and pathologic manifesta- which apoptotic neutrophils are cleared also warrants atten-
tions. Therefore, upon induction of cytokine release syndrome tion. Although researchers have often used LPS and/or IFN-␥
by anti-CD3 antibody, it is likely that IFN-␥ augments the re- as stimulants, bacteria may be phagocytosed and killed, and
sponse of DCs to apoptotic cells, which leads to the produc- therefore, IFN-␥ rather than LPS may be an adequate stimu-
tion of NO. This possibility may be worthy of further study. lant for such a study.
We have shown previously that macrophages cocultured with
early apoptotic cells produce neither proinflammatory nor
anti-inflammatory cytokines [36] but that such macrophages MOLECULAR MECHANISM UNDERLYING
produce a large quantity of NO to suppress any inflammatory
THE SUPPRESSION OF
PROINFLAMMATORY CYTOKINE
responses such as MIP-2 production [3]. We further showed
EXPRESSION BY NO
that upon induction of apoptosis in the thymus by means of
X-rays, iNOS-KO mice exhibited higher levels of neutrophil Various intracellular signaling molecules are regulated by NO,
infiltration and production of MIP-2 and KC in the thymus including MAPK, JAK, NF-␬B, and AP-1 [41]. These kinases
A immunoregulation B effector function

Figure 1. The immunoregulatory effect of NO IFN-γγ or other stimulants IFN-γ or other stimulants
versus the effector function of NO. (A) When
cells such as macrophages are activated with
IFN-␥ or other stimulants, they produce NO via
iNOS. A small amount of NO may enhance the
production of chemokines, such as MIP-2 and
MCP-1, whereas a large amount of NO may sup-
presses it. (B) When cells such as neutrophils iNOS iNOS
iNOS
are activated with IFN-␥ or other stimulants,
they produce a large amount of NO via iNOS,
which has a toxic effect to target cells (effector NO (high)
(hi h) NO (low)
(l ) NO (high)
function).
MIP-2, MCP-1 MIP-2, MCP-1
Anti-inflammation Inflammation Tissue Destruction

Kobayashi Role of NO in cytokine production
and transcription factors do not contain transition metals with different nitric oxide synthase isoforms in colonic injury. Am. J. Physiol.
Gastrointest. Liver Physiol. 286, G137–G147.
which NO can interact directly but contain critical cysteine 6. Bogdan, C. (2001) Nitric oxide and the immune response. Nat. Immunol.
residues that undergo S-nitrosylation and denitrosylation. NO 2, 907–916.
7. Poon, B. Y., Raharjo, E., Patel, K. D., Tavener, S. A., Kubes, P. (2003)
donors, such as SNP and SNAP, directly inhibited the DNA- Complexity of inducible nitric oxide synthase: cellular source determines
binding activity of rNF-␬B p50 and p65 homodimers and p50- benefit versus toxicity. Circulation 108, 1107–1112.
8. Kobzik, L. (2009) Translating NO biology into clinical advances. Am. J.
p65 heterodimers. Inhibition of NF-␬B p50 DNA binding in- Respir. Cell Mol. Biol. 41, 9 –13.
volved S-nitrosylation of the C62 residue [42]. SNP also caused 9. Videla, S., Vilaseca, J., Guamer, F., Salas, A., Treserra, F., Crespo, E., An-
Tyr nitration of p65 but not p50. Y66 and Y152 nitration of tolin, M., Malagelada, J. R. (1994) Role of intestinal microflora in
chronic inflammation and ulceration of the rat colon. Gut 35, 1090 –
p65 induces its dissociation from p50, its association with 1097.
I␬B␣, and subsequent sequestration of p65 in the cytoplasm 10. Farooq, S. M., Stillie, R., Svenson, M., Svanborg, C., Strieter, R. M., Stad-
nyk, A. W. (2009) Therapeutic effect of blocking CXCR2 on neutrophil
[43]. The NO donor SNAP also indirectly reduces NF-␬B DNA recruitment and dextran sodium sulfate-induced colitis. J. Pharmacol. Exp.
binding. LPS induced IL-12 p40 mRNA expression via IRAK- Ther. 329, 123–129.
11. Buanne, P., Di Carlo, E., Caputi, L., Brandolini, L., Mosca, M., Cattani,
dependent NF-␬B activation, whereas the NO donor inhibited F., Pllegrini, L., Biordi, L., Coletti, G., Sorrentino, C., Fedele, G., Colotta,
IRAK activation, causing attenuation of its interaction with F., Melillo, G., Bertini, R. (2007) Crucial pathophysiological role of
CXCR2 in experimental ulcerative colitis in mice. J. Leukoc. Biol. 82,
TRAF-6 and subsequent NF-␬B activation [44]. 1239 –1246.
Recent evidence indicates that the HO-1/carbon monoxide 12. Beck, P. L., Li, Y., Wong, J., Chen, C. W., Keenan, C. M., Sharkey, K. A.,
McCafferty, D. M. (2007) Inducible nitric oxide synthase from bone mar-
pathway is a key player in NO-mediated anti-inflammation row-derived cells plays a critical role in regulating colonic inflammation.
[45]. NO reacts with the heme group of soluble guanylyl cy- Gastroenterology 132, 1778 –1790.
13. Sparkman, L., Boggaram, V. (2004) Nitric oxide increases IL-8 gene tran-
clase, leading to enhanced production of cGMP and HO-1 scription and mRNA stability to enhance IL-8 gene expression in lung
production and an increase in LPS-induced IL-10 production epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L764 –L773.
14. Kostadinova, F. I., Baba, T., Ishida, Y., Kondo, T., Popivanova, B. K., Mu-
[46]. It is unknown, however, whether this pathway is involved kaida, N. (2010) Crucial involvement of the CX3CR1-CX3CL1 axis in
in NO-mediated suppression of chemokine production during dextran sulfate sodium-mediated acute colitis in mice. J. Leukoc. Biol. 88,
inflammation. 133–143.
15. Speyer, C. L., Neff, T. A., Warner, R. L., Guo, R. F., Sarma, J. V., Riede-
mann, N. C., Murphy, M. E., Murphy, H. S., Ward, P. A. (2003) Regula-
tory effects of iNOS on acute lung inflammatory responses in mice.
Am. J. Pathol. 163, 2319 –2328.
16. Kobayashi, Y. (2008) The role of chemokines in neutrophil biology.
Front. Biosci. 13, 2400 –2407.
17. Ajuebor, M. N., Virag, L., Flower, R. J., Perretti, M., Szabo, C. (1998)
CONCLUDING REMARKS Role of inducible nitric oxide synthase in the regulation of neutrophil
migration in zymosan-induced inflammation. Immunology 95, 625– 630.
As iNOS produces a large amount of NO, it has been assumed 18. Tsao, P. S., Wang, B., Buitrago, R., Shyy, J. Y., Cooke, J. P. (1997) Nitric
oxide regulates monocyte chemotactic protein-1. Circulation 96, 934 –940.
that such NO is harmful and plays an effector function. This is 19. Natarajan, R., Gupta, S., Fisher, B. J., Ghosh, S., Fowler, A. A. (2001) Ni-
true of neutrophils (Fig. 1B) and perhaps other cells such as tric oxide suppresses IL-8 transcription by inhibiting c-Jun N-terminal ki-
nase-induced AP-1 activation. Exp. Cell Res. 266, 203–212.
macrophages. However, as described in this review, it is evi- 20. Sonoki, K., Yoshinari, M., Iwase, M., Iino, K., Ichikawa, K., Ohdo, S.,
dent that NO also plays an immunoregulatory role in the in- Higuchi, S., Iida, M. (2002) Glycoxidized low-density lipoprotein en-
hances monocyte chemoattractant protein-1 mRNA expression in human
duction and resolution of inflammation. The immunomoregu- umbilical vein endothelial cells: relation to lysophosphatidylcholine con-
latory effect of NO appears to be determined by the NO level. tents and inhibition by nitric oxide donor. Metabolism 51, 1135–1142.
21. Ni, C. W., Wang, D. L., Lien, S. C., Cheng, J. J., Chao, Y. J., Hsieh, H. J.
A small amount of NO may enhance the production of che- (2003) Activation of PKC-␧ and ERK1/2 participates in shear-induced
mokines, such as MIP-2 and MCP-1, possibly through activa- endothelial MCP-1 expression that is repressed by nitric oxide. J. Cell.
tion of NF-␬B, whereas a large amount of NO may suppress it, Physiol. 195, 428 – 434.
22. Desai, A., Miller, M. J., Huang, X., Warren, J. S. (2003) Nitric oxide mod-
possibly through inhibition of NF-␬B (Fig. 1A). Therefore, ulates MCP-1 expression in endothelial cells: implications for the patho-
measurement of the NO level and identification of the NO genesis of pulmonary granulomatous vasculitis. Inflammation 27, 213–223.
23. Gilchrist, M., Befus, A. D. (2008) Interferon-␥ regulates chemokine ex-
producer cells should facilitate understanding of the effect of pression and release in the human mast cell line HMC1: role of nitric
NO in vivo. oxide. Immunology 123, 209 –217.
24. Muhl, H., Dinarello, C. A. (1997) Macrophage inflammatory protein-1 ␣
production in lipopolysaccharide-stimulated human adherent blood
mononuclear cells is inhibited by the nitric oxide synthase inhibitor
N(G)-monomethyl-L-arginine. J. Immunol. 159, 5063–5069.
25. Trifilieff, A., Fujitani, Y., Mentz, F., Dugas, B., Fuentes, M., Bertrand, C.
(2000) Inducible nitric oxide synthase inhibitors suppress airway inflam-
mation in mice through down-regulation of chemokine expression. J. Im-
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tial effects of apoptotic versus lysed cells on macrophage production of KEY WORDS:
cytokines: role of proteases. J. Immunol. 166, 6847– 6854. iNOS 䡠 NO donor 䡠 NF-␬B

Review Articles
Dietary long-chain n⫺3 fatty acids for the prevention of cancer:

a review of potential mechanisms1–3
Susanna C Larsson, Maria Kumlin, Magnus Ingelman-Sundberg, and Alicja Wolk
ABSTRACT has been highlighted by animal experiments and in vitro studies

Increasing evidence from animal and in vitro studies indicates that showing that these PUFAs suppress the development of major
nҀ3 fatty acids, especially the long-chain polyunsaturated fatty ac- cancers (26 –31). These experimental findings are supported by
ids eicosapentaenoic acid and docosahexaenoic acid, present in fatty results from clinical studies showing a reduction in intestinal
fish and fish oils inhibit carcinogenesis. The epidemiologic data on hyperproliferation after consumption of fish oil– derived nҀ3
the association between fish consumption, as a surrogate marker for PUFAs in subjects at elevated risk of colon cancer due to sporadic
nҀ3 fatty acid intake, and cancer risk are, however, somewhat less colonic adenomas (32, 33). Although a few previous reviews
consistent. This review highlights current knowledge of the potential have described some selected actions through which long-chain
mechanisms of the anticarcinogenic actions of nҀ3 fatty acids. nҀ3 fatty acids may play a role in carcinogenesis, such as bio-
Downloaded from ajcn.nutrition.org by guest on June 24, 2013

Moreover, a possible explanation of why some epidemiologic stud- synthesis of eicosanoids (34, 35), lipid peroxidation (36 –38),
ies failed to find an association between nҀ3 fatty acid intake and and some signal transduction pathways (34, 36), to our knowl-
cancer risk is provided. Several molecular mechanisms whereby edge, no comprehensive review that puts all these pieces and
nҀ3 fatty acids may modify the carcinogenic process have been further evidence together is available.
proposed. These include suppression of arachidonic acid– derived The present review focuses on several putative mechanisms
eicosanoid biosynthesis; influences on transcription factor activity, whereby long-chain nҀ3 fatty acids may modulate the carcino-
gene expression, and signal transduction pathways; alteration of genic process. Furthermore, a potential explanation of why sev-
estrogen metabolism; increased or decreased production of free rad- eral case-control studies and large cohort studies failed to con-
icals and reactive oxygen species; and mechanisms involving insulin firm a protective effect of long-chain nҀ3 fatty acids against
sensitivity and membrane fluidity. Further studies are needed to cancer development is briefly discussed. Moreover, we discuss
evaluate and verify these mechanisms in humans to gain more un- how knowledge of the mechanisms of action of PUFAs should be
derstanding of the effects of nҀ3 fatty acid intake on cancer taken into account in epidemiologic analyses.
risk. Am J Clin Nutr 2004;79:935– 45.
KEY WORDS nҀ3 Fatty acids, eicosapentaenoic acid, doco- MECHANISMS OF POTENTIAL CHEMOPREVENTIVE
sahexaenoic acid, ␣-linolenic acid, arachidonic acid, carcinogene- EFFECTS OF nⴚ3 FATTY ACIDS ON
sis, eicosanoids, gene expression, epidemiology CARCINOGENESIS
Mounting evidence shows that dietary nҀ3 PUFAs inhibit the
promotion and progression stages of carcinogenesis. Several mo-
INTRODUCTION lecular mechanisms whereby nҀ3 PUFAs potentially affect car-
We recently reviewed epidemiologic studies on the relation cinogenesis have been proposed. These mechanisms include 1)
between intakes of fish and marine fatty acids and the risks of suppression of arachidonic acid (AA, 20:4nҀ6)– derived eico-
breast and prostate cancers and of other hormone-related cancers sanoid biosynthesis, which results in altered immune response to
(1). In brief, ecologic studies have shown that high per capita fish cancer cells and modulation of inflammation, cell proliferation,
consumption is correlated with a lower incidence of cancer in the apoptosis, metastasis, and angiogenesis; 2) influences on tran-
population (2–5). Additionally, the decreased consumption of scription factor activity, gene expression, and signal transduc-
fish and increased intake of vegetable oils rich in nҀ6 fatty acids tion, which leads to changes in metabolism, cell growth, and
among Japanese women during the past decades have been ac- differentiation; 3) alteration of estrogen metabolism, which leads
companied by increased breast cancer rates (6). Nevertheless,
analytic epidemiologic studies having a case-control or cohort 1
From the Divisions of Nutritional Epidemiology (SCL and AW), Exper-
design have not yielded clear conclusions concerning the pro- imental Asthma and Allergy Research (MK), and Molecular Toxicology
tective effect of fish consumption or nҀ3 fatty acid intake against (MI-S), The National Institute of Environmental Medicine, Karolinska In-
cancer; although some studies showed an inverse association stitutet, Stockholm.
2
between the intake of nҀ3 fatty acids (7, 8) or fish (9 –16) and Supported by grants from the Swedish Cancer Society (to AW).
3
Reprints not available. Address correspondence to SC Larsson, Division
cancer risk, most did not (17–25).
of Nutritional Epidemiology, The National Institute of Environmental Med-
The role that the long-chain, marine nҀ3 polyunsaturated
icine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden. E-
fatty acids (PUFAs) eicosapentaenoic acid (EPA, 20:5nҀ3) and mail: susanna.larsson@imm.ki.se.
docosahexaenoic acid (DHA, 22:6nҀ3), which are present in Received September 22, 2003.
fatty cold-water fish and fish oils, play in the etiology of cancer Accepted for publication December 2, 2003.
Am J Clin Nutr 2004;79:935– 45. Printed in USA. © 2004 American Society for Clinical Nutrition 935
936 LARSSON ET AL

FIGURE 1. Overview of the metabolism of nҀ6 and nҀ3 polyunsaturated fatty acids (PUFAs) into eicosanoids involved in inflammation and carcino-
genesis. The names of these eicosanoids are shown in bold. LA, linoleic acid (18:2nҀ6); ␣-LNA, ␣-linolenic acid (18:3nҀ3); GLA, ␥-linolenic acid (18:3nҀ6);
DGLA, dihomo-␥-linolenic acid (20:3nҀ6); AA, arachidonic acid (20:4nҀ6); EPA, eicosapentaenoic acid (20:5nҀ3); DHA, docosahexaenoic acid (22:
6nҀ3); PLA2, phospholipase A2; LOX, lipoxygenase; COXs, cyclooxygenases (COX-1 and COX-2); 15-HETE, 15(S)-hydroxyeicosatetraenoic acid;
12-HETE, 12-hydroxyeicosatetraenoic acid; 5-HETE, 5-hydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; HPETE, hydroperoxyeicosa-
tetraenoic acid; HPEPE, hydroperoxyeicosapentaenoic acid; LT, leukotriene; HODE, hydroxyoctadecadienoic acid; PG, prostaglandin; TX, thromboxane.
to reduced estrogen-stimulated cell growth; 4) increased or de- The cyclooxygenases give rise to prostaglandins and thrombox-
creased production of free radicals and reactive oxygen species; anes, whereas the lipoxygenases produce leukotrienes, hydroxy
and 5) mechanisms involving insulin sensitivity and membrane fatty acids, and lipoxins. Cytochrome P450 monooxygenase–
fluidity. mediated oxidation of PUFAs generates hydroxyfatty acids, di-
hydroxyfatty acids, and epoxy fatty acids. The relative propor-
Inhibition of arachidonic acid– derived eicosanoid tions of PUFAs in cell membranes, as well as cell type, are the
biosynthesis primary factors in regulating which eicosanoid will be generated.
One of the more important functions of PUFAs (nҀ3 and nҀ6 Hydrolytic release of PUFAs from phospholipids appears to oc-
fatty acids) is related to their enzymatic conversion into eico- cur indiscriminately with nҀ3 and nҀ6 PUFAs. Because the
sanoids (Figure 1), which are short-lived, hormone-like lipids major PUFA in cell membranes is AA, most eicosanoids pro-
with chain lengths of 20 carbon atoms (eicosa ҃ 20). Eicosanoids duced will be of the 2-series prostanoids (prostaglandins and
are biologically potent and have a wide array of activities: they thromboxanes) and the 4-series leukotrienes, with 2 and 4 double
modulate inflammatory and immune responses and play a critical bonds, respectively, in the products. EPA is a substrate for
role in platelet aggregation, cellular growth, and cell differenti- 3-series prostanoids and 5-series leukotrienes. In general, AA-
ation. The precursor fatty acids for the formation of eicosanoids derived eicosanoids have proinflammatory effects (39 – 41)—
are dihomo-␥-linolenic acid (DGLA, 20:3nҀ6), AA, and EPA. although prostaglandin E2 (PGE2) has been suggested to also
Linoleic acid (LA, 18:2nҀ6) and ␣-linolenic acid (␣-LNA, have antiinflammatory properties (42)—whereas EPA-derived
18:3nҀ3) are the predominant plant-derived dietary PUFAs and eicosanoids have antiinflammatory effects. Eicosanoids gener-
are the precursors of DGLA and AA and of EPA, respectively. ated from AA, such as PGE2, leukotriene B4, thromboxane A2,
The production of eicosanoids begins with the liberation of PU- and 12-hydroxyeicosatetraenoic acid, have been positively
FAs from membrane phospholipids by the action of various linked to carcinogenesis (34). For example, PGE2 promotes tu-
phospholipases. Thereafter, these PUFAs serve as substrates for mor cell survival and is found at higher concentrations in cancer
cyclooxygenases (COX-1, which is a constitutive enzyme, and cells than in normal cells (43). The mechanisms whereby PGE2
COX-2, which is an inducible enzyme), lipoxygenases (5-, 12-, promotes tumor survival include inhibition of apoptosis and
and 15-lipoxygenase), or cytochrome P450 monooxygenases. stimulation of cell proliferation (44 – 46). It has also been re-
DIETARY nҀ3 FATTY ACIDS AND CANCER 937
ported that PGE2 increases tumor progression by promoting tu- Influence on transcription factor activity, gene
mor angiogenesis (47– 49). 12-Hydroxyeicosatetraenoic acid expression, and signal transduction
has been shown to suppress apoptosis (50, 51) and promote tumor Dietary PUFAs and their metabolites may exert some of their
angiogenesis (52) and tumor cell adhesion to endothelial cells antitumor effects by affecting gene expression or the activities of
(53, 54); the latter is an essential and early event in the initiation signal transduction molecules involved in the control of cell
of the metastatic cascade. Some lipoxygenase products gener- growth, differentiation apoptosis, angiogenesis, and metastasis.
ated from AA, such as leukotriene B4 and 5-hydroxyeico-
satetraenoic acid, also play a role in tumor cell adhesion (55) and Peroxisome proliferator-activated receptor
thus may augment metastatic potential. Leukotriene B4 further
The first transcription factor that was identified as being reg-
enhances generation of reactive oxygen species (40), which may
ulated by fatty acids was the peroxisome proliferator-activated
attack DNA and lead to cancer initiation. AA-derived eico-
receptor-␣ (PPAR␣) (77), a member of the PPAR family, which
sanoids synthesized by the action of cytochrome P450 monoox-
also comprises PPAR␦ (also referred to as PPAR␤) and PPAR␥
ygenase were recently shown to influence several biological
(3 isoforms: ␥1, ␥2, and ␥3). These ligand-activated transcrip-
processes, including cell proliferation, apoptosis, and inflamma-
tion factors were first found to be implicated in the regulation of
tion (56). For example, 14,15-epoxyeicosatrienoic acid inhibits
lipid metabolism and homeostasis but have recently appeared to
apoptosis (57) and increases cell proliferation (58). Although
be involved in cell proliferation, cell differentiation, and inflam-
several AA-derived eicosanoids have been suggested to promote matory responses (78, 79). The preferred natural ligands of
carcinogenesis, some of them, such as PGI2 (59), 15d-PGJ2 (me- PPAR␥ are PUFAs, including LA, ␣-LNA, AA, and EPA (80,
tabolite of PGD2) (60), and 15(S)-hydroxyeicosatetraenoic acid 81). Endogenous ligands include 15d-PGJ2, 9(S)-hydroxyocta-
(61), as well as the LA-derived 13(S)-hydroxyoctadecadienoic decadienoic acid, 13(S)-hydroxyoctadecadienoic acid, and 15-

acid (62, 63), have been found to suppress cell proliferation and hydroxyeicosatetraenoic acid (80, 82, 83). In addition to being a
induce apoptosis. PPAR␥ agonist, EPA, but not other fatty acids (␣-LNA, DHA,
The most salient mechanism by which nҀ3 fatty acids may and nҀ6 PUFAs), has been shown to significantly increase
lower the risk of cancer is through their suppressing effect on the PPAR␥1 messenger RNA concentrations in isolated adipocytes
biosynthesis of AA-derived eicosanoids. This effect is achieved (84). PPAR␣ can be activated by fibrates (hypolipidemic drugs)
at several levels. First, high intakes of nҀ3 fatty acids result in (79) and by various saturated and unsaturated fatty acids, includ-
their incorporation into membrane phospholipids, where they ing palmitic acid, oleic acid, LA, AA (85), conjugated LA (86),
partially replace AA (64). By decreasing the availability of AA and EPA (77). Known activators of PPAR␦ are DGLA, EPA,
precursors, this substitution suppresses the biosynthesis of AA- AA, palmitic acid, and the prostaglandins PGA1 (derived from
derived eicosanoids in favor of EPA-derived 3-series prosta- DGLA) and PGD3 (80). PPAR␥ is expressed in several epithelial
noids and 5-series leukotrienes. Second, nҀ3 PUFAs compete tissues that are important in human cancers (83). Agonists of
with nҀ6 PUFAs for desaturases and elongases, and nҀ3 PPAR␥ have been found to have antiproliferative effects both in
PUFAs have greater affinities for the enzymes than do nҀ6 vitro (87–92) and in vivo (93, 94). For instance, in a phase II
PUFAs. Thus, a higher intake of nҀ3 PUFAs reduces the de- clinical study in patients with advanced prostate cancer, the
saturation and elongation of LA to AA (34) and thus the produc- PPAR␥ agonist troglitazone blocked or reversed tumor progres-
tion of AA-derived eicosanoids. Third, nҀ3 fatty acids suppress sion, which led to a prolonged stabilization of or decrease in
COX-2 (65– 67) and compete with nҀ6 fatty acids for cyclooxy- prostate-specific antigen in 50% of the patients (93, 95). Further-
genases to form eicosanoids (68 –70). Compared with AA, EPA more, reduced concentrations of 15-hydroxyeicosatetraenoic
is the preferential substrate for lipoxygenase; hence an increased acid, an endogenous ligand for PPAR␥ in the prostate, contribute
EPA intake leads to higher formation of EPA-derived lipoxy- to increased proliferation of and reduced differentiation in pros-
genase products at the expense of AA-derived lipoxygenase tate carcinoma (96). DHA was found to induce apoptosis in
products when both fatty acids are simultaneously available (71). vascular smooth muscle cells by activation of PPAR␣, p38
Dietary nҀ6 PUFAs, in contrast with nҀ3 PUFAs, have been mitogen-activated protein kinases, bax, and cytochrome c (94).
reported to up-regulate the expression of rat COX-2 and, to some Murata et al (97) reported that EPA decreases the activity of
extent, COX-1 (72) and thus increase the production of prosta- mitogen-activated protein kinase and inhibits cell proliferation in
noids. Finally, nҀ3 PUFAs enhance eicosanoid catabolism, HepG2 cells. Both PPAR␣ and PPAR␥ have antiinflammatory
which is postulated to be mediated through induction of perox- properties and may thereby contribute to suppression of carci-
isomal enzymes (73). The formation of AA-derived eicosanoids nogenesis (79). PPAR␦ has been suggested to act as an inducer of
is decreased not only by nҀ3 PUFAs but also by eicosanoids cell proliferation and as a promoter of the progression of certain
derived from them, and some of these eicosanoids (eg, types of cancer (80). PPAR␦ antagonists may have a role in
15-hydroperoxyeicosapentaenoic acid) have an even more in- decreasing colon cancer risk (80) although this has not been
hibitory effect than does EPA (74). Taken together, these effects conclusively shown.
at different levels dramatically reduce the AA-derived eico-
sanoids that are linked to inflammation and carcinogenesis. Nuclear transcription factor ␬B
Note that the potency of dietary EPA and DHA is estimated to The nuclear transcription factor ␬B (NF-␬B) family of tran-
be approximately five-fold that of ␣-LNA for the suppression of scription factors is involved in cytokine gene expression, cellular
AA-derived eicosanoids (35). Similarly, the activities of ⌬5- and adhesion, cell cycle activation, apoptosis, and carcinogenesis
⌬6-desaturase are considerably lower in rats fed a fish-oil (rich in (98). Constitutive NF-␬B activation in cancer appears to play a
EPA and DHA) diet than in those fed a flaxseed-oil (rich in role in tumor growth (98). In an experimental study, nҀ3 fatty
␣-LNA) diet (75, 76). acids significantly decreased NF-␬B activation in murine
938 LARSSON ET AL
macrophages (99). Furthermore, cells treated with nҀ3 fatty (65, 117, 118). COX-2 expression has been shown to down-
acids showed a significant decrease in both messenger RNA and regulate the apoptotic pathway (34). Overexpression of COX-2
protein expression of tumor necrosis factor ␣ (decreases of 47% has been detected in many types of cancer, including cancer of
and 46%, respectively) (99). the breast, colon, and prostate (119, 120). Numerous epidemio-
logic studies found that long-term use of COX-2 inhibitors (non-
ras and protein kinase C steroidal antiinflammatory drugs) is associated with a lower risk
Collett et al (100) showed that, compared with LA, DHA of colorectal cancer, adenomatous polyps, and perhaps other
lowers the activation of ras oncogenes, which are frequently types of cancer (121). COX-2 catalyzes the conversion of pro-
activated in tumors, in mouse colon cells. ras activation by point carcinogens to carcinogens, and significant amounts of xenobi-
mutation or overexpression is associated with elevated concen- otics could be oxidized to mutagens by COX-2. Moreover, met-
trations of cellular diacylglycerol and thus down-regulation of abolic turnover of AA is sufficient to produce mutagens. For
protein kinase C (PKC) (101). Feeding rats dietary fish oil has example, malondialdehyde, a byproduct of the oxidation of AA,
been shown to block the azoxymethane (a carcinogen)-induced is highly reactive and forms adducts with DNA (122).
decrease in steady-state concentrations of PKC ⌬ and ␭-␨
isozymes, both of which have tumor suppressor functions (102). Nitric oxide
Unlike PKC ⌬ and ␭-␨, PKC ␤2, which is induced early during Nitric oxide (NO) and reactive products derived from it, such
colon carcinogenesis (103), promotes colon cancer (104, 105). as reactive nitrogen species, are mutagenic and have the potential
Murray et al (103) showed a significant decrease in the concen- to produce nitration, nitrosation, and deamination reactions on
tration of membrane-associated PKC ␤2 in the colonic epithe- DNA bases (123, 124). Excessive production of NO during
lium of rats fed fish oil. Furthermore, the fish-oil diet blocks PKC chronic inflammation is believed to cause DNA damage and
␤2–mediated hyperproliferation and enhances carcinogenesis in

impaired DNA repair (eg, mutation of the p53 tumor suppressor
intestinal epithelial cells (103). gene) and, in the long term, cancer (124 –126). Tumor-derived
NO promotes tumor growth and metastasis by enhancing the
Ornithine decarboxylase invasive, angiogenic, and migratory abilities of tumor cells (124,
Ornithine decarboxylase (ODC), the rate-limiting enzyme in 126, 127), which may also be triggered by activation of COX-2
polyamine biosynthesis, is intimately involved in normal cellular (124). Another mechanism whereby NO may stimulate tumor
proliferation. Both ODC activity and polyamine content are sig- growth is by increasing the production of PGE2 (128), which is
nificantly higher in most colorectal neoplasms than in normal, implicated in tumor progression. NO production in a macro-
adjacent, healthy control tissues (106, 107). Rao and Reddy (108) phage cell line was found to be suppressed by the nҀ3 PUFAs
investigated the modulating effect of high-fat diets rich in nҀ3, ␣-LNA, EPA, and DHA in a dose-dependent fashion (129). Sev-
nҀ6, and nҀ9 fatty acids on ODC activity in the liver, colon, and eral other studies provide additional evidence for a suppressing
small intestinal mucosa. The authors showed that high amounts effect of DHA on NO production (130 –132).
of corn oil (rich in nҀ6 fatty acids) in the diet increase the
activities of ODC and tyrosine-specific protein kinase in the Alteration of estrogen metabolism
colon and liver of male F344 rats, whereas high dietary amounts It is well known that estrogen has proliferative effects on
of fish oil and olive oil (rich in nҀ9 fatty acids) suppress these estrogen-sensitive tissues and that high estrogen concentrations
activities (108). These results were supported by those of Bar- may increase the risk of breast cancer and of some other
tram et al (109), who showed that, compared with corn oil, hormone-dependent cancers. The AA-derived eicosanoid PGE2
dietary fish oil suppresses ODC activity in healthy humans. has been shown to stimulate the activity of aromatase P450,
which converts 19-carbon steroids to estrogens (133). In con-
3-Hydroxy-3-methylglutaryl coenzyme-A reductase trast, PGE3, a product of EPA metabolism, does not activate
Several studies in rats showed that the long-chain nҀ3 fatty aromatase P450. Hence, an increased intake of EPA, which leads
acids reduce the activity and concentration of 3-hydroxy-3- to increased production of PGE3 and decreased production of
methylglutaryl coenzyme A (HMG-CoA) reductase (110 –113), PGE2, is expected to decrease estrogen production and thus re-
which catalyzes the biosynthesis of mevalonate. In addition to duce estrogen-stimulated cell growth. Although a high intake of
being essential for the biosynthesis of cholesterol and coenzyme nҀ3 PUFAs relative to that of nҀ6 PUFAs may decrease en-
Q, mevalonate is required for DNA synthesis and cell prolifer- dogenous estrogen production, no studies have yet directly ex-
ation (114). HMG-CoA reductase inhibitors (statins) have been amined this issue in humans.
shown to have antiangiogenic properties (115), which suggests
that HMG-CoA is involved in angiogenesis. However, unlike Increased or decreased production of free radicals and
statins, long-chain nҀ3 fatty acids have generally not been reactive oxygen species
shown to decrease cholesterol concentrations in humans (116). Free radicals and reactive oxygen species produced in cells
Thus, a potential effect of long-chain fatty acids on HMG-CoA may attack PUFAs to form lipid hydroperoxides, which decom-
reductase activity in humans remains speculative. pose in chain reactions to form more free radicals and reactive
aldehydes such as trans-4-hydroxy-2-nonenal and malondialde-
Cyclooxygenase-2 and lipoxygenases hyde. These metabolites potentially generate promutagenic exo-
Several studies indicate that although nҀ6 PUFAs promote cyclic DNA adducts in human cells, which lead to cancer (134,
colon and mammary carcinogenesis by up-regulating expression 135). Generally, the autooxidizability of various fatty acids in an
of p21ras and COX-2, nҀ3 PUFAs may exert one of their anti- air atmosphere is roughly proportional to the number of double
tumor effects by suppressing the expression of p21ras and COX-2 bounds in the molecule. The long-chain, highly unsaturated nҀ3

FIGURE 2. Hypothetical scheme showing potential mechanisms whereby nҀ6 polyunsaturated fatty acids (PUFAs) and nҀ3 PUFAs may promote and
suppress carcinogenesis, respectively. In initiated tumor cells, phospolipase A2, cyclooxygenase 2, and lipoxygenases are often overexpressed, which leads to
overproduction of arachidonic acid (AA, 20:4nҀ6)– derived eicosanoids that augment inflammation. Nitric oxide, which is elevated in inflammation, is
implicated in both the initiation and the progression stages of carcinogenesis. Nitric oxide may stimulate tumor growth and metastasis by enhancing the
angiogenic and migratory abilities of tumor cells. Dietary nҀ3 PUFAs reduce the desaturation and elongation of linoleic acid (18:2nҀ6) to AA, the
incorporation of AA into membranes, and the biosynthesis of AA-derived eicosanoids; suppress inflammation; stimulate apoptosis; up-regulate the expression
of genes coding for antioxidant enzymes; and thus inhibit tumor growth and metastasis. ѿ and solid arrows, stimulation; Ҁ and dashed arrows, suppression;
1, increase. EPA, eicosapentaenoic acid (20:5nҀ3); DHA, docosahexaenoic acid (22:6nҀ3); ROS, reactive oxygen species; RNS, reactive nitrogen species.
fatty acids are therefore believed to promote lipid peroxidation In this context, note that some researchers found that the in-
and thus carcinogenesis. These assumptions are based on the hibitory effects of fish oil on the growth of tumors in vitro are
results of investigations of the in vitro oxidation of unsaturated abolished by the concurrent addition of vitamin E (37, 141–143)
fatty acids in homogeneous systems (136). However, there is or vitamin C (143), which suggests that oxidized products of nҀ3
evidence that, compared with the intake of nҀ6 fatty acids, the PUFAs suppress cell growth. Generation of oxygen radicals ap-
intake of nҀ3 fatty acids suppresses so-called free radical dis- pears to be involved in the initiation of apoptosis and in the
eases, such as cancer, ageing, and atherosclerosis, which sug- natural defense against transformed or foreign cells (144). Thus,
gests that lipid peroxidation in vivo may not correspond with that the inhibitory effects of long-chain nҀ3 PUFAs on cell growth
in vitro (35). For instance, several studies found that increasing may, at least partly, be explained by their formation of oxidation
the dietary intakes of EPA and DHA does not increase the oxi- products, which leads to apoptosis and cell growth arrest. To
dative susceptibility of LDL cholesterol (137–139). Moreover, date, however, there is little direct evidence that nҀ3 PUFAs
Takahashi et al (140) reported that genes coding for some anti- influence the carcinogenic process by alteration of free radical
oxidant enzymes (eg, glutathione transferases and manganese- production in humans. Further studies on the role of lipid hy-
superoxide dismutase) were up-regulated in mice fed a fish-oil droperoxides in the modulation of tumor growth in vivo are
diet, which suggests a protective effect against the production of needed to elucidate their role in carcinogenesis.
reactive oxygen species and thus against cancer initiation. Stud-
ies in healthy humans also showed that consumption of a diet Other potential mechanisms
providing 쏜2.3 g EPA plus DHA/d decreases superoxide pro- In addition to the potential mechanisms described above, di-
duction (40). Inflammation has been hypothesized to increase the etary nҀ3 PUFAs may also modulate carcinogenesis through
production of free radicals and reactive oxygen species, which effects on insulin sensitivity and cell membrane fluidity, al-
leads to carcinogenesis. Although nҀ6 fatty acids augment these though these mechanisms have been less well studied. The nҀ3
events through the overproduction of AA-derived proinflammatory fatty acid EPA has been found to improve insulin sensitivity in
eicosanoids, the nҀ3 fatty acids suppress inflammation and thus the rats (145–147) and patients with type 2 diabetes (148). The effect
overproduction of free radicals and carcinogenesis (Figure 2). has been proposed as being mediated through PPAR␣ and
940 LARSSON ET AL
TABLE 1
Amounts of total fat (fatty acids), ␣-linolenic acid (␣-LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), and
arachidonic acid (AA) and ratios of nҀ3 to nҀ6 fatty acids in selected species of fish and in meat1
nҀ3 Fatty acids nҀ6 Fatty acids
Total fat ␣-LNA EPA DHA LA AA nҀ3:nҀ6 Fatty acids
g/100 g g/100 g g/100 g

Fish
Cod, Atlantic 0.7 Tr 0.06 (13.2) 0.17 (34.4) Tr 0.02 (4.6) 11.11
Haddock 0.6 Tr 0.05 (12.2) 0.10 (24.4) 0.01 (2.4) 0.01 (2.4) 7.67
Herring, Baltic 9.3 0.29 (3.5) 0.56 (6.7) 0.83 (9.9) 0.54 (6.5) 0.03 (0.4) 2.94
Herring, Pacific 18.5 0.32 (1.9) 1.03 (6.2) 1.63 (9.8) 0.43 (2.6) 0.07 (0.4) 5.88
Mackerel, Atlantic 16.0 0.29 (2.0) 0.89 (6.2) 1.56 (10.8) 0.30 (2.1) 0.07 (0.5) 7.14
Perch, all varieties 1.3 0.01 (1.6) 0.08 (8.7) 0.19 (21.4) 0.02 (2.1) 0.05 (6.0) 4.00
Pike 0.7 0.01 (1.1) 0.04 (7.6) 0.16 (33.0) 0.01 (2.2) 0.02 (3.7) 7.14
Salmon, Atlantic 12.0 0.18 (1.7) 0.49 (4.5) 1.33 (12.3) 0.41 (3.8) 0.11 (1.0) 3.85
Salmon, Pacific 5.2 0.05 (1.1) 0.63 (13.5) 0.88 (18.9) 0.07 (1.6) 0.03 (0.7) 16.67
Sardines, in tomato sauce 14.8 0.22 (1.6) 1.24 (8.8) 1.77 (12.6) 0.22 (1.6) 0.06 (0.4) 11.11
Trout, rainbow 9.6 0.15 (1.7) 0.60 (7.0) 1.76 (20.4) 0.41 (4.8) 0.07 (0.8) 5.26
Tuna, in water 1.2 0.01 (1.6) 0.09 (11.3) 0.16 (19.4) 0.01 (1.6) 0.03 (3.2) 6.67
Meat
Chicken, no skin 3.1 0.02 (0.9) 0.01 (0.3) 0.01 (0.6) 0.30 (12.2) 0.01 (0.5) 0.13

Beef, steak 8.8 0.03 (0.3) Tr Tr 0.18 (2.1) 0.03 (0.4) 0.14
Pork, fillet 1.6 0.01 (0.5) Tr 0.01 (0.4) 0.12 (8.1) 0.01 (0.5) 0.25
All values are x៮ ; percentage of total fatty acids in parentheses. Tr, trace (울0.005 g/100 g). The data for meat are from the Swedish National Food
1
Administration Database (152).
PPAR␥ (149) but may also involve modification of the phospho- adulthood. Thus, if exposure information is obtained at middle or
lipid components of skeletal muscle membranes (145, 150). Iigo old age when cancer is diagnosed, the association between nҀ3
et al (151) showed that treatment of colon carcinoma cells with fatty acid intake and cancer might be missed.
DHA resulted in altered tumor cell membrane characteristics and Another aspect that has to be taken into account when evalu-
a decreased ability to metastasize. ating results from epidemiologic studies is that most studies
examined the association between cancer risk and total fish in-
take rather than fatty fish intake, which may better mirror total
DISCUSSION intake of marine nҀ3 fatty acids. Total fat content in fish varies
Substantial evidence from experimental and animal studies widely between species, from 0.6 – 0.7 g/100 g in halibut and cod
indicates that long-chain nҀ3 fatty acids in fish and fish oils to 16.0 –18.5 g/100 g in mackerel and herring from the Pacific
inhibit carcinogenesis. Epidemiologic studies examining the as- (Table 1). The composition of the fat depends on the geographic
sociations of fish and marine nҀ3 fatty acids with the risk of area in which the fish live, the fish’s diet, and seasonal variations
development of cancer have, however, been inconclusive (1). (34) and on environmental factors, such as temperature, salinity,
About one-third to one-half of the studies that examined the and the depth at which the fish live, with the highest content of
relation between the intake of long-chain nҀ3 fatty acids or fish EPA and DHA in cold-water fish (152). In the future, the farming
and cancers of the breast, prostate, endometrium, or ovary re- industry may also have important influences on the fat compo-
ported a statistically significant reduction in the risks of these sition of the fish. The nҀ3 fatty acid ␣-LNA, which is found in
cancers. The remaining studies either found an inverse associa- dark green leafy vegetables, rapeseed oil (canola oil), flaxseed,
tion that was not statistically significant or failed to find any some nuts (especially walnuts), and soybeans, may also bias
association. results if only fish consumption is taken into account. Neverthe-
There are several possible explanations for these null findings. less, although humans can convert ␣-LNA to EPA, which can be
First, in comparison with the consistent protective effect of long- further elongated and desaturated to DHA, this conversion is not
chain nҀ3 fatty acids in animal and in vitro studies, the incon- very efficient. The extent of the conversion of ␣-LNA to EPA has
sistent associations observed in analytic epidemiologic studies not been fully characterized and may depend on intakes of total
may partly be due to the fact that the intake of long-chain nҀ3 fat, ␣-LNA, EPA, DHA, and LA (153–157). It has been reported
fatty acids in some of the studied populations was too low to that when the intake of LA is held constant at 15 g/d, the total
produce a protective effect. Other possible explanations include percentage of conversion of ␣-LNA to EPA and DHA is 11–
low within-population variability in the intake of fish or nҀ3 18.5%, but when the intake of LA is increased from 15 to 30 g/d,
fatty acids (which limits the statistical power to detect an asso- this conversion is reduced to 5–11% (155). A recent study
ciation) and nondifferential misclassification of estimated nҀ3 showed that 2.8% of the dietary ␣-LNA consumed was converted
fatty acid intake. Although most of the potential mechanisms by to EPA and that this conversion was down-regulated (2-fold) in
which long-chain nҀ3 fatty acids may inhibit carcinogenesis are subjects who consumed a diet high in EPA and DHA (154).
at the promotion and progression stages, the critical period for Pawlosky et al (157) reported an even more limited conversion of
dietary nҀ3 fatty acid exposure may be during childhood or early ␣-LNA to EPA in humans: only 앒0.2% of plasma ␣-LNA was
converted to EPA. Low-fat diets result in increased ⌬ - and 5
models, whereas long-term exposures to relatively low doses of
⌬6-desaturation (156), which may increase the conversion of long-chain nҀ3 PUFAs may not be as effective against cancer
␣-LNA to EPA. development in humans. Alternatively, the inconsistencies in
Another drawback is that most epidemiologic studies largely results between animal and epidemiologic studies may be due to
analyzed the intake of nҀ3 PUFAs without taking into account publication bias. Small animal studies, which take relatively little
the intake of nҀ6 PUFAs. Given the above-described mecha- effort and money, are more likely to suffer from publication bias
nisms through which EPA and DHA may decrease the risk of than are large, well-designed epidemiologic studies. Conse-
cancer development, the ratio of nҀ3 to nҀ6 PUFAs seems to be quently, the overall picture may be biased toward protective
more important than is the absolute intake of nҀ3 PUFAs. In- effects in animal studies.
deed, ratios of nҀ3 to nҀ6 PUFAs, but not absolute concentra- In the light of the above-mentioned methodologic difficulties
tions of these fatty acids, in adipose tissue biopsy specimens were and limitations of observational epidemiologic studies, it is not
found to be inversely associated with breast cancer risk in a surprising that the results reported from these studies to date on
multinational epidemiologic study (158). Experimental data in- the association between long-chain nҀ3 fatty acids and cancer
dicate that a ratio of nҀ3 to nҀ6 PUFAs of 1:1 or 1:2 is needed risk are inconsistent. Future epidemiologic studies have to take
for protection against the development of cancer (159). In most into account more aspects, as mentioned above, in the collection
Western countries, the ratio is 앒1:10 –1:20 (159); hence, no and analysis of data. In epidemiologic analyses, the biological
effect on carcinogenesis would be expected. Although dietary interplay— observed in experimental studies— between nҀ3
LA intake of up to 2–3% of energy intake increases tissue AA and nҀ6 fatty acids and other factors (eg, vitamin E and antiin-
concentrations, LA intake of 쏜3% of energy intake is poorly flammatory drugs) should be taken into account in appropriate
correlated with tissue AA concentrations (160, 161). Because the statistical analyses to address these issues.
average LA intake in the United States and Western Europe is In summary, several mechanisms whereby nҀ3 fatty acids

6 –7% of energy intake (162), a moderate change in dietary LA may modify the carcinogenic process were described. These
intake would not be expected to modulate tissue AA concentra- fatty acids can suppress AA-derived eicosanoid biosynthesis;
tions. However, LA intakes of 쏜12% of energy intake may influence transcription factor activity, gene expression, and sig-
actually decrease tissue AA concentrations because of inhibition nal transduction pathways; modulate estrogen metabolism; in-
of ⌬6-desaturase activity (156). On the other hand, dietary pre- crease or decrease the production of free radicals and reactive
formed AA, which is found in meat and fish (Table 1), is much oxygen species; and influence insulin sensitivity and membrane
more effective in enriching tissue phospholipid membranes than fluidity. On the basis of these multiple mechanisms, nҀ3 PUFAs
is LA (163). Thus, a low LA intake and a high nҀ3 fatty acid may have an important influence on carcinogenesis. Further
intake seems to be needed to suppress AA-derived eicosanoids, studies are needed to identify new mechanisms and to evaluate
and such a diet is not very common in Western societies. Because and verify these mechanisms in humans to gain more understand-
tissue concentrations of AA, in contrast with those of LA, are ing of the effects of marine nҀ3 fatty acid intake on cancer risk
strongly influenced by dietary intake, epidemiologic studies of in real-life situations. Epidemiologic studies with more detailed
the relation between the ratio of AA to nҀ3 PUFAs and cancer information about nҀ3 and nҀ6 fatty acid exposures and im-
risk may be warranted. proved analytic approaches that take into account the biological
The absence of an association between dietary long-chain fatty interplay between several nutritional factors in cancer develop-
acids and cancer risk in some epidemiologic studies may not ment are needed.
exclude the possibility of different effects in subgroups. The
The manuscript was drafted by SCL. MK and MI-S critically reviewed the
potential protective effect of dietary long-chain nҀ3 fatty acids manuscript. AW made intellectual contributions throughout the whole pro-
may be modified by intakes of antioxidants, such as vitamins E cess of manuscript preparation. All the authors reviewed the final version.
and C; such modification has been observed in experimental None of the authors had any conflicts of interest.
studies but has not been taken into account in epidemiologic
analyses to date.
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J Pain. Author manuscript; available in PMC 2010 PMCID: PMC2750819
September 1. NIHMSID:
Published in final edited form as: NIHMS143404
J Pain. 2009 September; 10(9): 895–926.
doi: 10.1016/j.jpain.2009.06.012
Copyright notice and Disclaimer
Central Sensitization: A Generator of Pain Hypersensitivity by Central Neural Plasticity
Alban Latremoliere and Clifford J. Woolf
Neural Plasticity Research Group, Department of Anesthesia and Critical Care,
Massachusetts General Hospital and Harvard Medical School, Charlestown,
Massachusetts
Address reprint requests to Dr Clifford J. Woolf, Neural Plasticity Research Group,
Department of Anesthesia and Critical Care, Massachusetts General Hospital and
Harvard Medical School, Charlestown, MA 02129. Email: cwoolf@partners.org
The publisher's final edited version of this article is available at J Pain
See other articles in PMC that cite the published article.
• Other Sections▼
o Abstract
o The Discovery of Central Sensitization
o Activity-Dependent Central Sensitization
o Triggers of Activity-Dependent Central Sensitization
o Signaling Pathways and Activity-Dependent Central Sensitization
o Effectors of Activity-Dependent Central Sensitization
o Global Features of Activity-Dependent Central Sensitization
o Activity-Dependent Central Sensitization and Synaptic Plasticity
o Central Sensitization in Pathological Settings
o Inflammatory Pain
o Neuropathic Pain
o Scaffolding Proteins, Synaptic Plasticity, and Central Sensitization
During Inflammation and After Nerve Injury
o PSD Proteins and AMPAR Recycling and Subunit Switch
o Conclusion
o References
Abstract
Central sensitization represents an enhancement in the function of neurons and circuits
in nociceptive pathways caused by increases in membrane excitability and synaptic
efficacy as well as to reduced inhibition and is a manifestation of the remarkable
plasticity of the somatosensory nervous system in response to activity, inflammation,
and neural injury. The net effect of central sensitization is to recruit previously
subthreshold synaptic inputs to nociceptive neurons, generating an increased or
augmented action potential output: a state of facilitation, potentiation, augmentation, or
amplification. Central sensitization is responsible for many of the temporal, spatial, and
threshold changes in pain sensibility in acute and chronic clinical pain settings and
exemplifies the fundamental contribution of the central nervous system to the
generation of pain hypersensitivity. Because central sensitization results from changes
in the properties of neurons in the central nervous system, the pain is no longer coupled,
as acute nociceptive pain is, to the presence, intensity, or duration of noxious peripheral
stimuli. Instead, central sensitization produces pain hypersensitivity by changing the
sensory response elicited by normal inputs, including those that usually evoke
innocuous sensations.
Perspective
In this article, we review the major triggers that initiate and maintain central
sensitization in healthy individuals in response to nociceptor input and in patients with
inflammatory and neuropathic pain, emphasizing the fundamental contribution and
multiple mechanisms of synaptic plasticity caused by changes in the density, nature, and
properties of ionotropic and metabotropic glutamate receptors.
Keywords: Central sensitization, inflammatory pain, neuropathic pain, scaffolding
protein, heterosynaptic facilitation
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Acute nociceptive pain is that physiological sensation of hurt that results from the
activation of nociceptive pathways by peripheral stimuli of sufficient intensity to lead to
or to threaten tissue damage (noxious stimuli).374 Nociception, the detection of noxious
stimuli,282 is a protective process that helps prevent injury by generating both a reflex
withdrawal from the stimulus and as a sensation so unpleasant that it results in complex
behavioral strategies to avoid further contact with such stimuli. An additional important
phenomenon that further enhances this protective function is the sensitization of the
nociceptive system that occurs after repeated or particularly intense noxious stimuli, so
that the threshold for its activation falls and responses to subsequent inputs are
amplified.132,376,380 In the absence of ongoing tissue injury, this state of heightened
sensitivity returns over time to the normal baseline, where high-intensity stimuli are
again required to initiate nociceptive pain; the phenomenon is long lasting but not
permanent. The nociceptor-induced sensitization of the somatosensory system is
adaptive in that it makes the system hyperalert in conditions in which a risk of further
damage is high, for example, immediately after exposure to an intense or damaging
stimulus. This sensitization is the expression of use-dependent synaptic plasticity
triggered in the central nervous system (CNS) by the nociceptor input and was the first
example of central sensitization, discovered 26 years ago.369 Since then, we have
learned that a number of different forms of functional, chemical, and structural plasticity
can sensitize the central nociceptive system to produce pain hypersensitivity under both
normal and pathological circumstances, some of which are persistent.
In many clinical syndromes, pain is no longer protective. The pain in these situations
arises spontaneously, can be elicited by normally innocuous stimuli (allodynia), is
exaggerated and prolonged in response to noxious stimuli (hyperalgesia), and spreads
beyond the site of injury (secondary hyperalgesia). Central sensitization has provided a
mechanistic explanation for many of the temporal, spatial, and threshold changes in
pain sensibility in acute and chronic clinical pain settings and has highlighted the
fundamental contribution of changes in the CNS to the generation of abnormal pain
sensitivity. Although phenomenologically central sensitization may appear to be
comparable to peripheral sensitization, it differs substantially, both in terms of the
molecular mechanisms responsible and its manifestation. Peripheral sensitization
represents a reduction in threshold and an amplification in the responsiveness of
nociceptors that occurs when the peripheral terminals of these high-threshold primary
sensory neurons are exposed to inflammatory mediators and damaged tissue46,105,124,242
and, in consequence, is restricted to the site of tissue injury.124 Although peripheral
sensitization certainly contributes to the sensitization of the nociceptive system and
thereby to inflammatory pain hypersensitivity at inflamed sites (primary hyperalgesia),
it nevertheless represents a form of pain elicited by activation of nociceptors, albeit one
with a lower threshold due to the increased peripheral transduction sensitivity, and
generally requires ongoing peripheral pathology for its maintenance. Peripheral
sensitization appears to play a major role in altered heat but not mechanical sensitivity,
which is a major feature of central sensitization.
Central sensitization, in contrast to peripheral sensitization, co-opts novel inputs to
nociceptive pathways including those that do not normally drive them, such as large
low-threshold mechanoreceptor myelinated fibers to produce Aβ fiber–mediated pain.376
It also produces pain hypersensitivity in noninflamed tissue by changing the sensory
response elicited by normal inputs and increases pain sensitivity long after the initiating
cause may have disappeared and when no peripheral pathology may be present. Because
central sensitization results from changes in the properties of neurons in the CNS, the
pain is no longer coupled, as acute nociceptive pain is, to the presence, intensity, or
duration of particular peripheral stimuli. Instead, central sensitization represents an
abnormal state of responsiveness or increased gain of the nociceptive system. The pain
is effectively generated as a consequence of changes within the CNS that then alter how
it responds to sensory inputs, rather than reflecting the presence of peripheral noxious
stimuli. In this respect, central sensitization represents a major functional shift in the
somatosensory system from high-threshold nociception to low-threshold pain
hypersensitivity. We all experience pain as arising from “out there,” and, in
consequence, imagine that it is actually triggered by noxious stimuli where we feel the
pain. Central sensitization reveals, however, that this in many cases is a sensory
illusion; specific alterations in the CNS can result in painful sensations occurring in the
absence of either peripheral pathology or noxious stimuli, and the target for treatment in
these situations must be the CNS not the periphery.
Central sensitization corresponds to an enhancement in the functional status of neurons
and circuits in nociceptive pathways throughout the neuraxis caused by increases in
membrane excitability, synaptic efficacy, or a reduced inhibition. The net effect is that
previously subthreshold synaptic inputs are recruited to generate an increased or
augmented action potential output, a state of facilitation, potentiation, or amplification.
The reason that these cellular changes alter the system so profoundly is that normally
only a small fraction of the synaptic inputs to dorsal horn neurons contribute to their
action potential output.373 Nociceptive-specific neurons, for example, although
dominated by large monosynaptic and polysynaptic synaptic potentials from nociceptors
in their receptive field, typically also have small-amplitude synaptic inputs from low-
threshold afferents and from nociceptor inputs outside their receptive fields, which
constitute a subliminal fringe that normally does not drive the output of the cells (Fig 1).
Recruiting these subthreshold inputs to the output of a neuron markedly alters its
receptive field properties, with profound changes in receptive field threshold, spatial,
and temporal properties (Fig 2). This provides an opportunity for rapid functional
plasticity that can be revealed experimentally by increasing the excitability of the
neuron or by blocking inhibitory transmitters. After administration of GABA or glycine
receptor antagonists, for example, Aβ inputs are recruited to neurons in the superficial
dorsal horn,17 and pain-like behavior can be elicited by movement of just a few hairs.289
The receptive field of somatosensory neurons are, therefore, not fixed or hard wired, but
are instead highly malleable. This malleability or plasticity is the substrate for the
functional effects of central sensitization, and the means is a change in synaptic
efficacy.
Figure 1
Subthreshold synaptic inputs. The substrate for receptive field plasticity.
Intracellular in vivo recordings from a nociceptive-specific rat dorsal
horn neuron revealing subthreshold synaptic inputs. The output of
somatosensory neurons is determined by (more ...)
Figure 2
Expansion of receptor fields during central sensitization. Recruiting
subthreshold synaptic inputs to the output of a nociceptive-specific
neuron can markedly alter its receptive field properties, producing
changes in receptive field threshold and spatial (more ...)
When neurons in the dorsal horn spinal cord are subject to central sensitization, they
exhibit some or all the following: development of or increases in spontaneous activity, a
reduction in the threshold for activation by peripheral stimuli, increased responses to
suprathreshold stimulation, and an enlargement of their receptive fields (Fig 2). Several
features appear particular to central sensitization: conversion of nociceptive-specific
neurons to wide-dynamic neurons that now respond to both innocuous and noxious
stimuli, progressive increases in the responses elicited by a standard series of repeated
innocuous stimuli (temporal windup), an expansion of the spatial extent of their input,
and changes that outlast an initiating trigger.132,368,369,372,376 These electrophysiological
changes correlate remarkably with the development in human experimental subjects
after a noxious conditioning input of allodynia (particularly dynamic tactile or brush-
evoked allodynia), the temporal summation of repeated low-intensity stimuli from an
innocuous sensation to pain, with “afterpain” on cessation of the stimulus, and
widespread secondary hyperalgesia. These changes can be elicited in human volunteers
by noxious stimulation of the skin (as with topical or intradermal capsaicin or repeated
heat stimuli340) and in the gastrointestinal tract by exposure to low pH solutions.272
Central sensitization contributes to neuropathic37 and inflammatory pain,26,274,395
migraine,35 and irritable bowel syndrome.253 In these patients, it is involved in
producing abnormal responsiveness to noxious and innocuous stimuli and a spread of
tenderness beyond lesion sites. Central sensitization may also play a fundamental role in
the abnormal and widespread pain sensitivity in patients with fibromyalgia.6,68,301-303
Given the major role of central sensitization in the generation of clinical pain
hypersensitivity, it is essential that we understand the triggers and mechanisms
responsible for the induction and maintenance of the switch in the somatosensory
system from the physiological state, in which the sensory experiences evoked by low-
intensity stimuli (innocuous sensations) and noxious stimuli (pain) are quite distinct and
separate, to a dysfunctional hypersensitive system in which this discrimination is lost.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
The Discovery of Central Sensitization

The first evidence for a central component to acute pain hypersensitivity was provided
in 1983.369 Electrophysiological recordings from single biceps femoris α-motoneuron
axons were used to measure the output of the nociceptive system, in this case the flexor
reflex withdrawal response elicited by noxious stimuli (Fig 3). These recordings
revealed, as expected, that under normal conditions there was no spontaneous activity in
the motor neurons and that their activation required a noxious mechanical or thermal
stimulus to the skin. These neurons had high-threshold nociceptive-specific receptive
fields restricted to the toes or hind paw, in keeping with their activation only as part of
the flexion withdrawal reflex. After repeated peripheral noxious heat stimuli sufficient
to generate mild inflammation of the hind paw, however, an increased excitability of the
motor neurons was detected that lasted for several hours and included a reduction in
threshold and enlargement of the cutaneous receptive fields. The flexor motor neurons
were now no longer nociceptive-specific but could be activated by low-intensity
(innocuous) peripheral inputs such as light touch.369 Three experiments showed that this
change in receptive field properties was due to alterations in the CNS and not the
periphery. First, electric stimulation of Aβ sensory fibers began to elicit responses in the
motor neurons after the conditioning noxious heat stimuli, whereas these inputs elicited
no response before. Second, a local anesthetic block of the site of the peripheral injury
did not result in collapse of the expanded receptive fields: The change was autonomous
once it was triggered by the peripheral input. Finally, the hypersensitivity produced by
the noxious heat could be mimicked in extent and duration by a brief 20-second low-
frequency electrical stimulation of the sural nerve only at C-fiber strength, which
produced changes lasting for tens of minutes. The interpretation of all these data was
that noxious heat stimulation, by activating C-fiber nociceptors, had induced a central
plasticity of the nociceptive system, which was thereafter capable of responding to
stimuli outside of the injury area and to low-threshold afferents that previously did not
activate the nociceptive system. This led to the articulation of a more general hypothesis
that brief trains of nociceptor C-fiber input could trigger or condition a long-lasting
sensitization of the nociceptive system (an effect termed central sensitization) by
producing activity-dependent changes in the functional properties of neurons in the
dorsal horn of the spinal cord and that this contributed both to postinjury flexor reflex
and pain hypersensitivity.
Figure 3
Schematic representation of the structures exhibiting central
sensitization. The first evidence for central sensitization was generated
in 1983 by revealing injury-induced changes in the cutaneous receptive
field properties of flexor motor neurons as (more ...)
Before the discovery of central sensitization, the receptive field properties of dorsal
horn neurons was thought to be fixed by the geometry of their dendrites relative to the
central terminals of sensory axons.33 Although plasticity of the receptive fields of dorsal
horn neurons had been shown to occur after peripheral nerve injury, this was thought to
be due to a loss of presynaptic inhibition increasing synaptic input from silent or
ineffective synapses and not to plasticity in dorsal horn neurons.71 After the first
demonstration of central sensitization in flexor motor neurons, essentially identical
changes were soon described in many studies in lamina I and V neurons in the dorsal
horn of the spinal cord55,79,173,176,192,287,365,372 (Fig 3) as well as in spinal nucleus pars
caudalis (Sp5c),36 thalamus78 (Fig 3), amygdala,219,220 and anterior cingulate cortex.364
More recently, functional magnetic resonance imaging, positron emission tomography,
and magnetoencephalography have revealed in human subjects that several other brain
structures implicated in pain (parabrachial nucleus, periaqueductal gray [PAG], superior
colliculus, prefrontal cortex) also exhibit changes compatible with increases in
excitability corresponding to central sensitization187,209,212,244,283 (Fig 3).
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Activity-Dependent Central Sensitization
The original description of central sensitization referred to an activity- or use-dependent
form of functional synaptic plasticity that resulted in pain hypersensitivity after an
intense noxious stimulus. This plasticity was triggered by the activity evoked in dorsal
horn neurons by input from C-nociceptors, as after repeated heat stimuli above 49°C,369
electrical stimulation of C-fibers (1 Hz for 10 to 20 seconds),358 and chemical activation
of nociceptors by irritant compounds such as allyl isothiocyanate (mustard oil)372 and
formalin, which both act through the TRPA1 channel135,199 as well as capsaicin, which
activates TRPV1 channels.158 To induce central sensitization, the noxious stimulus must
be intense, repeated, and sustained. Input from many fibers is required over tens of
seconds; a single stimulus, such as a pinch, is insufficient. Peripheral tissue injury is not
necessary, although the degree of noxious stimulation that produces frank tissue injury
almost always induces central sensitization, so that the phenomenon is very prominent
after post-traumatic or surgical injury. Interestingly, nociceptor afferents innervating
muscles or joints produce a longer-lasting central sensitization than those that innervate
skin.358
Once the phenomenon had been shown to be robust, easily activated, and detected in
both preclinical and human subjects, the issue then was what molecular mechanisms
were responsible. The first major mechanistic insight was that the induction and
maintenance of acute activity-dependent central sensitization was dependent on NMDA
receptors,379 revealing a key involvement of glutamate and its receptors. We now
appreciate from 2 decades of investigation by many labs that central sensitization
comprises 2 temporal phases, each with specific mechanisms. The early
phosphorylation-dependent and transcription-independent phase results mainly from
rapid changes in glutamate receptor and ion channel properties.376 The later, longer-
lasting, transcription-dependent phase drives synthesis of the new proteins responsible
for the longer-lasting form of central sensitization observed in several pathological
conditions.376 We will review the current understanding of these mechanisms.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Triggers of Activity-Dependent Central Sensitization

Glutamate, the fast transmitter of primary afferent neurons, binds to several receptors on
postsynaptic neurons in the dorsal horn of spinal cord, including ionotropic amino-3-
hydroxy-5-methyl-4-isoxazole propionate (AMPA), N-methyl-D-Aspartate (NMDA),
and Kainate (KA) receptors and several metabotropic (G-protein coupled) glutamate
receptor subtypes (mGluR). In the superficial laminae of the dorsal horn, AMPAR and
NMDAR are present in virtually every synapse and are arranged in a mosaic-like
manner, whereas mGluRs sit at the extremities of the postsynaptic density zone
(PSD).10,11,15,247 At the subunit level, NMDAR is a tetramer that contains 2 low-affinity
glycine-binding NR1 subunits and 2 subunits from the 6 different NR2A-D or NR3A/B
subunits.305 The most common NMDA complexes in the dorsal horn are composed of
NR1-NR2A/B subunits.202,215,243 AMPAR is also a tetramer, and its most abundant
subunits in the dorsal horn of the spinal cord are the calcium (Ca2+)-permeable subunits
GluR1 and GluR3 and the non–Ca2+-permeable GluR2 subunit.252 In basal conditions,
inhibitory interneurons appear to preferentially express GluR1, whereas the excitatory
neurons appear to express mostly GluR2.145,338 The AMPAR complex can also be a
GluR1/GluR2 heteromer,11 in which case the receptor displays mainly GluR2
properties.233 A subpopulation of lamina I neurons lacking the NK1 receptor and
expressing the GluR4 (Ca2+-permeable) subunit has been identified.248 The mGluR
family is composed of 8 receptors that form 3 groups, based on their sequence
similarities and their coupling with specific Gα-proteins.54 Group I mGluRs (mGluR1
and 5) are coupled with Gαq-proteins (whose activation causes an increase of [Ca2+]i),
whereas group II (mGluR 2 and 3) and group III (mGluR4, 6, 7 and 8) are coupled with
Gαi/o-proteins. All mGluRs except for mGluR6 and 8 are expressed in the spinal
cord,346 whereas only mGluR6 appears not to be expressed by primary afferent
neurons.39 In addition, a lamina-specific pattern of expression has been characterized for
mGluR1α (lamina V), mGluR5 (laminas I-II),10,247 and mGluR2/3 (lamina II inner),12
suggesting precise and distinct physiological roles for the different subtypes.
Activation of NMDAR is an essential step in both initiating and maintaining activity-
dependent central sensitization as its blockade by noncompetitive (MK801) or
competitive (D-CPP) NMDAR antagonists prevent and reverse the hyperexcitability of
nociceptive neurons induced by nociceptor conditioning inputs184,379 and conditional
deletion of NR1 abolishes NMDA synaptic inputs and acute activity-dependent central
sensitization.300 NMDAR is both a trigger and effector of central sensitization. Under
normal conditions, the NMDAR channel is blocked in a voltage-dependent manner by a
magnesium (Mg2+) ion sitting in the receptor pore.198 Sustained release by nociceptors
of glutamate and the neuropeptides substance P and CGRP leads to sufficient membrane
depolarization to force Mg2+ to leave the NMDAR pore, whereupon glutamate binding
to the receptor generates an inward current.198 Removal of this voltage-dependent block
is a major mechanism for rapidly boosting synaptic efficacy and allows entry of Ca2+
into the neuron, which then activates numerous intracellular pathways that then
contribute to the maintenance of central sensitization. In addition to the critical role of
NMDAR in increasing the excitability nociceptive neurons, activation of group I
mGluRs by glutamate also appear important for the development of central
sensitization. Although these receptors do not participate to basal nociception,221,392
their activation is necessary for activity-dependent central sensitization mediated by C-
fibers.14,67,165,296,392,393 In contrast, activation of group II-III mGluRs is associated with a
reduction of capsaicin-induced central sensitization.296
Substance P (SP), which is co-released with glutamate by unmyelinated peptidergic
nociceptors, is also involved in the generation of central sensitization.8,146,183,192,365
Substance P binds to the neurokinin-1 (NK1) G-protein–coupled receptor, which is
expressed by spinothalamic, spinoparabrachial, and spino-PAG neurons101 and causes a
long-lasting membrane depolarization,112 and contributes to the temporal summation of
C-fiber–evoked synaptic potentials80,388,389 as well as to intracellular signaling. Ablation
of NK1-positive neurons in the spinal cord leads to a reduction in capsaicin-evoked
central sensitization, confirming the importance of projecting neurons expressing the
substance P receptor in this phenomenon.146,192 Calcitonin gene-related peptide (CGRP),
also synthesized by small diameter sensory neurons, potentiates the effects of SP367 and
participates in central sensitization through postsynaptic CGRP1 receptors, which
activate PKA and PKC.307,308 CGRP also enhances release of brain-derived neurotrophic
factor (BDNF) from trigeminal nociceptors,34 which may contribute to its involvement
in migraine and other primary headaches.82,103
BDNF is a neurotrophic factor and synaptic modulator that is synthesized by nociceptor
neurons and released into the spinal cord404 in an activity-dependent manner,19 where it
also has a role in the production of central sensitization.113,144,330 On binding to its high-
affinity trkB receptor, BDNF enhances NMDAR-mediated C-fiber–evoked responses144
and causes activation of several signaling pathways in spinothalamic track neurons,
including ERK141,245,292 and PKC.293
The inflammatory kinin bradykinin is produced in the spinal cord in response to intense
peripheral noxious stimuli and acts through its Gq-coupled B2 receptor, which is
expressed by dorsal horn neurons41,359 and boosts synaptic strength by activating PKC,
PKA, and ERK.152 ERK is also activated by a serotoninergic (5-HT) descending input
involving the ionotropic 5-HT3 receptor143,310,313,396 and possibly the 5-HT7 GS-coupled
receptor.29 Fig 4 summarizes the key known synaptic triggers of central sensitization.
Figure 4
Central sensitization triggers: Schematic representation of key synaptic triggers of
central sensitization. (A), Model of the synapse between the central terminal of a
nociceptor and a lamina I neuron under control, basal conditions. mGluR receptors
sit (more ...)
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Signaling Pathways and Activity-Dependent Central Sensitization

An increase in intracellular Ca2+ beyond a certain threshold level appears to be the key
trigger for initiating activity-dependent central sensitization. Calcium influx through
NMDAR appears to be particularly prominent in the induction phase but can also occur
through calcium-permeable AMPARs,159,355 voltage-gated calcium channels,52,376 as
well as from release from intracellular microsomal stores in response to activation of
several metabotropic receptors106,182 (Fig 5, A through C). Why is the calcium-induced
activation of intracellular kinases so important? The reason is that ionotropic NMDA
and AMPA glutamate receptors can be phosphorylated on several key residues located
on their C-terminus,40,42 and this post-translational modification changes their activity as
well as their trafficking to or from the membrane,40,161 which with similar post-
translational changes in other ion channels, produces the functional changes that
manifest as central sensitization (Fig 6, A through C). AMPAR subunit GluR1 residue
Ser831 is phosphorylated by protein kinase C (PKC) and calcium-calmodulin-
dependent protein kinase II (CaM-KII); Ser845 is the phosphorylation target of PKA,
and GluR2 has 1 main site for phosphorylation by PKC, on Ser880.40 For the NR1
subunit of NMDAR, PKC phosphorylates Ser896, whereas PKA has 2 potential
phosphorylation sites on Ser890 and Ser897.168,333 NMDAR conductance properties are
modified by phosphorylation of tyrosine residues located on the NR2A and NR2B
subunits at 3 potential sites (Tyr1292, 1325, or 1387 for NR2A and Tyr1252, 1336, or
1472 for NR2B), through activation of nonreceptor tyrosine kinases such as Src or
Fyn.42,106,107,268
Figure 5
Sources of Ca2+ in the synapse of nociceptive neurons for inducing
central sensitization. (A), Model of a nociceptor–dorsal horn neuron
synapse under control, nonactivated conditions. After nociceptor input
(B), activation of NMDAR and mGluR result (more ...)
Figure 6
Contribution of PKC, CaMKII, PKA, and ERK activation to central
sensitization. (A), Phosphorylation by PKC, CaM-KII, PKA, and ERK
cause changes in the threshold and activation kinetics of NMDA and
AMPA receptors, boosting synaptic efficacy. ERK also produces (more
...)
Stimulation of AMPAR and group I mGluRs89,106,118,281 participate with NMDAR in the

activation of the intracellular pathways that sustain central sensitization. These include
the PLC/PKC pathway, through opening of Ca2+ channels on the endoplasmic
reticulum,85,391 the phosphatidylinositol-3-kinase (PI3 K) pathway,246 and the mitogen-
activated protein kinase (MAPK) pathway that involves the extracellular signal-
regulated kinases (ERK1 and ERK2), which are 44- and 42-kDa Ser/Thr kinases,
respectively, with 90% sequence identity, and the cAMP response element binding
protein (CREB).130,138,141,357,363 One way that ERK and CREB are activated is through
an elevation in intracellular Ca2+ sufficient to drive a calmodulin-induced stimulation of
adenylyl cyclases 1 and 8, whose cAMP production in turn activates PKA and
subsequent cascade(s).363
The activation of ERK by phosphorylation is regulated by a core signaling module that
consists of an apical MAPK kinase kinase (MAP3 K), a MAPK kinase (MEK or MKK),
and the downstream ERK. Many different signaling pathways can induce ERK
activation in addition to its canonical ras/raf pathway, and this can be readily detected
immunohistochemically in the dorsal horn within minutes of peripheral noxious
stimuli130 (Fig 7). The presence of phosphorylated ERK reveals the anatomical
distribution of those neurons whose intracellular signaling has been activated by the
nociceptor input and are presumably undergoing the synaptic changes that constitute
central sensitization120,131,141,152 (Fig 7). In the spinal cord, ERK is only activated in
neurons in response to intense peripheral noxious stimulation, effectively identical to
those stimuli that induce central sensitization,130,363 suggesting that ERK
phosphorylation is a better marker of the neural plasticity that mediates central
sensitization than c-Fos, which can be activated by low threshold stimuli that do not
induce central sensitization.126 Because the ERK pathway is composed of successive
protein kinases, each of which can be activated by several different signaling
pathways,131 most of the triggers of central sensitization such as NMDAR, group I
mGluR, TrkB, NK1, or CGRP1 converge to activate ERK120,130,131,171 (Fig 8). Once
activated, ERK produces translational and post-translational effects that participate in
the maintenance of central sensitization in spinal cord neurons.131,387 The post-
translational effects include an increase of NMDAR function through phosphorylation
of its NR1 subunit152,293 (Fig 6, A), recruitment of AMPAR to the membrane93,254 (Fig
6, B) leading to an increase in AMPAR, and NMDAR currents boosting synaptic
efficacy.152 Furthermore, ERK produces a decrease in K+ currents through
phosphorylation of the residue Ser616 of Kv4.2 channels leading to an increase in
membrane excitability118,119 (Fig 6, A). Transcriptional changes are mediated by
activation of CREB as well as other transcription factors, which drives expression of
several genes including c-Fos, NK1, TrkB, and Cox-2131 (Fig 6, C). Inhibition of ERK
activation using inhibitors of MEK reduces behavioral measures of activity-dependent
central sensitization.137,142
Figure 7
Key effectors of central sensitization in the dorsal horn. Subcutaneous
injection of capsaicin, a potent inducer of central sensitization, causes
rapid activation of ERK and CREB (upper panels) as well as a PKC-
induced phosphorylation of the NR1 subunit (more ...)
Figure 8
Key intracellular pathways contributing to the generation of central
sensitization. NMDAR activation causes activation of PKC, CaMKII,
and ERK (black arrows); GluR1-containing AMPAR activate PKC (red
arrow); NK1 and CGRP1 receptors activate PKC, PKA, (more ...)
Nitric oxide (NO) synthesized by either neuronal or inducible NO synthases in the
dorsal horn381 also has a role in central sensitization.381-383 Potential mechanisms for NO
actions include the cGMP synthesis cascade, nitrosylation of membrane channels, ADP-
ribosylation, and production of reactive species.64,278 The NO-cGMP pathway involves
soluble guanylate cyclase, which is expressed by NK1-positive spinothalamic neurons,
as well as inhibitory interneurons.73 Fig 8 summarizes key intracellular pathways whose
activation contributes to the generation of central sensitization.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Effectors of Activity-Dependent Central Sensitization

AMPAR and NMDAR phosphorylation during central sensitization increases the
activity/density of these receptors, leading to postsynaptic hyperexcitability.32,86-
88,134,178,345,394,407,408
The first phase of central sensitization is a fast augmentation of
excitatory glutamatergic synapses in the superficial dorsal horn that strengthens
nociceptive transmission and recruits non-nociceptive input to the pathway. This is
achieved by phosphorylation of numerous receptor and ion channel targets that lead to
changes in threshold, channel kinetics, and voltage dependence, as well as a
modification in the trafficking of the receptors to the synapse (Fig 6, A and B). On
noxious stimulation, PKA phosphorylates GluR1 subunits,87,88,214 leading to an insertion
of these receptors into the synapse84,93 and thereby an increase in synaptic strength.20
Phosphorylation of GluR1-containing AMPAR by PKC and CaMKII also increases the
excitability of nociceptive neurons.40,88
NR1 phosphorylation by PKA408 or PKC32,407 participates in the development of
hypersensitivity97,262,345 by increasing the response of NMDARs to glutamate.44,259
Phosphorylation of the NR2B subunit of NMDAR, mediated through Src activation,
increases the opening of the receptor channel268 and prevents endocytosis of activated
receptors by disrupting the binding site of AP-2, a protein involved in clathrin-coated
endocytosic vesicle formation.42 Decoupling Src interaction with NMDAR blocks
NR2B phosphorylation and reduces formalin-induced and inflammatory pain without
altering basal nociceptive pain.178
Activation of PKC contributes to hyperexcitability in nociceptive neurons by several
different pathways. First, PKC reduces the Mg2+ block of NMDAR and increases the
probability of channel opening, facilitating the activated state of NMDAR.44 Second,
activation of PKC decreases inhibitory transmission at the segmental level by reducing
GABA and glycine tonic inhibition174 and the descending inhibition driven from the
PAG.175 Disinhibition, mediated by whatever means, leaves dorsal horn neurons more
susceptible to activation by excitatory inputs including non-nociceptive A-fibers, and is
1 of the major mechanisms triggering and maintaining central sensitization.17,289,341,390
Finally, PKC contributes with PKA to the activation of ERK in a manner that requires
their coactivation and is triggered by the central release of bradykinin.152
Activation of guanylate cyclase seems to be the major way that NO contributes to the
induction of sensitization275,322,403 through increases in neuronal excitability and a
reduction in inhibition,173,176,275 although an NO-mediated activation of ADP-
ribosyltransferase may participate in the maintenance of central sensitization.403
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Global Features of Activity-Dependent Central Sensitization

The key features of acute activity-dependent central sensitization are that it is induced
with a short latency (seconds) by intense, repeated, or sustained nociceptor inputs and
typically lasts for tens of minutes to several hours in the absence of further nociceptor
input. It generally requires activation of NMDA receptors for its induction, and these
receptors contribute to its maintenance. Nevertheless, as reviewed above, multiple
different triggers can contribute to the establishment of this form of central
sensitization: glutamate acting on NMDAR, but also on AMPAR and mGluR, the
neuropeptides substance P and CGRP, the kinin bradykinin, as well as BDNF and NO
(Fig 4). The reason so many different transmitters, modulators, and their receptors are
involved is that it is not their specific action that is important but rather that they are
released directly from or induced in response to nociceptor afferent activity, and each
can separately or together initiate the activation of those multiple intracellular signaling
pathways that lead to the establishment of hyperexcitability in dorsal horn neurons (Figs
6 and and8).8). In other words, there are many parallel inputs to dorsal horn neurons
that can independently or cooperatively initiate central sensitization. Elevation in
intracellular calcium, by whatever means, is 1 major trigger, activating multiple
calcium-dependent kinases that act on receptors and ion channels to increase synaptic
efficacy (Figs 5 and and6).6). Many central sensitization-inducing inputs also activate
ERK, and this MAPK appears to have a pivotal role, contributing to increases in AMPA
and NMDA currents as well as reducing potassium currents (Fig 6, A). However, even
this kinase may not be essential. Other kinases such as PKC, PKA, and Src can,
independent of ERK, also modulate ionotropic receptors to lead to an increase in
synaptic efficacy (Fig 6, A).
What has become clear is that there is no single defining molecular mechanism of
central sensitization but rather that it is a general phenomenon, one that produces
distinct changes in somatosensory processing but nevertheless can be mediated by
several different processes that, in response to nociceptor input, can (1) increase
membrane excitability, (2) facilitate synaptic strength, or (3) decrease inhibitory
influences in dorsal horn neurons. Similarly, the effectors of this plasticity are multiple:
changes in the threshold and activation kinetics of NMDA and AMPA receptors and in
their trafficking to the membrane, alterations in ion channels to increase inward currents
and reduce outward currents, and reductions in the release or activity of GABA and
glycine (Fig 9). These changes produce dramatic alterations in function. However, they
are usually relatively short-lasting and reversible. Phosphatases will dephosphorylate
receptors and ion channels resetting their activity to baseline levels, trafficking to the
membrane will reverse by endocytosis, and, with time, the increased gain of the
nociceptive neurons will fade, at least in the absence of any further triggering
inputs.186,400-402 Different, transcription-dependent changes are required for longer-
lasting effects, and these generally do not occur in response only to nociceptor activity
but are the consequence of peripheral inflammation and nerve injury (see below).
Activity-dependent central sensitization, even though it increases pain sensitivity, is in
most situations an adaptive mechanism. Unlike nociceptive pain, which warns of
potential damage in response to noxious stimuli, central sensitization creates a situation
in which pain is elicited by innocuous stimuli. This change is protective, because it
helps healing by limiting use of an injured body part until the injury is fully repaired.
Central sensitization becomes pathological, however, if inflammation persists, as with
rheumatoid arthritis, in which no healing occurs, and in situations in which central
sensitization becomes autonomous and is maintained in the absence of active peripheral
pathology. Central sensitization represents not only a state in which pain can be
triggered by less intense inputs but in which the central sensitization itself can be
maintained by a lower level or different kind of input. Ongoing activity in C-fibers,
even at levels that do not elicit central sensitization in basal conditions, is sufficient to
maintain central sensitization once it has been induced for prolonged periods (days).153
Furthermore, although nociceptor input is required to trigger central sensitization,
phenotypic changes in myelinated fibers after inflammation and nerve injury can enable
these afferents to acquire the capacity to generate central sensitization (see later).
Figure 9
Multiple cellular processes lead to central sensitization. Central
sensitization is not defined by activation of a single molecular pathway
but rather represents the altered functional status of nociceptive neurons.
During central sensitization, these (more ...)
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Activity-Dependent Central Sensitization and Synaptic Plasticity

That the activity-dependent synaptic plasticity in the dorsal horn responsible for central
sensitization is reversible differs from the permanent activity-dependent synaptic
change in the cortex that leads to long-term memory, long-term potentiation (LTP), in
which the efficacy only of activated synapses is changed. Synaptic changes with some
resemblance to cortical LTP do occur in the spinal cord, that is, a form of homosynaptic
potentiation. However, the major synaptic alteration underlying activity-dependent
central sensitization is heterosynaptic potentiation, in which activity in 1 set of synapses
enhances activity in nonactivated synapses, typically by “sensitizing” the entire neuron,
something that never occurs with cortical LTP.
Homosynaptic potentiation is a type of use-dependent facilitation of a synapse evoked
by activation of that same synapse (Fig 10, A). Classic LTP in the CA1 region of the
hippocampus is formally defined as input-specific homosynaptic facilitation27,164,384 and
is dependent on NMDAR activation, Ca2+ influx, and activation of Ca2+-dependent
intracellular signaling pathways, notably the CaMKII pathway.164 Although the
increased Ca2+ is relatively widespread in neurons after tetanic conditioning stimulation
of afferents,128,182,260 only the stimulated synapse is potentiated.164,260 The development
of LTP by 2 independent synapses using asynchronous pairing stimulation has been
described in the hippocampus, but, once again, only conditioned synapses are
potentiated.123
Figure 10
Homo synaptic and heterosynaptic facilitation. (A), Homosynaptic
potentiation is a form of use-dependent facilitation of a synapse evoked
by activation of that same synapse (in red). A nonconditioned synapse
(green) is not potentiated. This type of potentiation (more ...)
One form of homosynaptic facilitation in spinal cord neurons is windup, in which the
action potential discharge elicited by a low-frequency (0.5 to 5 Hz) train of identical C-
fiber strength stimuli gets larger on each successive stimulus200 (Fig 11). Windup is the
result of the activation of NK1 and CGRP1 receptors after release of substance P and
CGRP from peptidergic nociceptors to produce a cumulative membrane depolarization
from the temporal summation of slow synaptic potentials.290 This then enables
activation of NMDAR by removal of the Mg2+ block, further boosting the responses in a
nonlinear fashion63,72,331,379 (Fig 11). The stimuli that induce windup (repeated C-fiber
stimulation) can lead to central sensitization,379 and, although windup is often
considered to be an aspect of central sensitization, it is instead the reflection of activity-
dependent excitability increases in neurons during a nociceptor conditioning paradigm
rather than changes that follow such inputs, which is when central sensitization
manifests. Windup disappears within tens of seconds of the end of the stimulus train as
the membrane potential returns to its normal resting level (Fig 11).
Figure 11
Action potential windup. Windup is the consequence of a cumulative
membrane depolarization resulting from the temporal summation of
slow synaptic potentials. Under normal conditions, low-frequency
stimulations of C-fibers (0.2 Hz) cause steady neuronal (more ...)
Another form of homosynaptic facilitation occurs in NK1-positive lamina I neurons in
the dorsal horn neuron. This has been termed LTP, although, unlike classic hippocampal
LTP, this form of homosynaptic facilitation appears not to be persistent, or at least the
functional effects on pain sensitivity are not permanent instead lasting, like central
sensitization for a few hours, with no evident change equivalent to long-term memory.
Perhaps, therefore, to avoid confusion with cortical plasticity, the term LTP should be
avoided for homosynaptic potentiation in the spinal cord because the changes in the
dorsal horn are long-lasting (hours) rather than long-term (persistent). The original
description of this long-lasting homosynaptic potentiation in the dorsal horn referred to
an activity-dependent facilitation of excitatory postsynaptic currents in
spinoparabrachial neurons in response to high-frequency (tetanic burst; 100 Hz)
stimulation of C-fibers.127,177,271 The physiological relevance of this phenomenon was
questionable because C-fibers do not fire at such high frequencies. Conditioning C-fiber
stimulation at a low frequency (2 Hz) was subsequently shown also to elicit a long-
lasting homosynaptic potentiation in lamina I spino-PAG neurons but not in
spinoparabrachial neurons.128 This low-frequency potentiation is dependent on
elevations in Ca2+, which activates PLC, PKC, CaMKII, and NOS.128 Capsaicin and
formalin injection also evoke a homosynaptic long-lasting potentiation, as manifested
by an enhancement of C-fiber–evoked synaptic potentials after the capsaicin/formalin
evoked conditioning input.128 Capsaicin is, of course, also a potent inducer of activity-
dependent central sensitization,294,340,383 characterized by the production of secondary
hyperalgesia and tactile allodynia. However, both of these particular forms of pain
hypersensitivity reflect heterosynaptic and not homosynaptic facilitation. Indeed,
heterosynaptic facilitation characterizes most major changes in the receptive field
properties of neurons and in pain sensitivity, in preclinical models, and human
subjects368,369,376 (Fig 10, B).
Interestingly, healthy human subjects receiving high-frequency stimulation of C-fibers
exhibit increased pain in the stimulated region, quite possibly caused by homosynaptic
facilitation, but also show evidence of heterosynaptic facilitation, as manifested by
dynamic mechanical allodynia in adjacent nonstimulated areas.149 The combination of
the homosynaptic potentiation of conditioning nociceptor inputs and the heterosynaptic
facilitation of nonconditioned fibers in the nociceptive pathway constitutes central
sensitization.
Heterosynaptic facilitation represents a form of activity-dependent facilitation where
activity in 1 set of synapses (the conditioning input) augments subsequent activity in
another nonactivated group of synapses (the test input) (Fig 10, B). For homosynaptic
potentiation, the test and conditioning inputs are the same; for heterosynaptic
facilitation they are different. “LTP”-like phenomena in spinobrachial neurons can only
account for the augmentation of the same C-fiber inputs that evoked the facilitation and
cannot contribute to either secondary hyperalgesia or tactile allodynia. Repeated
nociceptor input, such as that generated by capsaicin, will simultaneously generate both
a potentiation of the activated C-fiber synapses (homosynaptic), and, unlike LTP in the
hippocampus, also a potentiation of neighboring nonactivated synapses
(heterosynaptic). It seems likely, therefore, that long-lasting potentiation in projecting
dorsal horn neurons is simply a restricted aspect of the general widespread changes
induced in these neurons by nociceptor activity.
Heterosynaptic potentiation appears to dominate the functional sensory manifestations
of use-dependent central sensitization. After injection of capsaicin, for example, the
thresholds of sensory fibers innervating the area surrounding the injection site are not
modified,23,157,340,365 but pain hypersensitivity in these areas is prominent and depends
on centrally mediated heterosynaptic facilitation. The same argument holds for the
activation of pain in response to tactile stimulation or Aβ fiber inputs during central
sensitization. It is no surprise, then, that spinal “LTP” shares major mechanisms with
central sensitization (NMDAR, Ca2+, kinases, and NO) because it is very likely that the
phenomenon of central sensitization includes both homosynaptic and heterosynaptic
facilitations triggered by the same process; the major difference is that heterosynaptic
potentiation results from the spread of signaling from the conditioning synapse to other
synapses in the neuron182,270,371 (Fig 10, B). Homosynaptic changes will contribute with
peripheral sensitization to primary hyperalgesia,128,270 whereas heterosynaptic
facilitation alone is responsible for secondary hyperalgesia and allodynia.
Although several different forms of LTP have been characterized in the
hippocampus,188,224,384 “spreading” or heterosynaptic LTP has not been reported, even
though release of Ca2+ from intracellular stores can cause a spread of long-term
depression (LTD) to neighboring, unstimulated synapses.227 What, then, is responsible
for heterosynaptic facilitation in dorsal horn neurons? Two major candidates are the
activation of mGluRs and NO. mGluRs are coupled to the Ca2+ channels of the
endoplasmic reticulum85 and play an important role in central sensitization.7
Consequently, the release of intracellular Ca2+ in spinal cord neurons on mGluR
activation may participate in spreading facilitation from conditioned synapses to
neighboring test synapses. NO is also a major effector of spinal cord neuronal
plasticity211,309 and diffuses rapidly from the site of its production to produce multiple
effects at a distance via its downstream signaling pathways, and in this way may
contribute to the heterosynaptic facilitation characteristic of central sensitization. It is
certainly likely that these and other “spreading” signals cooperate to produce the
widespread synaptic facilitation so characteristic to central sensitization. Scaffolding
proteins play a major role in the addressing of specific kinases to the synapse and
represent another potential mechanism for widespread synaptic facilitation. A recent
study has shown that in the hippocampus, CaMKII activation is restricted to the
synaptic bouton of a conditioned synapse, thus only allowing homosynaptic facilitation
at that specific site.164 It is likely in the dorsal horn that CaMKII activation will be much
more widespread and indeed the dendrites of dorsal horn neurons lack synaptic boutons.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Central Sensitization in Pathological Settings

In addition to its role in rapidly and reversibly sensitizing the nociceptive system by
activity-dependent changes in synaptic strength and excitability, central sensitization
also contributes to the longer-lasting and sometimes persistent pain hypersensitivity
present in pathological situations involving inflammation and damage to the nervous
system. The molecular and cellular mechanisms involved include some that are also
responsible for activity-dependent central sensitization and others that are unique to
either inflammation or nerve injury. NMDAR,47,193,255,280,315 AMPAR,180,239 group I
mGluR,7,65,75,92,102,118,221,288,392,405 group II-III mGluR,45,104,194,207,286,398
BDNF,144,179,190,230 SP and CGRP,2,4,163 NO,50,316 and bradykinin241 have all been shown
to contribute both to the development of central sensitization and to pain
hypersensitivity in inflammatory and neuropathic pain models.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Inflammatory Pain
Peripheral inflammation induces a phenotypic switch in primary sensory neurons that
comprises a change in their neurochemical character and properties due to alterations in
transcription and translation. We will only discuss here those changes that relate
specifically to central sensitization by virtue of changes in the synaptic input produced
by the afferents and will not review the major changes that also alter peripheral
transduction sensitivity and membrane excitability (peripheral sensitization), although
of course, anything that increases nociceptor afferent input will also indirectly lead to
increased central sensitization. Large DRG neurons begin, unlike in their naive
condition, to express SP and BDNF when their peripheral terminals are exposed to
inflammatory signals and nerve growth factor (NGF).191,223 Consequently, activation of
the myelinated fibers by low-intensity innocuous stimuli now releases these
neuropeptides in the spinal cord, and conditioning stimulation of the afferents acquires
the capacity to generate central sensitization, something they normally cannot
do185,190,223 (Fig 12). After peripheral inflammation, Aβ-mediated synaptic input to
superficial dorsal horn neurons is substantially increased from the very low levels found
in noninflamed animals.16 TrkA-expressing nociceptors, instead of a phenotypic switch,
begin to express higher levels of neuropeptides and other NGF-dependent proteins as a
result of exposure to the increased NGF produced by inflammation.370,375
Figure 12
Central sensitization in pathological settings. A, Representation of the
superficial lamina of the dorsal horn of the spinal cord. Nociceptive
peptidergic fibers contact lamina I and II outer (I and IIo) neurons that
express GluR2-containing AMPAR (in (more ...)
A critical central pathway for the generation of inflammatory pain hypersensitivity

involves induction of cyclooxygenase-2 (Cox-2) in dorsal horn neurons, to drive
production of prostaglandin E2 (PGE2).269,349 PGE2 binds to its EP2 GPCR on dorsal
horn neurons to potentiate AM-PAR and NMDAR currents,152 activate nonselective
cation channels,18 and reduce in inhibitory glycinergic neurotransmission by blocking
glycinergic receptors with α3 subunits9,111,152,213 (Fig 12). PGE2 also acts on EP4
receptors on presynaptic terminals to increase transmitter release.350 The importance of
the central neuronal induction of COX-2 to inflammatory hyperalgesia is revealed by
conditional deletion of COX-2 only in neurons, which results in the retention of
peripheral inflammation and heat hyperalgesia but an almost complete loss of
mechanical pain hypersensitivity.350
Under normal conditions, microglia are the only immunocompetent cells of the nervous
system66,362 and constantly probe or survey the CNS parenchyma to maintain
homeostasis.62,225 After peripheral inflammation, some spinal cord microglial cells
change their shape, function, and chemical expression.115,258,311,312 In particular, p38
MAPK is activated311,312 and leads to the synthesis and release of pro-inflammatory
cytokines,115,258 among which, IL-1β and TNF-α contribute to the development of
central sensitization by enhancing excitatory and reducing inhibitory currents and by
activating induction of COX-2142,269 (Fig 12).
Neurons in the superficial lamina of the dorsal horn usually display a GluR2 AMPAR
phenotype (ie, are Ca2+-impermeable)252; however, peripheral inflammation triggers a
shift from GluR2/3 to GluR1-containing AMPARs at the membrane159,238,355 (see “PSD
Proteins and AMPAR Recycling and Subunit Switch,” below). Under these conditions,
activation of AMPAR elicits entry of Ca2+, which can then participate in the activation
of the signaling pathways that drive central sensitization. Ca2+-permeable AMPARs
appear to be a major source of the [Ca2+]i increase in inflammatory pain, generating as
much Ca2+ influx as with NMDAR activation.182 The functional state of NMDAR is
also modified in response to peripheral inflammation, with phosphorylation of NR2B
subunits by Src resulting in increased activity of the receptors106,107,178 and in their
maintenance at the synapse.42 Finally, peripheral inflammation also promotes group I
mGluR insertion into the membrane (mGluR5) and closer to the synapse (mGluR1),
thereby further clustering these receptors at the synapse.247
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Neuropathic Pain
After peripheral nerve injury, damaged and nondamaged A- and C-fibers begin to
generate spontaneous action potentials. Because these do not arise from the peripheral
terminal, it is a form of ectopic input.70,74 Such input in C-fibers can initiate and then
maintain activity-dependent central sensitization in the dorsal horn.154 However,
because of chemical and structural changes in A fibers,56,229,386 input in these afferents
can also begin to drive central sensitization.69 Injured, and to a much lesser extent,
noninjured sensory neurons in the dorsal root ganglion exhibit a massive change in
transcription that alter their membrane properties, growth, and transmitter
function.56,231,232,386 These changes are much greater than those that occur in response to
peripheral inflammation, where only a few tens of transcripts are altered in the DRG195
and involve altered expression of about 1000 transcripts, including ion channels,
receptors, transmitters, and the molecular machinery necessary for axon
regeneration.56,261 Among the many changes, large fibers begin to express new
transmitters and neuromodulators including substance P and BDNF and the synthetic
enzymes for tetrahydrobiopterin, an essential cofactor for NOS. Stimulation of non-
nociceptive fibers now triggers release of factors that can drive central
sensitization.24,56,91,229,327,386
Structural changes also contribute to altered synaptic function. Peripheral nerve injury
leads to a transganglionic degeneration of C-fiber terminals in lamina II.13,136 This loss
of presynaptic input, together with the triggering of increases in the intrinsic axonal
growth capacity as part of the regenerative response of the injured neurons, provides an
opportunity and the molecular means for myelinated A-β fibers to sprout from laminae
III-IV into laminae I-II and make contact with nociceptive-specific
neurons.166,191,285,377,378 The original experiments describing the sprouting phenomenon
were conducted using cholera toxin B subunit as a selective tracer for A-fibers as well
as single axonal label with HRP. The selectivity of this toxin after peripheral nerve
injury is somewhat controversial.125,339 Nevertheless, immunostaining for c-Fos
activation and electrophysio-logical recordings have clearly established that peripheral
nerve injury causes large myelinated fibers to begin to drive nociceptive neurons in
superficial lamina.24,151,235,366
A reduction in the synthesis, release, or activity of inhibitory transmitters leads to a state
of disinhibition, whose net functional effects are very similar to that produced by
increases in synaptic strength of excitatory synapses and in membrane
excitability.289,341,390 In neuropathic pain states, there is substantial disinhibition in the
superficial dorsal horn with loss of GABAergic and a reduction in glycinergic inhibitory
currents210 that can be attributed, at least in part, to apoptosis of inhibitory
interneurons.277 This neuronal death appears to be the result of an NMDAR-induced
excitotoxicity that develops over time rather than to the large amount of glutamate
released centrally at the time of nerve injury.277 One laboratory failed to find significant
loss of neurons or of GA-BAergic content in the dorsal horn of neuropathic pain animal
models.249-251 The reasons for this discrepancy are not clear but may reflect technical
differences in how the studies were performed. Interestingly, the reduction in
glycinergic neurotransmission caused by the activation of EP2 receptors after peripheral
inflammation does not appear to operate after nerve injury, further indicating that some
inflammatory and neuropathic pain mechanisms differ.117
Another mechanism contributing to the reduction in segmental inhibition in a
subpopulation of lamina I neurons in the spinal cord after nerve injury is dependent on
BDNF effects on an anion transporter, changing anion gradients across neuronal
membrane to alter the inhibitory efficacy of GABA. Under normal conditions, the
intracellular concentrations of Cl- are maintained by the opposed effects of Cl--
cotransporter K+-Cl- exporter 2 channels (KCC2) and Na+-K+-Cl- exporter 1 channels
(NKCC1). KCC2 drives Cl- ions out of the cells (along with K+) and NKCC1 is
responsible for an influx of K+, Na+, and Cl- into the cells. The net effect of these 2 co-
transporters is a steady-state Cl- concentration gradient in which opening of Cl- channels
(such as GABAA receptors) causes entry of Cl- into the neuron and hyperpolarizes the
neurons. After peripheral nerve injury, BDNF released by activated microglial cells
results in a reduction of KCC2 expression in a subset of neurons in the superficial
lamina of the dorsal horn.57,58,203 Consequently, activation of GABAA receptors by
GABA result in a diminution or absence of Cl- entry into the cell and thus a
disinhibition of these nociceptive neurons57,58,179,203 (Fig 12). As after peripheral
inflammation, there is also an increase in descending excitatory controls from the RVM
in the brainstem after peripheral nerve injury, as well as a reduction of descending
inhibitory controls.60,95,351,356
Peripheral nerve injury causes a massive activation of, and change in, glial cells in the
spinal cord as well as infiltration of peripheral immune-competent cells, notably
macrophages and T-cells.38,317,360 The extent and duration of the changes in microglia
and astrocytes is much greater than in response to peripheral inflammation. Activated
microglia produce and release trophic factors, neurotransmitters, cytokines, and reactive
oxygen species263,361 and appear to play an essential step in the development of pain
after nerve injury by triggering central sensitization through their interaction with
neurons.133,160,162,201,206,256,257,352 Numerous signals trigger microglial activation and
recruitment, including ATP and NO,62,81,225 cytokines, and chemokines, some of which
are released by injured sensory neurons and others by microglial cells themselves or by
astrocytes and T-cells.1,3,66,76,204,348,361,362 Release of cytokines by microglia increases
neuronal excitability through activation of ERK and CREB.131,142 Activated microglia
also release BDNF and NO,58,116 promoting segmental disinhibition.57 Finally, microglia
can also provoke neuronal death by producing ROS, pro-apoptotic cytokines such as
TNF,121 and by a diminished glutamate uptake.48,326,332 T-cells produce specific
cytokines such as IFN-γ, which reduce GABAergic currents in the dorsal horn354
through activation of IFN-γ receptors353 and also activate and recruit microglia.
Astrocytes also become activated after peripheral nerve injury,98,131,205 with a slower
onset and more prolonged time course than microglia, and may play more of a role in
the maintenance of neuropathic pain hypersensitivity than microglia.94,399,406 What
seems clear is that multiple different mechanisms operate after nerve injury to increase
excitability and reduce inhibition.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Scaffolding Proteins, Synaptic Plasticity, and Central Sensitization During

Inflammation and After Nerve Injury
The proteins that make up the PSD can drive a major functional reorganization of
synapses, modifying post-synaptic efficacy by altering receptor density at the membrane
and producing switches from Ca2+-impermeable to Ca2+-permeable AMPARs (Fig 13).
The PSD is not simply a structural landmark of the synapse but contains elements
essential both for the formation of the synapse and for changes in its properties.
Absence of scaffolding proteins or specific disruption of their binding sites results in a
dramatic reduction in synaptic plasticity because the proteins contribute both to
transcriptional and post-translational events. They initiate signaling cascades that lead to
the activation of transcription factors, traffic newly synthesized receptors to the PSD,
and “address” kinases and phosphatases to specific receptors in a stimulus-dependent
manner. Although the involvement of the PSD in synaptic plasticity in the cortex is
much better established than in the spinal cord, there is increasing evidence for a major
role for the PSD in changing synaptic efficacy in response to peripheral inflammation
and nerve injury.
Figure 13
Role of scaffolding proteins in central sensitization. Representation of
the post synaptic density (PSD) region of a synapse of a nociceptive
neuron in the superficial lamina of the spinal cord under basal
conditions (A) and during inflammatory pain (more ...)
The PSD consists of cytoskeletal proteins, signaling molecules, membrane receptors,
and scaffolding proteins.234 Scaffolding proteins are families of proteins characterized
by their ability to interact with numerous partners, and these proteins form the dense
molecular structure of the postsynaptic component of the synapse. A particularly
abundant component of the PSD are proteins containing a specific peptidergic domain
called PDZ, which is named after the protein in which the sequence was first identified
(postsynaptic density protein 95 [PSD-95]/discs large/zonula occludens 1). This family
of proteins includes, among hundreds of members, the 4 membrane-associated
guanylate kinases (MAGUK): PSD-95, PSD-93, synapse associated protein (SAP)-97,
SAP-102. The MAGUKs represent the most abundant scaffolding protein family in the
PSD234 and are characterized by 3 PDZ domains, an Src homology region (SH3)
domain, and a guanylate kinase-like (GK) domain,148 making them central elements of
the synapse scaffold. The prime binding protein for the MAGUK family is the NMDAR
subunit NR2,155 but it also binds to the transmembrane AMPAR regulatory proteins
(TARPs),43 nonreceptor tyrosine kinases,329 nNOS,30,31 GKAP,217 and AKAP79/150.53
MAGUKs can be seen as the functional scaffold of the PSD and are essential for the
structural integrity of synapses but also modulate the insertion of glutamate receptors
into the synapse and physically bring together key enzymes to the PSD.59,148,167,197,265
Knock-down of PSD-95 and PSD-93, as well as targeted mutagenesis of the residues
required for their protein:protein interaction, both prevent and reduce central
sensitization in normal conditions321 as well as in inflammatory319,323,397 and neuropathic
pain models99,320,323,397 but do not alter nociception or locomotor functions.319-321,323
Another member of the PDZ family, stargazin, is a 4-transmembrane domain protein
whose putative secondary structure is close to the Ca2+-channel γ subunit, and was
named γ2.170 Stargazin however, does not play an important role in neuronal Ca2+
channels170 but is instead highly concentrated in the PSD and co-immunoprecipitates
with GluR1, 2, and 4.43,335 The protein is a major AMPAR partner, along with the 4
other isoforms, γ3, γ4, γ7, and γ8,140,335 which form the TARP subgroup.335 Stargazin
traffics AMPAR from the endoplasmic reticulum to the extrasynaptic membrane.43,334
Once stargazin and AMPAR are addressed to the extrasynaptic membrane, their
recruitment to the synapse requires interaction of the C-terminus segment of stargazin
with PSD-95.21,276 Activity-dependent phosphorylation of stargazin by PKC and
CaMKII342 produces a massive insertion of AMPAR into the membrane,337 whereas
stargazin's dephosphorylation by PP1 or PP2B reduces the number of AMPAR at the
synapse.337 In addition, via the interaction between stargazin's ectodomain and the
glutamate binding region of AMPAR, stargazin modulates the activity of AMPAR by
slowing channel deactivation and desensitization and increasing the affinity of the
receptors for glutamate,49,334,336 thereby potentiating synaptic strength. Disruption of
stargazin in the spinal cord inhibits the second phase of formalin-induced pain and
reduces the heat hyperalgesia caused by intraplantar CFA injection.318 Recently,
cornichon homolog 2 (CNIH-2) and cornichon homolog 3 (CNIH-3) have been found to
be novel partner proteins for AMPAR in the CNS.279 Cornichon proteins bind to GluR1-
4 AM-PAR and, as for stargazin, they promote AMPAR surface expression and slow
their deactivation kinetics.279 Because their expression in the CNS is estimated to be in
70% of neurons, and because cornichon and stargazin appear to be mutually
exclusive,279 the determination of their presence in spinal cord neurons and their role in
central sensitization is something that needs to be investigated.
EphrinB-ephBR receptor interactions participate in NMDAR clustering at the PSD.90
EphBRs are receptor tyrosine kinases expressed by postsynaptic neurons, whereas
EphrinB is anchored to the presynaptic membrane.156 The kinase activity of EphBR is
not required for the initial clustering of NMDAR at the synapse but is essential for their
maintenance.61 Stimulation of EphBR potentiates NMDAR-induced Ca2+ influx and the
phosphorylation of CREB through the activation of the nonreceptor tyrosine kinase
src314 and recruits CaM-KII to the synapse236 to increase NMDAR activity.314
Activation of EphBR in the spinal cord induces thermal hyperalgesia (but not
allodynia),22,299 without modifying nociception.22 Inhibition of EphRB prevents or
reverses inflammatory22,291 and neuropathic150,299 pain and prevents establishment of
NMDAR-induced spinal cord “LTP.”299 More specifically, targeted disruption of the
coupling between EphRB-activated Src and NR2B also prevents the development of
central sensitization without altering basal nociceptive transmission.178 In addition,
sustained nociceptive activity leads to an upregulation and reorganization of presynaptic
EphB increasing EphB-EphRB interaction.22,298,299
The nonreceptor tyrosine kinase family also includes Fyn, which phosphorylates NR2
subunits268 and binds to PSD-93 to phosphorylate NR2A/B273 and could play a role in the
maintenance of neuropathic pain.5
NMDA receptors are structurally connected with group I metabotropic glutamate
receptors through a complex composed of PSD-95, GKAP, Shank1, and Homer1b/c.
GKAP binds to the GK domain of PSD-95147,217 and recruits Shank1 to the PSD via
their PDZ domain.216,264 Shank1 then binds to the EVH1 domain of homer1b/c.343
Homer1b/c proteins have a coiled-coil structure in their C-terminus region that enables
them to form homotetramers or heterotetramers.284 This assembly of Homers leaves 3
available EVH1 domains that can bind several other targets such as group I mGluRs
(but not group 2 or 3 mGluRs),28 inositol triphosphate receptors (IP3R), or the actin
cytoskeleton.284,344 The interaction between Homer1b and IP3R and between Shank1
and Homer1b/c controls local Ca2+ release from the endoplasmic reticulum upon
mGluRs activation.266,267 Homer1a, a short isoform of Homer1b/c that lacks the coiled-
coil structure, is an immediate early gene activated on neuronal activity and participates
in remodeling synapses in an activity-dependent manner.129 Homer1a is upregulated in
dorsal horn neurons after the subcutaneous injection of formalin or CFA324 as well as
transiently after peripheral nerve injury.208 Factors responsible for Homer1a activation
include NMDAR, ERK1/2 and Src.208,324 Knock-down of Homer1a increases pain-like
behaviors specific to central sensitization and not those associated with peripheral
sensitization,324 whereas overexpression reduced inflammatory pain-like behavior
without altering basal nociception.324 Homer1a probably causes a disruption of the
clustering properties of Homer1b/c protein and of Ca2+ release on NMDAR or mGluR
activation.324 In contrast, homer1b activation leads to an increase of AMPAR activity
after the stimulation of mGluR, whereas activation of Homer1a inhibits this.347 In
addition, Homer1b/c proteins could play an important role in clustering group 1
mGluRs at the synapse after CFA injection,247 an effect that is reduced by Homer1a
overexpression.385 Homer proteins are, therefore, convergent factors that potentially link
major glutamate receptors with Ca2+ stores in neurons, and the balance between
Homer1b/c and Homer1a may play an important role in the development and
maintenance of central sensitization.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
PSD Proteins and AMPAR Recycling and Subunit Switch

Stargazin may be important in creating the switch from GluR2- to GluR1-containing
AMPAR in response to peripheral inflammation159,355 by specifically addressing GluR1-
containing AMPARs to the synapse and then strengthening their activity via its
ectodomain.334 Peripheral inflammation leads via PKA to a phosphorylation of Ser831
and Ser845 on GluR1 in the spinal cord,180 which, in association with CaMKII
activation, promotes a transfer of GluR1-containing AMPARs to the membrane,84
where they are maintained by synaptic activity.189
GluR2-containing AMPARs are associated with high affinity to GRIP-1, which clusters
the receptors at the synapse,77,114 whereas PICK-1 is a critical element for activity-
dependent endocytosis of the receptor.51,96,226 NMDAR activation leads to a PKC-
mediated phosphorylation of GluR2 at Ser880,172,238 which decreases GluR2's affinity
for GRIP-1, whereas the increase in intracellular Ca2+ recruits PICK-1 to the
synapse,51,108,238 where it reduces the clustering of GluR2-containing AM-
PAR51,172,196,238,304,328 (Fig 14). On endocytosis, vesicles can either be inserted back into
the synapse under the action of NSF,122,226,228,297 which disrupts the [GluR2-PICK-1]
complex,109 or be maintained out of the synapse through PICK-1.172 During
inflammation, NSF expression is reduced in the spinal cord,139 thereby preventing
GluR2-containing AMPAR reinsertion into the synapse.
Figure 14
Synaptic scaffolding proteins and the switch to GluR1-containing
AMPAR after peripheral inflammation. Under basal conditions, GluR2-
containing AMPARs are associated in the synapse with stargazin and
GRIP-1. The C-terminus of stargazin binds to PSD-95 (more ...)
The net effect of these complex changes is an increase in GluR1 and a decrease in
GluR2 containing AMPAR at the synapse (Fig 14). Once inserted into the synapse,
GluR1-containing AMPAR remain there for as long as there is glutamate release,83,189
and their activation is potentially increased by stargazin's ectodomain, thereby further
promoting the Ca2+-dependent pathways required for maintenance of central
sensitization. In addition, the phosphorylation of stargazin's C-terminus domain by PKC
and CaMKII342 increases its affinity for PSD-95, thereby re-enforcing the clustering of
AMPAR with NMDAR at the synapse.276
After peripheral nerve injury, GluR2 and GRIP-1 are up-regulated in dorsal horn
neurons,100,110 whereas PICK-1 expression is not modified and NSF is
downregulated.100 Peptides that disrupt the binding of GRIP-1 or NSF with GluR2
partially decrease neuropathic pain-like behaviors.100 PICK-1 is also required for Gi/o-
coupled mGluR7 trafficking to the membrane.306 Activation of mGluR7 can block P/Q-
type Ca2+ channels240 and reduces the pain caused by injection of capsaicin218 or
peripheral nerve injury.237 The upregulation of GRIP-1 but not of PICK-1 in
neuropathic pain models would promote AM-PAR maintenance at the synapse but not
that of mGluR7, resulting in increased excitability in these cells.
Finally, the A kinase-anchoring protein 79/150 (AKAP79/150) binds to PSD-9553 and is
a scaffold for protein kinases and phosphatases,53,169 specifically trafficking enzymes
within the PSD to increase (kinases) or reduce (phosphatases) synaptic transmission.
When AKAP79/150 recruits PKA181,295 or PKC325 in the PSD, it promotes insertion of
new AMPAR to the membrane (via PKA) as well as increasing their activity (via PKC).
In contrast, recruitment of PP2B25,169 triggers AMPAR endocytosis.25 AKAP79/150
may function therefore as a “master switch” of central sensitization by promoting
phosphorylation or dephosphorylation of stargazin and AMPAR.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
Conclusion
Before central sensitization was discovered, there were 2 major models of pain. The
first was that it was a labeled-line system, in which specific “pain pathways” were
activated only by particular peripheral “pain stimuli” and that the amplitude and
duration of pain was determined solely by the intensity and timing of these inputs. The
second model evoked “gate controls” in the CNS, which, by opening or closing, enabled
or prevented pain. Neither model envisaged, however, that pain may arise as a result of
changes in the properties of neurons in the CNS: central sensitization. We now
appreciate that there are indeed specific nociceptive pathways and that these are subject
to complex facilitating and inhibitory controls; both models were in part correct. We
also know though, that changes in the functional properties of the neurons in these
pathways are sufficient to reduce pain threshold, increase the magnitude and duration of
responses to noxious input, and permit normally innocuous inputs to generate pain
sensations. Pain is not then simply a reflection of peripheral inputs or pathology but is
also a dynamic reflection of central neuronal plasticity. The plasticity profoundly alters
sensitivity to an extent that it is a -major contributor to many clinical pain syndromes
and represents a major target for therapeutic intervention. The past 26 years have seen
enormous advances both in our appreciation of the nature and manifestations of central
sensitization and in its underlying molecular mechanisms. We have great insight into
what triggers can induce central sensitization, through which signaling pathways and by
means of which effectors. The complexity is daunting because the essence of central
sensitization is a constantly changing mosaic of alterations in membrane excitability,
reductions in inhibitory transmission, and increases in synaptic efficacy, mediated by
many converging and diverging molecular players on a background of phenotypic
switches and structural alterations. Nevertheless, enormous progress has been made in
dissecting out where, when, and how the plasticity occurs, although clearly, more is still
waiting to be learned.
Acknowledgments
Supported by the National Institutes of Health and the Fondation pour la Recherche
Médicale.
Footnotes
Editor's Note: This article is 1 in a series of invited Critical Review articles designed to
celebrate The Journal of Pain's 10th year anniversary of publication.
o Abstract
o Inflammatory Pain
o Neuropathic Pain
o Conclusion
o References
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Pain
Pain: an overview
John D Loeser, Ronald Melzack
Until the 1960s, pain was considered an inevitable sensory response to tissue damage. There was little room for the
affective dimension of this ubiquitous experience, and none whatsoever for the effects of genetic differences, past
experience, anxiety, or expectation. In recent years, great advances have been made in our understanding of the
mechanisms that underlie pain and in the treatment of people who complain of pain. The roles of factors outside the
patient’s body have also been clarified. Pain is probably the most common symptomatic reason to seek medical
consultation. All of us have headaches, burns, cuts, and other pains at some time during childhood and adult life.
Individuals who undergo surgery are almost certain to have postoperative pain. Ageing is also associated with an
increased likelihood of chronic pain. Health-care expenditures for chronic pain are enormous, rivalled only by the costs
of wage replacement and welfare programmes for those who do not work because of pain. Despite improved knowledge
of underlying mechanisms and better treatments, many people who have chronic pain receive inadequate care.
Concepts of pain of the body upon which sensory data are played.6–8
In 1965, the Melzack-Wall gate control theory1 Perception of pain is, thus, generated by the output or the
emphasised the mechanisms in the central nervous neuromatrix as a function of sensory inputs that feed into
system that control the perception of a noxious stimulus, it, together with information from regions of the brain
and thus integrated afferent, upstream processes with involved in affective and cognitive activities. Moreover,
downstream modulation from the brain. However, this pain behaviours can be generated or perpetuated by
theory did not incorporate long-term changes in the previously conditioned cues in the environment or by
central nervous system to the noxious input and to other the expectation of pain and suffering. The figure shows
external factors that impinge upon the individual. It is how the widely distributed, parallel-processing neural
now widely recognised that nociceptor function is altered networks in the central nervous system generate the nerve
by the “inflammatory soup” that characterises a region of impulse patterns that produce the various somatic
tissue injury.2 experiences, including transient, acute, and chronic pain.
This dynamic feature is usually temporary and exists The output of the neuromatrix can be modified by
while the body heals a region of injury. The modifiers of multiple forms of treatment to change the inputs and
nociceptor activity are also transient. However, Dubner influences on the neuromatrix.
and Ruda3 showed that huge nociceptive input can— Injury does not only produce pain, it also leads to
through the mechanism of excitatory toxic effects of stress, which the body attempts to deal with by achieving
aminoacids—permanently change spinal-cord function homoeostatis. This process involves neural, hormonal,
and could, therefore, lead to chronic pain after an acute and behavioural activities derived from genetically
injury. determined repertoires.9 These homoeostatic repertoires
Physiological and behavioural studies have shown that exist at the injury site (cytokine release, for example), in
plasticity, or learning, has a role in pain. Hence, synaptic the adrenal cortex (release of corticosteroids), the
potentiation is facilitated by repetitive noxious immune system, and in widely distributed areas in the
stimulation, and, at the level of the brain, environmental brain.
influences alter the response to noxious stimulation.4,5 It
is clear from clinical pain states that the brain can Components of pain
generate pain in the absence of input from the peripheral The best definition of pain is that endorsed by the
nociceptors or the spinal cord—for example, in phantom International Association for the Study of Pain: “Pain is
limb pain and pain perceived by paraplegic patients below an unpleasant sensory and emotional experience
the level of a total spinal cord transection. A pattern- associated with actual or potential tissue damage, or
generating mechanism, or neuromatrix, must, therefore, described in terms of such damage”.10 The existence of
exist in the brain that is capable of sustaining an image many types of pain can be understood by the
identification of four broad categories: nociception,
Lancet 1999; 353: 1607–09
perception of pain, suffering, and pain behaviours.11
See Commentary page 1546
Behind each of these clinical categories are anatomical,
Department of Neurological Surgery, University of Washington, physiological, and psychological substrates.
Box 356470, Seattle, WA98195, USA (J D Loeser MD); and
Department of Psychology, McGill University, Montreal,
Nociception—is the detection of tissue damage by
Quebec, Canada
specialised transducers attached to A delta and C fibres.
Correspondence to: Dr John D Loeser These transducers may be biased by inflammatory and
(e-mail: jdloeser@u.washington.edu)
neural changes in their immediate environment. Aspirin,
THE LANCET • Vol 353 • May 8, 1999 1607

Patern-generating mechanism or neuromatrix modulated by multiple inputs and the internal milieu
Adapted from Melzack and Loeser7 and Melzack.9
acetaminophen, and non-steroidal anti-inflammatory Pain behaviours—result from pain and suffering and are
drugs act to change this “inflammatory soup” and the things a person does or does not do that can be
produce pain relief mainly by the restoration of ascribed to the presence of tissue damage. Examples of
nociceptive sensitivity to its resting state. Local and pain behaviours include saying “ouch”, grimacing,
regional anaesthesia can prevent nociception from limping, lying down, recourse to health care, refusing to
becoming pain, but so can downstream modulation, as work. Such behaviour is observable by others and can be
proposed in the Melzack-Wall theory.1 Such issues are quantified. All these behaviours are real and are also
routinely ignored by physicians. probably influenced by actual or expected environmental
consequences. From pain behaviours and the history
Perception of pain—is frequently triggered by a noxious and physical examination, we infer the existence of
stimulus, such as an injury or disease. Pain can also be nociception, pain, and suffering.
generated by lesions in the peripheral or central nervous
system, as seen in patients with diabetic neuropathy, Types of pain
spinal-cord injury, or stroke. When acute pain occurs, it Transient pain—is elicited by the activation of nociceptive
is initially associated with specific autonomic and somatic transducers in skin or other tissues of the body in the
reflexes, but these disappear in patients with chronic absence of any tissue damage. The function of such pain
pain. Many physicians and patients do not realise that to the individual is related to its speed of onset after
pain can occur without nociception. Furthermore, pain stimulation is applied and the speed of offset that
due to nerve injury does not respond to analgesics such as indicates that the offending physical disturbance is no
morphine as efficiently as pain caused by tissue damage, longer impinging upon the body. Presumably, transient
indicating the complex relation between injury and pain. pain evolved to protect man from physical damage by the
environment or by over stress of the body tissues. This
Finally, the intensity of chronic pain frequently bears
type of pain is ubiquitous in everyday life and is rarely a
little or no relation to the extent of tissue injury of other
reason to seek health care. In the clinical setting, transient
quantifiable pathology.
pain is seen only in incidental or procedural pain, such as
during a venepuncture or injection for immunisation.
Suffering—is a negative response induced by pain and Although the prevention of procedural pain is worthy of
also by fear, anxiety, stress, loss of loved objects, and study, it is not a major issue in clinical medicine.
other psychological states. The language of pain is used However, transient pain, which was the subject of most of
to describe suffering, irrespective of the cause, and thus the physiological experimentation during the first 75
misleads both the doctor and the patient as to the basis of years of this century, remains the common conceptual
the suffering. As stated by Cassell,12 “Suffering occurs model in health care.
when the physical or psychological integrity of the person
is threatened”. Not all suffering is caused by pain, but in Acute pain—is elicited by substantial injury of body tissue
our medicalised culture we describe suffering in the and activation of nociceptive transducers at the site of
language of pain. local tissue damage. The local injury alters the response
1608 THE LANCET • Vol 353 • May 8, 1999

characteristics of the nociceptors, their central more importantly, the inability of the body to restore its
connections, and the autonomic nervous system in the physiological functions to normal homoeostatic levels.
region. The local injury does not overwhelm the body’s
reparative mechanisms: “healing” can occur without Conclusions
medical intervention. The report of pain stops long The brain contains widely distributed neural networks
before healing has been completed. However, medical that create an image of self through genetic programmes
interventions may be useful in two respects: to prevent or and memories of past experience. Afferent inputs act on
reduce pain and to speed up the healing process by this neuromatrix and produce output patterns that lead to
shortening the duration of the injury. Most individuals the report of pain. Stress can change the interactions
with acute pain seek medical care. This type of pain is between the neuromatrix and peripheral stimuli, as can
also seen after trauma, surgical interventions, and some learned experiences and expectations. Clinically
diseases. Since the healing process usually takes a few significant acute pain always involves issue damage; the
days or a few weeks, pain that persists for months or years
central and peripheral nervous systems are dynamic, not
is not classified as acute. However, in malignant diseases
static, and are modulated by tissue damage and by
the invasion of body tissues can produce continuous
changes in the central nervous system and stress-
acute pain.
regulation systems that occur in response to such
damage. Although most of these modulations are of short
Chronic pains—such as low back pain, postherpetic duration, some may persist and lead to chronic pain
neuralgia, and fibromyalgia, are commonly triggered by states. In both clinical and basic research, we are rapidly
an injury or disease, but may be perpetuated by factors gaining useful information that will lead to more effective
other than the cause of the pain. The injury may exceed care for those who suffer pain.
the body’s capability for healing, because of the loss of
the body part, the extensiveness of the trauma and References
subsequent scarring, or the involvement of the nervous 1 Melzack R, Wall PD. Pain mechanisms: a new theory. Science 1965;
system in the injury itself. The nervous system may be 150: 971–79.
2 Levine J, Taiwo Y. Inflammatory pain. In Wall PD, Melzack R, eds.
damaged by the original injury in such a way as to be Textbook of pain. Edinburgh: Churchill Livingstone, 1994: 45–56.
unable to restore itself to a normal state. In addition to 3 Dubner R, Ruda MA. Activity-dependent neuronal plasticity following
chronic pain syndromes, in which the intensity of the pain tissue injury and inflammation. Trends Neurosci 1992; 15: 96–103.
is out of proportion to the original injury or tissue 4 Gracely RH, Lynch SA, Bennett GH. Painful neuropathy: altered
central processing maintained by peripheral input. Pain 1992; 51:
damage, other syndromes may occur spontaneously in the 175–94.
absence of any sign of injury. All types of chronic pain 5 Rainville P, Duncan GH, Price DD, Bushnell MC. Selective
lead people to seek health care, but they are often not modulation of pain unpleasantness alters activity in the human
treated effectively. Because chronic pain is unrelenting, it cerebral cortex. Neurosci Abstr 1996; 614: 2.
is likely that stress, environmental, and affective factors 6 Melzack R, Loeser JD. Phantom body pain in paraplegics: evidence
for a central “pattern generating mechanism” for pain. Pain 1978;
may be superimposed on the original damaged tissue and 4: 195–210.
contribute to the intensity and persistence of the pain. 7 Melzack R. Phantom limbs and the concept of a neuromatrix. Trends
Chronic pain differs from acute pain because therapies Neurosci 1990; 13: 88–92.
that provide only transient pain relief do not resolve the 8 Coderre T, Katz J, Vaccarino AL, Melzack R. Contribution of central
neuroplasty to pathological pain: review of clinical and experimental
underlying pathological process. Chronic pain will evidence. Pain 1992; 42: 259–85.
continue when treatment stops. Since the cause of a 9 Chrousos GP, Gold PW. The concepts of stress and stress system
person’s perception of pain may persist irrespective of disorders. JAMA 1992; 267: 1244–52.
medical treatments, psychological forms of therapy such 10 Merskey H, Bogduk N. Classification of chronic pain. Seattle:
International Association for the Study of Pain Press, 1994: 210.
as cognitive and behavioural treatments can be used to 11 Loeser JD. Perspectives on pain. In Turner P, ed. Procedings of the
alter the effect of the pain on the individual’s life. It is First World Congress on Clinical Pharmacology and Therapeutics.
possible that, just as the brain is modified by experience, London: Macmillan, 1980: 316–26.
especially in early life, the brain may be capable of 12 Cassell EJ. The nature of suffering and the goals of medicine. N Engl
J Med 1982; 306: 639–45.
altering the way pain-producing information is processed
13 Melzack R. Pain and stress: a new perspective. In Gatchel RJ,
to keep its impact to a minimum.13 It is not the duration Turk DC, eds. Psychological factors in pain. New York: Guilford
of pain that distinguishes acute from chronic pain but, Press, 1998.

Pain
Pain: an overview
John D Loeser, Ronald Melzack
Until the 1960s, pain was considered an inevitable sensory response to tissue damage. There was little room for the
affective dimension of this ubiquitous experience, and none whatsoever for the effects of genetic differences, past
experience, anxiety, or expectation. In recent years, great advances have been made in our understanding of the
mechanisms that underlie pain and in the treatment of people who complain of pain. The roles of factors outside the
patient’s body have also been clarified. Pain is probably the most common symptomatic reason to seek medical
consultation. All of us have headaches, burns, cuts, and other pains at some time during childhood and adult life.
Individuals who undergo surgery are almost certain to have postoperative pain. Ageing is also associated with an
increased likelihood of chronic pain. Health-care expenditures for chronic pain are enormous, rivalled only by the costs
of wage replacement and welfare programmes for those who do not work because of pain. Despite improved knowledge
of underlying mechanisms and better treatments, many people who have chronic pain receive inadequate care.
Concepts of pain of the body upon which sensory data are played.6–8
In 1965, the Melzack-Wall gate control theory1 Perception of pain is, thus, generated by the output or the
emphasised the mechanisms in the central nervous neuromatrix as a function of sensory inputs that feed into
system that control the perception of a noxious stimulus, it, together with information from regions of the brain
and thus integrated afferent, upstream processes with involved in affective and cognitive activities. Moreover,
downstream modulation from the brain. However, this pain behaviours can be generated or perpetuated by
theory did not incorporate long-term changes in the previously conditioned cues in the environment or by
central nervous system to the noxious input and to other the expectation of pain and suffering. The figure shows
external factors that impinge upon the individual. It is how the widely distributed, parallel-processing neural
now widely recognised that nociceptor function is altered networks in the central nervous system generate the nerve
by the “inflammatory soup” that characterises a region of impulse patterns that produce the various somatic
tissue injury.2 experiences, including transient, acute, and chronic pain.
This dynamic feature is usually temporary and exists The output of the neuromatrix can be modified by
while the body heals a region of injury. The modifiers of multiple forms of treatment to change the inputs and
nociceptor activity are also transient. However, Dubner influences on the neuromatrix.
and Ruda3 showed that huge nociceptive input can— Injury does not only produce pain, it also leads to
through the mechanism of excitatory toxic effects of stress, which the body attempts to deal with by achieving
aminoacids—permanently change spinal-cord function homoeostatis. This process involves neural, hormonal,
and could, therefore, lead to chronic pain after an acute and behavioural activities derived from genetically
injury. determined repertoires.9 These homoeostatic repertoires
Physiological and behavioural studies have shown that exist at the injury site (cytokine release, for example), in
plasticity, or learning, has a role in pain. Hence, synaptic the adrenal cortex (release of corticosteroids), the
potentiation is facilitated by repetitive noxious immune system, and in widely distributed areas in the
stimulation, and, at the level of the brain, environmental brain.
influences alter the response to noxious stimulation.4,5 It
is clear from clinical pain states that the brain can Components of pain
generate pain in the absence of input from the peripheral The best definition of pain is that endorsed by the
nociceptors or the spinal cord—for example, in phantom International Association for the Study of Pain: “Pain is
limb pain and pain perceived by paraplegic patients below an unpleasant sensory and emotional experience
the level of a total spinal cord transection. A pattern- associated with actual or potential tissue damage, or
generating mechanism, or neuromatrix, must, therefore, described in terms of such damage”.10 The existence of
exist in the brain that is capable of sustaining an image many types of pain can be understood by the
identification of four broad categories: nociception,
Lancet 1999; 353: 1607–09
perception of pain, suffering, and pain behaviours.11
See Commentary page 1546
Behind each of these clinical categories are anatomical,
Department of Neurological Surgery, University of Washington, physiological, and psychological substrates.
Box 356470, Seattle, WA98195, USA (J D Loeser MD); and
Department of Psychology, McGill University, Montreal,
Nociception—is the detection of tissue damage by
Quebec, Canada
specialised transducers attached to A delta and C fibres.
Correspondence to: Dr John D Loeser These transducers may be biased by inflammatory and
(e-mail: jdloeser@u.washington.edu)
neural changes in their immediate environment. Aspirin,

Patern-generating mechanism or neuromatrix modulated by multiple inputs and the internal milieu
Adapted from Melzack and Loeser7 and Melzack.9
acetaminophen, and non-steroidal anti-inflammatory Pain behaviours—result from pain and suffering and are
drugs act to change this “inflammatory soup” and the things a person does or does not do that can be
produce pain relief mainly by the restoration of ascribed to the presence of tissue damage. Examples of
nociceptive sensitivity to its resting state. Local and pain behaviours include saying “ouch”, grimacing,
regional anaesthesia can prevent nociception from limping, lying down, recourse to health care, refusing to
becoming pain, but so can downstream modulation, as work. Such behaviour is observable by others and can be
proposed in the Melzack-Wall theory.1 Such issues are quantified. All these behaviours are real and are also
routinely ignored by physicians. probably influenced by actual or expected environmental
consequences. From pain behaviours and the history
Perception of pain—is frequently triggered by a noxious and physical examination, we infer the existence of
stimulus, such as an injury or disease. Pain can also be nociception, pain, and suffering.
generated by lesions in the peripheral or central nervous
system, as seen in patients with diabetic neuropathy, Types of pain
spinal-cord injury, or stroke. When acute pain occurs, it Transient pain—is elicited by the activation of nociceptive
is initially associated with specific autonomic and somatic transducers in skin or other tissues of the body in the
reflexes, but these disappear in patients with chronic absence of any tissue damage. The function of such pain
pain. Many physicians and patients do not realise that to the individual is related to its speed of onset after
pain can occur without nociception. Furthermore, pain stimulation is applied and the speed of offset that
due to nerve injury does not respond to analgesics such as indicates that the offending physical disturbance is no
morphine as efficiently as pain caused by tissue damage, longer impinging upon the body. Presumably, transient
indicating the complex relation between injury and pain. pain evolved to protect man from physical damage by the
environment or by over stress of the body tissues. This
Finally, the intensity of chronic pain frequently bears
type of pain is ubiquitous in everyday life and is rarely a
little or no relation to the extent of tissue injury of other
reason to seek health care. In the clinical setting, transient
quantifiable pathology.
pain is seen only in incidental or procedural pain, such as
during a venepuncture or injection for immunisation.
Suffering—is a negative response induced by pain and Although the prevention of procedural pain is worthy of
also by fear, anxiety, stress, loss of loved objects, and study, it is not a major issue in clinical medicine.
other psychological states. The language of pain is used However, transient pain, which was the subject of most of
to describe suffering, irrespective of the cause, and thus the physiological experimentation during the first 75
misleads both the doctor and the patient as to the basis of years of this century, remains the common conceptual
the suffering. As stated by Cassell,12 “Suffering occurs model in health care.
when the physical or psychological integrity of the person
is threatened”. Not all suffering is caused by pain, but in Acute pain—is elicited by substantial injury of body tissue
our medicalised culture we describe suffering in the and activation of nociceptive transducers at the site of
language of pain. local tissue damage. The local injury alters the response
1608 THE LANCET • Vol 353 • May 8, 1999

characteristics of the nociceptors, their central more importantly, the inability of the body to restore its
connections, and the autonomic nervous system in the physiological functions to normal homoeostatic levels.
region. The local injury does not overwhelm the body’s
reparative mechanisms: “healing” can occur without Conclusions
medical intervention. The report of pain stops long The brain contains widely distributed neural networks
before healing has been completed. However, medical that create an image of self through genetic programmes
interventions may be useful in two respects: to prevent or and memories of past experience. Afferent inputs act on
reduce pain and to speed up the healing process by this neuromatrix and produce output patterns that lead to
shortening the duration of the injury. Most individuals the report of pain. Stress can change the interactions
with acute pain seek medical care. This type of pain is between the neuromatrix and peripheral stimuli, as can
also seen after trauma, surgical interventions, and some learned experiences and expectations. Clinically
diseases. Since the healing process usually takes a few significant acute pain always involves issue damage; the
days or a few weeks, pain that persists for months or years
central and peripheral nervous systems are dynamic, not
is not classified as acute. However, in malignant diseases
static, and are modulated by tissue damage and by
the invasion of body tissues can produce continuous
changes in the central nervous system and stress-
acute pain.
regulation systems that occur in response to such
damage. Although most of these modulations are of short
Chronic pains—such as low back pain, postherpetic duration, some may persist and lead to chronic pain
neuralgia, and fibromyalgia, are commonly triggered by states. In both clinical and basic research, we are rapidly
an injury or disease, but may be perpetuated by factors gaining useful information that will lead to more effective
other than the cause of the pain. The injury may exceed care for those who suffer pain.
the body’s capability for healing, because of the loss of
the body part, the extensiveness of the trauma and References
subsequent scarring, or the involvement of the nervous 1 Melzack R, Wall PD. Pain mechanisms: a new theory. Science 1965;
system in the injury itself. The nervous system may be 150: 971–79.
2 Levine J, Taiwo Y. Inflammatory pain. In Wall PD, Melzack R, eds.
damaged by the original injury in such a way as to be Textbook of pain. Edinburgh: Churchill Livingstone, 1994: 45–56.
unable to restore itself to a normal state. In addition to 3 Dubner R, Ruda MA. Activity-dependent neuronal plasticity following
chronic pain syndromes, in which the intensity of the pain tissue injury and inflammation. Trends Neurosci 1992; 15: 96–103.
is out of proportion to the original injury or tissue 4 Gracely RH, Lynch SA, Bennett GH. Painful neuropathy: altered
central processing maintained by peripheral input. Pain 1992; 51:
damage, other syndromes may occur spontaneously in the 175–94.
absence of any sign of injury. All types of chronic pain 5 Rainville P, Duncan GH, Price DD, Bushnell MC. Selective
lead people to seek health care, but they are often not modulation of pain unpleasantness alters activity in the human
treated effectively. Because chronic pain is unrelenting, it cerebral cortex. Neurosci Abstr 1996; 614: 2.
is likely that stress, environmental, and affective factors 6 Melzack R, Loeser JD. Phantom body pain in paraplegics: evidence
for a central “pattern generating mechanism” for pain. Pain 1978;
may be superimposed on the original damaged tissue and 4: 195–210.
contribute to the intensity and persistence of the pain. 7 Melzack R. Phantom limbs and the concept of a neuromatrix. Trends
Chronic pain differs from acute pain because therapies Neurosci 1990; 13: 88–92.
that provide only transient pain relief do not resolve the 8 Coderre T, Katz J, Vaccarino AL, Melzack R. Contribution of central
neuroplasty to pathological pain: review of clinical and experimental
underlying pathological process. Chronic pain will evidence. Pain 1992; 42: 259–85.
continue when treatment stops. Since the cause of a 9 Chrousos GP, Gold PW. The concepts of stress and stress system
person’s perception of pain may persist irrespective of disorders. JAMA 1992; 267: 1244–52.
medical treatments, psychological forms of therapy such 10 Merskey H, Bogduk N. Classification of chronic pain. Seattle:
International Association for the Study of Pain Press, 1994: 210.
as cognitive and behavioural treatments can be used to 11 Loeser JD. Perspectives on pain. In Turner P, ed. Procedings of the
alter the effect of the pain on the individual’s life. It is First World Congress on Clinical Pharmacology and Therapeutics.
possible that, just as the brain is modified by experience, London: Macmillan, 1980: 316–26.
especially in early life, the brain may be capable of 12 Cassell EJ. The nature of suffering and the goals of medicine. N Engl
J Med 1982; 306: 639–45.
altering the way pain-producing information is processed
13 Melzack R. Pain and stress: a new perspective. In Gatchel RJ,
to keep its impact to a minimum.13 It is not the duration Turk DC, eds. Psychological factors in pain. New York: Guilford
of pain that distinguishes acute from chronic pain but, Press, 1998.

Psychological Bulletin Copyright 2005 by the American Psychological Association
2005, Vol. 131, No. 2, 202–223 0033-2909/05/$12.00 DOI: 10.1037/0033-2909.131.2.202
Why Does Social Exclusion Hurt? The Relationship Between Social

and Physical Pain
Geoff MacDonald Mark R. Leary

University of Queensland Wake Forest University
The authors forward the hypothesis that social exclusion is experienced as painful because reactions to
rejection are mediated by aspects of the physical pain system. The authors begin by presenting the theory
that overlap between social and physical pain was an evolutionary development to aid social animals in
responding to threats to inclusion. The authors then review evidence showing that humans demonstrate
convergence between the 2 types of pain in thought, emotion, and behavior, and demonstrate, primarily
through nonhuman animal research, that social and physical pain share common physiological mecha-
nisms. Finally, the authors explore the implications of social pain theory for rejection-elicited aggression
and physical pain disorders.
The physical pain alone was terrible. I always used to think the past, we propose that threats to one’s social connections are
expression “a broken heart” was just a metaphor. But it felt as if I was processed at a basic level as a severe threat to one’s safety. In fact,
having a heart attack. we argue that such threats are partly mediated by the same system
—Bob Geldof, on the end of his 19-year relationship
that processes physical pain because the pain system was already
In a recent documentary about the death penalty, a camera crew in place when social animals evolved adaptations for responding to
was present in the home of a woman whose son was to be executed social exclusion.
that day. Although she was not present at the execution itself, at In this article, we use the term social pain to refer to a specific
the time the penalty was to be exacted she burst out of her front emotional reaction to the perception that one is being excluded
door and fell to the ground screaming and crying. Friends and from desired relationships or being devalued by desired relation-
family followed her outside and tried to help her up, as if her being ship partners or groups. Exclusion may be a result of a number of
on the ground was the problem. However, whenever people would factors, including rejection, death of a loved one, or forced sepa-
try to touch her, she would scream at them with fury to keep away. ration. In everyday life, extreme social pain may be experienced as
Her behavior was akin to that of a wounded animal, scaring others the deep aching of homesickness, grief, abandonment, or longing
away because her pain was so great. for a loved one. Relational devaluation refers to feeling less valued
In reflecting on the most agonizing moments in one’s life, as a relational partner (e.g., friend, romantic partner, group mem-
events involving severe physical pain (e.g., serious injuries, labor ber) than one desires (Leary & Springer, 2001). We argue that such
pain, kidney stones) quickly come to mind. But other events, such devaluation is experienced as aversive because it signals an in-
as the example above, may be as severely distressing, if not creased probability of ultimate exclusion. The acute emotional
painful, despite the lack of any tangible threat to one’s personal distress felt in response to relational devaluation is known as hurt
health or safety. Most people have experiences in which socially feelings (Leary & Springer, 2001). However, other affective states
mediated pain is so great that they are not only in agony but are such as embarrassment, shame, guilt, or jealousy can also serve as
overwhelmed or incapacitated. In this article, we argue that refer- signs that one is not living up to the standards of valued others, and
ring to these responses to social exclusion, rejection, or loss as
thus we consider these emotions to be aspects of social pain as
pain is more than just a metaphor. Because inclusion in social
well.
groups has been a key to survival for social animals deep into the
The concept of social pain was first suggested by Panksepp and
colleagues. They provided evidence that the social attachment
system was built up from more primitive regulation systems such
Geoff MacDonald, School of Psychology, University of Queensland, St.
as those involved in place attachment, thermoregulation, and phys-
Lucia, Queensland, Australia; Mark R. Leary, Department of Psychology,
Wake Forest University.
ical pain (Panksepp, 1998). Herman and Panksepp (1978) sug-
Work on this article was supported in part by a Social Sciences and gested specifically that “it is conceivable that brain circuits for
Humanities Research Council of Canada Post-Doctoral Fellowship separation distress represent an evolutionary elaboration of an
awarded to Geoff MacDonald. I (Geoff MacDonald) am extremely grateful endorphin-based pain network” (p. 219), and Nelson and Panksepp
for the opportunities provided for me by the Canadian government via this (1998) stated, “The pain components made stronger contributions
fellowship. We thank Rod Ashton, Terry Blumenthal, Matthew Hornsey, to the subcomponents which aroused emotional distress during
Winnifred Louis, Mike Smith, and Valerie Stone for their insightful com-
social absence” (p. 438). In this article, we attempt to extend
ments on drafts of this article. Thanks also to our colleagues and friends
who provided translations of “hurt feelings.”
Panksepp’s ideas with the goal of tying social pain more strongly
Correspondence concerning this article should be addressed to Geoff to human reaction to perceived social exclusion and by considering
MacDonald, School of Psychology, University of Queensland, St. Lucia the implications of social pain for the important problems of
QLD 4072, Australia. E-mail: geoff@psy.uq.edu.au relationship aggression and pain disorders.
202
SOCIAL AND PHYSICAL PAIN 203
We argue that the aversive emotional state of social pain is the more likely to survive to 1 year of age than infants of less socially
same unpleasantness that is experienced in response to physical integrated mothers, even controlling for the mothers’ dominance
pain. Others before us have proposed the existence of nonphysical rank (Silk, Alberts, & Altmann, 2003). Further, vervets and rhesus
forms of pain such as “emotional pain,” “mental pain,” and “psy- monkeys who showed low interest in social contact (a result of
chological pain.” Thornhill and Thornhill (1989) proposed a theory receiving amygdala and other brain site lesions) after rerelease to
of emotional pain, suggesting that its function is analogous to that the wild were excluded from the social group and died without the
of physical pain. That is, they proposed that such pain focuses protection of their conspecifics (Kling, Lancaster, & Benitone,
attention on significant social events and promotes correction and 1970). Such data support the premise that, over evolutionary time,
avoidance of such events in the future. They further theorized that social animals who formed strong relationships and were inte-
the causes of emotional pain would be circumstances that had grated most strongly into group living were most likely to survive,
influenced inclusive fitness in the environment of evolutionary reproduce, and raise offspring to reproductive age (Baumeister &
adaptiveness such as the death of genetic relatives or close asso- Leary, 1995). Phrased differently, for social animals, being so-
ciates, loss of status, sexual jealousy, childlessness, and rape. In cially excluded was often equivalent to death. As a result, the
the current article, we restrict our analysis to a very specific process of natural selection favored those who were motivated to
evolutionary adaptation—the desire to avoid social exclusion. It is be included, meaning such animals were more likely to leave
important to make clear that we are not suggesting that social pain viable descendants.
is the only viable form of nonphysical pain. It is more accurate to To adapt to changing conditions vis-à-vis social inclusion and
suggest that social pain may be one form of emotional pain. In fact, exclusion, social animals required mechanisms that allowed them
in our analysis, it is most accurate to say that the affective re- to recognize and react to threats of exclusion in an efficient
sponses to physical trauma usually described as physical pain are manner. In particular, cues such as physical distance from impor-
themselves a subcategory of emotional pain, albeit a fundamental tant conspecifics may have been reliably correlated with eventual
one. Given Gray’s (1971) suggestion that the same punishment exclusion. In evolutionary terms, genetic mutations that provided
mechanism underlies both fear and frustration, it seems reasonable learning mechanisms for associating such cues with response
to suggest that feelings of pain may be associated with a wide mechanisms that helped avoid exclusion would have facilitated
variety of stimuli that either lead to harm or block a highly desired survival (cf. Krebs, Stephens, & Sutherland, 1983). As a result,
goal. In this article, we do not claim to provide an exhaustive such mutations would have provided an important evolutionary
analysis of all possible forms of emotional pain but rather of one advantage for social animals and would have been likely to be
specific form—social pain. passed on to future generations. For early social animals, such cues
To begin, we forward our theory of why social and physical pain would have included factors such as physical distance from im-
overlap as they do. We argue that social animals require a system portant conspecifics or absence of those conspecifics. For exam-
that punishes individuals who do not avoid social exclusion and ple, social inclusion took on particular importance for mammals
motivates quick responses to signs of exclusion. In line with the because they nurse their young, meaning parent– offspring inter-
work of Panksepp, we propose that at the point in evolutionary dependence is a critical survival issue. The mammalian infant’s
history when such a system developed, existing physical pain reliance on its mother for nourishment, as well as protection from
mechanisms provided its foundation. To support this hypothesis, predators and other dangers, means that any prolonged separation
we provide evidence that social and physical pain overlap in the of an infant from its mother is potentially disastrous (MacLean,
attitudes, behaviors, and cognitions of humans, reviewing evidence 1993).
that the two types of pain correlate similarly with factors such as This high degree of dependence on the mother set the stage for
extraversion, social support, anxiety, aggression, and depression. adaptations that maintained the infant–mother bond (Bowlby,
From there, we present physiological evidence that social and 1973; Panksepp, 1998). Over time, some social animals gradually
physical pain operate via shared mechanisms. Specifically, both developed more complex cooperative social structures that even-
types of pain have been shown to involve the anterior cingulate tually blossomed into high degrees of interdependence, making
cortex and periaqueductal gray brain structures and the opioid and inclusion crucial for survival across the life span (Gilbert, 1992;
oxytocin neuroendocrine systems. We then move to discuss the MacLean, 1993; Whiten & Byrne, 1989). This increase in social
implications of social pain theory, focusing on its implications for complexity would have been accompanied by new cues of exclu-
understanding rejection-elicited aggression such as violence in close sion threat, such as averted eye gaze. Again, genetic mutations that
relationships and pain disorders such as somatoform pain. We con- tied such cues to appropriate warning and response mechanisms
clude by suggesting future directions for research on social pain. (or that facilitated the learning of such associations) would have
conferred an important advantage, and thus would have been likely
to be retained across generations. Because social exclusion has
Why Is Social Exclusion Painful?
been such an important threat to survival from the earliest days of
The pain of separation slams down, the guillotine. social speciation, it seems reasonable to suggest that exclusion
—Lucy Gwin, “Normal zone: You in or out?”, Adbusters: Journal cues recognized by modern humans have the potential to be
of the Mental Environment, 2002 processed as a basic and severe threat to existence in the same way
as do other primitive threats (e.g., snakes or spiders; Öhman &
Social pain theory is based on the idea that the possibility of Mineka, 2001).
being separated from important social entities posed a critical The question, then, is what kind of warning and response
challenge to the survival of our ancestors, dating back at least to mechanisms these cues of social distance came to trigger. Evolu-
the earliest mammals (and likely beyond). For example, infants of tionary theory suggests that such cues were likely to become
highly socially integrated female baboons have been shown to be associated with the activation of existing threat defense mecha-
204 MACDONALD AND LEARY
nisms. Such “preadaptations” are considered to be a common decreases in painful feelings reward an organism for moving
means of responding quickly to new survival challenges, including toward safety (i.e., away from danger and/or toward safety). Fur-
social ones (D. C. Craig, Gilbert-MacLeod, & Lilley, 2000; ther, the aversiveness of pain can condition an organism to avoid
Keverne, Nevison, & Martel, 1999; Öhman & Mineka, 2001; situations in which a quick response is needed. After organisms
Panksepp, 1998; Rozin, Haidt, & McCauley, 1993). For example, learn to associate certain situational cues with pain, these cues
the negative emotional and physical reaction provoked by moral trigger relevant approach/avoidance tendencies so that pain is
offenses such as incest appears to tap the physical disgust re- avoided or minimized. As a result, the organism learns to fear not
sponse, leading to grimacing, flared nostrils, and nausea (Rozin et just pain itself but also cues that indicate the possibility of pain
al., 1993). We propose that cues of social distance came to activate (Bowlby, 1973). For example, a child who is bitten by a dog may
threat-defense responses that originally functioned to help organ- become fearful in the future upon seeing or hearing the dog,
isms avoid physical danger, as such mechanisms predated the regardless of whether the dog actually bites the child again. This
evolution of social animals. In particular, we propose that social conditioned fear can motivate movement away from the fear-
exclusion cues accessed threat-defense responses by stimulating evoking stimulus and movement toward a helpful attachment
the same painful feelings associated with physical injury. figure, both of which have the potential to reduce the threat of a
In the remainder of this section, we lay out our argument as to harmful stimulus.
why pain would have provided an excellent mechanism for the We propose that painful feelings triggered by social exclusion
regulation of social inclusion (Panksepp, 1998). In order to de- also provide a mechanism useful for learning effective approach/
scribe how social experience may have come to be mediated by avoidance regulation to avoid exclusion. People who reject, ex-
feelings of pain, it is important to note that the experience of pain clude, or ignore an individual are not likely to be safe or stable
consists of two separate components—pain sensation and pain sources of support for that person and, in fact, may be inclined to
affect (Melzack & Wall, 1996; Price, 1999; Rainville, 2002). Pain cause harm to that person. Thus, experiencing painful emotions in
sensation provides information about ongoing tissue damage, in- connection with social exclusion guides an individual away from
formation that is gathered by the body’s specialized pain receptors sources of rejection and toward sources of acceptance. Indeed,
and transmitted to the brain for processing via the dorsal horn of people are highly attuned to social cues indicating that social pain
the spinal cord (K. D. Craig, 1999; Melzack & Wall, 1965). We do is likely and work to avoid social pain when such cues are
not propose that social exclusion directly taps into this circuitry, detected. Just as a person is less likely to approach a dog who bit
although we do discuss the possibility of indirect influence later. him or her, that person is also less likely to seek the company of
Pain affect consists of the feelings of unpleasantness that are an individual who has insulted, ostracized, or otherwise hurt him
associated with pain sensation, as well as emotions associated with or her. Indeed, research has shown that people tend to distance
the future implications of those sensations (Price, 2000). It is this themselves from others if they feel that rejection, and hence hurt
affective experience of pain that signals an aversive state and feelings, is likely (Bourgeois & Leary, 2001; Feeney, Noller, &
motivates behavior to terminate, reduce, or escape exposure to the Roberts, 2001; Murray, Holmes, MacDonald, & Ellsworth, 1998;
source of the noxious stimulation (Melzack & Casey, 1968; Price, Vangelisti, 2001). For example, individuals who feel unsure of
1999). Because this affect component is separate from the sensa- acceptance from a romantic partner often devalue the importance
tion component, it is possible to experience painful feelings in the of the relationship as a means of protecting themselves from the
absence of a signal of tissue damage (Fields, 1999; Rainville, hurt of rejection (Murray et al., 1998). Furthermore, social pain
2002). Thus, our suggestion is that social exclusion triggers these experienced in the context of a specific relationship can motivate
same painful feelings, leading to an emotional experience of pain people to seek support from trusted others or to pursue new
without accompanying physical pain sensation. relationships (Leary & Springer, 2001). Overall, both physical and
We believe that pain affect came to underlie social regulation social pain appear to serve a similar function in promoting adap-
needs because it serves at least two functions crucial for the tive approach and avoidance behavior in response to physical and
avoidance of social exclusion. First, learning that promotes avoid- social threats, respectively.
ance of inclusion-threatening situations is needed to minimize the Another important point of overlap between social exclusion
number of exclusion threats that one faces. Feelings of pain can and physical pain relevant to learning is that certain early-life
provide a strong sense of aversiveness that, when paired with sensory experiences, particularly gentle touch, are involved in the
exclusion-threatening situations, can motivate avoidance of such alleviation of both physical pain and social separation. Physical
situations. Second, quick action in response to exclusion warnings touch provides the basis for attachment (Harlow, 1958). Human
(e.g., ceasing an offending behavior) is needed to help sustain infants appear prepared to learn associations between attachment
inclusionary status. Because of the strong relation between pain behaviors and parental responses as mediated by physical contact
and threat-defense response mechanisms (Gray & McNaughton, (Bowlby, 1973). When babies express physical discomfort (e.g.,
2000; Panksepp, 1998), pain affect should provide a pathway by through crying), their distress can be alleviated through physical
which social exclusion cues could trigger quick, defensive reac- contact by the attachment figure such as holding or patting
tions to regulate inclusionary status. (Bowlby, 1973). In essence, this is the key to attachment theory;
children learn about the reliability of social support from the
Aversiveness of Pain attachment figure on the basis of that attachment figure’s physical
The affective component of physical pain aids organisms in responsiveness to their distress. At a more basic level, however,
avoiding threats to their physical safety by serving as a source of the baby also learns that isolation and physical pain go hand in
punishment, and it functions to guide organisms toward safety by hand. Because the child learns that uncomfortable states such as
serving as a source of negative reinforcement. Increases in painful hunger pangs and gas pain often continue until the attachment
feelings motivate organisms to avoid dangerous stimuli, whereas figure is in physical contact with the child, the association between
physical and social pain occurs at a very early age. (Interestingly, predator), a panic response promotes fight/flight/freezing behavior
when we feel another person has helped us in a special, supportive as a means of providing a quick route to safety. It is important to
way, we may say that we feel “touched.”) All told, then, attach- note that when we refer to panic responses throughout this article,
ment regulation may have intertwined with pain mechanisms be- we specifically mean the motivation for undirected escape from
cause these mechanisms would have already been highly respon- threat, or the fight/flight/freezing response. Such panic behavior
sive to the crucial cues of distress and physical touch. can be highly reactive and relatively undirected as high levels of
Overall, we propose that social pain hurts because social inclu- coordination and planning are sacrificed for a quick response to
sion was and is key for human survival. In accordance with this danger. The panic response is facilitated by a set of physiological
view, people do appear to take social pain very seriously. For changes designed to prepare an organism for urgent action such as
example, Kaplan and Bratman (2000) showed that people judge increased heart rate, increased blood clotting factor, and analgesia
doctor-assisted suicide as more moral and easier to understand (Gray & McNaughton, 2000). Factors that have been shown to
when the patient is in emotional pain than in matched cases trigger the panic response include immediate predators, high levels
without emotional pain. This study also showed that participants of carbon dioxide, and physical pain (Gray & McNaughton, 2000).
viewed doctor-assisted suicide as more justifiable and understand- Physical pain can be an important signal of immediate threat, as it
able when both emotional and physical pain were present than often accompanies tissue damage. In this way, pain serves to
when the patient experienced only physical pain. Thus, people activate and regulate avoidance responses including fight, flight, or
realize that emotional pain can be excruciating. Further, K. D. freezing (Berkowitz, 1993; Berkowitz, Cochran, & Embree, 1981;
Williams (1997) has observed that many people would prefer to be Merskey, 2000).
hit than ostracized, suggesting that the pain of social exclusion Social relationships also require approach/avoidance regulation.
may be more aversive than the pain of physical injury in many Whereas the need to belong (Baumeister & Leary, 1995) and
instances. In fact, simply thinking about separation from close sexual desire provide approach motivation, the dangers of rejection
others has been shown to increase the accessibility of death-related and exclusion provide avoidance motivation. In fact, people often
thoughts (Florian, Mikulincer, & Hirschberger, 2002; Mikulincer react to threats to social inclusion as if they were as important as
& Florian, 2000; Mikulincer, Florian, Birnbaum, & Malishkevich, threats to physical safety, if not more so (K. D. Williams, 1997).
2002), suggesting a strong link between attachment and a sense of Perhaps not surprisingly, then, rejection appears to lead to re-
physical safety. As MacLean (1993) aptly put it, “A sense of sponses consistent with Gray and McNaughton’s (2000) model.
separation is a condition that makes being a mammal so painful” For example, Vangelisti and Crumley (1998) asked participants to
(p. 74). recall instances when their feelings had been hurt and to describe
their responses to the incident. A factor analysis of participants’
Pain and Quick Reaction to Threat responses to feeling hurt classified these responses into three
categories. The first, “acquiescent,” consisted of behaviors such as
Another benefit of tying social exclusion cues to pain is the apologizing that appear to facilitate safety from hurt via cautious
ability to capitalize on the strong relation between pain and the approach. The second, labeled “invulnerable,” consisted of behav-
threat-defense system (Gray & McNaughton, 2000; Panksepp, iors such as ignoring the source of hurt that serve to help one avoid
1998). This way, social pain would not only lead to exclusion cues or withdraw from a hurtful exchange. Finally, the response labeled
being perceived as aversive, but it would also promote timely “active verbal” consisted of behaviors such as verbally attacking
response to such cues. As sketched by Gray and McNaughton the source of hurt that seem to reflect more aggressive responses.
(2000), the physical defense system regulates behavior in response These classes of responses appear to map well onto the anxiety,
to threat on the basis of the state of two key variables. The first fear, and panic components of the physical defense system, re-
variable, defensive distance, refers to the degree of perceived spectively. As with physical pain, we believe that the panic re-
threat in a given situation (Blanchard & Blanchard, 1990). That is, sponse to perceived exclusion should occur only when defensive
the more threatening a stimulus is perceived to be to well-being, distance from exclusion is perceived to be low. That is, reactions
and the more imminent that threat is perceived to be, the more the to social stimuli should most resemble reactions to acute physical
defense system promotes active, self-protective behavior. The sec- pain when strong relational devaluation by another is perceived,
ond variable, defensive direction, refers to whether motivation especially when there is a strong desire to maintain a relationship
exists to approach a potentially dangerous stimulus (Gray & Mc- with the devaluation source (Leary & MacDonald, 2003; Leary,
Naughton, 2000). For example, a mouse may perceive moving Springer, Negel, Ansell, & Evans, 1998).
onto an open field as threatening (as it would be exposed to Thus, like physical pain, social pain leads animals to approach
predation), but it may need to do so to acquire food. friendly conspecifics, avoid threats to separation when possible,
According to Gray and McNaughton’s (2000) model, approach- and attack unavoidable threats to separation (Alexander, 1986;
ing a potentially threatening stimulus results in anxiety, promoting Carter, 1998). Unlike physical pain, however, the emotional dis-
cautious approach behavior such as initially making brief forays tress of social pain serves a protective function in social contexts
onto the open field followed by quickly returning to a safe posi- (Baumeister & Tice, 1990; Leary & Springer, 2001; Miller &
tion. The intensity of anxious emotion and behavior should in- Leary, 1992; Thornhill & Thornhill, 1989; Vangelisti & Crumley,
crease as defensive distance is reduced. When a potentially dan- 1998). Because the need to belong is a fundamental aspect of
gerous stimulus is detected and is not accompanied by a human experience, a system to protect social well-being has great
motivation to approach the stimulus, the resulting response is adaptive value for human beings (Baumeister & Leary, 1995;
fearful avoidance of the stimulus when defensive distance is high Baumeister & Tice, 1990; Leary, Tambor, Terdal, & Downs,
(e.g., the faint odor of a predator is detected). However, when 1995). In support of these ideas, separation from attachment fig-
defensive distance is low (e.g., the presence of an immediate ures in primates activates major behavioral and stress response
systems, according to a review of relevant literature (Mason & The most obvious connection between the two is that similar
Mendoza, 1998). Such separation has been shown to lead to words are used to describe both experiences. The phrase “I am
reactions similar to those seen in human beings, including in- hurt” could just as easily refer to the result of a physical injury as
creased anxiety and depression-like behavior (E. O. Johnson et al., to one’s reaction to a relationship dissolution. In fact, many of the
1996; Levine & Stanton, 1990), increased plasma cortisol (E. O. terms used to describe social pain, if taken literally, would be great
Johnson et al., 1996; Rilling et al., 2001), decreased norepineph- sources of physical pain. For example, people may say that they
rine (Kraemer, Ebert, Schmidt, & McKinney, 1991), and overt were “broken hearted,” “cut to the core,” or “emotionally scarred”
crying (E. O. Johnson et al., 1996; Panksepp, 1998). For example, by a rejection or other loss of social connection. Similarly, a
marmosets placed in isolation for a 2-week period evidenced person may say that being rejected “ripped out my heart” or was
increases in plasma cortisol concentrations (a stress-related hor- like a “slap in the face.” More generally, people report feeling
mone involved in preparation for physical defense) and submissive “crushed,” “deeply hurt,” or “wounded.” It should be noted that
crying, weight loss averaging 10% of body mass, and frequent counterexamples, where pain is used as a metaphor for positive
crouching that the authors likened to freezing behavior in rats social experience, can also be brought to mind, such as “having a
(E. O. Johnson et al., 1996). Social stressors have been shown to crush on someone” or “getting a kick out of someone.” However,
evoke similarly strong physiological responses in humans. A meta- unlike most other emotional states, the English language contains
analysis by Dickerson and Kemeny (2004) showed that the threat no direct synonym for the term hurt feelings, the emotion that
of social evaluation is unique among psychological stressors in accompanies perceived relational devaluation by other people
stimulating the release of high levels of cortisol (this relation was (Leary & Springer, 2001). Thus, English speakers not only de-
particularly strong when stress was uncontrollable). scribe social pain using images connoting physical pain, but at
Further, separation from caregivers and isolation from conspe- least in the case of hurt feelings, they have constructed no other
cifics has been shown to lead to another aspect of the fight/flight/ way to describe that common and important experience except
freezing response in nonhuman mammals—analgesia (or reduced with reference to pain. Further, as can be seen in Table 1, examples
pain sensitivity). Analgesia in response to short-term isolation has of a linguistic link between exclusion and pain can be found across
been demonstrated in rat pups (Kehoe & Blass, 1986a, 1986b; a wide variety of languages and cultures.
Naranjo & Fuentes, 1985; Spear, Enters, Aswad, & Louzan, 1985), Beyond these linguistic associations, if social and physical pain
mice (Konecka & Sroczynska, 1990), cows (Rushen, Boissy, Ter- share a common psychological and/or physiological basis in hu-
louw, & de Passillé, 1999), and chicks (Sufka & Hughes, 1990; mans, then both should be similarly related to a number of com-
Sufka & Weed, 1994). For example, as we discuss in more detail mon factors. Because both types of pain serve to promote avoid-
later, rat pups isolated from their mother (or “dam”) and littermates ance of pain-eliciting stimuli, both types of pain should be
have been shown to have longer response latencies to heat stimuli, associated with higher degrees of caution and defensiveness. Fur-
suggesting a decreased sensitivity to pain (Kehoe & Blass, 1986b). thermore, there should be evidence of crossover between the two
Analogous results have been found with human participants. Mac- types of pain. That is, higher degrees of physical pain should be
Donald, Kingsbury, and Shaw (in press) randomly assigned par- associated with increased social caution or isolation and vice versa.
ticipants to be either included or excluded from an online ball- In this section, we offer evidence supporting these two postulates
tossing game they believed they were playing with other from research on extraversion–introversion, social support, anxiety
participants (who were actually controlled by a computer sched- and fear, defensive aggression, and depression.
ule). Before the game, participants’ proneness to experience hurt
feelings (i.e., the ease with which an individual’s feelings are hurt)
was measured. Following the game, participants’ physical pain Table 1
sensitivity was tested by having them place an arm in cold water International Terms for Hurt Feelings
(the “cold pressor” task) and report how quickly they felt pain (i.e.,
pain threshold). The results showed that individuals whose feelings Language Native term English translation
were more easily hurt and who were excluded from the game
reported higher pain thresholds (i.e., slower onset of pain) than German verletzt sein hurt or wounded
French blessé hurt
hurt-prone individuals included in the game. That is, individuals Dutch gekwetst hurt
who were sensitive to rejection and who experienced social exclu- Spanish sentirse herido feel injured or harmed
sion demonstrated an analgesic response to the cold water. Indi- Italian ferito hurt
viduals less prone to hurt feelings did not differ across conditions. Greek pligomenos hurt
Hebrew he pag’ah baregashot shelo she hit/damaged his feelings
These data provide direct evidence that social exclusion can influ- Hungarian megsertoedni being hurt
ence physical pain detection mechanisms. This supports the notion Armenian zkatsoumnires tsavtsoutsir you hurt my feelings
that reactions to social exclusion are regulated by a general threat- Mandarin shang liao kan ching hurt feelings
defense system that prepares an organism for potentially harmful Cantonese siong sum hurt heart
situations and is responsive to “stimulation that is intense, painful, Tibetan snying la phog hit the heart
Bhutanese sems lu phog hit the mind
[italics added] or unexpected” (Mason & Mendoza, 1998, p. 771). Inuktitut anniqtuq hurt by harsh words
Note. These terms were solicited from colleagues and friends with the
Shared Psychological Correlates of Social and Physical following e-mail: “In English, we refer to a person’s emotional reaction to
Pain being rejected as ‘hurt feelings.’ We are interested in what word or words
native speakers of other languages use. We would appreciate it if you could
We now move to review evidence of the link between social and tell me the expression used to describe the emotional reaction to rejection
physical pain in the thoughts, feelings, and behavior of humans. in any other language.”
Extraversion and Introversion addition, people who are socially alienated are more prone to
physical ailments (Bockian, Meager, & Millon, 2000), and people
Extraversion–introversion appears to have an important relation experiencing marital dissatisfaction and conflict show poor adjust-
to both social and physical pain. Extraverts are more sociable and ment to chronic pain (Robinson & Riley, 1999). The link between
outgoing than introverts (Pervin, 1996), partly because they are physical pain and social support has also been demonstrated ex-
less afraid of being rejected and hurt in social settings. Indeed, perimentally. Brown, Sheffield, Leary, and Robinson (2003) tested
extraversion is negatively related to rejection sensitivity (Downey pain sensitivity with the cold pressor task after randomly assigning
& Feldman, 1996). Further, extraversion is positively related to participants to receive active social support (verbal support), pas-
self-esteem (Halamandaris & Power, 1997; Kwan, Bond, & Sin- sive social support (presence of other with no communication),
gelis, 1997), a variable strongly tied to the belief that one is distracting interaction (verbal interaction without support instruc-
acceptable to other people (Leary & MacDonald, 2003). tions), or no support. Relative to those with no support or distrac-
Extraversion is also related to physical pain. A review of re- tion, those with active or passive social support reported less pain
search by Phillips and Gatchel (2000) showed that extraverts from the task. Social support, then, appears to play a role in
demonstrated both higher pain thresholds (the point at which pain buffering both social and physical pain.
is detected) and higher pain tolerance (the degree of pain that can The data reviewed here support the notion that perceptions of
be withstood). It is interesting to note, however, that extraverts are social support are associated with perceptions of reduced levels of
more likely than introverts to express that they are in physical pain physical pain. However, they also appear to be in conflict with
(Phillips & Gatchel, 2000; Wade & Price, 2000). If one considers research presented earlier suggesting that isolation leads to anal-
that expressing injury could be taken as a sign of weakness, it gesia in nonhuman animals (e.g., Kehoe & Blass, 1986b) and that
appears that introverts, who are more wary of being rejected exclusion can lead to decreased pain sensitivity for hurt-prone
(Downey & Feldman, 1996), may want to hide their hurt. As a humans (MacDonald et al., in press). Why would both inclusion
result, introverts may express less pain than extraverts even while and exclusion lead to decreased pain sensitivity? One answer may
experiencing it more intensely. In fact, as chronic pain continues be that inclusion and exclusion affect different aspects of sensi-
over time, pain sufferers become more introverted (Phillips & tivity to pain that follow different time courses. For example, in the
Gatchel, 2000), demonstrating increased social anxiety and avoid- Brown et al. (2003) study, socially supported participants did not
ance of social situations (Sharp & Harvey, 2001). In general, then, begin reporting lower pain sensitivity until 1 min into the cold
introverts appear to have a higher level of reactivity to both pressor task, an effect that remained significant for the duration of
physical and social pain than extraverts, supporting the notion that the task (the task stopped after 3 min). Correlational studies
the two types of pain operate via common mechanisms. relating social support to lower pain sensitivity in human partici-
pants tend to encompass a relatively long time frame, with mea-
Social Support sures of social support tapping perceptions of long-term support.
On the other hand, excluded hurt-prone individuals in Mac-
The common-sense notion that meaningful support from close Donald et al.’s (in press) study reported initial pain less quickly
others is strongly tied to social pain and hurt feelings is supported than included hurt-prone individuals, but this effect did not last
by the literature (Leary, 1990; Leary, Koch, & Hechenbleikner, beyond the early stages of the cold pressor task. It is important to
2001). Indeed, we have defined social pain in terms of separation note that much of the work associating separation with analgesia in
from valued others, and hurt feelings as a perception of suboptimal nonhuman animals has been conducted following relatively short-
valuation by other people. In essence, then, a perceived lack of term isolation periods (e.g., separating a rat pup from its dam for
adequate social connections is the sine qua non of social pain. In 5 min).1 Thus, measures of pain sensitivity in these studies are
support of this notion, hurt feelings have been shown to arise from taken during a relatively short time span when analgesia is still
the perception that one is less valued by another person or group active. Indeed, this short-term focus is consistent with the role of
than one wishes (Leary et al., 1998, 2001). For example, Leary et analgesia in response to physical injury as a mechanism that blocks
al. (1998) asked participants to recount instances when their feel- attention to injury until safety is achieved. Analgesia that extended
ings had been hurt and found that 99% of these instances involved for long periods of time would be less functional, as injury would
relational devaluation. Further, the feeling of being valued that not be associated with the discomfort of pain and thus not promote
comes from meaningful social support helps to soothe social pain; avoidance learning. Further, it is unclear from these studies
people regularly derive a great deal of solace from other people whether inclusion and exclusion were related to pain intensity,
when they are distressed (Buunk & Verhoeven, 1991; Finch, pain affect, or both, highlighting our limited knowledge about the
Okun, Pool, & Ruehlman, 1999; Haley, 1997; Schachter, 1959). mechanisms by which these studies found their effects. Finally, it
For example, in a meta-analysis of relevant studies, Finch et al. seems potentially useful to test for a moderating role for hurt
(1999) found that both social support and negative social interac-
tion had significant (and oppositely valenced) relations with psy-
1
chological distress. Some researchers have demonstrated reduced pain sensitivity in adult
What is perhaps less intuitive is that social support is also rats that were raised in isolation and thus experienced long-term social
separation (Gentsch, Lichtsteiner, Frischknecht, Feer, & Siegfried, 1988;
related to physical pain. Research has shown that higher levels of
Schwandt, 1993). However, as these rats were raised from an early age
social support are associated with lower levels of chronic pain without the presence of any conspecifics, it seems likely the analgesia
(Phillips & Gatchel, 2000), labor pain (Klaus, Kennel, Robertson, demonstrated in these studies is related to developmental difficulties. Thus,
& Sosa, 1986; Niven, 1985), cardiac pain (Chalmers, Wolman, we do not view these studies as comparable with the long-term social
Nikodem, Gulmezoglu, & Hofmeyer, 1995; Cogan & Spinnato, support data from correlational studies involving nondevelopmentally chal-
1988), and postoperative pain (Lidderdale & Walsh, 1998). In lenged human populations.
proneness in the support–analgesia link found by Brown et al. controlled, hurt feelings proneness was related to evaluating the
(2003), as the exclusion–analgesia effect found by MacDonald et painful clips as more aversive and less humorous, suggesting that
al. (in press) was limited to hurt-prone individuals. Again, these hurt-prone individuals are relatively vigilant for physical threat.
data support a clear link between social exclusion and physical Clearly, both physical pain and social exclusion are important
pain mechanisms, but clearly more research is needed to investi- correlates of anxiety. In fact, Baumeister and Tice (1990) proposed
gate the nature of this relationship and the mechanisms by which that all instances of anxiety arise from either the threat of physical
it operates. pain or the threat of social exclusion. In both cases, anxiety signals
a potentially dangerous stimulus or situation, necessitating cau-
Anxiety and Fear tious approach or avoidance of the stimulus (Gray & McNaughton,
2000; Frijda, 1986). However, there is a problematic aspect to a
Anxiety and fear are strongly tied to physical pain (Robinson & long-term avoidant response common to both social and physical
Riley, 1999; Turk & Flor, 1999; Weisberg & Keefe, 1999). For pain. One common strategy for avoiding social pain in romantic
example, data from a survey of a representative sample of the relationships is described by Murray and Holmes’s dependency
population of the United States indicate that individuals experi- regulation model (Murray, Holmes, & Griffin, 2000; Murray,
encing the chronic pain of arthritis are more likely to experience Holmes, Griffin, Bellavia, & Rose, 2001; Murray et al., 1998).
anxiety and panic disorders, even when a wide range of sociode- According to the model, individuals who fear rejection from inti-
mographic variables and medical conditions were controlled (Mc- mate others tend to avoid creating situations where the expected
Williams, Cox, & Enns, 2003). Arthritis was also associated with rejection might materialize. Thus, such individuals will keep emo-
social phobia in this study controlling for the sociodemographic tionally distant from their partners, limiting the risks they take to
variables, but this relation was nonsignificant when the other increase intimacy such as self-disclosure. As discussed earlier,
medical conditions were controlled. Further, people who score such a self-protective stance can be functional in the short-term by
highly on measures of trait anxiety and neuroticism have lower limiting rejection. The problem with this approach is that by not
thresholds for physical pain than those who are less anxious exposing oneself to the potential for rejection, one’s fears of
(Phillips & Gatchel, 2000; Wade & Price, 2000; D. A. Williams, rejection are never disconfirmed. The emotional distance moti-
1999). Similarly, longitudinal research has shown that neuroticism vated by these rejection fears undermines relationship closeness
predicts the experience of neck pain and migraine headaches 3 (Murray et al., 1998, 2000, 2001), often instigating the feared
years later (Wade & Price, 2000). Chronic pain sufferers who fear hurtful behavior from others (Ayduk, Downey, Testa, Yen, &
abandonment from close others (i.e., have a more anxious attach- Shoda, 1999; Murray, Holmes, & Griffin, 1996) and leading to
ment style) have been shown to experience their physical pain as eventual dissolution of the relationship (Karney & Bradbury, 1995;
more threatening and distressing than those with more secure Kelly & Conley, 1987; Kurdek, 1997).
attachment (Mikulincer & Florian, 1998). Not surprisingly, anx- A similar process appears to occur for those with chronic
ious attachment is related to neuroticism (Shaver & Brennan, physical pain. Chronic pain patients often decrease physical activ-
1992), and people who score high in neuroticism are more prone ity, especially activity that might increase pain in the affected
to death-related thoughts when they are reminded of their corpo- somatic region (Sharp & Harvey, 2001; E. P. Simon & Folen,
real nature (Goldenberg, Pyszczynski, McCoy, Greenberg, & Sol- 2001). However, analogous to the results of dependency regula-
omon, 1999), suggesting that anxiousness is associated with more tion, such inactivity means that individuals’ beliefs about their pain
accessible cognitions related to threats to survival. are never tested, despite the fact that such activity may lead to no
Neuroticism is also related to the propensity to experience hurt increase in pain or increased pain that is easily tolerable (Sharp &
feelings and other negative emotional reactions to social exclusion. Harvey, 2001). Ultimately, decreased physical activity contributes
People who are more neurotic are more prone to feel hurt when to weakened muscle tissue and weight gain, both of which can
they do not feel valued (Leary & Springer, 2001) and are generally exacerbate chronic pain (E. P. Simon & Folen, 2001). In both the
more rejection sensitive (Downey & Feldman, 1996; Downey, cases of social and physical pain, then, an avoidant response may
Feldman, & Ayduk, 2000). Individuals high in anxious attachment be functional for preventing short-term pain but dysfunctional for
evidence higher levels of anxiety and distress in response to meeting long-term interpersonal and health goals. This sacrifice of
separation, conflict, and breakup in close relationships than those long-term goals for short-term relief highlights the extremely
with more secure attachment (Feeney, 1999; Fraley & Shaver, aversive nature of both types of pain. More generally, the literature
1999; Mikulincer & Florian, 1998; Rholes, Simpson, & Stevens, reviewed here suggests that both social exclusion and physical
1998). Those with higher levels of neuroticism are more likely to pain are related to the activation of emotional states related to
feel socially anxious and embarrassed when they become con- cautious approach (anxiety) and avoidance (fear).
cerned about social approval and acceptance (Leary & Kowalski,
1993). In a survey of chronic pain patients and other community Defensive Aggression
members, MacDonald et al. (in press) showed that the tendency to
experience hurt feelings was associated with higher reports of Although fleeing physical harm often provides the best chance
physical pain and that both of these factors were related to higher for an animal’s survival, when escape is difficult or impossible,
levels of anxiety. In addition, physical pain reports partially me- defensive aggression often minimizes the likelihood of injury or
diated the relation between hurt feelings and anxiety, suggesting death. Physical pain is a primary elicitor of aggression (C. A.
that one mechanism by which hurt feelings increase anxiety is Anderson & Bushman, 2002; Berkowitz, 1993; Vernon, 1965)
through feelings of pain. Finally, in another study, MacDonald et because pain frequently indicates a highly proximal threat that
al. (in press) presented individuals with video clips of painful and requires immediate action (Bowlby, 1973). Although it is unclear
nonpainful events. When the ratings of the nonpainful clips were whether pain can directly prime aggressive tendencies or whether
this effect is mediated by cognition, what is clear is that pain initiate the breakup themselves (Ayduk, Downey, & Kim, 2001).
frequently triggers defensive behavior that is quick and highly Thus, hurt feelings and depression appear related only as a result
reactive (Berkowitz, 1993). In this way, aggression in response to of unwanted separation. This point is important, given that emo-
pain is an important aspect of the fight response, aiding in rapid tional reactions to physical pain are different from the sensory
response to threats to safety. However, research with both animals experience of pain and depend heavily on the meaning or impor-
and humans suggests that this highly defensive stance can lead to tance given to the painful stimulus (K. D. Craig, 1999; Engel,
aggression against others who are not related to the cause of the 1959). That is, emotional reactions to physical pain depend heavily
pain (Berkowitz, 1993; Ulrich, Hutchinson, & Azrin, 1965). For on the implications of the injury. Although injury is typically
example, fighting can be induced in rats by delivering electric associated with negative emotion, some injuries (e.g., a battlefield
footshock (e.g., O’Kelly & Steckle, 1939). wound that earns a soldier the right to go home) may result in
Social exclusion has also been shown to cause aggression positive emotion. Analogously, then, social pain may only be
(Buckley, Winkel, & Leary, 2003; Leary & Springer, 2001; emotionally distressing to the extent that the social bond being
Twenge, Baumeister, Tice, & Stucke, 2001; Vangelisti, 2001; threatened is considered valuable. In MacDonald et al.’s (in press)
Vangelisti & Crumley, 1998). Researchers who study aggression research involving chronic pain patients and community members,
have long capitalized on the link between socially aversive, hurtful both hurt feelings proneness and physical pain were significantly
stimuli and aggression by using insults, criticism, slights, and other related to depression. As with anxiety, the relation between hurt
stimuli that connote exclusion to make participants angry feelings and depression was partially mediated by reports of phys-
(Berkowitz, 1993; Bushman, Baumeister, & Phillips, 2001; Don- ical pain. Again, it appears that one mechanism by which hurt
nerstein, Donnerstein, & Evans, 1975; Harmon-Jones & Sigelman, feelings may influence depression is by leading to increased feel-
2001; Scheier, 1976). In a series of studies, participants who were ings of pain.
randomly assigned to receive feedback that they had been ex- This review suggests that both physical and social pain can
cluded by other participants or that they would have a lonely future induce depression, depressed people have a lower threshold for
exhibited higher levels of aggression such as administering un- experiencing both physical and social pain, and physical pain
pleasant noise blasts to others than did those who did not receive partially mediates the link between hurt feelings and depression.
exclusion feedback (Twenge et al., 2001). This was true even when Overall, depression appears to be intimately related to physical and
the victim of aggression was not involved in the rejection episode social pain, and it may be a mechanism to increase cautiousness or
in any way. This research suggests that, like physical pain, hurt to downregulate behavior to reduce the risk of further injury or
feelings sometimes lead to aggression that is not limited to the exclusion (Allen & Badcock, 2003).
source of threat. Although this pattern may seem interpersonally
maladaptive (would not rejected individuals wish to foster rela- Summary
tionships with other people rather than alienate them through
aggression?), it parallels the findings regarding pain-elicited ag- Overall, social and physical pain correlate similarly with a
gression. Overall, both physical and social pain appear to induce a number of important variables. Both types of pain are related to
general defensive stance that can lead to defensive aggression. extraversion and perceptions of social support, providing evidence
that social inclusion has a strong relation to sensitivity to physical
Depression pain. Further, both types of pain are related to emotional reactions
indicative of increased caution and defensiveness such as anxiety
Both physical pain and hurt feelings are related to higher levels and depression, suggesting that both exclusion and injury are
of sadness and depression (Fine & Olson, 1997; Leary et al., related to general threat-defense mechanisms. These emotional
2001). Physical pain and depression overlap significantly. Accord- reactions are consistent with Chapman’s (1991) depiction of the
ing to a literature review of studies on the subject (Fishbain, emotional response of chronic pain patients to their malady, which
Cutler, Rosomoff, & Rosomoff, 1997), higher levels of depression he described as “an adaptation to loss” (p. 411). This character-
are related to a higher likelihood of experiencing pain, higher pain ization suggests that emotional reactions to both physical and
severity, and more frequent pain. In fact, responses to chronic pain social pain represent a kind of reorganization in response to the
and depression appear so similar that they are often confused by loss of vitally important personal assets—physical and social in-
medical professionals (Weisberg, & Keefe, 1999; Seville & Rob- tegrity, respectively.
inson, 2000; Turk & Flor, 1999). Social pain is also often associ- There also appears to be ample evidence of overlap between the
ated with sadness and depression (Allen & Badcock, 2003; Leary two types of pain: Extraverts are less sensitive to physical pain,
et al., 2001). For example, widows and widowers evidence rates of and physical pain increases introversion, social anxiety, and avoid-
clinical depression twice that of base rates even 24 to 30 months ance of social situations (Phillips & Gatchel, 2000; Sharp &
after their partner has passed (Fraley & Shaver, 1999). Allen and Harvey, 2001); people with low social support are more prone to
Badcock (2003) argued that depression is a mechanism that de- physical pain (Phillips & Gatchel, 2000); research participants
creases social risk when the likelihood of exclusion is perceived as randomly assigned to receive social support experienced less ex-
high. These authors presented evidence from a review of the perimental pain (Brown et al., 2003); fears of abandonment in-
literature indicating that depression is associated with increased crease the distress of physical pain (Mikulincer & Florian, 1998);
sensitivity to social threat, the instigation of support-eliciting be- those more prone to hurt feelings find presentations of physically
haviors, and a decrease in potentially risky social behaviors. Re- painful situations more aversive (MacDonald et al., in press); and
search on romantic relationship dissolution suggests that romantic physical harm is used in retaliation for rejection (Buckley et al.,
rejection-related depression occurs mainly when individuals have 2003; Twenge et al., 2001). Furthermore, the relation between hurt
been rejected by their partners but not when these individuals feelings and both anxiety and depression has been shown to be
partially mediated by reports of physical pain (MacDonald et al., in erized schedule. Participants were scanned first while watching
press). These findings support the notion that both social and others play the game (implicit exclusion), again while being in-
physical pain are managed by similar psychological and physio- cluded in a game (inclusion), and finally while being excluded by
logical systems in humans. 2 other players who did not throw the ball to the participant
(explicit exclusion). Heightened activity in the dorsal ACC was
Shared Physiological Mechanisms of Social and Physical found when participants were either implicitly or explicitly ex-
cluded, relative to the inclusion condition. These researchers also
Pain
found increased activation in the right ventral prefrontal cortex, a
On the face of it, the notion that reactions to physical harm and site associated with negative affect regulation, during explicit
social rejection are mediated by a similar physiological system (though not implicit) exclusion. Eisenberger et al. (2003) described
may seem odd because these two types of threats are typically these reactions to social exclusion as “a pattern of activations very
encountered through different sensory modalities. That is, whereas similar to those found in studies of physical pain” (p. 291). Like
physical pain is most frequently registered via direct touch, stimuli the PET data, the functional magnetic resonance imaging data are
that create social pain typically come in the form of sights or unable to shed light on whether ACC activation causes the expe-
sounds, often through stimuli with purely symbolic meaning (i.e., rience of social pain. However, given the activation of the ACC in
words, gestures, facial expressions). In this way, the two types of relation to the affective component of physical pain, this does
pain can seem quite different. At what point, then, do these provide evidence that reactions to both social and physical pain are
different types of signals come to be processed and experienced in related to similar neurologic components. Moreover, this evidence
similar ways? suggests that the ACC is a viable candidate to be involved partic-
Panksepp (1998) has suggested that the physiology of the at- ularly in processing the aversiveness of social pain.
tachment system may be composed of two separate components:
one component devoted to regulating reactions to social absence The Periaqueductal Gray (PAG)
(what we call social pain), the other to regulating the pursuit of
social engagement. As discussed, Panksepp proposed that this The PAG receives input from the body’s injury detection mech-
attachment system has been built up from older physiological anism, the nociceptive system (A. D. Craig & Dostrovsky, 1999),
systems including those that function to regulate basic needs such as well as from the ACC (An, Bandler, Öngür, & Price, 1998;
as energy balance, thermoregulation, place attachments, and pain Floyd, Price, Ferry, Keay, & Bandler, 2000), and has been shown
perception. Indeed, there is reasonable evidence to suggest that an to be active in connection with physical pain. For example, it has
absence-regulation system is a part of mammalian physiology been linked to analgesia, as it is part of a circuit that controls
(Mason & Mendoza, 1998; Panksepp, 1998). In this section, we nociceptive neurons in the dorsal horn of the spinal cord, with
present evidence for physiological mechanisms that underlie the stimulation of the PAG inhibiting pain transmission by the dorsal
aversiveness and threat response aspects of both social and phys- horn via the release of endogenous opioids (Fields, 2000). Re-
ical pain. search with rat pups has shown that isolation from a dam and
littermates can trigger such analgesia (Kehoe & Blass, 1986b;
Anterior Cingulate Cortex (ACC) Spear et al., 1985). This effect appears to be mediated by the PAG.
In one study, lesions to the lateral or ventrolateral PAG in rat pups
The most direct evidence for the social pain hypothesis comes were shown to disrupt the decreased pain sensitivity following
from work involving the ACC. The ACC has been well established social isolation that was demonstrated by pups assigned to a sham
as an important site for processing physical pain signals (Rainville, (or “placebo”) lesion condition (Wiedenmayer, Goodwin, & Barr,
2002). Specifically, pain affect, but not pain intensity (i.e., sensa- 2000).
tion), appears to be associated with activation in the ACC (Rain- The PAG has also been shown to be related to bonding behavior.
ville, Carrier, Hofbauer, Bushnell, & Duncan, 1999; Rainville, First, evidence suggests that the PAG is involved in regulating
Duncan, Price, Carrier, & Bushnell, 1997; Singer et al., 2004; maternal behavior in rats such as kyphosis (optimal nursing pos-
Tölle et al., 1999). For example, Rainville et al. (1997) manipu- ture), retrieval and transport of pups to the nest, and defense of the
lated the unpleasantness of a painful sensation through hypnotic pups against outsiders (Lonstein, Simmons, & Stern, 1998;
suggestion. Positron emission tomography (PET) revealed that Miranda-Paiva, Ribeiro-Barbosa, Canteras, & Felicio, 2003; Stack,
activation in the ACC was associated with changes in perceived Balakrishnan, Numan, & Numan, 2002). For example, lesions to
unpleasantness, but activity in other brain areas related to pain the caudal intercollicular PAG have been shown to disrupt the
perception (i.e., primary and secondary somatosensory cortices nursing posture of rat pup mothers, resulting in 10% less weight
and rostral insula) did not covary with unpleasantness ratings. gain for the pups of lesioned as opposed to unlesioned dams
Such data suggest that the ACC is involved in the processing of (Lonstein et al., 1998). Second, the PAG has been shown to be
pain affect, although the correlational nature of PET data leaves involved in infant proximity-seeking behavior. During the first 2.5
open the question of whether pain affect is caused by ACC weeks of life, rats have been shown to emit ultrasonic vocaliza-
activation. tions when separated from their dam and littermates that can be
A functional magnetic resonance imaging study of participants reduced by reintroducing an anesthetized dam or littermate (Car-
experiencing social exclusion has shown the ACC to be active in den & Hofer, 1990a; Hofer & Shair, 1978; Kehoe & Blass, 1986b).
response to social exclusion (Eisenberger, Lieberman, & Williams, These “separation distress” cries appear to serve the function of
2003). In this study, participants were told they would be partic- assisting in the reunification of a separated infant with its mother.
ipating in an online ball-tossing game with 2 other participants. Direct stimulation of the PAG can elicit these separation distress
However, the other players were actually controlled by a comput- cries (Panksepp, 1998), and lesions to the PAG appear to decrease
such cries (Wiedenmayer et al., 2000). In fact, Panksepp (1998), after removal from the nest, those removed for 5 min evidenced
on the basis of the physical proximity of PAG areas that can be longer response latencies, indicating that separation prompted an
stimulated to produce distress vocalizations and physical pain analgesic response. Further, response latencies were reduced to
responses, suggested that separation distress emerged anatomically baseline for pups treated with naltrexone (an opioid blocker) and
from more basic pain systems. He concluded that “this affirms that exaggerated for pups treated with morphine, supporting the notion
separation distress is related to perceptions of [physical] pain” that opioids mediate the isolation–analgesia relation.
(Panksepp, 1998, p. 267). There is also strong evidence from animal research that opioids
In general, the PAG is considered an important site for the are involved in signaling the adequacy of social conditions. Low
integration of homeostatic control and limbic motor output in doses of morphine have been shown to reduce the separation
response to threats (Fanselow, 1991; Gray & McNaughton, 2000; distress cries of isolated rat pups (Carden, Hernandez, & Hofer,
Lonstein & Stern, 1998). Gray and McNaughton (2000) argued 1996; Carden & Hofer, 1990b; Kehoe & Blass, 1986b; Kehoe &
specifically that the PAG serves as the coordinator of the panic Boylan, 1994; but see Winslow & Insel, 1991a). Similar results
response and is thus at the base of the hierarchically organized have been reported with other vertebrates including primates (Ka-
neuroanatomical threat-defense system. That is, it is the structure lin, Shelton, & Barksdale, 1988), dogs (Panksepp, Herman, Con-
at the lowest level of the defense system capable of coordinating a ner, Bishop, & Scott, 1978), guinea pigs (Herman & Panksepp,
variety of physiological changes and behaviors to produce a rela- 1978), and birds (Panksepp, Vilberg, Bean, Coy, & Kastin, 1978).
tively organized reaction to potential harm. Indeed, activation of Further, reductions in crying of isolated rat pups as a result of the
the lateral PAG in nonhuman mammals (e.g., through injection of introduction of a dam or littermate can be reversed by administra-
excitatory amino acids) leads to key aspects of the panic response, tion of opioid antagonists such as naltrexone (Carden et al., 1996;
including defensive behavior, hypertension, tachycardia, and non- Carden & Hofer 1990a, 1990b), suggesting that drops in opioid
opioid analgesia (Bandler & Shipley, 1994). In particular, stimu- levels may signal an unsatisfactory social environment. In partic-
lation of the caudal lateral PAG appears to lead to preparation for ular, ␮-opioid receptors (responsive to endorphins) and, to a lesser
flight through physiological mechanisms such as increased blood extent, ␦-opioid receptors (responsive to enkephalins), both of
flow to the limbs and decreased blood flow to the face. Stimulation which are powerfully related to reductions in pain, also appear to
of the intermediate lateral PAG appears to lead to preparation for be generally effective in reducing separation distress vocalizations
confrontational defense, through mechanisms such as decreased (Carden, Barr, & Hofer, 1991; Carden et al., 1996; Kehoe &
blood flow to the limbs and increased blood flow to the face Boylan, 1994; Panksepp, 1998). Administration of morphine has
(suggesting a possible link with blushing). Further, stimulation of also been shown to reduce the pursuit of social interaction in
the ventrolateral PAG appears to lead to reactions with similarities primates (Keverne, Martensz, & Tuite, 1989; Martel, Nevison,
to responses to social defeat and depression, including quiescence Simpson, & Keverne, 1995), guinea pigs (Herman & Panksepp,
and hyporeactivity (Bandler & Shipley, 1994). Overall, the PAG 1978), and rats (Panksepp, Najam, & Soares, 1980). For example,
appears responsive to both separation cues and physical pain in young rhesus monkeys treated with naloxone (another opioid
nonhuman animals and appears to contribute to coordinated re- blocker) pursued more contact with their mothers, and mature
sponses to both. In particular, these responses seem to be quick, naloxone-treated monkeys solicited and received more grooming
reactive impulses such as defense, escape, and downregulation from conspecifics (Martel et al., 1995). This again suggests that
regardless of whether the initial input was social or physical in opioids may comprise one way the body regulates response to
nature. Thus, at least for nonhuman animals, both social exclusion social distress, with low levels of opioids signaling an unsatisfac-
and physical pain appear intimately related to the most base level tory social environment and motivating the pursuit of social inter-
of the threat-defense system. This supports the notion that social action. It is important to note that such a withdrawal of opioid
exclusion is processed as a primal threat for animals who rely on activity from ␮- and ␦-opioid receptors can create an aversive,
interdependent relationships with conspecifics. Functionally, the painful state as in the case of withdrawal from opiate addiction
PAG appears to provide at least one mechanism by which signals (e.g., heroin addiction; Panksepp, 1998). Thus, another potential
of social exclusion may facilitate quick action in response to mechanism for the aversiveness of social pain is the reduction of
inclusion threats. opioid activity experienced during rejection, separation, or loss.
Opioids Oxytocin
Another point of overlap between social and physical pain is the The neuropeptide oxytocin provides a further link between
opioid neuroendocrine system (Panksepp, 1998; Taylor, Dicker- social and physical pain. Oxytocin is perhaps most widely known
son, & Klein, 2002). Endogenous opioids have long been recog- for its roles in lactation and parent– child bonding. For example,
nized as an important regulator of physical pain, with exogenous administration of oxytocin has been shown to induce maternal
forms such as morphine used to treat pain complaints (Panksepp, behavior (e.g., following and cleaning young) in virgin rats (Ped-
1998; Smith, Stevens, & Caldwell, 1999). Research with both rats ersen, Ascher, Monroe, & Prange, 1982) and sheep (Kendrick,
and mice suggests that opioids play an important role in isolation- Keverne, & Baldwin, 1987). This peptide has also been shown to
induced analgesia (Kehoe & Blass, 1986a, 1986b; Konecka & reduce distress vocalizations in rat pups isolated from their dam
Sroczynska, 1990; Naranjo & Fuentes, 1985; Spear et al., 1985). and littermates (Insel & Winslow, 1991). Oxytocin has also been
For example, Kehoe and Blass (1986b) measured the response tied to a wider range of social behaviors. One avenue of investi-
latencies of 10-day-old rat pups when a paw was placed on a gation has involved comparing two closely related species, the
heated metal surface either immediately after isolation from the prairie vole and the montane vole. These two species are highly
nest or 5 min after removal. Relative to pups tested immediately similar, except for their social behavior. The prairie vole tends to
be monogamous and affiliative and cares for its young, whereas Social pain theory argues that physiological social pain mecha-
the montane vole tends to be highly solitary (Carter, DeVries, & nisms should be shared across a wide variety of social animals, as
Getz, 1995). This difference has been partially attributed to the such mechanisms should date back to the early days of social
different distribution of oxytocin receptor sites in the brains of the speciation. However, there are also crucial differences between
two types of voles (Insel & Shapiro, 1992). In addition, adminis- humans and other mammals. For example, more sophisticated
tration of oxytocin has been shown to facilitate social contact and processing ability in humans relative to other mammals allows for
selective preference of mates in prairie voles, with oxytocin an- self-awareness and projection of the self into the future (Leary &
tagonists blocking such partner preferences (Cho, DeVries, Wil- Buttermore, 2003; Suddendorf, 1999). Thus, social exclusion can
liams, & Carter, 1999; Insel & Hulihan, 1995; Witt, Carter, & have implications for humans, not just in terms of their current
Walton, 1990). For example, Cho et al. (1999) treated prairie voles social status, but also for beliefs about acceptability to others in the
with either oxytocin (1, 10, or 100 ng) or a placebo, then placed future. This may well influence how social pain is processed by
them with an opposite sex conspecific for 1 hr. Following this, the humans. Further, although the nonhuman animal data are useful
experimental voles were placed in a cage permitting access to for investigating behavioral reactions to separation and injury, they
either the familiar or an unfamiliar conspecific. Those treated with are incapable of speaking to the phenomenological experience of
higher levels of oxytocin (100 ng) were significantly more likely to pain in response to exclusion. Finally, much of the nonhuman
place themselves in contact with the familiar vole, suggesting that animal research focuses on infants (e.g., rat pups), while social
oxytocin positively reinforced time spent with a partner, thus pain theory is concerned with regulation of social behavior across
promoting a partner preference. Oxytocin administration has also the life span. Nevertheless, the scant research on physiological
been shown to facilitate social behavior in rats (Witt, Winslow, & reactions to rejection in humans conducted thus far appears con-
Insel, 1992) and squirrel monkeys (Winslow & Insel, 1991b), and sistent with the nonhuman animal data. Specifically, rejection has
gentle stroking has been shown to lead to the release of oxytocin been shown to lead to ACC activation (Eisenberger et al., 2003),
in rats (Stock & Uvnäs-Moberg, 1988). increased blood pressure and cortisol (Stroud, Tanofsky-Kraff,
Uvnäs-Moberg and colleagues have argued that oxytocin also Wilfley, & Salovey, 2000), and analgesia for individuals prone to
functions to regulate physical pain. In a series of studies conducted hurt feelings (MacDonald et al., in press). Clearly, significant work
with rats, administration of oxytocin was shown to reduce sensi- remains to be done to clarify physiological responses to social
tivity to pain (Ågren, Lundeberg, Uvnäs-Moberg, & Sato, 1995; exclusion in humans. Nevertheless, future research examining
Lundeberg, Meister, Björkstrand, & Uvnäs-Moberg, 1993; Lunde- whether such responses are mediated by the mechanisms listed in
berg, Uvnäs-Moberg, Ågren, & Bruzelius, 1994; Uvnäs-Moberg, this review, and/or other mechanisms, seems warranted.
Bruzelius, Alster, Bileviciute, & Lundeberg, 1992), whereas oxy-
tocin antagonists, but not opioid antagonists, were shown to block
Implications of Social Pain Theory for Aggressive
this analgesic effect (Ågren et al., 1995; Lundeberg et al., 1994;
Uvnäs-Moberg, Bruzelius, Alster, & Lundeberg, 1993; Uvnäs-
Behavior
Moberg et al., 1992). For example, Ågren et al. (1995) demon- It is the denial of our intrinsic biological and psychological need for
strated that rats treated with oxytocin showed slower response the “other” that may partly explain the length of time that it has taken
latencies to thermal stimuli up to 90 min posttreatment, whereas to begin to understand the origins of human violence. (de Zulueta,
rats treated with saline returned to baseline responses at 15 min. 1996, p. 176)
Rats treated with oxytocin antagonists demonstrated faster than
baseline response latencies 45 and 75 min posttreatment. Thus, In this section, we strive to demonstrate one important implica-
oxytocin appeared to promote analgesia, while hyperalgesia was tion of conceptualizing social exclusion in terms of pain. Specif-
demonstrated in rats whose oxytocin receptors had been blocked. ically, we propose that if exclusion is perceived as a serious,
Furthermore, oxitonergic neurons project from the paraventricular primal threat, then it should motivate an individual to adopt a
nucleus of the hypothalamus to a number of pain-related brain sites highly defensive stance. Further, because the role of the panic
including the PAG and the dorsal horn of the spinal cord response is to provide quick action in the face of any imminent
(Sawchenko & Swanson, 1982), suggesting a possible role for threat, we propose that social pain should lead to a preparedness to
oxytocin in the regulation of pain. However, there is controversy defend against not just social, but physical threats as well. In line
over this conclusion, as not all researchers agree that oxytocin has with the notion of fight/flight/freezing, such a stance should in-
true analgesic properties (e.g., Xu & Wiesenfeld-Hallin, 1994). clude a preparedness to escape and a preparedness to aggress. In
fact, research has shown that excluded individuals exhibit many
Summary features of the panic response, including aggressiveness (Buckley
et al., 2003; Twenge et al., 2001), analgesia (MacDonald et al., in
Overall, strong evidence for a physiological connection between press), high blood pressure and plasma cortisol concentrations
responses to physical pain and social exclusion has been found (Stroud et al., 2000), and disruptions in higher order cognitive
across a variety of physiological markers. Our analysis suggests processing without accompanying disruptions in more automatic
that the ACC, the PAG, opioids, and oxytocin may all underlie mental tasks (Baumeister, Twenge, & Nuss, 2002). These findings
both physical pain and social behavior regulation. However, some suggest that exclusion taps into relatively basic systems that are
caution must be taken in evaluating the applicability of the phys- oriented toward response to generalized threat, rather than social
iological evidence to human social behavior. Specifically, only the threat in particular. The review that follows aims to demonstrate
evidence relating to the ACC is derived from studies with human that both social and physical pain can prime aggressive action
participants. Thus, the extent to which the PAG, opioids, and tendencies and that both types of pain-elicited aggression are
oxytocin are involved in human social pain is an open question. moderated by perceptions of defensive distance. Overall, the goal
of this section is to provide a more comprehensive understanding who hurt his or her feelings proximal (as in the case of romantic
of exclusion-elicited aggression by noting its similarities with couples or good friends), aggressive responses to social pain are
defensive aggression provoked by physical pain. likely to be interpersonally dysfunctional in the long run.3
Few people would be surprised to learn that those whose feel- Although it may be difficult to conceive of social exclusion
ings are hurt often desire to inflict hurt in return (Leary & Springer, eliciting automatic reactions such as aggression, there is one com-
2001). Research has confirmed this intuition; as discussed earlier, mon automatic response to both exclusion and physical pain al-
rejection often elicits aggressive behavior (Buckley et al., 2003; ready known— crying (D. C. Craig et al., 2000; Vingerhoets,
Twenge et al., 2001). In some instances, more forceful forms of Cornelius, Van Heck, & Becht, 2000; Vangelisti & Crumley,
behavior (i.e., assertiveness) in response to hurt feelings can be 1998). Although crying can be controlled, or at least delayed
useful because actively confronting sources of hurt can help to (Leary et al., 1998), the urge to cry is often involuntary. As a result
resolve troubling relational issues (Fine & Olson, 1997; Vangelisti of its involuntary nature, crying is an expression that may elicit
& Crumley, 1998). However, aggression, especially physical ag- support from empathic onlookers without the individual needing to
gression, in response to hurt may be destructive and counterpro- approach anyone or explicitly seek support (Gross, Fredrickson, &
ductive by ultimately adding to the person’s interpersonal prob- Levenson, 1994). This elicitation of support is obviously beneficial
lems and creating more, rather than less, social pain (Buckley et
in the case of both physical injury and social loss.4 Furthermore, its
al., 2003; Twenge et al., 2001). Specifically, given that hurt
automatic nature is important given that people who are hurt may
feelings arise when people feel less relationally valued than they
not approach others readily because their pain encourages them to
desire (Leary & Springer, 2001; Leary et al., 1998), aggression
take a defensive stance. We suggest, then, that aggression may be
seems like an odd response. A person who feels devalued is
a relatively automatic response to both social exclusion and phys-
unlikely to increase others’ acceptance by insulting, abusing, or
ical pain in much the same way as is crying.
attacking them.2 From an early age, children who aggress are
rejected (Cantrell & Prinz, 1985; Dodge & Coie, 1987; McDou- Of course, social exclusion does not always result in aggressive
gall, Hymel, Vaillancourt, & Mercer, 2001), suggesting that it is behavior. An important question, then, is when social pain is most
unlikely that rejected aggressors expect to win interpersonal ac- likely to trigger an aggressive response. In general, we expect that
ceptance from their victims. Furthermore, even if the experience of aggression would be especially likely when defensive distance
hurt feelings reduces individuals’ desires to be accepted by the from exclusion is low. Thus, aggression in response to social pain
source of the hurt (Bourgeois & Leary, 2001; Leary et al., 1995), should be more likely when higher degrees of rejection or rela-
aggression still does not seem like an appropriate response. If you tional devaluation are perceived. However, as with physical pain,
have given up on being accepted by someone, why not just walk there are a number of cognitive moderators that can impact on
away rather than risk the social, physical, and legal consequences
of a verbal or physical attack? 2
Perhaps the saddest and clearest example of this paradox comes from
We suggest that hurt feelings may contribute to aggressive
the recent trend of school and work shootings that appear to be largely
behavior on the basis of our proposals that social pain can activate motivated by perceived rejection (Leary, Kowalski, Smith, & Phillips,
the generalized threat-defense system and that when ongoing re- 2003).
jection or high degrees of relational devaluation are perceived (i.e., 3
This speculation on relatively automatic aggressive reactions to social
when defensive distance is low), reactions are motivated by panic pain is consistent with Twenge and her colleagues’ studies that showed that
response mechanisms. Specifically, aggressive responses to rejec- self-reports of negative emotion did not mediate the rejection–aggression
tion mimic behavior under conditions of physical pain. A great link (Twenge et al., 2001). If it is true that conscious negative feelings are
deal of research has demonstrated that physical pain reliably elicits not the proximal cause of rejection-elicited aggression, then automatically
aggression in both human beings and other animals (Berkowitz, primed aggressive tendencies in response to social pain may be key.
1989; Scott, 1966; Ulrich et al., 1965). When pain results from However, the Twenge et al. (2001) work is peculiar in that excluded
physical attack, a quick counterattack is often very effective in individuals did not report more negative emotion than individuals who
were not excluded (see also Twenge, Catanese, & Baumeister, 2003),
stopping the threat (Öhman & Mineka, 2001). In fact, some re-
despite the fact that exclusion has often been shown to evoke negative
searchers have suggested that painful stimuli may automatically
affective reactions (Baumeister & Tice, 1990; Leary et al., 2001). Thus,
prime action tendencies associated with pain such as aggressive Twenge et al.’s (2001, 2003) failure to find mediation may reflect a more
responding (C. A. Anderson & Bushman, 2002; Berkowitz, 1989; general difficulty with capturing self-reported negative affective reactions
da Gloria, Pahlavan, Duda, & Bonnet, 1994). For example, Izard to exclusion in their studies. This issue is complicated by the finding that
(1991) showed that 90% of infants aged 2 to 7 months displayed individuals high in proneness to hurt feelings may experience analgesia, or
an angry facial expression after a painful inoculation. Indeed, pain numbing, in response to rejection (MacDonald et al., in press; Twenge et
signals have been shown to reach reflexive motor circuits before al., 2003), suggesting negative affect may be blunted for some individuals.
the pain is realized consciously (Panksepp, 1998), suggesting that Overall, we consider there to be too little data currently to resolve the
pain-induced aggression may be difficult to control. Thus, to the question of whether negative affect mediates the rejection–aggression link,
but pursuit of this question should help shed light on the possible existence
extent that social exclusion taps into panic mechanisms, such
of automatic, aggressive reactions to social pain.
exclusion may also prime a preparedness to aggress. The diffi- 4
The fact that crying also occurs in response to joyful feelings may be
culty, however, is that such quick, aggressive reactions are likely
considered to weaken this argument. However, we suggest that crying may
to be much less functional in warding off social as opposed to be a signal for the elicitation of closeness from others associated with both
physical threats. For example, hurt feelings from a lover’s insult negative and positive affective states. Indeed, happy individuals desire
may lead to a quick counterattack, such as a shout, shove, or closeness from others as a means of maintaining or enhancing positive
punch, triggered by a sense of threat. However, because the at- affect, and closeness associated with joyful times may be a mechanism for
tacker in this instance likely has an interest in keeping the person cementing interpersonal bonds.
perceptions of defensive distance from exclusion. In this section, we suggest that factors that decrease perceptions of defensive
we explore these factors by reviewing commonalities between distance (from injury or exclusion) increase the risk of aggression.
conditions that lead to physical pain-elicited aggression and social
pain-elicited aggression. That is, we searched the literature to Situation Appraisal and Reappraisal
determine factors that make aggression especially likely as a result
of physical pain and then looked for analogues in the realm of According to C. A. Anderson and Bushman’s (2002) general
social pain. In particular, we draw heavily on the domestic vio- aggression model, aggressive behavior can be based either on a
lence literature to help elucidate our ideas regarding social pain relatively automatic appraisal of whether the situation calls for
and aggression. aggression or on a more deliberate reappraisal of one’s initial
Partner abuse is an important topic in the consideration of a action tendency. Appraisal of a stressful situation, such as the
hurt–aggression link for two reasons. First, it is well known that experience of pain, changes according to an individual’s percep-
hurt feelings can lead to violence (Leary & Springer, 2001), and tions of the consequences of the experience, the extent to which
the highly interdependent nature of close relationships provides a one’s well-being is threatened, and the resources available for
fertile breeding ground for such hurt (Levitt, Silver, & Franco, coping with the threat (Weisenberg, 1999). As discussed earlier,
1996; Vangelisti & Maguire, 2002). Indeed, there is a wide range we propose that social exclusion can lead to aggression because
of evidence suggesting that partner abuse may largely stem from such exclusion is processed at a basic level as a primal threat to
abusers’ perceptions or fears of rejection. Literature reviews indi- one’s well-being. For example, K. D. Williams, Case, and Govan
cate that abusers report higher fears of rejection and abandonment (in press) demonstrated that participants who were ostracized
(Dutton, 2002; Holtzworth-Munroe, Bates, Smutzler, & Sandin, during an online ball-toss game evidenced higher levels of implicit
1997), attribute more negative intentions to their partners (Eck- racial prejudice, as measured by a reaction time task, than those
hardt & Dye, 2000; Holtzworth-Munroe, Bates, et al., 1997), who were not ostracized (self-reports of prejudice were not af-
experience more jealousy and less secure attachment (Dutton, fected). This suggests that exclusion may prime an automatic
2002; Holtzworth-Munroe, Smutzler, & Bates, 1997; Schumacher, defensive stance against perceived threat, even if such defensive-
Smith Slep, & Heyman, 2001), have their demandingness met with ness is not recognized consciously. Thus, social exclusion may
lead to an initial situation appraisal that indicates a high degree of
withdrawal or ostracism (Holtzworth-Munroe, Smutzler, & Bates,
threat, possibly priming an aggressive response.
1997; Schumacher et al., 2001), report higher levels of depression
C. A. Anderson and Bushman (2002) argued that a reappraisal
and anxiety (Gleason, 1997; Holtzworth-Munroe, Smutzler, &
of such a situation will occur only if two conditions are met: (a)
Bates, 1997; Schumacher et al., 2001), and are more likely to have
there are sufficient cognitive resources to permit reappraisal and
been rejected by their parents (Schumacher et al., 2001). Further,
(b) the outcome of the initial appraisal is both important and
those individuals who kill their spouses usually do so during
unsatisfying. Both social and physical pain may place pressure on
periods of perceived or actual abandonment (Dutton, 2002).
reappraisal efforts because a strong perception of threat can shift
Second, aggression in relationships is depressingly common,
an individual into a more defensive, reactive mode. Such a reactive
with estimates of the incidence of violence in relationships ranging
stance is likely to augment the importance of automatic as opposed
from 12% to 57% (Arriaga & Oskamp, 1998). Figures this high
to controlled processing, limiting the cognitive resources available
suggest that such violence is too common to attribute its cause for reappraisal. Literature reviews suggest that physical pain dis-
simply to some “abnormality” in abusers. Thus, understanding rupts cognitive functioning, particularly decreasing attentional ca-
family violence is an important research goal in its own right. In pacity and processing speed (Eccleston & Crombez, 1999; Hart,
fact, one would hope that if people could restrain aggressive Martelli, & Zasler, 2000). Response to social exclusion appears to
impulses against any target, it would be their relationship partners, have a similar effect. Baumeister et al. (2002) randomly assigned
but too often this is not the case. Instead, family violence appears participants to receive feedback that they would have a lonely
to be a poignant example of an area in which aggressive responses future or that they would have an accident-prone future. Partici-
to rejection not only create tremendous trauma for the victim of pants told they would be lonely scored lower on IQ and Graduate
aggression but also make further rejection of the aggressor more Record Examination tests, but did not differ from control partici-
likely. pants on more automatic tasks such as memory recall. These
Because of the extensive literature on family violence, we were results suggest that threats to social inclusion interfere with higher
able to examine parallels between such violence and aggression order cognitive functioning, possibly indicating a bias toward more
resulting from physical pain in some detail. This review highlights automatic processing under these circumstances. Thus, one re-
the fact that both social pain-elicited aggression and physical sponse to social or physical pain appears to be a heavier reliance
pain-elicited aggression appear to stem from difficult-to-control on more automatic cognitive processing, suggesting that both types
impulses that arise quickly, possibly automatically, from painful of pain are treated as highly threatening, requiring a quick
feelings. We suggest that the relatively rapid activation of social response.
pain-elicited aggression is a result of the strong sense of threat that Given the possibility that defensive aggression may be an au-
social pain evokes. That is, evolution has equated exclusion with tomatic response to social exclusion, such a decrease in higher
extinction, meaning rejection may be treated as a mortal danger at cognitive functioning means that exclusion should be especially
the motivational level. This high level of perceived threat should likely to lead to aggression when further burdens are placed on
lead individuals to adopt a protective posture, bypassing complex processing capacity. Research on relationship aggression backs up
cognitive processing in favor of efficient, defensive behavior. We this notion. Higher risk of abuse has been connected with a number
note further that both types of aggression are moderated by a of variables related to deficits in cognitive capacity and impulse
strikingly similar constellation of cognitive moderators. In general, control such as alcohol consumption (Gleason, 1997; Leonard &
Senchak, 1996; MacDonald, Zanna, & Holmes, 2000) and head power and control (Dutton, 2002; Jackson, 1999; Schumacher et
injury (Holtzworth-Munroe, Bates, et al., 1997). Further, male al., 2001). This point is especially important in light of the afore-
abusers asked to imagine themselves in scenarios in which they are mentioned meta-analysis showing that the combination of social
rejected by their wives react with more irrational ideas than non- evaluative stressors and lack of control is strongly predictive of
abusers (Eckhardt & Dye, 2000; Schumacher et al., 2001). That is, cortisol release (Dickerson & Kemeny, 2004). As those who more
abusers in this research exhibited a less sophisticated cognitive strongly desire control are more likely to perceive unsatisfactory
response to rejection than nonabusers, suggesting they may be less levels of control in a given situation, such individuals may be
able to control aggressive action tendencies spurred by exclusion. especially reactive when perceiving rejection.
The notion that relationship aggression involves attempts at
Attributions control is not new. K. D. Williams et al. (in press) suggested that
social exclusion can induce a need to regain control of the situation
If sufficient cognitive resources are available for more complex that motivates behavior to gain the attention of the excluder,
processing, attributing causality for physical pain to a specific whether that behavior (including aggression) is prosocial or not.
source can either reduce or exacerbate aggressiveness (Berkowitz, Further, some arguments from the feminist tradition suggest that
1993). Specifically, attributions that decrease defensive distance relationship aggression may represent men’s efforts to exert the
(i.e., increase perception of threat) should more strongly prime power granted them by a patriarchal society (Renfrew, 1997),
aggressive impulses. For example, if an individual determines that although feminist scholars also point to other control motives
a coworker spilled hot coffee on him or her intentionally instead of (M. P. Johnson & Ferraro, 2000). The latter point is important,
accidentally, an aggressive response is more likely. In this case, the given a recent review suggesting that women are aggressive in
incident signals a greater threat of future harm than if the act was relationships as often (though not as severely) as men (Archer,
accidental and thus warrants a more hostile response. Reviews of 2000). Our theory of social pain suggests that control-oriented
the partner abuse literature suggest that attributions of causality aggression may reflect a basic motive to increase defensive dis-
also play an important role in partner abuse, as abusers tend to tance and reduce the sense of threat signaled by social pain. In this
attribute more hostile intentions and traits to their partners (Eck- way, our theory of social pain suggests that control-oriented ag-
hardt & Dye, 2000; Holtzworth-Munroe, Bates, et al., 1997). Thus, gression may not be limited to those whom society grants power.
abusers may be aggressive, in part, because their negative attribu- However, this is not to say that issues such as patriarchy are
tions indicate to them a higher degree of rejection and an increased irrelevant to aggression. As a loss of control may be related to
likelihood of future pain. increased pain (Staub et al., 1971), men who believe in patriarchal
structure may experience the threat of social pain especially
Control acutely in relationships if they feel their “rightful” control is
slipping.
Another important factor in both physical and social pain is
control. Literature reviews suggest that perceiving that one has Feeling Trapped
control over physical pain, particularly that one can terminate the
painful stimulus, is related to a lower subjective experience of pain Aggression is more likely if individuals feel trapped in a painful
and increased pain tolerance (Arntz & Schmidt, 1989; Seville & situation. For example, animals generally prefer to avoid physical
Robinson, 2000). Thus, perceptions of control appear to decrease conflict when possible but will fight in response to physical pain
aggressiveness by increasing defensive distance from threat. Feel- when no escape is available and a threat is perceived as imminent
ings of lost control over painful stimuli may be especially painful. (Blanchard & Blanchard, 1990; Gray & McNaughton, 2000; Ul-
In one study (Staub, Tursky, & Schwartz, 1971), some participants rich et al., 1965). Thus, we would expect hurt feelings to be more
were given control over the administration of an initial regimen of likely to result in aggression when the hurt individual believes that
electric shocks they were to receive, then lost that control during significant barriers to exiting a relationship exist. Phrased differ-
a second regimen. The other participants in the study were not ently, when hurt feelings signal a threat and the individual feels
given control during either the first or second set of shocks but trapped in that situation, the likelihood of aggression may be
were yoked to the “in-control” participants such that they received higher than when the individual feels free to walk away. Like the
shocks of the same intensity and temporal sequence. Relative to different sources of physical and social pain, being physically
those who never had control, those who lost control were quicker trapped appears on the surface to be much different from being
to report discomfort as shock intensity increased and ended the emotionally trapped. However, one commonality between the two
shock regimen after a smaller number of shocks. is a lack of control in that escape from pain seems impossible.
Given the relationship between control and perceived pain, a When physically trapped, inaction in response to threat inevitably
potentially useful way of explaining pain-induced aggression is as leads to physical harm. When emotionally dependent on another,
a behavioral tendency that can be helpful in increasing control over withdrawal of the other inevitably leads to social pain (Panksepp,
a potentially threatening stimulus. In the case of physical pain, 1998). Indeed, a review of the spousal violence literature suggests
achieving such control may be relatively straightforward. How- that relationship aggression is more likely when the aggressor feels
ever, by definition, a source of social pain is some social entity. dependent on the relationship (Holtzworth-Munroe, Bates, et al.,
Thus, exerting control over the source of hurt feelings or other 1997). Further, violence often follows increases in commitment to
social pain may mean attempting to control another person. Ag- the relationship such as marriage (Henton, Cate, Koval, Lloyd, &
gression is certainly one way to exert such power and control Christopher, 1983; Rounsaville, 1978), times when increased de-
(Tedeschi & Felson, 1994), and reviews of the literature suggest pendence is highly salient. Finally, in a review of spousal homicide
that relationship aggressors tend to demonstrate higher needs for literature, Dutton (2002) suggested that a large percentage of
perpetrators of spousal homicide experience a catathymic crisis, or surprising, then, that reviews of the literature indicate that insecure
aversive emotional tension that is perceived as inescapable and attachment is a common aspect of those who abuse their romantic
caused by the spouse. Thus, aggression against romantic partners partners (Holtzworth-Munroe, Bates, et al., 1997; Roberts & Nol-
by individuals who are emotionally dependent on the relationship ler, 1998; Schumacher et al., 2001). That is, those who closely
may be motivated by a desire for control similar to that demon- attend to cues connoting the threat of social pain are likely to
strated with physical pain-elicited aggression. experience that pain more intensely, decreasing defensive distance
and increasing the possibility of rejection-elicited aggression.
Hostility
Summary
Hostility, or trait anger, is an important personality variable
influencing the likelihood of aggression in response to pain. Angry The literature reviewed in this section supports the usefulness of
and aggressive thoughts and feelings can be primed by exposure to conceptualizing social pain-elicited aggression as a reaction to low
physical pain (C. A. Anderson & Bushman, 2002; Berkowitz et al., perceived defensive distance from rejection. Such a relation ap-
1981), especially for those with generally hostile dispositions pears analogous to physical pain-elicited aggression that stems
(K. B. Anderson, Anderson, Dill, & Deuser, 1998). Participants in from low perceived defensive distance from injury. Further, this
a study by K. B. Anderson et al. (1998) were randomly assigned to review suggests that cognitive factors that influence perceptions of
hold their arm in either a painful or comfortable position while defensive distance including appraisals, attributions, perceived
evaluating the similarity between pairs of words. Some of these control, hostility, and pain expectancies moderate the likelihood of
words were explicitly related to aggression (e.g., choke), while aggressive responses to both social and physical threats. This
others were more ambiguously related to aggression (e.g., stick). analysis helps to resolve the question of why aggression is a
Participants high in hostility who were experiencing pain evalu- frequent response to rejection, despite the fact that such aggression
ated the ambiguous-aggressive and aggressive-aggressive word appears to be nonfunctional in winning acceptance. Particularly, if
pairs as highly similar, suggesting that aggressive thoughts were rejection is indeed a primal threat, then the panic motivation
highly accessible for these individuals. Reviews of the domestic spurred by low defensive distance may lead to defensive aggres-
violence literature suggest that trait hostility is also a strong sion that is functional in physical threat contexts but less functional
correlate of domestic violence (Gleason, 1997; Holtzworth- in social threat contexts. Of course, some caution is warranted in
Munroe, Bates, et al., 1997; Schumacher et al., 2001). Thus, accepting this conclusion. Whereas much of the physical pain-
people with hostile personalities appear to be under a relatively related data comes from experimental research, much of the do-
chronic state of threat, perceiving defensive distance from both mestic violence literature reviewed is based on correlational work.
injury and rejection as low. As a result, hostile individuals appear Thus, although we have drawn parallels between the two it is
to react to both physical and social pain cues with more aggressive difficult to be certain that the results are directly comparable.
thoughts and behavior. Nevertheless, it is striking that results from these two very differ-
ent approaches share these parallels. It is our belief that social pain
theory provides a parsimonious framework through which to view
Anticipation of Pain
the commonalities between aggression provoked by social and
Expecting a stimulus to be physically painful has been associ- physical threat.
ated with an increase in felt pain and more aggression in response
to that pain (Berkowitz & Thome, 1987; Leventhal, Brown, Implications of Social Pain for Chronic Pain and Pain
Shacham, & Engquist, 1979). For example, Berkowitz and Thome Disorders
(1987) instructed participants to place an arm in water, telling
some to expect the water to be painful while others did not receive Our contention that social pain stems from the same emotional
this information. Half of those told to expect pain placed their arm unpleasantness associated with physical pain implies that feelings
in uncomfortably cold water, whereas the other half placed their of exclusion and relational devaluation may contribute to pain-
arm in more comfortable room temperature water. All participants related disorders. Indeed, clinical views of somatoform pain dis-
not told to expect pain placed their arm in cold water. Participants order (pain complaints that cannot be adequately explained by a
were then asked to deliver rewards (nickels) and punishments known medical disorder) suggest that drawing a distinct line
(noise blasts) based on the performance of a “worker” (a confed- between physical and social pain is inaccurate and potentially
erate). Relative to those not expecting the cold water to be painful, harmful (Roth, 2000; G. E. Simon, 1998; Sullivan, 2000). Like
those in the cold water condition who did expect pain reported the other forms of physical pain, somatoform pain is strongly related
water to be more painful, unpleasant, and annoying. In addition, to both anxiety and depression, and treatments for anxiety and
those expecting and experiencing pain delivered fewer rewards depression, including cognitive– behavioral therapy and tricyclic
and more noise blasts relative to the other two groups. These data antidepressants, help to alleviate somatoform pain (Fishbain, Cut-
suggest that those expecting to experience pain may exhibit the ler, Rosomoff, & Rosomoff, 1998; G. E. Simon, 1998; Sullivan,
confirmation bias, selectively searching for and attending to threat- 2000).
ening pain cues (Sharp & Harvey, 2001), thus increasing their Social pain theory may be useful in understanding aspects of
experience of pain, decreasing defensive distance, and enhancing somatoform pain and other pain disorders. For example, individ-
aggressive action tendencies. Analogously, people with insecure uals who experience any type of chronic pain are prone to feelings
attachment styles are highly wary of rejection from close others of embarrassment resulting from conflict with others who do not
and thus tend to be highly sensitive to cues that may signal the understand their pain, including medical professionals (Chapman,
potential for hurt feelings (Downey & Feldman, 1996). It is not 1991; E. P. Simon & Folen, 2001). Given that such social pain may
lead to activation of pain circuits in the ACC and right ventral that future research in this vein also feature control groups in
prefrontal cortex (Eisenberger et al., 2003), perceived relational which participants experience noxious but nonexclusion-related
devaluation resulting from a pain disorder might add directly to an stimuli. For example, participants could be exposed to socially
individual’s suffering. Further, individuals who perceive rejection disgusting stimuli to test whether brain areas involved in pain
readily may find their body’s pain management systems taxed. For processing are independent of this type of aversive social stimulus.
example, chronic activation of analgesia in response to social This analysis also suggests that research investigating the neuroen-
threat (MacDonald et al., in press) may lead to the depletion of docrine basis of reactions to exclusion in humans would be
resources over time, leaving rejection-sensitive individuals vulner- valuable.
able to increased pain. Indeed, low levels of opioids create a higher Despite the fact that much of this article focuses on pain in
level of vulnerability to physical pain because of opioids’ impor- response to social exclusion, its focus on the role of the general
tance in regulating physical discomfort (Panksepp, 1998). This threat-defense system in responding to social conditions is equally
notion may help account for the fact that individuals with low important. We believe that future research examining what condi-
social support are more pain sensitive (Phillips & Gatchel, 2000). tions lead to particular physiological reactions to social exclusion
Thus, encouraging feelings of social acceptance may help to is needed to determine with increased clarity the role of the
alleviate pain complaints (Brown et al., 2003). Along these lines, defense system. Given that fight/flight/freezing appear to underlie
an important aspect of current treatment for somatoform pain is response to exclusion, it becomes increasingly important to under-
support and validation that the pain is real (G. E. Simon, 1998), stand what the human analogue of these three reactions are in
and treatment for chronic pain often involves family members in social situations. At least on the surface, fight and flight responses
the therapeutic process (Chapman, 1991). Such affirmation may seem relatively easy to understand. Social fight motivation appears
help to break a cycle in which feelings of exclusion contribute to to manifest in assertiveness and aggression, and social flight mo-
inexplicable pain, the expression of which alienates the pain suf- tivation in withdrawal from interactions. Although the freezing
ferer from others and leads to increased exclusion that then exac- response in humans appears less intuitive, it may well be repre-
erbates the pain further (Sullivan, 2000). sented by depressive affect and behavior. An important follow-up
question is under what conditions social threat will lead to each of
Research Directions the three responses. In physical contexts, flight is the preferred
option in response to threat and is instigated if an escape route
Although the evidence reviewed in this article has clearly drawn from the threatening stimulus is available (Gray & McNaughton,
a link between social and physical pain, it has also highlighted 2000). Freezing occurs when escape is impossible but the threat
some apparent discrepancies that require further research attention. can be avoided by becoming motionless. Fight is the least pre-
As discussed earlier, some work suggests that social exclusion is ferred option but is selected when escape is not possible and a
related to decreased pain sensitivity, whereas other work suggests threat is actively manifested. Considering these factors in a social
that social inclusion is related to decreased pain sensitivity. context provokes questions such as: What leads to a perception
Clearly, future research is needed both to replicate the experimen- that a social threat cannot be avoided? How is an “escape route”
tal results with human participants and to help understand the perceived in social space? Is fight also the least preferred option in
mechanisms by which perceptions of social standing can influence social interaction? In general, it is not entirely clear how the
sensitivity to pain. One consideration that may be important for situational cues highlighted in analyses of response to physical
future research is the notion of framing inclusion and exclusion as threat map onto a social context. This question is important, as
separate factors rather than as opposite ends of a continuum. As answering it should help in understanding why some individuals
discussed, the pursuit of social relationships involves both an aggress in response to rejection while others pursue other courses
approach and an avoidance component. Comfort with closeness in of behavior. More generally, research is needed to investigate
relationships is relatively weakly related to the fear of rejection physiological reactions to exclusion, as this would help in under-
(Feeney, Noller, & Hanrahan, 1994), suggesting that motivation standing the relation of the panic response to exclusion experi-
for inclusion may be tied to the behavioral approach system ences. For example, physical pain puts large demands on attention
whereas concerns over exclusion may be tied to the avoidance (Eccleston & Crombez, 1999), suggesting that exclusion may also
(defense) system. Given that analgesia can be achieved through a have a similar effect. In general, we suggest that viewing exclu-
variety of mechanisms (Panksepp, 1998), it is possible that the sion, not just as a generic stressor, but as a major threat to
mechanisms through which inclusion (approach) relates to anal- perceptions of safety will help researchers understand the seem-
gesia may be different than the mechanisms through which exclu- ingly extreme reactions that exclusion can provoke.
sion (avoidance) relates to analgesia. Thus, it seems potentially More generally, although the focus of this article has been to
important for future research to compare the effects of both inclu- make a case for social pain, it is important to reemphasize our
sion and exclusion against a neutral control condition. belief that social pain is only one aspect of a more general
Further, investigating the specific mechanisms by which social emotional pain mechanism. Given that our analysis suggests that
conditions influence pain sensitivity is also of importance. For pain affect is the key mechanism behind social pain, it seems likely
example, the work of Eisenberger et al. (2003) mapping brain that pain affect may be experienced in many situations other than
areas active in response to social exclusion is an extremely im- those connoting physical or social injury. An important question,
portant contribution. Future research should continue to investigate then, is how to determine what other forms of behavior regulation
which pain-related brain areas are active in response to exclusion, may involve pain affect. One approach, suggested by our linguistic
and which are not, in order to help determine what aspects of pain analysis, is to consider the possibility that usage of terms such as
are involved in the experience of rejection. Our review suggests hurt and pain in noninjury contexts may, to some degree, accu-
the PAG as one brain site deserving of attention. It is important rately reflect the activation of pain affect. Thus, an examination of
what situations most consistently evoke the use of pain terms depressed mood: Evolutionary, psychosocial, and neurobiological per-
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Psychological Bulletin Copyright 2005 by the American Psychological Association
2005, Vol. 131, No. 2, 202–223 0033-2909/05/$12.00 DOI: 10.1037/0033-2909.131.2.202
Why Does Social Exclusion Hurt? The Relationship Between Social

and Physical Pain
Geoff MacDonald Mark R. Leary

University of Queensland Wake Forest University
The authors forward the hypothesis that social exclusion is experienced as painful because reactions to
rejection are mediated by aspects of the physical pain system. The authors begin by presenting the theory
that overlap between social and physical pain was an evolutionary development to aid social animals in
responding to threats to inclusion. The authors then review evidence showing that humans demonstrate
convergence between the 2 types of pain in thought, emotion, and behavior, and demonstrate, primarily
through nonhuman animal research, that social and physical pain share common physiological mecha-
nisms. Finally, the authors explore the implications of social pain theory for rejection-elicited aggression
and physical pain disorders.
The physical pain alone was terrible. I always used to think the past, we propose that threats to one’s social connections are
expression “a broken heart” was just a metaphor. But it felt as if I was processed at a basic level as a severe threat to one’s safety. In fact,
having a heart attack. we argue that such threats are partly mediated by the same system
—Bob Geldof, on the end of his 19-year relationship
that processes physical pain because the pain system was already
In a recent documentary about the death penalty, a camera crew in place when social animals evolved adaptations for responding to
was present in the home of a woman whose son was to be executed social exclusion.
that day. Although she was not present at the execution itself, at In this article, we use the term social pain to refer to a specific
the time the penalty was to be exacted she burst out of her front emotional reaction to the perception that one is being excluded
door and fell to the ground screaming and crying. Friends and from desired relationships or being devalued by desired relation-
family followed her outside and tried to help her up, as if her being ship partners or groups. Exclusion may be a result of a number of
on the ground was the problem. However, whenever people would factors, including rejection, death of a loved one, or forced sepa-
try to touch her, she would scream at them with fury to keep away. ration. In everyday life, extreme social pain may be experienced as
Her behavior was akin to that of a wounded animal, scaring others the deep aching of homesickness, grief, abandonment, or longing
away because her pain was so great. for a loved one. Relational devaluation refers to feeling less valued
In reflecting on the most agonizing moments in one’s life, as a relational partner (e.g., friend, romantic partner, group mem-
events involving severe physical pain (e.g., serious injuries, labor ber) than one desires (Leary & Springer, 2001). We argue that such
pain, kidney stones) quickly come to mind. But other events, such devaluation is experienced as aversive because it signals an in-
as the example above, may be as severely distressing, if not creased probability of ultimate exclusion. The acute emotional
painful, despite the lack of any tangible threat to one’s personal distress felt in response to relational devaluation is known as hurt
health or safety. Most people have experiences in which socially feelings (Leary & Springer, 2001). However, other affective states
mediated pain is so great that they are not only in agony but are such as embarrassment, shame, guilt, or jealousy can also serve as
overwhelmed or incapacitated. In this article, we argue that refer- signs that one is not living up to the standards of valued others, and
ring to these responses to social exclusion, rejection, or loss as
thus we consider these emotions to be aspects of social pain as
pain is more than just a metaphor. Because inclusion in social
well.
groups has been a key to survival for social animals deep into the
The concept of social pain was first suggested by Panksepp and
colleagues. They provided evidence that the social attachment
system was built up from more primitive regulation systems such
Geoff MacDonald, School of Psychology, University of Queensland, St.
as those involved in place attachment, thermoregulation, and phys-
Lucia, Queensland, Australia; Mark R. Leary, Department of Psychology,
Wake Forest University.
ical pain (Panksepp, 1998). Herman and Panksepp (1978) sug-
Work on this article was supported in part by a Social Sciences and gested specifically that “it is conceivable that brain circuits for
Humanities Research Council of Canada Post-Doctoral Fellowship separation distress represent an evolutionary elaboration of an
awarded to Geoff MacDonald. I (Geoff MacDonald) am extremely grateful endorphin-based pain network” (p. 219), and Nelson and Panksepp
for the opportunities provided for me by the Canadian government via this (1998) stated, “The pain components made stronger contributions
fellowship. We thank Rod Ashton, Terry Blumenthal, Matthew Hornsey, to the subcomponents which aroused emotional distress during
Winnifred Louis, Mike Smith, and Valerie Stone for their insightful com-
social absence” (p. 438). In this article, we attempt to extend
ments on drafts of this article. Thanks also to our colleagues and friends
who provided translations of “hurt feelings.”
Panksepp’s ideas with the goal of tying social pain more strongly
Correspondence concerning this article should be addressed to Geoff to human reaction to perceived social exclusion and by considering
MacDonald, School of Psychology, University of Queensland, St. Lucia the implications of social pain for the important problems of
QLD 4072, Australia. E-mail: geoff@psy.uq.edu.au relationship aggression and pain disorders.
202
We argue that the aversive emotional state of social pain is the more likely to survive to 1 year of age than infants of less socially
same unpleasantness that is experienced in response to physical integrated mothers, even controlling for the mothers’ dominance
pain. Others before us have proposed the existence of nonphysical rank (Silk, Alberts, & Altmann, 2003). Further, vervets and rhesus
forms of pain such as “emotional pain,” “mental pain,” and “psy- monkeys who showed low interest in social contact (a result of
chological pain.” Thornhill and Thornhill (1989) proposed a theory receiving amygdala and other brain site lesions) after rerelease to
of emotional pain, suggesting that its function is analogous to that the wild were excluded from the social group and died without the
of physical pain. That is, they proposed that such pain focuses protection of their conspecifics (Kling, Lancaster, & Benitone,
attention on significant social events and promotes correction and 1970). Such data support the premise that, over evolutionary time,
avoidance of such events in the future. They further theorized that social animals who formed strong relationships and were inte-
the causes of emotional pain would be circumstances that had grated most strongly into group living were most likely to survive,
influenced inclusive fitness in the environment of evolutionary reproduce, and raise offspring to reproductive age (Baumeister &
adaptiveness such as the death of genetic relatives or close asso- Leary, 1995). Phrased differently, for social animals, being so-
ciates, loss of status, sexual jealousy, childlessness, and rape. In cially excluded was often equivalent to death. As a result, the
the current article, we restrict our analysis to a very specific process of natural selection favored those who were motivated to
evolutionary adaptation—the desire to avoid social exclusion. It is be included, meaning such animals were more likely to leave
important to make clear that we are not suggesting that social pain viable descendants.
is the only viable form of nonphysical pain. It is more accurate to To adapt to changing conditions vis-à-vis social inclusion and
suggest that social pain may be one form of emotional pain. In fact, exclusion, social animals required mechanisms that allowed them
in our analysis, it is most accurate to say that the affective re- to recognize and react to threats of exclusion in an efficient
sponses to physical trauma usually described as physical pain are manner. In particular, cues such as physical distance from impor-
themselves a subcategory of emotional pain, albeit a fundamental tant conspecifics may have been reliably correlated with eventual
one. Given Gray’s (1971) suggestion that the same punishment exclusion. In evolutionary terms, genetic mutations that provided
mechanism underlies both fear and frustration, it seems reasonable learning mechanisms for associating such cues with response
to suggest that feelings of pain may be associated with a wide mechanisms that helped avoid exclusion would have facilitated
variety of stimuli that either lead to harm or block a highly desired survival (cf. Krebs, Stephens, & Sutherland, 1983). As a result,
goal. In this article, we do not claim to provide an exhaustive such mutations would have provided an important evolutionary
analysis of all possible forms of emotional pain but rather of one advantage for social animals and would have been likely to be
specific form—social pain. passed on to future generations. For early social animals, such cues
To begin, we forward our theory of why social and physical pain would have included factors such as physical distance from im-
overlap as they do. We argue that social animals require a system portant conspecifics or absence of those conspecifics. For exam-
that punishes individuals who do not avoid social exclusion and ple, social inclusion took on particular importance for mammals
motivates quick responses to signs of exclusion. In line with the because they nurse their young, meaning parent– offspring inter-
work of Panksepp, we propose that at the point in evolutionary dependence is a critical survival issue. The mammalian infant’s
history when such a system developed, existing physical pain reliance on its mother for nourishment, as well as protection from
mechanisms provided its foundation. To support this hypothesis, predators and other dangers, means that any prolonged separation
we provide evidence that social and physical pain overlap in the of an infant from its mother is potentially disastrous (MacLean,
attitudes, behaviors, and cognitions of humans, reviewing evidence 1993).
that the two types of pain correlate similarly with factors such as This high degree of dependence on the mother set the stage for
extraversion, social support, anxiety, aggression, and depression. adaptations that maintained the infant–mother bond (Bowlby,
From there, we present physiological evidence that social and 1973; Panksepp, 1998). Over time, some social animals gradually
physical pain operate via shared mechanisms. Specifically, both developed more complex cooperative social structures that even-
types of pain have been shown to involve the anterior cingulate tually blossomed into high degrees of interdependence, making
cortex and periaqueductal gray brain structures and the opioid and inclusion crucial for survival across the life span (Gilbert, 1992;
oxytocin neuroendocrine systems. We then move to discuss the MacLean, 1993; Whiten & Byrne, 1989). This increase in social
implications of social pain theory, focusing on its implications for complexity would have been accompanied by new cues of exclu-
understanding rejection-elicited aggression such as violence in close sion threat, such as averted eye gaze. Again, genetic mutations that
relationships and pain disorders such as somatoform pain. We con- tied such cues to appropriate warning and response mechanisms
clude by suggesting future directions for research on social pain. (or that facilitated the learning of such associations) would have
conferred an important advantage, and thus would have been likely
to be retained across generations. Because social exclusion has
Why Is Social Exclusion Painful?
been such an important threat to survival from the earliest days of
The pain of separation slams down, the guillotine. social speciation, it seems reasonable to suggest that exclusion
—Lucy Gwin, “Normal zone: You in or out?”, Adbusters: Journal cues recognized by modern humans have the potential to be
of the Mental Environment, 2002 processed as a basic and severe threat to existence in the same way
as do other primitive threats (e.g., snakes or spiders; Öhman &
Social pain theory is based on the idea that the possibility of Mineka, 2001).
being separated from important social entities posed a critical The question, then, is what kind of warning and response
challenge to the survival of our ancestors, dating back at least to mechanisms these cues of social distance came to trigger. Evolu-
the earliest mammals (and likely beyond). For example, infants of tionary theory suggests that such cues were likely to become
highly socially integrated female baboons have been shown to be associated with the activation of existing threat defense mecha-
nisms. Such “preadaptations” are considered to be a common decreases in painful feelings reward an organism for moving
means of responding quickly to new survival challenges, including toward safety (i.e., away from danger and/or toward safety). Fur-
social ones (D. C. Craig, Gilbert-MacLeod, & Lilley, 2000; ther, the aversiveness of pain can condition an organism to avoid
Keverne, Nevison, & Martel, 1999; Öhman & Mineka, 2001; situations in which a quick response is needed. After organisms
Panksepp, 1998; Rozin, Haidt, & McCauley, 1993). For example, learn to associate certain situational cues with pain, these cues
the negative emotional and physical reaction provoked by moral trigger relevant approach/avoidance tendencies so that pain is
offenses such as incest appears to tap the physical disgust re- avoided or minimized. As a result, the organism learns to fear not
sponse, leading to grimacing, flared nostrils, and nausea (Rozin et just pain itself but also cues that indicate the possibility of pain
al., 1993). We propose that cues of social distance came to activate (Bowlby, 1973). For example, a child who is bitten by a dog may
threat-defense responses that originally functioned to help organ- become fearful in the future upon seeing or hearing the dog,
isms avoid physical danger, as such mechanisms predated the regardless of whether the dog actually bites the child again. This
evolution of social animals. In particular, we propose that social conditioned fear can motivate movement away from the fear-
exclusion cues accessed threat-defense responses by stimulating evoking stimulus and movement toward a helpful attachment
the same painful feelings associated with physical injury. figure, both of which have the potential to reduce the threat of a
In the remainder of this section, we lay out our argument as to harmful stimulus.
why pain would have provided an excellent mechanism for the We propose that painful feelings triggered by social exclusion
regulation of social inclusion (Panksepp, 1998). In order to de- also provide a mechanism useful for learning effective approach/
scribe how social experience may have come to be mediated by avoidance regulation to avoid exclusion. People who reject, ex-
feelings of pain, it is important to note that the experience of pain clude, or ignore an individual are not likely to be safe or stable
consists of two separate components—pain sensation and pain sources of support for that person and, in fact, may be inclined to
affect (Melzack & Wall, 1996; Price, 1999; Rainville, 2002). Pain cause harm to that person. Thus, experiencing painful emotions in
sensation provides information about ongoing tissue damage, in- connection with social exclusion guides an individual away from
formation that is gathered by the body’s specialized pain receptors sources of rejection and toward sources of acceptance. Indeed,
and transmitted to the brain for processing via the dorsal horn of people are highly attuned to social cues indicating that social pain
the spinal cord (K. D. Craig, 1999; Melzack & Wall, 1965). We do is likely and work to avoid social pain when such cues are
not propose that social exclusion directly taps into this circuitry, detected. Just as a person is less likely to approach a dog who bit
although we do discuss the possibility of indirect influence later. him or her, that person is also less likely to seek the company of
Pain affect consists of the feelings of unpleasantness that are an individual who has insulted, ostracized, or otherwise hurt him
associated with pain sensation, as well as emotions associated with or her. Indeed, research has shown that people tend to distance
the future implications of those sensations (Price, 2000). It is this themselves from others if they feel that rejection, and hence hurt
affective experience of pain that signals an aversive state and feelings, is likely (Bourgeois & Leary, 2001; Feeney, Noller, &
motivates behavior to terminate, reduce, or escape exposure to the Roberts, 2001; Murray, Holmes, MacDonald, & Ellsworth, 1998;
source of the noxious stimulation (Melzack & Casey, 1968; Price, Vangelisti, 2001). For example, individuals who feel unsure of
1999). Because this affect component is separate from the sensa- acceptance from a romantic partner often devalue the importance
tion component, it is possible to experience painful feelings in the of the relationship as a means of protecting themselves from the
absence of a signal of tissue damage (Fields, 1999; Rainville, hurt of rejection (Murray et al., 1998). Furthermore, social pain
2002). Thus, our suggestion is that social exclusion triggers these experienced in the context of a specific relationship can motivate
same painful feelings, leading to an emotional experience of pain people to seek support from trusted others or to pursue new
without accompanying physical pain sensation. relationships (Leary & Springer, 2001). Overall, both physical and
We believe that pain affect came to underlie social regulation social pain appear to serve a similar function in promoting adap-
needs because it serves at least two functions crucial for the tive approach and avoidance behavior in response to physical and
avoidance of social exclusion. First, learning that promotes avoid- social threats, respectively.
ance of inclusion-threatening situations is needed to minimize the Another important point of overlap between social exclusion
number of exclusion threats that one faces. Feelings of pain can and physical pain relevant to learning is that certain early-life
provide a strong sense of aversiveness that, when paired with sensory experiences, particularly gentle touch, are involved in the
exclusion-threatening situations, can motivate avoidance of such alleviation of both physical pain and social separation. Physical
situations. Second, quick action in response to exclusion warnings touch provides the basis for attachment (Harlow, 1958). Human
(e.g., ceasing an offending behavior) is needed to help sustain infants appear prepared to learn associations between attachment
inclusionary status. Because of the strong relation between pain behaviors and parental responses as mediated by physical contact
and threat-defense response mechanisms (Gray & McNaughton, (Bowlby, 1973). When babies express physical discomfort (e.g.,
2000; Panksepp, 1998), pain affect should provide a pathway by through crying), their distress can be alleviated through physical
which social exclusion cues could trigger quick, defensive reac- contact by the attachment figure such as holding or patting
tions to regulate inclusionary status. (Bowlby, 1973). In essence, this is the key to attachment theory;
children learn about the reliability of social support from the
Aversiveness of Pain attachment figure on the basis of that attachment figure’s physical
The affective component of physical pain aids organisms in responsiveness to their distress. At a more basic level, however,
avoiding threats to their physical safety by serving as a source of the baby also learns that isolation and physical pain go hand in
punishment, and it functions to guide organisms toward safety by hand. Because the child learns that uncomfortable states such as
serving as a source of negative reinforcement. Increases in painful hunger pangs and gas pain often continue until the attachment
feelings motivate organisms to avoid dangerous stimuli, whereas figure is in physical contact with the child, the association between
physical and social pain occurs at a very early age. (Interestingly, predator), a panic response promotes fight/flight/freezing behavior
when we feel another person has helped us in a special, supportive as a means of providing a quick route to safety. It is important to
way, we may say that we feel “touched.”) All told, then, attach- note that when we refer to panic responses throughout this article,
ment regulation may have intertwined with pain mechanisms be- we specifically mean the motivation for undirected escape from
cause these mechanisms would have already been highly respon- threat, or the fight/flight/freezing response. Such panic behavior
sive to the crucial cues of distress and physical touch. can be highly reactive and relatively undirected as high levels of
Overall, we propose that social pain hurts because social inclu- coordination and planning are sacrificed for a quick response to
sion was and is key for human survival. In accordance with this danger. The panic response is facilitated by a set of physiological
view, people do appear to take social pain very seriously. For changes designed to prepare an organism for urgent action such as
example, Kaplan and Bratman (2000) showed that people judge increased heart rate, increased blood clotting factor, and analgesia
doctor-assisted suicide as more moral and easier to understand (Gray & McNaughton, 2000). Factors that have been shown to
when the patient is in emotional pain than in matched cases trigger the panic response include immediate predators, high levels
without emotional pain. This study also showed that participants of carbon dioxide, and physical pain (Gray & McNaughton, 2000).
viewed doctor-assisted suicide as more justifiable and understand- Physical pain can be an important signal of immediate threat, as it
able when both emotional and physical pain were present than often accompanies tissue damage. In this way, pain serves to
when the patient experienced only physical pain. Thus, people activate and regulate avoidance responses including fight, flight, or
realize that emotional pain can be excruciating. Further, K. D. freezing (Berkowitz, 1993; Berkowitz, Cochran, & Embree, 1981;
Williams (1997) has observed that many people would prefer to be Merskey, 2000).
hit than ostracized, suggesting that the pain of social exclusion Social relationships also require approach/avoidance regulation.
may be more aversive than the pain of physical injury in many Whereas the need to belong (Baumeister & Leary, 1995) and
instances. In fact, simply thinking about separation from close sexual desire provide approach motivation, the dangers of rejection
others has been shown to increase the accessibility of death-related and exclusion provide avoidance motivation. In fact, people often
thoughts (Florian, Mikulincer, & Hirschberger, 2002; Mikulincer react to threats to social inclusion as if they were as important as
& Florian, 2000; Mikulincer, Florian, Birnbaum, & Malishkevich, threats to physical safety, if not more so (K. D. Williams, 1997).
2002), suggesting a strong link between attachment and a sense of Perhaps not surprisingly, then, rejection appears to lead to re-
physical safety. As MacLean (1993) aptly put it, “A sense of sponses consistent with Gray and McNaughton’s (2000) model.
separation is a condition that makes being a mammal so painful” For example, Vangelisti and Crumley (1998) asked participants to
(p. 74). recall instances when their feelings had been hurt and to describe
their responses to the incident. A factor analysis of participants’
Pain and Quick Reaction to Threat responses to feeling hurt classified these responses into three
categories. The first, “acquiescent,” consisted of behaviors such as
Another benefit of tying social exclusion cues to pain is the apologizing that appear to facilitate safety from hurt via cautious
ability to capitalize on the strong relation between pain and the approach. The second, labeled “invulnerable,” consisted of behav-
threat-defense system (Gray & McNaughton, 2000; Panksepp, iors such as ignoring the source of hurt that serve to help one avoid
1998). This way, social pain would not only lead to exclusion cues or withdraw from a hurtful exchange. Finally, the response labeled
being perceived as aversive, but it would also promote timely “active verbal” consisted of behaviors such as verbally attacking
response to such cues. As sketched by Gray and McNaughton the source of hurt that seem to reflect more aggressive responses.
(2000), the physical defense system regulates behavior in response These classes of responses appear to map well onto the anxiety,
to threat on the basis of the state of two key variables. The first fear, and panic components of the physical defense system, re-
variable, defensive distance, refers to the degree of perceived spectively. As with physical pain, we believe that the panic re-
threat in a given situation (Blanchard & Blanchard, 1990). That is, sponse to perceived exclusion should occur only when defensive
the more threatening a stimulus is perceived to be to well-being, distance from exclusion is perceived to be low. That is, reactions
and the more imminent that threat is perceived to be, the more the to social stimuli should most resemble reactions to acute physical
defense system promotes active, self-protective behavior. The sec- pain when strong relational devaluation by another is perceived,
ond variable, defensive direction, refers to whether motivation especially when there is a strong desire to maintain a relationship
exists to approach a potentially dangerous stimulus (Gray & Mc- with the devaluation source (Leary & MacDonald, 2003; Leary,
Naughton, 2000). For example, a mouse may perceive moving Springer, Negel, Ansell, & Evans, 1998).
onto an open field as threatening (as it would be exposed to Thus, like physical pain, social pain leads animals to approach
predation), but it may need to do so to acquire food. friendly conspecifics, avoid threats to separation when possible,
According to Gray and McNaughton’s (2000) model, approach- and attack unavoidable threats to separation (Alexander, 1986;
ing a potentially threatening stimulus results in anxiety, promoting Carter, 1998). Unlike physical pain, however, the emotional dis-
cautious approach behavior such as initially making brief forays tress of social pain serves a protective function in social contexts
onto the open field followed by quickly returning to a safe posi- (Baumeister & Tice, 1990; Leary & Springer, 2001; Miller &
tion. The intensity of anxious emotion and behavior should in- Leary, 1992; Thornhill & Thornhill, 1989; Vangelisti & Crumley,
crease as defensive distance is reduced. When a potentially dan- 1998). Because the need to belong is a fundamental aspect of
gerous stimulus is detected and is not accompanied by a human experience, a system to protect social well-being has great
motivation to approach the stimulus, the resulting response is adaptive value for human beings (Baumeister & Leary, 1995;
fearful avoidance of the stimulus when defensive distance is high Baumeister & Tice, 1990; Leary, Tambor, Terdal, & Downs,
(e.g., the faint odor of a predator is detected). However, when 1995). In support of these ideas, separation from attachment fig-
defensive distance is low (e.g., the presence of an immediate ures in primates activates major behavioral and stress response
systems, according to a review of relevant literature (Mason & The most obvious connection between the two is that similar
Mendoza, 1998). Such separation has been shown to lead to words are used to describe both experiences. The phrase “I am
reactions similar to those seen in human beings, including in- hurt” could just as easily refer to the result of a physical injury as
creased anxiety and depression-like behavior (E. O. Johnson et al., to one’s reaction to a relationship dissolution. In fact, many of the
1996; Levine & Stanton, 1990), increased plasma cortisol (E. O. terms used to describe social pain, if taken literally, would be great
Johnson et al., 1996; Rilling et al., 2001), decreased norepineph- sources of physical pain. For example, people may say that they
rine (Kraemer, Ebert, Schmidt, & McKinney, 1991), and overt were “broken hearted,” “cut to the core,” or “emotionally scarred”
crying (E. O. Johnson et al., 1996; Panksepp, 1998). For example, by a rejection or other loss of social connection. Similarly, a
marmosets placed in isolation for a 2-week period evidenced person may say that being rejected “ripped out my heart” or was
increases in plasma cortisol concentrations (a stress-related hor- like a “slap in the face.” More generally, people report feeling
mone involved in preparation for physical defense) and submissive “crushed,” “deeply hurt,” or “wounded.” It should be noted that
crying, weight loss averaging 10% of body mass, and frequent counterexamples, where pain is used as a metaphor for positive
crouching that the authors likened to freezing behavior in rats social experience, can also be brought to mind, such as “having a
(E. O. Johnson et al., 1996). Social stressors have been shown to crush on someone” or “getting a kick out of someone.” However,
evoke similarly strong physiological responses in humans. A meta- unlike most other emotional states, the English language contains
analysis by Dickerson and Kemeny (2004) showed that the threat no direct synonym for the term hurt feelings, the emotion that
of social evaluation is unique among psychological stressors in accompanies perceived relational devaluation by other people
stimulating the release of high levels of cortisol (this relation was (Leary & Springer, 2001). Thus, English speakers not only de-
particularly strong when stress was uncontrollable). scribe social pain using images connoting physical pain, but at
Further, separation from caregivers and isolation from conspe- least in the case of hurt feelings, they have constructed no other
cifics has been shown to lead to another aspect of the fight/flight/ way to describe that common and important experience except
freezing response in nonhuman mammals—analgesia (or reduced with reference to pain. Further, as can be seen in Table 1, examples
pain sensitivity). Analgesia in response to short-term isolation has of a linguistic link between exclusion and pain can be found across
been demonstrated in rat pups (Kehoe & Blass, 1986a, 1986b; a wide variety of languages and cultures.
Naranjo & Fuentes, 1985; Spear, Enters, Aswad, & Louzan, 1985), Beyond these linguistic associations, if social and physical pain
mice (Konecka & Sroczynska, 1990), cows (Rushen, Boissy, Ter- share a common psychological and/or physiological basis in hu-
louw, & de Passillé, 1999), and chicks (Sufka & Hughes, 1990; mans, then both should be similarly related to a number of com-
Sufka & Weed, 1994). For example, as we discuss in more detail mon factors. Because both types of pain serve to promote avoid-
later, rat pups isolated from their mother (or “dam”) and littermates ance of pain-eliciting stimuli, both types of pain should be
have been shown to have longer response latencies to heat stimuli, associated with higher degrees of caution and defensiveness. Fur-
suggesting a decreased sensitivity to pain (Kehoe & Blass, 1986b). thermore, there should be evidence of crossover between the two
Analogous results have been found with human participants. Mac- types of pain. That is, higher degrees of physical pain should be
Donald, Kingsbury, and Shaw (in press) randomly assigned par- associated with increased social caution or isolation and vice versa.
ticipants to be either included or excluded from an online ball- In this section, we offer evidence supporting these two postulates
tossing game they believed they were playing with other from research on extraversion–introversion, social support, anxiety
participants (who were actually controlled by a computer sched- and fear, defensive aggression, and depression.
ule). Before the game, participants’ proneness to experience hurt
feelings (i.e., the ease with which an individual’s feelings are hurt)
was measured. Following the game, participants’ physical pain Table 1
sensitivity was tested by having them place an arm in cold water International Terms for Hurt Feelings
(the “cold pressor” task) and report how quickly they felt pain (i.e.,
pain threshold). The results showed that individuals whose feelings Language Native term English translation
were more easily hurt and who were excluded from the game
reported higher pain thresholds (i.e., slower onset of pain) than German verletzt sein hurt or wounded
French blessé hurt
hurt-prone individuals included in the game. That is, individuals Dutch gekwetst hurt
who were sensitive to rejection and who experienced social exclu- Spanish sentirse herido feel injured or harmed
sion demonstrated an analgesic response to the cold water. Indi- Italian ferito hurt
viduals less prone to hurt feelings did not differ across conditions. Greek pligomenos hurt
Hebrew he pag’ah baregashot shelo she hit/damaged his feelings
These data provide direct evidence that social exclusion can influ- Hungarian megsertoedni being hurt
ence physical pain detection mechanisms. This supports the notion Armenian zkatsoumnires tsavtsoutsir you hurt my feelings
that reactions to social exclusion are regulated by a general threat- Mandarin shang liao kan ching hurt feelings
defense system that prepares an organism for potentially harmful Cantonese siong sum hurt heart
situations and is responsive to “stimulation that is intense, painful, Tibetan snying la phog hit the heart
Bhutanese sems lu phog hit the mind
[italics added] or unexpected” (Mason & Mendoza, 1998, p. 771). Inuktitut anniqtuq hurt by harsh words
Note. These terms were solicited from colleagues and friends with the
Shared Psychological Correlates of Social and Physical following e-mail: “In English, we refer to a person’s emotional reaction to
Pain being rejected as ‘hurt feelings.’ We are interested in what word or words
native speakers of other languages use. We would appreciate it if you could
We now move to review evidence of the link between social and tell me the expression used to describe the emotional reaction to rejection
physical pain in the thoughts, feelings, and behavior of humans. in any other language.”
Extraversion and Introversion addition, people who are socially alienated are more prone to
physical ailments (Bockian, Meager, & Millon, 2000), and people
Extraversion–introversion appears to have an important relation experiencing marital dissatisfaction and conflict show poor adjust-
to both social and physical pain. Extraverts are more sociable and ment to chronic pain (Robinson & Riley, 1999). The link between
outgoing than introverts (Pervin, 1996), partly because they are physical pain and social support has also been demonstrated ex-
less afraid of being rejected and hurt in social settings. Indeed, perimentally. Brown, Sheffield, Leary, and Robinson (2003) tested
extraversion is negatively related to rejection sensitivity (Downey pain sensitivity with the cold pressor task after randomly assigning
& Feldman, 1996). Further, extraversion is positively related to participants to receive active social support (verbal support), pas-
self-esteem (Halamandaris & Power, 1997; Kwan, Bond, & Sin- sive social support (presence of other with no communication),
gelis, 1997), a variable strongly tied to the belief that one is distracting interaction (verbal interaction without support instruc-
acceptable to other people (Leary & MacDonald, 2003). tions), or no support. Relative to those with no support or distrac-
Extraversion is also related to physical pain. A review of re- tion, those with active or passive social support reported less pain
search by Phillips and Gatchel (2000) showed that extraverts from the task. Social support, then, appears to play a role in
demonstrated both higher pain thresholds (the point at which pain buffering both social and physical pain.
is detected) and higher pain tolerance (the degree of pain that can The data reviewed here support the notion that perceptions of
be withstood). It is interesting to note, however, that extraverts are social support are associated with perceptions of reduced levels of
more likely than introverts to express that they are in physical pain physical pain. However, they also appear to be in conflict with
(Phillips & Gatchel, 2000; Wade & Price, 2000). If one considers research presented earlier suggesting that isolation leads to anal-
that expressing injury could be taken as a sign of weakness, it gesia in nonhuman animals (e.g., Kehoe & Blass, 1986b) and that
appears that introverts, who are more wary of being rejected exclusion can lead to decreased pain sensitivity for hurt-prone
(Downey & Feldman, 1996), may want to hide their hurt. As a humans (MacDonald et al., in press). Why would both inclusion
result, introverts may express less pain than extraverts even while and exclusion lead to decreased pain sensitivity? One answer may
experiencing it more intensely. In fact, as chronic pain continues be that inclusion and exclusion affect different aspects of sensi-
over time, pain sufferers become more introverted (Phillips & tivity to pain that follow different time courses. For example, in the
Gatchel, 2000), demonstrating increased social anxiety and avoid- Brown et al. (2003) study, socially supported participants did not
ance of social situations (Sharp & Harvey, 2001). In general, then, begin reporting lower pain sensitivity until 1 min into the cold
introverts appear to have a higher level of reactivity to both pressor task, an effect that remained significant for the duration of
physical and social pain than extraverts, supporting the notion that the task (the task stopped after 3 min). Correlational studies
the two types of pain operate via common mechanisms. relating social support to lower pain sensitivity in human partici-
pants tend to encompass a relatively long time frame, with mea-
Social Support sures of social support tapping perceptions of long-term support.
On the other hand, excluded hurt-prone individuals in Mac-
The common-sense notion that meaningful support from close Donald et al.’s (in press) study reported initial pain less quickly
others is strongly tied to social pain and hurt feelings is supported than included hurt-prone individuals, but this effect did not last
by the literature (Leary, 1990; Leary, Koch, & Hechenbleikner, beyond the early stages of the cold pressor task. It is important to
2001). Indeed, we have defined social pain in terms of separation note that much of the work associating separation with analgesia in
from valued others, and hurt feelings as a perception of suboptimal nonhuman animals has been conducted following relatively short-
valuation by other people. In essence, then, a perceived lack of term isolation periods (e.g., separating a rat pup from its dam for
adequate social connections is the sine qua non of social pain. In 5 min).1 Thus, measures of pain sensitivity in these studies are
support of this notion, hurt feelings have been shown to arise from taken during a relatively short time span when analgesia is still
the perception that one is less valued by another person or group active. Indeed, this short-term focus is consistent with the role of
than one wishes (Leary et al., 1998, 2001). For example, Leary et analgesia in response to physical injury as a mechanism that blocks
al. (1998) asked participants to recount instances when their feel- attention to injury until safety is achieved. Analgesia that extended
ings had been hurt and found that 99% of these instances involved for long periods of time would be less functional, as injury would
relational devaluation. Further, the feeling of being valued that not be associated with the discomfort of pain and thus not promote
comes from meaningful social support helps to soothe social pain; avoidance learning. Further, it is unclear from these studies
people regularly derive a great deal of solace from other people whether inclusion and exclusion were related to pain intensity,
when they are distressed (Buunk & Verhoeven, 1991; Finch, pain affect, or both, highlighting our limited knowledge about the
Okun, Pool, & Ruehlman, 1999; Haley, 1997; Schachter, 1959). mechanisms by which these studies found their effects. Finally, it
For example, in a meta-analysis of relevant studies, Finch et al. seems potentially useful to test for a moderating role for hurt
(1999) found that both social support and negative social interac-
tion had significant (and oppositely valenced) relations with psy-
1
chological distress. Some researchers have demonstrated reduced pain sensitivity in adult
What is perhaps less intuitive is that social support is also rats that were raised in isolation and thus experienced long-term social
separation (Gentsch, Lichtsteiner, Frischknecht, Feer, & Siegfried, 1988;
related to physical pain. Research has shown that higher levels of
Schwandt, 1993). However, as these rats were raised from an early age
social support are associated with lower levels of chronic pain without the presence of any conspecifics, it seems likely the analgesia
(Phillips & Gatchel, 2000), labor pain (Klaus, Kennel, Robertson, demonstrated in these studies is related to developmental difficulties. Thus,
& Sosa, 1986; Niven, 1985), cardiac pain (Chalmers, Wolman, we do not view these studies as comparable with the long-term social
Nikodem, Gulmezoglu, & Hofmeyer, 1995; Cogan & Spinnato, support data from correlational studies involving nondevelopmentally chal-
1988), and postoperative pain (Lidderdale & Walsh, 1998). In lenged human populations.
proneness in the support–analgesia link found by Brown et al. controlled, hurt feelings proneness was related to evaluating the
(2003), as the exclusion–analgesia effect found by MacDonald et painful clips as more aversive and less humorous, suggesting that
al. (in press) was limited to hurt-prone individuals. Again, these hurt-prone individuals are relatively vigilant for physical threat.
data support a clear link between social exclusion and physical Clearly, both physical pain and social exclusion are important
pain mechanisms, but clearly more research is needed to investi- correlates of anxiety. In fact, Baumeister and Tice (1990) proposed
gate the nature of this relationship and the mechanisms by which that all instances of anxiety arise from either the threat of physical
it operates. pain or the threat of social exclusion. In both cases, anxiety signals
a potentially dangerous stimulus or situation, necessitating cau-
Anxiety and Fear tious approach or avoidance of the stimulus (Gray & McNaughton,
2000; Frijda, 1986). However, there is a problematic aspect to a
Anxiety and fear are strongly tied to physical pain (Robinson & long-term avoidant response common to both social and physical
Riley, 1999; Turk & Flor, 1999; Weisberg & Keefe, 1999). For pain. One common strategy for avoiding social pain in romantic
example, data from a survey of a representative sample of the relationships is described by Murray and Holmes’s dependency
population of the United States indicate that individuals experi- regulation model (Murray, Holmes, & Griffin, 2000; Murray,
encing the chronic pain of arthritis are more likely to experience Holmes, Griffin, Bellavia, & Rose, 2001; Murray et al., 1998).
anxiety and panic disorders, even when a wide range of sociode- According to the model, individuals who fear rejection from inti-
mographic variables and medical conditions were controlled (Mc- mate others tend to avoid creating situations where the expected
Williams, Cox, & Enns, 2003). Arthritis was also associated with rejection might materialize. Thus, such individuals will keep emo-
social phobia in this study controlling for the sociodemographic tionally distant from their partners, limiting the risks they take to
variables, but this relation was nonsignificant when the other increase intimacy such as self-disclosure. As discussed earlier,
medical conditions were controlled. Further, people who score such a self-protective stance can be functional in the short-term by
highly on measures of trait anxiety and neuroticism have lower limiting rejection. The problem with this approach is that by not
thresholds for physical pain than those who are less anxious exposing oneself to the potential for rejection, one’s fears of
(Phillips & Gatchel, 2000; Wade & Price, 2000; D. A. Williams, rejection are never disconfirmed. The emotional distance moti-
1999). Similarly, longitudinal research has shown that neuroticism vated by these rejection fears undermines relationship closeness
predicts the experience of neck pain and migraine headaches 3 (Murray et al., 1998, 2000, 2001), often instigating the feared
years later (Wade & Price, 2000). Chronic pain sufferers who fear hurtful behavior from others (Ayduk, Downey, Testa, Yen, &
abandonment from close others (i.e., have a more anxious attach- Shoda, 1999; Murray, Holmes, & Griffin, 1996) and leading to
ment style) have been shown to experience their physical pain as eventual dissolution of the relationship (Karney & Bradbury, 1995;
more threatening and distressing than those with more secure Kelly & Conley, 1987; Kurdek, 1997).
attachment (Mikulincer & Florian, 1998). Not surprisingly, anx- A similar process appears to occur for those with chronic
ious attachment is related to neuroticism (Shaver & Brennan, physical pain. Chronic pain patients often decrease physical activ-
1992), and people who score high in neuroticism are more prone ity, especially activity that might increase pain in the affected
to death-related thoughts when they are reminded of their corpo- somatic region (Sharp & Harvey, 2001; E. P. Simon & Folen,
real nature (Goldenberg, Pyszczynski, McCoy, Greenberg, & Sol- 2001). However, analogous to the results of dependency regula-
omon, 1999), suggesting that anxiousness is associated with more tion, such inactivity means that individuals’ beliefs about their pain
accessible cognitions related to threats to survival. are never tested, despite the fact that such activity may lead to no
Neuroticism is also related to the propensity to experience hurt increase in pain or increased pain that is easily tolerable (Sharp &
feelings and other negative emotional reactions to social exclusion. Harvey, 2001). Ultimately, decreased physical activity contributes
People who are more neurotic are more prone to feel hurt when to weakened muscle tissue and weight gain, both of which can
they do not feel valued (Leary & Springer, 2001) and are generally exacerbate chronic pain (E. P. Simon & Folen, 2001). In both the
more rejection sensitive (Downey & Feldman, 1996; Downey, cases of social and physical pain, then, an avoidant response may
Feldman, & Ayduk, 2000). Individuals high in anxious attachment be functional for preventing short-term pain but dysfunctional for
evidence higher levels of anxiety and distress in response to meeting long-term interpersonal and health goals. This sacrifice of
separation, conflict, and breakup in close relationships than those long-term goals for short-term relief highlights the extremely
with more secure attachment (Feeney, 1999; Fraley & Shaver, aversive nature of both types of pain. More generally, the literature
1999; Mikulincer & Florian, 1998; Rholes, Simpson, & Stevens, reviewed here suggests that both social exclusion and physical
1998). Those with higher levels of neuroticism are more likely to pain are related to the activation of emotional states related to
feel socially anxious and embarrassed when they become con- cautious approach (anxiety) and avoidance (fear).
cerned about social approval and acceptance (Leary & Kowalski,
1993). In a survey of chronic pain patients and other community Defensive Aggression
members, MacDonald et al. (in press) showed that the tendency to
experience hurt feelings was associated with higher reports of Although fleeing physical harm often provides the best chance
physical pain and that both of these factors were related to higher for an animal’s survival, when escape is difficult or impossible,
levels of anxiety. In addition, physical pain reports partially me- defensive aggression often minimizes the likelihood of injury or
diated the relation between hurt feelings and anxiety, suggesting death. Physical pain is a primary elicitor of aggression (C. A.
that one mechanism by which hurt feelings increase anxiety is Anderson & Bushman, 2002; Berkowitz, 1993; Vernon, 1965)
through feelings of pain. Finally, in another study, MacDonald et because pain frequently indicates a highly proximal threat that
al. (in press) presented individuals with video clips of painful and requires immediate action (Bowlby, 1973). Although it is unclear
nonpainful events. When the ratings of the nonpainful clips were whether pain can directly prime aggressive tendencies or whether
this effect is mediated by cognition, what is clear is that pain initiate the breakup themselves (Ayduk, Downey, & Kim, 2001).
frequently triggers defensive behavior that is quick and highly Thus, hurt feelings and depression appear related only as a result
reactive (Berkowitz, 1993). In this way, aggression in response to of unwanted separation. This point is important, given that emo-
pain is an important aspect of the fight response, aiding in rapid tional reactions to physical pain are different from the sensory
response to threats to safety. However, research with both animals experience of pain and depend heavily on the meaning or impor-
and humans suggests that this highly defensive stance can lead to tance given to the painful stimulus (K. D. Craig, 1999; Engel,
aggression against others who are not related to the cause of the 1959). That is, emotional reactions to physical pain depend heavily
pain (Berkowitz, 1993; Ulrich, Hutchinson, & Azrin, 1965). For on the implications of the injury. Although injury is typically
example, fighting can be induced in rats by delivering electric associated with negative emotion, some injuries (e.g., a battlefield
footshock (e.g., O’Kelly & Steckle, 1939). wound that earns a soldier the right to go home) may result in
Social exclusion has also been shown to cause aggression positive emotion. Analogously, then, social pain may only be
(Buckley, Winkel, & Leary, 2003; Leary & Springer, 2001; emotionally distressing to the extent that the social bond being
Twenge, Baumeister, Tice, & Stucke, 2001; Vangelisti, 2001; threatened is considered valuable. In MacDonald et al.’s (in press)
Vangelisti & Crumley, 1998). Researchers who study aggression research involving chronic pain patients and community members,
have long capitalized on the link between socially aversive, hurtful both hurt feelings proneness and physical pain were significantly
stimuli and aggression by using insults, criticism, slights, and other related to depression. As with anxiety, the relation between hurt
stimuli that connote exclusion to make participants angry feelings and depression was partially mediated by reports of phys-
(Berkowitz, 1993; Bushman, Baumeister, & Phillips, 2001; Don- ical pain. Again, it appears that one mechanism by which hurt
nerstein, Donnerstein, & Evans, 1975; Harmon-Jones & Sigelman, feelings may influence depression is by leading to increased feel-
2001; Scheier, 1976). In a series of studies, participants who were ings of pain.
randomly assigned to receive feedback that they had been ex- This review suggests that both physical and social pain can
cluded by other participants or that they would have a lonely future induce depression, depressed people have a lower threshold for
exhibited higher levels of aggression such as administering un- experiencing both physical and social pain, and physical pain
pleasant noise blasts to others than did those who did not receive partially mediates the link between hurt feelings and depression.
exclusion feedback (Twenge et al., 2001). This was true even when Overall, depression appears to be intimately related to physical and
the victim of aggression was not involved in the rejection episode social pain, and it may be a mechanism to increase cautiousness or
in any way. This research suggests that, like physical pain, hurt to downregulate behavior to reduce the risk of further injury or
feelings sometimes lead to aggression that is not limited to the exclusion (Allen & Badcock, 2003).
source of threat. Although this pattern may seem interpersonally
maladaptive (would not rejected individuals wish to foster rela- Summary
tionships with other people rather than alienate them through
aggression?), it parallels the findings regarding pain-elicited ag- Overall, social and physical pain correlate similarly with a
gression. Overall, both physical and social pain appear to induce a number of important variables. Both types of pain are related to
general defensive stance that can lead to defensive aggression. extraversion and perceptions of social support, providing evidence
that social inclusion has a strong relation to sensitivity to physical
Depression pain. Further, both types of pain are related to emotional reactions
indicative of increased caution and defensiveness such as anxiety
Both physical pain and hurt feelings are related to higher levels and depression, suggesting that both exclusion and injury are
of sadness and depression (Fine & Olson, 1997; Leary et al., related to general threat-defense mechanisms. These emotional
2001). Physical pain and depression overlap significantly. Accord- reactions are consistent with Chapman’s (1991) depiction of the
ing to a literature review of studies on the subject (Fishbain, emotional response of chronic pain patients to their malady, which
Cutler, Rosomoff, & Rosomoff, 1997), higher levels of depression he described as “an adaptation to loss” (p. 411). This character-
are related to a higher likelihood of experiencing pain, higher pain ization suggests that emotional reactions to both physical and
severity, and more frequent pain. In fact, responses to chronic pain social pain represent a kind of reorganization in response to the
and depression appear so similar that they are often confused by loss of vitally important personal assets—physical and social in-
medical professionals (Weisberg, & Keefe, 1999; Seville & Rob- tegrity, respectively.
inson, 2000; Turk & Flor, 1999). Social pain is also often associ- There also appears to be ample evidence of overlap between the
ated with sadness and depression (Allen & Badcock, 2003; Leary two types of pain: Extraverts are less sensitive to physical pain,
et al., 2001). For example, widows and widowers evidence rates of and physical pain increases introversion, social anxiety, and avoid-
clinical depression twice that of base rates even 24 to 30 months ance of social situations (Phillips & Gatchel, 2000; Sharp &
after their partner has passed (Fraley & Shaver, 1999). Allen and Harvey, 2001); people with low social support are more prone to
Badcock (2003) argued that depression is a mechanism that de- physical pain (Phillips & Gatchel, 2000); research participants
creases social risk when the likelihood of exclusion is perceived as randomly assigned to receive social support experienced less ex-
high. These authors presented evidence from a review of the perimental pain (Brown et al., 2003); fears of abandonment in-
literature indicating that depression is associated with increased crease the distress of physical pain (Mikulincer & Florian, 1998);
sensitivity to social threat, the instigation of support-eliciting be- those more prone to hurt feelings find presentations of physically
haviors, and a decrease in potentially risky social behaviors. Re- painful situations more aversive (MacDonald et al., in press); and
search on romantic relationship dissolution suggests that romantic physical harm is used in retaliation for rejection (Buckley et al.,
rejection-related depression occurs mainly when individuals have 2003; Twenge et al., 2001). Furthermore, the relation between hurt
been rejected by their partners but not when these individuals feelings and both anxiety and depression has been shown to be
partially mediated by reports of physical pain (MacDonald et al., in erized schedule. Participants were scanned first while watching
press). These findings support the notion that both social and others play the game (implicit exclusion), again while being in-
physical pain are managed by similar psychological and physio- cluded in a game (inclusion), and finally while being excluded by
logical systems in humans. 2 other players who did not throw the ball to the participant
(explicit exclusion). Heightened activity in the dorsal ACC was
Shared Physiological Mechanisms of Social and Physical found when participants were either implicitly or explicitly ex-
cluded, relative to the inclusion condition. These researchers also
Pain
found increased activation in the right ventral prefrontal cortex, a
On the face of it, the notion that reactions to physical harm and site associated with negative affect regulation, during explicit
social rejection are mediated by a similar physiological system (though not implicit) exclusion. Eisenberger et al. (2003) described
may seem odd because these two types of threats are typically these reactions to social exclusion as “a pattern of activations very
encountered through different sensory modalities. That is, whereas similar to those found in studies of physical pain” (p. 291). Like
physical pain is most frequently registered via direct touch, stimuli the PET data, the functional magnetic resonance imaging data are
that create social pain typically come in the form of sights or unable to shed light on whether ACC activation causes the expe-
sounds, often through stimuli with purely symbolic meaning (i.e., rience of social pain. However, given the activation of the ACC in
words, gestures, facial expressions). In this way, the two types of relation to the affective component of physical pain, this does
pain can seem quite different. At what point, then, do these provide evidence that reactions to both social and physical pain are
different types of signals come to be processed and experienced in related to similar neurologic components. Moreover, this evidence
similar ways? suggests that the ACC is a viable candidate to be involved partic-
Panksepp (1998) has suggested that the physiology of the at- ularly in processing the aversiveness of social pain.
tachment system may be composed of two separate components:
one component devoted to regulating reactions to social absence The Periaqueductal Gray (PAG)
(what we call social pain), the other to regulating the pursuit of
social engagement. As discussed, Panksepp proposed that this The PAG receives input from the body’s injury detection mech-
attachment system has been built up from older physiological anism, the nociceptive system (A. D. Craig & Dostrovsky, 1999),
systems including those that function to regulate basic needs such as well as from the ACC (An, Bandler, Öngür, & Price, 1998;
as energy balance, thermoregulation, place attachments, and pain Floyd, Price, Ferry, Keay, & Bandler, 2000), and has been shown
perception. Indeed, there is reasonable evidence to suggest that an to be active in connection with physical pain. For example, it has
absence-regulation system is a part of mammalian physiology been linked to analgesia, as it is part of a circuit that controls
(Mason & Mendoza, 1998; Panksepp, 1998). In this section, we nociceptive neurons in the dorsal horn of the spinal cord, with
present evidence for physiological mechanisms that underlie the stimulation of the PAG inhibiting pain transmission by the dorsal
aversiveness and threat response aspects of both social and phys- horn via the release of endogenous opioids (Fields, 2000). Re-
ical pain. search with rat pups has shown that isolation from a dam and
littermates can trigger such analgesia (Kehoe & Blass, 1986b;
Anterior Cingulate Cortex (ACC) Spear et al., 1985). This effect appears to be mediated by the PAG.
In one study, lesions to the lateral or ventrolateral PAG in rat pups
The most direct evidence for the social pain hypothesis comes were shown to disrupt the decreased pain sensitivity following
from work involving the ACC. The ACC has been well established social isolation that was demonstrated by pups assigned to a sham
as an important site for processing physical pain signals (Rainville, (or “placebo”) lesion condition (Wiedenmayer, Goodwin, & Barr,
2002). Specifically, pain affect, but not pain intensity (i.e., sensa- 2000).
tion), appears to be associated with activation in the ACC (Rain- The PAG has also been shown to be related to bonding behavior.
ville, Carrier, Hofbauer, Bushnell, & Duncan, 1999; Rainville, First, evidence suggests that the PAG is involved in regulating
Duncan, Price, Carrier, & Bushnell, 1997; Singer et al., 2004; maternal behavior in rats such as kyphosis (optimal nursing pos-
Tölle et al., 1999). For example, Rainville et al. (1997) manipu- ture), retrieval and transport of pups to the nest, and defense of the
lated the unpleasantness of a painful sensation through hypnotic pups against outsiders (Lonstein, Simmons, & Stern, 1998;
suggestion. Positron emission tomography (PET) revealed that Miranda-Paiva, Ribeiro-Barbosa, Canteras, & Felicio, 2003; Stack,
activation in the ACC was associated with changes in perceived Balakrishnan, Numan, & Numan, 2002). For example, lesions to
unpleasantness, but activity in other brain areas related to pain the caudal intercollicular PAG have been shown to disrupt the
perception (i.e., primary and secondary somatosensory cortices nursing posture of rat pup mothers, resulting in 10% less weight
and rostral insula) did not covary with unpleasantness ratings. gain for the pups of lesioned as opposed to unlesioned dams
Such data suggest that the ACC is involved in the processing of (Lonstein et al., 1998). Second, the PAG has been shown to be
pain affect, although the correlational nature of PET data leaves involved in infant proximity-seeking behavior. During the first 2.5
open the question of whether pain affect is caused by ACC weeks of life, rats have been shown to emit ultrasonic vocaliza-
activation. tions when separated from their dam and littermates that can be
A functional magnetic resonance imaging study of participants reduced by reintroducing an anesthetized dam or littermate (Car-
experiencing social exclusion has shown the ACC to be active in den & Hofer, 1990a; Hofer & Shair, 1978; Kehoe & Blass, 1986b).
response to social exclusion (Eisenberger, Lieberman, & Williams, These “separation distress” cries appear to serve the function of
2003). In this study, participants were told they would be partic- assisting in the reunification of a separated infant with its mother.
ipating in an online ball-tossing game with 2 other participants. Direct stimulation of the PAG can elicit these separation distress
However, the other players were actually controlled by a comput- cries (Panksepp, 1998), and lesions to the PAG appear to decrease
such cries (Wiedenmayer et al., 2000). In fact, Panksepp (1998), after removal from the nest, those removed for 5 min evidenced
on the basis of the physical proximity of PAG areas that can be longer response latencies, indicating that separation prompted an
stimulated to produce distress vocalizations and physical pain analgesic response. Further, response latencies were reduced to
responses, suggested that separation distress emerged anatomically baseline for pups treated with naltrexone (an opioid blocker) and
from more basic pain systems. He concluded that “this affirms that exaggerated for pups treated with morphine, supporting the notion
separation distress is related to perceptions of [physical] pain” that opioids mediate the isolation–analgesia relation.
(Panksepp, 1998, p. 267). There is also strong evidence from animal research that opioids
In general, the PAG is considered an important site for the are involved in signaling the adequacy of social conditions. Low
integration of homeostatic control and limbic motor output in doses of morphine have been shown to reduce the separation
response to threats (Fanselow, 1991; Gray & McNaughton, 2000; distress cries of isolated rat pups (Carden, Hernandez, & Hofer,
Lonstein & Stern, 1998). Gray and McNaughton (2000) argued 1996; Carden & Hofer, 1990b; Kehoe & Blass, 1986b; Kehoe &
specifically that the PAG serves as the coordinator of the panic Boylan, 1994; but see Winslow & Insel, 1991a). Similar results
response and is thus at the base of the hierarchically organized have been reported with other vertebrates including primates (Ka-
neuroanatomical threat-defense system. That is, it is the structure lin, Shelton, & Barksdale, 1988), dogs (Panksepp, Herman, Con-
at the lowest level of the defense system capable of coordinating a ner, Bishop, & Scott, 1978), guinea pigs (Herman & Panksepp,
variety of physiological changes and behaviors to produce a rela- 1978), and birds (Panksepp, Vilberg, Bean, Coy, & Kastin, 1978).
tively organized reaction to potential harm. Indeed, activation of Further, reductions in crying of isolated rat pups as a result of the
the lateral PAG in nonhuman mammals (e.g., through injection of introduction of a dam or littermate can be reversed by administra-
excitatory amino acids) leads to key aspects of the panic response, tion of opioid antagonists such as naltrexone (Carden et al., 1996;
including defensive behavior, hypertension, tachycardia, and non- Carden & Hofer 1990a, 1990b), suggesting that drops in opioid
opioid analgesia (Bandler & Shipley, 1994). In particular, stimu- levels may signal an unsatisfactory social environment. In partic-
lation of the caudal lateral PAG appears to lead to preparation for ular, ␮-opioid receptors (responsive to endorphins) and, to a lesser
flight through physiological mechanisms such as increased blood extent, ␦-opioid receptors (responsive to enkephalins), both of
flow to the limbs and decreased blood flow to the face. Stimulation which are powerfully related to reductions in pain, also appear to
of the intermediate lateral PAG appears to lead to preparation for be generally effective in reducing separation distress vocalizations
confrontational defense, through mechanisms such as decreased (Carden, Barr, & Hofer, 1991; Carden et al., 1996; Kehoe &
blood flow to the limbs and increased blood flow to the face Boylan, 1994; Panksepp, 1998). Administration of morphine has
(suggesting a possible link with blushing). Further, stimulation of also been shown to reduce the pursuit of social interaction in
the ventrolateral PAG appears to lead to reactions with similarities primates (Keverne, Martensz, & Tuite, 1989; Martel, Nevison,
to responses to social defeat and depression, including quiescence Simpson, & Keverne, 1995), guinea pigs (Herman & Panksepp,
and hyporeactivity (Bandler & Shipley, 1994). Overall, the PAG 1978), and rats (Panksepp, Najam, & Soares, 1980). For example,
appears responsive to both separation cues and physical pain in young rhesus monkeys treated with naloxone (another opioid
nonhuman animals and appears to contribute to coordinated re- blocker) pursued more contact with their mothers, and mature
sponses to both. In particular, these responses seem to be quick, naloxone-treated monkeys solicited and received more grooming
reactive impulses such as defense, escape, and downregulation from conspecifics (Martel et al., 1995). This again suggests that
regardless of whether the initial input was social or physical in opioids may comprise one way the body regulates response to
nature. Thus, at least for nonhuman animals, both social exclusion social distress, with low levels of opioids signaling an unsatisfac-
and physical pain appear intimately related to the most base level tory social environment and motivating the pursuit of social inter-
of the threat-defense system. This supports the notion that social action. It is important to note that such a withdrawal of opioid
exclusion is processed as a primal threat for animals who rely on activity from ␮- and ␦-opioid receptors can create an aversive,
interdependent relationships with conspecifics. Functionally, the painful state as in the case of withdrawal from opiate addiction
PAG appears to provide at least one mechanism by which signals (e.g., heroin addiction; Panksepp, 1998). Thus, another potential
of social exclusion may facilitate quick action in response to mechanism for the aversiveness of social pain is the reduction of
inclusion threats. opioid activity experienced during rejection, separation, or loss.
Opioids Oxytocin
Another point of overlap between social and physical pain is the The neuropeptide oxytocin provides a further link between
opioid neuroendocrine system (Panksepp, 1998; Taylor, Dicker- social and physical pain. Oxytocin is perhaps most widely known
son, & Klein, 2002). Endogenous opioids have long been recog- for its roles in lactation and parent– child bonding. For example,
nized as an important regulator of physical pain, with exogenous administration of oxytocin has been shown to induce maternal
forms such as morphine used to treat pain complaints (Panksepp, behavior (e.g., following and cleaning young) in virgin rats (Ped-
1998; Smith, Stevens, & Caldwell, 1999). Research with both rats ersen, Ascher, Monroe, & Prange, 1982) and sheep (Kendrick,
and mice suggests that opioids play an important role in isolation- Keverne, & Baldwin, 1987). This peptide has also been shown to
induced analgesia (Kehoe & Blass, 1986a, 1986b; Konecka & reduce distress vocalizations in rat pups isolated from their dam
Sroczynska, 1990; Naranjo & Fuentes, 1985; Spear et al., 1985). and littermates (Insel & Winslow, 1991). Oxytocin has also been
For example, Kehoe and Blass (1986b) measured the response tied to a wider range of social behaviors. One avenue of investi-
latencies of 10-day-old rat pups when a paw was placed on a gation has involved comparing two closely related species, the
heated metal surface either immediately after isolation from the prairie vole and the montane vole. These two species are highly
nest or 5 min after removal. Relative to pups tested immediately similar, except for their social behavior. The prairie vole tends to
be monogamous and affiliative and cares for its young, whereas Social pain theory argues that physiological social pain mecha-
the montane vole tends to be highly solitary (Carter, DeVries, & nisms should be shared across a wide variety of social animals, as
Getz, 1995). This difference has been partially attributed to the such mechanisms should date back to the early days of social
different distribution of oxytocin receptor sites in the brains of the speciation. However, there are also crucial differences between
two types of voles (Insel & Shapiro, 1992). In addition, adminis- humans and other mammals. For example, more sophisticated
tration of oxytocin has been shown to facilitate social contact and processing ability in humans relative to other mammals allows for
selective preference of mates in prairie voles, with oxytocin an- self-awareness and projection of the self into the future (Leary &
tagonists blocking such partner preferences (Cho, DeVries, Wil- Buttermore, 2003; Suddendorf, 1999). Thus, social exclusion can
liams, & Carter, 1999; Insel & Hulihan, 1995; Witt, Carter, & have implications for humans, not just in terms of their current
Walton, 1990). For example, Cho et al. (1999) treated prairie voles social status, but also for beliefs about acceptability to others in the
with either oxytocin (1, 10, or 100 ng) or a placebo, then placed future. This may well influence how social pain is processed by
them with an opposite sex conspecific for 1 hr. Following this, the humans. Further, although the nonhuman animal data are useful
experimental voles were placed in a cage permitting access to for investigating behavioral reactions to separation and injury, they
either the familiar or an unfamiliar conspecific. Those treated with are incapable of speaking to the phenomenological experience of
higher levels of oxytocin (100 ng) were significantly more likely to pain in response to exclusion. Finally, much of the nonhuman
place themselves in contact with the familiar vole, suggesting that animal research focuses on infants (e.g., rat pups), while social
oxytocin positively reinforced time spent with a partner, thus pain theory is concerned with regulation of social behavior across
promoting a partner preference. Oxytocin administration has also the life span. Nevertheless, the scant research on physiological
been shown to facilitate social behavior in rats (Witt, Winslow, & reactions to rejection in humans conducted thus far appears con-
Insel, 1992) and squirrel monkeys (Winslow & Insel, 1991b), and sistent with the nonhuman animal data. Specifically, rejection has
gentle stroking has been shown to lead to the release of oxytocin been shown to lead to ACC activation (Eisenberger et al., 2003),
in rats (Stock & Uvnäs-Moberg, 1988). increased blood pressure and cortisol (Stroud, Tanofsky-Kraff,
Uvnäs-Moberg and colleagues have argued that oxytocin also Wilfley, & Salovey, 2000), and analgesia for individuals prone to
functions to regulate physical pain. In a series of studies conducted hurt feelings (MacDonald et al., in press). Clearly, significant work
with rats, administration of oxytocin was shown to reduce sensi- remains to be done to clarify physiological responses to social
tivity to pain (Ågren, Lundeberg, Uvnäs-Moberg, & Sato, 1995; exclusion in humans. Nevertheless, future research examining
Lundeberg, Meister, Björkstrand, & Uvnäs-Moberg, 1993; Lunde- whether such responses are mediated by the mechanisms listed in
berg, Uvnäs-Moberg, Ågren, & Bruzelius, 1994; Uvnäs-Moberg, this review, and/or other mechanisms, seems warranted.
Bruzelius, Alster, Bileviciute, & Lundeberg, 1992), whereas oxy-
tocin antagonists, but not opioid antagonists, were shown to block
Implications of Social Pain Theory for Aggressive
this analgesic effect (Ågren et al., 1995; Lundeberg et al., 1994;
Uvnäs-Moberg, Bruzelius, Alster, & Lundeberg, 1993; Uvnäs-
Behavior
Moberg et al., 1992). For example, Ågren et al. (1995) demon- It is the denial of our intrinsic biological and psychological need for
strated that rats treated with oxytocin showed slower response the “other” that may partly explain the length of time that it has taken
latencies to thermal stimuli up to 90 min posttreatment, whereas to begin to understand the origins of human violence. (de Zulueta,
rats treated with saline returned to baseline responses at 15 min. 1996, p. 176)
Rats treated with oxytocin antagonists demonstrated faster than
baseline response latencies 45 and 75 min posttreatment. Thus, In this section, we strive to demonstrate one important implica-
oxytocin appeared to promote analgesia, while hyperalgesia was tion of conceptualizing social exclusion in terms of pain. Specif-
demonstrated in rats whose oxytocin receptors had been blocked. ically, we propose that if exclusion is perceived as a serious,
Furthermore, oxitonergic neurons project from the paraventricular primal threat, then it should motivate an individual to adopt a
nucleus of the hypothalamus to a number of pain-related brain sites highly defensive stance. Further, because the role of the panic
including the PAG and the dorsal horn of the spinal cord response is to provide quick action in the face of any imminent
(Sawchenko & Swanson, 1982), suggesting a possible role for threat, we propose that social pain should lead to a preparedness to
oxytocin in the regulation of pain. However, there is controversy defend against not just social, but physical threats as well. In line
over this conclusion, as not all researchers agree that oxytocin has with the notion of fight/flight/freezing, such a stance should in-
true analgesic properties (e.g., Xu & Wiesenfeld-Hallin, 1994). clude a preparedness to escape and a preparedness to aggress. In
fact, research has shown that excluded individuals exhibit many
Summary features of the panic response, including aggressiveness (Buckley
et al., 2003; Twenge et al., 2001), analgesia (MacDonald et al., in
Overall, strong evidence for a physiological connection between press), high blood pressure and plasma cortisol concentrations
responses to physical pain and social exclusion has been found (Stroud et al., 2000), and disruptions in higher order cognitive
across a variety of physiological markers. Our analysis suggests processing without accompanying disruptions in more automatic
that the ACC, the PAG, opioids, and oxytocin may all underlie mental tasks (Baumeister, Twenge, & Nuss, 2002). These findings
both physical pain and social behavior regulation. However, some suggest that exclusion taps into relatively basic systems that are
caution must be taken in evaluating the applicability of the phys- oriented toward response to generalized threat, rather than social
iological evidence to human social behavior. Specifically, only the threat in particular. The review that follows aims to demonstrate
evidence relating to the ACC is derived from studies with human that both social and physical pain can prime aggressive action
participants. Thus, the extent to which the PAG, opioids, and tendencies and that both types of pain-elicited aggression are
oxytocin are involved in human social pain is an open question. moderated by perceptions of defensive distance. Overall, the goal
of this section is to provide a more comprehensive understanding who hurt his or her feelings proximal (as in the case of romantic
of exclusion-elicited aggression by noting its similarities with couples or good friends), aggressive responses to social pain are
defensive aggression provoked by physical pain. likely to be interpersonally dysfunctional in the long run.3
Few people would be surprised to learn that those whose feel- Although it may be difficult to conceive of social exclusion
ings are hurt often desire to inflict hurt in return (Leary & Springer, eliciting automatic reactions such as aggression, there is one com-
2001). Research has confirmed this intuition; as discussed earlier, mon automatic response to both exclusion and physical pain al-
rejection often elicits aggressive behavior (Buckley et al., 2003; ready known— crying (D. C. Craig et al., 2000; Vingerhoets,
Twenge et al., 2001). In some instances, more forceful forms of Cornelius, Van Heck, & Becht, 2000; Vangelisti & Crumley,
behavior (i.e., assertiveness) in response to hurt feelings can be 1998). Although crying can be controlled, or at least delayed
useful because actively confronting sources of hurt can help to (Leary et al., 1998), the urge to cry is often involuntary. As a result
resolve troubling relational issues (Fine & Olson, 1997; Vangelisti of its involuntary nature, crying is an expression that may elicit
& Crumley, 1998). However, aggression, especially physical ag- support from empathic onlookers without the individual needing to
gression, in response to hurt may be destructive and counterpro- approach anyone or explicitly seek support (Gross, Fredrickson, &
ductive by ultimately adding to the person’s interpersonal prob- Levenson, 1994). This elicitation of support is obviously beneficial
lems and creating more, rather than less, social pain (Buckley et
in the case of both physical injury and social loss.4 Furthermore, its
al., 2003; Twenge et al., 2001). Specifically, given that hurt
automatic nature is important given that people who are hurt may
feelings arise when people feel less relationally valued than they
not approach others readily because their pain encourages them to
desire (Leary & Springer, 2001; Leary et al., 1998), aggression
take a defensive stance. We suggest, then, that aggression may be
seems like an odd response. A person who feels devalued is
a relatively automatic response to both social exclusion and phys-
unlikely to increase others’ acceptance by insulting, abusing, or
ical pain in much the same way as is crying.
attacking them.2 From an early age, children who aggress are
rejected (Cantrell & Prinz, 1985; Dodge & Coie, 1987; McDou- Of course, social exclusion does not always result in aggressive
gall, Hymel, Vaillancourt, & Mercer, 2001), suggesting that it is behavior. An important question, then, is when social pain is most
unlikely that rejected aggressors expect to win interpersonal ac- likely to trigger an aggressive response. In general, we expect that
ceptance from their victims. Furthermore, even if the experience of aggression would be especially likely when defensive distance
hurt feelings reduces individuals’ desires to be accepted by the from exclusion is low. Thus, aggression in response to social pain
source of the hurt (Bourgeois & Leary, 2001; Leary et al., 1995), should be more likely when higher degrees of rejection or rela-
aggression still does not seem like an appropriate response. If you tional devaluation are perceived. However, as with physical pain,
have given up on being accepted by someone, why not just walk there are a number of cognitive moderators that can impact on
away rather than risk the social, physical, and legal consequences
of a verbal or physical attack? 2
Perhaps the saddest and clearest example of this paradox comes from
We suggest that hurt feelings may contribute to aggressive
the recent trend of school and work shootings that appear to be largely
behavior on the basis of our proposals that social pain can activate motivated by perceived rejection (Leary, Kowalski, Smith, & Phillips,
the generalized threat-defense system and that when ongoing re- 2003).
jection or high degrees of relational devaluation are perceived (i.e., 3
This speculation on relatively automatic aggressive reactions to social
when defensive distance is low), reactions are motivated by panic pain is consistent with Twenge and her colleagues’ studies that showed that
response mechanisms. Specifically, aggressive responses to rejec- self-reports of negative emotion did not mediate the rejection–aggression
tion mimic behavior under conditions of physical pain. A great link (Twenge et al., 2001). If it is true that conscious negative feelings are
deal of research has demonstrated that physical pain reliably elicits not the proximal cause of rejection-elicited aggression, then automatically
aggression in both human beings and other animals (Berkowitz, primed aggressive tendencies in response to social pain may be key.
1989; Scott, 1966; Ulrich et al., 1965). When pain results from However, the Twenge et al. (2001) work is peculiar in that excluded
physical attack, a quick counterattack is often very effective in individuals did not report more negative emotion than individuals who
were not excluded (see also Twenge, Catanese, & Baumeister, 2003),
stopping the threat (Öhman & Mineka, 2001). In fact, some re-
despite the fact that exclusion has often been shown to evoke negative
searchers have suggested that painful stimuli may automatically
affective reactions (Baumeister & Tice, 1990; Leary et al., 2001). Thus,
prime action tendencies associated with pain such as aggressive Twenge et al.’s (2001, 2003) failure to find mediation may reflect a more
responding (C. A. Anderson & Bushman, 2002; Berkowitz, 1989; general difficulty with capturing self-reported negative affective reactions
da Gloria, Pahlavan, Duda, & Bonnet, 1994). For example, Izard to exclusion in their studies. This issue is complicated by the finding that
(1991) showed that 90% of infants aged 2 to 7 months displayed individuals high in proneness to hurt feelings may experience analgesia, or
an angry facial expression after a painful inoculation. Indeed, pain numbing, in response to rejection (MacDonald et al., in press; Twenge et
signals have been shown to reach reflexive motor circuits before al., 2003), suggesting negative affect may be blunted for some individuals.
the pain is realized consciously (Panksepp, 1998), suggesting that Overall, we consider there to be too little data currently to resolve the
pain-induced aggression may be difficult to control. Thus, to the question of whether negative affect mediates the rejection–aggression link,
but pursuit of this question should help shed light on the possible existence
extent that social exclusion taps into panic mechanisms, such
of automatic, aggressive reactions to social pain.
exclusion may also prime a preparedness to aggress. The diffi- 4
The fact that crying also occurs in response to joyful feelings may be
culty, however, is that such quick, aggressive reactions are likely
considered to weaken this argument. However, we suggest that crying may
to be much less functional in warding off social as opposed to be a signal for the elicitation of closeness from others associated with both
physical threats. For example, hurt feelings from a lover’s insult negative and positive affective states. Indeed, happy individuals desire
may lead to a quick counterattack, such as a shout, shove, or closeness from others as a means of maintaining or enhancing positive
punch, triggered by a sense of threat. However, because the at- affect, and closeness associated with joyful times may be a mechanism for
tacker in this instance likely has an interest in keeping the person cementing interpersonal bonds.
perceptions of defensive distance from exclusion. In this section, we suggest that factors that decrease perceptions of defensive
we explore these factors by reviewing commonalities between distance (from injury or exclusion) increase the risk of aggression.
conditions that lead to physical pain-elicited aggression and social
pain-elicited aggression. That is, we searched the literature to Situation Appraisal and Reappraisal
determine factors that make aggression especially likely as a result
of physical pain and then looked for analogues in the realm of According to C. A. Anderson and Bushman’s (2002) general
social pain. In particular, we draw heavily on the domestic vio- aggression model, aggressive behavior can be based either on a
lence literature to help elucidate our ideas regarding social pain relatively automatic appraisal of whether the situation calls for
and aggression. aggression or on a more deliberate reappraisal of one’s initial
Partner abuse is an important topic in the consideration of a action tendency. Appraisal of a stressful situation, such as the
hurt–aggression link for two reasons. First, it is well known that experience of pain, changes according to an individual’s percep-
hurt feelings can lead to violence (Leary & Springer, 2001), and tions of the consequences of the experience, the extent to which
the highly interdependent nature of close relationships provides a one’s well-being is threatened, and the resources available for
fertile breeding ground for such hurt (Levitt, Silver, & Franco, coping with the threat (Weisenberg, 1999). As discussed earlier,
1996; Vangelisti & Maguire, 2002). Indeed, there is a wide range we propose that social exclusion can lead to aggression because
of evidence suggesting that partner abuse may largely stem from such exclusion is processed at a basic level as a primal threat to
abusers’ perceptions or fears of rejection. Literature reviews indi- one’s well-being. For example, K. D. Williams, Case, and Govan
cate that abusers report higher fears of rejection and abandonment (in press) demonstrated that participants who were ostracized
(Dutton, 2002; Holtzworth-Munroe, Bates, Smutzler, & Sandin, during an online ball-toss game evidenced higher levels of implicit
1997), attribute more negative intentions to their partners (Eck- racial prejudice, as measured by a reaction time task, than those
hardt & Dye, 2000; Holtzworth-Munroe, Bates, et al., 1997), who were not ostracized (self-reports of prejudice were not af-
experience more jealousy and less secure attachment (Dutton, fected). This suggests that exclusion may prime an automatic
2002; Holtzworth-Munroe, Smutzler, & Bates, 1997; Schumacher, defensive stance against perceived threat, even if such defensive-
Smith Slep, & Heyman, 2001), have their demandingness met with ness is not recognized consciously. Thus, social exclusion may
lead to an initial situation appraisal that indicates a high degree of
withdrawal or ostracism (Holtzworth-Munroe, Smutzler, & Bates,
threat, possibly priming an aggressive response.
1997; Schumacher et al., 2001), report higher levels of depression
C. A. Anderson and Bushman (2002) argued that a reappraisal
and anxiety (Gleason, 1997; Holtzworth-Munroe, Smutzler, &
of such a situation will occur only if two conditions are met: (a)
Bates, 1997; Schumacher et al., 2001), and are more likely to have
there are sufficient cognitive resources to permit reappraisal and
been rejected by their parents (Schumacher et al., 2001). Further,
(b) the outcome of the initial appraisal is both important and
those individuals who kill their spouses usually do so during
unsatisfying. Both social and physical pain may place pressure on
periods of perceived or actual abandonment (Dutton, 2002).
reappraisal efforts because a strong perception of threat can shift
Second, aggression in relationships is depressingly common,
an individual into a more defensive, reactive mode. Such a reactive
with estimates of the incidence of violence in relationships ranging
stance is likely to augment the importance of automatic as opposed
from 12% to 57% (Arriaga & Oskamp, 1998). Figures this high
to controlled processing, limiting the cognitive resources available
suggest that such violence is too common to attribute its cause for reappraisal. Literature reviews suggest that physical pain dis-
simply to some “abnormality” in abusers. Thus, understanding rupts cognitive functioning, particularly decreasing attentional ca-
family violence is an important research goal in its own right. In pacity and processing speed (Eccleston & Crombez, 1999; Hart,
fact, one would hope that if people could restrain aggressive Martelli, & Zasler, 2000). Response to social exclusion appears to
impulses against any target, it would be their relationship partners, have a similar effect. Baumeister et al. (2002) randomly assigned
but too often this is not the case. Instead, family violence appears participants to receive feedback that they would have a lonely
to be a poignant example of an area in which aggressive responses future or that they would have an accident-prone future. Partici-
to rejection not only create tremendous trauma for the victim of pants told they would be lonely scored lower on IQ and Graduate
aggression but also make further rejection of the aggressor more Record Examination tests, but did not differ from control partici-
likely. pants on more automatic tasks such as memory recall. These
Because of the extensive literature on family violence, we were results suggest that threats to social inclusion interfere with higher
able to examine parallels between such violence and aggression order cognitive functioning, possibly indicating a bias toward more
resulting from physical pain in some detail. This review highlights automatic processing under these circumstances. Thus, one re-
the fact that both social pain-elicited aggression and physical sponse to social or physical pain appears to be a heavier reliance
pain-elicited aggression appear to stem from difficult-to-control on more automatic cognitive processing, suggesting that both types
impulses that arise quickly, possibly automatically, from painful of pain are treated as highly threatening, requiring a quick
feelings. We suggest that the relatively rapid activation of social response.
pain-elicited aggression is a result of the strong sense of threat that Given the possibility that defensive aggression may be an au-
social pain evokes. That is, evolution has equated exclusion with tomatic response to social exclusion, such a decrease in higher
extinction, meaning rejection may be treated as a mortal danger at cognitive functioning means that exclusion should be especially
the motivational level. This high level of perceived threat should likely to lead to aggression when further burdens are placed on
lead individuals to adopt a protective posture, bypassing complex processing capacity. Research on relationship aggression backs up
cognitive processing in favor of efficient, defensive behavior. We this notion. Higher risk of abuse has been connected with a number
note further that both types of aggression are moderated by a of variables related to deficits in cognitive capacity and impulse
strikingly similar constellation of cognitive moderators. In general, control such as alcohol consumption (Gleason, 1997; Leonard &
Senchak, 1996; MacDonald, Zanna, & Holmes, 2000) and head power and control (Dutton, 2002; Jackson, 1999; Schumacher et
injury (Holtzworth-Munroe, Bates, et al., 1997). Further, male al., 2001). This point is especially important in light of the afore-
abusers asked to imagine themselves in scenarios in which they are mentioned meta-analysis showing that the combination of social
rejected by their wives react with more irrational ideas than non- evaluative stressors and lack of control is strongly predictive of
abusers (Eckhardt & Dye, 2000; Schumacher et al., 2001). That is, cortisol release (Dickerson & Kemeny, 2004). As those who more
abusers in this research exhibited a less sophisticated cognitive strongly desire control are more likely to perceive unsatisfactory
response to rejection than nonabusers, suggesting they may be less levels of control in a given situation, such individuals may be
able to control aggressive action tendencies spurred by exclusion. especially reactive when perceiving rejection.
The notion that relationship aggression involves attempts at
Attributions control is not new. K. D. Williams et al. (in press) suggested that
social exclusion can induce a need to regain control of the situation
If sufficient cognitive resources are available for more complex that motivates behavior to gain the attention of the excluder,
processing, attributing causality for physical pain to a specific whether that behavior (including aggression) is prosocial or not.
source can either reduce or exacerbate aggressiveness (Berkowitz, Further, some arguments from the feminist tradition suggest that
1993). Specifically, attributions that decrease defensive distance relationship aggression may represent men’s efforts to exert the
(i.e., increase perception of threat) should more strongly prime power granted them by a patriarchal society (Renfrew, 1997),
aggressive impulses. For example, if an individual determines that although feminist scholars also point to other control motives
a coworker spilled hot coffee on him or her intentionally instead of (M. P. Johnson & Ferraro, 2000). The latter point is important,
accidentally, an aggressive response is more likely. In this case, the given a recent review suggesting that women are aggressive in
incident signals a greater threat of future harm than if the act was relationships as often (though not as severely) as men (Archer,
accidental and thus warrants a more hostile response. Reviews of 2000). Our theory of social pain suggests that control-oriented
the partner abuse literature suggest that attributions of causality aggression may reflect a basic motive to increase defensive dis-
also play an important role in partner abuse, as abusers tend to tance and reduce the sense of threat signaled by social pain. In this
attribute more hostile intentions and traits to their partners (Eck- way, our theory of social pain suggests that control-oriented ag-
hardt & Dye, 2000; Holtzworth-Munroe, Bates, et al., 1997). Thus, gression may not be limited to those whom society grants power.
abusers may be aggressive, in part, because their negative attribu- However, this is not to say that issues such as patriarchy are
tions indicate to them a higher degree of rejection and an increased irrelevant to aggression. As a loss of control may be related to
likelihood of future pain. increased pain (Staub et al., 1971), men who believe in patriarchal
structure may experience the threat of social pain especially
Control acutely in relationships if they feel their “rightful” control is
slipping.
Another important factor in both physical and social pain is
control. Literature reviews suggest that perceiving that one has Feeling Trapped
control over physical pain, particularly that one can terminate the
painful stimulus, is related to a lower subjective experience of pain Aggression is more likely if individuals feel trapped in a painful
and increased pain tolerance (Arntz & Schmidt, 1989; Seville & situation. For example, animals generally prefer to avoid physical
Robinson, 2000). Thus, perceptions of control appear to decrease conflict when possible but will fight in response to physical pain
aggressiveness by increasing defensive distance from threat. Feel- when no escape is available and a threat is perceived as imminent
ings of lost control over painful stimuli may be especially painful. (Blanchard & Blanchard, 1990; Gray & McNaughton, 2000; Ul-
In one study (Staub, Tursky, & Schwartz, 1971), some participants rich et al., 1965). Thus, we would expect hurt feelings to be more
were given control over the administration of an initial regimen of likely to result in aggression when the hurt individual believes that
electric shocks they were to receive, then lost that control during significant barriers to exiting a relationship exist. Phrased differ-
a second regimen. The other participants in the study were not ently, when hurt feelings signal a threat and the individual feels
given control during either the first or second set of shocks but trapped in that situation, the likelihood of aggression may be
were yoked to the “in-control” participants such that they received higher than when the individual feels free to walk away. Like the
shocks of the same intensity and temporal sequence. Relative to different sources of physical and social pain, being physically
those who never had control, those who lost control were quicker trapped appears on the surface to be much different from being
to report discomfort as shock intensity increased and ended the emotionally trapped. However, one commonality between the two
shock regimen after a smaller number of shocks. is a lack of control in that escape from pain seems impossible.
Given the relationship between control and perceived pain, a When physically trapped, inaction in response to threat inevitably
potentially useful way of explaining pain-induced aggression is as leads to physical harm. When emotionally dependent on another,
a behavioral tendency that can be helpful in increasing control over withdrawal of the other inevitably leads to social pain (Panksepp,
a potentially threatening stimulus. In the case of physical pain, 1998). Indeed, a review of the spousal violence literature suggests
achieving such control may be relatively straightforward. How- that relationship aggression is more likely when the aggressor feels
ever, by definition, a source of social pain is some social entity. dependent on the relationship (Holtzworth-Munroe, Bates, et al.,
Thus, exerting control over the source of hurt feelings or other 1997). Further, violence often follows increases in commitment to
social pain may mean attempting to control another person. Ag- the relationship such as marriage (Henton, Cate, Koval, Lloyd, &
gression is certainly one way to exert such power and control Christopher, 1983; Rounsaville, 1978), times when increased de-
(Tedeschi & Felson, 1994), and reviews of the literature suggest pendence is highly salient. Finally, in a review of spousal homicide
that relationship aggressors tend to demonstrate higher needs for literature, Dutton (2002) suggested that a large percentage of
perpetrators of spousal homicide experience a catathymic crisis, or surprising, then, that reviews of the literature indicate that insecure
aversive emotional tension that is perceived as inescapable and attachment is a common aspect of those who abuse their romantic
caused by the spouse. Thus, aggression against romantic partners partners (Holtzworth-Munroe, Bates, et al., 1997; Roberts & Nol-
by individuals who are emotionally dependent on the relationship ler, 1998; Schumacher et al., 2001). That is, those who closely
may be motivated by a desire for control similar to that demon- attend to cues connoting the threat of social pain are likely to
strated with physical pain-elicited aggression. experience that pain more intensely, decreasing defensive distance
and increasing the possibility of rejection-elicited aggression.
Hostility
Summary
Hostility, or trait anger, is an important personality variable
influencing the likelihood of aggression in response to pain. Angry The literature reviewed in this section supports the usefulness of
and aggressive thoughts and feelings can be primed by exposure to conceptualizing social pain-elicited aggression as a reaction to low
physical pain (C. A. Anderson & Bushman, 2002; Berkowitz et al., perceived defensive distance from rejection. Such a relation ap-
1981), especially for those with generally hostile dispositions pears analogous to physical pain-elicited aggression that stems
(K. B. Anderson, Anderson, Dill, & Deuser, 1998). Participants in from low perceived defensive distance from injury. Further, this
a study by K. B. Anderson et al. (1998) were randomly assigned to review suggests that cognitive factors that influence perceptions of
hold their arm in either a painful or comfortable position while defensive distance including appraisals, attributions, perceived
evaluating the similarity between pairs of words. Some of these control, hostility, and pain expectancies moderate the likelihood of
words were explicitly related to aggression (e.g., choke), while aggressive responses to both social and physical threats. This
others were more ambiguously related to aggression (e.g., stick). analysis helps to resolve the question of why aggression is a
Participants high in hostility who were experiencing pain evalu- frequent response to rejection, despite the fact that such aggression
ated the ambiguous-aggressive and aggressive-aggressive word appears to be nonfunctional in winning acceptance. Particularly, if
pairs as highly similar, suggesting that aggressive thoughts were rejection is indeed a primal threat, then the panic motivation
highly accessible for these individuals. Reviews of the domestic spurred by low defensive distance may lead to defensive aggres-
violence literature suggest that trait hostility is also a strong sion that is functional in physical threat contexts but less functional
correlate of domestic violence (Gleason, 1997; Holtzworth- in social threat contexts. Of course, some caution is warranted in
Munroe, Bates, et al., 1997; Schumacher et al., 2001). Thus, accepting this conclusion. Whereas much of the physical pain-
people with hostile personalities appear to be under a relatively related data comes from experimental research, much of the do-
chronic state of threat, perceiving defensive distance from both mestic violence literature reviewed is based on correlational work.
injury and rejection as low. As a result, hostile individuals appear Thus, although we have drawn parallels between the two it is
to react to both physical and social pain cues with more aggressive difficult to be certain that the results are directly comparable.
thoughts and behavior. Nevertheless, it is striking that results from these two very differ-
ent approaches share these parallels. It is our belief that social pain
theory provides a parsimonious framework through which to view
Anticipation of Pain
the commonalities between aggression provoked by social and
Expecting a stimulus to be physically painful has been associ- physical threat.
ated with an increase in felt pain and more aggression in response
to that pain (Berkowitz & Thome, 1987; Leventhal, Brown, Implications of Social Pain for Chronic Pain and Pain
Shacham, & Engquist, 1979). For example, Berkowitz and Thome Disorders
(1987) instructed participants to place an arm in water, telling
some to expect the water to be painful while others did not receive Our contention that social pain stems from the same emotional
this information. Half of those told to expect pain placed their arm unpleasantness associated with physical pain implies that feelings
in uncomfortably cold water, whereas the other half placed their of exclusion and relational devaluation may contribute to pain-
arm in more comfortable room temperature water. All participants related disorders. Indeed, clinical views of somatoform pain dis-
not told to expect pain placed their arm in cold water. Participants order (pain complaints that cannot be adequately explained by a
were then asked to deliver rewards (nickels) and punishments known medical disorder) suggest that drawing a distinct line
(noise blasts) based on the performance of a “worker” (a confed- between physical and social pain is inaccurate and potentially
erate). Relative to those not expecting the cold water to be painful, harmful (Roth, 2000; G. E. Simon, 1998; Sullivan, 2000). Like
those in the cold water condition who did expect pain reported the other forms of physical pain, somatoform pain is strongly related
water to be more painful, unpleasant, and annoying. In addition, to both anxiety and depression, and treatments for anxiety and
those expecting and experiencing pain delivered fewer rewards depression, including cognitive– behavioral therapy and tricyclic
and more noise blasts relative to the other two groups. These data antidepressants, help to alleviate somatoform pain (Fishbain, Cut-
suggest that those expecting to experience pain may exhibit the ler, Rosomoff, & Rosomoff, 1998; G. E. Simon, 1998; Sullivan,
confirmation bias, selectively searching for and attending to threat- 2000).
ening pain cues (Sharp & Harvey, 2001), thus increasing their Social pain theory may be useful in understanding aspects of
experience of pain, decreasing defensive distance, and enhancing somatoform pain and other pain disorders. For example, individ-
aggressive action tendencies. Analogously, people with insecure uals who experience any type of chronic pain are prone to feelings
attachment styles are highly wary of rejection from close others of embarrassment resulting from conflict with others who do not
and thus tend to be highly sensitive to cues that may signal the understand their pain, including medical professionals (Chapman,
potential for hurt feelings (Downey & Feldman, 1996). It is not 1991; E. P. Simon & Folen, 2001). Given that such social pain may
lead to activation of pain circuits in the ACC and right ventral that future research in this vein also feature control groups in
prefrontal cortex (Eisenberger et al., 2003), perceived relational which participants experience noxious but nonexclusion-related
devaluation resulting from a pain disorder might add directly to an stimuli. For example, participants could be exposed to socially
individual’s suffering. Further, individuals who perceive rejection disgusting stimuli to test whether brain areas involved in pain
readily may find their body’s pain management systems taxed. For processing are independent of this type of aversive social stimulus.
example, chronic activation of analgesia in response to social This analysis also suggests that research investigating the neuroen-
threat (MacDonald et al., in press) may lead to the depletion of docrine basis of reactions to exclusion in humans would be
resources over time, leaving rejection-sensitive individuals vulner- valuable.
able to increased pain. Indeed, low levels of opioids create a higher Despite the fact that much of this article focuses on pain in
level of vulnerability to physical pain because of opioids’ impor- response to social exclusion, its focus on the role of the general
tance in regulating physical discomfort (Panksepp, 1998). This threat-defense system in responding to social conditions is equally
notion may help account for the fact that individuals with low important. We believe that future research examining what condi-
social support are more pain sensitive (Phillips & Gatchel, 2000). tions lead to particular physiological reactions to social exclusion
Thus, encouraging feelings of social acceptance may help to is needed to determine with increased clarity the role of the
alleviate pain complaints (Brown et al., 2003). Along these lines, defense system. Given that fight/flight/freezing appear to underlie
an important aspect of current treatment for somatoform pain is response to exclusion, it becomes increasingly important to under-
support and validation that the pain is real (G. E. Simon, 1998), stand what the human analogue of these three reactions are in
and treatment for chronic pain often involves family members in social situations. At least on the surface, fight and flight responses
the therapeutic process (Chapman, 1991). Such affirmation may seem relatively easy to understand. Social fight motivation appears
help to break a cycle in which feelings of exclusion contribute to to manifest in assertiveness and aggression, and social flight mo-
inexplicable pain, the expression of which alienates the pain suf- tivation in withdrawal from interactions. Although the freezing
ferer from others and leads to increased exclusion that then exac- response in humans appears less intuitive, it may well be repre-
erbates the pain further (Sullivan, 2000). sented by depressive affect and behavior. An important follow-up
question is under what conditions social threat will lead to each of
Research Directions the three responses. In physical contexts, flight is the preferred
option in response to threat and is instigated if an escape route
Although the evidence reviewed in this article has clearly drawn from the threatening stimulus is available (Gray & McNaughton,
a link between social and physical pain, it has also highlighted 2000). Freezing occurs when escape is impossible but the threat
some apparent discrepancies that require further research attention. can be avoided by becoming motionless. Fight is the least pre-
As discussed earlier, some work suggests that social exclusion is ferred option but is selected when escape is not possible and a
related to decreased pain sensitivity, whereas other work suggests threat is actively manifested. Considering these factors in a social
that social inclusion is related to decreased pain sensitivity. context provokes questions such as: What leads to a perception
Clearly, future research is needed both to replicate the experimen- that a social threat cannot be avoided? How is an “escape route”
tal results with human participants and to help understand the perceived in social space? Is fight also the least preferred option in
mechanisms by which perceptions of social standing can influence social interaction? In general, it is not entirely clear how the
sensitivity to pain. One consideration that may be important for situational cues highlighted in analyses of response to physical
future research is the notion of framing inclusion and exclusion as threat map onto a social context. This question is important, as
separate factors rather than as opposite ends of a continuum. As answering it should help in understanding why some individuals
discussed, the pursuit of social relationships involves both an aggress in response to rejection while others pursue other courses
approach and an avoidance component. Comfort with closeness in of behavior. More generally, research is needed to investigate
relationships is relatively weakly related to the fear of rejection physiological reactions to exclusion, as this would help in under-
(Feeney, Noller, & Hanrahan, 1994), suggesting that motivation standing the relation of the panic response to exclusion experi-
for inclusion may be tied to the behavioral approach system ences. For example, physical pain puts large demands on attention
whereas concerns over exclusion may be tied to the avoidance (Eccleston & Crombez, 1999), suggesting that exclusion may also
(defense) system. Given that analgesia can be achieved through a have a similar effect. In general, we suggest that viewing exclu-
variety of mechanisms (Panksepp, 1998), it is possible that the sion, not just as a generic stressor, but as a major threat to
mechanisms through which inclusion (approach) relates to anal- perceptions of safety will help researchers understand the seem-
gesia may be different than the mechanisms through which exclu- ingly extreme reactions that exclusion can provoke.
sion (avoidance) relates to analgesia. Thus, it seems potentially More generally, although the focus of this article has been to
important for future research to compare the effects of both inclu- make a case for social pain, it is important to reemphasize our
sion and exclusion against a neutral control condition. belief that social pain is only one aspect of a more general
Further, investigating the specific mechanisms by which social emotional pain mechanism. Given that our analysis suggests that
conditions influence pain sensitivity is also of importance. For pain affect is the key mechanism behind social pain, it seems likely
example, the work of Eisenberger et al. (2003) mapping brain that pain affect may be experienced in many situations other than
areas active in response to social exclusion is an extremely im- those connoting physical or social injury. An important question,
portant contribution. Future research should continue to investigate then, is how to determine what other forms of behavior regulation
which pain-related brain areas are active in response to exclusion, may involve pain affect. One approach, suggested by our linguistic
and which are not, in order to help determine what aspects of pain analysis, is to consider the possibility that usage of terms such as
are involved in the experience of rejection. Our review suggests hurt and pain in noninjury contexts may, to some degree, accu-
the PAG as one brain site deserving of attention. It is important rately reflect the activation of pain affect. Thus, an examination of
what situations most consistently evoke the use of pain terms depressed mood: Evolutionary, psychosocial, and neurobiological per-
across cultures may provide important clues as to where else pain spectives. Psychological Bulletin, 129, 887–913.
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479.
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a preadaptive mechanism and could involve emotional pain as an interactive relations between trait hostility, pain, and aggressive
important part of behavior regulation. Such work would help thoughts. Aggressive Behavior, 24, 161–171.
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Summary and Conclusion interpersonal attachments as a fundamental human motivation. Psycho-
Evidence from a wide range of psychological disciplines con- logical Bulletin, 117, 497–529.
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Baumeister, R. F., Twenge, J. M., & Nuss, C. K. (2002). Effects of social
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and are extremely emotionally aversive. Both types of pain share 827.
common psychological correlates and physiological pathways. Fi- Berkowitz, L. (1989). Situational influences on aggression. In J. Groebel &
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mechanisms. (pp. 91–100). New York: Cambridge University Press.
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gled. We have focused specifically on how individuals’ feelings Berkowitz, L., Cochran, S. T., & Embree, M. C. (1981). Physical pain and
for other people may stem in part from the same pain that keeps the goal of aversively stimulated aggression. Journal of Personality and
Social Psychology, 40, 687–700.
them physically safe. We also believe that social pain theory helps
Berkowitz, L., & Thome, P. R. (1987). Pain expectation, negative affect,
emphasize the vital role of connection with others in human
and angry aggression. Motivation and Emotion, 11, 183–193.
behavior. Those of us living in individualistic societies are inun- Blanchard, R. J., & Blanchard, D. C. (1990). An ethoexperimental analysis
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social pain to be one such example of our deep, physical need for the Millon Behavioral Health Inventory, the Millon Behavioral Medicine
each other. Diagnostic, and the Millon Clinical Multiaxial Inventory: Locus of
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[Frontiers in Bioscience 14, 5291-5338, June 1, 2009]
Vladimir Maletic1 Charles L. Raison2

1
University of South Carolina, School of Medicine, Department of Neuropsychiatry and Behavioral Sciences, Columbia, SC,
2
Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Atlanta, GA
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Epidemiology and Comorbidity of Major Depression, Fibromyalgia and Neuropathic Pain
4. Clinical Relevance of Neurobiological Understanding of MDD, FM and NeP
5. Genetics of MDD, FM and NeP
5.1. Genetics of MDD
5.1.1. Genes Involved in Regulation of Serotoninergic Function
5.1.2. The Role of Genes Regulating Neurotrophic Factors
5.1.3. The Role of Genetic Epistasis in MDD
5.1.4. Polymorphism of Genes Regulating MAO
5.1.5. COMT Gene Polymorphism
5.1.6 Polymorphism of Genes Involved in Regulating Inflammatory Response
5.2 Genetics of Nociception and FM
6.Functional and Structural Alterations in Central Nervous System Circuitry Involved in Regulation of Emotion and Pain
6.1. Structural and Functional Brain Changes in Depression
6.1.1. Abnormalities in Prefrontal Cortex (PFC)
6.1.2. Abnormalities in Anterior Cingulate Cortex (ACC)
6.1.3. Hippocampal Changes
6.1.4. Amygdala Alterations
6.1.5. Insular Abnormalities
6.1.6. Abnormalities in Subcortical Areas
6.1.7. Abnormalities in Connections Between Brain Regions in MDD
6.2. The Role of Peripheral and Central Nervous System in FM and Chronic Pain
6.2.1. Abnormalities in Peripheral Nerves and Spinal Cord Pathways
6.2.2. The Role of Supraspinal Structures in Pain
6.2.3. Descending Pain Modulation in Chronic Pain Disorders
6.2.4. Structural CNS Changes in FM and NeP
6.3. Implications of CNS Overlap of Depression and Pain
7. Neuroendocrine, Autonomic and Immune Dysregulation in MDD, FM and NeP
7.1. Abnormalities of HPA axis in Depression and Pain
7.2. Autonomic Nervous System Abnormalities in Depression and Pain
7.3. Inflammatory Signaling in Mood and Pain Disorders
8. Neurotransmitters Implicated in MDD, FM and NeP
8.1.1. Glutamate and GABA in MDD
8.1.2. Monoamine Neurotransmitters in MDD
8.1.3. Endogenous Opioids and Other Peptide Neurotransmitters in MDD
8.2.1. Glutamate and GABA in Pain Disorders
8.2.2. Monoaminergic Neurotransmitters in Pain Disorders
8.2.4. Endogenous Opioids in Chronic Pain
8.2.5. Contribution of SP to Conditions of Chronic Pain
9. Cellular, Subcellular and Neurotrophic Changes in MDD, FM and NeP
9.1. An Overview of Glial Architecture and Function
9.2. Contributions of Glial Pathology to MDD
9.3. Neuron-Glia Interactions in Chronic Pain
10.Conclusion
11. Acknowledgements
12. References
1. ABSTRACT from the fact that these disorders share multiple biological
and environmental underpinnings. This perspective
This article synthesizes recent data suggesting suggests that these biologically complex conditions result
that the high rates of comorbidity observed between major from similar genetic vulnerabilities interacting with
depression, fibromyalgia and neuropathic pain likely result environmental adversity. Shared genetic determinants
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include poorly functional alleles regulating monoaminergic, Given these findings, it should perhaps not be
glutamatergic, neurotrophic, opioid and inflammatory surprising that recent developments in fields as diverse as
cytokine signaling. Chief among environmental risk factors social neuroscience and psychoimmunology point to the
are psychosocial stress and illness, both of which promote, fact that pain and depression co-exist symptomatically
in vulnerable individuals, relative resistance to because they are driven by largely overlapping
glucocorticoids, increased sympathetic/decreased pathophysiological processes in the brain and body. This
parasympathetic activity and increased production and review will attempt to review current scientific
release of proinflamnmatory mediators. Dysregulation of understandings of these shared processes and to
stress/inflammatory pathways promotes alterations in brain demonstrate how recent advances in our knowledge
circuitry that modulates mood, pain and the stress response. regarding the epidemiology, phenomenology and etiology
Over time, these functional changes likely promote of FM, chronic pain and major depression (MDD) provide
disruptions in neurotrophic support and disturbances of important clues for how the clinician may best approach
glia-neuronal communication. These changes, in turn, have these challenging clinical issues. Indeed, we believe that
been associated with the related processes of central gaining a better understanding of the shared
sensitization in pain disorders and “kindling” in depression, neurobiological bases of MDD, FM and neuropathic pain
both of which may account for the progressive and self- (NeP) will provide the clinician with important tools for
perpetuating nature of these disorders, especially when improving clinical decision making, and—by extension—
inadequately treated. patient outcomes.
2. INTRODUCTION 3. EPIDEMIOLOGY AND COMORBIDITY OF

MAJOR DEPRESSION, FIBROMYALGIA AND
Nowhere are the limitations of current psychiatric NEUROPATHIC PAIN
diagnostic schemas more apparent than at the interface of
Major Depressive Disorder (MDD) and chronic pain. Although 10-12% of the population worldwide
Indeed, one might emerge from a search through the DSM- and in the US endorse chronic widespread pain, only about
IV-TR with the impression that depression and pain had 2% (3.4% of women and 0.5% of men) of individuals meet
little in common. Pain is not listed as a symptom of any the American College of Rheumatology (ACR) criteria for
mood disorder, and depressive and anxiety complaints are Fibromyalgia (FM). FM is characterized by chronic
strikingly marginalized in the list of symptoms required to widespread pain (tenderness in at least 11 of 18 pre-defined
meet criteria for a chronic pain disorder. Unfortunately, points), lasting at least three months, typically accompanied
several decades of research demonstrate that this officially by fatigue and sleep disturbance (9, 10). While no single
sanctioned segregation of mood and pain maps poorly onto etiology has been identified for the condition, a unifying
both clinical and neurobiological reality. hypothesis that continues to receive increasing scientific
support suggests that FM is a consequence of sensitization
In reality, comorbidity between depression and of the central nervous system (9).
pain appears to be more the rule than the exception, with a
30-60% co-occurrence rate reported in a recent review (1). Neuropathic pain (NeP) is typically a
Similarly high rates of comorbidity have been repeatedly consequence of a direct nerve injury or damage to neural
observed between fibromyalgia (FM) and major tissue, resulting in abnormal sensory processing (11).
depression, leading to many years of debate between Common features of NeP include hyperalgesia (augmented
researchers as to whether the conditions are most sensitivity to painful stimuli), allodynia (abnormal pain
parsimoniously considered as separate illnesses with high response to non-noxious stimuli) and paresthesias (11, 12).
comorbidity or as differential symptom presentations of a Shared clinical features, consistent with a role for central
single underlying condition (2). Moreover, overwhelming sensitization, have prompted authors such as Martinez-
evidence suggests that chronic pain and depression do more Lavin and others to suggest that FM and NeP share an
than co-occur—they also promote the development of each overlapping pathophysiology (12, 13). Estimates suggest
other, such that chronic pain is strong predictor of that various forms of NeP (diabetic neuropathy,
subsequently developing major depression, and vice versa. postherpetic neuralgia, trigeminal neuralgia, spinal cord
When comorbid, pain and depression also significantly injury, radiculopathy, etc.) afflict up to 26 million people
complicate the treatment of each condition. For example, worldwide, including approximately 1.5% of the US
once treatment of depression is initiated, pain is a major population (11, 12). Consistent with these estimates, a large
obstacle to achieving remission (3, 4) and a significant risk survey of 6000 adults in the United Kingdom reported an
factor for relapse (5). In a three-year longitudinal study by 8% prevalence of chronic pain, most of which was of
Geerlings et al. the presence of painful symptoms neuropathic origin. As with FM, women were significantly
substantially reduced the chance of recovery in a group of more likely than men to be affected (14).
elderly depressed patients (n=327). Significantly more
patients with MDD alone attained recovery (47%), Many patients suffering with either FM or NeP
compared with only 9% of patients with MDD combined (or both) will also meet diagnostic criteria for major
with painful symptoms (6). Finally, a higher level of pain depressive disorder (MDD). Moreover, patients with FM
may delay the onset of remission in the treatment of MDD are at significantly increased risk of subsequently
(7), thereby decreasing the chances for an optimal outcome developing MDD and related psychiatric conditions. For
(8). example, Arnold et al reported that patients with FM were
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4.3 times more likely than healthy control subjects to precipitated or aggravated by stress (19, 28, 30, 33-36). In
develop MDD at some point in their lives and 4.7 times addition to peripheral and central sensitization, FM and
more likely to develop an anxiety disorder (15). NeP are also characterized by altered limbic and cortical
Surprisingly, Weir et al. found that although women were function and structure (28, 37, 38). The circuitry involved
much more likely to develop fibromyalgia in the first place, in modulating pain (typically altered in FM and NeP) has
once FM had developed the risk of males and females common elements with the circuits regulating stress
subsequently developing MDD was comparable: with a risk response and mood (36, 39-43). Remarkably, fMRI studies
ratio of 2.91 (95% CI 2.15-3.94) vs. 2.85 (95% CI 2.38-3.42) have demonstrated that brain areas (i.e. dorsal anterior
(16). Overall depression and anxiety are among the most cingulate) central to experience of negative affect in response
common comorbidities of FM, with prevalence rates ranging to physical pain also mediate distress in response to the “pain”
in studies from 20-80% and 13-63.8%, respectively (17). of social exclusion (44). These findings strongly suggest that
emotional and physical pain co-occur so often because they
The high comorbidity between depression and share the same central nervous system pathways (45).
pain is not restricted to subjects with FM, but is also Consistent with this, similar functional and structural changes
relevant for patients suffering with NeP. For example, in an in amygdala and hippocampus have been described in MDD,
Austrian study that surveyed 7707 individuals (18), the FM and NeP (46-51). Dysfunction of these limbic formations
prevalence of NeP was 3.3%, with two-thirds of the sample is believed to contribute to perturbations in neuroendocrine,
suffering from pain for longer than a year. Depression was autonomic and immune functioning that may further contribute
reported in 34% of the sample, anxiety in 25% (18). The to the generation and/or worsening of mood and pain
severity of pain tended to be enduring and associated with symptoms (28, 37, 42, 52). In this regard, increasing data
significant impairment of functioning. demonstrate that excessive sympathetic activation,
combined with elevated proinflammatory cytokine
4. CLINICAL RELEVANCE OF A production and release, likely plays a role in the etiology of
NEUROBIOLOGICAL UNDERSTANDING OF MDD, MDD, FM and NeP (19, 42, 53). Finally, at the cellular level
FM and NeP all three conditions are associated with disturbed neuron-glia
relationship, glutamatergic dysregulation and alterations in
Relationships between MDD, FM and NeP are intracellular signaling cascades and neurotrophic trafficking
complex and intricate. Based on clinical and (Figure 1) (54-61).
neurobiological similarities, some researches have even
made a claim that FM and NeP may be variations of the One clear clinical implication of the
same condition (13, 19). However, other researchers have neurobiological links between depression and pain is that
pointed out that, unlike FM which is characterized each set of symptoms worsens the other and/or makes the
exclusively by altered neural function, NeP has a other more likely to develop. A second implication arises
discernable neuro-pathologic substrate, such as a lesion of from the many empirical studies demonstrating that the
the peripheral nerve or CNS (e.g. central pain due to brain core symptoms of pain disorders (i.e. pain, chronic fatigue,
tumor) (20). Diagnostic quandaries aside, it is increasingly sleep disturbance and cognitive complaints) significantly
apparent that, like MDD, conditions characterized by complicate the treatment of MDD because these symptoms
chronic pain share a common a progressive course that is tend to be especially non-responsive to conventional
reflected in cognitive alterations, as well as structural treatments (4, 62-64). Conversely, both depression and
changes within the brain itself (21-27). anxiety are extremely common in FM and NeP (17, 18) and
may intensify the experience of pain (43, 65). In summary,
As noted above, a preponderance of current available evidence suggests that MDD and FM/NeP
scientific evidence suggests that NeP and FM are mutually amplify each other, thus contributing significantly
characterized by central sensitization (28, 29). Relevant to to treatment resistance in both depressive and pain
its relationship with pain, MDD is widely considered to disorders. Consistent with this, timely treatment of MDD
represent a “kindling” phenomenon. In the context of may optimize the chance of remission and decrease the
depression, kindling implies that each episode of chances of enduring structural changes (8, 21, 66-68). Full
depression makes subsequent depressive episodes more and sustained remission may also decrease the chance of
likely and less dependent upon an external impetus such as future recurrences (69). In this regard, the comorbidity of
stress or sickness (30). Robert Post—who first proposed MDD and pain may hinder an early and appropriate
kindling as a construct for understanding the tendency of diagnosis of MDD (70, 71), thereby delaying treatment and
mood disorders to worsen over time—has recently subsequent benefits of remission. Consistent with this,
suggested that kindling and sensitization may have similar resolution of painful symptoms doubled the remission rate
neurobiological underpinnings, such as neuroplastic in a recent study of depressed patients: 36.2% of the
changes and alterations in gene expression (31). In this subjects who had >50% reduction of pain on a visual
vein, some authors have gone so far as to posit “neuro- analogue scale (VAS) attained remission vs. only 17.8% of
sensitization” as a common etiology for chronic pain, the individuals who had <50% reduction on the VAS (3).
depression and anxiety disorders (i.e. PTSD) (32).
5. GENETICS OF MDD, FM AND NEP
Consistent with the predictions of a sensitization
model for pain and mood disorders, FM, NeP and MDD Given the high degree of overlap between MDD,
have many common features. All 3 conditions are either FM and NeP in terms of symptom profiles, disease course
5293
Figure 1. An integrated view of the shared neurobiological underpinnings of major depression (MDD), fibromyalgia (FM) and
neuropathic pain (NeP) .
and underlying neurobiology, it should perhaps not come as 5.1. Genetics of MDD
a surprise that these conditions also appear to share a Dozens of different genes have been implicated
number of genetic determinants. Interestingly, many of in the development of MDD. Among the many genes that
these shared genes contribute to the structure and/or have been identified as conferring vulnerability to the
functioning of pathways in the brain and body that evolved disorder, studies have most consistently supported a role
to respond to danger signals from the internal and external for polymorphisms in genes that regulate the serotonin
environment, whether these signals arise from transporter promoter locus (5HTTPR), the serotonin
environmental threat (i.e. stress), tissue damage (i.e. pain) 5HT2A receptor, catechol-O-methyl transferase (COMT),
or infection (i.e. inflammation). One result of the monoamine oxidase (MAO) and brain-derived
tremendous complexity and interdependency of these neurotrophic factor (BDNF). More recently, genes
systems is that MDD, FM and NeP are clearly genetically regulating the synthesis and/or activity of CRF and
heterogeneous conditions to which multiple polymorphic glutamate receptors have also been implicated (75). An
genes contribute to various degrees and in various exciting development in the field of psychiatric genetics is
combinations. On the other hand, because CNS and the recognition that genes are more likely to code for
peripheral danger pathways tend to respond in a “endophenotypic traits” that increase the risk of
coordinated and highly stereotyped manner to a variety of psychiatric morbidity, than they are to code directly for
environmental threats (72), it should also come as no any specific psychiatric disorder (74). For mood and pain
surprise that alterations in genes linked to very different disorders, these endophenotypic factors center primarily
physiological elements in this circuitry (i.e. neurotransmitters on circuitry essential for affective function, cognition and
and inflammatory cytokines) are capable of producing similar threat appraisal, as well as for stress and immune system
abnormalities in emotional state and pain perception, reflecting activity.
what is often referred to as a “final common pathway”
phenomenon. Nonetheless, even this level of analysis fails to 5.1.1. Genes involved in regulation of serotonergic
do justice to the full complexity of genetic contributions to function
MDD, FM and NeP, as demonstrated by epistatic interactions As noted above, “depression genes” frequently
in which two “bad” genes—rather than additively contributing do not contribute directly to the development of the
to disease development—actually cancel each other out (73). disorder, but rather confer vulnerability to developing
Finally, even the notion that genes specifically exist for MDD, depression in response to environmental threat. For
FM and NeP reifies these states of emotional distress and example, a study by Caspi et al. demonstrated that the
physical pain in a way that does violence to emerging scientific inconsistently observed association between depression
data demonstrating that risk factor genes do not cause these and the short allele of 5HTTPR was likely explained by
conditions, but rather predispose the brain and body to the fact that the allele increased the likelihood of
physiologically respond to internal and external environmental developing both depression and suicidal ideation in
conditions in ways that lead to symptom production (74). individuals exposed to life stressors, but not in individuals
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spared environmental adversity (76). This finding has allele of the 5HTTPR and the “met” variant of the val66met
been replicated several times. For example, Kendler et al BDNF was recently reported by Pezawas and colleagues. In
observed identical interactions between the 5HTTPR short a volumetric MRI study of 111 healthy, non-depressed,
variant (“s” allele), stressful life events and the risk of individuals, researchers found that—contrary to
developing MDD. In addition they described a gender expectations—the met BDNF allele (which increases
effect, such that women with the short 5HTTPR allele depression risk) provided protection to circuitry linking
were especially likely to develop depression in response to amygdala and rostral ACC (rACC). As noted above, this
stress (odds ratio of 7 compared to males with the long circuitry, which is involved in emotional modulation and
allele) (77). stress responses, tends to function sub-optimally and be
reduced in volume in 5HTTPR “s” carriers. However, these
The “s” allele of the 5HTTLPR gene has been abnormalities are significantly muted in 5HTTPR “s”
associated with a number of alterations in brain function carriers who also carry the met BDNF allele, because this
and morphology, including compromised functional and allele made amygdala-rACC circuitry relatively insensitive
structural integrity of amygdala-ACC circuitry involved in to 5HT signaling, thereby protecting these individuals from
regulation of emotional, behavioral and endocrine loss of rACC gray matter volume (73). This finding may
response to stress, as well as in processing the experience have significant clinical implications, given that rACC also
of pain (40, 41, 78-81). The short allele has also been plays a role in pain signaling and predicts treatment
linked to reduced anterior cingulate cortex (ACC), response to antidepressants (91).
amygdala and hippocampal volumes (78). These structural
changes, in turn, may result in aberrant connectivity Similarly, studies have reported that 5HTT,
between ACC and amygdala which is believed to interfere COMT, and MAOA polymorphisms and gender have a
with emotional homeostasis and effective modulation of convergent effect on the HPA-axis response to
the stress response (78, 79, 82). Gotlib et al. have psychological stress and endocrine challenges (92).
proposed relationships between the “s” 5HTT allele, Individuals with less functional 5HTT, COMT and MAOA
responses to stress, HPA reactivity and susceptibility to alleles had blunted baseline ACTH and cortisol responses
MDD, given data that girls homozygous for the “s” allele to DEX/CRH challenge, reflective of potential
reacted to stress with prolonged elevation of cortisol, susceptibility towards MDD. This study also implies that
compared to ones with “l” allele, indicating greater monoamines play an important role in neuroendocrine
reactivity to stress and possible increased susceptibility homeostasis and maintenance of health (92).
towards depression (83).
5.1.4. Polymorphism of genes regulating MAO
Of course, genes such as the “s” 5HTTPR allele Polymorphisms in the MAOA gene have been
don’t exist in isolation but interact in myriad ways with shown to interact with maltreatment with adverse impact
other genes—a process known as epistasis. For example, on children’s mental health. Depressed MAO-H (higher
the combination of the 5HTTPR “s” allele with a activity) carriers, tend to have compromised amygdala-
functionally less competent allele of the 5HT1A receptor prefrontal connectivity and greater illness severity (93).
gene has been associated with exaggerated amygdala
reactivity in medicated MDD patients (84). 5.1.5. COMT gene polymorphisms
The COMT Val108/158 Met genotype has a
5.1.2. The role of genes regulating neurotrophic factors significant impact on hippocampal and prefrontal gray
The activity of genes known to modulate matter volume (94). Beyond structural differences, Met
neurotrophic factors, cellular resilience, neuroplasticity and 158 COMT allele has been associated with increased
neurogenesis may be compromised in MDD patients (85, limbic reactivity to unpleasant stimuli and altered
86). For example, the val66met allele confers reduced connectivity between amygdala and PFC (95).
BDNF functioning and has been associated with structural
brain changes common in MDD, including reduced gray 5.1.6. Polymorphisms in genes involved in regulating
matter volume in dorsolateral prefrontal cortex (DLPFC), the inflammatory response
lateral-orbital prefrontal cortex (LOPFC) and hippocampus Polymorphisms in inflammation-related genes
(87, 88). Dwivedi et al reported altered genetic expression have been reported to confer susceptibility to major
of BDNF in hippocampus and PFC of depressed suicide depression and to effect antidepressant response (96, 97).
subjects, further supporting the relevance of neurotrophic Inflammatory reactions are an integral component of the
factors in MDD (89). stress response. Interestingly, proinflammatory cytokines
have a profound impact on mood, HPA axis regulation,
5.1.3. The Role of genetic epistasis in MDD monoamine signaling, as well as regulation of neurotrophic
Epistasis has been reported with the 5HTTPR factors and pain modulation (42, 53).
gene and a variant of the gene for BDNF known as
val66met. In a seminal study, Kaufman et al. reported that In summary, genes regulating monoamine
the “s” allele of 5HTTPR and the “met” variant of the receptors, transporters and enzymes involved in their
val66met BDNF allele increase the risk of developing metabolism, all seem to contribute to vulnerability towards
depression in an additive fashion in the context of MDD (98). These genes combined with ones regulating
environmental adversity early in life (90). On the other corticosteroid, neurotrophic and inflammatory signaling
hand, a very different epistatic interaction between the “s” influence structural integrity and functional connectivity in
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areas involved in generating adaptation to stress (86, 99, a risk for FM development, genetically-linked alterations
100). Consistent with this, a recent review suggested that of 5HTTLPR density in FM patients may be associated
genes associated with depression may act in unison by with symptom severity (108).
accelerating sensitization to stress (101).
Other genes implicated in mood and anxiety
5.2. Genetics of nociception and FM disorders that may also contribute to chronic pain states,
Significant data suggest that FM is genetically include COMT, as well as the 5HT2A and D4 dopamine
related to a wide range of conditions subsumed under the receptors. Zubieta et al reported an association between a
rubric of “affective spectrum disorders” (ASD), including common val158met polymorphism of COMT (catechol-
depressive and anxiety disorders, premenstrual dysphoric O-methyl-transferase, an enzyme involved in
disorder, attention deficit hyperactivity disorder (ADHD), catecholamine metabolism) and sensory and affective pain
FM, irritable bowel syndrome (IBS) as well as migraine ratings (116). Met homozygous carriers showed a
and cataplexy conditions. For example, a recent family diminished mu-opioid receptor response to pain, and a
study concluded that patients with FM were twice as stronger subjective experience of pain when compared to
likely to have at least one of these other conditions, heterozygous subjects. Other studies have found that this
compared with individuals without FM (OR 2.0, 95% CI COMT polymorphism may play a role in the stress
1.2-3.2, p = 0.004) (102). A recent study has refined our response, the trait of novelty seeking, cognition, MDD,
understanding of genetic links between mood and schizophrenia, anxiety disorders and ADHD (92, 117,
pain/somatic disorders by suggesting intriguing patterns of 118). The 5HT2A receptor gene is also a candidate for
genetic overlap and environmental specificity for these involvement in FM, given a recent study reporting that
conditions. Specifically, in a large twin study of the carriers of the T/T allele, while under-represented in the
relationship between two psychiatric disorders (MDD and FM population, had significantly higher pain scores than
generalized anxiety disorder [GAD]) and somatic FM carriers of the C/C allele or healthy controls (119).
syndromes such as FM, chronic fatigue, IBS and recurrent Interestingly, a separate study noted that the same T/T
headache, multivariate analyses suggested the influence of allele, in the presence of high maternal nurturance, was
two factors: one, most likely genetic, shared between associated with lower depressive symptoms than the C/C
somatic disorders, MDD and GAD and a second one, genotype, consistent with the notion that “vulnerability”
more specific to somatic conditions, that was more genes may have been maintained in the human gene pool
environmentally based. Thus, these conditions are a because they are “opportunity” genes given the proper
product of interaction of genes, some shared, some more environmental exposure (120). Finally, some reports have
specific to individual conditions, and environmental established a connection between a gene coding for the
factors (103). D4 dopamine receptor and a vulnerability towards FM
(119, 121). Other studies link the D4 receptor gene to a
It is not surprising that genetic studies of personality trait called “novelty seeking” and to ADHD
nociception are in their infancy given the genetic (118, 119).
heterogeneity of pain disorders, as well as the complexity
of relevant gene/environment interactions (104). In conclusion, genetic studies of depression, pain
Nonetheless, a number of genes known to modulate and FM have noted alterations in genes regulating (likely in
human nociception have emerged as potential risk factors a convergent manner) monoamine and inflammatory
for impaired pain processing, including genes coding for signaling (92, 115, 119). It is tempting to speculate that
opioid receptors, transient receptor potential cation shared genetic vulnerabilities towards depression and pain
channels (TRPV1), fatty acid amino hydrolase (FAAH) states may be reflected in dysregulation of circuitry
and GTP cyclohydrolase 1 (104, 105). Genes implicated involved in modulating stress responses, pain and
in mood disorders have also been identified as risk factors emotional states.
for FM and related pain states. These genes include
5HTTPR, the 5HT2A receptor, COMT and the dopamine 6. FUNCTIONAL AND STRUCTURAL
D4 receptor, as well the proinflammatory cytokines IL-1 ALTERATIONS IN CENTRAL NERVOUS SYSTEM
and IL-6 (105-110). Although not found by all studies CIRCUITRY INVOLVED IN REGULATION OF
(107), the association between FM and the “s” allele of EMOTION AND PAIN
5HTTLPR is particularly interesting given its association
with a wide range of conditions that are either risk factors Because emotional reality appears to be actively
for—or frequent concomitants to—FM, including anxiety, construed, rather than just passively experienced (122), a
neuroticism (best conceptualized as a tendency towards person’s world literally changes with changes in brain
excessive emotional reactivity to stressful stimuli), MDD, functioning. In the case of depression, these changes warp
Bipolar Disorder, Psychosis and even ADHD (111-114). reality into a frightening or empty realm that calls forth a
Given the significant role played by stress in the initiation depth of emotional pain that often defies verbal
of chronic pain and fatigue states, it is interesting that the description (123).This emotional pain frequently
“s” 5HTTLPR allele has also been shown to affect stress engenders an equally disabling experience of physical
responses and to be a risk factor for the development of pain, even when no peripheral source for such pain can be
depression and fatigue in response to chronic identified. Conversely, anyone who has long struggled
inflammatory exposure during interferon-alpha treatment with chronic physical pain knows that the dark emergence
for hepatitis C (115). In addition to potentially conferring of emotional despair commonly compounds the physical
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ache with a sense of affective terror. Given the numerous structures (134, 137) (Figure 2). Decreased activity in
links between depression and pain conditions such as FM DLPFC in MDD may contribute to the compromised
or NeP outlined thus far, it should come as no surprise that working memory, impaired sustained attention and
the different disorders also resemble each other in terms executive dysfunction seen in the disorder (137). In
of abnormalities in CNS structure and function. It is to addition to functional abnormalities in MDD, a recent
these abnormalities that we now turn. three-year prospective study reported significantly greater
DLPFC gray matter decline in un-remitted MDD patients
6.1. Structural and functional brain changes in compared to control subjects and to subjects with MDD
depression who attained remission (21).
6.1.1. Abnormalities in prefrontal cortex (PFC)
Prefrontal cortical (PFC) abnormalities are a 6.1.2. Abnormalities in anterior cingulate cortex
common finding in MDD. However, because different (ACC)
subregions of the PFC have profoundly different The anterior cingulate cortex (ACC) is probably
functional roles, a more detailed description of these areas the brain area most often implicated in the
and their activities is required to gain a sense of how this pathophysiology of MDD, with most studies emphasizing
brain region contributes to MDD. In this discussion we the subgenual ACC (sgACC) as being especially relevant.
will focus on three subregions most often implicated in The sgACC has a role in assessing the salience of emotional
MDD: ventromedial prefrontal cortex (VMPFC), lateral and motivational information and making necessary
orbital prefrontal cortex (LOPFC) and dorsolateral adjustments in behavior. It is also involved in modulation of
prefrontal cortex (DLPFC). sympathetic and neuroendocrine responses. Functional
imaging studies suggest increased metabolism in this area in
VMPFC tends to demonstrate increased activity depressed patients (when corrected for reduced volume)
in MDD (26, 124). VMPFC has rich reciprocal (138). Structural studies have noted significantly decreased
connections with limbic formations and the hypothalamus volume of sgACC in MDD subjects. For example, Drevets et
(125-127). VMPFC not only serves as a major recipient of al. noted that sgACC had 48% lesser volume in individuals
limbic projections, but also modulates amygdala and with familial depression than in healthy controls (130). These
hippocampal activity through complex feedback alterations likely contribute to disturbances of motivation,
connections (126, 128). VMPFC also plays a key role in limbic regulation (especially amygdala), and neuroendocrine
regulating appetitive drives and pain responses (129). function, all of which are commonly seen in patients with
Increased VMPFC activity in MDD patients has been MDD (48, 80, 138). Although more anterior areas of the
associated with melancholy ruminations and intensity of ACC have been a primary focus of research, more dorsal
negative affect (130, 131). These observations suggest areas of the ACC have received increasing attention for
that this prefrontal subregion, together with anterior their potential role in MDD. For example, in a study of
cingulate cortex (ACC) and limbic areas, is a component patients with MDD, Chen and colleagues reported that
of an integrated network involved in processing reduced gray matter volume of dorsal ACC and DLPFC were
emotionally relevant information for the purpose of correlated with increased symptom severity in MDD patients
guiding behavior and orchestrating adaptive autonomic (91). This report resonates with the work of Matthews et al.
and endocrine responses (130). Given the key regulatory who noted reciprocal connectivity between supragenual ACC
role of VMPFC, it is intriguing that several authors have and amygdala in unmedicated depressed patients (48). In
found significant VMPFC volume reduction (up to 32%) summary, recent research lends credence to the hypothesis
in MDD patients compared to healthy controls (132, 133). that these “executive network” areas have a role in “top-
down” modulation of limbic areas and that when this
Lateral orbital prefrontal cortex (LOPFC) seems modulation fails, depression is likely to ensue (134, 139).
to be involved in suppressing maladaptive and Finally, several lines of evidence suggest that ACC
perseverative emotional responses. LOPFC may also have abnormalities are associated with treatment response.
a major role in volitional regulation of emotion and Mayberg et al. has noted that activity in sgACC normalizes in
cognitive reappraisal (122, 134). LOPFC activity is patients who respond to either an active antidepressant or
enhanced in depression (26), most likely in a placebo (137), and diminished function and volume of pre-
compensatory role to modulate excessive limbic activity. genual ACC has also been associated with a delayed
Lacerda et al have found a significant reduction in LOPFC antidepressant treatment response (91).
gray matter volume of MDD patients compared with
healthy subjects (133). Medial dorsal PFC (MDPFC), 6.1.3. Hippocampal changes
associated with automatic emotional and attentional The hippocampus is a key limbic area located at
control and self-appraisal (134, 135), also suffers a greater the “crossroads” of circuitry that regulates the stress
gray matter decline over time in MDD patients compared response by providing inhibitory feedback to the HPA
to healthy controls (20). axis, in addition to its role in mood modulation and
memory formation (68, 125). However, its central
Dorsolateral prefrontal cortex (DLPFC) is a location may render the hippocampus vulnerable to
primary component of an executive function network in functional dysregulation that accompanies extreme
the brain that also includes dorsal ACC (dACC) and parts stress and mood disorders (125). Reflecting this
of the parietal cortex (130, 314, 136). DLPFC tends to vulnerability, alterations in hippocampal volume are
have a “top-down” regulatory influence over limbic among the most common imaging findings in
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Figure 2. Cortical and subcortical areas involved in voluntary and automatic regulation of mood and the stress response.
Reprinted with permission from (134).
MDD patients (68). Recent data strongly suggest that investigators found a significant association between
physiological abnormalities, inherent to depression, may hippocampal increases in N-acetylaspartate (NAA) (a
actually lead to reductions in hippocampal volume. marker of neuronal density, function and myelination) and
Indeed, Frodl et al found a significant decline over a choline compounds (Cho) (a marker of membrane
three-year period in hippocampal gray matter volume in integrity and metabolism) and treatment response. These
MDD patients compared to healthy controls (21). authors also reported reduced glutamine (Gln) in
Successful treatment appeared to have a protective effect, depressed subjects. Glutamate (Glu), a major excitatory
given that remitted patients had a significantly greater neurotransmitter is metabolized into Gln, after being taken
hippocampal density than non-remitted ones (21). up by astrocytes. Gln has major significance for
Consistent with this, one post hoc analysis (68) has found maintenance of Glu/GABA balance in neuron-glia
a significant increase in hippocampal volume (21%, signaling. This is the first study providing evidence that
p=0.004) after 6-7 months of antidepressant treatment in a successful treatment with two different antidepressants, a
small subset of patients with atypical depression. predominantly noradrenergic and a predominantly
Similarly, Sheline et al found an inverse relationship serotonergic one, may lead to functional and structural
between the days of untreated depression and restoration of hippocampal neurons and astroglia (142).
hippocampal volume (140), and Colla and colleagues
noted a negative correlation between duration of 6.1.4. Amygdala alterations
depression and hippocampal volume, corrected for age As with other relevant brain regions,
and intracranial volume (141). The potential importance neuroimaging studies suggest that functional
of restoration of hippocampal structure and function for abnormalities of the amygdala may contribute to
successful treatment of depression was further highlighted depressive symptom development and that
by a study conducted by Block et al (142). Using pathophysiological processes inherent to depression may,
magnetic resonance spectroscopy (MRS), these in turn, damage this extremely important brain structure.
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Functional neuroimaging studies have, for the most part, 6.1.7. Abnormalities in connections between brain
found increased activity in the amygdala of depressed regions in MDD
individuals (143). Because the amygdala plays an Several studies have found altered connectivity
important role in rapidly assessing and assigning between limbic and paralimbic prefrontal areas in MDD
emotional value to surprising and ambiguous stimuli, it is (144, 157-159). Combined with studies that have found
not surprising that patients with MDD respond to angry white matter abnormalities in patients with MDD (158),
and fearful faces with increased amygdala activity, even these studies point to a compromise in the integrity of
when these faces are presented below the level of fronto-subcortical and prefrontal-limbic circuits in the
conscious awareness (143-145). It is intriguing to disorder (48, 157, 159). Additional involvement of fronto-
consider that this type of amygdala over-activity may cerebellar-thalamic circuitry is likely (151). Therefore,
translate into the increased anxiety and emotional cumulative evidence suggests that disruption of circuitry
misattribution, both of which are commonly observed that provides cognitive-emotional integration and control
in patients with MDD (146, 147). Because the of stress responses may be responsible for complex
amygdala and other limbic structures have significant manifestations of MDD. In summary, structural and
bidirectional connections with the hypothalamus, it is functional studies support an organic basis for the
also not surprising that sympathetic and emotional, cognitive and neuroendocrine symptomatology
neuroendocrine dysregulation are a frequent of MDD (128, 138, 158). Unfortunately, current research
concomitant of mood disorders (125, 128). Structural indicates that cognitive impairments in MDD tend to be
neuroimaging studies of the amygdala suggest that this persistent, non-specific and progressive, with a greater
brain region is negatively impacted by physiological number of episodes being associated with increasing
processes inherent to depression. The same prospective cognitive burden (160, 161).
study that recently reported an association between
ongoing depression and loss of brain volume in DLPFC 6.2. The role of the peripheral and central nervous
and hippocampus also noted a decline in amygdala system in FM and chronic pain
gray matter (esp. left) over time in MDD patients, 6.2.1. Abnormalities in peripheral nerves and spinal
relative to control subjects (21). cord pathways
Pain pathways implicated in NeP and FM have
6.1.5. Insular abnormalities peripheral and central components—in this section we
Insular cortex has emerged in recent years as trace how pain signals originate in the periphery and reach
another limbic structure of tremendous relevance to the the brain
regulation of both affect and pain. Insula has rich
connections with other limbic (e.g., amygdala and Pain signals are detected by peripheral
hippocampus) and paralimbic cortical areas (ACC and nociceptive nerve endings and conveyed to neurons
LOPFC). Because of this, it is widely considered to be located in dorsal root ganglia (DRG). From the DRG pain
a primary target of thalamo-cortical projections. As information is conducted by lightly myelinated A-delta
such, the insula plays an essential role in sensory- and un-myelinated slow C-fibers to secondary sensory
affective integration that creates a bodily sense of self. neurons localized in the dorsal column of the spinal cord.
The anterior insula is also involved in modulating the Aside from functional alterations in nerve membranes and
influence of sensory and emotional distractors (148- endocellular signaling (discussed elsewhere), peripheral
150). Consistent with these roles and interconnections, sensitization may occur as a result of alterations in
altered activation of insula in MDD subjects has been synaptic connectivity resulting from sprouting of
described in several studies (26, 150, 151). In general, sympathetic axons within DRG (which may further
MDD patients appear to have decreased activity of augment pain transmission), ectopic discharges and
insula, which tends to improve with antidepressant ephaptic (direct electrical transfer of signal)
treatment (26, 150). Neuroimaging studies, utilizing communication. Central sensitization in NeP may in part
labeling of 5HT2 receptors, have also found evidence be mediated by collateral sprouting (whereby non-
of altered sertononergic transmission in insula in MDD nociceptive A-beta fibers form new connections with
subjects (148). nociceptive neurons in the dorsal horn), as well as damage
to inhibitory GABA inter-neurons (29, 162, 163).
6.1.6. Abnormalities in sub-cortical areas
Functional and structural changes have been Damage to these initial components of pain
noted in basal ganglia and thalamus of patients with mood processing circuitry (e.g. nerve injury) is the primary
disorders (152). Functional studies have noted increased cause of altered pain signaling in NeP. On the other hand,
thalamic activity in depressed individuals. Thalamic there are fewer data to support an important role for
hyper-metabolism is associated with increased activity in abnormalities in peripheral or spinal cord pain signaling in
sgACC and decreased metabolic activity in dACC (153), FM. Nonetheless, some evidence does indicate potential
both of which have been observed in MDD. Some, but not peripheral contributions to the disorder. For example,
all, studies have found alterations in cerebellar function in Salemi et al. have performed skin biopsies in 53 FM
patients with MDD (26, 152). This is of particular interest patients and found mononuclear and fibroblast-like cells
because the vermis of the cerebellum has been implicated adjacent to nociceptive neuronal fibers that stained
in generating automatic emotional responses—including positive for inflammatory cytokines, suggesting a role for
empathy—to facial expressions (154-156). neurogenic inflammation in the etiology of the FM (164).
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Other authors have found evidence of changes in Schwann fMRI studies like these provide objective
cell morphology in the skin (165) and altered blood flow evidence of altered cerebral processing of painful stimuli
in the muscles of FM patients (166, 167). in FM patients. More recently, abnormalities in some of
the same brain regions have also been observed using
6.2.2. The role of supraspinal structures in pain magnetic resonance spectroscopy (MRS). For example,
After synaptic processing in the dorsal horn of Petrou et al. noted altered choline compounds (markers of
the spinal cord, pain signals are propagated via membrane metabolism and myelination) in DLPFC of FM
spinothalamic (paleo-spinothalamic) and patients compared to healthy controls (173) Alterations in
spinoparabrachial (neo-spinothalamic) tracts to higher choline significantly correlated with subjective pain
CNS pain centers. Spinothalamic signals are relayed intensity. Another MRS study found evidence of increased
through thalamus to somatosensory cortices I and II (SI glutamatergic activity in the insula of FM patients that
and SII) and associated areas, including insula, ACC correlated with measures of clinical pain (174). In an
and posterior cingulate cortex (PCC). ACC, in turn, has earlier study, the same group described decreased mu-
close bidirectional connections with amygdala and opiate receptor binding potential in the cingulate cortex,
hippocampus. Spinoparabrachial fibers convey amygdala and nucleus accumbens of FM individuals
information to the parabrachial nucleus in the compared to controls, a finding that may explain why
brainstem and then on to amygdala, hippocampus and opiate medications are often ineffectual in reducing FM
hypothalamus. Ascending pain signals and information pain (50).
from supraspinal pain circuitry are integrated in the
mesencephalic periaqueductal gray area (PAG), which Temporal summation of “second pain” (TSSP) is
also has a pivotal role in regulating descending pain one of the landmark clinical features of FM. TSSP results
pathways (163, 168). DLPFC and LOPFC appear to from repetitive stimulation of C-fibers and is believed to
initiate the descending pain modulatory sequence, reflect a summation mechanisms in dorsal horn neurons
explaining how attention and anticipation may (i.e., “windup”), therefore it is a basic substrate for
influence the intensity of pain. These prefrontal areas, widespread central sensitization in FM (175). Consistent
richly innervated by dopamine fibers, can trigger opioid with this, Staud et al found that TSSP correlated with
release in PAG, substantially reducing the intensity of elevated activity in S1, S2, thalamus, insula and ACC in
experienced pain (descending pain modulatory system FM subjects relative to healthy controls (176). It appears
will be discussed in more detail, later in the text (38, 169). that enhanced pain sensitivity in the context of FM may
arise more from altered activity of the entire “pain
Imaging studies have consistently identified matrix”, rather than from individual components.
several brain areas as having a major role in pain
processing, including primary and secondary 6.2.3. Descending pain modulation in chronic pain
somatosensory cortices (S1 and S2), thalamus, insula, disorders
ACC and PFC. Together these brain areas are commonly Imaging studies have consistently suggested that
referred to as the “pain matrix” (170) and many studies both FM and NeP are characterized by dysregulation of
indicate that function is disrupted in this matrix in the pain processing circuitry involving ACC and insula. These
context of chronic pain states, including FM and NeP. For two formations appear to have a significant role in
example, Bailiki et al. utilized fMRI to study chronic back regulating descending pain modulatory pathways that
pain patients. These authors reported an association include PAG and rostro-ventral medulla (RVM) (177-
between the intensity of spontaneous pain and activation 179). Insular cortex appears to exert regulatory control
of medial PFC (mPFC) (49), an area known to have a over descending inhibitory tracts that project from the
role in automatic emotional regulation (134). On the RVM to the dorsal horn of the spinal cord via the
other hand, in this population, duration of pain was dorsolateral funiculi (DLF). ACC has a principal role in
most strongly associated with increased activity in the modulating descending pain facilitatory pathways, which
insula (49). A second fMRI study noted a greater project from RVM via the ventrolateral funiculi (VLF) to
activation in DLPFC and ACC in response to non- the dorsal columns. NE, 5HT and Ach are the principal
painful stimuli in patients with FM relative to control neurotransmitters in inhibitory descending pathways
subjects, a finding likely to reflect alterations in (177). Primary neurotransmitters in facilitatory
processes central to the cognitive and emotional descending pain pathways include glutamate, 5HT,
aspects of pain, such as attention and anticipation neurotensin, cholecystokinins and BDNF (177, 178, 180).
(171). In response to an equivalent pressure stimulus, Dysregulation of descending pain pathways, as a result of
patients with FM have been shown to demonstrate inadequate activity in the inhibitory pathway, excessive
increased activity in several areas of the CNS pain activity in the facilitatory pathway or both, has been
matrix when compared to normal control subjects, proposed as an important etiological factor for the
including S2, insula, posterior cingulate cortex (PCC), secondary hyperalgesia/ allodynia often observed in FM
ACC, superior temporal gyrus and inferior parietal and NeP (178, 180, 181).
lobule (172). Moreover, mild pressure applied to
subjects with FM elicited subjective pain and cerebral 6.2.4 Structural brain changes in FM and NeP
responses similar to the responses seen in normal In addition to functional differences, several
subjects when twice as much pressure was applied studies have found significant structural changes in the
(172). brains of FM patients. Kuchinad et al. reported
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significantly reduced gray matter density in the cingulate in ways that cause depression and pain conditions to
cortex, insula, mPFC and the para-hippocampal lobe of worsen each other in a feed-forward circuit. For example,
FM patients when compared to a control group (24). As multiple lines of evidence (i.e. electrophysiological,
with depression, the physiological changes that biochemical, and imaging) are indicative of compromised
accompany FM may themselves damage brain structures function and structure of the amygdala in chronic pain
over time, given that in this study duration of illness (43). Depressive and anxious feelings, as well as chronic
correlated with greater gray matter changes, such that stress, may enhance amygdala responsivity to pain (43).
each year of disease had an impact equivalent to 9.5 times Rainville et al. have demonstrated that negative emotions,
the loss due to normal aging. Changes in these areas a defining feature of MDD, enhance the perception of pain
appear to contribute to the compromised pain regulation, and associated autonomic responses to painful stimuli
emotional modulation, stress responsivity and cognitive (65). The very similar functional and morphological
functioning, often described in FM patients (24). For alterations of DLPFC in MDD and chronic pain (21, 22,
example, Luerding et al. reported that neurocognitive 26), may conspire to disrupt the “top-down” regulation of
deficits in FM patients correlated with reduced gray depression and pain, eventually impacting the function of
matter volume in DLPFC and ACC (areas typically descending pain pathways (while at the same time
associated with executive function), additionally pain compromising cognition, and therefore, coping abilities)
scores were noted to be negatively correlated with gray (182).
matter volume in mPFC (25). A recent MRS study noted
lower NAA levels in hippocampus of FM patients Ventral striatum/ nucleus accumbens (N.Acc.) is
compared to controls. NAA levels are commonly a critical component of both stress/pain response and
considered to be a marker of neuronal and axonal integrity reward/analgesic systems (183-185). N.Acc. receives
and function. Hippocampal alterations may be associated cognitive information from the PFC, emotionally relevant
with impaired pain regulation, cognition, sleep and signals from amygdala, and contextual information from
neuroendocrine function in FM (47). hippocampus (183). It is richly innervated with
dopaminergic and opiate fibers (183, 184, 186). Feedback
Fewer neuroimaging studies have been conducted fibers from N.Acc have a key role in modulating VTA
in neuropathic pain than in either depression or FM. dopaminergic output, and therefore play an important role
Nonetheless, fMRI studies of neuropathic pain have in maintaining the balance between meso-cortical and
implicated brain areas known to be functionally abnormal meso-limbic dopaminergic activity (183). Neuroimaging
in FM and chronic non-neuropathic pain, including PFC, evidence suggests that N.Acc/VTA regulation may be
thalamus, insula and ACC (38, 41). Apkarian et al. disrupted in MDD and chronic pain states (187, 188).
utilized volumetric MRI to assess gray matter changes in a Excessive activation of amygdala/sgACC/vmPFC in
group of chronic back pain (CBP) sufferers, the majority chronic stress, pain and depression may interrupt VTA
of who had neuropathic pain (22). The investigators found dopaminergic transmission leading to impaired reward
significantly reduced gray matter volume in DLPFC and perception, cognitive dysfunction, decreased interest in
thalamus of CBP patients when compared to controls. novelty, compromised pain regulation and possibly,
Moreover, decreased gray matter density in DLPFC was vulnerability towards substance abuse (184-188).
correlated with pain intensity, duration and negative
affective characteristics in this population. Disturbingly, In summary, MDD, pain and chronic stress
the magnitude of gray matter reduction in CBP patients synergistically activate limbic circuitry, mutually
was equivalent to 10-20 years of normal aging. Wiech et amplifying the distress signal, while simultaneously
al. have reviewed the evidence that establishes a degrading the regulatory influence of prefrontal cortical
supporting role for DLPFC and dorsal ACC in volitional structures.
pain control via activation of descending pain modulatory
pathways (182). Considering the well-established role of Depressed patients activate DLPFC and VLPFC
DLPFC in top-down regulation of limbic and paralimbic in response to pain in a significantly greater manner than
prefrontal areas, it is conceivable that morphological healthy controls, which may be interpreted as a
changes in DLPFC may contribute to the compromised compensatory attempt to suppress negative emotions
emotional and pain modulation apparent in NeP patients (189). Consistent with this view, Graff- Guerrero et al.
(22). Prefrontal structures, through processes of attention, reported decreased cerebral blood flow (CBF) in areas
expectation and reappraisal, also appear to play an associated with emotional response to pain, such as
important role in the cognitive modulation of pain (182). amygdala, hippocampus, insula, ACC and PCC after
antidepressant treatment. Changes in CBF coincided with
6.3. Implications of the CNS overlap of depression and increased experimental pain threshold and tolerance (190).
pain Depressed patients may also have an impaired ability to
A striking feature of neuroimaging studies, modulate pain due to heightened emotional reactivity. For
reviewed thus far, is the significant overlap in brain areas example, Strigo at al. noted increased activation of insula,
that are functionally and/or structurally abnormal in dACC, and amygdala in MDD subjects anticipating pain
MDD, FM and NeP. These brain areas include DLPFC, compared to controls (191). Depressed patients also had
mPFC, LOPFC, insula, ACC, amygdala, hippocampus decreased activation of rACC and PAG, implicating
and thalamus. As would be expected from this overlap, compromised functioning in descending pain modulatory
symptoms of pain and depression also ramify each other pathways. Greater activation of amygdala in this study
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was associated with increased levels of perceived nervous system and inflammatory response system in
helplessness, indicating a possible bidirectional major depression, FM, NeP and other conditions of
relationship between depression and pain (191). Changes chronic pain and distress.
in ACC function and structure have been noted in MDD,
FM and NeP (21, 24, 41, 91). Some authors have 7.1. Abnormalities of the HPA axis in depression and
proposed that altered ACC function may result in pain
increased activity of facilitatory descending pathways 7.1.1. HPA abnormalities in depression
(179, 192). Similarly, changes in insula in MDD and FM Hyperactivity of the HPA axis (with resultant
may negatively impact inhibitory descending pathways hypercortisolism) is one of the most replicated
(24, 26, 177, 181). physiological abnormalities seen in patients with major
depression (193). Many studies suggest that this
In summary, when viewed as a continuum MDD, hyperactivity commences with overproduction of
FM and NeP overlap significantly in terms of associated corticotropin releasing hormone (CRH) (the primary
brain changes. When viewed as separate diagnostic secretagogue for the HPA axis), which is released from
conditions they clearly exist in complicated bidirectional the paraventricular nucleus (PVN) of the hypothalamus,
relationships with each other, such that depression may whenever higher brain areas (or signals arising in the
give rise to altered pain processing and alterations in pain body) indicate potential threat to the organism (194).
processing may promote affective states conducive to the Findings indicative of CRH over production in MDD
development of depression. Shared patterns of include 1.) elevated cerebrospinal fluid (CSF)
dysregulation in circuitry involved in modulating emotion, concentrations of CRH; 2.) increased CRH mRNA and
pain and the stress response, especially as manifested by protein in the PVN (postmortem samples); 3.) blunted
excessive activation of amygdala and compromised ACTH response to CRH challenge (likely reflecting
hippocampal function and structure in MDD and FM, may ACTH receptor downregulation secondary to increased
also have neuroendocrine, autonomic and immune CRH), and 4.) downregulation of CRH receptors in the
repercussions, to which we turn next. frontal cortex of suicide victims (many of whom were
presumably depressed) (195-200). Reflecting the fact that
7. NEUROENDOCRINE, AUTONOMIC AND CRH drives production of adrenocorticotropin (ACTH)
IMMUNE DYSREGULATION IN MDD, FM AND and the subsequent release of glucocorticoids (i.e. cortisol
NEP in primates, corticosterone in rodents) from the adrenal
glands, MDD is also frequently associated with
A central thesis of this article is that disorders of hypercortisolemia. Evidence for this in MDD includes 1)
mood and pain share so many commonalities in symptoms increased number of cortisol and ACTH secretory pulses
and disease course precisely because these conditions are across the circadian cycle resulting in elevated
characterized by similar patterns of dysregulation in the concentrations of cortisol in the peripheral circulation,
activity and structure of brain regions and circuits that urine and CNS; 2) an exaggerated cortisol response to
play a primary role in sensing, evaluating and responding adrenocorticotropin (ACTH); and 3) enlargement of
to danger, whether the danger be from the external (e.g. a pituitary and adrenal glands, suggestive of hypertrophy
predator or social threat) or internal (e.g. infection, tissue secondary to HPA overdrive.(201-205)
damage) environment. This perspective provides clarity to
the otherwise rather remarkable finding that disorders as The notion that MDD is characterized by
diagnostically diverse as major depression, post-traumatic insufficient glucocorticoid signaling seems at first blush to
stress disorder, FM and chronic fatigue syndrome share be directly contradicted by the multiple findings indicative
similar abnormalities in neuroendocrine, autonomic and of increased—rather than decreased—HPA axis activity in
immune function. At the most basic level, we would the disorder. However, the signaling strength of any
suggest that these brain-body commonalities reflect the hormonal system depends not just on hormone levels, but
fact that many conditions currently parsed into separate also on how effectively the signal “gets through”, which is
DSM diagnoses can be understood as states of CNS and also dependent upon receptor sensitivity, as well as
peripheral danger system hyperactivity. Moreover, even ancillary factors such as binding proteins (206). And in the
the patterns of hyperactivity observed across these context of MDD, many years of data demonstrate that
multifarious conditions are strikingly stereotyped and glucocorticoid receptors (GR) have reduced activity,
have as their primary features insufficient glucocorticoid leading to a relative failure in cortisol signaling, even in the
signaling, increased sympathetic and/or reduced context of increased cortisol levels (207, 208). Indeed, a
parasympathetic tone and activation of innate immune strong association exists between increased cortisol blood
inflammatory pathways (42). Increasing evidence suggests levels and decreased GR sensitivity in MDD (209),
that these patterns of stress—immune system derangement suggesting that rather than being indicative of excessive
not only purvey a heightened risk for the development of glucocorticoid tone, the hypercortisolism of depression is
medical illness, but may also provide physiological more often a reflection of relative GR resistance and hence
feedback to the brain that promotes the development of inadequate glucocorticoid signaling.
both depression and chronic pain. In this section we will
attempt to enlarge upon the role of danger system In addition to being a primary mediator of the
dysregulation in depression and pain by describing and stress response, cortisol is also the body’s primary anti-
comparing abnormalities in the HPA axis, autonomic stress and anti-inflammatory molecule, so one would
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expect that insufficient glucocorticoid signaling would lead reduced sensitivity is highlighted by findings that high
to an inability of neuroendocrine stress pathways to levels of cortisol following the DST (i.e. glucocorticoid
terminate activity and to a release of inflammatory resistance) strongly predicted the future development of
processes from regulatory control (206). Both CWP over a 15-month period in a group of 267 at-risk
abnormalities are seen in MDD and strongly argue for the individuals based on a somatasizing personality style (226).
fact that glucocorticoid signaling is insufficient in the Also suggesting that reduced glucocorticoid sensitivity may
disorder. Rates of impaired glucocorticoid responsiveness contribute to symptom development are findings from a
in the HPA axis (as a result of the axis being insensitive to trial of a multidisciplinary intervention in FM. The
cortisol-mediated inhibitory feedback control) vary from investigators found that symptomatic improvement over a
approximately 25 to 80% (201), depending on depressive 3-week period was associated with increased mRNA
symptomatology (highest rates are found for melancholic, expression for the active form of the glucocorticoid
or endogenous, subtypes) (210), age (older subjects are receptor (which would be expected to enhance
more likely to exhibit glucocorticoid resistance) and the glucocorticoid signaling) (227).
technique used for assessment (the DEX-CRH stimulation
test is more sensitive than the traditional dexamethasone To resolve whether the end products of the HPA
suppression test [DST]).(201, 211) Of note, both the DST axis (i.e. glucocorticoids) contribute to depression, fatigue
and the DEX-CRH test have been shown to powerfully and pain primarily through overactivity or under-activity
predict clinical response and relapse (212-215). And in the (i.e. insufficient signaling), several lines of evidence can be
case of the DEX-CRH test, there is evidence that impaired marshaled based on known effects of glucocorticoids on a
glucocorticoid responsiveness represents a genetically- variety of relevant physiological and behavioral endpoints.
based risk factor for the development of depression (216). Because glucocorticoids exert profound anti-inflammatory
Complimenting in vivo findings, in vitro studies have effects in the brain and body, one would expect evidence
demonstrated that peripheral immune cells from patients for reduced inflammatory activity in MDD, FM and CFS if
with major depression exhibit decreased sensitivity to the these conditions were states of excessive glucocorticoid
well-known immunosuppressive effects of glucocorticoids activity. As will be reviewed in some detail below, the
(207). Whereas normal subjects show a marked inhibition opposite appears to be the case as evidence for increased
of in vitro natural killer (NK) cell activity, lymphocyte inflammation in these conditions mounts. Similarly, if
proliferation or cytokine production following increased glucocorticoid signaling contributes to symptom
glucocorticoid (usually DEX) exposure, patients with major development in these disorders, one would expect that
depression, especially DST non-suppressors, consistently adding exogenous corticosteroids would worsen symptoms.
show an attenuated inhibitory response (217-220). In fact, quite the opposite appears to be the case, given
studies showing that hydrocortisone, although not widely
7.1.2. HPA abnormalities in FM and chronic pain used due to risk for adverse events, demonstrates
Although not entirely consistent, most studies antidepressant efficacy and improves symptoms in patients
report that conditions of chronic pain, fatigue and other with CFS and PTSD, both of which are frequently
somatic symptoms (i.e. FM, chronic fatigue syndrome comorbid with FM (228-231). If cortisol routinely
[CFS]) are characterized not by the hypercortisolism of contributed to the high rate of FM development that occurs
depression but by decreased cortisol production and after motor vehicle accidents, one would expect high levels
release, both at baseline and in response to a variety of of the hormone to predict subsequent symptom
stressors (206, 221). Interestingly, depressive conditions development, but in fact the opposite appears to be the
characterized by atypical symptoms (i.e. increased sleep, case, with several studies showing that reduced cortisol
increase appetite and profound lethargy) also appear to be responses to motor vehicle accidents strongly predicts
associated with reduced HPA axis activity (blunted subsequent PTSD, which is, in turn, a significant risk factor
cerebrospinal CRH) (222), perhaps reflecting the fact that for the development of FM-type symptoms. Consistent with
pain and exhaustion feature prominently in this type of a protective effect for cortisol, patients who receive stress
depressive presentation. As with MDD, the pressing doses of hydrocortisone as part of their treatment while in a
question revolves around whether decreased cortisol medical intensive care unit (ICU) have reduced rates of
production and release leads to glucocorticoid insufficiency ICU-related PTSD symptoms (232), and subjects
in conditions of chronic pain and fatigue or whether administered cortisol or prednisone prior to exposure to
changes in glucocorticoid receptor activity compensate (or standardized laboratory stressors report less distress and
even over-compensate) for the reduced levels of hormone. less fatigue (233, 234). Finally, the administration of
The relevance of this question is highlighted by the fact that hydrocortisone to aging veterans with PTSD was shown to
the idea of glucocorticoid receptor supersensitivity in FM, enhance both episodic and working memory (235).
CFS (and PTSD) has gained significant currency in recent
years based on studies showing increased sensitivity to Less controversial than whether glucocorticoid
feedback inhibition of both the HPA axis and the immune signaling is excessive or impaired in the context of pain and
system in these frequently comorbid conditions (223, 224). depression is the consistent finding that the diurnal rhythm
However, although not as widely considered, a significant of the HPA axis is flattened in MDD, anxiety and
number of studies support the opposite conclusion—that conditions associated with fatigue and pain, such as FM
(as with MDD) FM, chronic widespread pain (CWP) and and CFS.(236-238) In healthy individuals, cortisol
CFS are conditions of reduced glucocorticoid sensitivity production peaks around wake time and then falls thereafter
(225). The potential pathophysiological relevance of this (excepting “blips” of increased cortisol in response to
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meals) until the early morning hours when it commences its As with MDD, disturbances in
climb toward wake-time (239). In a variety of conditions SNS/parasympathetic balance have been frequently
linked to stress, pain and emotional misery, this pattern is reported in FM and highly comorbid conditions such as
disrupted, such that cortisol levels are lower around PTSD and CFS and may contribute to disease pathology
wakening, show more disorganized variability during the by perpetuating neuroimmune dysregulation and
day and do not fall sufficiently in the evening. Such facilitating neurogenic inflammation (42, 263). In one of
flattening is an ominous development indeed, given that it the rare studies in men with FM, Cohen et al. conducted
predicts the following: subsequent development of chronic power spectral analysis of HRV and found that orthostatic
widespread pain in medically healthy individuals (226), intolerance in FM patients may result from sympathetic
development of fatigue and depression in patients receiving hyperactivity and concomitant reduced parasympathetic
interferon (IFN)-alpha (240) and increased mortality in tone (264). Several other authors have identified
patients with cancer (241). In animal models, flattening of sympathetic overactivity as a component of the stress
the cortisol rhythm impairs the production and release of response that both precipitates and perpetuates FM and
BDNF and impairs neurogenesis in brain regions in which NeP symptoms such as fatigue, sleep disturbance, anxiety,
such neurogenic processes are tightly linked to depression, vasomotor instability and gastrointestinal
antidepressant response (242, 243). Behavioral complaints (19, 265). In an intricately designed,
interventions that improve pain symptoms in FM patients controlled, study, Torpy et al. observed excessive
also steepen the cortisol slope.(227). sympathetic and HPA activity in FM patients after an IL-6
injection, supporting the notion that FM may, at least in
7.2. Autonomic nervous system abnormalities in part, be disorder of the stress system dysregulation (266).
depression and pain A number of studies report increased heart rate in FM
Given the known role of glucocorticoids in (267). HRV is also reduced and shows abnormalities in
modulating autonomic nervous system (ANS) activity circadian patterning (268-270), although the association
(206), if the hypercortisolism of depression and the between reduced HRV and FM may be stronger in women
hypocortisolism of FM, CWP and CFS were really than men (271). One study noted that FM patients show
functionally different, one would expect to find these enhanced SNS and reduced parasympathetic activity when
disorders reliably associated with very different types of recumbent, but fail to adequately activate SNS activity or
ANS abnormalities. In fact, the opposite is the case. withdraw vagal tone upon standing (272). This lack of
Although inconsistencies in the data exist, the majority of reactivity has also been documented in response to
available evidence suggests that depressive and pain psychological stressors (224). Of note, biofeedback
disorders are characterized by a common ANS signature training that enhanced HRV (i.e. reduced SNS activity and
comprised of increased SNS signaling and diminished increased vagal tone) significantly improved pain and
parasympathetic (or cholinergic) tone. other symptoms in patients with FM (262), suggesting
that—as with MDD—ANS abnormalities may actually
Evidence supporting increased SNS/decreased contribute to symptom development and might be an
parasympathetic tone in MDD includes increased heart effective target for intervention in FM. Similar patterns of
rate at rest and in response to stress, increased blood ANS disturbance have been reported in conditions that are
pressure, increased systemic vascular resistance, increased highly comorbid with FM, such as CFS and PTSD (236,
whole body sympathetic activity based on measures of 273, 274).
postganglionic norepinephrine (NE) release and NE
clearance, reduced overall heart rate variability (HRV), 7.3. Inflammatory signaling in mood and pain disorders
reduced high frequency HRV (a measure of Interestingly, abnormalities in HPA axis and
parasympathetic tone), impaired autonomic ANS functioning frequently observed in both mood and
information flow (AIF) on 24-h ambulatory pain disorders have in common a proclivity for activating
electrocardiograms, impaired baroreflex, higher the same peripheral innate immune inflammatory
ventricular repolarization time and increased incidence pathways that are activated in response to pathogen
of multiple firing within a sympathetic burst based on invasion and/or tissue trauma (206). Several studies link
single-unit muscle sympathetic nerve analysis (244- glucocorticoid resistance with increased inflammation
254), Conversely, although not entirely consistent, data (220, 275). An even larger literature demonstrates a tie
suggest that various forms of treatment, including between SNS activation and/or parasympathetic
repeated transcranial magnetic stimulation, ECT, withdrawal and enhanced inflammatory tone (248, 275-
antidepressants and psychotherapy correct 283). Not surprisingly, therefore, a rapidly increasing
sympathetic/parasympathetic imbalances by literature indicates that mood and pain disorders are
attenuating SNS activity and/or increasing vagal tone associated with increased inflammation.
(255-260). Epidemiologic studies link intake of omega-
3 fatty acid with a reduced depression risk and When compared to non-depressed individuals, as
improved heart rate variability (261). Interestingly, a a group patients with major depression exhibit all of the
recent study suggests that applying biofeedback to cardinal features of inflammation, including elevations in
directly induce these ANS changes improves inflammatory cytokines and their soluble receptors in
depressive symptoms, strongly suggesting that ANS peripheral blood and cerebrospinal fluid (CSF), as well as
activity may be as much a cause of depressive elevations in blood concentrations of acute phase proteins,
symptomatology as a result (262). chemokines, adhesion molecules and inflammatory
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mediators such as prostaglandins (284-304). Associations have also recently been reported in FM and chronic pelvic
between depression and increased proinflammatory pain (330, 331). Although microglial activation in the
cytokines and/or c-reactive protein (CRP) have been CNS of humans with chronic pain has not been directly
apparent across the adult life span, whether comparisons demonstrated, it is intriguing that several studies find
are made between clinically depressed patients and elevations in proinflammatory cytokines in the
matched controls (284, 285, 288) or whether they derive cerebrospinal fluid of patients with chronic pain (332,
from large population-based studies (289, 291, 305-310). 333). In further support of the important role of immune
Recent investigations have reported positive correlations factors in etiopathogenesis of disorders characterized by
between levels of various inflammatory mediators and chronic pain and fatigue, researchers at the Centers for
depressive symptom severity (284, 285, 304, 305, 309, Disease Control and Prevention have recently reported
311-315); however, the association between immune that subjects with CFS or with CFS symptoms such as
activation and depression appears to be robust enough to fatigue and pain, had significantly higher levels of plasma
be detectable in the context of mild depressive symptoms CRP than did well controls (334). This study utilized a
that do not meet criteria for major depression (314). large population-based sample and found correlations with
Indeed, even single depression-related symptoms—such unwellness symptoms such as pain, fatigue and sleep
as fatigue, insomnia, fear and anger/hostility—appear to disturbance even after adjustment for multiple potential
increase the likelihood of inflammatory activation in confounding factors such as age, sex, race, body mass
otherwise healthy individuals (314, 316-321). index and depressive symptom status. In this study,
Inflammation has generally been assessed at a single time increased CRP was associated with increased physical, but
point; however, a recent study found that IL-6 production not emotional symptoms, highlighting the especially close
was abnormal across the entire circadian cycle in patients link between somatic symptoms and inflammatory
with major depression (284). Although most studies have activity.
focused on cytokine production/release, emerging data
indicate that depression may also be associated with In summary, altered neuroimmune,
activation of “downstream” inflammatory second neuroendocrine and autonomic regulation may interact to
messenger signaling pathways, such as the NF-kappa-B perpetuate states of pain and depression (53, 335-337).
pathway (208). Although recent research has focused Peripheral mediators of an aberrant stress response
primarily on cytokines which mediate the innate immune (cortisol, NE, proinflammatory cytokines) may not only
response, including IL-1, tumor necrosis factor (TNF)- be responsible for many of the clinical symptoms of
alpha, and IL-6, findings of increased markers of T cell MDD, FM and NeP, but may also have a role in
activation (e.g. soluble IL-2 receptor) in depressed perpetuating disturbed homeostasis in cortico-limbic
patients raises the specter that both acquired (e.g. T and B circuitry, therefore maintaining a vicious cycle (42, 53,
cell) and innate (e.g. macrophage) immune responses may 335-337) (Figure 3). In this sense MDD, FM and NeP
participate in inflammation in depression (322). may be seen as composed not just of psychosomatic
components (i.e. brain driving bodily dysregulation), but
Perturbation of neuroimmune control has also also of somato-psychic components (i.e. bodily processes
repeatedly been cited as a shared feature of conditions promoting CNS dysregulation). On a deeper level, one
such as FM, NeP and CFS that are characterized by pain, may even question the utility of such causal dichotomies
fatigue, sleep disturbances and cognitive complaints (53, between mind/brain and body.
164, 323, 324). Although results have not always been
consistent, several authors have reported elevations of 8. NEUROTRANSMITTERS IMPLICATED IN
TNF-alpha, IL-8 and IL-6 in FM (324-326). The MDD, FM AND NEP
inflammatory cytokine IL-8 is released by the pain
promoting peptide substance P, modulates HPA axis 8.1.1. Glutamate and GABA in MDD
activity and is involved in the induction of Multiple lines of evidence implicate aberrant
sympathetically mediated pain (324-327). Consistent with glutamate (Glu) and GABA transmission in the
these effects, elevation of IL-8 appears to be one of the etiopathogenesis of MDD, NeP and FM. As the principal
more consistent findings in FM (325, 326, 328). excitatory neurotransmitter in the circuitry linking limbic
Moreover, several studies found associations between and cortical areas, Glu is virtually ubiquitous in the brain.
blood levels of IL-8 and subjective pain ratings in FM Cortical glutamatergic projections have a principal role in
(325, 328). In addition to elevations in proinflammatory modulating activity in the source neuronal regions for
activity, data suggest that chronic pain conditions may norepinephrine (locus ceruleus [LC]), serotonin (nuclei
also be characterized by reductions in anti-inflammatory raphe [NR]) and dopamine (substantia nigra [SN] and
activity. For example, chronic widespread pain has been ventral tegmental area [VTA]) (338, 339). Glutamatergic
shown to be associated with reduced gene expression and neurotransmission relies on several classes of receptors.
blood protein levels of the anti-inflammatory cytokines Ionotropic receptors include N-methyl-D-aspartate
IL-4 and IL-10 (329). Elevations in inflammatory activity (NMDA-R), alpha-amino-3-hydroxyl-5 methyl-4-
may be a widespread phenomenon in FM, given a recent isoxazole-propionate (AMPA-R) and kainite receptors
study that reported TNF-alpha, IL-1 and IL-6 in the skin (KR). In order for NMDA-R to be activated, neural
of a majority of FM patients but in none of the controls membranes must also express AMPA-R. NMDA –R
(164). Elevations in peripheral levels of the chemokine “uncoupled” from AMPA-R are also referred to as “silent
MCP-1, which is known to activate microglia in the CNS, NMDA” receptors (340). Increased neural activity leads to
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Figure 3. Mood disorders, fibromyalgia (FM) and neuropathic pain (NeP) may have shared systemic consequences.
Compromised homeostatic function of prefrontal cortical-limbic circuitry in MDD, FM and NeP appears to disrupt autonomic,
neuroendocrine and neuroimmune regulation. Stress, pain and depression lead to excessive and untimely release of corticotropin-
releasing hormone (CRH), adenocorticotropic hormone (ACTH) and glucocorticoids. Sympathetic over-activity, combined with
diminished parasympathetic tone, contributes to immune activation and release of proinflammatory cytokines (e.g. TNF-alpha,
IL-1, IL-6) from macrophages and other immune cells. Inflammatory cytokines further interfere with monoaminergic and
neurotrophic signaling. They may also down-regulate central glucocorticoid receptor sensitivity, leading to further disruption of
feedback control of the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. In depression and pain states,
disturbances of serotonin (5HT), norepinephrine (NE) and dopamine (DA) transmission may impair regulatory feedback loops
that turn off the stress response, with a resultant compromise in the function of descending pain modulatory pathways. Elevated
mediators of the inflammatory response, combined with excessive sympathetic tone may further impact dorsal column processing
of pain signals by contributing to activation of microglia and astroglia. Activated microglia exchange signals with astrocytes and
nociceptive neurons, amplifying pain-related transmission of glutamate (Glu), substance P (SP), adenosine triphosphate (ATP),
brain-derived neurotrophic factor (BDNF), pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-alpha, nitrogen oxide (NO) and
prostaglandins (PGs). PVN= Paraventricular nucleus of hypothalamus; IL = interleukin; TNF-alpha = Tumor necrosis factor-
alpha; Ach=acetyl choline
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[Frontiers in Bioscience 14, 5291-5290, January 1, 2009]
repeated activation of synaptic NMDA-R, promoting clinical studies noted that antidepressants and ECT
synthesis of brain derived neurotrophic factor (BDNF). improve GABA deficits (350, 351).
Binding of BDNF to tyrosine kinase-B (TrkB) receptors
facilitates synaptic delivery of AMPA-R, thereby mediating 8.1.2. Monoamine Neurotransmitters in MDD
neural plasticity and long-term potentiation (LTP), which Despite decades of elaboration, the “monoamine
are key mechanisms for translating experience into hypothesis” of depression remains beset with
enduring modification of synaptic transmission (340, 341). controversies. Prompted by the evident treatment efficacy
Additionally, BDNF modulates dendritic arborization and of medications that modulate 5HT, dopamine (DA) and
synaptic spine density and morphology, contributing to a norepinephrine (NE), it has long been held that
micro- feed-forward circuit that allows the brain to “re- insufficient monoamine signaling may play a cardinal role
wire” itself in response to increased neural activity (341). in the etiopathogenesis of MDD (352, 353). In support of
Conversely, Glu binding to extra-synaptic NMDA-Rs, has this hypothesis, researches have noted alterations of NE
an opposite effect and suppresses BDNF synthesis (338). and 5HT receptor density in cortical and limbic
Glu also binds to three different classes of pre-synaptic and formations of depressed patients (353). In a PET imaging
post-synaptic metabotropic receptors (mGlu-Rs), which study comparing dopamine type-1 receptor (D1) binding
have a role in modulating neurotransmitter release and potential (BP) in MDD patients vs. healthy subjects,
post-synaptic activation of AMPA-Rs and NMDA-Rs(342). researchers found significantly reduced D1 BP in the
depressed group. This reduction in D1 BP correlated
Early studies that examined serum and plasma negatively with illness duration and anhedonia ratings
glutamate levels in MDD subjects had equivocal findings (354). We have already provided evidence implicating
(338). More recently, MRS studies have noted decreased genes that code for 5HTT, COMT, MAO and monoamine
Glx (a measure combining glutamate, homocarnosine, receptors in perpetuating vulnerability towards depression.
GABA and glutamine spectroscopic signatures) in ACC Meyer et al. have reported elevated levels of MAO-A in
and amygdala of depressed adults, children and elderly the brains of depressed patients. Since MAO-A is a
patients. In one of these studies, this alteration normalized primary monoamine-lowering enzyme in the nerve cells,
with successful ECT treatment (see (338) and (343) for a its excessive activity may contribute to a disturbance of
review). A significantly higher Glu/GABA ratio was monoamine signaling in MDD (355). Changes in platelet
reported in the occipital cortex of depressed individuals 5HT and NE receptor density have also been noted in
compared to controls. Additional reports indicate changes depressed and suicidal patients (353). Patho-histological
in the NMDA receptor glycine binding site in depressed studies have reported a decrease in the number of NE
suicide victims and NMDA subunits in LC of depressed neurons in LC of depressed patients whose death was
subjects (338). Altered function of glutamatergic fibers unrelated to suicide when compared to matched controls
originating from vmPFC and projecting to sympathetic (356). The same authors also noted a decrease in the number
and parasympathetic brainstem centers has been described of 5HT neurons in dorsal nuclei raphe of depressed patients
in MDD patients (344). This abnormality may provide a compared to healthy controls (356). Neuromelanine-sensitive
link between emotional dysregulation and excessive stress MRI imaging has revealed an attenuated signal in LC of
reactivity, repeatedly noted in depressed individuals (344). depressed patients (357). On the other hand, research
Open label studies of lamotrigine and rizulole (both assessing NE and 5HT metabolites in the CSF has yielded
inhibitors of glutamate release), as well as a controlled inconsistent results. Levels of 5-hydroxyindoleacetic acid (5-
randomized trial of ketamine (an NMDA antagonist) in HIAA) have been reported to be lower in depressed patients
treatment resistant depressed patients provide preliminary and especially low in victims of violent suicide (353).
evidence of efficacy of glutamatergic modulators in the However, because MDD is a biologically heterogeneous
treatment of MDD (345). syndrome, it is not surprising that there are differing reports
on the levels of monoamine biomarkers. For example, 3-
Several studies have reported that MDD is methoxy-4-hydroxyphenylglycol (MHPG), a major NE
associated with reduced plasma GABA levels (see (346) metabolite, was found to be elevated in plasma of agitated
for a review), as well as decreased GABA neurons in and anxious depressed patients, while depressed individuals
lateral orbital PFC (LOPFC) and DLPFC (347). This is a with psychomotor retardation were found to have lower
particularly interesting finding, given the previously MHPG and homovanillic acid (HVA) (a primary dopamine
described role of DLPFC and LOPFC in cognition, metabolite) levels than healthy controls. Patients with
voluntary regulation of emotion and suppression of psychotic depression had elevated plasma levels of HVA.
maladaptive affect. Some preliminary data suggest that (358). Another study, using catheters placed in the internal
altered cortical GABA and Glu concentrations may not jugular vein noted reduced NE and DA release in the brains
only differentiate MDD patients from controls but also of patients suffering from refractory depression relative to
differentiate depressive subtypes from each other healthy volunteers (359). In contrast, using a similar
(melancholy and atypical) (348). A recent MRS study technique, brain 5HT turnover was found to be elevated in
found reductions in GABA levels in dmPFC, DLPFC and depressed patients when compared to healthy subjects (360).
ACC in MDD. It should be noted that these are the same
brain areas in which patho-histological studies established Monoamine transporters also seem to be
alterations in glial density in MDD patients compared to influenced by the disease process. For example, the
healthy controls (349). Finally, several preclinical and dopamine transporter (DAT) has a 15% lower binding
potential in depressed patients compared to controls (361),
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and 5HT transporter (5-HTT) binding potential (which example, dopaminergic input tends to up-regulate
can be considered as a marker of 5-HTT density) tends to serotonergic and down-regulate noradrenergic activity.
be elevated during a depressive episode, especially a more Elevated serotonergic activity has a mostly inhibitory
severe one (362). Pharmacologic strategies that deplete effect on NE and DA, while increases in NE tend to
monoamines suggest that the therapeutic effect of suppress DA but can modulate 5HT transmission in either
antidepressants may be selectively reversed by depleting direction (368, 369). Complex cross talk between
the monoamine affected by that particular antidepressant dopaminergic and noradrenergic systems appears to take
(363). place in VTA, LC and dorsal hippocampus (370).
Extracellular DA in the prefrontal cortex originates not
However, a converging body of evidence brings only from dopamine but also from NE terminals (371).
into question simplistic interpretations of the “monoamine Furthermore, not only do monoamines influence each
theory”. If monoamines are the primary abnormality in other via complex interactions but GABA and Glu tend to
MDD, it is hard to understand why in the STAR*D trial have a bidirectional regulatory influence on monoamines
50% of the patients failed to respond to the first line SSRI (351, 372-375). In summary, extant evidence supports the
treatment, 65% did not achieve remission and more than a view of a complex dysregulation of interrelated
half of those who did still had two or more residual neurotransmitter systems and distributed brain networks
symptoms (364). While in vivo preclinical evidence involved in regulation of mood, cognition and the stress
describes prompt LC activation following SSRI response, rather than a simple deficit of monoamine
application (365), clinical studies have shown that it may signaling (122, 376).
take as long as five to eight weeks until optimal
antidepressant response is attained (366). Mayberg et al. 8.1.3. Endogenous Opioids and other peptide
evaluated SSRI responses utilizing PET imaging. These neurotransmitters in MDD
investigators found no difference between responders and The role of the peptide neurotransmitters galanin
non-responders following a week of antidepressant and substance-P (SP) and endogenous opiates in the
treatment. Rather, the pattern of activity associated with pathophysiology of MDD has been a focus of recent
depression was reversed only after six weeks of treatment, research (377-379). Galanin coexists in serotonergic DNR
suggesting an adaptation to chronic antidepressant neurons and noradrenergic LC neurons. Galanin receptors
administration as the basis of therapeutic effect (137). are located in PFC, amygdala, hippocampus,
Wong et al. measured CSF NE and plasma cortisol levels hypothalamus and the brainstem nuclei LC and DNR
in a group of patients suffering from melancholic MDD. (379). Galanin receptor modulators are showing early
Samples obtained around the clock showed that depressed promise in preclinical and clinical studies as
patients had significantly higher levels of NE and cortisol antidepressant, anxiolytic and neurogenesis-promoting
than healthy subjects. NE and cortisol levels were strongly agents (379).
correlated to each other (200). A variety of antidepressant
medications and ECT have been reported to suppress the A recent review emphasized the pivotal role of
activity of tyrosine hydroxylase (TH), a key enzyme SP in communicating peripheral inflammation and stress
regulating NE synthesis (367), while remarkably response to the brain. SP appears to occupy a key position
consistent evidence shows reductions in CSF levels of in bidirectional communication between the brain and the
MHPG (the main NE metabolite) in depressed patients body (377). Additionally, SP fibers and the main
taking antidepressants (331). Imaging studies have neurokinin-1 receptor (NK-1), are well represented in the
uncovered insufficient activity and reduced volume of prefrontal cortical and limbic areas, involved in the
vmPFC in depressed individuals (26, 124, 132, 137, 344). regulation of mood, anxiety and stress response (380).
Because vmPFC plays a key role in regulating amygdala Recent studies have demonstrated that emotional stress
activity and sympathetic/parasympathetic balance, it is not results in SP efflux in amygdala and septal areas (380). A
surprising that depressed patients tend to exhibit excessive preclinical study indicates that stress-induced release of
sympathetic activation in response to stressful stimuli SP in LC, facilitates neurotransmission via NE projections
(221, 344). A recent preclinical study demonstrated a to mPFC, thus relaying the stress signal to this area (380).
significant reduction in LC electrophysiological activity SP also modulates the firing rate of serotonergic DNR
following 14 days of treatment with a diverse group of neurons and dopaminergic VTA neurons, as has been
antidepressants, including desipramine (a predominantly demonstrated in studies utilizing systemic administration
noradrenergic TCA), paroxetine and citalopram (SSRIs) of NK-1 receptor antagonists (381).
and mirtazepine, an alpha-2 antagonist (331).
Kennedy et al. utilized PET imaging to assess
Barton et al. recently reported elevated 5HT endogenous opioid transmission in female MDD patients.
turnover in unmedicated MDD patients. Marked reduction These authors reported a reduction in mu-opioid binding
in brain 5HT turnover, accompanied by clinical in ACC, ventral basal ganglia, hypothalamus and
improvement, ensued after twelve weeks of SSRI amygdala of MDD patients compared to healthy subjects
treatment (360), suggesting a relatively insufficient 5HT (378). Supporting data in depressed patients, demonstrate
transmission in MDD. perturbation of opioid transmission in the aforementioned
areas involved in emotional and neuroendocrine
Strong evidence suggests that monoamines also regulation.
regulate each other through complex interactions. For
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8.2.1. Glutamate and GABA in pain disorders GABA has an important modulatory role in pain
Glutamate is the principal neurotransmitter in transmission, from interneurons in dorsal column to
both ascending and descending pathways involved in complex cerebral circuitry involved in pain processing and
inhibiting and facilitating pain transmission in the CNS, regulation of descending pain pathways (168, 395).
including the dorsal column, thalamo-cortical and cortico- Alterations in peripheral benzodiazepine receptors (PBR)
limbic circuitry involved in pain processing (174, 177, on platelets (396) and monocytes (291) have been
180, 382, 386). Excessive Glu transmission leads to reported in FM patients relative to healthy controls. For
conjoined activation of AMPA and NMDA receptors all example, upregulation of PBRs, a proxy indicator of
along the neuroaxis involved in pain perception and GABA-ergic function, correlated with severity of FM
modulation, which tends to promote increased synthesis of symptoms (396). Insular dysfunction associated with
neurotrophic factors (177, 180, 382). Such an GABA, DA and opiate neurotransmission has been
enhancement of neurotrophic signaling has been documented in both clinical and preclinical models of FM
associated with enduring neuroplastic changes, which (397). Moreover, extensive preclinical studies support a
are—in turn—the substrate for long-term potentiation role for GABA in the mediation of neuropathic pain (395).
(LTP) and “central sensitization” (177, 180, 382). Similarly, dysfunction of dorsal horn GABA interneurons
Additionally, activation of metabotropic Glu receptors due to injury may play a key role in NeP pathogenesis
(mGluR) has been implicated in both peripheral and (163, 398). Directly supporting a role for GABA in the
central pain sensitization (387). development of chronic pain, preclinical models suggest
that early GABA administration to spinal cord may
Based on concomitant increases in Glu, nerve prevent subsequent development of neuropathic pain
growth factor (NGF) and BDNF in the CSF of FM (395). Consistent with these data, pharmacologic
patients, investigators have recently hypothesized that manipulation of GABA-A receptors successfully
increased Glu neurotransmission may facilitate NGF and prevented NeP and alleviated pain in a preclinical model
BDNF synthesis in pain relevant pathways, which in turn involving sciatic nerve crush injury (399). On the other
may promote the expression of NMDA receptors in neural hand, inadequate GABA-opiate system regulation in PAG
membranes, effectively closing a positive feed-forward may result in compromised function of the descending
circuit that enhances pain signaling (388). Temporal pain pathway in models of NeP (162, 395).
summation of second pain (TSSP) and “windup”, also
believed to reflect a summation process characteristic of 8.2.3. Monoaminergic neurotransmitters in pain
dorsal horn neurons, are cardinal manifestations of central disorders
sensitization in FM. Repeated release of Glu and SP from Monoaminergic nuclei in mesencephalon and
C-fiber terminals, followed by excessive NMDA brainstem have rich projections that innervate cortical,
activation, appears to be the neural substrate for these limbic and thalamic areas involved in regulating mood,
phenomena (175). For example, a study that measured stress responses and pain processing (see previous text).
the concentration of excitatory amino acids in CSF of Additionally, serotonergic and noradrenergic brainstem
FM patients reported a relationship between elevated nuclei provide innervation for descending pain-
Glu and examination-based measures of pain intensity modulating tracts (180),(177), (400). Several authors have
in FM patients, including the tender point index (TPI) reported decreased CSF levels of the monoamine
(389). Harris et al, using fMRI imaging to study the metabolites (5-HIAA, MHPG and HVA) in FM patients
effect of a non-pharmacologic treatment in a group of relative to healthy controls (401, 402). Serotonergic
FM patients demonstrated a relationship between abnormalities in FM are of particular importance given the
changes in Glu levels in insula and improvements in role played by 5HT in regulating SP transmission, another
multiple pain domains (174). Thus, alterations in Glu mediator heavily implicated in pain regulation (402).
transmission at both cortical and dorsal column levels Serum concentrations of 5HT were found to be lower,
may play an important role in the etiology of FM. while 5-HT receptor density on circulating platelets was
Consistent with this, alterations in spinal glutamatergic higher, in FM patients compared to controls (401, 403). In
signaling and subsequent NMDA activation, as well as vivo microanalytical studies have reported elevated levels
excessive limbic NMDA mediated transmission, has of 5-HT, NE, SP, TNF-alpha and IL-1 in skeletal muscle
also been documented in preclinical models of of myofascial pain sufferers (404). FM patients were also
neuropathic pain (11, 390-392). noted to have lower plasma levels of tryptophan, which is
the metabolic substrate for 5HT (405). Although not all
Elevated glutamatergic transmission in NeP may, the studies support serotonergic involvement in FM,
in part, be attributable to upregulation of the alpha-2-delta alterations in 5HT signaling may contribute to the HPA
subunit of voltage-gated calcium channels. A group of axis and sleep dysregulation commonly observed in
authors reported a 17-fold increase in alpha-2-delta studies of FM (9. 405). In the context of NeP, 5-HT
subunit in dorsal root ganglia of animals subjected to an appears to have a dual role, participating in
experimental model of neuropathic pain (393). Such antinociceptive descending modulation (400, 406) on the
increased expression of alpha-2-delta subunits may one hand, and contributing to signaling in facilitatory
produce an excessive influx of calcium into the nerve descending fibers, on the other. Moreover, together with
cells, resulting in augmented release of excitatory amino NE, 5HT participates in sensitizing peripheral C-fiber
acids and SP, with a resultant amplification of pain (394). axons in preclinical models of NeP (407).
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We have previously described elevated receptor availability in amygdala of FM patients compared

sympathetic tone in FM and NeP, and recent studies have to controls (50). Reduced mu-opioid receptor availability
confirmed that sympathetic manipulation enhances pain correlated with affective pain ratings, possibly explaining
sensitivity in a subgroup of heat sensitive NeP patients the reduced efficacy of exogenous opiates in FM patients
(408). Decreased CSF concentrations of MHPG and HVA and elevated CSF and plasma levels of endogenous
(primary metabolites of NE and DA) in FM patients may opioids in this population (50, 405). Preliminary evidence
be related to compromised neurotransmission in inhibitory provides an intriguing view of the role that glial cells may
descending pathways (401, 402). A recent study utilizing play in the modulation of pain. In preclinical models,
PET imaging assessed dopaminergic function in FM morphine promoted release of Glu and inflammatory
patients (409). Unlike healthy controls, FM patients were mediators from astrocytes, leading to paradoxical increase
not able to mount a release of DA in basal ganglia in in pain transmission. Nonetheless, the role of this
response to painful stimulation. While the amount of mechanism in the clinical setting remains to be
released DA correlated with pain intensity in normal determined (415).
controls, no such relationship was noted in FM patients
(262). Because DA has a role in motivation and cognition, Preclinical models of NeP suggest an intriguing
it is intriguing to speculate that it may mediate at least dissociation between supraspinal and spinal opioid
some psychiatric symptoms that are common in the nociceptive systems. In an animal model of NeP, an
context of FM (39). Finally, monoamines may play a intrathecal injection of morphine had a significantly
synergistic role in modulating NeP, given a recent reduced analgesic effect, whereas supraspinal
preclinical study that found that combined 5-HT and NE administration had a potent antinociceptive effect (386).
uptake inhibition conferred greater analgesic benefits than Additionally, pain-facilitating neurons that originate from
uptake inhibition of a single monoamine. Interestingly the rostral ventromedial medulla (RVM) and are typically
addition of DA uptake inhibition further ameliorated NeP modulated by the opioid system seem to be sensitized
pain in this animal model (410). after nerve injury in preclinical models of NeP. This
sensitization likely contributes to allodynia and
8.2.4. Endogenous opioids in chronic pain hyperalgesia in NeP (416).
The dopaminergic system is closely linked to the
opioidergic system, which is probably the best established 8.2.5. Contributions of SP to conditions of chronic pain
antinociceptive pathway in the CNS (38). Opioid release Much like opioid and monoamine projections, the
in cortical and limbic areas involved in emotional substance-P (SP) system is present in several cortical and
processing and the stress response appears to regulate limbic regions involved in the stress response and the
pain-associated cognition, effectively attenuating emotive regulation of emotion and pain (380, 417). In preclinical
aspects of pain and sadness (411, 412). Sustained pain models, both chronic pain and chronic stress have been
reflexively activates the endogenous opioid system in associated with down-regulation of SP NK-1 receptors
ACC, PFC, insula, thalamus and hypothalamus (413). and BDNF synthesis in hippocampus, suggesting
Cognitive modulation of pain, mediated by ACC and similarities in mechanisms underlying chronic pain and
DLPFC activity, is conveyed by mu-opioid receptors depression (417). Activation of nociceptive fibers leads to
(414). The coupling of rACC and PAG opioid analgesic a concomitant release of glutamate and SP in dorsal horn
systems may have a crucial role in placebo anesthesia and synapses (9, 418). NK-1 receptors are expressed by
alteration of pain perception by change in expectation lamina-I dorsal horn neurons which project to brainstem
(413). Subcortical opioid circuits are more involved in and thalamus, modulating descending inhibitory and
modulating the sensory component of pain. Moreover, facilitatory pathways (418). It is no surprise that altered
activation of the endogenous opioid system in conditions SP transmission and subsequent dorsal horn neuron
of stress and danger may modulate affective and sensory sensitization has been implicated in the
components of pain independently (411). The etiopathogenesis of both FM and NeP (9, 419, 420). SP
hyperalgesic effect of psychological distress appears to be release seems to be promoted by elevations of
suppressed by endogenous opioid release, particularly in proinflammatory mediators and reduced by
women (411). Activation of descending pain inhibitory administration of dexamethasone in preclinical models
tracts leads to opioid release in the dorsal horn of the of NeP (421). C-fibers are also capable of retrograde
spinal column, where a hyperpolarizing analgesic effect is release of SP into the injured tissue, which then
mediated by mu- , delta- and kappa- opioid receptors contributes to “neurogenic inflammation” mediated by
(400). Glu acting through NMDA receptors, and SP acting proinflammatory substances, especially IL-8 (168,
through NK-1 activation, both modulate endogenous 326). This reiterative loop may amplify and perpetuate
opioids, which in turn have an inhibitory influence on NE chronic pain. Several authors have reported elevations
release (400). of SP in CSF of FM patients relative to healthy
controls, often associated with a reduction of
Evidence suggests altered opioid signaling in FM monoamine metabolites and endogenous opiates (401,
and NeP (12, 405). A recent PET imaging study has 422-425). However, elevated levels of SP in the CSF do
revealed decreased availability of mu-opioid receptors in not appear to be specific to FM. Given its association with
nucleus accumbens, dACC and amygdala of FM patients other painful conditions, it may be better considered as a
relative to healthy controls (50). A significant relationship biological marker of chronic pain than as a marker of any
was also detected between depression and mu-opioid particular diagnostic condition (37).
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Glu and SP have a synergistic pro-nociceptive Moreover, at times of peak neuronal activity glia cells
effect. Proinflammatory cytokines enhance the release of release lactate in response to increased energy needs.
both transmitters, and are in turn, themselves stimulated
by SP. IL-8, whose release is facilitated by SP, promotes Recent research suggests that in addition to
sympathetic pain (326, 400). Pro-nociceptive effects of SP myelinating axons, Schwann cells and oligodendroglia are
are opposed by endogenous opiates and galanin, a peptide instrumental in the long term maintenance of axonal
over-expressed in sensory neurons following peripheral functional integrity, with this role being independent of
nerve damage that is also known to promote a sustained myelin formation (431). Neurotrophic factors, including
inhibitory effect on dorsal horn synapses (400). BDNF that are generated and released by oligodendroglia
have a significant role in axonal resilience and recovery
Only selected neurotransmitters and mediators of (432). Loss of glial support appears to promote axonal
pain have been addressed in this article. A more degeneration and local inflammation (431).
exhaustive review of substances involved in pain Oligodendroglia appear to have a dominant role in human
modulation is beyond the scope of this review. In white matter Glu clearance. An altered ability to remove
summary, there is a remarkable similarity and overlap in Glu, likely mediated by TNF-alpha, may be the
how multiple neurotransmitter systems, utilizing underpinning of Glu excitotoxicity (433).
glutamate, GABA, monoamines, endogenous opioids and
SP, through complex supraspinal and spinal interactions, Astrocytes and oligodendrocytes most likely have
modulate the stress response, emotions and pain. a common glial precursor, with local microenvironments
during development determining the eventual fate of the
9. CELLULAR, SUBCELLULAR AND cell (434). On the other hand, microglia are the only cell
NEUROTROPHIC CHANGES IN MDD, FM AND population in the brain of mesodermal origin (435).
NeP Microglia have a complex role in the CNS. They act as
resident macrophages, constantly screening the CNS
9.1. An overview of glial architecture and function environment. Depending on circumstances, microglia
Accumulating evidence suggests that MDD may perform a variety of functions, including the apparently
be associated with significant CNS cellular pathology, opposite functions of neuroprotection and perpetuation of
especially in glia cells. The human nervous system has cellular damage, as well as the support of
approximately 100 billion neurons and one trillion glia oligodendrogliagenesis (436). Microglia may be
cells (426). Traditionally, glia cells have been cast as a conceived as the principal CNS recipients of peripheral
passive supportive matrix for neurons (426). However, in stress signals . Under usual circumstances, microglia have
dramatic contrast to prior assumptions, contemporary a protective role; however, in pathologic conditions these
research has established astroglia and oligodendroglia as cells amplify and perpetuate stress signals, contributing to
full-fledged neuronal partners in neurotransmission (54). oligodendroglial and neuronal damage (437, 438). Like
Indeed, brain architecture is defined by astrocytes. Each astrocytes and oligodendroglia, microglia are endowed
protoplasmic astrocyte occupies its own territory, with a wide variety of receptors, including those for
covering all the neural elements within its domain (427). chemokines, cytokines, GABA, glutamate, Ach, NE,
Each human astrocyte contacts and encapsulates dopamine, SP and opioids. They are capable of
approximately two million synapses (427). In addition to synthesizing and releasing prostaglandins,
managing the content of the synaptic cleft, astroglia may proinflammatory cytokines, oxygen- and nitrogen-reactive
have a role in synchronizing the activity of all neurons species (ROS and NRS), neurotrophic factors, Glu and
within their domains (428). Brain connectivity is quinolinic acid (439).
effectively shaped by astroglia through regulation of
synaptogenesis, synaptic strength and plasticity (427). 9.2. Contributions of glial pathology to MDD
Almost all classes of serotonin, norepinephrine, Glial cell pathology has been reported in the
dopamine, cholinergic, GABA, glutamate, neurotrophin sgACC, DLPFC, orbitofrontal cortex and the amygdala of
and cytokine receptors are expressed on glial cell unmedicated MDD patients (54, 440, 441)) It appears that
membranes (54, 55, 428). Additionally, astroglial both astroglia and oligodendroglia may be affected.
membranes express monoamine (5HTT, NAT, DAT) and Uranova et al. describe a prominent 19% reduction in
glutamate transporters. Evidence suggests that oligodendroglia in the DLPFC (BA 9) of MDD patients
neurotrophic factors, such as brain-derived and glia- (441). Having in mind the crucial role that DLPFC plays in
derived neurotrophic factors (BDNF and GDNF), as well executive function and “top-down” limbic regulation, the
as cytokines (i.e. TNF-alpha) are synthesized within glia implications of this finding are striking, because it may
cells (54, 55). However, unlike neurons, which release provide a neurobiological substrate for both the emotional
neurotransmitters in response to action potentials, glia dysregulation and cognitive dysfunction commonly
cells discharge the content of Glu vesicles in response to observed in MDD. Hamidi et al. have described reductions
graded increases in cytoplasmic Ca++. Astroglia also have in oligodendroglia density in amygdala of MDD patients
a role in regulating neuronal energy supply. Cerebral (442). Genetic factors may contribute to these
perfusion is significantly influenced by astroglia. On one oligodendroglia abnormalities, given a recent
end astroglial extensions “sense” synaptic activity, on the transcriptional profiling study of MDD subjects that has
other end their distal processes, or “feet”, modulate identified aberrant expression of 17 genes related to
vascular tone and capillary permeability (429, 430). oligodendroglia function (443). Rajkowsa et al. have noted
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a significant reduction of glia density in DLPFC and OPFC amplifies cytokine signals from the periphery (55, 429,
of MDD subjects (444). Further supporting glial 437, 448, 452, 453), Multiple effects are likely to result
involvement in mood disorders (445), Stockmeier et al. from this feed-forward proinflammatory, pro-oxidant
observed a significant decrease in glial density in the activity. For example, oligodendroglia are likely to suffer
dentate gyrus of the hippocampus in MDD subjects (446). oxidative damage due to overexposure to reactive oxygen
Additionally, age dependant decrements in the astroglial and nitrogen species (ROS and RNS). TNF-alpha released
markers were reported in a group of MDD subjects (447). by microglia and astrocytes in the context of diminished
neurotrophic support has a direct toxic impact on
A study using immunohistochemistry assessed oligodendroglia, consequently contributing to
microglia density in DLPFC, ACC, mediodorsal thalamus demyelination. Because oligodendroglia also play a role in
and hippocampus of depressed patients. The authors Glu uptake, their damage may add to excessive
suggest that significant microgliosis (i.e. increased number accumulation of this neurotoxic transmitter (455),
of microglia) in depressed patients who committed suicide (433),(456).
relative to healthy controls, might be a marker of pre-
suicidal stress. Proinflammatory mediators released from Proinflammatory cytokines (e.g. IL-1, IL-6, TNF-
microglia may have altered 5HT and NE transmission and alpha) and prostaglandins (e.g. PGE-2) synergistically
contributed to suicidality (448). induce indoleamine 2,3- dioxygenase (IDO), an enzyme
that converts tryptophan to kynurenine, thereby
In contradistinction to widespread glial diminishing its availability for 5-HT and melatonin
abnormalities, neuronal changes appear to be subtler and synthesis (56, 457). Proinflammatory cytokines, such as
more discrete in MDD. For example, some authors have IL-1and TNF-alpha, may also compromise serotonergic
noted decreased pyramidal somal size in hippocampus neurotransmission by increasing reuptake of the
(446), ACC (449), DLPFC (BA 9) and OPFC (BA 47) in neurotransmitter from the synaptic cleft (90). For example,
postmortem studies of MDD patients (440). The mRNA expression for these cytokines was associated with
distribution of this cellular pathology overlaps remarkably elevated 5HT transporter activity in MDD patients (458).
with findings from structural and functional imaging
studies. Si et al. investigated the influence of age and MDD In addition to reducing serotonin availability,
on packing density of glial fibrillary acedic protein IDO may contribute to depression directly as a result of the
(GFAP)-positive astrocytes in DLPFC. Individuals afflicted production of kynurenine and its downstream metabolites.
with MDD had a much steeper correlation between For example, the downstream metabolite quinolinic acid
reduction of GFAP levels and age relative to healthy (QUIN) is a potent NMDA agonist and stimulator of Glu
controls, suggesting a synergy between the aging process release. While the complete enzymatic pathway for QUIN
and disease state in reducing glia density in DLPFC (286). production is only present in microglia, activation of IDO
in astrocytes may aid in the conversion of tryptophan into
Microglia-Astroglia-Oligodendroglia-Neuron kynurenic acid (KA), a compound known to down-regulate
“units” may be conceptualized as neural microsystems that dopaminergic transmission and NMDA activity (56, 459-
interface with peripheral macrosystems, including 461). Whether KA is more likely to protect against
autonomic, immune and endocrine signals, providing an depression via its inhibitory effects on NMDA transmission
ongoing integration of these peripheral regulatory systems or to promote depression via its ability to down-regulate
with cerebral activity. We have previously described dopamine signaling is an important, and unanswered,
circumstances leading to neuroendocrine, autonomic and question.
neuroimmune dysregulation in MDD, with concomitant
elevations of proinflammatory cytokines, catecholamines Astrocytes have recently been found to be a
and circulating corticosteroids (sections 7.1-7.3). Peripheral source of GABA in the CNS and may be important
mediators of the inflammatory response propagate their modulators of GABA activity in hippocampus (462). In the
influence on the brain via several pathways, including 1.) context of MDD, a shift in the microglia/astroglia balance,
afferent neural fibers (i.e. vagal nerve); 2.) stimulation of favoring microglial activity, may lead to excessive Glu
immune cells in circumventricular organs (e.g. area transmission relative to GABA output (56, 459, 462); a
postrema, eminentia mediana, pineal gland), leading to dominance of T-helper-1 (Th-1) signaling (mostly
release of proinflammatory cytokines; and 3.) induction of proinflammatory) over Th-2 activity (predominantly
blood-brain barrier (BBB) cells (ependymal, endothelial associated with release of the anti-inflammatory cytokines
and choroid plexus cells), which respond by releasing IL-1, IL-4 and IL-10); and inadequate neurotrophic support for
IL-6, TNF-alpha, prostaglandins, NO and MCP-1. Limited oligodendroglia, contributing to demyelination (456, 459).
evidence suggests that transport of proinflammatory
cytokines across BBB may also be possible. (450),(451). Astrocytes contain serine racemase, an enzyme
responsible for the conversion of L-serine to D-serine
Microglia are the chief recipients of (463). Compromised astrocytic function in MDD may
inflammatory signals conveyed from the periphery. therefore lead to altered D-serine release. D-serine is an
Activated microglia respond by releasing additional endogenous ligand of the glycine receptor and therefore a
amounts of IL-1, IL-6, TNF-alpha, PGs, NO and H2O2, co-modulator of NMDA function and synaptic plasticity
which in turn induce astroglia to release more of these (463). Astroglial coverage of the synapse determines the
inflammatory mediators. This positive feedback loop extent of D-serine available to synaptic NMDA receptors.
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If astrocytic processes retract from the synapse, the amount of Multiple signaling pathways have a convergent
D-serine is reduced, resulting in long term depression of influence on neurotrophic factor synthesis. We have already
synaptic function (430). Conversely, excessive astrocytic suggested that altered microglia-astroglia-neuron
release of D-serine and consequent NMDA over-stimulation communication may lead to increased NO and prostaglandin
may be toxic to neurons (429). In support of this view, a recent production which may, in turn, have a negative impact on
study has reported that elevation of plasma L-serine and Glu in neurotrophic signaling. Preclinical data also demonstrate that a
depressed patients was directly associated with symptom loss of diurnal rhythm of corticosterone secretion impairs
severity, possibly hinting at astrocytic insufficiency of BDNF function (242). Altered astroglia function in MDD may
converting L-serine to D-serine (464) (Figure 4). have significant ramifications on neurotrophic signaling.
Stimulated astroglia release Glu almost exclusively into the
The disturbance in sympathetic/parasympathetic extrasynaptic space where it binds to extrasynaptic NMDA
balance associated with MDD may have significant receptors (430). Unlike activation of synaptic NMDA
repercussions on glia-neuron communication. Parasympathetic receptors, which promote BDNF release, activation of
activity has a stabilizing effect on microglia activity, while extrasynaptic NMDA receptors powerfully suppress BDNF
sympathetic input can play a dual role, either inhibiting or synthesis through a CREB-dependent mechanism (478). Glia-
activating microglia (439). In stressful situations it appears that derived neurotrophic factor (GDNF) is an important regulator
sympathetic discharge induces microglial release of of neuronal health and cognitive function. A recent study
proinflammatory cytokines (465). Consistent with this, a recent reported decreased serum levels of GDNF in depressed
study that reported increased nuclear factor- kappa B (NF- patients compared to healthy controls. Moreover, eight weeks
kappa B) activity in human volunteers after stress also of antidepressant treatment was associated with a significant
observed that NF-kappa B responses to stress in animals was elevation of serum GDNF in depressed patients (479). Juric et
obviated by alpha-1 receptor blockade (277). NF-kappa B al. demonstrated a significant increase in BDNF in astroglial
plays a pivotal role in catecholamine-induced synthesis of cultures treated with NE, DA and 5HT, establishing an
proinflammatory cytokines (466). Proinflammatory cytokines important relationship between monoaminergic neuronal
and glucocorticoids have opposing effects on NF-kappa-B activity and astroglial neurotrophic support (480).
regulation (208). Under usual circumstances glucocorticoids
have an immunosuppressant effect. MDD and sustained stress In summary, disturbed neuron-glia relationships
are accompanied by insufficiency of glucocorticoid signaling in MDD may result in diminished neurotrophic support
and glucocorticoid receptor (GR) resistance (42. 206). (BDNF and GDNF), altered energy supply and oxidative
Glucocorticoid resistance, in turn, may be associated with a regulation, massive releases of glutamate accompanied by
“permissive state”, leading to an augmented inflammatory compromised uptake, accumulation of ROS and RNS, all of
response (42, 206). However, in the context of aberrant stress which may jointly contribute to neurotoxicity (56, 452,
responses, glucocorticoids have also been reported to increase 481).
NMDA activation in microglia and neurons (467, 468).
Regardless, it appears that stress-MDD-HPA-sympathetic- 9.3. Neuron-Glia Interactions in chronic pain
inflammatory dysregulation may be a self-perpetuated As with MDD, alterations in neuron-glia
vicious cycle with the potential to negatively impact relationships may be of fundamental importance in the
glia-neuron signaling. etiopathogenesis of chronic pain conditions (482-484). In
the case of NeP, preclinical models are the primary source
Proinflammatory cytokines have been reported to of information about the cascade of interactions between
induce nitric oxide synthase (NOS), a pivotal enzyme spinal glia cells and neurons, and the role of these
regulating nitrogen oxide (NO) synthesis. NOS induction interactions in the development of NeP-associated
following exposure to inflammatory cytokines has been symptoms, such as allodynia and hyperalgesia. Microglia
reported in microglial, astrocytic and neuronal preparations have been reported to respond to a range of pathologic
(469-471). NMDA signaling is also associated with increased conditions conducive to development of NeP, such as
release of NO. A recent postmortem study reported elevated ischemia, infection and mechanical damage. These
NOS in hippocampal neurons of depressed patients (471). responses include morphological transformation,
Additionally, Xiong et al. reported a suppressant effect of NO proliferation and migration to the site of the injury (398). In
on BDNF synthesis (472). pathologic circumstances, a repetitive barrage of synaptic
firing by nociceptive A-delta and C-fibers induces
Neural injury caused by proinflammatory cytokines increased responsiveness of the dorsal horn pain-projecting
may in part be attributable to induction of cyclooxygenase neurons, a phenomenon also known as “central
(COX) and ensuing prostaglandin (PG) synthesis and release sensitization” (482). In this context, an intricate co-release
(473). Direct PG injection into rodent dorsal hippocampus was pattern of glutamate, SP, calcitonin gene-related peptide
sufficient to impair memory and reduce post-conditioning (CGRP), fractalkine, BDNF and ATP ensues (163, 330,
BDNF levels (473). Other consequences of COX-2 activation 482, 484). BDNF signaling, mediated by TrkB receptors,
include NMDA and QUIN mediated neurodegeneration (474, perpetuates the release of glutamate, CGRP and SP, and
475) and induction of NOS with ensuing accumulation of NO stimulates noradrenergic fiber sprouting following nerve
(476). Finally, preclinical studies suggest that age-related injury, thus adding to the development of pathologic pain
increases in hippocampal IL-1, TNF-alpha and PGE-2 can be (485, 486). Both astrocytes and microglia release D-serine
prevented by a selective COX-2 inhibitor (477).
5313
Figure 4. Microglia are the primary recipients of peripheral inflammatory signals as they reach the brain. Activated microglia
initiate an inflammatory cascade by releasing cytokines, chemokines, prostaglandins and reactive nitrogen and oxygen species
(RNS and ROS, respectively). Bi-directional exchanges between microglia and astroglia amplify inflammatory signals within the
central nervous system (CNS). Cytokines including interleukin (IL)-1, IL-6, tumor necrosis (TNF)-alpha and interferon (IFN)-
gamma induce indoleamine 2,3 dioxygenase (IDO), the enzyme responsible for degrading tryptophan, the primary precursor of
serotonin (5-HT), into kynurenine, which is eventually metabolized into quinolinic acid (QUIN), a potent NMDA agonist and
stimulator of glutamate (Glu) release. Multiple astrocytic functions are compromised due to the excessive exposure to cytokines,
prostaglandins, QUIN and RNS/ROS, ultimately leading to downregulation of glutamate transporters, impaired glutamate
reuptake, excessive glutamate release and compromised synthesis and release of neurotrophic factors. Oligodendroglia suffer
damage due to toxic overexposure to cytokines such as TNF-alpha, and diminished neurotrophic support, both of which promote
apoptosis and demyelination. Copious amounts of glutamate are released from astrocytes in the vicinity of extrasynaptic NMDA
receptors, whose activation leads to inhibition of BDNF synthesis. Excessive NMDA activation, caused by QUIN and D-serine,
is compounded by diminished glutamate reuptake by astrocytes and oligodendroglia. NMDA-mediated excitotoxicity, combined
with a consequent decline in neurotrophic support, and an increase in oxidative stress, synergistically disrupts neural plasticity
and induces apoptosis.
5314
in response to peripheral inflammation, further up- gap-junction propagation of calcium waves that will
regulating NMDA mediated transmission (487). Glutamate activate glia on the contra-lateral side, leading to a release
mediated NMDA receptor activation combined with of pain mediators in an otherwise unaffected area (323).
voltage-gated Ca 2+ currents (VGCCs) cumulatively alter New data suggest that glia may also interfere with opioid
intracellular signaling (482). The subsequent activation of analgesia in pathologic pain states (415). In response to
mitogen-activated protein kinase-1 (MAPK-1) pathways morphine, glia cells release neuroexcitatory substances that
(e.g. extracellular signal-regulated kinase [ERK], p38, c- oppose its analgesic effect (415). It appears that microglial
Jun N-terminal kinase [JNK]) and NF-kappa B pathways in activation is necessary for the initiation of pathological
spinal glia and neurons plays an important role in pain, while astroglia have a major role in propagation and
development of NeP (482-484). Neuronal ERK further maintenance of pain states (493, 494). Consistent with this
sensitizes AMPA and NMDA receptors, thus augmenting view, administration of minocycline (an inhibitor of
excitatory neurotransmission (482). Enhanced purinergic microglial activation) prevented the development of
transmission, mediated by ATP binding to P2X3 receptors, neuropathic pain, but failed to suppress acute pain or
neurokinin-1 (NK-1) receptor activation by SP, glutamate already entrenched pathologic pain (495, 496).
binding to mGlu receptors and BDNF release cumulatively Accumulating preclinical evidence suggests that disturbed
augment nociceptive transmission (482). Synaptic neuron-glia relationships in the context of NeP may extend
glutamate transporters, including glutamate transporter-1 to the supraspinal regions (497-499). For example, a sciatic
(GLT-1) and glutamate-aspartate transporter (GLAST) nerve ligation model of chronic pain produced a significant
(both principal regulators of synaptic glutamate) become astrocyte activation in the cingulate gyrus (497). Similarly,
dysregulated following prolonged and excessive exposure a recent report indicated that spinal cord injury induced
to glutamate (482). Under usual circumstances, GABA microglial activation and ensuing release of
released by inhibitory neurons modulates glutamatergic proinflammatory cytokines in remote locations, such as
transmission in the dorsal horn synapse (488). Following “below-level” spinal cord (i.e. below the level of injury)
peripheral nerve injury, ATP stimulates BDNF release via and the thalamus (500). Pain induced by peripheral
P2X4 receptors in microglia (488). Interestingly, microglia inflammation was associated with elevated BDNF in PAG
are not the only source of BDNF, as oligodendroglia and and subsequent NMDA-mediated descending pain
astrocytes have also been found to release neurotrophins facilitation, via RVM (rostral ventromedial medulla),
following spinal cord injury (489). BDNF signaling suggesting a potential role for the supraspinal
through Trk-B receptors reduces the levels of anion inflammation-neurotrophic factor-glutamate interactions in
transporter KCC2, ultimately resulting in increased persistent pain (498). In a separate study, the same group of
intracellular Cl – concentrations (488). In these pathological authors reported elevations of IL-1 and TNF-alpha in
circumstances, GABA activation paradoxically leads to Cl – RVM, resulting in excessive NMDA activation and
efflux, further depolarizing the dorsal horn neuron (488). allodynia following chronic constriction injury, a
Thus, BDNF has a role in perpetuating “central preclinical model of neuropathic pain. Injection of the
sensitization” by both facilitating excitatory transmission microglial and astroglial inhibitors abolished hyperalgesia
and by interfering with inhibitory regulation (488, 490). and allodynia, providing further proof of supraspinal
astrocyte and microglia involvement in perpetuating the
Activation of MAPK and NF-kappa-B in descending pain facilitation (499).
microglia and astrocytes ultimately leads to increased
synthesis and eventual release of proinflammatory factors, Evidence supporting the neuron-glia pathology in
such as IL-1, IL-6, IL-18, TNF-alpha, PGE-2 and NO (482- the genesis of FM is modest at best. Kim et al. conducted
484). Recent research suggests that the dorsal root ganglia an electron microscopic assessment of the skin biopsy
(DRG) nociceptive neurons also express IL-1, possibly samples from fibromyalgia patients and compared them
contributing to the inflammatory response seen in with healthy controls (165). In 9/13 of FM patients
pathologic pain states (491). Release of proinflammatory unmyelinated Schwann cells were “ballooned”, whereas
mediators leads to a self-perpetuating activation of glia none of the control samples displayed this type of
cells and excessive stimulation of dorsal horn neurons, pathology. Also, in most of the FM patients, axons were
producing sensory abnormalities typically associated with localized in the periphery of the unmyelinated Schwann
nerve injury (483, 484). Additionally, proinflammatory cell sheets (165). This preliminary finding suggests that
mediators may directly impact axons or become transported altered glia morphology and function may play a role in
in a retrograde fashion to the cell bodies in dorsal root FM. Despite the paucity of hard evidence, some authors
ganglia, where they alter gene expression in the primary have posited that dysregulated neuron-glia relationships
nociceptive neurons, producing “neurogenic inflammation” may play a role in FM, based on similarities in biochemical
and contributing to “peripheral sensitization” (163, 168, markers between NeP, other chronic pain states and FM
492). (60, 420). In summary, multiple lines of evidence implicate
disturbances in neuron-glia relationships with concomitant
Altered neuron-glia interactions have recently disruption in the regulation of glutamatergic, neurotrophic
been implicated in the genesis of “mirror pain”, whereby and inflammatory signaling as principal mediators of
pain is propagated to the side contra-lateral to the site of chronic pain states, including NeP, and perhaps FM. Some
pathology (323). Hypothetically, robust glia activation on authors have even characterized chronic pain as a
one side, due to pathological pain processes, can trigger “gliopathy” (501).
5315
10. CONCLUSION all associated with altered sympathetic/parasympathetic

balance, neuroendocrine disturbances, characterized by
Multiple lines of evidence suggest that the co- insufficient HPA regulation and altered immune function.
occurrence of depression and pain is more the rule than the In turn, these peripheral responses signal back to the neural
exception. For example, recent studies demonstrate that 30- structures to further drive the CNS danger pathway
60% of depressed patients also suffer from a painful activation, leading to a maladaptive feedforward circuit,
condition (1), and at least an equal percentage of people which increasingly appears to be implicated in the
with chronic pain also meet criteria for a mood disorder production and maintenance of symptoms.
diagnosis. (502-506). Of course, pain is one of many
syndromes/symptoms that co-occur regularly with MDD. Microglia seem to be the principal recipients of
For example, there is also a considerable overlap between bodily distress/pain signals. Differences in the micro-
MDD and anxiety disorders (507, 508). Some authors have environments in the dorsal column of the spinal cord and
gone so far as to suggest that they are dual manifestations brain areas involved in processing of pain in MDD, NeP
of the same underlying patho-physiological condition (354, and FM may be reflected in the different patterns of
509). Manchikanti et al. even reported a “dose-response” interaction between microglia, astroglia and neurons, in
relationship amongst pain, and depression and anxiety. In these respective conditions. Excessive firing of nociceptive
this study, 14% of the control group experienced GAD and neurons in the context of NeP and FM tends to be
4% experienced MDD. Among the patients who associated with increased release of substance-P, IL-8
experienced chronic pain involving one bodily region, the (which promotes sympathetic pain), IL-18, fractalkine and
prevalence of GAD and MDD was 30% and 20%, CGRP, in addition to chemokines produced by macrophage
respectively. If patients experienced pain in two or more and mononuclear cells (324, 482-484, 492), none of which
regions, the prevalence of GAD and MDD rose to 54% and are part of the usual spinal cord cellular milieu in MDD.
32% (504). Given this overlap, it is no surprise that the Nonetheless, excessive excitatory glutamatergic
presence of pain is a major predictor of depression and transmission and compromised GABA mediated inhibition
anxiety. appear to be common features of depression and pain
disorders. Dysregulation in monoamine, substance P,
It is becoming increasingly clear that galanin and opiate signaling also characterizes both pain
relationships between MDD, anxiety and pain run even syndromes and MDD. On the other hand, depression and
deeper than the surface similarities shared by these pain disorders have been reported to have different
conditions. For example, although not completely patterns of abnormality in the production of
consistent, studies point to a shared genetic underpinning neurotrophic factors. MDD is characterized by the
for these disorders, especially in relation to genes involved reduction of the serum BDNF levels, while serum
in the regulation of monoaminergic and peptide BDNF levels tend to be increased in FM. . Nonetheless,
transmission, inflammatory response, diurnal rhythm and pain, stress and depression have a similar, if not
neurotrophic signaling. All of these are important synergistic, impact on neurotrophic signaling in
modulators of pain, emotional tone and the stress response. hippocampus, with all three conditions being associated
Stress, in turn, is a major precipitant, perpetuant and an with reduced BDNF synthesis. This finding is of
aggravating factor in all three conditions. Consistent with particular interest, given that hippocampus represents a
this, brain circuitry involved in the regulation of mood and veritable “intersection” of pathways involved in
the stress response overlaps to a significant degree with emotional regulation, memory and coordination of the
components of the “pain matrix”, involved in emotional stress response.
and cognitive aspects of pain processing.
MDD, NeP and FM are all associated with
From an evolutionary perspective, it is apparent neuroplastic changes in the CNS. In pathologic pain states,
that both negative emotions and physical pain have a facilitation of pain signaling, presumably based on
tremendous survival value, given that both provide a clear neuroplastic changes in pain pathways, is often designated
signal that current conditions are a threat to an organism’s as the “central sensitization”. Similarly, the recurrent and
goals and/or survival. Thus, it is not surprising, that, both most likely progressive nature of MDD is often ascribed to
peripheral and CNS depression and pain pathways overlap “kindling”, which—like central sensitization—reflects
significantly, nor that these pathways are built into the warp neuroplastic changes. Given this, MDD, NeP and FM may
and woof of the mammalian stress and immune response all be characterized by adaptive processes gone awry as a
systems. While important differences exist in the result of complex interactions between genetic
processing of physical pain, emotional pain and stress vulnerabilities and environmental factors. In this scenario,
responses at the level of pain sensory areas (e.g. thalamus, persistent aberrant processing of emotional, painful and
SI and SII), striking similarities are apparent in the stressful signals eventually becomes “ossified”, presumably
involvement of limbic and paralimbic prefrontal cortical due to ensuing neuroplastic changes. In some regards,
areas (amygdala, hippocampus, insula, ACC, vmPFC), as depression, FM and NeP share dysfunctional
well as more “cognitive” and integrative brain areas, such psychosomatic and somatopsychic communication. It
as rACC, dACC dmPFC and DLPFC. Moreover, pain and should be no surprise, then, that overlapping pathology
depressed mood appear to have an overlapping capacity to gives rise to similar phenomenological manifestations. If
engage autonomic, neuroendocrine and neuroimmune we assume that shared biological underpinnings give rise to
components of the stress response. MDD, FM and NeP are the clinical symptoms of MDD, NeP and FM, it is clear that
5316
a full understanding of this “synergy” has critical treatment 12. Offenbaecher M and M. Ackenheil: Current trends in
implications. neuropathic pain treatments with special reference to
fibromyalgia. CNS Spectr, 10(4), 285-97 (2005)
11. ACKNOWLEDGEMENTS
13. Martinez-Lavin M, S. Lopez, M. Medina and A. Nava:
The authors would like to thank Eli Lilly and Use of the leeds assessment of neuropathic symptoms and
Company, Mathew Iype and Christopher Wikoff of signs questionnaire in patients with fibromyalgia. Semin
Indegene, for financial and technical assistance in Arthritis Rheum, 32(6), 407-11 (2003)
preparation of illustrations. We would also like to
acknowledge Eli Lilly and Company for assistance with 14. Torrance N, B. H. Smith, M. I. Bennett and A. J. Lee:
publication costs. The epidemiology of chronic pain of predominantly
neuropathic origin. Results from a general population
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Key Words: Neurobiology, Depression, Neuropathic Pain,

Fibromyalgia, Hypothalamic-Pituitary-Adrenal Axis,
Inflammation, Glia, Neurotrophic Factors, Review
Send correspondence to: Vladimir Maletic, 38 Parkway

Commons Way, Greer, SC 29650, Tel: 864-848-4448, Fax:
864-848-4428, E-mail: vmaletic@bellsouth.net
http://www.bioscience.org/current/vol14.htm
5338
Nutritional Support
for Wound Healing
Douglas MacKay, ND,
and Alan L. Miller, ND
Abstract
Introduction
Healing of wounds, whether from accidental
Wound healing involves a complex series
injury or surgical intervention, involves the
of interactions between different cell types,
activity of an intricate network of blood cells,
cytokine mediators, and the extracellular matrix.
tissue types, cytokines, and growth factors.
The phases of normal wound healing include he-
This results in increased cellular activity, which
mostasis, inflammation, proliferation, and remod-
causes an intensified metabolic demand for
eling. Each phase of wound healing is distinct,
nutrients. Nutritional deficiencies can impede
although the wound healing process is continu-
wound healing, and several nutritional factors
ous, with each phase overlapping the next. Be-
required for wound repair may improve healing
cause successful wound healing requires adequate
time and wound outcome. Vitamin A is required
blood and nutrients to be supplied to the site of
for epithelial and bone formation, cellular
damage, the overall health and nutritional status
differentiation, and immune function. Vitamin
of the patient influences the outcome of the dam-
C is necessary for collagen formation, proper
aged tissue. Some wound care experts advocate a
immune function, and as a tissue antioxidant.
holistic approach for wound patients that consid-
Vitamin E is the major lipid-soluble antioxidant
ers coexisting physical and psychological factors,
in the skin; however, the effect of vitamin E on
including nutritional status and disease states such
surgical wounds is inconclusive. Bromelain
as diabetes, cancer, and arthritis. Keast and Orsted1
reduces edema, bruising, pain, and healing
wittily state, “Best practice requires the assess-
ment of the whole patient, not just the hole in the
Glucosamine appears to be the rate-limiting
patient. All possible contributing factors must be
substrate for hyaluronic acid production in the
explored.”
wound. Adequate dietary protein is absolutely
Wound repair must occur in a physiologic
essential for proper wound healing, and tissue
environment conducive to tissue repair and regen-
levels of the amino acids arginine and
eration. However, several clinically significant
glutamine may influence wound repair and
factors are known to impede wound healing, in-
immune function. The botanical medicines
cluding hypoxia, infection, tumors, metabolic dis-
Centella asiatica and Aloe vera have been used
orders such as diabetes mellitus, the presence of
for decades, both topically and internally, to
debris and necrotic tissue, certain medications, and
enhance wound repair, and scientific studies
are now beginning to validate efficacy and
explore mechanisms of action for these Douglas J. MacKay, ND – Technical Advisor, Thorne
botanicals. To promote wound healing in the Research, Inc; Senior Editor, Alternative Medicine Review;
private practice, Sandpoint, ID.
shortest time possible, with minimal pain, Correspondence address: Thorne Research, PO Box 25,
discomfort, and scarring to the patient, it is Dover, ID 83825 E-mail: duffy@thorne.com
important to explore nutritional and botanical Alan L. Miller, ND – Technical Advisor, Thorne Research,
influences on wound outcome. Inc; Senior Editor, Alternative Medicine Review.
Correspondence address: Thorne Research, PO Box 25,
(Altern Med Rev 2003;8(4):359-377) Dover, ID 83825 E-mail: alanm@thorne.com

a diet deficient in protein, vitamins, or minerals. tissue. The neutrophils engulf debris and
In addition, increased metabolic demands are microorganisms, providing the first line of defense
made by the inflammation and cellular activity in against infection. Neutrophil migration ceases
the healing wound, which may require increased after the first few days post-injury if the wound is
protein or amino acids, vitamins, and minerals.2 not contaminated. If this acute inflammatory phase
The objective in wound management is persists, due to wound hypoxia, infection,
to heal the wound in the shortest time possible, nutritional deficiencies, medication use, or other
with minimal pain, discomfort, and scarring to the factors related to the patient’s immune response,
patient. At the site of wound closure a flexible and it can interfere with the late inflammatory phase.3
fine scar with high tensile strength is desired. In the late inflammatory phase, monocytes
Understanding the healing process and nutritional converted in the tissue to macrophages, which di-
influences on wound outcome is critical to suc- gest and kill bacterial pathogens, scavenge tissue
cessful management of wound patients. Research- debris and destroy remaining neutrophils. Mac-
ers who have explored the complex dynamics of rophages begin the transition from wound inflam-
tissue repair have identified several nutritional mation to wound repair by secreting a variety of
cofactors involved in tissue regeneration, includ- chemotactic and growth factors that stimulate cell
ing vitamins A, C, and E, zinc, arginine, glutamine, migration, proliferation, and formation of the tis-
and glucosamine. Botanical extracts from Aloe sue matrix.
vera, Centella asiatica, and the enzyme brome- The subsequent proliferative phase is
lain from pineapple have also been shown to im- dominated by the formation of granulation tissue
prove healing time and wound outcome. Eclectic and epithelialization. Its duration is dependent on
therapies, including topical application of honey, the size of the wound. Chemotactic and growth
sugar, sugar paste, or Calendula succus to open factors released from platelets and macrophages
wounds, and comfrey poultices and hydrotherapy stimulate the migration and activation of wound
to closed wounds are still in use today. Although fibroblasts that produce a variety of substances
anecdotal reports support the efficacy of these essential to wound repair, including glycosami-
eclectic therapies, scientific evidence is lacking. noglycans (mainly hyaluronic acid, chondroitin-
4-sulfate, dermatan sulfate, and heparan sulfate)
The Four Phases of Wound Healing and collagen.2 These form an amorphous, gel-like
Tissue injury initiates a response that first connective tissue matrix necessary for cell migra-
clears the wound of devitalized tissue and foreign tion.
material, setting the stage for subsequent tissue New capillary growth must accompany
healing and regeneration. The initial vascular re- the advancing fibroblasts into the wound to pro-
sponse involves a brief and transient period of vide metabolic needs. Collagen synthesis and
vasoconstriction and hemostasis. A 5-10 minute cross-linkage is responsible for vascular integrity
period of intense vasoconstriction is followed by and strength of new capillary beds. Improper
active vasodilation accompanied by an increase cross-linkage of collagen fibers has been respon-
in capillary permeability. Platelets aggregated sible for nonspecific post-operative bleeding in
within a fibrin clot secrete a variety of growth fac- patients with normal coagulation parameters.4
tors and cytokines that set the stage for an orderly Early in the proliferation phase fibroblast activity
series of events leading to tissue repair. is limited to cellular replication and migration.
The second phase of wound healing, the Around the third day after wounding the growing
inflammatory phase, presents itself as erythema, mass of fibroblast cells begin to synthesize and
swelling, and warmth, and is often associated with secrete measurable amounts of collagen. Collagen
pain. The inflammatory response increases levels rise continually for approximately three
vascular permeability, resulting in migration of weeks. The amount of collagen secreted during
neutrophils and monocytes into the surrounding this period determines the tensile strength of the
wound.

Figure 1. Nutrient Impacts on the Phases of Wound Healing
Wounding
Calendula succus – topical antimicrobial
Hemostasis
Drugs, herbs, vitamins, amino acids, or minerals that effect blood-clotting

mechanisms should be avoided prior to surgery.
Inflammatory Phase
Vitamin A – enhances early inflammatory phase
Bromelain and adequate protein intake – prevent prolonging inflammatory phase
Vitamin C – enhances neutrophil migration and lymphocyte transformation
Proliferative Phase
Vitamin C – necessary for collagen synthesis
Centella asiatica – promotes type-1 collagen synthesis
Glucosamine – enhances hyaluronic acid production
Vitamin A – promotes epithelial cell differentiation
Zinc – required for DNA synthesis, cell division, and protein synthesis
Calendula succus and Aloe vera – support formation of granulation tissue
Remodeling
Protein deficiency – inhibits wound remodeling
The final phase of wound healing is Figure 1 summarizes the phases of wound
wound remodeling, including a reorganization of healing and nutrients that impact the various
new collagen fibers, forming a more organized lat- phases.
tice structure that progressively continues to in-
crease wound tensile strength. The remodeling
process continues up to two years, achieving 40-
70 percent of the strength of undamaged tissue at
four weeks.2

Vitamins and Minerals Essential to with fractures, tendon damage, or vitamin A defi-
ciency may also benefit from perioperative vita-
Wound Healing min A supplementation. Additional research is
Vitamin A necessary to establish the effectiveness of univer-
Vitamin A is required for epithelial and sal perioperative vitamin A supplementation in
bone tissue development, cellular differentiation, healthy individuals.
and immune system function. Substantial evidence Concern among some practitioners re-
supports the use of vitamin A as a perioperative garding the potential toxicity of higher doses of
nutritional supplement.5 In addition to facilitating vitamin A has led to uneasiness about using it
normal physiological wound repair, Ehrlich and perioperatively. The vast majority of toxicity cases
Hunt have shown vitamin A reverses the cortico- have occurred at daily vitamin A dosages of
steroid-induced inhibition of cutaneous and fas- 50,000-100,000 IU in adults over a period of
cial wound healing.6-8 Vitamin A has also corrected weeks to years.15 Short-term supplementation of
non-steroid induced, post-operative immune de- 25,000 IU daily appears to be safe for most non-
pression9 and improved survival in surgically-in- pregnant adults. Caution must be exercised in
duced abdominal sepsis.10 Levenson et al suggest supplementing vitamin A in patients for whom the
vitamin A benefits the wound by enhancing the anti-inflammatory effect of steroids is essential,
early inflammatory phase, including increasing the such as in rheumatoid arthritis or organ transplants,
number of monocytes and macrophages at the as well as in pregnant women and women of child-
wound site, modulating collagenase activity, sup- bearing age.5
porting epithelial cell differentiation, and improv-
ing localization and stimulation of the immune Vitamin C
response.10,11 Ascorbic acid is an essential cofactor for
Animal studies show vitamin A may in- the synthesis of collagen, proteoglycans, and other
crease both collagen cross-linkage and wound- organic components of the intracellular matrix of
breaking strength. Greenwald et al inflicted sur- tissues such as bones, skin, capillary walls, and
gical flexor profundus damage and immediate re- other connective tissues. Ascorbic acid deficiency
pair on adult chickens. They found chickens that causes abnormal collagen fibers and alterations
ate a diet supplemented with vitamin A (150,000 of the intracellular matrix that manifests as cuta-
IU/kg chicken chow) demonstrated wound-break- neous lesions, poor adhesion of endothelium cells,
ing strength more than double that of controls fed and decreased tensile strength of fibrous tissue.16
standard chicken chow.12 In addition, rats with Clinical manifestations of ascorbic acid deficiency
dorsal skin incisions and concurrent comminuted include bleeding gums, poor immunity, easy bruis-
femoral fractures exhibited delayed cutaneous ing and bleeding, and slow healing of wounds and
healing. Supplemental vitamin A enhanced wound fractures.17 Ascorbic acid is necessary for the hy-
healing in these animals, demonstrated by in- droxylation of proline and lysine residues in
creased breaking strength of the dorsal skin inci- procollagen, which is necessary for its release and
sions in rats fed supplemental vitamin A compared subsequent conversion to collagen. Hydroxypro-
to the non-supplemented group. The authors be- line also stabilizes the collagen triple-helix struc-
lieve the improved wound healing is a result of an ture.18 In addition to collagen production, ascor-
increased rate of collagen cross-linkage.13 bic acid enhances neutrophil function,19 increases
Levenson and Demetrio recommend vi- angiogenesis,20 and functions as a powerful anti-
tamin A supplementation of 25,000 IU daily be- oxidant.21
fore and after elective surgery.14 Research supports Although ascorbic acid is required for
perioperative vitamin A supplementation in pa- reparation of damaged tissue, researchers have
tients known to be immune depleted or steroid demonstrated the benefit of vitamin C only in vi-
treated. Surgical patients with sepsis and those tamin C-deficient individuals using low doses of

ascorbic acid.22 In a study by Hodges et al, four Events leading to wounds, including trauma and
subjects (ages 33-44) were depleted of vitamin C surgery, are perceived as physiological stressors
for 99 days to induce scurvy. On day 100, a 5-cm that have also been correlated with a decrease in
incision was made in the left thigh of each subject plasma ascorbic acid.34,35 Thus, the acute stress
and they began the oral administration of 4, 8, 16, experienced by trauma or surgery patients may
or 32 mg ascorbic acid daily. Healing was mea- unmask marginal vitamin C deficiencies, leading
sured by histological and electron microscope to deficiency symptoms.
technique. It was shown that 4 mg daily of vita- Cutaneous healing wounds have been
min C was just as effective as 32 mg daily for found to have lower ascorbic acid content than
wound healing in these vitamin C-deficient sub- intact tissue. Levels of vitamin C were compared
jects.22 The efficacy of using vitamin C to improve to normal skin in two-, four-, seven-, and 14-day-
wound healing in non-deficient individuals re- old wounds in animals. Vitamin C levels decreased
mains uncertain. It should be noted, however, that approximately 60 percent post-wound and had not
even the highest dose in this study (32 mg) is be- exhibited full recovery by day 14.36 In addition,
low the RDA for vitamin C. Higher doses and low levels of antioxidants, including ascorbic acid,
larger differences between doses might have accompanied by elevated levels of markers of free
yielded more significant differences. radical damage have been detected in elderly rat
Humans lack the ability to store vitamin cutaneous wounds exhibiting delayed healing.
C, and certain populations are more likely to be Eighteen-month-old wounded male rats were com-
deficient in ascorbic acid, including the elderly, pared to 3-4 month-old rats pre-wound and seven
alcoholics, drug abusers, and under-nourished in- days post-wound. Normal skin of aged and young
dividuals.23 Subclinical vitamin C deficiency is rats showed no difference in ascorbic acid con-
being recognized increasingly in the general popu- tent; however, a 59-percent decrease in ascorbic
lation. Published cases show that restricted eating acid content was observed in wound tissues of
patterns, prolonged hospitalization, severe ill- aged animals compared to its content in young
nesses, and poor dietary intake in both children adult wounds.37 Rasik and Shukla propose the de-
and adults cause deficiency with significant clini- lay in wound healing of older rats is at least par-
cal consequences.4,24-26 In one study 12 patients tially a result of increased free radical damage.37
with post-surgical diffuse hemorrhage, each ex- The programmed sequences of the cellu-
hibiting normal coagulation parameters, were lar and molecular processes occurring during
found to have low plasma ascorbic acid levels. wound repair are also dependent on immune func-
Each patient received 250-1,000 mg oral vitamin tion. Infection resulting from impaired immunity
C daily. Within 24 hours of vitamin C administra- is one of the most commonly encountered and
tion there was no further evidence of bleeding or clinically significant impediments to wound heal-
need for subsequent blood transfusions in any pa- ing. 3 In addition, cellular immunity and
tient. The authors concluded vitamin C deficiency dysregulation of cytokines can impair wound heal-
should be included in the differential diagnosis for ing.38 Ascorbic acid has been shown to improve
nonspecific bleeding in surgical patients.4 immune function in humans.39-42 Human volun-
In mammals, ascorbic acid is necessary teers who ingested 2-3 g ascorbate daily for sev-
for a normal response to physiological stressors, eral weeks exhibited enhanced neutrophil motil-
with the need for ascorbic acid increasing during ity to chemotactic stimulus and stimulation of lym-
times of injury or stress.27 Studies have shown the phocyte transformation.43 Neutrophil motility and
physiological stress of intense exercise generates lymphocyte transformation were also stimulated
excess reactive oxygen species (ROS), increasing by 1 g intravenous ascorbic acid in six healthy
the demand on the antioxidant defense system.28- volunteers. Alterations in these activities were re-
30
A similar elevation of ROS has been noted within lated to serum ascorbic acid levels.
wounds; therefore, substances that increase tissue
antioxidants are thought to benefit healing.31-33

The combined effect of ascorbic acid on Perioperative zinc supplementation is re-

collagen synthesis, antioxidant status, and commended for zinc-depleted patients.23 Data is
immunomodulation make it an appropriate supple- lacking to show zinc supplementation improves
ment for wound repair protocols. Research pro- healing in non-deficient individuals; however, zinc
vides evidence for the use of low doses of vitamin deficiency in humans is widespread, and injured
C in vitamin C-deficient individuals, but many and stressed individuals are more prone to devel-
practitioners believe larger doses of ascorbic acid oping deficiencies. Ehrlich et al suggest zinc is
in non-deficient individuals are indicated for op- lost in significant amounts after surgery because
timal wound repair. Levenson and Demetriou re- of fistulas, stress, and diarrhea.50 Zinc deficien-
commend supplementing 1-2 g ascorbic acid daily cies have also been identified in individuals with
from wound onset until healing is complete.14 Such deep partial- or full-thickness burns and chronic
doses may be justified due to the lack of adverse venous leg ulceration.51,52
effects at these levels44 combined with the poten- Further research is needed on the efficacy
tial for deficiency in certain individuals. In addi- of zinc supplements for wound healing. Justifica-
tion, the transient increase in metabolic require- tion for perioperative zinc supplementation in-
ments for vitamin C resulting from the physiologic cludes the absence of adverse effects at moderate
stress of trauma or surgery and the metabolic re- doses (15-30 mg daily) and evidence that zinc
quirement of vitamin C for collagen synthesis are deficiency impairs wound healing. Zinc supple-
indications for higher doses of vitamin C in non- mentation of 15-30 mg daily is recommended
deficient individuals. perioperatively to prevent unmasking of marginal
deficiencies. Higher levels of zinc supplementa-
Zinc tion may be necessary in patients with malnutri-
Approximately 300 enzymes require zinc tion, malabsorption, chronic diarrhea, or other risk
for their activities. Zinc is an essential trace min- factors of zinc deficiency.
eral for DNA synthesis, cell division, and protein
synthesis,45 all necessary processes for tissue re- Vitamin E
generation and repair. Zinc deficiency has been Vitamin E is popular among consumers
associated with poor wound healing and decreased for skin care and to prevent scar formation. It func-
breaking strength of animal wounds,46 which can tions as the major lipophilic antioxidant, prevent-
result from decreased protein and collagen syn- ing peroxidation of lipids and resulting in more
thesis during healing found in zinc-deficient ani- stable cell membranes. The antioxidant-membrane
mals.47 Senapati and Thompson found zinc levels stabilizing effect of vitamin E also includes stabi-
were 50-percent higher in muscle and skin from lization of the lysomal membrane, a function
abdominal wounds of rats during wound healing, shared by glucocorticoids.53 Systemic vitamin E
but mild deficiency reduced this accumulation.48 and glucocorticoids inhibit the inflammatory re-
Zinc demands are thought to be the high- sponse and collagen synthesis, thereby possibly
est from time of wounding throughout the early impeding the healing process. The effect of vita-
inflammatory phase. Sequential changes in zinc min E on wound healing is complex; it may have
concentrations were studied in the incisional alternate effects in different types of wounds and
wound model in the rat. Zinc levels increased from in the presence of other nutrients, as well as dif-
wounding and peaked on the fifth day – at a time ferent functions for water soluble versus lipid
of high inflammation, granulation tissue forma- soluble preparations of vitamin E.
tion, and epidermal cell proliferation.49 Zinc con- Animal studies of vitamin E supplemen-
centrations returned to normal by the seventh day, tation on surgical wounds show conflicting results.
when inflammation had regressed. It has been sug- Greenwald et al showed flexor tendon repair in
gested that increased local demand for zinc result- chickens treated with vitamin E had breaking
ing from surgery and wounding exposes otherwise strength less than half that of controls measured
marginal zinc deficiencies in humans.48

after days 7 and 45 from surgical repair.12 Another time critical to interpretation of this study. It was
animal study showed impaired collagen synthesis also noted that breakdown products and contami-
in rats treated with vitamin E after wounding.54 nants could account for the inflammatory response
The researchers cite the glucocorticoid-like effect encountered.59 In a second, larger blinded study,
of vitamin E as the cause of the negative results. the effects of topical steroids, vitamin E, or the
However, these effects are mitigated by vitamin base cream carrier for these substances on scar
A, as vitamin A is a lysomal destabilizer that re- outcome of 159 post-operative patients were
verses several of the deleterious effects of gluco- evaluated. Both topical steroids and topical vita-
corticoids.8 min E failed to impact scar thickness, range of
Paradoxical results found by Galeano et motion, or ultimate cosmetic appearance.60
al showed a hydrophilic vitamin E preparation The available data on vitamin E and
positively impacted delayed wound healing in dia- wound healing could lead to several possible con-
betic mice. Increased breaking strength and col- clusions: (1) systemic vitamin E may have a nega-
lagen content of the wound was found in treated tive impact on surgical wounds due to its lysoso-
animals. These authors speculate inhibition of lipid mal-stabilizing properties; (2) vitamin A may miti-
peroxidation accounted for the positive results.55 gate these negative effects; and (3) hydrophilic and
In addition, prophylactic administration of vita- hydrophobic preparations of vitamin E may have
min E has been shown to increase breaking different actions related to wounds. The benefit
strength and normalize healing of wounds exposed of topical vitamin E on surgical wound healing
to preoperative irradiation56 and to decrease the and scar formation remains inconclusive and, al-
development of intraperitoneal adhesions in ani- though anecdotal reports support topical use of
mals.57 vitamin E for scar therapy, research shows it may
Since the discovery of vitamin E as the have a negative effect on scarring and wound out-
major lipid-soluble antioxidant in skin, it has been come.
used topically for a wide variety of skin lesions.
Anecdotal reports claim topical vitamin E is valu- Other Dietary Supplements and
able for speeding wound healing and improving
cosmetic outcome of burns and other wounds, in-
Wound Healing
cluding surgical scars. Such claims are disputed Bromelain
by two human clinical trials. In a double-blind Bromelain is a general name given to a
study of 15 patients with surgically-induced family of proteolytic enzymes derived from
wounds, emollient lotion and emollient lotion Ananas comosus, the pineapple plant. Through-
mixed with vitamin E were applied to healing out the 1960s and 1970s a series of studies found
wounds. The wounds were randomly divided into the effects of orally administered bromelain in-
two parts and the different topical applications clude the reduction of edema, bruising, pain, and
were applied to the same half of each wound twice healing time following trauma and surgical pro-
daily. Physicians and patients independently evalu- cedures.61-64 More recently, researchers from the
ated the scars for cosmetic appearance on weeks Czech Republic found that patients with long bone
1, 4, and 12. In 90 percent of cases, topical vita- fractures administered a proteolytic enzyme com-
min E either had no effect, or actually worsened bination containing 90 mg bromelain per tablet
the cosmetic appearance of scars.58 In addition, had less post-operative swelling compared to pa-
33 percent of the patients studied developed con- tients given placebo.65 Fractures were treated by
tact dermatitis to topical vitamin E. A response to surgically inserting rods through the long axis of
this study, published in Dermatologic Surgery, the fractured bone (intramedullary fixation) or by
pointed out that d-alpha tocopherol is an extremely constructing an external framework of pins and
unstable compound, rendering details of its source, rods going through the skin and muscle to con-
formulation, storage condition, and stability over nect to the fractured bone (external fixators). The
treatment group was given three 90-mg tablets

three times daily for three days after surgery and, Tassman et al noted that, while post-sur-
subsequently, two tablets three times daily for two gical oral bromelain administration was effective
weeks. On the fourteenth post-operative day the in reducing pain, swelling, and healing time, a
limb volume of the treatment group was reduced protocol using pre- and post-surgical bromelain
by 17 percent compared with nine percent in the is recommended.63 Studies have shown bromelain
control group. The total number of analgesics con- prevents aggregation of blood platelets in patients
sumed by the treatment group was also signifi- with high platelet aggregation values, which has
cantly reduced in comparison to the control led to recommendations by physicians and sur-
group.65 geons to avoid oral bromelain prior to any surgi-
Studies by Tassman et al show bromelain cal procedure. In one human trial, bromelain was
reduced swelling, bruising, pain, and healing time administered orally to 20 volunteers with a his-
in patients following dental surgeries.63,66 In a tory of heart attack or stroke, or with high platelet
double-blind study of dental surgery patients, bro- aggregation values. Bromelain decreased platelet
melain was found to decrease swelling to 3.8 days, aggregation in 17 of the subjects and normalized
compared with seven days in patients given pla- values in eight of the nine subjects who previously
cebo. In addition, duration of pain was reduced to had high aggregation values.72 Contrary to this,
five days in the treatment group, compared to eight other human studies have shown oral bromelain
days in the placebo group.63 to be free of any significant effects on clotting
In an uncontrolled trial, bromelain was parameters.73,74 In one study, 47 patients with vari-
reported to positively influence swelling, pain at ous disorders leading to edema and inflammation
rest and during movement, and tenderness in pa- found no significant effects of oral bromelain (40
tients with blunt injuries to the musculoskeletal mg four times daily for one week) on bleeding,
system.67 Although bromelain has been shown to coagulation, and prothrombin time.
reduce post-operative and trauma-related pain, this It is noteworthy that the studies pertain-
is probably related to its anti-inflammatory action ing to bromelain and platelet aggregation are over
rather than a direct analgesic effect.68 30 years old. The potential benefit of pre- and post-
Aside from its documented anti-inflam- surgical oral bromelain on hematoma resorption,
matory activity, bromelain is of interest to surgeons pain, inflammation, and healing time justifies the
because of its ability to increase resorption rate of need for concise, well-designed clinical trials
hematomas. Bromelain’s influence on hematoma evaluating different doses of bromelain on clot-
resorption was demonstrated using artificially in- ting parameters. Until further data is available re-
duced hematomas in humans. Hematomas in the garding bromelain’s action on platelets, oral bro-
treatment group resolved significantly faster than melain administration should be withheld or used
controls when oral bromelain was given at the time with caution before surgery.
of hematoma induction and for seven days there-
after.69 Glucosamine
Seltzer investigated two different doses of Hyaluronic acid is an important part of
bromelain in patients undergoing rhinoplasty. the extracellular matrix and one of the main gly-
Fifty-three patients were randomized to receive cosaminoglycans secreted during tissue repair.
either one of two doses of bromelain or placebo. Production of hyaluronic acid by fibroblasts dur-
In patients receiving placebo, swelling and ecchy- ing the proliferative stage of wound healing stimu-
mosis persisted for seven days, compared to two lates the migration and mitosis of fibroblasts and
days in both bromelain groups.70 However, a ran- epithelial cells. Glucosamine appears to be the
domized trial of 154 facial plastic surgery patients rate-limiting substrate for hyaluronic acid synthe-
receiving either 400 mg bromelain daily or pla- sis.75 In vitro studies suggest the mechanism of
cebo for one day before and four days after sur- glucosamine on repair processes involves stimu-
gery found no statistically significant differences lation of the synthesis of glycosaminoglycans and
in edema between the two groups.71

Table 1. Perioperative Nutritional Protocol
Nutrient Dose Action
Vitamin A** 25,000 IU daily Enhances early inflammatory

phase of wound healing;
supports epithelial cell
differentiation;
improves localization and
stimulation of immune response.
Vitamin C** 1-2 g daily Synthesis of collagen,

proteoglycans, and other
organic components of the
intracellular matrix;
tissue antioxidant;
supports immune response.
Zinc** 15-30 mg daily Required for DNA synthesis, cell

division, and protein synthesis.
Glucosamine** 1,500 mg daily Enhances hyaluronic acid

production in the wound.
Protein** Minimum of 0.8 g/kg body Prevents delayed healing and

weight daily surgical complications.
Bromelain (use 500-1,000 mg daily Reduces edema, bruising, pain,

post-surgery only) and healing time.
** Use from two weeks prior to surgery until healing is complete
collagen.76 Animal studies have shown the con- well as the first few days after surgery or trauma
tent of glycosaminoglycans within the site of par- might enhance hyaluronic acid production in the
tially ruptured muscles increased maximally five wound, promoting swifter healing and possibly
days after trauma and decreased thereafter.77 This fewer complications related to scarring.
suggests the timing of glucosamine supplementa-
tion may determine its therapeutic impact on Protein and Wound Healing
wounds. Adequate protein intake is essential for
Clinical trials using glucosamine for proper wound healing. Protein depletion appears
perioperative support are lacking. However, the to delay wound healing by prolonging the inflam-
administration of oral glucosamine both before as matory phase; by inhibiting fibroplasia, collagen

and proteoglycan synthesis, and neoangiogenesis stress and have the greatest impact on protein re-
(proliferation phase); and by inhibiting wound quirements, increasing protein need 75-100 per-
remodeling.78,79 cent.86
Experimental protein depletion in animals Table 1 summarizes nutrients recom-
caused a decrease in the tensile strength of wounds. mended for perioperative nutritional support.
Rats fed a diet deficient in protein exhibited de-
creased wound integrity and strength versus con- Amino Acids in Wound Healing
trol animals.80 In a study of 108 human patients It is well accepted that sufficient protein
with experimental wounds, individuals with either is necessary for wound healing. This appears to
low serum protein or serum albumin had signifi- be due to the increased overall protein need for
cantly weaker wounds than those with normal pro- tissue regeneration and repair. Researchers have
tein values.81 investigated the effects of specific amino acids on
Protein calorie malnutrition increases the healing process and determined that arginine
morbidity and mortality in the surgical/trauma and glutamine appear to be necessary for proper
patient. Many studies have found hospitalized wound healing.
patients in a state of malnutrition at admission.
Thus, it is important to increase protein intake to
optimize healing and immune function, and to
Arginine
prevent post-surgical complications in these indi- Arginine is a non-essential amino acid that
viduals.82-84 plays a key role in protein and amino acid synthe-
Protein supplementation of elderly pa- sis. It is acquired from the diet and derived en-
tients with liquid protein formulas significantly dogenously from citrulline in a reaction catalyzed
enhanced healing of pressure ulcers. The change by the enzyme arginine synthetase. Adequate tis-
in ulcer area was significantly correlated with the sue arginine appears to be essential for efficient
amount of protein in the diet.85 wound repair and immune function.87
The surgical or trauma patient exists in a Arginine (17 g/day) was given to 30 eld-
state of metabolic stress, with the severity of the erly patients (>65 years of age) who sustained an
stress depending on the severity of the wounded experimental surgical injury. Supplemented pa-
state. An injured patient requires more protein than tients demonstrated significantly greater hydroxy-
a non-injured patient because of the increased proline (a sign of collagen deposition) and pro-
metabolic activity of wound healing, acute-phase tein accumulation at the wound site, compared to
protein production in response to stress, and amino non-supplemented controls. Lymphocyte re-
acid mobilization from muscle used for hepatic sponse, signifying greater immune activity, was
gluconeogenesis. elevated in the supplemented group, as was insu-
In a non-injured state, adults require ap- lin-like growth factor-1, which is a control mol-
proximately 0.8 g dietary protein/kg body wt/day. ecule for wound repair.88 Other studies have found
Elderly patients have a higher protein requirement similar results.89,90
(1-1.2 g/kg body wt/ day) due to a decreased abil-
ity to synthesize proteins. The surgical/trauma Glutamine
patient can require significantly more protein. Glutamine is used by inflammatory cells
Minor surgery may not significantly increase the within the wound for proliferation and as a source
protein requirement; however, if the patient is al- of energy.91,92 Fibroblasts use glutamine for these
ready protein malnourished, wound healing will same purposes, as well as for protein and nucleic
be adversely affected unless dietary protein intake acid synthesis. Because optimal functioning of
is increased. Major surgery can increase protein these cells is paramount to the healing process,
requirements 10 percent, while a patient with glutamine is a necessary component of the pro-
multiple traumas may need 75-percent more process of tissue repair. Glutamine is a non-essential
tein. Burn wounds cause tremendous metabolic amino acid that can become a “conditionally es-

sential” amino acid in

certain circumstances,
including tissue injury.93 Table 2. Post-surgery or Trauma Protocol
Glutamine is released
from skeletal muscle fol-
lowing injury or surgery, Nutrient Dose
which can cause a rela-
tive deficiency of Bromelain 500-1,000 mg daily
glutamine in skeletal
muscle and the gut, as Vitamin A 25,000 IU daily
intestinal uptake is fre-
quently diminished as
well.
Vitamin C 1-2 g daily
Studies utilizing
oral glutamine pre- and Zinc 15-30 mg daily
post-surgery, and in burn
patients, have shown Protein Minimum 0.8 g/kg body weight daily
mixed results. Oral feed-
ing of glutamine in sur- Glucosamine 1,500 mg daily
gery patients did not af-
fect plasma glutamine or
nitrogen turnover. Intra- Recommended from wounding until healing is complete.
venous glutamine in sur-
gery patients as an ala-
nine-glutamine dipeptide Botanical Medicines in Wound
showed consistently better post-operative results,
as seen by significantly decreased length of hos- Healing
92
pital stays (average of four days or less). A sig- Centella asiatica and Aloe vera
nificantly smaller incidence of pneumonia, bacte- Centella asiatica and Aloe vera have been
remia, and sepsis was noted in patients with mul- used for decades as folk remedies for burns,
tiple trauma given enteral glutamine feedings.94 wounds, and scars. Improved wound healing has
Whether glutamine supplementation will enhance been reported from topical or internal application
wound healing in less severely injured individu- of these two botanical medicines. Continued use
als is not known. of these plants as healing agents has led to scien-
A mixture of arginine (14 g/day), tific investigation of their efficacy as wound heal-
glutamine (14 g/day), and beta-hydroxy-beta- ing agents.
methylbutyrate (HMB) (3 g/day) was given to 18 Centella asiatica (gotu kola) has been
elderly (>70 years) individuals who then under- documented to aid wound healing in several sci-
went experimental implantation of sterile entific studies.96-99 One of the primary mechanisms
polytetrafluoroethylene tubes that could later be of action of Centella appears to be the stimulation
excised and studied for fibroblastic migration and of type-1 collagen production.100 Animal studies
collagen deposition. Supplementation with this have consistently shown topical application of
mixture resulted in significantly greater wound Centella asiatica to a sutured wound significantly
collagen deposition than in 17 controls not supple- increased the breaking strength of the
mented.95 wound.96,99,101,102 Asiaticoside, a saponin extracted
Table 2 summarizes nutrients recom- from Centella asiatica, is thought to be one of its
mended for post-surgery or trauma care.

active constituents. Shukla et al showed a 0.2-per- Eclectic Wound Therapies

cent asiaticoside solution applied topically twice Humans have always been faced with the
daily for seven days to punch wounds in guinea dilemma of how to treat wounds. Many diverse
pigs resulted in 56-percent increase in hydroxypro- and interesting approaches to wound management
line, 57-percent increase in tensile strength, in- have been applied throughout medical history.
creased collagen content, and better epithelializa- Thirty years ago physicians believed pus in a
tion compared to controls. Using the same punch wound was laudable and anxiously awaited its
wound model the researchers demonstrated an oral arrival;108 surgeons today attempt every conceiv-
dose of 1 mg/kg for seven days produced a 28- able means to prevent its presence. Although sci-
percent reduction in wound area and a significant entific validation is absent, some wound-care
increase of tensile strength and hydroxyproline therapies applied by eclectic physicians are still
content of the wound.102 considered valuable and effective therapies today.
Topical treatment with Aloe vera has been Honey and sugar or sugar paste have been
shown to improve healing in frostbite and electri- used to treat wounds for decades. Both are con-
cal injury in animals.103,104 In addition, Aloe vera sidered to be antimicrobial and have been associ-
has improved the healing of wounds in both nor- ated with scarless healing in some cavity
mal and diabetic rats.105,106 Topical application and wounds.109 Hyaluronic acid consists of disaccha-
oral administration of Aloe vera to rats with heal- ride chains made from modifications of the
ing dermal wounds increased the collagen con- monosaccharide glucose. One possible mechanism
tent of the granulation tissue as well as the degree in scar prevention is that glucose in honey or de-
of cross-linkage. Collagen increased 93 percent rived from sugar may be converted into hyaluronic
with topical treatment and 67 percent with oral acid at the wound surface, forming an extracellu-
treatment compared to controls. The increase was lar matrix that promotes wound healing.109 Fetal
attributed to increased stimulation by Aloe vera wounds heal without scar formation and the ex-
of collagen synthesis or increased proliferation of tracellular matrix of fetal wounds is rich with hy-
fibroblast synthesis of collagen, or both.107 In a aluronic acid and lacks excessive collagen.109 The
similar study, the effects of oral and topical Aloe glucose in honey or derived from sugar may fa-
vera on full thickness dermal wounds in rats ex- cilitate a balance between hyaluronic acid and
hibited an increase in glycosaminoglycan compo- collagen, similar to that found in fetal wounds.
nents of the extracellular matrix and, in particu- Preparations of fresh juice from Calen-
lar, hyaluronic acid and dermatan sulphate lev- dula officinalis preserved in alcohol, known as
els.107 Calendula succus, are used topically to promote
Aloe vera and Centella asiatica have been wound healing. Naturopathic doctors utilize Cal-
widely used for a host of curative purposes in- endula succus to cleanse wounds after minor sur-
cluding facilitating wound repair. In spite of their gical procedures and throughout the healing pro-
wide use as folk remedies the biochemical basis cess. External Calendula succus is listed in The
for their action or influence on tissue repair is just Complete German Commission E Monographs for
beginning to be understood. Human clinical trials promoting wound healing. Topical application is
are needed to determine safety and benefits of thought to have anti-inflammatory and granulatory
perioperative oral administration of these botani- action.110
cals. Topical application of both Aloe vera and Knitbone and bruisewort are common
Centella asiatica extracts to healing wounds or names for Symphytum officinalis (comfrey) that
surgical scars appears to be safe and facilitates give clues to its traditional uses. The active ingre-
improved wound repair. dient in comfrey is thought to be allantoin, which
is reported to promote cell division and the growth
of connective tissue, bone, and cartilage. Com-
frey poultices are applied externally on intact skin

for bruises, sprains, and

fractures. Medical litera-
ture regarding comfrey is Table 3. Topical Wound Care
limited to its potential
liver toxicity when taken
internally. However, Topical preparation Action
many anecdotal reports
claim comfrey is ex- Aloe vera Increases collagen content and degree of
tremely effective at procollagen cross-linkage within the wound.
moting swift healing in
bruises, sprains, and frac- Centella asiatica Stimulates type-1 collagen production.
tures. External applica-
tion to intact skin does Honey or sugar paste Glucose converted into hyaluronic acid at
not appear to have the the wound surface forming an extracellular
same toxicity concerns as matrix that promotes wound healing; also
internal consumption. considered antimicrobial.
Table 3 summa-
rizes botanicals and other Calendula succus Anti-inflammatory and promotes
topical treatments for granulation.
wound healing.
Adequate tissue Symphytum officinale Promotes cell division and the growth of
perfusion, blood flow, bone, cartilage, and other connective
and oxygen levels are re- tissues; applied topically to closed wounds.
quired for wound heal-
ing. Tissue perfusion de-
livers oxygen and nutri-
ents to regenerating tissue. The synthesis of fibro- influences the biochemical processes necessary for
blasts and the enzymatic hydroxylation of proline the phases of normal healing to occur. Undernour-
and lysine residues on the forming collagen chains ished or malnourished individuals heal less effi-
are dependent, in part, on the availability of oxy- ciently and are at greater risk for complications
111
gen. Hydrotherapy utilizes external hot and cold during and after surgery. Part of treating the whole
applications of water to manipulate the quantity patient and not just the “hole in the patient” is
of blood flow through a given tissue. Adequate appreciating the complex interactions and the nu-
blood flow brings oxygen, nutrients, and red and trients involved in the wound-healing process. The
white blood cells to target tissues. This basic physi- relationship between malnutrition and poor wound
ological manipulation of blood flow can support healing is well documented,112-114 while the impact
the wound healing process. Hydrotherapy is an of optimal levels of dietary and supplemental nu-
inexpensive and powerful adjunct to wound care; trient intakes for wound healing is relatively un-
however, there are some limitations to applying known.
hydrotherapy to open wounds, burns, and in pa- Promotion of good nutrition is recom-
tients with peripheral neuropathies. mended, particularly in populations at risk for
marginal and frank nutritional deficiencies, includ-
ing the elderly,115 severely injured,116 smokers,117,118
Discussion patients with maldigestion or poor assimilation,3
Wound healing proceeds quickly and ef- and hospitalized patients119 before elective surgery.
ficiently in a physiologic environment conducive Evidence supporting supplementation of nutrients
to tissue regeneration and repair. Nutritional sta- known to benefit the healing process in healthy
tus of patients at the time of trauma or surgery

individuals is lacking. Several journal reviews cite 2. Stadelmann WK, Digenis AG, Tobin GR.
a high prevalence of complementary and alterna- Physiology and healing dynamics of chronic
cutaneous wounds. Am J Surg 1998;176:26S-
tive medicine (CAM) use by surgical patients.120-
126
38S.
The authors of these articles caution against
3. Stadelmann WK, Digenis AG, Tobin GR.
the use of CAM therapies because of potential Impediments to wound healing. Am J Surg
adverse reactions, the most common being poten- 1998;176:39S-47S.
tial vitamin, mineral, herb, or amino acid interac- 4. Blee TH, Cogbill TH, Lambert PJ. Hemor-
tions with platelet aggregation or anesthetics or rhage associated with vitamin C deficiency in
other pharmaceuticals given perioperatively.124-126 surgical patients. Surgery 2002;131:408-412.
The potential benefit of nutrients is seldom dis- 5. Petry JJ. Surgically significant nutritional
cussed. supplements. Plast Reconstr Surg
Evidence exists that vitamins A and C, 1996;97:233-240.
zinc, arginine, glutamine, glucosamine, bromelain, 6. Ehrlich HP, Hunt TK. Effects of cortisone and
Aloe vera, and Centella asiatica may be benefi- vitamin A on wound healing. Ann Surg
1968;167:324-328.
cial to wounded or surgical patients; however,
7. Hunt TK, Ehrlich HP, Garcia JA, Dunphy JE.
many patients will be advised to avoid them. More
Effect of vitamin A on reversing the inhibitory
extensive, well-defined, blinded clinical trials to effect of cortisone on healing of open wounds
evaluate the safety, efficacy, and drug interactions in animals and man. Ann Surg 1969;170:633-
of these potential beneficial substances are needed. 641.
From the current available data it would 8. Ehrlich HP, Tarver H, Hunt TK. Effects of
appear that an adequate protein supply, as well as vitamin A and glucocorticoids upon inflamma-
supplementation of 25,000 IU vitamin A, 1-2 g tion and collagen synthesis. Ann Surg
1973;177:222-227.
vitamin C, 15-30 mg zinc, 3-15 g arginine, 3-15 g
glutamine, and 1,500 mg glucosamine per day 9. Cohen BE, Gill G, Cullen PR, Morris PJ.
Reversal of postoperative immunosuppression
prior to and after surgery would benefit adult pain man by vitamin A. Surg Gynecol Obstet
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complete. Post-operative topical application of of vitamin A and beta carotene on intra-
Aloe vera and Centella asiatica extracts may fa- abdominal sepsis. Arch Surg 1984;119:161-
cilitate the creation of a flexible, fine scar with 165.
high tensile strength at the wound site. In addi- 11. Levenson SM, Gruber CA, Rettura G, et al.
tion, 750-1,000 mg bromelain post-operatively Supplemental vitamin A prevents the acute
radiation-induced defect in wound healing.
may reduce edema, bruising, pain, and healing
Ann Surg 1984;200:494-512.
12. Greenwald DP, Sharzer LA, Padawer J, et al.
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Influence of vitamin A on wound healing in
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Neuron. 2006 October 5; 52(1): 77–92.
Mechanisms of Neuropathic Pain
James N. Campbell1,* and Richard A. Meyer1

1 Johns Hopkins University School of Medicine Baltimore, Maryland 21287
Abstract
Neuropathic pain refers to pain that originates from pathology of the nervous system. Diabetes,
infection (herpes zoster),nerve compression, nerve trauma, “channelopathies,” and autoimmune
disease are examples of diseases that maycause neuropathic pain. The development ofbothanimal
models and newer pharmacological strategies has led to an explosion of interest in the underlying
mechanisms. Neuropathic pain reflects both peripheral and central sensitization mechanisms.
Abnormal signals arise not only from injured axons but also from the intact nociceptors that share
the innervation territory of the injured nerve. This review focuses on how both human studies and
animal models are helping to elucidate the mechanisms underlying these surprisingly common
disorders. The rapid gain in knowledge about abnormal signaling promises breakthroughs in the
treatment of these often debilitating disorders.
Chronic pain has been estimated to affect one-sixth of the population. The phrase “neuropathic
pain” came into common use only in the last decade and increasingly has been appreciated as
a frequent source of chronic pain, perhaps trailing only osteoarthritis as a cause. Neuropathic
pain results from pathology in the nervous system. Consider the following clinical presentation:
A 47-year-old woman presented for consultation for complaints of pain on the right chest wall.
The patient underwent a mastectomy on the right side as a treatment for cancer 5 years
previously. As the surgical pain faded, the patient noted increasing chest-wall pain that
extended well beyond the surgical borders. Clothing lightly touching the skin increased the
pain. Reconstructive surgery was deferred because of concerns about the ongoing pain. In
addition to the ongoing burning pain, the patient also complained of sudden “pain attacks” one
to several times a day. These attacks lasted seconds to minutes and were described as
debilitating. Examination revealed a well-healed surgical scar. Light stroking of the skin
provoked significant pain in an area from the clavicle down to the T8 dermatome. Despite the
pain to light tactile stimuli, the patient also had areas of decreased sensibility as demonstrated
by the inability to detect a fine probe applied to the skin.
The above case represents a classical presentation of a patient with neuropathic pain. The
notable features that point to neuropathic processes are as follows:
• Widespread pain not otherwise explainable
• Evidence of sensory deficit
• Burning pain
• Pain to light stroking of the skin
• Attacks of pain without seeming provocation
The liability for pain appears to vary from person to person, from nerve to nerve, between
males and females, and even with age. What appears to be the same lesion may induce no pain
*Correspondence: jcampbel@jhmi.edu.
in one person but severe pain in another. In addition to ongoing pain (i.e., stimulus-independent
pain), patients may have heightened pain to stimuli applied to their skin. This enhanced
stimulus-dependent pain is called hyperalgesia. In some patients, lightly stoking the skin may
evoke pain. This pain to light touch is often called allodynia (Treede et al., 1992).
In this review, we will focus on the mechanisms of neuropathic pain. Lesions of the CNS (e.g.,
spinal cord) and peripheral nerves may lead to pain, but the majority of experimental studies
have focused on consequences of lesions of peripheral nerves, and therefore, we will focus on
this area in this review.
Anatomical Considerations
Neuropathic pain is distinguished from other pain conditions where the pain generator begins
with disease of nonneural tissues. These nonneuropathic pain entities are said to be nociceptive
and include conditions such as osteoarthitis and inflammatory pain. By definition, neuropathic
pain originates from a lesion of the nervous system. Innumerable diseases may be the culprits.
Examples include autoimmune disease (e.g., multiple sclerosis), metabolic diseases (e.g.,
diabetic neuropathy), infection (e.g., shingles and the sequel, postherpetic neuralgia), vascular
disease (stroke), trauma, and cancer. A rule without apparent exception is that the lesion leading
to pain must directly involve the nociceptive pathways (Boivie et al., 1989). Accordingly, for
example, lesions of the medial lemniscal system (e.g., dorsal columns) do not induce pain
(Cook and Browder, 1965).
Whereas evidence supports the hypothesis that pain-generating lesions of the nervous system
must involve the nociceptive pathways, the converse clearly does not hold up. Namely, not all
lesions of nociceptive pathways induce pain. A lesion of the peripheral nerve may induce pain,
but simply severing dorsal roots seems to have little chance of creating lasting pain (Li et al.,
2000). For example, dorsal roots are cut to treat spasticity and sometimes to remove tumors.
The authors know of no case of neuropathic pain arising from these types of lesions in man.
Here, we do not take up the issue of CNS lesions, but it is nevertheless worth noting that though
spinal cord lesions carry a substantial risk of inducing pain, some evidence suggests that the
lesion must include the gray matter. Vireck has found that simple cordotomy where only the
white matter is lesioned does not induce abnormal pain behavior in a primate model, whereas
lesions that include the gray matter may (Vierck and Light, 1999). Lesions of the brainstem
and thalamus carry a risk of causing pain if the nociceptive pathways are involved (Boivie,
2006). In nearly all of these cases, there is the paradoxical juxtaposition of ongoing pain and
a sensory deficit to noxious stimulation (even when hyperalgesia is present). Lesions confined
to the cortex appear not to be associated with abnormal pain (but it is also unclear whether
lesions confined to the cerebral cortex induce deficits of pain sensibility [Head and Holmes,
1911]).
Nervous System Diseases Associated with Pain

As noted above, a rich variety of diseases of the nervous system are associated with pain (Table
1) (Scadding and Koltzenburg, 2006). In the introduction, a case of traumatic painful
neuropathy was presented (Campbell, 2001). The offending lesion may involve a simple
axotomy (cutting of the nerve) or merely nerve entrapment. Ongoing pain may be the only
manifestation, but in many cases, hyperalgesia with pain to light-stroking stimuli (allodynia)
may be manifest. In some cases, there is striking cooling hyperalgesia, where mild lowering
of skin temperature exacerbates the pain. Weir Mitchell described a striking variant of
neuropathic pain that he termed causalgia (Lau and Chung, 2004). He noted this condition in
patients who had missile lesions of an extremity. Patients presented with striking edema,
profound autonomic features (abnormal sweating and either hot or cold skin), and devastating
pain. A similar presentation can occur without a clear nerve injury, and presently, this is referred

to as “complex regional pain syndrome (CRPS), type 1.” Where a clear nerve lesion is manifest,
the condition is referred to as CRPS, type 2 (Stanton-Hicks et al., 1995).
One of the classic examples of neuropathic pain is Tic douloureux. Without treatment, this is
a debilitating disorder that involves attacks of severe pain in the facial area (also referred to as
trigeminal neuralgia). Often there is little or no pain between attacks. The lightening-like
attacks are referred to one of the dermatomes (V1, V2, or V3). Light touching of the skin in a
so-called trigger zone suffices to evoke an attack. The disease appears to be associated with
mechanical distortion at the entry zone of the nerve root to the brainstem. Demyelination may
be seen at the compression site. Nerve compression from an aberrant blood vessel is one of the
more common causes (Elias and Burchiel, 2002).
Another classical neuropathic pain condition is painful diabetic neuropathy (Dyck et al.,
2000). Diabetes often causes a length-dependent neuropathy (meaning that the longest axons
in the peripheral nerve are most vulnerable). Patients report bilateral burning pain in the toes
and feet. Quantitative sensory testing reveals decreased pain sensibility (with or without
decreased touch sensibility).
Postherpetic neuralgia is a complication of shingles and is an example of how an infection can

lead to pain. Shingles results from an activation of the herpes zoster virus that takes up residence
in the dorsal root ganglion after a chickenpox infection. The shingles eruption consists of
blisters that follow the dermatome(s) of one or more spinal nerves. The blisters heal in time,
but the pain may continue. Allodynia is a particularly prominent feature of postherpetic
neuralgia. This allodynia may be present even with loss of C-fiber innervation of the epidermis.
The clinical manifestations vary with the type of disease (Table 1). These variations suggest
different mechanisms. These differences in mechanism may also be reflected in responses to
therapy. Tic douloureux responds nicely to treatment with the anticonvulsant carbamazepine.
Responses to carbamazepine for other conditions are typically disappointing. Painful diabetic
neuropathy leads to ongoing pain, but allodynia is distinctly unusual, and cooling may relieve
pain. In contrast, allodynia is prominent in traumatic neuropathy and cooling often causes
severe pain.
Animal Models of Neuropathic Pain

Whereas the distinction between nociceptive and neuropathic pain has utility, in the actual
clinical setting, the mechanisms are often intertwined (e.g., back pain with radiculopathy).
Fortunately, however, this need not be the case with regard to animal models. A great advance
in the study of neuropathic pain emanated from the discovery that placement of loose chromic
ligatures on the sciatic nerve in rat brought about behavior that appeared analogous to human
neuropathic pain conditions (Bennett and Xie, 1988). The rats engaged in protective behavior
and had lowered thresholds to heat, cooling, and mechanical stimuli.
Subsequent work indicated that an idiosyncratic immune-mediated response to the chromic

suture played a major role in the development of the model (Maves et al., 1993). The nerve
swelled, leading to nerve compression and axotomy. But can an axotomy by itself induce pain?
The answer is unequivocally yes. Chung and colleagues (Kim and Chung, 1992) devised a now
frequently used model in rat whereby one or more spinal nerves that innervates the foot is cut
(SNL model in Figure 1A). A substantial innervation of the foot remains, reflecting input from
the adjacent spinal nerves. This remaining innervation allows tests for hyperalgesia to be
undertaken. A simple axotomy provides a foundation by which to study mechanism in diseases
where axotomy is part of the disease process. Some of the common traumatic nerve injury
models are illustrated in Figure 1A. These injuries lead to hyperalgesia (Figures 1B and 1C).

Trauma has not been the only model used in studies of neuropathic pain. One of the leading
causes of pain in humans is diabetic neuropathy. Injection of streptozotocin leads to an animal
model of diabetes and is associated with the development of neuropathy, similar to what is
seen in humans. Hyperalgesia can be measured in rodent models, thus providing a means to
study treatments and mechanisms of pain in this neuropathy model (Ahlgren and Levine,
1993). One of the dose-limiting problems in chemotherapy is neuropathy, and these
neuropathies are often associated with pain. Animal models of paclitaxel and vincristine-
induced painful neuropathies have been developed (Flatters and Bennett, 2004).
Though hyperalgesia is relatively easy to demonstrate in animal models, the measurement of

ongoing pain is more problematic. This issue is important because ongoing pain without clear
hyperalgesia appears to be a common occurrence in humans (Basbaum et al., 2006). Substantial
denervation of a limb in rat often leads to autotomy behavior. Some have advocated that
autotomy is an indication of ongoing pain, the presumed rationale being that the animal desires
to eliminate the painful body part (Devor, 1991). An alternative explanation is that the animal
chews an anesthetic part for reasons unrelated to pain. Some have argued that spontaneous foot
lifting may provide a behavioral measure of spontaneous pain (Djouhri et al., 2006). Alternative
measures of ongoing pain involve the use of cellular markers of increased neuronal activity.
Increased expression of the immediate early gene protein, c-Fos, in the dorsal horn (and perhaps
other more rostral sites) is an example (Bullitt, 1990). Small-animal functional magnetic
resonance imaging (fMRI) and/or PET imaging may in the future offer alternative measures.
Animal models of neuropathic pain in some cases have been inconsistent with human models.
Allodynia in painful diabetic neuropathy in humans is infrequent yet appears to be robust in
rat models. NK1 antagonists appeared to have promise for treatments of pain based on animal
models yet to date have not proven useful in patients. Vierck (2006) argues that “reflex”
measures of pain in animal neuropathic models are intrinsically flawed and are neither sensitive
nor specific predictors of drug efficacy in man. For example, he points out that the paw-
withdrawal threshold method tests motor neuron response rather than simply providing a
measure of pain. Moreover, he indicates that rostral signaling pathways may be ignored when
one merely measures the paw-withdrawal threshold. His solution is to utilize operant models.
However, operant measures may be flawed as well in that they introduce other complexities
such as motivational factors that may be extraneous to the question of how much pain is felt.
Furthermore, the simple paw-withdrawal technique has in fact demonstrated some consistency
with human pain studies. In a recent pharmacological study that used a paw-withdrawal method
in rodents with the SNL model (LaBuda and Little, 2005), hyperalgesia was reversed with
gabapentin, amitriptyline, and fluoxetine, but not indomethacin, treatment. This correlates with
the observation that the first three drugs are useful in treatment of neuropathic pain in humans
but that nonsteroidal drugs are in general not useful.
A careful comparison of operant and reflex models in terms of their predictive capacity has
yet to be performed. For drugs with a predominantly supraspinal action, operant measures may
be more appropriate, whereas simple paw-withdrawal thresholds may be sufficient for drugs
that work at peripheral or dorsal horn targets. Though the particulars of how pain is assessed
remain an issue, a greater concern may relate to substantial inherent differences in the biology
of nociception in rodent and man. Regardless of this, new drugs for treatment of neuropathic
pain have been developed, and their utility in animal models played an influential role in
moving these treatments into clinical trials.
Secondary Hyperalgesia and Central Sensitization

A starting place in understanding neuropathic pain is to consider what happens with injury to
nonneural tissues. Skin injury produces ongoing pain and two types of hyperalgesia: primary

and secondary (Meyer et al., 2006). Primary hyperalgesia occurs at the site of tissue injury and
is mediated in part by sensitization of primary afferent nociceptors. This is reflected by
increased responses to heat stimuli, for example. Secondary hyperalgesia occurs in the
uninjured tissue surrounding the site of injury and is thought to be due to sensitization in the
central nervous system. Secondary hyperalgesia is characterized by hyperalgesia to
mechanical, but not heat, stimuli. This mechanical hyperalgesia is comparable to the
hyperalgesia seen in patients with neuropathic pain. Two types of mechanical hyperalgesia are
observed: pain to light-stroking stimuli (i.e., allodynia) and enhanced pain to punctate stimuli.
Two psychophysical studies in human volunteers provide strong evidence that secondary
hyperalgesia is due to sensitization in the central nervous system. In both studies, intradermal
injection of capsaicin, the algesic substance in hot peppers, was used to produce a large zone
of secondary hyperalgesia. In the first study, the nerve supplying the area to be injected was
anesthetized with a local anesthetic (LaMotte et al., 1991). Injection of capsaicin still produced
a large flare, consistent with the concept that the flare reflects a peripherally mediated release
of vasoactive peptides subsequent to antidromic spread of action potentials in the nociceptive
terminals. In the opposite control arm, capsaicin injection at the same time produced intense
pain and a large zone of secondary hyperalgesia. When the anesthesia wore off, no zone of
mechanical hyperalgesia was found in the test arm, but hyperalgesia persisted in the control
arm. This experiment provides evidence that the barrage of nociceptor activity associated with
the capsaicin injection leads to an altered central processing of input from mechanosensitive
afferents.
In the second study, a fine electrode was placed into the superficial peroneal nerve (Torebjörk
et al., 1992). Electrical intraneural microstimulation produced a non-painful tactile percept that
was referred to a small zone on the top of the foot. Capsaicin was injected adjacent to this area
so that the zone of secondary hyperalgesia overlapped the area of referred sensation.
Microstimulation after the injection produced a painful percept. Because this stimulation
bypasses any peripheral sensitization processes at the cutaneous receptor, this experiment also
provides evidence for altered central processing of mechanoreceptor input. Additional studies
have shown that the tactile pain arises from central sensitization to the inputs of Aβ fibers,
whereas the punctate hyperalgesia is due to a central sensitization to the inputs of capsaicin-
insensitive Aδ nociceptors (Magerl et al., 2001).
The tactile fibers have known convergence onto dorsal horn cells that in addition receive inputs
from nociceptive primary afferents. The inputs from the nociceptors in the injury zone are
presumed to sensitize these so-called wide-dynamic range neurons and thereby enhance the
synaptic efficacy of the tactile fibers. Nociceptive-specific neurons (dorsal horn neurons in
lamina I) may be sensitized in similar fashion.
Central sensitization to the inputs of tactile afferents in patients has been demonstrated in
patients with neuropathic pain. A blood pressure cuff inflated in the proximal extremity leads
to an orderly loss of sensation beginning first with loss of tactile function. When this was done
in patients, the loss of tactile function coincided with loss of allodynia (Campbell et al.,
1988). It was also noted that the detection of pain with tactile stimuli was fast and indicated
that primary afferents transmitting the stimulus were likely in the A-β range.
Where Do the Abnormal Signals in Neuropathic Pain Originate?

As shown in Figure 2, multiple sites along the neural axis are altered after nerve injury.
Abnormalities occur in the injured and uninjured afferents supplying the affected region.
Central sensitization, similar to that discussed above following tissue injury, is observed. In
addition, changes in descending control systems have been reported. Finally, an immune
response, both peripherally and centrally, is observed.

A Role for Primary Afferents

The importance of primary afferent inputs in neuropathic pain is strongly suggested by several
pharmacological studies. For example, systemic administration of AM1241, a selective CB2

cannabinoid receptor agonist, results in reversal of signs of mechanical and thermal
hyperalgesia following an SNL lesion (Ibrahim et al., 2003). Because CB2 is not expressed in
the central nervous system, the effects are likely due to a peripheral mechanism.
Systemic administration of artemin dose-dependently reversed the behavioral signs of

neuropathic pain in rats with an SNL injury (Gardell et al., 2003). Artemin is a member of the
glial-derived neurotrophic factor (GDNF) family that signals through a common tyrosine
kinase receptor (RET) and GFRα3. GFRα3 is an accessory protein that is predominantly
expressed in small-diameter, unmyelinated neurons (Orozco et al., 2001). The artemin-induced
changes in neuropathic pain behavior are therefore most likely due to its action on nociceptive
primary afferents.
Antisense oligodeoxynucleotides (ODNs) directed against Nav 1.8 reversed signs of

mechanical hyperalgesia (Porreca et al., 1999). Nav 1.8 is a tetrodotoxin-resistant sodium
channel that is primarily expressed in small-diameter primary afferent neurons. This treatment
was effective even when applied 6–14 days after nerve injury, demonstrating that ongoing
peripheral neuronal input is critically involved in the maintenance of neuropathic pain.
The Injured Afferent Hypothesis

The above evidence makes a clear case for the role of primary afferents, and one might assume
that the culprit is the injured afferent itself. Indeed, much evidence favors this hypothesis. When
nerve is injured a neuroma forms. The spontaneous activity and ectopic sensitivity to
mechanical, thermal, and chemical stimuli that originate from the traumatic neuroma have been
well documented (Blumberg and Jänig, 1984; Devor, 2006a). Clinical evidence has suggested
that local-anesthetic blockade of an injured nerve in patients relieves pain, though no study,
surprisingly, has really addressed this issue in a well-designed blinded fashion (Arner et al.,
1990; Burchiel et al., 1993; Gracely et al., 1992; Koltzenburg et al., 1994). Additional support
for the role of injured afferents comes from experiments in the rat L5 SNL model in which
anesthetics (Sukhotinsky et al., 2004) or tetrodotoxin (TTX) (Lyu et al., 2000) directed at the
L5 ganglia reversed the neuropathic behavior.
Regardless of the reasonableness of the so-called injured afferent hypothesis, several lines of
evidence suggest that there is more to the story.
Surprisingly, several authors report that the L5 SNL model in rat leads to spontaneous activity
in A fiber, but not C fiber, afferents as recorded in the L5 dorsal root (Boucher et al., 2000;
Liu et al., 2000). This spontaneous activity in myelinated fibers appears to be produced mainly
in afferents that (preinjury) serve muscle and joint, but not skin (Michaelis et al., 2000; Proske
et al., 1995). These findings produce a dilemma because activity in C fibers is thought to be
necessary to evoke the central sensitization mechanisms that account for the hyperalgesia (Ji
and Woolf, 2001). One possible explanation is that A fiber-spontaneous activity can initiate
central sensitization after nerve injury, if the afferent undergoes a phenotypic switch. A de
novo expression of neuropeptides normally only expressed in nociceptive afferents could
occur. For example, substance P immunoreactivity increases in large- and medium-size DRG
neurons after axotomy (Noguchi et al., 1994). However, studies investigating substance P
release from myelinated afferents following nerve injury are inconclusive (Allen et al., 1999;
Malcangio et al., 2000). Another explanation is that the spontaneous activity occurs in “A-β”
nociceptors, the existence of which has been documented in rodent and primate (Djouhri and
Lawson, 2004; Treede et al., 1998).

Other behavioral data suggest that signals originating from the injured spinal nerve are not
essential for hyperalgesia to occur. An L5 dorsal rhizotomy immediately before or after an L5
spinal nerve ligation did not prevent or reverse neuropathic behavior (Eschenfelder et al.,
2000; Li et al., 2000). The counter to this observation is that dorsal rhizotomy by itself may
produce signs of mechanical hyperalgesia (Colburn et al., 1999; Eschenfelder et al., 2000).
More importantly, neuropathic pain behavior is observed after an L5 ganglionectomy, where
the injured afferents are removed altogether (Sheth et al., 2002). The prevailing message seems
to be that hyperalgesia can develop in the absence of neural activity from the injured nerve.
The Intact Nociceptor Hypothesis

According to this hypothesis, the intact nociceptors that survive injury and that innervate the
region affected by the transected nerve fibers sensitize and have spontaneous activity. These
changes in the intact nociceptors may induce ongoing pain and may account for certain aspects
of hyperalgesia.
Following peripheral nerve lesions in primate and rodent models, spontaneous activity
developed in uninjured, unmyelinated nociceptive afferents that shared the same innervation
terrritory of the transected fibers (Ali et al., 1999; Djouhri et al., 2006; Wu et al., 2001).
Although the average discharge frequency was low (seven action potentials/5 min), the
incidence of spontaneously active fibers was high (50%). Low rates of spontaneous activity
may therefore assume importance if this phenomenon is occurring in large numbers of C fibers,
in particular given the high convergence of C fiber input in the CNS. Consistent with this
hypothesis is the observation that low-frequency electrical stimulation in C fibers can lead to
hyperalgesia in humans (Klede et al., 2003) and behavioral signs of hyperalgesia in rats (Wu
et al., 2002). The development of spontaneous activity has also been observed in uninjured,
myelinated afferents in the L4 dorsal root (Boucher et al., 2000), and this activity originated
within the dorsal root ganglion.
Sensitization has also been reported in the intact nociceptors. Following peripheral or spinal
nerve lesion in rat or rabbit, uninjured afferents develop adrenergic sensitivity (Sato and Perl,
1991) and an increased sensitivity to tumor necrosis factor-α (TNFα) (Schäfers et al., 2003).
One study has suggested that sensitization to mechanical and heat stimuli occurs in the
uninjured, unmyelinated afferents as well (Shim et al., 2005). Increased responsiveness to heat
stimuli could be due to the observed increases in expression of mRNA and protein for the
transient receptor potential receptor V1 (TRPV1) in the DRG of uninjured afferents (Fukuoka
et al., 2000; Hudson et al., 2001). In addition, the incidence of cold-responsive neurons in the
L4 DRG is increased after an L5 SNL (Djouhri et al., 2004), and there is an increase in
expression of the putative cold-sensitive channel TRPA1 (Katsura et al., 2006). These
observations may account for the occurrence of cooling hyperalgesia seen in patients after
nerve trauma.
One compelling line of evidence in favor of the intact nociceptor hypothesis stems from
experiments where local treatments are applied to the partly denervated skin. Local treatments
can only affect the intact fibers. In patients, local treatment with capsaicin may significantly
relieve ongoing pain. This is of interest because the effects of capsaicin are specific to
nociceptors. Experiments with animal models point to similar conclusions. Mibefradil, a T-
type calcium-channel blocker, reversed mechanical and thermal hyperalgesia (Dogrul et al.,
2003), and local application of a glutamate receptor antagonist reversed signs of thermal
hyperalgesia (Dogrul et al., 2000). In both studies, contralateral injections were without effects.
Intraplantar injection of morphine on the ipsilateral, but not contralateral, side significantly
reversed mechanical allodynia (Pertovaara and Wei, 2001).

The intact L4 dorsal root ganglion has been studied extensively to determine changes in
phenotype after injury to the adjacent L5 spinal nerve (L5 SNL). In addition to increased
expression of TRPV1 and TRPA1, mRNA for calcitonin gene-related peptide (CGRP) is
upregulated (Fukuoka et al., 1998). Furthermore, the number of uninjured DRG neurons
expressing brain-derived neurotrophic factor (BDNF) is increased (Fukuoka et al., 2001). In
the SNI model, there is an upregulation of P2X3 mRNA in uninjured neurons (Tsuzuki et al.,
2001).
Sympathetically Maintained Pain and the Intact Nociceptor Hypothesis

CRPS is a striking disorder manifest by severe pain typically in an extremity. Patients typically
have edema, hyperalgesia, and may even have a motor disability that is difficult to explain
from a purely electrophysiological perspective. In certain of these patients, selective anesthetic
blockade of the sympathetic nervous system leads to dramatic relief of pain (sympathetically
maintained pain, or SMP). Blockade of α -adrenergic receptor function with intravenous
infusion of the antagonist phentolamine also leads to pain relief (Raja et al., 1991). In patients
with SMP, in whom a sympathetic ganglion block was done to relieve pain and block release
of norepinephrine, injection of physiological concentrations of norepinephrine evoked
substantial pain (Figure 3A).
In a primate model of neuropathic pain, an L6 SNL leads to hyperalgesia just as it does in

rodent models (Carlton et al., 1994). About one-half of the afferents to the top of the foot are
lost, and the other half reach the foot from the adjoining and putatively normal spinal nerves
(Ali et al., 1999). More than 60% of the intact nociceptors had spontaneous activity (normally
infrequently seen), and more than 50% had sensitivity to the select α -adrenergic agonist
phenylephrine (Figure 3B). These studies indicate that the mechanisms of SMP relate to an
adrenergic sensitization of nociceptors (Figure 3C). Moreover, this chemical sensitization
occurs in the intact nociceptors that survive a proximal nerve injury. Thus, SMP is a specific
example of the importance of the intact nociceptors in the pathogenesis of a particular
neuropathic pain disorder.
The Role of Growth Factors

Trophic factors in the target tissue have an ongoing influence on sensory and motor fibers.
Nerve injury induces changes in growth-factor expression (Griffin, 2006). The change in
expression occurs in the tissue deprived of innervation (e.g., the skin), the Schwann cells
affected by Wallerian degeneration (denervated Schwann cells), the DRG, and the dorsal horn.
Of note, in the SNL model, the L5 root is lesioned, but the L4 root shares the innervation
territory, the same Schwann cells, and has convergent input to dorsal horn cells served by the
L5 afferents. Nerve injury leads to increased levels of NGF in the skin supplied by L4. The
binding of this NGF to the Trk-A receptors on the L4 fibers leads to transport of NGF back to
the L4 DRG. Evidence suggests that this increased level of NGF in the L4 DRG affects factors
such as BDNF. Indeed, the changes in gene expression in the intact L4 DRG and the lesioned
L5 DRG are quite different.
Griffin has introduced the terms “undertrophed” and “overtrophed” to describe a putative
mechanism by which growth factors may dually affect both the injured and uninjured afferents
(Griffin, 2006). The injured axons by losing connectivity with the peripheral tissues lose trophic
influences. The increased expression of trophic factors in the denervated tissues on the other
hand “overtroph” the remaining (intact) fibers. NGF might be one of the villains in the case of
overtrophing, whereas GDNF might be the missing growth factor, absence of which generates
the pain-signaling mechanisms in the injured L5 spinal nerve (Boucher and McMahon,
2001).

Transduction, Channel Function, and Ectopic Discharge

Primary afferents transduce different forms of energy into generator potentials. If of sufficient
magnitude, these generator potentials lead to action potentials. The action potentials propagate
along the axon to the CNS. Pathological pain may result from disorders involved in any of
these steps.
There are two ways nociceptors may be sensitized. One is for the transduction channel to
sensitize (i.e., a bigger generator potential to a given natural stimulus). The other mechanism
regards a decrease in threshold of the sodium channels responsible for spike initiation. It is
logical therefore to ask whether primary afferent sensitization may account for features of
neuropathic pain. NGF regulates TRPV1 expression, and thus, over-trophing mechanisms
could link growth factors to transduction-channel function (Cortright and Szallasi, 2004). A
role for TRPV4, a channel that may be involved in transduction of mechanical stimuli, was
suggested in a neuropathic pain model produced by administration of taxol (Alessandri-Haber
et al., 2004). Abnormal transduction, at least in part, likely underlies the pathophysiology of
the “intact nociceptor.” Sensitization of the “intact” nociceptor in the SNL model may be
responsible for the hyperalgesia (Ringkamp and Meyer, 2006). Another mechanism relates to
the acquisition of transduction capacity for stimuli that ordinarily do not activate nociceptors.
This has been clearly demonstrated in the case of SMP, where intact nociceptors acquire
sensitivity to norepinephrine. Cooling hyperalgesia is prominent in neuropathic pain.
Abnormal expression of transduction channels such as TRPA1, or TRPM8 in nociceptors,
could account for this phenomenon.
Transduction can be displaced to a point of nerve injury as well. This misplaced transduction
could account for the so-called “Tinel sign,” a useful clinical finding, wherein patients
experience paresthesias (tingling sensation) or dysthesthesia (an unpleasant or frankly painful
sensation) when the area over the nerve injury is tapped. The Tinel sign may be present at a
point of entrapment where continuity of the axon has not necessarily been interrupted. The
presumed mechanism is that a disturbance of fast axonal transport (which serves as delivery
mechanism for newly synthesized transduction proteins) leads to ectopic expression of the
transduction proteins. Axotomy leads to neuroma formation, where budding regenerative nerve
sprouts devoid of growth guidance from denervated Schwann cells form an entangled mass.
Many neuromas are “painful,” but for unclear reasons (genetic factors, location?) many are
not. Areas of nerve injury may be tethered to adjacent moving structures (e.g., tendons), such
that otherwise normal movements evoke an increase in pain presumably by activating
nociceptive afferents through activation of mechanoreceptive transduction channels.
Nociceptor sensitization could also relate to changes in voltage-gated sodium channels,

particularly in the region of spike initiation (Devor, 2006b). For example, if the threshold for
sodium-channel activation decreases, the response to a given generator potential will be

enhanced.
One reason for interest in sodium-channel function centers on clinical evidence that drugs that
affect sodium-channel function may be very effective in relieving neuropathic pain. For
example, the anticonvulsant carbamazepine, which works by stabilizing sodium channels in
an inactive state, is considered the treatment of choice for trigeminal neuralgia. The effects are
consistent enough that clinicians are trained to question the diagnosis if the patient does not
have at least some response to the drug. As a class, each of the anticonvulsants that work
through a sodium-channel mechanism (lamotrigine, phenytoin, in addition to carbamazepine)
have some level of efficacy in neuropathic pain, though side effects often preclude usefulness.
Another class of drugs that is effective in neuropathic pain is the local anesthetics (Tremont-
Lukats et al., 2006). At doses that have no effect on normal sensibility, local anesthetics may
favorably affect neuropathic pain.

There are multiple types of voltage-gated sodium channels that are expressed in the dorsal root
ganglion and may have a role in neuropathic pain. The channels that have received greatest
interest are Nav 1.3, 1.7, 1.8, and 1.9. These channels are found in small DRG cells and therefore
are likely involved in action potential generation and conduction in nociceptors. Channels
Nav 1.3 and 1.7 are TTX sensitive, whereas Nav 1.8 and 1.9 are TTX insensitive. Some animal
data suggest that TTX given systemically may relieve hyperalgesia without adversely affecting
normal functions (Marcil et al., 2006). Of the channels tested, only the TTX-sensitive channel
Nav 1.3 is upregulated in the DRG of the injured axons (Black et al., 1999). This channel has
kinetic properties that could favor repetitive spiking.
Whereas prior efforts have concentrated on the α -subunit expression, other complexities affect
sodium-channel function. Expression of the β subunits could be part of this complexity. β2
subunits regulate channel gating, assembly, and cell-surface expression of TTX-sensitive
channels. Expression of these subunits is increased in injured sensory afferents (Pertin et al.,
2005), and in β2 null mice, mechanical hyperalgesia is reduced in a spared nerve injury model.
β2 null mice display marked reduction in TTX-sensitive sodium current in small DRG neurons
(Lopez-Santiago et al., 2006).
That channel function may strongly affect nociception has been further emphasized by the
discovery of the genetic underpinnings of a striking neuropathic pain disorder known as
erythromelalgia. Here, patients present with profound heat hyperalgesia and ongoing burning
pain typically affecting the feet. The feet may also be intensely red, indicating profound
vasodilation. Point mutations in the TTX-sensitive channel Nav1.7 account for the disorder
(Figure 4). These mutations lead to a hypo-excitability in sympathetic neurons (accounting for
the increased perfusion in the feet) and a hyperexcitability in small-sensory neurons
(accounting for the pain and hyperalgesia) (Rush et al., 2006). Release of vasoactive peptides
in the skin from the tonically active nociceptors may also contribute to the vasodilatation.
The Role of Central Sensitization

Central sensitization refers to the augmented response of central signaling neurons. Though
thalamic and cortical levels may be involved, most attention has focused on the dorsal horn
and in this review we will concentrate on this area.
When it occurs as a result of inflammation, central sensitization is strongly dependent on

ongoing input from nociceptors. Thus, if nociceptive input is blocked in the injury zone,
secondary hyperalgesia abates promptly (LaMotte et al., 1991). This same dependence on
peripheral input probably also applies to central sensitization associated with neuropathic pain,
but this issue requires further clarification. This distinction is of great importance clinically. If
mechanisms of pain are completely centralized, it might be argued that attention to peripheral
mechanisms is unlikely to be therapeutically beneficial.
Central sensitization involves homosynaptic and heterosynaptic mechanisms (Magerl et al.,

1998; Woolf et al., 1988). Homosynaptic sensitization means that the conditioning stimulus
and the test stimulus involve the same input. In dorsal horn nociceptive neurons, this is evident
in a phenomenon referred to as “windup,” where continual low-frequency stimulation of C
fiber afferents leads to an increasing response in the dorsal horn cell. Windup lasts tens of
seconds and represents a short-term form of sensitization.
Herterosynaptic sensitization means that the conditioning stimulus and the test stimulus involve
different sets of afferents. Heterosynaptic sensitization accounts for allodynia. Here,
nociceptive inputs alter synaptic efficacy such that A-β mechanoreceptors acquire the capacity
to evoke responses. Electrophysiological evidence for heterosynaptic sensitization has been

shown in primate studies of dorsal horn cells where the response to light stroking of the skin
was enhanced after capsaicin injection (Simone et al., 1991).
Ample evidence indicates that heterosynaptic and homosynaptic sensitization occur in

neuropathic pain conditions (Campbell et al., 1988; Ji et al., 2003). Figure 5 provides a model
for how this may occur in the L5 SNL model of neuropathic pain. Injured myelinated fibers
from the L5 root develop spontaneous activity. Some of this activity could be in A-β
mechanoreceptors (that have acquired the capacity to release substance P) or in A-δ
nociceptors. The input of these fibers onto cells in the L5 segment could lead to homosynaptic
sensitization and enhanced ongoing activity. The enhanced activity in L5 spinal thalamic tract
cells could play a role in ongoing pain sensation. The injured L5 afferents could also project
to the adjacent L4 segment where they could produce heterosynaptic sensitization to input from
the L4 segment. This could account for the hyperalgesia to mechanical and heat stimuli that is
signaled by activity in the intact L4 afferents.
Uninjured nociceptors in L4 root can develop spontaneous activity after an L5 SNL. This
spontaneous activity can lead to homosynaptic sensitization such that stimulus-evoked activity
in these nociceptors results in an augmented response of the dorsal horn cells. The spontaneous
activity in uninjured L4 nociceptors could also produce heterosynaptic sensitization such that
the response of L4 spinal thalamic cells to L4 mechanoreceptor input is enhanced. A brush
stimulus applied to the foot activates the A-β fibers in the L4 root and acquires the capacity to
drive activity in the L4 dorsal horn cell, consistent with heterosynaptic sensitization.
Homosynaptic and heterosynaptic sensitization involve many mechanisms, but fundamentally

these mechanisms come down to two processes: (1) increased release of excitatory
neurotransmitter (e.g., glutamate, substance P) and/or (2) enhanced synaptic efficacy. These
changes in turn may relate to several cellular mechanisms (Basbaum, 1999). These mechanisms
may be considered in five categories: (1) presynaptic changes, (2) postsynaptic changes, (3)
interneuron changes, (4) changes in descending modulation, and (5) immune/microglial
mechanisms (discussed in the following section). Evidence suggests a role for each of these
mechanisms in neuropathic pain. Long-term potentiation (LTP), a phenomenon that has
received intensive study in the hippocampus, appears to apply to the dorsal horn to some degree.
However, given the abnormal ongoing input of primary afferents documented after nerve
injury, the acute central-sensitizing effects of primary afferents may have an ongoing role.
Thus, both short-term sensitization and LTP mechanisms likely apply. A role for long-term
depression (LTD) in inhibitory cells is also considered below.
Presynaptic Mechanisms
Release of glutamate is inhibited presynaptically by several metabotropic G protein-coupled
receptors, including μ-opioid receptors, GABA-b, and adenosine receptors. Downregulation

of μ-opioid receptors has been documented both pre- and postsynaptically after nerve injury
(Kohno et al., 2005) and could lead to an enhanced release of glutamate.
Neurotransmitter release is triggered by voltage-gated calcium channel activity on the central

terminals of primary afferents. Nerve injury leads to an upregulation of the α -2-δ subunit of
these channels in the DRG and spinal cord (Li et al., 2004). This could be associated with
increased calcium entry and augmented release of glutamate. The anticonvulsant drugs
gabapentin and pregabalin are effective in neuropathic pain presumably because they bind to
and block this subunit (Bian et al., 2006; Freynhagen et al., 2005). Although these drugs reverse
hyperalgesia in neuropathic models, there is no apparent effect on normal pain sensibility
(Suzuki et al., 2005). This argues for a special role of the α -2 subunit in neuropathic pain.

There is also evidence to suggest that under conditions of nerve injury, A-β fibers undergo a
phenotypic switch, such that they begin to express substance P (Noguchi et al., 1994). As a
consequence, stimulation of these fibers leads to substance P release, providing an additional
potential mechanism for sensitization of dorsal horn neurons.
Postsynaptic Mechanisms
There is excellent documentation of the role of postsynaptic mechanisms in central
sensitization. Slow depolarization induced by substance P and other peptides leads to an
opening of the NMDA glutamate-gated channel, which in turn leads to calcium entry
(Dougherty et al., 1993; Duggan, 1995; Yoshimura and Yonehara, 2006). Calcium may gain
entry through AMPA receptors that do not contain the GluR2 subunit, as well, and this
mechanism may play a role in dorsal horn LTP (Gu et al., 1996). A conditional knockdown of
one NMDA subunit, NR1, abolished hyperalgesia to formalin but had no effect on normal pain
sensibility (South et al., 2003). Evidence exists for a multitude of intracellular events linked
to postsynaptic sensitization that result from calcium entry. Protein kinases A and C and other
transcriptional factors likely play important roles in these events (Kawasaki et al., 2004).
Another possible mechanism of postsynaptic sensitization involves an increased trafficking of

AMPA receptors to the cell surface. The expression of AMPA receptors is increased in the
superficial dorsal horn after an SNL lesion (Harris et al., 1996). In addition, upregulation of
the mRNAs for both AMPA and NMDA receptors has been demonstrated in diabetes
(Tomiyama et al., 2005).
We have suggested that allodynia stems from sensitization to the inputs of tactile afferents in
the dorsal horn. However, certain evidence suggests that the “Aβ sensitization” phenomenon
may actually arise from alternative pathways. A lesion of the ipsilateral dorsal column
abolished mechanical hyperalgesia but, interestingly, not heat hyperalgesia (Ossipov et al.,
2002; Sun et al., 2001). These data suggest that dorsal horn trafficking may account for heat
hyperalgesia but that other centers, such as the thalamus, may actually account for the allodynia.
Role of Disinhibition Mechanisms

Inhibitory neurons play an important role in governing the sensitivity of dorsal horn neurons
(Gu et al., 1996). Decreased expression of inhibitory receptors (on primary afferent terminals
and postsynaptic neurons) has been noted following nerve injury, and sensitization could result
from this disinhibition mechanism (Kohno et al., 2005). Several other mechanisms involving
inhibitory interneurons have been suggested. We have discussed LTP. The reciprocal
phenomenon is LTD. Salter has suggested that LTD may be evoked by activation of NMDA
receptors present on GABAergic cells due to differential expression of the subunits in the
NMDA receptor (Salter, 2006). LTD in inhibitory cells may lead to central sensitization
(Randic et al., 1993). To what extent this mechanism applies to the dorsal horn is unclear.
Nerve injury also induces a downregulation of the potassium-chloride transporter KCC2. This
trans-synaptic effect on superficial dorsal horn cells (presumed projection neurons of the
spinothalamic tract), leads to a less-negative equilibrium potential for chloride, such that
opening the chloride channel with GABA induces depolarization sufficient to induce
excitation. Consistent with this finding, knockdown of the expression of KCC2 leads to
hyperalgesia (Coull et al., 2003).
Further evidence indicates that nerve injury induces a selective apoptosis of inhibitory
GABAergic inter-neurons (Moore et al., 2002). Apoptosis of inhibitory GABAergic neurons
argues for a hard-wire change in circuitry (Scholz et al., 2005). This is a critical therapeutic
issue. If central sensitization evolves such that nociceptive signaling becomes independent of

inputs from primary afferents, then treating the peripheral nerve may no longer be
therapeutically useful. Moreover, the relative importance of peripheral and central mechanisms
may well depend on the underlying disease. Some diseases may involve primary afferents as
well as the CNS. An example is postherpetic neuralgia where damage may extend from the
primary afferents to the dorsal horn.
Descending Modulatory Mechanisms

Descending modulatory pathways also appear to influence dorsal horn sensitization
mechanisms in neuropathic pain. Cortical, thalamic, and periaqueductal inputs converge on
the rostroventromedial medulla (RVM). This center gives rise to both inhibitory and excitatory
inputs to the dorsal horn via an ipsilateral pathway in the dorsolateral funiculus (DLF). Many
of the cells in the RVM express μ-opioid receptors. Selective ablation of these cells either
before or after establishment of the SNL model can eliminate hyperalgesia (Porreca et al.,
2001). Lidocaine microinjection (Burgess et al., 2002) and hemisection experiments (Bian et
al., 1998) have yielded similar results.
Supraspinal Mechanisms
Recent fMRI studies reveal changes in the processing of cutaneous stimuli at supraspinal
centers in conditions of hyperalgesia and allodynia. Experimental studies of secondary
hyperalgesia allow the comparison of fMRI images before and after the development of
hyperalgesia. In these studies, mechanical stimulation of the skin produced enhanced pain in
the zone of secondary hyperalgesia and led to enhanced activation in areas of the brain
associated with pain signaling (Baron et al., 1999; Maihofner et al., 2004). Whether these
changes in supraspinal processing are passive or whether they reflect additional plasticity
mechanisms is difficult to know. Functional imaging studies in patients with neuropathic pain
reveal that light stroking that produces pain recruits a complex cortical network, including
nociceptive, motor, and cognitive areas (Maihofner et al., 2006; Schweinhardt et al., 2006).
Functional imaging may lead to new insights into the mechanisms of neuropathic pain as well
as to objective measures of altered sensations (Borsook et al., 2004).
The Immune System and Neuropathic Pain

Clearly the immune system is critical to the pain associated with inflammatory pain. The
immune system appears to play a critical role in neuropathic pain as well (Marchand et al.,
2005). Nude rats that lack mature T cells had diminished hyperalgesia (Moalem et al., 2004).
Passive transfer of type 1, but not type 2, T cells led to similar levels of hyperalgesia as those
seen in rats with intact T cell function. In addition, cytokines, II-1, II-6, and TNFα all have
demonstrated roles in neuropathic pain (Manning, 2006). Several variables are worth noting
in approaching the question of immune mechanisms.

• One must distinguish the immune mechanisms associated with a particular disease
from the immune mechanisms that are particular to the issue of neuropathic pain.
• Immune mechanisms need to be considered separately in terms of what happens in
the peripheral nervous system versus those that are important in the central nervous
system.
• Finally, immune mechanisms may play a role in initiating neuropathic pain,
maintaining neuropathic pain acutely, and/or play a role in chronic neuropathic pain.
Peripheral Immune Mechanisms

Nociceptors have a rich variety of immune receptors, and evidence exists for roles of the
interleukins (IL-1β and IL-6), TNFα, bradykinin, prostanoids, and others (Opree and Kress,

2000). In the CCI model of neuropathic pain, a “neuritis” develops, likely reflecting the
inflammatory response to the suture material (Kleinschnitz et al., 2004). In this model, immune
mechanisms might be a particular issue simply because of the foreign suture material. A more
interesting question is what happens with simple surgical axotomy (e.g., the SNL model).
Nerve damage evokes a cascade of immune responses. Nerve damage leads to macrophage
infiltration, T cell activation, and increased expression of proinflammatory cytokines. With
axotomy, Schwann cells are in a sense denervated. These denervated Schwann cells (as well
as cells in the partially denervated target tissue) may communicate with intact fibers that pass
within the same nerve. This provides a mechanism by which to explain changes in the intact
nociceptor (see above). Schwann cell denervation recruits macrophages via secretion of
leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 (MCP-1) (Sugiura
et al., 2000). MCP-1 serves as a ligand for CCR2 (Tofaris et al., 2002). CCR2 knockout mice
do not develop mechanical hyperalgesia after a partial nerve ligation injury (Abbadie et al.,
2003). Cytokine IL-1β leads to increased expression of nerve growth factor (NGF), and NGF
may sensitize nociceptors (Kanaan et al., 1998). Knockout of the IL-1 receptor type I and
genetically mediated overexpression of an IL-1 receptor antagonist decreased hyperalgesia.
That the effect was peripheral was supported by the observation that spontaneous activity
recorded from the dorsal root fibers was reduced (Wolf et al., 2006).
TNFα has received much attention as a culprit in neuropathic pain. Endoneurial administration
induces hyperalgesia (Wagner and Myers, 1996). Preemptive treatment with the TNF-
sequestering drug etanercept decreases hyperalgesia but has no effect once hyperalgesia is
established (Sommer et al., 2001). TNFα can initiate activity in nociceptors (Sorkin et al.,
1997). Inhibition of TNFα blocks phosphorylation of the MAP kinase p38 in DRG and
hyperalgesia but again only when given preemptively (Schäfers et al., 2003). Another cytokine
that has received attention is IL-6. A role in the CCI model has been suggested (Okamoto et
al., 2001), but other peripheral actions are unclear at present.
Central Immune Mechanisms

Somewhat surprising is the mounting evidence that central immune mechanisms play a role in
neuropathic pain (Figure 6) (Watkins and Maier, 2005). As with peripheral mechanisms, much
evidence supports the view that initiation, but not maintenance, of neuropathic pain behaviors
is associated with immune mechanisms (Xu et al., 2006). Microglia serve as the macrophages
of the CNS. Nerve injury distal to the dorsal root ganglia leads to microglial activation.
However, activation after dorsal rhizotomy is much less, in keeping with the observation that
dorsal rhizotomy leads to less-impressive hyperalgesia compared to spinal nerve lesions
involving the identical root (Willis et al., 2006). Interthecal delivery of the antibiotic
minocycline, an inhibitor of microglial activation, attenuates neuropathic pain (Raghavendra
et al., 2003).
The mechanisms are far from clear. Nociceptor activity leads to rapid activation of the MAP
kinase ERK in microglia and initially not in other central cells (Tsuda et al., 2005). Inhibition
of this activation attenuates pain behavior. The mechanisms for this activation might be multi-
factorial. Evidence exists for a role of the ATP receptors P2X4 and/or P2X7, both of which
are expressed on microglia. The chemokine fractalkine may also be involved, as blockade of
its receptor, CX3CR1, attenuates hyperalgesia in neuropathic pain models (Milligan et al.,
2005). The activated microglia produce the toxic cytokines IL-1, IL-6, and TNFα as well as
nitric oxide, excitatory amino acids, ATP, and prostaglandins (Inoue, 2006).
The toll-like receptor-4 (TLR4) is a molecule involved in the innate immune response and is
mainly expressed on microglia. Spinal mRNA for TLR4 is increased after L5 SNL. A knockout
or point mutation in TLR4, as well as downregulation through intrathecal delivery of anti-sense

ODN, has a significant effect on neuropathic pain behavior, but only when given in the early
stages (Tanga et al., 2005).
Conclusion
We began this review by presenting a patient with chest-wall pain following a mastectomy.
The simultaneous finding of decreased sensation and ongoing pain was suggested to be a
paradox. Animal models, as well as studies in humans, however, have taken us a long way
toward understanding this case of neuropathic pain. Ongoing pain likely represents
spontaneous discharge in afferents. The injured afferents are an obvious source of abnormal
input.
Sodium-channel dysfunction likely plays an important role in leading to ectopic generation of

action potentials. The patient had episodes of abrupt “pain attacks.” These attacks may
correspond to bursts of spontaneous activity in the injured afferents related to sodium-channel
dysfunction. Somewhat surprisingly, however, the intact afferents that share the innervation
territory of the injured afferents also discharge spontaneously. Studies of their cell bodies in
the DRG reveal striking phenotype changes. Trophic factors released from the partly
denervated skin working through their receptors on the peripheral terminals may account for
these abnormalities. The abnormalities seen in these “intact” nociceptors likely account for the
fact that patients such as this may respond to therapies applied at the level of the skin. Central
changes also play a role in neuropathic pain. Many of the mechanisms probably are the same
as those observed with inflammatory pain. The patient had pain and hyperalgesia well outside
the region of nerve injury. This is precisely the case in inflammatory pain, where secondary
hyperalgesia extends well beyond the injury site. The patient had substantial pain when the
skin was lightly stroked (allodynia). Multiple lines of evidence indicate that this allodynia is
due to central sensitization, such that tactile afferents acquire synaptic efficacy, which enables
them to trigger activity in central pain signaling neurons. Prevailing work has focused on the
dorsal horn as the site for this sensitization, but more rostral pathways may be involved as well.
Though not easily understood as yet, peripheral nerve injury induces some striking transynaptic
effects. One is apoptosis that appears to preferentially affect GABA inhibitory cells. Several
lines of evidence indicate that immune mechanisms are involved both peripherally and
centrally. Activation of microglial cells occurs in the dorsal horn, and this activation may play
a vital role in initiating central sensitization. The role of this activation in ongoing neuropathic
pain is less clear. The sensation of pain begins with a simple thesis: nociceptors encode
information about noxious stimuli and propagate these messages to the CNS and pain is felt.
In the case of neuropathic pain, however, we see that a rich biology is at play. Studies are
quickly uncovering the mechanisms, and no longer can we consider the presence of this
problem quite so mysterious.
Acknowledgements
We greatly appreciated the suggestions from Drs. Jasenka Borzan, Michael Caterina, and Matthias Ringkamp on earlier
drafts of this manuscript and the technical assistance of Cheryl Carmona. This research was supported by the National
Institutes of Health (NS-014447 and NS-041269) and the Blaustein Pain Treatment Center.
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Figure 1. Animal Models of Neuropathic Pain

(A) Four different nerve injury models are shown. In the spinal nerve ligation (SNL) model,
one or more spinal nerves going to the foot are ligated and cut (Kim and Chung, 1992). In the
partial sciatic ligation (PSL) model, a portion of the sciatic nerve is tightly ligated (Seltzer et
al., 1990). The chronic constriction injury (CCI) model involves placement of four loose
chromic-gut ligatures on the sciatic nerve. An immune response to the sutures leads to nerve
swelling and nerve constriction. In the spared nerve injury (SNI) model, the common peroneal
and tibial nerves are cut, sparing the sural nerve (Decosterd and Woolf, 2000). In each model,
only a portion of the afferents going to the foot are lesioned.
(B and C) Each of these nerve injury models leads to hyperalgesia, which is manifest by
enhanced responses to mechanical, heat, and/or cooling stimuli. (B) To test for mechanical
hyperalgesia, Von Frey monofilaments with different bending forces are applied to the plantar
surface of the foot. The threshold force for paw withdrawal decreases dramatically after the
nerve injury (adapted with permission [Li et al., 2000])
(C) To test for heat hyperalgesia, a radiant heat source is focused onto the plantar surface of
the foot, and the reaction time for paw withdrawal is measured. The difference in reaction time
between the ipsilateral and contralateral foot is calculated. After the SNL, the withdrawal of
the ipsilateral foot is faster than the contralateral foot (negative latency difference), indicating
the presence of heat hyperalgesia (adapted from Kim and Chung [1992] reprinted from Pain,
pp. 355–363, copyright 1992, with permission from the International Association for the Study
of Pain).

Figure 2. A Spinal Nerve Injury Leads to Alterations at Many Sites along the Neural Axis for Pain
Eight different sites of pathophysiological changes are shown. (1) Spontaneous neural activity
and ectopic sensitivity to mechanical stimuli develops at the site of nerve injury. (2) The
expression of different molecules in the dorsal root ganglion of the injured nerve is up- or
downregulated, reflecting the loss of trophic support from the periphery. Spontaneous neural
activity develops in the dorsal root ganglia. (3) The distal part of the injured nerve undergoes
Wallerian degeneration, exposing the surviving nerve fibers from uninjured portions of the
nerve to a milieu of cytokines and growth factors. (4) Partial denervation of the peripheral
tissues leads to an excess of trophic factors from the partly denervated tissue that can lead to
sensitization of primary afferent nociceptors. (5) The expression of different molecules in the
dorsal root ganglion of the uninjured nerve is up- or downregulated, reflecting the enhanced
trophic support from the periphery. (6) Sensitization of the postsynaptic dorsal horn cell
develops, leading to an augmentation of the response to cutaneous stimuli. (7) Activated
microglial cells contribute to the development of this dorsal horn sensitization. (8) Changes in
descending modulation of dorsal horn neurons also may contribute to the enhanced
responsiveness of dorsal horn neurons.

Figure 3. A Model for Sympathetically Maintained Pain

(A) After acutely relieving pain by performing a sympathetic block, norepinephrine in
physiological concentrations was injected intra-dermally in a blinded fashion into the
previously hyperalgesic area. The norepinephrine injections into the affected, but not the
unaffected, extremity produced pain. Norepinephrine does not induce pain in control subjects.
These data suggest that the nociceptors are sensitized to catechols in patients with SMP
(adapted with permission [Ali et al., 2000]). ACUC, area under curve.
(B) In primates, normal nociceptors do not respond to catechol administration. However, in a
monkey-spinal nerve ligation model, nociceptors developed a response to the α 1 adrenergic
agonist phenylephrine administered topically to their receptive field (adapted and used with

permission [Ali et al., 1999]). AP, action potentials. (C) A model to account for SMP. After a
partial nerve lesion, some afferent fibers still remain in the skin. Factors released in the skin
induce the sympathetic efferents to sprout into more superficial areas of the skin (Yen et al.,
2006). These factors also lead the nociceptors to express α 1 adrenergic receptors. Now, the
release of norepinephrine from the sympathetic terminals leads to activation of the nociceptive
terminals, which accounts for the coupling of sympathetic activity with nociceptor responses.

Figure 4. A Genetic Basis for Erythromelalgia

(A) Schematic of the Nav 1.7 voltage-gated sodium channel that is found exclusively in sensory
and sympathetic neurons. A number of point mutations have been identified in this sodium
channel in families of patients with erythromelalgia (Waxman and Dib-Hajj, 2005). The two
mutations shown here produced single amino acid substitutions at the sites indicated.
(B) Whole-cell-patch-clamp recordings in transfected HEK293 cells revealed that these point
mutations lead to an augmentation of the response of the channel to a slow depolarizing ramp
(100 mV in 500 ms). These mutations would be expected to increase the excitability of the
peripheral sensory neuron. (Adapted with permission [Cummins et al., 2004]. Copyright 2004
by the Society for Neuroscience.).

Figure 5. Central Sensitization Mechanisms Involved in the Spinal Nerve Ligation Model
Spontaneous activity from the injured afferents (L5) and intact nociceptors (L4) may sensitize
central pain-signaling neurons. The spontaneous activity in the L5 fibers is restricted to
myelinated afferents. Nociceptive C fibers from L4 spontaneously discharge and may
themselves be sensitized. The enhanced discharge of the primary afferents leads to augmented
response of dorsal horn cells to nociceptor input and increased synaptic efficacy of inputs from
mechanoreceptors (mechanism for allodynia). Alterations in descending modulation and
inhibitory interneuron function also likely play a role.

Figure 6.
Microglial Cells in the CNS Are Activated following Peripheral Nerve Injury and Release
Cytokines that Alter the Responses of Dorsal Horn Cells

Table 1
Examples of Neuropathic Pain Conditions
Disease Pathology Symptoms Hyperalgesia Special Features

Cooling Heat Mechanical
Traumatic neuropathy Axotomy distal to Ongoing pain ++ + ++ Great variety of

DRG presentations
Tic douloureux Compression (often Lightening- ? ? +++ Mechanical
vascular) of like attacks of stimuli evoke
trigeminal nerve near pain attacks of pain
brainstem
Painful diabetic neuropathy Length- Burning pain − + +/− Neuropathy
dependent neuropathy in the feet sometimes
affects small
fibers exclusively
Postherpetic neuralgia Results from damage Burning pain +/− +/− ++ Pain follows the
by herpes zoster distribution of the
infection of peripheral affected
nerve dermatomes

PAIN
Pain Supplement 6 ( 1999) S 121 -S 126
From the gate to the neuromatrix

Ronald Melzack
Department of Psychology. McGill University, 1205 Dr. Penjkld Avenue. Montreal, Quebec H3A IBI, Canada
Abstract
The gate control theory’s most important contribution to understanding pain was its emphasis on central neural mechanisms. The theory
forced the medical and biological sciences to accept the brain as an active system that filters, selects and modulates inputs. The dorsal horns,
too, were not merely passive transmission stations but sites at which dynamic activities (inhibition, excitation and modulation) occurred. The
great challenge ahead of us is to understand brain function. I have therefore proposed that the brain possesses a neural network - the body-
self neuromatrix - which integrates multiple inputs to produce the output pattern that evokes pain. The body-self neuromatrix comprises a
widely distributed neural network that includes parallel somatosensory, limbic and thalamocortical components that subserve the sensory-
discriminative, affective-motivational and evaluative-cognitive dimensions of pain experience. The synaptic architecture of the neuromatrix
is determined by genetic and sensory influences. The ‘neurosignature’ output of the neuromatrix - patterns of nerve impulses of varying
temporal and spatial dimensions - is produced by neural programs genetically build into the neuromatrix and determines the particular
qualities and other properties of the pain experience and behavior. Multiple inputs that act on the neuromatrix programs and contribute to the
output neurosignature include, (1) sensory inputs (cutaneous, visceral and other somatic receptors); (2) visual and other sensory inputs that
influence the cognitive interpretation of the situation; (3) phasic and tonic cognitive and emotional inputs from other areas of the brain; (4)
intrinsic neural inhibitory modulation inherent in all brain function; (5) the activity of the body’s stress-regulation systems, including
cytokines as well as the endocrine, autonomic, immune and opioid systems. We have traveled a long way from the psychophysical concept
that seeks a simple one-to-one relationship between injury and pain. We now have a theoretical framework in which a genetically determined
template for the body-self is modulated by the powerful stress system and the cognitive functions of the brain, in addition to the traditional
sensory inputs. 6 1999 International Association for the Study of Pain. Published by Elsevier Science B.V.
Keywords; Gate control theory: Neuromatrix; Neurosignature
1. Introduction apparatus that resembled the catapult in the comic-strip

‘Hager the Horrible’ and flung cans of food against a cement
In 1959, Patrick Wall had already achieved a reputation surface. Happily for me, Pat convinced them that they could
as a brilliant young scientist who had done important dispense with some of their space to allow me to continue
research on spinal cord physiology. As a result, when I my research on the effects of early sensory deprivation on
arrived at the Massachusetts Institute of Technology in the perception of pain. Thus began a life-long friendship that
September 1959 as an Assistant Professor of Psychology, has been one of the highlights of my life.
I was eager to meet Pat, who was a Professor in M.I.T.‘s During periodic visits to Pat’s lab, where he was invari-
prestigious Department of Biology. We met more quickly ably doing an experiment on the spinal cord, he and I talked
than I expected because I was appalled to discover on the often about our interests in somesthesis and the particularly
day of my arrival that research with animals could not be challenging problem of pain. W.K. Livingston visited me
done in my building and I would have to go elsewhere to do from time to time, and he was delighted to join Pat and me
it. A colleague suggested that I call Pat Wall, who might be during one of our discussions on alternatives to specificity
able to help me. theory. This topic - the need for a new theory - was
Pat detected the desperation in my voice and invited me foremost in our minds, and after a year or so, Pat and I
over. After a warm, friendly conversation, Pat said that decided to write a paper together.
some research space might be available for me. Members When we began our discussions that led to the gate
of the Department of Food Technology in Pat’s building had control theory of pain, we were convinced that (1) brain
funds from a U.S. Space Agency to discover what might processes had to be integrated into the theory, including
happen to cans of food that make a hard landing on the feedforward and feedback transmission; and (2) the new
moon’s surface. To find out, these investigators used an hypothetical spinal cord mechanism would need sufficient
0304-3959/99/$20.00 0 1999 International Association for the Study of Pain. Published by Elsevier Science B.V
PII: s0304-3959(99)00 145- 1
explanatory power to challenge spinal-cord physiologists
and entice them away from the concept of specificity.
How the theory actually came into being involves an
amusing sequence of events. My early research in psychol-
ogy and physiology led me to speculate that the brain exerts
a powerful, continuous descending inhibitory control over
the input that is transmitted through the dorsal horns
(Melzack et al.. 1958). But this notion of modulation of
input by the brain does not constitute a conceptual model
of pain. It could be part of one, but more was needed. In
19.59. Pat was examining the different nerve impulse
patterns evoked in dorsal horn cells by various stimuli and
the way in which vibration modulated the pattern evoked by
noxious stimulation. In I96 1 1 published an article in Scien-
tific American which reviewed the psychology and physiol-
ogy of pain as it was understood at the time. It emphasized
4
patterning, modulation in the dorsal horns, multiple ascend-
ing pathways and the multidimensional qualities of pain
L
I GATE CONTROL SYSTEM
experience. But it was not a cohesive, succinct theory. In

1962. Pat and I (Melzack and Wall, 1962) proposed a
general theory of somesthesis in the form of eight proposi- INPUT ACTION -)
SYSTEM
tions. The paper, published in Brain. evoked some interest
but had little impact. We then toyed with the idea of using
S
this general ‘theory’ as the basis for a theory that dealt
exclusively with pain but we made no headway and put
the project aside.
Then, things unexpectedly and suddenly started to fall Fig. I. The evolution of the gate control theory. (A) show Noordenbos’
into place. It began in the fall of 1962. when I first stumbled model in which large. fast-conductin g fibers Inhibit small. multisynaptic.
onto William Noordenbos’ 1959 book on pain (Noordenbos, slowly conducting fibers. Noordenbos ( 1959) wy\ of the circle that repre-
sents the dorsal horns: ‘In this circle which includes the wbstantia gelati-
1959). That brilliant little book led me to have a ‘flash of
nosa of Rolandi and its immediate adjacent parts. the multitiher pattern of
insight’. afferent impulses is modified... The nature of this inhibitory interaction will
Fig. IA shows Noordenbos’ concept of pain. He did not not be further discussed...’ (B) An early development that led to the pate
fill in the circle in the dorsal horns to show how large fibers control theory in which the large fiber system is shown to actiwtc psycho-
inhibit small ones. He just said that they did, and showed a lopical variables (such as meanin g and past experience) that then project
down to the dorsal horns and modulate the input. (C) Show:, a further
picture of the substantia gelatinosa to illustrate the complex-
development toward the gate control theory which comprises a theoretical
ity of dorsal horn anatomy. He then went on to explain presynaptic inhibitton exerted by the sutxtantin gelatinosa. The idea\
temporal and spatial summation, referred pain, and other gradually evolved into the model of the gate control theory shown ;Ltthe
properties of pain after nerve injury. However, Noordenbos’ bottom.
story stops at the thalamus - the T at the top. My idea was
to put a cortex on Bill’s thalamus, show the dorsal column by mail or during my many visits to Boston where we
projection as a rapid, precise feedforward system to activate consumed large amounts of duty-free whiskey and talked
psychological processes, with a feedback to the circle to late into the night at Pat’s home.
modulate the input (Fig. IB). Here, at last, was the begin- When the gate control theory of pain was published in
ning of a conceptual model in which brain processes can 1965, we were astonished by the reception. The theory
select, filter and modulate pain signals. generated vigorous (sometimes vicious) debate as well as
When I discussed all this with Pat, he began to have ideas a great deal of research to disprove or support the theory.
too. He soon developed a concept, based on his research on The search for specific pain fibers and spinal-cells by our
the substantia gelatinosa, for a hypothetical mechanism to opponents now became almost frantic. It was not until the
put in the circle. A few weeks later he gave me his picture mid-1970’s that the gate control theory was presented in
(Fig. 1C). It may seem an easy step from our two pictures to almost every major textbook in the biological and medical
the final gate model, but it was not. We invented and sciences. At the same time there was an explosion in
rejected a variety of names for the theory and the compo- research on the physiology and pharmacology of the dorsal
nents of the model. It took countless drafts, changes and horns and the descending control systems. The theory’s
compromises to produce the final paper (Melzack and emphasis on the modulation of inputs in the spinal dorsal
Wall. 1965). I moved to McGill University in 1963, so horns and the dynamic role of the brain in pain processes
that most of the paper was written by exchanging drafts had a clinical as well as a scientific impact. Psychological
R. Mel:nck / Pair? Sup~~lmtcwt 6 (1999) S121-SIX s123
factors, which were previously dismissed as ‘reactions to and is identified as the ‘self’, distinct from other people and
pain’ were now seen to be an integral part of pain processing the surrounding world. The experience of a unity of such
and new avenues for pain control were opened. Similarly, diverse feelings, including the self as the point of orientation
cutting nerves and pathways was gradually replaced by a in the surrounding environment. is produced by central
host of methods to modulate the input. Physical therapists neural processes and cannot derive from the peripheral
and other healthcare professionals who use a multitude of nervous system or spinal cord. Fourth, the brain processes
sensory modulation techniques were brought into the that underlie the body-self are, to an important extent which
picture, and TENS became an important modality for the can no longer be ignored, ‘built-in’ by genetic specification,
treatment of chronic and acute pain. although this built-in substrate must, of course, be modified
A major force in this exciting epoch was John Bonica, by experience. These conclusions provide the basis of a new
who had been trying valiantly to convince his medical conceptual model.
colleagues that pain is a syndrome in its own right that How can we explain our experience of the body? I
merits special attention, research and funding. The arrival propose that a genetically built-in matrix of neurons for
of the gate control theory encouraged John to pursue his the whole body produces characteristic nerve-impulse
cause even more vigorously. At the same time, he promoted patterns for the body and the myriad somatosensory quali-
the gate control theory as a focus for new medical ties we feel. 1 have termed the network, whose spatial distri-
approaches. Out of all this ferment of theory, research and bution and synaptic links are initially determined
clinical advances, John brought together a host of scientists genetically and are later sculpted by sensory inputs, a
and clinicians and formed the International Association for ‘neuromatrix’. Thalamocortical and limbic loops that
the Study of Pain (Bonica. 1974). At the same time, the comprise the neuromatrix diverge to permit parallel proces-
journal Pain was created, with Pat as its founding Editor. sing in different components of the neuromatrix and
He has done, and continues to do, a brilliant job of it. The converge to permit interactions between the output products
journal helped establish the held of pain as a major specialty of processing. The cyclical processing and synthesis of
in the health sciences and professions. nerve impulses in the neuromatrix imposes a characteristic
What was the gate control theory’s most important contri- output pattern or ‘neurosignature’.
bution to biological and medical science? I believe it was Loeser and I (Melzack and Loeser, 1978) have presented
the emphasis on CNS mechanisms. Never again, after 1965, a model. consistent with the gate control theory of pain,
could anyone try to explain pain exclusively in terms of which proposes that synaptic areas along the transmission
peripheral factors. The theory forced the medical and biolo- routes of the major sensory projection systems - from the
gical sciences to accept the brain as an active system that dorsal horns to the somatosensory projection areas in the
filters. selects and modulates inputs. The dorsal horns, too, thalamus and cortex - may become pattern generating
were not merely passive transmission stations but sites at mechanisms. Their activity is capable of producing patterns
which dynamic activities - inhibition, excitation and of nerve impulses which exceed a critical firing level per
modulation - occurred. This then was the revolution: we unit time (or have a particular pattern, or both) and project to
highlighted the central nervous system as an essential other areas that subserve pain experience and the localiza-
component in pain processes. tion of pain at specific sites.
This concept is consistent with the fact that injury may
produce high firing levels that signal pain as well as with the
2. The neuromatrix observation that loss of input to central structures by deaf-
ferentation after amputation, root section or cord transection
Where do we go from here? I believe the great challenge also produce high firing levels and abnormal bursting
ahead of us is to understand brain function. My analysis of patterns that may provide the necessary conditions for
phantom limb phenomena (Melzack, 1989; Melzack et al.. pain. Thus. any input to the hyperactive central cells -
1997) has led to four conclusions which point to a new from nearby injured tissues, from visceral sensory nerve,
conceptual nervous system. First. because the phantom from small afferents in the sympathetic chain and from
limb (or other body part) feels so real. it is reasonable to higher psychoneuronal processes - can trigger abnormal.
conclude that the body we normally feel is subserved by the prolonged firing and produce severe, persistent pains in
same neural processes in the brain. These brain processes discrete areas of the denervated limbs or other body parts.
are normally activated and modulated by inputs from the
body but they can act in the absence of any inputs. Second,
all the qualities we normally feel from the body, including 3. Pain and stress
pain, are also felt in the absence of inputs from the body.
From this we may conclude that the origins of the patterns We are so accustomed to considering pain as a purely
that underlie the qualities of experience lie in neural sensory phenomenon that we have ignored the obvious
networks in the brain: stimuli may trigger the patterns but fact that injury does not merely produce pain: it also disrupt
do not produce them. Third, the body is perceived as a unity the brain’s homeostatic regulation systems, thereby produ-
S124 R. Melzack / Pain Supplement 6 (1999) S1214126
cing ‘stress’ and initiating complex programs to reinstate released within minutes, their initial function may be simply
homeostasis. By recognizing the role of the stress system to inhibit or modulate the release of cortisol. Experiments
in pain processes, we discover that the scope of the puzzle of with animals suggest that their analgesic effects may not
pain is vastly expanded and new pieces of the puzzle appear until as long as 30 min after injury.
provide valuable clues in our quest to understand chronic Cortisol, together with noradrenergic activation, sets the
pain (Melzack, 1998, 1999). stage for response to life-threatening emergency. If the
Hans Selye, who founded the field of stress research, dealt output of cortisol is prolonged, excessive or of abnormal
with stress in the biological sense of physical injury, infec- patterning, it may produce destruction of muscle, bone
tion and pathology, and also recognized the importance of and neural tissue and produce the conditions for many
psychological stresses. In recent years, the latter sense of the kinds of chronic pain.
word has come to dominate the field. However, it is impor- Cortisol is an essential hormone for survival after injury
tant for the purpose of understanding pain to keep in mind because it is responsible for producing and maintaining high
that stress is a biological system that is activated by physical levels of glucose for rapid response after injury, threat or
injury, infection or any threat to biological homeostasis as other emergency. However, cortisol is potentially a highly
well as by psychological threat and insult of the body-self. destructive substance because, to ensure a high level of
Both are correct and important. glucose, it breaks down the protein in muscle and inhibits
The disruption of homeostasis by injury activates the ongoing replacement of calcium in bone. Sustained
programs of neural, hormonal and behavioral activity cortisol release, therefore, can produce myopathy, weak-
aimed at a return to homeostasis. The particular programs ness, fatigue and decalcification of bone. It can also accel-
that are activated are selected from a genetically determined erate neural degeneration of the hippocampus during aging.
repertoire of programs and are influenced by the extent and Furthermore, it suppresses the immune system.
severity of the injury. A major clue to the relationships between injury, stress
When injury occurs, sensory information rapidly alerts and pain is that many autoimmune diseases, such as rheu-
the brain and begins the complex sequence of events to matoid arthritis and scleroderma, are also pain syndromes.
reinstate homeostasis. Cytokines are released within Furthermore, more women than men suffer from autoim-
seconds after injury. These substances, such as gamma- mune diseases as well as chronic pain syndromes. Among
interferon, interleukins 1 and 6, and tumour necrosis factor, the 5% of adults who suffer from an autoimmune disease.
enter the bloodstream in l-4 min and travel to the brain. The two out of three are women. Pain diseases also show a sex
cytokines, therefore, are able to activate fibers that send difference, as Berkley (1997) has argued, with the majority
messages to the brain and, concurrently, to breach the prevalent in women, and a smaller number prevalent in men.
blood-brain barrier at specific sites and have an immediate Of particular importance is the concurrent change in sex
effect on hypothalamic cells. The cytokines together with ratios with changes in sex hormone output as a function of
evaluative information from the brain rapidly begin a age. Estrogen increases the release of peripheral cytokines.
sequence of activities aimed at the release and utilization such as gamma-interferon, which in turn produce increased
of glucose for necessary actions, such as removal of debris, cortisol. This may explain why more females than males
the repair of tissues and (sometimes) fever to destroy suffer from most kinds of chronic pain as well as painful
bacteria and other foreign substances. At sufficient severity autoimmune diseases such as multiple sclerosis and lupus.
of injury, the noradrenergic system is activated. Adrenalin is I propose that some forms of chronic pain may occur as a
released into the blood stream and the powerful locus coer- result of the cumulative destructive effect of cortisol on
uleus/norepinephrine (LUNE) system in the brainstem muscle, bone and neural tissue. Furthermore, loss of fibers
projects information upward throughout the brain and in the hippocampus due to aging reduces a natural brake on
downward through the descending efferent sympathetic cortisol release which is normally exerted by the hippocam-
nervous system. Thus the whole sympathetic system is acti- pus. As a result, cortisol is released in larger amounts,
vated to produce readiness of the heart, blood vessels and producing a greater loss of hippocampal fibers and a cascad-
other viscera for complex programs to reinstate homeostasis ing deleterious effect. This is found in aging primates and
(Chrousos and Gold, 1992; Sapolsky, 1992). presumably also occurs in humans. It could explain the
At the same time, the perception of injury activates the increase of chronic pain problems among older people.
hypothalamic-pituitary-adrenal (HPA) system, in which Cortisol output by itself may not be sufficient to cause any
corticotropin-releasing hormone (CRH) produced in the of these problems, but rather it provides the conditions so
hypothalamus enters the local blood stream which carries that other contributing factors may, all together, produce
the hormone to the pituitary, causing the release of adreno- them. Sex-related hormones, genetic predispositions,
corticotropic hormone (ACTH) and other substances. The psychological stresses derived from social competition
ACTH then activates the adrenal cortex to release cortisol, and the hassles of every day life may act together to influ-
which must inevitably play a powerful role in determining ence cortisol release, its amount and pattern, and the effects
chronic pain. Cortisol also acts on the immune system and of the target organs.
the endogeneous opioid system. Although these opioids are These speculations are supported by strong evidence.
R. Melzack / Pain Supplement 6 (1999) S121S126 S125
Chrousos and his colleagues (Chrousos and Gold, 1992) by multiple influences. These influences range from the
have documented the effects of dysregulation of the cortisol existing synaptic architecture of the neuromatrix - which
system: effects on muscle and bone, to which they attribute is determined by genetic and sensory factors - to influ-
fibromyalgia, rheumatoid arthritis and chronic fatigue ences from within the body and from other areas in the
syndrome. They propose that they are caused by hypocorti- brain. Genetic influences on synaptic architecture may
solism, which could be due to depletion of cortisol as a determine, or predispose toward, the development of
result of prolonged stress. Indeed, Sapolsky (1992) attri- chronic pain syndromes. Fig. 2 summarizes the factors
butes myopathy, bone decalcification, fatigue and acceler- that contribute to the output pattern from the neuromatrix
ated neural degeneration during aging to prolonged that produce the sensory, affective and cognitive dimensions
exposure to stress. of pain experience and behavior.
Clearly, consideration of the relationship between stress- We have traveled a long way from the psychophysical
system effects and chronic pain leads directly to examina- concept that seeks a simple one-to-one relationship between
tion of the effects of suppression of the immune system and injury and pain. We now have a theoretical framework in
the development of autoimmune effects. The fact that which a genetically determined template for the body-self is
several autoimmune diseases, such as Crohn’s disease, modulated by the powerful stress system and the cognitive
multiple sclerosis, rheumatoid arthritis, scleroderma and functions of the brain, in addition to the traditional sensory
lupus, are also classified as chronic pain syndromes suggests inputs.
that the study of these syndromes in relation to stress effects The neuromatrix theory of pain - which places genetic
and chronic pain could be fruitful. Immune suppression, contributions and the neural-hormonal mechanisms of stress
which involves prolonging the presence of dead tissue, on a level of equal importance with the neural mechanisms
invading bacteria and viruses, could produce a greater of sensory transmission - has important implications for
output of cytokines, with a consequent increase in cortisol research and therapy. An immediate recommendation is that
and its destructive effects. Furthermore, prolonged immune interdisciplinary pain clinics should expand to include
suppression may diminish gradually and give way to a specialists in endocrinology and immunology. Such a colla-
rebound, excessive immune response. The immune boration may lead to insights and new research strategies
system’s attack on its own body’s tissues may produce auto-
immune diseases that are also chronic pain syndromes.
Thorough investigation may provide valuable clues for
understanding at least some of the terrible chronic pain
syndromes that now perplex us and are beyond our control.
4. The multiple determinants of pain

ViSUSl, luditory nnd
The neuromatrix theory of pain proposes that the neuro- other sensory input
signature for pain experience is determined by the synaptic Tonic somstic input
(trigger points.
architecture of the neuromatrix, which is produced by
genetic and sensory influences. The neurosignature pattern
is also modulated by sensory inputs and by cognitive events,
such as psychological stress. It may also occur because
stressors, physical as well as psychological, act on stress- Fig. 2. The body-self nemomatrix. The body-self neuromatrix, which
regulation systems, which may produce lesions of muscle, comprises a widely distributed neural network that includes somatosensory,
limbic. and thalamocortical components, is schematically depicted as a
bone, and nerve tissue, thereby contributing to the neuro-
circle containing smaller parallel networks that contribute to the sensory-
signature patterns that give rise to chronic pain. In short, the discriminative (S), affective-motivational (A), and evaluative-cognitive (E)
neuromatrix, as a result of homeostasis-regulation patterns dimensions of pain experience. The synaptic architecture of the neuroma-
that have failed, produces the destructive conditions that trix is determined by genetic and sensory influences. The ‘neurosignature’
may give rise to many of the chronic pains that so far output of the neuromatrix-patterns of nerve impulses of varying temporal
and spatial dimensions-is produced by neural programs genetically built
have been resistant to treatments developed primarily to
into the neuromatrix and determines the particular qualities and other prop-
manage pains that are triggered by sensory inputs. The stress erties of the pain experience and behavior. Multiple inputs that act on the
regulation system, with its complex, delicately balanced neuromatrix programs and contribute to the output neurosignature include
interactions, is an integral part of the multiple contributions (1) sensory inputs from somatic receptors (phasic cutaneous, visceral and
that give rise to chronic pain. tonic somatic inputs); (2) visual and other sensory inputs that influence the
cognitive interpretation of the situation; (3) phasic and tonic cognitive and
The neuromatrix theory guides us away from the Carte-
emotional inputs from other areas of the brain; (4) intrinsic neural inhibitory
sian concept of pain as a sensation produced by injury, modulation inherent in all brain function; and (5) the activity of the body’s
inflammation, or other tissue pathology and toward the stress-regulation systems, including cytokines as well as the endocrine.
concept of pain as a multidimensional experience produced autonomic. immune and opioid systems.
S126 R. Melzack/ Pain Supplement 6 (1999) S12/-S126
that may reveal the underlying mechanisms of chronic pain Melzack R, Wall PD. On the nature of cutaneous sensory mechanisms.
and give rise to new therapies to relieve the tragedy of Brain 1962;85:331-356.
Melzack R, Wall PD. Pain mechanisms: a new theory. Science
unrelenting suffering.
1965;150:971-979.
Melzack R, Stotler WA, Livingston WK. Effects of discrete brainstem
lesions in cats on perception of noxious stimulation. J Neurophysiol
Acknowledgements 1958;21:353-367.
Melzack R, Israel R, Lacroix R, Schultz G. Phantom limbs in people with
This paper was supported by grant #A7891 from the congenital limb deficiency or amputation in early childhood. Brain
Natural Sciences and Engineering Research Council of 1997:120:1603-1620.
Melzack R. Pain and stress: clues toward understanding chronic pain. In:
Canada.
Sabourin M. Craik F, Robert M, editors. Advances in psychological
science, Vol. 2. Biological and Cognitive Aspects. Hove: Psychology
Press, 1998. pp. 63-85.
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central ‘pattern generating mechanism’ for pain. Pain 1978;4:195-210. Cambridge, MA: MIT Press, 1992. pp. 287-324.
Molecular mechanisms of nociception

David Julius* & Allan I. Basbaum†
*Department of Cellular and Molecular Pharmacology, and †Departments of Anatomy and Physiology and W. M. Keck Foundation Center for
Integrative Neuroscience, University of California San Francisco, San Francisco, California 94143, USA (e-mail: julius@socrates.ucsf.edu)
The sensation of pain alerts us to real or impending injury and triggers appropriate protective responses.
Unfortunately, pain often outlives its usefulness as a warning system and instead becomes chronic
and debilitating. This transition to a chronic phase involves changes within the spinal cord and brain,
but there is also remarkable modulation where pain messages are initiated — at the level of the primary
sensory neuron. Efforts to determine how these neurons detect pain-producing stimuli of a thermal,
mechanical or chemical nature have revealed new signalling mechanisms and brought us closer to
understanding the molecular events that facilitate transitions from acute to persistent pain.
J
ust as beauty is not inherent in a visual image, root ganglia (DRG), respectively, and can be categorized into
pain is a complex experience that involves not three main groups based on anatomical and functional crite-
only the transduction of noxious environmental ria (Fig. 1a). Cell bodies with the largest diameters give rise to
stimuli, but also cognitive and emotional myelinated, rapidly conducting A primary sensory fibres.
processing by the brain. Progress has been made Most, but not all4, A fibres detect innocuous stimuli applied
in identifying cortical loci that process pain messages, but to skin, muscle and joints and thus do not contribute to pain.
far greater advances have been made in understanding the Indeed, stimulation of large fibres can reduce pain, as occurs
molecular mechanisms whereby primary sensory neurons when you activate them by rubbing your hand. By contrast,
detect pain-producing stimuli, a process referred to as small- and medium-diameter cell bodies give rise to most of
nociception. These insights have arisen predominantly the nociceptors, including unmyelinated, slowly conducting
from the analysis of sensory systems in mammals, as well C fibres and thinly myelinated, more rapidly conducting A
as from studies of invertebrates. Of course, invertebrate fibres, respectively. It has long been assumed that A and C
organisms do not experience pain per se, but they do have nociceptors mediate ‘first’ and ‘second’ pain, respectively,
transduction mechanisms that enable them to detect and namely the rapid, acute, sharp pain and the delayed, more
avoid potentially harmful stimuli in their environment. diffuse, dull pain evoked by noxious stimuli5 (Fig. 1b).
These signalling pathways can be regarded as the There are two main classes of A nociceptor6; both
evolutionary precursors of nociceptive processing in respond to intense mechanical stimuli, but can be
vertebrates, and genetic studies have facilitated the distinguished by their differential responsiveness to intense
identification and functional characterization of molecules heat or how they are affected by tissue injury. Most C-fibre
and signalling pathways that contribute to the detection of nociceptors are also polymodal, responding to noxious
noxious stimuli in animals. Indeed, many of the receptors thermal and mechanical stimuli6. Others are mechanically
and ion channels we refer to here are related to molecules insensitive, but respond to noxious heat. Importantly, most
highlighted in the accompanying reviews on C-fibre nociceptors also respond to noxious chemical stimuli,
mechanosensation, vision or olfaction in flies or worms. such as acid or capsaicin, the pungent ingredient in hot chilli
peppers. Finally, the natural stimulus of some nociceptors is
The primary afferent nociceptor difficult to identify. These so-called ‘silent’ or ‘sleeping’ noci-
Nearly a century ago, Sherrington proposed the existence of ceptors are responsive only when sensitized by tissue injury7.
the nociceptor, a primary sensory neuron that is activated by These nociceptor profiles derive largely from analysis of
stimuli capable of causing tissue damage1. According to this fibres that innervate skin. But very different features charac-
model, nociceptors have characteristic thresholds or sensi- terize nociceptors in other tissues6. For example, although
tivities that distinguish them from other sensory nerve corneal afferents can be activated by capsaicin and sensitized
fibres. Electrophysiological studies have, in fact, shown the by inflammatory mediators, pain is normally produced by
existence of primary sensory neurons that can be excited by innocuous tactile stimulation. In teeth, almost any stimulus
noxious heat, intense pressure or irritant chemicals, but not produces pain. Visceral pain is unique in that there are no
by innocuous stimuli such as warming or light touch2. In this first (fast) and second (slow) components; instead, pain is
respect, acute pain can be regarded as a sensory modality often poorly localized, deep and dull8. Tissue damage is also
much like vision or olfaction, where stimuli of a certain not required for visceral pain to occur; it can result from
quality or intensity are detected by cells with appropriately excessive distension (for example, of the colon). And the pain
tuned receptive properties. of ischaemia may have unique features that reflect innerva-
tion of vasculature by distinct subsets of acid-sensitive
Many types of nociceptors for many types of pain primary sensory nociceptors. These features illustrate the
Pain is unique among sensory modalities in that electrophysi- difficulty of defining a nociceptor based only on activation
ological recordings of single primary sensory fibres have been threshold or on whether its activation evokes pain.
made in awake humans, allowing simultaneous measure-
ment of psychophysical responses when regions of the head The neurochemistry of nociceptors
and body are stimulated3. Fibres that innervate regions of the Glutamate is the predominant excitatory neurotransmitter in
head and body arise from cell bodies in trigeminal and dorsal all nociceptors. Histochemical studies of adult DRG, however,
a b
Primary afferent axons Aα β
Thermal threshold
Aα and Aβ fibres
Voltage
Myelinated
Large diameter None
Proprioception, light touch
Aδ C
Aδ Fibre
Lightly myelinated Time
Medium diameter ~ 53 °C Type I
Nociception
(mechanical, thermal, chemical) ~ 43 °C Type II First pain
C fibre Second pain

Unmyelinated
Small diameter
Innocuous temperature, itch ~ 43 °C
Nociception
(mechanical, thermal, chemical)
Figure 1 Different nociceptors detect different types of pain. a, Peripheral nerves action potential recording from a peripheral nerve. Most nociceptors are either A or C
include small-diameter (A) and medium- to large-diameter (A,) myelinated afferent fibres, and their different conduction velocities (6–25 and ~1.0 m s–1, respectively)
fibres, as well as small-diameter unmyelinated afferent fibres (C). b, The fact that account for the first (fast) and second (slow) pain responses to injury. Panel b adapted
conduction velocity is directly related to fibre diameter is highlighted in the compound from ref. 75.
reveal two broad classes of unmyelinated C fibre. The so-called pep- spinal cord ‘pain’ transmission neurons (central sensitization), or
tidergic population contains the peptide neurotransmitter substance lowering of nociceptor activation thresholds (peripheral sensitiza-
P, and expresses TrkA, the high-affinity tyrosine kinase receptor for tion). With central sensitization, pain can be produced by activity in
nerve growth factor (NGF)9. The second population does not express non-nociceptive primary sensory fibres. Peripheral sensitization is
substance P or TrkA, but can be labelled selectively with the -D-galac- produced when nociceptor terminals become exposed to products of
tosyl-binding lectin IB4, and expresses P2X3 receptors, a specific tissue damage and inflammation, referred to collectively as the
subtype of ATP-gated ion channel. This categorization is a first approx- ‘inflammatory soup’ (Fig. 3). Such products include extracellular
imation at best — as additional molecular markers become available, protons, arachidonic acid and other lipid metabolites, serotonin,
new subsets are likely to be recognized. It is, however, unclear whether bradykinin, nucleotides and NGF, all of which interact with receptors
these neurochemically distinct groups represent different functional or ion channels on sensory nerve endings. Because nociceptors can
classes of nociceptor. Moreover, because expression of neurotransmit- release peptides and neurotransmitters (for example, substance P,
ters, their receptors and other signalling molecules are dramatically calcitonin-gene-related peptide and ATP) from their peripheral
altered after tissue or nerve injury10,11, both the significance and the terminals when activated by noxious stimuli, they are able to facilitate
complexity of nociceptor neurochemistry are increased. production of the inflammatory soup by promoting the release of
factors from neighbouring non-neuronal cells and vascular tissue, a
Diversity of nociceptor signalling phenomenon known as neurogenic inflammation5.
All sensory systems must convert environmental stimuli into electro- In contrast to vision, olfaction or taste, sensory nerve endings that
chemical signals. In the case of vision or olfaction, primary sensory detect painful stimuli are not localized to a particular anatomical
neurons need only detect one type of stimulus (light or chemical odor- structure, but are instead dispersed over the body, innervating skin,
ants) and use redundant and convergent biochemical mechanisms to muscle, joints and internal organs. Although this has made the bio-
accomplish this goal (Fig. 2a). In this regard, nociception is unique chemical analysis of nociceptive pathways particularly challenging,
because individual primary sensory neurons of the ‘pain pathway’ have the combined application of electrophysiological, pharmacological
the remarkable ability to detect a wide range of stimulus modalities, and genetic methods are generating significant progress in
including those of a physical and chemical nature. Compared with understanding the molecular basis of nociceptor signalling. In some
sensory neurons of other systems, nociceptors must therefore be respects, the field can be compared to the state of cellular immunolo-
equipped with a diverse repertoire of transduction devices (Fig. 2b). At gy some 25 years ago, when one of the main goals was to define
the same time, markedly different stimuli of a chemical (capsaicin and biochemical markers for specific subsets of lymphocytes. The goal for
acid) or physical (heat) variety can excite nociceptors by activating a nociceptor research is to elucidate signalling functions for key
single receptor, enabling the cell to integrate information and respond cell-surface markers and to assign physiological roles to molecularly
to complex changes in the physiological environment. defined sub-populations of sensory neurons.
Primary afferent nociceptors are also unique in the extent to
which their receptive properties can be modulated. Thus, nocicep- Detectors of noxious stimuli
tors not only signal acute pain, but also contribute to persistent and Response to heat
pathological pain conditions (allodynia) that occur in the setting of Remarkably, many functional characteristics of nociceptors are
injury, wherein pain is produced by innocuous stimuli5,12. Allodynia retained when sensory ganglia are dissociated and placed into cul-
can result from two different conditions: increased responsiveness of ture13. Thus, ~45% of small- to medium-diameter neurons exhibit
Figure 2 Polymodal nociceptors use a greater diversity of a

signal-transduction mechanisms to detect physiological
Light Odorants
stimuli than do primary sensory neurons in other systems.
a, In mammals, light or odorants are detected by a
convergent signalling pathway in which G-protein-coupled
receptors modulate the production of cyclic nucleotide
second messengers, which then alter sensory neuron
excitability by regulating the activity of a single type of
cation channel. b, In contrast, nociceptors use different cGMP cAMP
signal-transduction mechanisms to detect physical and
chemical stimuli. Recent studies suggest that TRP-
b
channel family members (VR1 and VRL-1) detect noxious
heat, and that ENaC/DEG-channel family detect
mechanical stimuli. Molecular transducers for noxious
cold remain enigmatic. Noxious chemicals, such as ?
capsaicin or acid (that is, extracellular protons) may be
detected through a common transducer (VR1), illustrating
aspects of redundancy in nociception. At the same time, a A
N A N
single type of stimulus can interact with multiple detectors,
C C C
as shown by the ability of extracellular protons to activate A N
not only VR1, but also ASICs, which are also members of
the ENaC/DEG-channel family.
heat-evoked membrane currents with a ‘moderate’ threshold of and C fibres, but exhibited markedly reduced pain behaviour at
~45 C, whereas another 5–10% of cells respond with a ‘high’ higher temperatures (>50 C). In other words, VR1–/– animals recog-
threshold of ~52 C and are insensitive to capsaicin14,15. The former nize a noxious heat stimulus, but discriminate poorly among stimuli
corresponds presumably to C and type II A nociceptors, and the of different noxious intensities. Perhaps the threshold for a behav-
latter to type I A nociceptors. What is the molecular basis by which ioural pain response is determined by the thermal threshold of a
specific thermal thresholds are established in these nociceptor sub- small number of nociceptors, whereas discrimination among
types? The answer came from a well-proven path of success in the pain suprathreshold temperatures requires information from a larger
field, namely identifying the molecular targets through which natural cohort of nociceptors that adequately encode stimulus intensity. In
products (for example, morphine from the opium poppy or aspirin some respects, the phenotype of the VR1–/– mouse illustrates the
from willow bark) produce or modulate our sensation of pain. futility of a long-lasting debate as to whether pain is generated by
In the case of moderate thermal nociception by C and type II A activity in specific nociceptors that are connected to the spinal cord
afferents, a transducer was revealed with the cloning and functional through a dedicated pathway, or whether it is the pattern of activity
characterization of the vanilloid receptor VR1 (Fig. 2b), which is across afferents that determines the pain response. Both models are
activated by capsaicin and other vanilloid compounds16. VR1 is a likely to be relevant, depending on the magnitude of the stimulus and
non-selective plasma-membrane cation channel possessing a very steep the context in which it is measured. Moreover, because most nocicep-
temperature dependence (Q1020.6) and a thermal activation thresh- tors are polymodal, our ability to distinguish pain sensations
old of ~43 C, characteristics that are shared with native heat-evoked resulting from heat, cold or pressure must involve decoding of
currents in sensory neurons17,18. The strong correlation between nociceptive signals within the central nervous system.
moderate heat and capsaicin sensitivity, and the similarity of the Another striking and significant phenotype of the VR1–/– mouse is
non-selective cationic currents and pharmacology underlying these its failure to develop increased sensitivity to heat in the context of
responses14,15 support the hypothesis that heat and capsaicin activate a tissue injury20,21. This deficit suggests that VR1 is modulated by one or
common transducer. Heat-evoked single-channel currents are more components of the inflammatory soup, which act on the
observed in membrane patches excised from sensory neurons or VR1- nociceptor to increase its sensitivity to heat.
expressing cells, indicating that VR1 is an intrinsically heat-sensitive What mediates high-threshold heat responses in type I A affer-
channel that functions as a molecular thermometer at the cell surface17. ents? One candidate transducer is the vanilloid receptor-like
The high correlation that exists between heat and capsaicin (VRL-1) channel (Fig. 2b) that shares ~50% sequence identity with
sensitivity in sensory neurons at the whole-cell level is less pronounced VR1 (ref. 22). Functional analysis of VRL-1 in transfected
at the single-channel level19. Although this observation could be mammalian cells and frog oocytes showed that this channel is not
explained by different functional states of the channel, it has raised responsive to vanilloid compounds, but can be activated by noxious
some controversy regarding the link between the activities of native thermal stimuli with a threshold of ~52 C. VRL-1 is most
and cloned channels. But studies of mice lacking functional VR1 chan- prominently expressed by a subset of medium- to large-diameter
nels20,21 clearly show that cultured DRG neurons from VR1-null mice myelinated neurons within the DRG. But because VRL-1 transcripts
are severely deficient in moderate heat-evoked responses, whereas are also expressed outside the sensory nervous system, this channel
high-threshold heat responses persist. VR1–/– mice also have signifi- probably serves multiple physiological functions22,23.
cantly fewer (>threefold) heat-sensitive C fibres, and total heat-evoked VR1 and VRL-1 belong to the larger family of transient receptor
C-fibre output may be decreased by ~85% compared with wild-type potential (TRP) channels, whose core transmembrane structure
mice. Thus, although VR1 is not the only detector of moderate-thresh- resembles that of voltage-gated potassium or cyclic nucleotide-gated
old heat stimuli, it accounts for the majority of such responses and channels (reviewed in ref. 24). The prototypical TRP channel was
must contribute significantly to thermal coding in normal animals. discovered in the Drosophila phototransduction pathway, where it is
Somewhat paradoxically, VR1–/– mice showed normal behaviour- activated downstream of phospholipase C (PLC)-coupled rhodopsin
al responses at temperatures near the threshold for activation of VR1 (see review in this issue by Hardie and Raghu, pages 186–193). Some
mammalian TRP channels are also activated by G-protein-coupled One way to detect pressure or tissue deformation is through acti-
or tyrosine kinase receptors that stimulate PLC, but as in the fly, the vation of a mechanically gated protein. Another mechanism might
underlying gating mechanism remains enigmatic. Recent studies involve a ‘mechanochemical’ process whereby stretch evokes the
indicate that PLC-mediated hydrolysis of the membrane phospho- release of a diffusible chemical messenger that then excites nearby
lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and primary sensory nerve terminals. Extracellular ATP is of particular
the consequent production of lipid second messengers constitute interest because large- and small-diameter sensory neurons express
important steps in TRP channel activation25 (see below). G-protein-coupled ATP receptors or ATP-gated ion channels (P2Y
and P2X receptors, respectively), and because extracellular ATP
Detection of noxious cold excites primary sensory neurons33. Using frog oocytes as a model
The definition of cold sensitivity is less stringent compared with heat system, Nakamura and Strittmatter34 showed that mechanical stimu-
sensitivity, in large part because thermal thresholds for activation of lation can release ATP from the cell, promoting autocrine activation
cold-sensitive fibres may not be as distinct or precipitous as they are of P2Yreceptors on the cell’s surface. In vivo, mechanical force might
with heat, and because the threshold for cold-evoked pain is not as therefore promote ATP release from one or more cell types in the
precise. Whereas noxious heat (47 C) activates ~50% of C fibres that periphery, where it could activate purinergic receptors (for example,
innervate the hindpaw of a rodent, noxious cold (4 C) may excite P2X3) on nearby nociceptor terminals.
only 10–15% of fibres within the same cutaneous receptive field20. In fact, P2X3-deficient mice show reduced urination stemming
However, a significantly greater proportion of C and A fibres are from a failure to empty a full bladder35. Filling and stretching of the
classified as cold-sensitive when the range of stimulus intensities urinary bladder promotes the release of ATP from epithelial cells that
extends below 0 C (ref. 26). Few, if any, cold-sensitive (4 C) afferents lie beneath the smooth muscle layer. Once released, ATP may excite
are heat sensitive, and VR1–/– mice show a normal prevalence of sensory nerve endings embedded within the bladder wall to initiate
cold-responsive fibres in the hindpaw20, indicating that noxious heat the voiding reflex. The deficit observed in P2X3–/– mice provides
and cold are detected by distinct mechanisms. It is not known compelling evidence that ATP transduces signals of mechanical
whether noxious cold depolarizes nerve fibres by inhibiting a tissue distension into depolarization of primary sensory neurons, in
(Na+K+)ATPase or background potassium current27, or by pro- this case through direct activation of an ion channel.
moting calcium and/or sodium influx28,29.
Chemical transducers to make the pain worse
Pressure and mechanical stress As described above, injury heightens our pain experience by increas-
Nociceptors can be activated by mechanical stress resulting from ing the sensitivity of nociceptors to both thermal and mechanical
direct pressure, tissue deformation or changes in osmolarity, stimuli. This phenomenon results, in part, from the production and
enabling the detection of touch, deep pressure, distension of a viscer- release of chemical mediators from the primary sensory terminal and
al organ, destruction of bone, or swelling. Functional, mechanically from non-neural cells (for example, fibroblasts, mast cells,
gated channels have yet to be identified at the molecular level in neutrophils and platelets) in the environment36 (Fig. 3). Some com-
eukaryotes, although model genetic organisms such as bacteria, ponents of the inflammatory soup (for example, protons, ATP,
worms and flies have provided important leads for identifying serotonin or lipids) can alter neuronal excitability directly by inter-
mechanosensory transducers in mammals (see review in this issue by acting with ion channels on the nociceptor surface, whereas others
Gillespie and Walker, pages 194–202). One such ion channel of the (for example, bradykinin and NGF) bind to metabotropic receptors
degenerin (DEG/ENaC) family, called MDEG (alternatively, brain and mediate their effects through second-messenger signalling
sodium channel 1 (BNC1) or acid-sensing ion channel 2 (ASIC2); cascades11. Considerable progress has been made in understanding
Fig. 2b)30, has attracted particular interest in the pain field because its the biochemical basis of such modulatory mechanisms.
messenger RNA is expressed in primary sensory neurons31. Respons-
es of primary sensory fibres from BNC1-deficient mice to a range of Extracellular protons and tissue acidosis
mechanical stimuli identified a specific class of rapidly adapting Local tissue acidosis is a hallmark physiological response to injury,
mechanoreceptors that showed reduced sensitivity to hair move- and the degree of associated pain or discomfort is well correlated with
ment32. Other types of afferents, including C fibres, showed normal the magnitude of acidifica-
responses in these mutant mice, indicating that BNC1 is involved in tion37. Application of acid Representative
Stimulus
some aspects of innocuous mechanical (touch) sensation, but not in (pH 5) to the skin produces receptor
the detection of noxious mechanical stimuli. sustained discharges in a third NGF TrkA
Bradykinin BK2
Serotonin 5-HT3
Mast cell or ATP P2X3
Figure 3 The molecular complexity of the primary afferent nociceptor is illustrated H+ ASIC3/VR1
neutrophil
by its response to inflammatory mediators released at the site of tissue injury. Lipids PGE2/CB1/VR1
Some of the main components of the ‘inflammatory soup’ are shown, including Heat VR1/VRL-1
Substance Pressure
peptides (bradykinin), lipids (prostaglandins), neurotransmitters (serotonin (5-HT) P DEG/ENaC ?
and ATP) and neurotrophins (NGF). The acidic nature of the inflammatory soup is
Histamine DRG cell body
also indicated. Each of these factors sensitize (lower the threshold) or excite the Bradykinin NGF
terminals of the nociceptor by interacting with cell-surface receptors expressed by Tissue 5-HT
these neurons. Examples of these factors and representative molecular targets injury
Prostaglandin
are indicated in the box. Activation of the nociceptor not only transmits afferent ATP H+
messages to the spinal cord dorsal horn (and from there to the brain), but also CGRP
Substance P
initiates the process of neurogenic inflammation. This is an efferent function of the
nociceptor whereby release of neurotransmitters, notably substance P and Blood
calcitonin gene related peptide (CGRP), from the peripheral terminal induces vessel
vasodilation and plasma extravasation (leakage of proteins and fluid from
postcapillary venules), as well as activation of many non-neuronal cells, including Spinal cord
mast cells and neutrophils. These cells in turn contribute additional elements to
the inflammatory soup. Figure adapted from refs 75,76.
206 © 2001 Macmillan Magazines Ltd NATURE | VOL 413 | 13 SEPTEMBER 2001 | www.nature.com
Figure 4 When nociceptors are exposed to Ca2+

Na+
products of injury and inflammation, their BK
VR1
excitability is altered by a variety of intracellular TrkA H+
signalling pathways. The figure highlights the
vanilloid receptor (VR1) and tetrodotoxin-resistant AEA
(TTX-R) voltage-gated sodium channels (Nav1.8
and 1.9) as downstream targets of modulation. PLC-γ
(+)
Responses of VR1 to heat can be potentiated by PLC-β Na+
Gq
direct interaction of the channel with extracellular PKC-ε TTX-R
(+)
protons (H+) or lipid metabolites, such as PKA (Nav1.8
anandamide (AEA). VR1 activity can also be and 1.9)
(-) AC (+)
heightened by agents such as NGF or bradykinin, Gi Gs
which bind to their own cell-surface receptors (TrkA
and BK, respectively) to stimulate phospholipase C
(PLC- or PLC-) signalling pathways. This, in
turn, leads to hydrolysis of plasma membrane lipids AEA PGE2
and the subsequent stimulation of protein kinase C
isoforms, such as PKC-
. Both of these actions CB (or opiate) PG
have been proposed to potentiate VR1 function.
Prostaglandins (PGE2) and other inflammatory products that activate adenylyl cyclase (AC) through Gs-coupled receptors also enhance nociceptor excitability. This occurs, in part, by
a cyclic AMP-dependent protein kinase (PKA)-dependent phosphorylation of Nav1.8 and/or Nav1.9. By activating Gi-coupled receptors, opiates and cannabinoids can counteract
these increases in excitability of the nociceptor, and produce a peripherally mediated analgesia.
or more of polymodal nociceptors that innervate the receptive field20. do indeed show a marked reduction in sustained proton (pH 5)-
At the cellular level, protons depolarize sensory neurons by directly evoked membrane currents. But proton-evoked responses, particu-
activating a non-selective cationic current38,39. In many DRG larly those of a transient nature, are not eliminated completely in
neurons, this response consists of a transient, rapidly inactivating VR1–/– mice and may be mediated by members of the ASIC channel
current that is carried predominantly by Na+ ions, followed by a family.
sustained non-selective cationic current. Responses of a sustained Lazdunski and colleagues44 described a family of two-transmem-
nature have been proposed to underlie persistent pain associated brane-domain proteins that are related to the putative mechanosen-
with tissue acidosis37, but this may not occur in all physiological sory DEG/ENaC channels. These novel cation-channel subunits
settings. For example, during cardiac ischaemia, DRG neurons that were named ASICs because of their ability to be gated by reductions
innervate the epicardium show very large, but transient, response to in extracellular pH when expressed in heterologous systems (Fig. 2b).
extracellular protons40. A number of proton-sensitive channels are Including splice variants, there are at least five ASIC subtypes (1a, 1b,
found on sensory neurons and thus an important goal has been to 2a, 2b and 3) in rats, each having a unique profile of pH sensitivity,
determine which, if any, of these molecules contributes to proton activation and desensitization rates, ionic permeability and tissue
sensitivity of nociceptors in vivo. Two main candidates have emerged distribution. Most subtypes are expressed in DRG, with ASIC1b and
— VR1 and a family of ASICs. ASIC3 (known also as ASIC- and DRASIC, respectively) showing
The similarity between native proton (pH 5)-evoked and exclusive or preferential expression within sensory ganglia. Can
capsaicin-evoked currents in dissociated DRG neurons41 is well ASIC channels account for any aspect of transient or sustained pro-
established, and low extracellular pH can augment responses of ton-evoked currents observed in DRG neurons? Heterologous
cultured DRG neurons to capsaicin42. These observations suggested expression of most ASIC subunits produces currents consisting of a
that protons and vanilloid compounds interact with the same ion- single transient phase, or one having a sustained component that is
channel complex on the nociceptor (perhaps providing a cellular Na+-selective or observed only at non-physiological proton
rationale for the culinary appeal of ‘hot’ and sour soup). Analysis of concentrations (pH 5). However, co-expression of ASIC3 with
the cloned vanilloid receptor in heterologous expression systems has ASIC2b (a splice variant of MDEG, also called MDEG2) generates a
substantiated these observations. Protons have two main effects on more native-like current containing a sustained component with
VR1 function17. First, VR1 can be activated at room temperature non-selective cation permeability30. McCleskey and colleagues40 have
when the extracellular pH drops below 6, producing currents that provided strong evidence that ASIC3/2b heteromeric channels,
resemble the sustained component of proton-evoked responses rather than VR1, underlie the unusually large and mostly transient
observed in sensory neurons. Second, protons potentiate responses proton-evoked currents observed in the relatively small subpopula-
to capsaicin or heat, and do so over a concentration range (pH 6–8) tion (2–3%) of DRG afferents that innervate the heart. This makes
that matches the extent of local acidosis associated with various good physiological sense because occlusion of a cardiac artery
forms of tissue injury. These changes in VR1 activity would be produces modest acidosis (to just below pH 7), but this is sufficient to
expected to increase nociceptor excitability, even at normal body activate cardiac nociceptors or ASIC3/2b.
temperature. Structure–function studies have now identified several By contrast, genetic studies indicate that MDEG (BNC1) gene
negatively charged residues within putative extracellular loops of products (ASIC2a and 2b) do not contribute to acid sensitivity in
VR1 that are important for mediating these effects18,43, supporting most non-cardiac nociceptors32. Thus, BNC1–/– mice showed no
the idea that protons interact directly with the vanilloid receptor to obvious decrement in pH 5-evoked responses among large- or
allosterically modulate channel function. small-diameter DRG neurons. Moreover, acid produced normal
Although proton-evoked changes in VR1 thermal sensitivity sustained discharges in cutaneous C fibres from these animals. Gene
closely resemble those exhibited by nociceptors during inflamma- knockouts of other members of this family will be needed to clarify
tion, does VR1 actually contribute to pH sensitivity of nociceptors in the contribution of ASICs to proton detection and nociceptor
vivo? DRG neurons or sensory nerve fibres from VR1-deficient mice sensitization at various physiological sites.
Peptides and growth factors pro-algesic agents that activate PLC. TRP channels in the fly eye exist
Tissue damage promotes the release or production of bioactive in a complex with other components of the phototransduction
peptides from non-neural cells and plasma proteins at the site of machinery (see review by Hardie and Raghu), an arrangement that
injury45. Principal among these is the nonapeptide bradykinin, influences sensitivity and kinetics of the signalling process57,58.
which, when applied to primary sensory nerve terminals or cultured Interestingly, VR1 forms a signalling complex with TrkA and PLC-
sensory neurons, produces immediate membrane depolarization as in heterologous systems50. Whether a similar signalling scaffold exists
well as sensitization to other noxious, or even innocuous stimuli46. in nociceptors remains to be determined, but phylogenetic compar-
Bradykinin activates G-protein-coupled (BK2) receptors on these isons suggest that this might well be the case.
cells to stimulate PLC-catalysed hydrolysis of PtdIns(4,5)P2,
consequently releasing Ca2+ from intracellular stores and activating Sensitization and activation by lipids
protein kinase C (PKC) (Fig. 4). Treatment of cultured DRG neurons The efficacy of non-steroidal anti-inflammatory agents, such as
with bradykinin augments heat-evoked currents in these cells47, an aspirin, is attributed generally to blockade of cyclooxygenase (COX)
effect that may be mediated through direct or indirect modification enzymes that convert arachidonic acid, a lipid messenger, into pro-
of VR1 by the
-isoform of PKC48 (Fig. 4). PKC-
-knockout mice inflammatory prostanoid products, notably prostaglandin E2
exhibit reduced thermal and mechanical hypersensitivity after (PGE2). Most studies indicate that PGE2 contributes to peripheral
treatment with adrenaline or acetic acid49, but effects of bradykinin sensitization by binding to G-protein-coupled receptors that
have not been reported. In any event, molecular validation of this increase levels of cyclic AMP within nociceptors45. However, it now
pathway will require biochemical proof that VR1 is phosphorylated seems likely that cyclooxygenase products are also present in the
in response to BK2-receptor activation and that mutation of one or spinal cord, where they could interact with receptors on the central
more phosphate-accepting residues abrogates channel modulation. terminals of nociceptors59. This idea has aroused great interest
Bradykinin might also heighten VR1 sensitivity through a PKC- because it argues that COX inhibitors may exert their pain-relieving
independent process50. In this mechanism, channel modulation effects by modulating nociception at both peripheral and central
occurs as a direct consequence of PLC-mediated PtdIns(4,5)P2 sites60.
hydrolysis, in effect releasing VR1 from PtdIns(4,5)P2-mediated Recent studies have provided important information on a likely
inhibition. Precedence for such a regulatory mechanism comes from molecular target through which PGE2 sensitizes primary sensory
studies of cyclic nucleotide-gated channels51 and G-protein-gated fibres. Nociceptors express a specific subclass of voltage-gated
inwardly rectifying potassium channels52,53. Moreover, as mentioned sodium channel that is resistant to blockade by tetrodotoxin61. These
above, several members of the TRP channel family are activated TTX-R Na+ channels are believed to contribute significantly to
downstream of PLC-coupled receptors, and recent evidence from action-potential firing rate and duration in small-diameter sensory
both vertebrate and invertebrate systems suggests that PtdIns(4,5)P2 neurons. Electrophysiological studies suggest that PGE2 increases
hydrolysis also is important in modulating the activity of these excitability of DRG neurons, in part by shifting the voltage depen-
channels24,25. Exogenously applied lipids, such as anandamide, dence of TTX-R Na+-channel activation in the hyperpolarizing
arachidonate or diacylglycerol, have been shown to activate VR1 (see direction. This reduces the extent of membrane depolarization
below) or other TRP channels (see review by Hardie and Raghu, needed to initiate an action potential and favours repetitive spiking.
pages 186–193), raising the possibility that these hydrophobic Pharmacological and biochemical studies indicate that PGE2-depen-
agonists exert their effects by displacing PtdIns(4,5)P2 or other dent modulation of the TTX-R Na+ current involves phosphoryla-
membrane lipids from an inhibitory site on the channel complex. tion of the channel protein by cAMP-dependent protein kinase A62–64
NGF is best known as a survival factor for embryonic neurons and (Fig. 4). It is not known which of the two known TTX-R Na+-channel
is essential for the development of all primary sensory nociceptors. subtypes within DRG (Nav1.8 or Nav1.9)65–67 are targeted by lipids,
But in the adult, NGF serves a very different function, being released and under what conditions this occurs. Behavioural analysis of
by mast cells, fibroblasts and other cell types at sites of injury and Nav1.8-deficient mice revealed modest deficits in acute sensation to
inflammation, where it acts on primary sensory nerve terminals to noxious stimuli68. These animals also showed a delayed onset of
promote thermal hypersensitivity54. Consistent with this action, inflammatory thermal hypersensitivity, but the maximal
Mendell and colleagues55 showed that exposure of cultured DRG response was equivalent to that of wild-type animals. Such observa-
neurons to NGF for several minutes produces acute sensitization of tions support the involvement of Nav1.8 in tissue injury-evoked
capsaicin-evoked responses. Although NGF elicits long-term hypersensitivity, but also suggest that there is redundancy among
changes in gene expression, its ability to promote short-term voltage-gated Na+ channel subtypes in nociceptor function.
nociceptor sensitization suggests that post-translational mecha- Capsaicin is a hydrophobic molecule that bears structural
nisms also are involved. similarity to several lipid second messengers (Fig. 5), leading many to
NGF binds to TrkA tyrosine kinase receptors on peptidergic sen- suggest that lipid metabolites might serve as endogenous vanilloid-
sory neurons to activate mitogen-activated protein (MAP) kinase receptor agonists. The hunt for such ligands has been further
and PLC- signalling pathways56. When frog oocytes expressing TrkA advanced by the realization that the vanilloid receptor belongs to the
and VR1 are treated with NGF for several minutes, proton-, vanil- TRP channel family, some members of which can be activated by
loid- or heat-evoked responses are potentiated, recapitulating the polyunsaturated fatty acids or other lipid metabolites (see review by
sensitization that occurs in cultured DRG neurons or primary senso- Hardie and Raghu, pages 186–193). Indeed, we and others have
ry fibres. A TrkA mutant that specifically disrupts coupling of the shown that the endogenous cannabinoid-receptor agonist anan-
receptor to PLC- abrogates NGF enhancement of VR1, consistent damide (arachidonylethanolamide), or lipoxygenase products of
with the potentiation mechanism outlined for bradykinin50. The arachidonic-acid metabolism (for example, 12- and 15-(S)-
importance of the MAP-kinase pathway in the regulation of gene hydroperoxyeicosatetraenoic acid), activate native or cloned vanil-
expression is well appreciated, but the physiological significance of loid receptors when applied to whole cells or excised membrane
PLC activation to neurotrophin action has been enigmatic. These patches containing VR1 channels69–71. These lipid messengers are
findings now suggest that PLC signalling contributes to NGF-medi- admittedly weak as VR1 agonists (EC50 ~ 1–10 M at 25 C, pH 7.6),
ated thermal hypersensitivity and possibly to other forms of at least when compared to capsaicin, and this has fuelled some debate
short-term, post-translational nociceptor modulation (Fig. 4). as to whether they should be considered ‘endovanilloids’72. But the
VR1–/– mice do not develop thermal hypersensitivity in response to physiological setting in which these molecules are likely to act as VR1
NGF or bradykinin treatment50, indicating that this channel is a agonists is one involving inflammation, and thus the relevant
probable downstream target for modulation by these and other question should be whether they act synergistically with other
nociceptor. Functional presynaptic purinergic receptors have also
Capsaicin been described and the source of ATP can be identified73. By contrast,
O presynaptic vanilloid receptors will never be exposed to noxious
H 3CO
N
thermal temperatures or to the magnitude of pH changes that
H regulate VR1 gating at the peripheral terminal of the nociceptor, and
HO thus other ligands must be considered. Lipid mediators, such as
Olvanil
anandamide, may be relevant. There is evidence for presynaptic reg-
O ulation of neurotransmitter release through an action of anandamide
H3CO
N at both CB1 cannabinoid and vanilloid receptors in the dorsal horn74.
H Similarly, the function of central ASICs is unclear, nor is it known
HO whether novel endogenous ligands for these channels exist.
AM404
Future directions
HO
O One of the main challenges is to understand how both the specific
physiological properties of nociceptors and the circuits that they
N
H engage in the central nervous system determine pain perception and
resultant behaviour. Molecular markers make it possible to identify
Anandamide and manipulate the activity of subsets of nociceptors, so facilitating
O the mapping of spinal cord and brainstem circuits that are engaged by
HO
N
specific nociceptor populations. It is important to understand the
H function of the many neurotransmitters, receptors and transducers
that are expressed by nociceptors and the significance of their tran-
15-HPETE O
scriptional and post-translational regulation in the setting of injury.
Although opioids and non-steroidal anti-inflammatory agents are
HO analgesic drugs of choice for the treatment of pain, their utility is
often limited by unacceptable side-effects due to actions at identical
HOO receptors outside of the pain pathway. Because many of the channels
and receptors described in this review seem to be unique to the
Figure 5 An alignment of natural and synthetic vanilloid receptor agonists illustrates nociceptor (for example, TTX-R Na+ channels, P2X3 and VR1), they
their structural similarity. Olvanil is a synthetic, non-pungent capsaicin analogue that represent promising targets for the development of new and highly
activates VR1 with relatively slow kinetics. Anandamide is an endogenous lipid selective local anaesthetics and analgesics for treating a wide variety
metabolite (similar in structure to arachidonic acid) that was initially discovered as a of persistent pain conditions. ■
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ARTICLE IN PRESS
Medical Hypotheses (2006) x, xxx–xxx
http://intl.elsevierhealth.com/journals/mehy
Chronic pain: A non-use disease

L. Pruimboom *, A.C. van Dam
Faculty of Human Sciences, University of Gerona, C/Francesc Masia 65 Salt, Spain
Received 20 July 2006; accepted 1 August 2006
Summary One of the major problems in modern medicine is to find remedies for the group of people with chronic
pain syndromes. Low back pain is one of the most frequent syndromes and perhaps the most invalidating of all of them.
Chronic pain seems to develop through several pathways affecting the spinal cord and the brain: (1) neuro-anatomical
reorganisation, (2) neuro-physiological changes, and (3) activation of glia cells (immune reaction in the central nervous
system). Although all of these pathways seem to provide a (partial) plausible explanation for chronic pain, treatments
influencing these pathways often fail to alleviate chronic pain patients. This could be because of the probability that
chronic pain develops by all three mechanisms of disease. A treatment influencing just one of these mechanisms can
only be partially successful. Other factors that seem to contribute to the development of chronic pain are
psychosocial. Fear, attention and anxiety are part of the chronic pain syndrome being cause or consequence. The three
pathways and the psycho-emotional factors constitute a psycho-neuro-immunological substrate for chronic pain
syndromes; a substrate which resembles the substrate for phantom pain and functional invalidity after stroke. Both
phantom pain and functional invalidity are considered non-use syndromes. The similarity of the substrate of both these
two neurological disorders and chronic pain makes it reasonable to consider chronic pain a non-use disease (the
hypothesis).
To test this hypothesis, we developed a ‘‘paradoxal pain therapy’’. A therapy which combines the constraint induced
movement therapy and strategies to dissociate pain from conditioning factors like fear, anxiety and attention. The aim
of the therapy is to establish a behaviour perpendicular on the pathological pain-behaviour. Clinically, the treatment
seems promising, although we just have preliminary results. Further clinical and laboratory studies are needed to
measure eventual changes at neuro-anatomical and neuro-psychological level using modern neuro-imaging instruments
(PET, SPECT, fMRI). Randomised clinical trials should be carried out to test our hypothesis for all-day use in clinical
practice. The hypothesis: chronic pain is a non-use disease produced by psycho-emotional factors like fear, attention
and anxiety. Optimal treatment should be based on physiological use, and dissociation of pain and the mentioned
psycho-emotional factors. Paradoxal pain therapy could serve these treatment conditions.
c 2006 Published by Elsevier Ltd.
Introduction
Finding remedies for patients with chronic pain is

* Corresponding author. Present address: Faculty of Human
one of the most difficult and at the same time
Sciences, University of Gerona, C/Los Helechos 5a, 35500
Arrecife, Las Palmas, Spain. Tel.: +34 928844959.
one of the most intruding objectives of modern sci-
E-mail address: leo.pruimboom@fudgif.udg.es entific investigation. The last decade has brought a
(L. Pruimboom). lot of new insides in the mechanisms of disease for

0306-9877/$ - see front matter c 2006 Published by Elsevier Ltd.
doi:10.1016/j.mehy.2006.08.036
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
people with chronic pain syndrome. Neurological lated to changes of the homeostasis, ranging from
imaging techniques like positron evoked tomogra- tissue damage to hypoxia and acidosis. The intero-
phy (PET) and functional MRI (fMRI) have made it ceptive afferents enter the spinal cord via lamina
possible to visualise changes at neurological, histo- I neurons (in the so called heat-pinch-cold neurons),
logical and immunological level. Resuming the re- cross to the other side of the spinal cord and ascend
sults of the most revealing work of the last as a spinothalamic tract through homeostatic cen-
decade [1–3] it seems obvious that mechanisms tres in the brain stem and thalamus, ending up in
of disease for developing chronic pain include: the insular cortex and gyrus cingularis. Their theory
is that chronic pain is caused by central sensitisat-
1. Neuro-anatomical reorganisation in the spinal ion of the so called ‘‘neuromatrix of homeostasis’’
cord and the brain. and involves neuro-anatomical and neuro-physio-
2. Neuro-physiological changes in the spinal cord logical pathways. For an excellent review of la-
and the brain. belled line interoceptive afferents and pain, I
3. Activation of glia cells (immune reaction in the would like to refer to [5].
central nervous system). Wieseler-Frank et al. [6,7] describe chronic pain
as a process of activation of glia cells in the spinal
Craig et al. [2,4] have developed a reasonable cord. Glia cells (microglia and astrocytes) normally
amount of evidence that pain is an efferent homeo- exhibit minor (astrocytes) or no activity (microglia)
static feeling produced as a reaction on labelled in physiological circumstances. Several factors,
line interoceptive afferent information. Interocep- like long term use of morphine, virus, trauma and
tive afferents transport all kind of information re- ischemia, can activate glia cells (Fig. 1). Once glia
Figure 1 Glia-cell activation as a direct cause for enhanced activity of excitatory neurons in the spinal cord,
provoking a pain sensation. Interoceptive afferents can be activated by a great deal of peripheral homeostatic
imbalances. All of them can activate glia cells in lamina I of the spinal cord. Glia cell activation can continue with
absence of the initial activator.
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
cells are activated they produce pro-inflammatory behaviour characteristics are anxiety and continu-
cytokines like interleukin 6 (IL6), interleukin 1 ous focusing on the pain. Fear, anxiety and chronic
(IL1) and tumor necrosis factor alpha (TNFa). These focusing can be considered ‘‘homeostatic feeling’’.
substances are able to activate excitatory neurons All these homeostatic feeling activate the so called
in the spinal cord responsible for afferent pain interoceptive cortex (insular cortex) and the gyrus
(interoceptive) transmission. Chronic activation of cingularis [4,5]. When fear, anxiety, pain and pain-
glia cells can produce long term changes in neuro- focusing activate the interoceptive cortex at the
nal activity in spinal cord and brain neurons respon- same time, in the end they will be neurologically
sible for pain transmission; changes which could be connected:
responsible for the development of chronic pain
‘‘When two neurons fire together they wire
syndromes.
together and if they don’t they won’t’’
Results of clinical trials using antagonistic drugs
of pro-inflammatory cytokines (glia-modulator) The described process of classical and operant
show that these kind of treatments are promising conditioning will lead to the typical neuro-psycho-
for people with chronic pain syndromes [8–11]. logical substrate for chronic pain patients:
Current treatment of patients with chronic pain
syndrome is based on the neuro-immunological There is no pain without fear [21];
pathways of developing chronic pain. Tricyclic There is no pain without anxiety [21];
antidepressants [12,13], gabapentin and morphine There is no pain without attention [22] and;
[14] are the most frequently used drugs combined There is no pain without gain [21].
with anti-inflammatory drugs (NSAID), glia-modula-
tor drugs and cognitive psychotherapy. Effective- This pain-behaviour reduces the use of the af-
ness of these treatments has been proven, fected area. Reduced or non-use of a part of the
although there still is a group of non-reacting pa- body results in a neuro-anatomical reorganisation
tients, and secondary effects of pain-drugs are in various parts of the brain [15,23]; a situation sim-
considerable. ilar to patients with phantom pain. Patients with
For these reasons we have developed a new phantom pain are successfully treated with con-
therapy for patients with chronic pain based on straint induced movement [(CIMT,15,23)]. A therapy
the similarity of the scientific substrate of both based on limitation of the non-affected side of the
phantom pain and chronic pain [15,16]. Phantom body and massive use (6 h a day) of a functional pros-
pain is considered a non-use syndrome. If chronic thesis adapted to the amputated extremity.
pain patients present a similar substrate as pa- ‘‘Paradoxal pain therapy’’ is based on a combi-
tients with non-use syndromes, than chronic pain nation of CIMT and dissociation-techniques of neu-
could also be considered a non-use syndrome. If ronal connections.
so then:
First Chronic pain patients should hardly use The hypothesis

there affected part of the body;
Second Constraint induced use of the affected Chronic pain is a non-use disease provoked by psy-
part of their body should change the pain cho-emotional factors like fear, attention and anx-
intensity and function and; iety [25]. Treatment should be based on 1.
Third Deep learning and massive training of the physiological use, and 2. on dissociation of pain
affected part of the body should provide and the mentioned psycho-emotional factors.
a re-reorganisation of neuro-anatomical ‘‘Paradoxal pain therapy’’ could serve these treat-
structure in the brain and spinal cord. ment conditions.
Neuro-physiological changes should
include neurotransmitters and glia-cell
activation. Paradoxal pain therapy
The theory 1. Deep learning therapy.

2. Dissociation-techniques Inform the environ-
People with chronic pain tend to ‘‘fly’’ from their ment to ‘‘forget’’ the pain of the affected
affected part of the body. Fear for pain and more patient. Pain-associated behaviour of the
damage seems to be the mayor reason for this part patient is not rewarded anymore (loss of gain
of the typical pain-behaviour [17–20]. Other pain- and attention).
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
3. Constraint induced use of affected area (from Hence, these conditions for constraint
fearful non-use to relaxed massive use). induced movement training in patients
4. Illusion technique as an early pain-distraction with chronic pain vary compared with the
strategy. CIMT for patients with phantom pain or
stroke. The adaptation is based on the fact
Ad. 1. Patients are informed about the influence that the affected part of the body is still
of emotions (fear, anxiety) and attention there and tissue overload has to be
on their pain syndrome. Further informa- avoided. Studies of Moseley [24] made it
tion is given about the pathways that clear that overload can be prevented if
lead to the neuro-immunological changes the patient chooses intensity (load, the
in the spinal cord and brain [24]. It is number of repetitions, pause between
important to state that the patient’s the repetitions and the context in which
problem is not a psychological but a neu- the task is executed). The CIMT period
rological problem. The profound explana- duration in chronic pain patients varies
tion (deep learning) activates from 8 days to 14 days. After one month
motivational areas in the brain; areas a new training program starts (if neces-
which belong to the so called central sary) adapted to the actual functions of
reward circuit [25]. Several parts of the the patient. Task difficulty depends on
central reward circuit are able to pro- function and not on pain. Task may not
duce a serial of pain-inhibiting neuro- provoke fear or anxiety.
transmitters like endorphins, dopamine Restoration of function is probably the
and GABA. Activation of these areas by missing link treating patients with chronic
deep learning enhances pain-killing pro- pain. Current cognitive/behavioural- and
cesses and diminishes fear and anxiety medical therapies to often focus on pain
by rational understanding of the pain pro- killing alone [26]. The CIMT recovers nor-
cess. Deep learning explanation has to be mal function, offering physiological infor-
profound and understandable for every mation to the brain areas responsible for
individual and includes neuro-physiologi- interoceptive/homeostatic analysis.
cal, neuro-anatomical and psycho-emo- These areas can produce the homeostatic
tional pathways related with chronic feeling ‘‘pain’’ but would not do so if the
pain. information, which enters these areas is
Ad. 2. Patient and the direct environment are completely normal.
informed about the influence of attention Ad. 4. Patients with chronic pain often (almost
related to pain and pain-behaviour. Pain- always) start a new day focusing on their
behaviour should be avoided (for pain. ‘‘How will I feel today? Will I be in
instance, hand supporting the low back agony? This means that although they feel
in patients with chronic low back pain) no pain at that moment, their brain (and
by the patient and ignored by his/her spinal cord) is already occupied with pain
environment. and fear for pain. To prevent this focusing
Ad. 3. Patient trains her/his affected part of the moment (with a huge impact on pain-con-
body using a massive training program of ditioning processes) at the beginning of
difficult motor tasks, constraining other the day, patients have to carry out a so
parts of the body. The therapist can help called ‘‘illusion therapy’’. This therapy
to execute the tasks if necessary. Before (therapy conditions see frame) has proven
starting the program the therapist valo- its effects in laboratory investigations of
rises the actual functions of the patient. patients with pain in our clinic. Effects
After inventorying the different tasks, seem to be mediated by changes in endor-
training begins. The amount of repetitions phin and dopamine production in the
depends on resistance declared by the reward circuit of the brain. Both natural
patient. This means that the patient and neurotransmitters are able to regulate
the therapist choose respectively, the neurological activation in pain transmis-
intensity of training and the task. A train- sion neurons [27]. Another goal is to dis-
ing session lasts not more than 60– tract patient’s attention to the pain,
90 min. The constraint part of the body being distraction-therapy one of the most
has to be constraint for at least 50% of promising treatments for chronic pain
wakeful daytime. patients [28–30].
(2006), doi:10.1016/j.mehy.2006.08.036
ARTICLE IN PRESS
[3] Taub E et al. A placebo-controlled trial of constraint-

Illusion therapy induced movement therapy for upper extremity after
stroke. Stroke 2006;37(4):1045–9.
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2005;14(4):166–74.
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Review Articles, Systematic Reviews and Meta-Analyses
Eur Surg Res 2012;49:35–43 Received: March 22, 2012

Accepted after revision: April 28, 2012
DOI: 10.1159/000339613
Published online: July 11, 2012
Wound Repair and Regeneration

J.M. Reinke H. Sorg
Department of Plastic, Hand and Reconstructive Surgery, Hannover Medical School, Hannover, Germany
Key Words tion of granulation tissue and the restoration of the vascular
Angiogenesis ⴢ Epithelialization ⴢ Granulation tissue ⴢ network. Therefore, next to the immigration of local fibro-
Inflammation ⴢ Scarring blasts along the fibrin network and the beginning of reepi-
thelialization from the wound edges, neovascularization
and angiogenesis get activated by capillary sprouting. The
Abstract formation of granulation tissue stops through apoptosis of
The skin is the biggest organ of the human being and has the cells, characterizing a mature wound as avascular as well
many functions. Therefore, the healing of a skin wound dis- as acellular. During the maturation of the wound the compo-
plays an extraordinary mechanism of cascading cellular nents of the extracellular matrix undergo certain changes.
functions which is unique in nature. As healing and regen- The physiological endpoint of mammalian wound repair dis-
eration processes take place in all parts of the human body, plays the formation of a scar, which is directly linked to the
this review focuses on the healing processes of the skin and extent of the inflammatory process throughout wound
highlights the classical wound healing phases. While regen- healing. Copyright © 2012 S. Karger AG, Basel
eration describes the specific substitution of the tissue, i.e.
the superficial epidermis, mucosa or fetal skin, skin repair
displays an unspecific form of healing in which the wound
heals by fibrosis and scar formation. The first stage of acute Introduction
wound healing is dedicated to hemostasis and the formation
of a provisional wound matrix, which occurs immediately af- An intact outer sheath is one of the most important
ter injury and is completed after some hours. Furthermore, features from simple bacteria to complex multicellular
this phase initiates the inflammatory process. The inflamma- organisms. Furthermore, the ability of organisms to re-
tory phase of the wound healing cascade gets activated dur- pair or regenerate tissues in order to restore organ func-
ing the coagulation phase and can roughly be divided into tions has been and still is a selective advantage and a
an early phase with neutrophil recruitment and a late phase survival factor in nature. As most of the organisms are
with the appearance and transformation of monocytes. In subject to a continuous renewal process throughout life,
the phase of proliferation the main focus of the healing pro- the ability to heal is developed differently throughout di-
cess lies in the recovering of the wound surface, the forma- verse species from simple tissue repair to the regenera-
62.151.188.247 - 6/1/2013 9:12:52 AM
© 2012 S. Karger AG, Basel Heiko Sorg, MD

0014–312X/12/0491–0035$38.00/0 Department of Plastic, Hand and Reconstructive Surgery, Hannover Medical School
Fax +41 61 306 12 34 Carl-Neuberg-Strasse 1
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E-Mail karger@karger.ch Accessible online at: DE–30625 Hannover (Germany)

www.karger.com www.karger.com/esr Tel. +49 511 5320, E-Mail sorg.heiko @ mh-hannover.de
tion of complete organs as shown in axolotls (Ambysto- process of skin wound healing is often described as a
ma mexicanum). Wound healing, mostly meaning the playing orchestra or as the acts of a drama [3, 4], whose
healing of the skin, has been recognized as important to interplay of cells, growth factors and cytokines ends up
health since the beginning of mankind. Papyrus scrolls in a closure of the skin. However, even when this sensitive
from old Egypt (3,200–300 BC) already describe wound balance between cells and mediators might be disrupted,
care procedures with the use of compression for hemo- recent data suggest that the deficiency of a cell type or the
stasis. They also describe wound dressing techniques by absence of a mediator can be compensated by others that
Hippocrates, indicating the importance of pus drainage are involved in wound healing so that the repair can still
from the wound (‘Ubi pus, ibi evacua’), and Galen, de- occur [5]. The process of wound healing can artificially
picting the principles of wound healing by primary and be divided into three to five phases which overlap in time
secondary intention. Much of this knowledge, however, and space [6]. The purpose of this review is to highlight
got lost over time and has been newly or rediscovered in in detail the actual distinct processes during skin wound
the modern era by Brunschwig, von Gersdorff and Para- healing.
celsus. In the late 19th century the development of anti-
sepsis by Lister and Semmelweis, the detection of patho-
genic microorganisms by Koch, and most notably the The Vascular Response: Hemostasis and Coagulation
discovery of penicillin by Fleming and of sulfonamides
by Domagk [1] had an enormous impact on the under- The first stage of physiological or acute wound healing
standing, therapy and outcome of wound healing. Now- is dedicated to hemostasis and the formation of a provi-
adays, research highlights a deeper understanding of the sional wound matrix, which occurs immediately after in-
complex interplay of cells and the distinct influence of jury and is completed after some hours (fig. 1). Further-
the different cytokines and growth factors, and unfolds more, this phase initiates the inflammatory process.
the molecular biology of skin wound healing. But after Sometimes this phase is also described as the ‘lag-phase’,
5,000 years of wound therapy the goals have not changed. in which the organism has to manage the recruitment of
The patient still deserves a fast, uncomplicated and anti- the many cells and factors for the healing process in the
septic wound closure, but also claims an aesthetic out- absence of the mechanical strength of the wound [1].
come with unimposing scar formation. As healing and With a skin injury outreaching the epidermal layer, blood
regeneration processes take place in all parts of the hu- and lymphatic vessels are traumatized, flushing the
man body, this review will focus on the healing process- wound to remove microorganisms and antigens [7]. The
es of the skin and will highlight the classical wound heal- different clotting cascades are then initiated by clotting
ing phases. factors from the injured skin (extrinsic system), and
thrombocytes get activated for aggregation by exposed
collagen (intrinsic system). At the same time the injured
Physiology of Adult Skin Wound Healing vessels follow a 5- to 10-min vasoconstriction, triggered
by the platelets, to reduce blood loss and fill the tissue gap
Skin wound healing is a dynamic and highly regulated with a blood clot comprised of cytokines and growth fac-
process of cellular, humoral and molecular mechanisms tors [8]. Furthermore, the blood clot contains fibrin mol-
which begins directly after wounding and might last for ecules, fibronectin, vitronectin and thrombospondins,
years. Every tissue disruption of normal anatomic struc- forming the provisional matrix as a scaffold structure for
ture with consecutive loss of function can be described as the migration of leukocytes, keratinocytes, fibroblasts
a wound [2]. Integumental injuries are defined as open or and endothelial cells and as a reservoir of growth factors.
outer wounds, whereas inner or closed wounds describe The life-saving vasoconstriction with clot formation ac-
injuries or ruptures of inner organs and tissues with a still counts for a local perfusion failure with a consecutive lack
intact skin. The closure of a skin wound can be realized of oxygen, increased glycolysis and pH-changes [9]. The
by regeneration or repair. While regeneration describes vasoconstriction is then followed by a vasodilation in
the specific substitution of the tissue, i.e. the superficial which thrombocytes invade the provisional wound ma-
epidermis, mucosa or fetal skin, skin repair displays an trix [1]. In addition, platelets influence the infiltration of
unspecific form of healing in which the wound heals by leukocytes by the release of chemotactic factors. Both
fibrosis and scar formation. The latter, unfortunately, platelets and leukocytes release cytokines and growth
presents the main form in adult skin wound healing. The factors to activate the inflammatory process (IL-1␣, IL-
62.151.188.247 - 6/1/2013 9:12:52 AM
36 Eur Surg Res 2012;49:35–43 Reinke/Sorg

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Color version available online
Phase 1: inflammatory phase (days 1–3)
Cutaneous wound
Epidermis Platelet
invasion
for
hemostasis
Artery
Vein
Dermis
Vasoconstriction
Neutrophil
Hemostasis
Hair follicle invasion for Invasion of
Extrinsic
clotting phagocytosis and macrophages
system secretion of IL-1␣,
Subcutis IL-1␤, IL-6 and
Blood vessels TNF-␣
Fig. 1. Inflammatory phase after a cutane-
ous cut; hemostasis and invasion of in-
flammatory cells.
1␤, IL-6 and TNF-␣), stimulate the collagen synthesis Neutrophils release mediators such as TNF-␣, IL-1␤
(FGF-2, IGF-1, TGF-␤), activate the transformation of fi- and IL-6, which amplify the inflammatory response and
broblasts to myofibroblasts (TGF-␤), start the angiogen- stimulate VEGF and IL-8 for an adequate repair response.
esis (FGF-2, VEGF-A, HIF-1␣, TGF-␤) and already sup- Furthermore, they start their debridement by releasing
port the reepithelialization process (EGF, FGF-2, IGF-1, highly active antimicrobial substances (cationic peptides
TGF-␣) [10]. The vasodilation can also be recognized by and eicosanoids) and proteinases (elastase, cathepsin G,
a local redness (hyperemia) and by an edema of the proteinase 3 and a urokinase-type plasminogen activa-
wound. tor) [5]. In vitro studies further showed that neutrophils
could change the phenotype and cytokine profile expres-
sion of macrophages, which leads to an innate immune
The Cellular Response: Inflammation response during healing [11].
Approximately 3 days after injury macrophages enter
The inflammatory phase of the wound healing cas- the zone of injury and support the ongoing process by
cade gets activated during the hemostasis and coagula- performing phagocytosis of pathogens and cell debris
tion phase and can roughly be divided into an early phase [12, 13] as well as by the secretion of growth factors, che-
with neutrophil recruitment and a late phase with the ap- mokines and cytokines. Apart from their actual support
pearance and transformation of monocytes (fig. 1). Due in wound healing, these molecules keep the healing pro-
to the response of the activated complement pathway, de- cess intact, as some of them are able to activate the next
granulated platelets and by-products of bacterial degra- phase of wound healing (proliferative phase) [14]. The in-
dation, neutrophils are recruited to the site of the skin flammatory response to injury is essential for supplying
injury and are present for 2–5 days unless the wound gets growth factor and cytokine signals that are responsible
infected. The work of the neutrophils is crucial within the for cell and tissue movements, which are crucial for the
first days after injury because their ability in phagocyto- subsequent repair mechanisms in adult mammalians [5,
sis and protease secretion kills local bacteria and helps to 15]. There is evidence that the amount of inflammation
degrade necrotic tissue. Furthermore, they act as che- determines the extent of scar formation. The lack of in-
moattractants for other cells that are involved in the intrauterine inflammation is mentioned as evidence for the
flammatory phase [5]. theory of scarless wound healing in fetuses [16, 17].
62.151.188.247 - 6/1/2013 9:12:52 AM
Wound Repair and Regeneration Eur Surg Res 2012;49:35–43 37

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Macrophages have many functions including host de- the intercellular desmosomes via collagenase and elas-
fense, the promotion and resolution of inflammation, the tase, activated keratinocytes migrate along the preformed
removal of apoptotic cells and the support of cell prolif- fibrin blood clot in the higher layers of the granulation
eration and tissue restoration following injury [18]. Be- tissue. This process is called the ‘shuffling’ of keratino-
side their immunological functions as antigen-present- cytes [27] and describes the ability of these cells to mi-
ing cells and phagocytes during wound repair, macro- grate competitively along a chemotactic gradient estab-
phages supposedly play an integral role in a successful lished by mediators such as IL-1, and over a fibronectin-
healing response through the synthesis of numerous po- rich matrix into the center of the wound [28, 29].
tent growth factors such as TGF-␤, TGF-␣, basic FGF, Lamellipodial crawling performs the migration itself and
PDGF and VEGF, which promote cell proliferation and is directed into the defect site via the polymerization of
the synthesis of extracellular matrix (ECM) molecules by cytoskeletal actin fibers in the excrescence and the for-
resident skin cells [19]. mation of a new focal adhesion at the ECM which is me-
diated by integrins. These cytoskeletal mechanisms are
regulated by RhoGTPases (Rho, Rac, Cdc42) [30, 31].
Proliferation and Repair Small GTPases are the shifters of the intracellular orga-
nization of fibers and are essential for an orchestrated
Reepithelialization/Resurfacing epithelialization process as well as the termination of mi-
In the phase of proliferation (approx. 3–10 days after gration. This process proceeds until the migrating cells
wounding) the main focus of the healing process lies in touch each other. Then, the GTPases will probably be
covering the wound surface, the formation of granulation turned off leading to a reorganization of the cytoskeleton
tissue and restoring the vascular network. Therefore, [27]. The fusion of the opposing epithelia is realized by a
next to the immigration of local fibroblasts along the fi- degradation of the actin fibers in filopodia, which are re-
brin network and the beginning of reepithelialization placed by intercellular adherence contacts to finally close
from the wound edges, neovascularization and angiogen- the wound like a zipper [27].
esis get activated by capillary sprouting [1, 7, 20–22]. Un-
der the control of regulating cytokines like IFN-␥ and Neovascularization/Angiogenesis
TGF-␤, the synthesis of collagen, fibronectin and other The restoration of the vascular system of the skin is a
basic substances needed for wound healing by fibroblasts complex cascade of cellular, humoral and molecular
represents the basis for the new matrix of connective tis- events in the wound bed to reconnect to the nutritive per-
sue, serving for the closure of tissue gaps and the restora- fusion. Initiators are growth factors, e.g. VEGF, PDGF,
tion of the mechanical strength of the wound. Subse- bFGF and the serine protease thrombin. The first step in
quently, the synthesis of collagen increases throughout new vessel formation is the binding of growth factors to
the wound, while the proliferation of fibroblasts declines their receptors on the endothelial cells of existing vessels,
successively, adjusting a balance between synthesis and thereby activating intracellular signaling cascades. The
degradation of the ECM [23]. activated endothelial cells secrete proteolytic enzymes
The reepithelialization process is ensured by local ke- which dissolve the basal lamina. Thus, the endothelial
ratinocytes at the wound edges and by epithelial stem cells are now able to proliferate and migrate into the
cells from hair follicles or sweat glands [8, 24–26]. This wound, a process also known as ‘sprouting’. The endothe-
process is activated by signaling pathways of epithelial lial cells orientate themselves at superficial adhesion mol-
and nonepithelial cells at the wound edges, which release ecules, e.g. integrins (␣v␤3, ␣v␤5, ␣5␤1). Furthermore,
a myriad of different cytokines and growth factors, e.g. they release matrix metalloproteinases at the front of pro-
EGF, KGF, IGF-1, and NGF [10]. Furthermore, the aboli- liferation, lysing the surrounding tissue for the ongoing
tion of the contact inhibition and physical tension at des- endothelial proliferation. The newly built sprouts form
mosomes and hemidesmosomes produces lipid media- small tubular canals which interconnect to others form-
tors and activates membrane-associated kinases (SRC ing a vessel loop. Thereafter, the new vessels differentiate
kinases) resulting in an increased permeability of the into arteries and venules and mature by a further stabili-
membranes for ions, e.g. calcium. This displays an initiat- zation of their vessel wall via the recruitment of pericytes
ing signal to the cells at the wound edges with a retraction and smooth muscle cells. Finally, the initial blood flow
and reorganization of their intracellular tonofilaments in completes the angiogenic process. Within full dermal
the direction of migration. By the enzymatic loosening of thickness wounds the neovascularization process follows
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Phase 2: proliferative phase (days 4–21)
Macrophage
PDGF TGF-␤1
Framework for endothelial

proliferation and angiogenesis
Fibroblast
VEGF, PDGF, bFGF, thrombin
Granulation tissue:
fibroblasts, granulocytes,
macrophages, capillaries and
loosely organized collagen
bundles
Collagen III
Fig. 2. Proliferative phase; organization of
the thrombus, secretion of growth factors,
synthesis of collagen III and the beginning
of angiogenesis.
a distinct pattern in time and shape. At the beginning the glycans and hyaluronic acid). The formation of the ECM
vessels form an inner ring of circularly arranged vessels represents another important step as it provides a scaffold
at the wound margin followed by outer radially arranged for cell adhesion and critically regulates and organizes
vessels supplying the inner ones. Because the design of the growth, movement and differentiation of the cells
the vessels is similar to the sun, this has also been called within it [34, 35]. The fibroblast is, therefore, the precur-
‘sola cutis se reficientis’ [32]. As the wound closure pro- sor of the provisional wound matrix on and in which the
ceeds, the inner vascular ring shrinks, resulting in the respective cell migration and organization takes place
complete disappearance of the vessel ring. The radially [27]. At the end of this phase the number of maturing fi-
arranged vessels, however, interconnect with each other broblasts is reduced by myofibroblast differentiation and
in time, forming a new dermal vascular network [32]. terminated by consecutive apoptosis [36].
Granulation Tissue Formation

The last step in the proliferation phase is the develop- Remodeling
ment of the acute granulation tissue (fig. 2). At the same
time the remodeling phase is already initiated. As a tran- Remodeling is the last phase of wound healing and oc-
sitional tissue it replaces the fibrin-/fibronectin-based curs from day 21 to up to 1 year after injury. The forma-
provisional wound matrix and might produce a scar by tion of granulation tissue stops through apoptosis of the
maturation [1, 3, 4, 7, 8, 14, 33]. Furthermore, it is charac- cells. A mature wound is, therefore, characterized as
terized by a high density of fibroblasts, granulocytes, avascular as well as acellular [37]. During the maturation
macrophages, capillaries and loosely organized collagen of the wound the components of the ECM undergo cer-
bundles (fig. 2). Due to this high amount of cellular com- tain changes. Collagen III, which was produced in the
pounds it is called granulation tissue. Also, as the angio- proliferative phase, is now replaced by the stronger col-
genesis is not completely finished yet, this tissue is highly lagen I (fig. 3). This type of collagen is oriented in small
vascular. As a result it appears with a classic redness and parallel bundles and is, therefore, different from the bas-
might be traumatized easily. However, the dominating ket-weave collagen in healthy dermis [38]. Later on the
cell in this phase is the fibroblast, which fulfils different myofibroblasts cause wound contractions by their mul-
functions such as the production of collagen and ECM tiple attachment to collagen and help to decrease the sur-
substances (i.e. fibronectin, glycosaminoglycans, proteo- face of the developing scar [12, 13, 38]. Furthermore, the
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Phase 3: remodeling phase (day 21 to 1 year)
Collagen III
Myofibroblast
Contractile response
decreasing scar surface
Collagen I
Angiogenesis
blood flow
Hair follicles or sweat glands have

Fig. 3. Remodeling phase; regenerative no potential to heal
processes fade and are followed by reorga- Apoptosis
nization of the connective tissue and con-
tractile response.
angiogenic processes diminish, the wound blood flow de- 45, 46]. The exact day is determined by the wound size;
clines, and the acute wound metabolic activity slows bigger injuries might leave a scar earlier than small
down and finally stops. scratches [44]. Fetal skin structure and histology change
Contrary to fetal wound healing, there are certain skin rapidly during pregnancy. Within the first 20 days the
components that will never fully recover after wound clo- fetus is only covered in a primitive epidermis, which de-
sure. Subepidermal appendages such as hair follicles or velops into a two-layered periderm and a basal cell layer
sweat glands have no potential to heal or grow back after within 4–8 weeks. At week 9 the stratification of the fetal
serious injury. The epidermis of the resultant scar differs epidermis begins and keratinization begins at week 14. By
from uninjured skin after wound healing due to the lack the time the fetus reaches week 16 the fetal epidermis al-
of rete pegs that are normally anchored into the underly- ready has many of the components of the adult epidermis:
ing connective tissue matrix and are responsible for the a basal layer, an intermediate layer, hair follicles, sweat
tight connection of the epidermis to the dermis [1]. glands and follicular keratinization. After 24 weeks of
gestation rapid growth and maturation start to dominate
the process and by the time of birth the neonatal skin is
Fetal Wound Healing indistinguishable from adult skin [47, 48]. In response to
tissue injury, the fetal dermis has the ability to regenerate
In contrast to the adult human tissue reaction to in- a nondisrupted collagen matrix that is identical to the
jury the early gestation fetus has the ability to heal with- original tissue [49, 50]. Interestingly, even dermal ap-
out scarring. This unique healing process was first dis- pendages such as sebaceous glands and hair follicles heal
covered in 1979 [39] and was confirmed in many animal normally after fetal injury [51]. There is evidence that the
and human trials [40, 41]. Scarless wound healing was reasons for scarless wound healing in fetal tissue is due to
observed in the fetuses of rats, mice, pigs, monkeys and the differences in the ECM, inflammatory response, cel-
humans [42] and is age-dependent in the different species lular mediators, differential gene expression and stem cell
[43, 44]. In humans, scarless wound healing stops at ap- function [52–55]. The inflammatory response in an em-
proximately 24 weeks of gestation, whereas in mice it bryonic wound consists of a lower number of less-differ-
stops at day 18.5 (gestation period of mice: 20 days) [42, entiated cells (neutrophils, monocytes and macrophages),
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which in combination with a very different profile of more, mechanical irritation in the early proliferative phase
growth factors contributes to the much better wound leads to hypertrophic scars by inhibiting apoptosis [64].
healing in fetal tissue. Furthermore, the length of presen- Changes in the ECM and the epithelium seem to be
tation of an inflammatory cell is reduced compared to involved in excessive scarring also [65–67]. A neurogenic
adult wounds [56]. Molecular changes involve low levels inflammation hypothesis has been suggested [68]: me-
of TGF-␤1, TGF-␤2 and PDGF and high levels of TGF-␤3 chanical stress stimulates mechanosensitive nociceptors
[41]. Due to the rapidly developing and growing skin vol- in skin sensory fibers that release neuropeptides involved
ume, the fetal tissue consists of high levels of morphoge- in vessel modifications and fibroblast activation. Exces-
netic factors which are involved in skin growth, remodel- sive scarring is a fibrotic disorder resulting from the dis-
ing and morphogenesis. These two principals – reduced ruption of the normal wound healing process [69].
inflammatory response and altered skin morphogene-
sis – lead to a qualitatively, quantitatively and temporally
different growth factor profile compared to adult wound Conclusion and Outlook
healing [41, 49, 56, 57].
The regulation of cellular functions during skin repair
following injury is complex and critically dependent on
Scarring the interaction of cells with the ECM, which can be im-
paired at every step and time point of the wound healing
Scar formation is the physiological endpoint of mam- process. Many cellular or cytokine actions can be adopt-
malian wound repair. There are different situations which ed by others without severe interference with the healing
provide evidence that inflammation during the process process. However, some singular actions might be crucial
of wound healing is directly linked to the extent of scar or the sum of many healing insufficiencies might lead to
formation [5]. First, there is the fact that fetal wound heal- chronic or nonhealing wounds. Research in this special-
ing, which shows a lack of the typical inflammatory re- ized field is still ongoing and many questions are still un-
sponse, is scarless up to a certain age [16, 17]. In addition, answered. However, a better understanding of this com-
scar formation seems to be extended when inflammation plex interplay provides the basis for designing new and
in fetal wounds is induced [49]. A second example indi- effective wound healing therapies. Knowledge gained
cating the role of inflammation on the formation of scars from studying the genetic and molecular pathways and
is the influence of reproductive hormones on this pro- from cytokine interactions and influences, as well as the
cess. Studies showed that low estrogen levels in mice re- enormous development of functional new wound dress-
sulted in an impaired rate of healing including excessive ings for the conservative wound therapy, might further
inflammation and scarring [5, 58, 59]. help to decrease the incidence of nonhealing wounds and
Most nonhealing wounds fail to progress through the support the healing process under poor conditions. Fur-
normal phases of wound repair, but remain in a chronic thermore, the auspicious options in stem cell therapy as
inflammatory state [60] which leads to abnormal wound well as in tissue engineering offer new therapy possibili-
repair, e.g. to hypertrophic or keloid scars. Keloids con- ties in skin wound healing.
tain thick collagen fibers, whereas hypertrophic scars
contain thin fibers which are organized into nodules [61,
62]. Changes in collagen maturation are fundamental
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Int J Clin Exp Pathol 2010;3(7):643-653
www.ijcep.com /IJCEP1007002
Review Article
Skin wound healing modulation by macrophages
Mathieu P. Rodero, Kiarash Khosrotehrani
University of Queensland Centre for Clinical Research, Experimental Dermatology Group, Brisbane, Australia
Received July 5, 2010; accepted July 23, 2010; available online July 25, 2010
Abstract: Skin wound healing is a multi stage phenomenon that requires the activation, recruitment or activity of nu-
merous cell types as keratinocytes, endothelial cells, fibroblast and inflammatory cells. Among the latter, macro-
phages appear to be central to this process. They colonize the wound at its very early stage and in addition to their
protective immune role seem to organize the activity of other cell types at the following stages of the healing. Their
benefit to this process is however controversial, as macrophages are described to promote the speed of healing but
may also favour the fibrosis resulting from it in scars. Moreover wound healing defects are associated with abnormali-
ties in the inflammatory phase. In this review, we summarise our knowledge on what are the Wound Associated
Macrophages, and how they interact with the other cell types to control the reepithelialisation, angiogenesis and the
extracellular matrix remodelling. We believe this knowledge may open new avenues for therapeutic intervention on
skin wounds.
Keywords: Macrophage, skin wound, healing, inflammation, cytokines, growth factors, M1, alternatively activated
Introduction also the fibrosis associated with scar formation.

Wound Associated Macrophages (WAM) have a
Macrophages are mature myeloid cells, mostly central role in the control of wound inflamma-
derived from the differentiation of circulating tion. Here, we will present an overview of the
monocytes after tissue infiltration. Far to be a main functions of WAM and their influence on
homogeneous population, macrophages display the other major cell types present in the wound
a wide range of phenotypes and physiological during the healing process and how modulating
properties depending on the cytokines inducing these processes might result in better wound
their maturation [1]. Moreover, macrophages healing.
are very plastic cells, able to switch from one
functional sub-population to another depending Wound healing
on the stimulus received [2]. Another aspect of
macrophage heterogeneity is the tissue speciali- In humans, and more widely in all mammalian
sation of resident macrophages, as microglial species, the wound healing process can be sub-
cells in the brain, Kupffer cells in the liver or divided in three consecutive and overlapping
alveolar macrophages in the lung. However, it is stages: inflammation, new tissue formation and
still not clear if these resident macrophages are remodelling [3]. The transition from one stage
derived in steady state condition from circulat- to another depends on the maturation and dif-
ing monocytes and if they are terminally differ- ferentiation of the main cell populations in-
entiated cells [1]. After birth, macrophages are volved, among which the keratinocytes, the fi-
known to affect different stages of skin wound broblasts and the macrophages.
healing, modulating the function of the different
cell types involved in this process. The benefit of Inflammation
inflammation and inflammatory cells in the
wound healing is a matter of debate as in nor- The first event occurring after injury is the for-
mal conditions it promotes wound closure but mation of a blood clot by activated platelets.
The blood plug will be composed of various cell Macrophages and wound healing
types including platelet, red and white blood
cells. The initial plug is stabilised by fibrin fibres Wound macrophage phenotypes
and will be a scaffold for the various infiltrating
cells. The first inflammatory cells recruited are Macrophages used to be divided in several sub-
the neutrophils [4]. They infiltrate massively the populations depending of the way they had
wound during the first 24h post injury [5] at- been activated, their cell surface markers or
tracted by the numerous inflammatory cytokines their functionality. [1, 10,11]. Schematically,
produced by the activated platelets, endothelial macrophages activated by microbial agents and
cells, as well as by the degradation products cytokines like Interferon gamma (IFNγ) are clas-
from pathogens. Neutrophils enter apoptosis sified as M1 macrophages. These macrophages
soon after infiltrating the wound and the release produce an important level of Nitric Oxide (NO)
of cytokines during this apoptotic process is an and pro inflammatory cytokines such as Tumor
important component in macrophage recruit- Necrosis Factor alfa (TNFα), IL-1β, IL-6, or IL-12,
ment. Macrophages infiltrate the wound mas- and overexpress MHC class II molecules. They
sively 2 days post injury and exacerbate at this bear microbicidal and antitumoral properties.
stage an intense phagocytic activity [6]. M2 macrophages are a much more heterogene-
ous population composed of all macrophages
New tissue formation that do not correspond to M1 characteristics
[12]. M2 macrophages could be divided in 3
The reepithelialisation process begins few hours sub-populations. The alternatively activated
after the wound formation. Keratinocytes from macrophages or M2a, that promote a Th2 type
the wound edges migrate over the wound bed at of inflammation resulting in increased IgE as
the interface between the wound dermis and observed in allergy and parasite immunity, the
the fibrin clot. This migration is facilitated by the M2b macrophages, that promote Th2 inflamma-
production of specific proteases such as the tion and bear some immunoregulation proper-
collagenase by the epidermal cells to degrade ties, and the deactivated macrophages or M2c,
the extracellular matrix [7]. Activated fibroblasts able to control the inflammation and implicated
also migrate to the wound bed and form, with in tissue remodelling [12]. In another classifica-
the macrophages, the granulation tissue. A tion, M1 macrophages are considered as the
massive angiogenesis allowing the supply of classically activated macrophages as compared
oxygen and nutrients necessary for the healing to the alternatively activated macrophages. The
process also occurs within this tissue [8]. Later, best described alternative activation consists in
some of the fibroblasts differentiate into myofi- the stimulation of the IL-4R by IL-4 and IL-13
broblasts. These contractile cells will help that induces pro Th2 macrophages [13]. Of
bridge the gap between the wound edges [9]. course, all these sub populations should not be
During the same time, growth factors produced considered as distinct populations in vivo but
by the granulation tissue will favour proliferation more as different stages of a continuum of acti-
and differentiation of epithelial cells restoring vation and differentiation of macrophage popu-
the epithelial barrier integrity. lations [14]. The phenotype of skin wound infil-
trating macrophages is not yet fully character-
Remodelling ised, but it already appears that it changes dur-
ing the healing process suggesting that macro-
The last stage of the wound healing process phages have different roles in the diverse
consists in a gradual involution of the granula- phases of skin repair [15, 16].
tion tissue and dermal regeneration. This step is
associated with the apoptosis of myofibroblasts, Several reviews refer to wound macrophages as
endothelial cells and macrophages. The remain- the IL4 alternatively activated macrophages
ing tissue is therefore composed mostly of ex- because of numerous markers of tissues re-
tracellular matrix proteins, essentially collagen modelling, such as YM1, Resistin Like Molecule
type III that will be remodelled by the metallo- Alfa (RELMα), Insulin Growth Factor 1 (IGF1),
proteinase produced by the epidermal cells, factor XIII-A or arginase that are expressed after
endothelial cells, fibroblasts and the macro- in vitro activation of macrophages with IL4. [13,
phages remaining in the scar and be replaced 17-20]. Recent work on aseptic wounds using
by collagen type I [8]. an implantable sponge model described wound
644 Int J Clin Exp Pathol 2010;3(7):643-653

Table 1. Impact of macrophages depletion strategy on wound healing

Depletion stage Depletion Wound closure Gr T fromation / Collagen / Ref
process Angiogenesis fibrosis/αSMA
Early Macrophage an- ↓ ND ↓ [6]
tiserum
Early CD11b DTR ↓ ↓ ↓ [22]
Early lysM-Cre DTR ↓ ↓ ↓ [23]
Early lysM-Cre DTR ↓ ↓ ↓ [16]
Middle lysM-Cre DTR ↓ ↓ ND [16]
Late lysM-Cre DTR = = = [16]
Gr T: Granulation tissue
macrophages as cells bearing both classical not needed for wound closure [21]. However,
activation markers, like TNFα expression, and several recent models of specific inducible
alternatively activated markers like the man- macrophage depletion, based on genetically
nose receptor [15]. The authors also described modified mice resulted in detrimental effect of
the evolution of the macrophage phenotype. pre-injury depletion of macrophages [16, 22,
Day 1 macrophages produced more TNFα and 23]. Mice depleted before injury typically show a
IL6 and less Tumor Growth Factor beta (TGFβ) defect in re-epithelisation, granulation tissue
compared to day 7 macrophages suggesting a formation, angiogenesis, wound cytokine pro-
transition from an inflammatory to an immu- duction and myofibroblast associated wound
noregulatory or tissue remodelling state. How- contraction. Recently, the effect of a post-injury
ever, it is important to consider that neither IL4 depletion of the WAM has also been investi-
nor IL13 were detectable in this model, perhaps gated. Macrophage depletion during the granu-
because of the aseptic experimental condition. lation tissue formation, about 3 days after in-
However, it is not clear to what extent the bacte- jury, is associated with vascularisation defect,
rial colonization of the wound would modify delay in wound closure as well as in granulation
Wound Associated Macrophage phenotypes and tissue maturation [16]. Finally, in their study,
healing functions, as it is clear that parasitic Lucas et al did not find any morphological or
and bacterial component are major macro- biological differences between mice that were
phage activators [13]. or were not depleted 9 days after injury suggest-
ing no further implication of macrophages at
Macrophage importance in wound healing later stages.
In order to show the implication of macrophages During the healing process numerous cytokines
in the control of wound healing, several studies and growth factors are produced by the various
have analysed skin wound healing upon macro- cell types present in the wound or at the wound
phage depletion (Table 1). The first attempt con- edge [24]. The level of production of the differ-
sisted in corticosteroids associated with anti ent cytokines depends on the regulation of the
macrophage sera to deplete macrophages in cross talk between the major cell populations in
wounded guinea pigs. This treatment resulted in the wound: epithelial cells, endothelial cells,
a delayed infiltration of the wound by fibroblasts fibroblasts and inflammatory cells. However, the
and decreased fibrosis [6]. These results are in literature clearly lacks in vivo characterisation of
accordance with observations that fetal wounds the cytokine production kinetics by the various
heal without fibrosis and are not infiltrated by cell types during skin wound healing. Most of
macrophages. In a model of PU.1 null mice our current knowledge concerning the ability of
where animals lacked macrophages, mast cells a cell population to produce or to respond to a
and functional neutrophils due to defective mye- specific cytokine is based on in vitro studies.
lopoiesis, wounds performed on newborns addi-
tionally treated with antibiotic healed at the Cross talk with keratinocyte
same speed as wild types, but without scar for-
mation, suggesting that inflammatory cells are Injured and inflamed keratinocytes produce

several cytokines allowing the recruitment and tracted on day 1 produced more TNFα com-
activation of the WAM, including chemokines, pared to day 7 [15]. Finally, the other cytokine
interleukins and growth factors [25, 26]. On the known to be massively produced by macro-
other hand, the ability of immune cells to pro- phages, and highly implicated in reepithelialisa-
duce factors able to regulate keratinocyte tion, is TGFβ, although its effect on keratinocyte
growth in vitro has been described since 1988 proliferation and migration remains controver-
[27]. Interestingly, conditioned medium from co- sial [36, 40-43]. Cultured WAM extracted from
culture of macrophages and allogenic T cells implanted sponges start producing TGFβ from
were much more efficient in inducing keratino- the very first day of the wound [15].
cyte proliferation compared to conditioned me-
dium from macrophages alone. However, the Influence on fibroblasts and myofibroblast
direct implication of WAM in producing cyto-
kines or growth factors to influence directly or Fibroblasts begin to infiltrate the wound during
indirectly keratinocyte migration and prolifera- the first steps of granulation tissue formation.
tion is not clear in vivo. Several cytokines and Fibroblasts are involved in several processes of
growth factors, that can be produced by macro- the wound healing. They contribute to the
phages, have been associated with reepitheliali- granulation tissue formation, produce the cyto-
sation, mostly in vitro. Among the Epithelial kines that favour keratinocyte proliferation and
growth Factor family (EGF), the main members migration [44], and finally differentiate in myofi-
involved in wound healing are EGF, TGFα and broblasts to promote wound closure. Several
Heparin Bound EGF (EGF-HB) [28-32]. The acti- molecules, such as PDGF-bb, TNFα, IL-1 or IL-6,
vation of the EGF Receptor (EGFR) on keratino- produced by activated macrophages are able to
cytes promotes cell migration and proliferation. induce in vitro the production of pro-
Although, only TGFα and EGF-HB have been reepithelialisation molecules by fibroblasts such
described to be produced by macrophages in as KGF [45-48]. Interestingly, the effect of most
vitro, their production by WAM in vivo is more of these molecules is dependent on the pres-
enigmatic [33, 34]. TGFα has been detected in ence of serum in the fibroblast culture media,
macrophages collected from sub epidermal indicating that the stimulation resulting in their
wound cylinders 6 days after implantation in production is a cooperative process [49]. Mori
mice [35]. However, no significant wound heal- et al demonstrated nicely that macrophage se-
ing abnormalities were observed in TGFα-/- cretion of PDGF-bb isoform induced the produc-
mice [36]. In addition, all EGFR ligands are syn- tion of osteopontin by fibroblasts [50]. The inhi-
thetised as membrane-anchored forms, which bition of osteopontin was associated with lower
can be released as a soluble form after prote- scar fibrosis [50]. Finally, the molecule pro-
olysis by MMP [37]. Therefore, another possible duced by macrophages that has been the most
role for WAM might be the regulation of EGF extensively studied for its actions on wound
family release by the production of MMPs. Other fibroblasts is TGFβ. It is a family of three cyto-
cytokines as IL-6, IL1 and TNF-α produced by kines, TGFβ1, 2 and 3, produced by several cell
macrophages are also associated with reepi- types during the healing process, including
thelisation. Even if the secretion of these cyto- platelets and epidermal cells, but the main and
kines by WAM has not been clearly demon- sustained source of production are the WAM
strated yet, the ability of macrophages activated [16, 51]. All TGFβ family members are secreted
by the inflammatory cytokine IFNγ and/or bacte- coupled to a “latent complex” which has the
rial products via Toll Like Receptor 4 (TLR4) to ability to bind the Extra Cellular Matrix (ECM).
produce IL-6, IL1β and TNFα makes this hy- The latent forms have to be activated via vari-
pothesis probable [13]. The implication of IL-1 ous mechanisms to release the mature TGFβ,
and IL-6 in the reepithelialisation is indirect and including proteolysis by MMPs known to be ex-
is mediated by other cell types present in the tensively secreted by macrophages as reviewed
wound, at least the fibroblast [38, 39]. On the by Annes et al [52]. While TGFβ1 and 2 promote
contrary, TNFα directly stimulates transcription the inflammation within the wound and are as-
of genes associated with numerous cell func- sociated with scar formation, TGFβ3 is associ-
tions including inflammation, mobility, cell divi- ated with scar free wound healing [53]. How-
sion and survival in keratinocytes [25]. Of note, ever, the expression of TGFβ3 in adult wound is
in a model of sponge implanted under aseptic rare while the two other forms are highly ex-
conditions in the dermis, macrophages expressed [53]. TGFβ1 is chemotactic for fibro-

Table 2. Targeting of macrophages to control healing of pathological wound

Molecules / Delivery Stage Pathological Wound Effect on MΦ Effect on Ref
cells model model healing
MALP-2 local Early to Diabetic Acute ↑ recruitment ↑ [89]
middle C57Bl6 db/db
GM-CSF local NA Human CVLU Chronic ↑ VEGF pro- ↑ [88]
duction
Anti TNFα Systemic Early to Diabetic Acute ↓ recruitment ↑ [90]
late ob/ob
IL1β acti- Local Early Diabetic Acute ↑ ↑ [72]
vated db/db C57Bl6 db/db VEGFC/
MΦ VEGFR3 tran-
scription
GM-CSF local Early Strpz induce Acute ↑ Recruit- ↑ [86]
diabetic mice ment
CVLU: Chronic Veinous Leg Ulcer. Strpz : streptozotocin

blasts [54, 55]. It induces the production of sev- Eming et al [63]. VEGF promotes endothelial
eral growths factors by fibroblasts, including cell and precursor recruitment [64] but it also
Connective Tissue Growth Factor (CTGF) [56], acts as a mitogen and survival factor on the
that results in their proliferation by an autocrine endothelial cells [65-67]. The increase of VEGF
loop [57]. But the main action of TGFβ on fibro- production in the wound is described in migra-
blast is to promote their differentiation into myo- tory keratinocytes and in macrophages infiltrat-
fibroblast and to favour collagen production ing the granulation tissue [62]. The keratinocyte
[58]. The secretion of TGFβ and MMP by the production of VEGF is indirectly promoted by
macrophages is critical in the control of the macrophages as well through the secretion of
ECM composition. Accordingly, macrophage cytokines as TNFα or TGFβ [68]. CTGF produced
depletion is associated with a defect of Alpha by fibroblasts [56], also favours endothelial cell
Smouth Muscle Actin (αSMA) positive cell in the proliferation and new vessel formation [69, 70].
granulation tissue [23]. However, it is estimated VEGF family members, mostly VEGF-C and VEGF
that 30 to 50% of the granulation tissue αSMA+ -D, have also been recognized for their ability to
cells do not derive from fibroblasts, but more modulate lymphangiogenesis [71]. Decreased
likely from bone marrow derived fibrocytes or macrophage numbers and activation has been
mesenchymal stem cells [59-61]. The implica- associated with reduced lymphatic vessel for-
tion of macrophage produced cytokines in the mation in diabetic mouse wounds [72]. It is im-
recruitment, activation and differentiation of portant to note that double positive F4/80/Lyve
these marrow derived fibrocytes in the wound -1 cells have been described to be integrated in
bed is unknown and has to be further investi- lymphatic vessels in several models of neo-
gated. lymphangiogenesis suggesting that macro-
phages could be directly implicated in their for-
Cross talk with endothelial cell mation [72, 73].
Neo-angiogenesis in the granulation tissue is an The other main pro-antigenic molecule during
important process allowing the supply of nutri- the wound healing is the Placental Growth Fac-
ents necessary for the healing process. The tor (PlGF), a member of the VEGF family [67]
main angiogenic factor in the wound is the Vas- [74]. PlGF expression is induced in vitro by TGFα
cular Endothelial Growth Factor (VEGF) and its and TGFβ, and is produced during the angio-
reduced expression results in a wound healing genic stage of the healing process [75]. The
defect [62]. VEGF promotes wound vascularisa- effects of PlGF are similar to those of VEGF,
tion by multiple mechanisms targeting directly promoting monocytes and endothelial precursor
or indirectly endothelial cells as reviewed by cell migration [76] and favouring endothelial

cell survival [77]. PlGF stimulates also the se- Macrophages and defective wound healing
cretion of VEGF by the monocyte/macrophages
[78]. Interestingly, even if PlGF is described to Despite their heterogeneous aetiology, most
have chemotactic properties by his own on chronic wounds have in common a defect in the
VEGFR1+ expressing cells, the synergy between progression from the inflammatory to the tissue
PlGF and VEGF is important in angiogenesis formation stage. Loots et al reported increased
[79]: i) VEGFR1 homodimers may be a decoy infiltration of chronic and diabetic wounds by
receptor. Binding of PlGF homodimer to VEGFR1 WAM compared to control acute wounds. In
homodimer make more VEGF available to bind addition they observed higher amounts of ECM
to activated VEGFR2. ii) VEGF/PlGF het- in the wound edge [81]. Similarly, in diabetic
erodimers bind to VEGFR1/VEGFR2 het- db/db mice, wounds are characterising by a
erodimer and induce more potent angiogenic prolonged expression of inflammatory cytokines
signals iii) binding of PlGF homodimers to and larger infiltration and persistence of the
VEGFR1 homodimers favours VEGFR2 WAM [82]. However, these cells seem to have
homodimer phosphorylation [80]. Overall, PlGF altered sensitivity towards exogenous signals
seems to potentiate the effect of VEGF in wound such as VEGF or IGF1 [83, 84] as well as al-
angiogenesis. These results altogether point to tered ability to release cytokine [85]. Several
the important role of macrophages in coordinat- studies have targeted macrophages, by differ-
ing the angiogenic signal during wound healing. ent strategies in order to improve defective
wound healing (Table 2). In the obese diabetic
Figure 1. Direct and indirect control of reepithelialisation, angiogenesis and fibroblast activation by macrophages.

db/db mouse model, the injection of peritoneal population or fulfilled by specialised sub popula-
macrophages activated by IL-1β at the wound tions at different time points? Should the future
site was associated with an increased produc- therapeutic strategies consist in the control of
tion of prolymphangiogenic molecules such as the WAM polarisation or in the specific recruit-
VEGF-C, resulting in improved lymphangiogene- ment or depletion of sub populations? These
sis and wound healing [72]. Local application of strategies will also have to define the appropri-
GM-CSF is associated with better healing both ate timing for their effectiveness as WAM bear
in human and mouse pathological wounds in a different functions during the healing time
clinical setting. In Streptozotocin induced dia- course [16].
betic mice, GM-CSF is associated with a
stronger infiltration of the wound by macro- Acknowledgement
phages, increased angiogenesis and a better
healing, while no effect was observed in normal This work was supported by the Ramaciotti
wound healing [86] [87]. Similarly, in a pilot foundation.
study in humans, GM-CSF treatment improves
chronic vascular ulcers, probably by promoting Please address correspondence to: Kiarash Khosro-
the secretion of VEGF by macrophages [88]. tehrani, MD, PhD, University of Queensland Centre for
With a similar strategy, Deiters et al injected Clinical Research, Building 71/918, Royal Brisbane &
Macrophage-Activating Lipopeptide-2 (MALP-2) Women's Hospital Campus, Herston, QLD, 4029, Tel:
+6173346 6077, Fax : +61733465598, E-mail:
in wounds on obese diabetic db/db mice. MALP- k.khosrotehrani@uq.edu.au
2 induced the transcription of several genes
associated with wound healing, including GM- References
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Activation of Spinobulbar Lamina I Neurons by Static
Muscle Contraction
L. B. Wilson, D. Andrew and A. D. Craig
J Neurophysiol 87:1641-1645, 2002.
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J Neurophysiol
RAPID COMMUNICATION 87: 1641–1645, 2002; 10.1152/jn.00609.2001.
Activation of Spinobulbar Lamina I Neurons by Static Muscle

Contraction
L. B. WILSON,1 D. ANDREW,2 AND A. D. CRAIG2

1
Department of Physiology, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2Atkinson Pain
Research Laboratory, Division of Neurosurgery, Barrow Neurological Institute, Phoenix, Arizona 85013
Received 24 July 2001; accepted in final form 14 November 2001
Wilson, L. B., D. Andrew, and A. D. Craig. Activation of spino- afferents terminate in the dorsal horn of the spinal cord, par-
bulbar lamina I neurons by static muscle contraction. J Neurophysiol ticularly within lamina I (Craig and Mense 1983). Spinobulbar
87: 1641–1645, 2002; 10.1152/jn.00609.2001. Spinal lamina I neu- lamina I neurons have never been recorded. Spinothalamic
rons are selectively activated by small-diameter somatic afferents, and lamina I neurons excited by group III muscle afferents and
they project to brain stem sites that are critical for homeostatic control.
Because small-diameter afferent activity evoked by contraction of
noxious muscle stimulation were identified previously (Craig
skeletal muscle reflexly elicits exercise-related cardiorespiratory acti- and Kniffki 1985), but activation of lamina I neurons by

vation, we tested whether spinobulbar lamina I cells respond to muscle contraction has never been demonstrated; this requires
muscle contraction. Spinobulbar lamina I neurons were identified in maintaining microelectrode recordings from these small spinal
chloralose-anesthetized cats by antidromic activation from the ipsilat- neurons during hindlimb muscle contractions strong enough to
eral caudal ventrolateral medulla. Static contractions of the ipsilateral elicit blood pressure changes in an unparalyzed animal under
triceps surae muscle were evoked by tibial nerve stimulation using anesthesia. The purpose of this study was to determine whether
parameters that avoid afferent activation, and arterial blood pressure excitation of identified spinobulbar lamina I neurons by static
responses were recorded. Recordings were maintained from 13 of 17 contraction of skeletal muscle can be demonstrated.
L7 lamina I spinobulbar neurons during static muscle contraction, and
5 of these neurons were excited. Three were selectively activated only
by muscle afferents and did not have a cutaneous receptive field. METHODS
Spinobulbar lamina I neurons activated by muscle contraction provide
an ascending link for the reflex cardiorespiratory adjustments that Five adult cats (3.5– 4.2 kg) were premedicated with ketamine (25
accompany muscular work. This study provides an important first step mg/kg im) and anesthetized with ␣-chloralose (80 mg/kg) and urethan
in elucidating an ascending afferent pathway for somato-autonomic (100 mg/kg) administered via a cannula in the cephalic vein. Cannulae
reflexes. were inserted into the carotid artery (to record blood pressure) and
into the trachea (for artificial respiration). The animals were paralyzed
during surgery with pancuronium (400 ␮g/h) and ventilated; end-tidal
CO2 was maintained near 4.0%. Body temperature was monitored
INTRODUCTION
using a rectal thermistor and maintained at 37.5°C.
Lamina I, the most superficial layer of the spinal or trigem- A laminectomy was performed to expose the lumbosacral spinal
inal dorsal horn, receives modality-selective A␦- and C-fiber cord, and a pool filled with warm (38°C) Tyrode’s solution was
afferent input from somatic and visceral tissues (Craig 2000). constructed. The medulla was exposed by enlarging the foramen
magnum. A bipolar electrode (Rhodes NE- or NEX-100) was inserted
Anatomic findings indicate that lamina I neurons are the major into the left caudal ventrolateral medulla (CVLM) to antidromically
source of spinobulbar projections (Krout and Craig 1996) and activate spinobulbar neurons. The left calcaneal bone was cut, and the
project to specific brain stem sites involved in homeostatic Achilles tendon was connected to a transducer (Grass model FT10) to
regulation (Craig 1995). Thus lamina I may provide an impor- measure the tension developed during contraction of the triceps surae
tant link for the somato-autonomic reflex responses evoked by muscle. The patellar tendon was secured to a post to ensure an
somatic and visceral small-diameter afferent input (Sato and isometric contraction. The popliteal fossa was exposed, and the tibial
Schmidt 1973). nerve was placed on platinum wire electrodes (LeDoux and Wilson
The brain stem plays a pivotal role in homeostatic responses, 2001). A pool made around the exposed nerves and muscles was filled
including cardiovascular and respiratory responses to muscle with warm mineral oil. The preparation is shown diagrammatically in
work (Iwamoto et al. 1985). Static contraction of skeletal Fig. 1. Once the preparation was complete, the paralytic was allowed
to wear off.
muscle activates small-diameter afferents that evoke a reflex Glass-insulated tungsten microelectrodes were used to record ex-
increase in sympathetic nerve activity and cardiovascular func- tracellularly from single lamina I neurons, identified by ongoing unit
tion (Kaufman and Forster 1996; Mitchell and Schmidt 1983; activity or by antidromic search stimuli (see Craig et al. 2001).
Wilson and Hand 1997). Activation of spinobulbar dorsal horn Bipolar or monopolar electrical stimuli were delivered from the elec-
neurons is required for this somato-autonomic reflex, the “ex- trode in the CVLM to determine whether a neuron projected to the
ercise pressor reflex” (Wilson 2001). Small-diameter muscle brain stem by antidromic activation. Two stimulus pulses of 400 ␮A
Address for reprint requests: A. D. Craig, Atkinson Pain Research Labora- The costs of publication of this article were defrayed in part by the payment
tory, Div. of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas of page charges. The article must therefore be hereby marked ‘‘advertisement’’
Rd., Phoenix, AZ 85013 (E-mail: bcraig@chw.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
www.jn.org 0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society 1641
and 1-ms duration were delivered at 100 Hz; stronger stimuli that average central conduction velocity of the spinobulbar lamina
evoked movement were avoided unless pancuronium could be admin- I neurons was 11.3 ⫾ 6.2 (SD) m/s (n ⫽ 17; range, 2.2–20.8),
istered. Every unit that followed two pulses at 100 Hz also followed which is significantly faster than spinothalamic lamina I neu-
a train of six antidromic stimuli at 250 Hz when tested (Fig. 2A); rons (mean, 4.7 ⫾ 2.2 m/s, n ⫽ 186, P ⬍ 10 ⫺7, unpaired t-test)
collision was also observed between naturally and antidromically
evoked impulses (Fig. 2B). Each unit was characterized using me-
(Andrew and Craig 2001). Unitary recordings from 13 spino-
chanical and thermal stimulation applied over the entire hindlimb bulbar lamina I neurons and 4 other lamina I neurons were
(Craig et al. 2001), and each cell was tested for excitation by muscle successfully maintained during muscle contraction.
contraction. Muscle contraction was evoked by stimulation of the Figure 2 shows the response to muscle contraction of an
tibial nerve with a 30-Hz train of 25-␮s pulses at 1.3 times motor antidromically identified and histologically confirmed lamina I
threshold for 10 –30 s. These stimulus parameters excite motor axons spinobulbar neuron. This unit began to respond ⬃15 s after the
but do not excite A or C afferent fibers (Degtyarenko and Kaufman onset of static contraction of the triceps surae muscle (Fig. 2E);
2000; Kaufman and Forster 1996). Only moderate contractions that its activity was maintained ⬎1 min following the contraction.
evoked moderate blood pressure changes were evoked because of the It did not respond to cutaneous mechanical or thermal innoc-
need to maintain stable single-unit microelectrode recordings. uous or noxious stimulation or to blood pressure changes these
Electrolytic lesions (⫹20 –50 ␮A, 10 s) were made at spinal re-
stimuli evoked or to tapping or moving the hindlimb, but it did
cording sites and at stimulation sites in the medulla. The tissue blocks
were fixed in formalin, and the lesions were recovered in 50-␮m respond to strong squeezing and stretching of the triceps surae
frozen sections that were stained with thionin (Fig. 2, C and D). muscle. It received time-locked (monosynaptic) input from
group III (A␦) tibial afferent fibers (conduction velocity, 18.5
RESULTS
m/s) but not from group IV (C) fibers (determined during
paralysis with paired 1-mA, 1-ms pulses at 120 Hz).
Recordings were obtained from 17 lamina I neurons identi- The responses of lamina I neurons to static muscle contrac-

fied as spinobulbar neurons by antidromic activation from the tion varied; 9 of the 17 neurons tested were excited, 2 were
CVLM and 5 lamina I cells with unidentified projections. The inhibited and 6 were unaffected. Of the 13 spinobulbar lamina
I neurons tested, 5 were excited by muscle contraction. Of
these, 3 had no detectable cutaneous receptive field, whereas 1
was a polymodal nociceptive neuron (HPC) (Craig et al. 2001)
that responded to noxious heat and pinch and noxious cold on
the hindpaw, and one was a wide-dynamic-range (WDR) neu-
ron that responded to low- and high-threshold stimulation of
the foot. Of the four lamina I neurons with unidentified pro-
jections that were excited by contraction, one received input
only from muscle afferents and three had HPC cutaneous
receptive properties. Most (4/6) of the neurons that were un-
affected by contraction were nociceptive-specific (NS) cells,
all of which projected to the CVLM. (Two had receptive fields
on the leg near the muscle exposure, indicating that muscle-
responsive cells without cutaneous fields did not result from
surgical damage to hindlimb cutaneous afferents.) The mean
central conduction velocity of the spinobulbar neurons excited
by contraction (9.7 m/s; range, 2.2–19.3, SD 7.4) was not
different from that of the other spinobulbar neurons (mean
10.9; range, 4.1–16.9, SD 6.0; P ⬎ 0.7, unpaired t-test).
Various responses of spinobulbar and other lamina I neurons
to muscle contraction are shown in Figs. 2 and 3. These
neurons responded during the contraction (Fig. 3A), after the
contraction (Fig. 3B) or both (Figs. 2E and 3C), and their
responses paralleled the tension developed in the muscle (Fig.
3, A and C), the centrally evoked cardiovascular reflex (Fig.
3A), or both (Fig. 3A). Interestingly, rhythmic muscle contrac-
tion (5 Hz), which is a potent stimulus for group IV (C)
afferents (Adreani et al. 1997), was the most effective stimulus
for activation of the cell shown in Fig. 3C.
DISCUSSION
FIG. 1. Diagram of the preparation. The activity of single lamina I neurons These are the first records of lamina I responses to static
was recorded extracellularly with microelectrodes (R). Electrical stimuli (S) contraction of skeletal muscle as well as the first recordings
were applied to the caudal ventrolateral medulla (CVLM) to test for neuronal from identified spinobulbar lamina I neurons. These technical
projections to the brain stem and to the axons of motoneurons in the tibial
nerve to evoke a static contraction of the triceps surae muscle. A tension advances notwithstanding, the most striking and novel result is
transducer (T) attached to the free end of the muscle was used to measure the the documentation of spinobulbar lamina I neurons that re-
force developed during a contraction. sponded to muscle contraction, particularly those which re-

FIG. 2. Example of the properties of a lamina I spinobulbar

neuron and its response to static contraction. A: a pair of traces
showing reproducible 1-for-1 following of a train of 6 anti-
dromic stimuli (180 ␮A, 1 ms; ●) delivered at 250-Hz from an
electrode in the ipsilateral CVLM. B: collision of the 1st
impulse of a train of 3 antidromic action potentials (200 Hz, ●)
with a preceding spontaneously occurring impulse (*). 1, the
time at which the 1st antidromic response would have oc-
curred. C: reconstruction of the recording site in the L6 seg-
ment of the spinal cord showing an electrolytic lesion local-
ized to lamina I. D: diagram of the stimulating site in the
medulla: Cu, cuneate nucleus; EC, external cuneate nucleus;
G, gracile nucleus; LR, lateral reticular formation; IO, inferior
olive; V, trigeminal nucleus, X, vagal nucleus, XII, hypoglos-
sal nucleus. E: response of the same neuron to static contrac-
tion of the triceps surae muscle. The traces are, from the top

downward: arterial blood pressure; tension in the triceps surae,
the neural record and a histogram of the unit’s activity (1-s
bins). The thickening of the neural recording is due to stimulus
artifacts during muscle stimulation (1.3⫻ motor threshold, 25
95 s, 30 Hz for 30 s). This neuron received input only from
group III muscle afferents; its central conduction velocity was
2.2 m/s; and, it had no cutaneous receptive field.
sponded selectively and had no cutaneous receptive field. Such solitary tract, the retrotrapezoidal region, and other homeo-
neurons provide an ascending pathway for the cardiovascular static sites in the brain stem (Craig 1995; Westlund and Craig
and respiratory responses that are reflexly elicited by muscular 1996). The neurons we antidromically activated from the ipsi-
work. This evidence directly supports the proposal that modal- lateral CVLM could have projected to any of these sites (and
ity-selective lamina I neurons are an afferent link for homeo- are therefore termed spinobulbar rather than spinomedullary),
static responses to changes in the physiologic condition of the and the anatomic data indicate that they probably did not
body, consistent with a fundamental role in homeostasis and project any further rostrally (Craig 1995). Therefore a role for
interoception, of which pain, temperature, itch and muscular these neurons in somato-autonomic reflexes (Li et al. 2001;
sensations are particular aspects (Andrew and Craig 2001; Stornetta et al. 1989) and homeostatic integration is highly
Craig 1996, 2000; Craig et al. 2001; Sherrington 1900). likely.
Anatomic evidence indicates that lamina I receives dense A recent study reported contraction-evoked activation of
input from small-diameter muscle afferents and also that lam- neurons in the deep dorsal horn (Degtyarenko and Kaufman
ina I provides the predominant spinal input to specific homeo- 2000) that was reduced by stimulation of the mesencephalic
static brain stem sites bilaterally (Craig 1995, 2000; Craig and locomotor region, similar to the effects of motor commands on
Mense 1983; Krout and Craig 1996). We identified these afferent-induced cardiovascular responses. In contrast to mo-
spinobulbar lamina I projection neurons by using antidromic dality-selective lamina I neurons, deep dorsal horn neurons are
activation from the ipsilateral CVLM because lamina I axons modality-ambiguous and integrate nearly all somatic afferent
that ascend to the mesencephalon or thalamus are almost all inflow (Carstens 1997). Further, the axonal projections of such
contralateral (Craig 2000). The lamina I neurons we recorded cells were not identified, and thus their role in brain stem
that had unidentified projections (i.e., not antidromically acti- somato-autonomic integration is unclear.
vated from the ipsilateral CVLM nor in some cases from the The present findings show that spinobulbar lamina I neurons
contralateral thalamus) could also have included contralateral have appropriate characteristics to engage homeostatic re-
spinobulbar cells. Lamina I spinobulbar axons terminate in the sponses to muscle exercise. Exercise demands rapid cardiovas-
caudal and rostral ventrolateral medulla, the nucleus of the cular and respiratory adjustments. Exercise-evoked cardiores-

indirectly excited by metabolites released from the contracting

muscle (metabo-receptive; Kaufman and Forster 1996; Kauf-
man et al. 1983; Kniffki et al. 1981; McCloskey and Mitchell
1972). The central neural correlates of both mechanisms were
observed in the present study. Some lamina I neurons were
excited immediately following the onset of contraction and
their activity paralleled muscle tension, similar to mechano-
sensitive small-diameter muscle afferents. Other neurons were
excited late during a contraction or after the contraction, sim-
ilar to metabo-receptive muscle afferents.
In addition, most (3/5) of the spinobulbar lamina I neurons
in this initial sample that responded to muscle contraction had
no cutaneous input, and conversely, the spinobulbar cells that
responded selectively to cutaneous nociceptors (NS cells) did
not respond to contraction. This observation supports the pos-
sibility that the muscle afferents that initiate reflex cardiores-
piratory changes produced by muscular work are “ergorecep-
tors” that are distinct from nociceptors (see Kniffki et al. 1981;
Mitchell and Schmidt 1983), an idea that has received recent
experimental support (LeDoux and Wilson 2001). This obser-
vation is also consistent with the general modality-selectivity

of lamina I neurons (Craig 2000). On the other hand, muscle-
responsive lamina I cells that did have cutaneous input were
nearly all HPC cells, consistent with the possibility that the
activity in such polymodal nociceptive cells generally repre-
sents increased regional metabolic needs (Craig 1996). Further
investigations can address these issues more fully.
Our observations demonstrate that spinobulbar lamina I neu-
rons associated with homeostasis can be investigated. Detailed
analyses of this unexplored pathway are needed. Only lamina
I neurons that project to the mesencephalon (Hylden et al.
1986) or thalamus (Craig et al. 2001) have been characterized
before. Lamina I receives dense afferent input not only from
skin and muscle but also from the viscera and other tissues of
the body, and it receives descending controls from homeostatic
brain stem sites and from the hypothalamus (Craig 2000). Thus
lamina I spinobulbar neurons could be involved in many so-
mato-autonomic and homeostatic reflexes. Significantly, recent
retrograde labeling results indicate that contralateral spinotha-
lamic and ipsilateral spinobulbar lamina I neurons are com-
pletely separate populations (Andrew and Craig 2000). Our
present physiologic observations support this finding, because
spinobulbar lamina I neurons have characteristics not observed
in spinothalamic lamina I neurons (i.e., contraction-responsive,
FIG. 3. Examples of the variety of responses of lamina I neurons that were wide-dynamic-range, faster conduction velocities), and be-
excited by muscle contraction. The traces show from the top downward: the cause spinobulbar lamina I neurons are not antidromically
tension developed in the triceps surae muscle, the discharge of the single activated from the contralateral thalamus (0 of 10 tested)
neuron histogrammed in 1 s bins and either mean blood pressure (A) or the neural
recording (B and C). A: response from an HPC lamina I neuron with unidentified
(Andrew and Craig, unpublished observations).
projections that was excited by contraction that paralleled the tension devel- To conclude, we demonstrated that spinobulbar lamina I
oped in the muscle and the centrally-evoked cardiovascular reflex. B: response neurons can be identified that are activated by muscle contrac-
of another HPC lamina I neuron with unidentified projections that was only tion; such neurons provide an ascending pathway for the au-
excited after the contraction. C: response of a spinobulbar lamina I neuron tomatic cardiorespiratory adjustments to muscular work, that
(central CV 2.2 m/s) that was excited during and after the contraction; this
neuron had no identifiable cutaneous receptive field but responded to stretching is, the exercise pressor reflex. This study provides the founda-
the triceps muscle and also to rhythmic contractions at 5 Hz. tion for investigations of spinobulbar mechanisms that mediate
homeostatic adjustments elicited by small-diameter afferent
piratory responses are mediated in part by central motor com- activity from all tissues of the body. It remains to be estab-
mands and in part by the “exercise pressor reflex,” which lished how other types of somatic and visceral afferent activity
originates in small-diameter group III and IV (A␦ and C) are represented in this unexplored pathway.
afferents from active muscles (Iwamoto et al. 1985; Mitchell
and Schmidt 1983; Wilson and Hand 1997). These receptors We thank M. Tatum and S. Jordan for excellent technical assistance.
are either directly excited by contraction (mechanosensitive) or This work was supported by National Institute of Neurological Disorders

and Stroke Grant NS-25616 (A. D. Craig) and by the American Heart Asso- KAUFMAN MP AND FORSTER HV. Reflexes controlling circulatory, ventilatory
ciation–Southeast Affiliate (L. B. Wilson). and airway responses to exercise. In: Handbook of Physiology. Exercise:
Regulation and Integration of Multiple Systems, edited by Rowell LB and
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KAUFMAN MP, LONGHURST JC, RYBICKI KJ, WALLACH JH, AND MITCHELL JH.
ADREANI CM, HILL JM, AND KAUFMAN MP. Responses of group III and IV Effects of static muscular contraction on impulse activity of groups III and
muscle afferents to dynamic exercise. J Appl Physiol 82: 1811–1817, 1997. IV afferents in cats. J Appl Physiol 55: 105–112, 1983.
ANDREW D AND CRAIG AD. Lamina I spinothalamic and spinobulbar neurons KNIFFKI KD, MENSE S, AND SCHMIDT RF. Muscle receptors with fine afferent
are anatomically distinct. Soc Neurosci Abstr 30: 157.12, 2000. fibers which may evoke circulatory reflexes. Circ Res 48: 125–131, 1981.
ANDREW D AND CRAIG AD. Spinothalamic lamina I neurons selectively sen- KROUT K AND CRAIG AD. Retrograde identification of lamina I neurons that
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lamina I. Prog Brain Res 107: 225–242, 1996. (SRF) of cats. Brain Res 894: 249 –254, 2001.
CRAIG AD. The functional anatomy of lamina I and its role in post-stroke MCCLOSKEY DI AND MITCHELL JH. Reflex cardiovascular and respiratory
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CRAIG AD AND KNIFFKI KD. Spinothalamic lumbosacral lamina I cells respon- from skeletal muscle receptors. In: Handbook of Physiology. The Cardio-
sive to skin and muscle stimulation in the cat. J Physiol (Lond) 365: vascular System. Peripheral Circulation and Organ Blood Flow. Bethesda,
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CRAIG AD, KROUT K, AND ANDREW D. Quantitative response characteristics of SATO A AND SCHMIDT RF. Somatosympathetic reflexes: afferent fibers, central
thermoreceptive and nociceptive lamina I spinothalamic neurons in the cat. pathways, discharge characteristics. Physiol Rev 53: 916 –947, 1973.
J Neurophysiol 86: 1459 –1480, 2001. SHERRINGTON CS. Cutaneous sensations. In: Text Book of Physiology, edited by
CRAIG AD AND MENSE S. The distribution of afferent fibers from the gastroc- Schäfer EA. Edinburgh, UK: Pentland, 1900, p. 920 –1001.
nemius-soleus muscle in the dorsal horn of the cat, as revealed by the STORNETTA RL, MORRISON SF, RUGGIERO DA, AND REIS DJ. Neurons of rostral
transport of horseradish peroxidase. Neurosci Lett 41: 233–238, 1983. ventrolateral medulla mediate somatic pressor reflex. Am J Physiol Regu-
DEGTYARENKO AM AND KAUFMAN MP. Stimulation of the MLR inhibits the latory Integrative Comp Physiol 256: R448 –R462, 1989.
discharge of dorsal horn neurons responsive to muscular contraction. Brain WESTLUND KN AND CRAIG AD. Association of spinal lamina I projections with
Res 880: 178 –182, 2000. brainstem catecholamine neurons in the monkey. Exp Brain Res 110: 151–
HYLDEN JL, HAYASHI H, DUBNER R, AND BENNETT GJ. Physiology and mor- 162, 1996.
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505–515, 1986. pressor reflex. J Appl Physiol 90: 919 –925, 2001.
IWAMOTO GA, WALDROP TG, KAUFMAN MP, BOTTERMAN BR, RYBICKI KJ, WILSON LB AND HAND GA. The pressor reflex evoked by static contraction:
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PERSPECTIVES
Second, astrocytes, microglia and — in
OPINION
some regions of the CNS — mast cells are
the innate, parenchymal immune cells
Neurogenic neuroinflammation: of the CNS5–8. Their activation is actively
depressed under non-pathological condi-
inflammatory CNS reactions in tions. Finally, the permeability of microves-
sels in the CNS for extravasation of large
response to neuronal activity molecules and blood cells is reduced in
comparison to the rest of the body by the
‘blood–CNS barrier’. Hence, it is much
Dimitris N. Xanthos and Jürgen Sandkühler more difficult to activate complement
cascades and to recruit cells involved in
Abstract | The CNS is endowed with an elaborated response repertoire termed the adaptive immunity response, such as
‘neuroinflammation’, which enables it to cope with pathogens, toxins, traumata leucocytes, into the CNS parenchyma. With
and degeneration. On the basis of recent publications, we deduce that the notable exception of activated T cells,
orchestrated actions of immune cells, vascular cells and neurons that constitute which readily penetrate the intact blood–
CNS barrier, CNS innate immune cells thus
neuroinflammation are not only provoked by pathological conditions but can also
do not as efficiently recruit the machin-
be induced by increased neuronal activity. We suggest that the technical term ery of the adaptive immune response as
‘neurogenic neuroinflammation’ should be used for inflammatory reactions in the do dendritic cells in peripheral tissues9.
CNS in response to neuronal activity. We believe that neurogenic neuro- Therefore, resident innate immune cells
inflammation maintains homeostasis to enable the CNS to cope with enhanced of the CNS must often deal directly with
pathogens and tissue damage, and it is only
metabolic demands and increases the computational power and plasticity of CNS
under severe conditions that inflammatory
neuronal networks. However, neurogenic neuroinflammation may also become cells such as infiltrating T cells are involved
maladaptive and aggravate the outcomes of pain, stress and epilepsy. (see REF. 10 for a review).
The mild inflammatory tissue reac-
The integrity of all body tissues is endan- of exogenous or endogenous antigens and tions in the CNS protect neurons — with
gered by microbial pathogens, toxins, trau- activation of the complement system. In their low regenerative capacity — from the
mata and degeneration. In response to such peripheral tissues, dendritic cells provide destructive inflammatory responses that are
situations, innate and adaptive immune information to cells of the adaptive immune readily induced in regenerating peripheral
cells, vascular cells and neurons take con- system, leading to vigorous inflamma- tissues. This has led to the introduction of
certed and finely tuned defence actions to tory responses, such as phagocytosis (and the term ‘neuroinflammation’ to distinguish
maintain or restore tissue integrity. Initially, eventually necrosis), the formation of new inflammatory reactions in the CNS from
innate immune cells, such as macrophages, connective tissue and granulomas. Diverse inflammation in other tissues. From the pre-
mast cells and dendritic cells, are activated communication channels link the immune sent literature, it is not always clear which
and respond in a nonspecific manner to system to the CNS and enable it to support criteria must be met to qualify for the label
exogenous or endogenous danger signals. host defence by promoting fever, increased neuroinflammation. Numerous studies have
This leads to tissue reactions that range sleep and enhanced pain sensitivity (hyper- assessed individual responses such as the
from mild homeostatic responses (some- algesia)2. The spectrum of actions and production and the release of pro-inflam-
times known as ‘para-inflammation’) that responses that occur strongly depends matory cytokines or disturbances of the
are close to the basal, non-stressed state to upon the type, intensity and duration of blood–brain barrier. For example, it is well
a transition into full-scale inflammation1. the initial trigger signal, the tissue affected established that epileptic seizures lead to the
In the full inflammatory response, the and the phase of the reaction. Collectively, release of cytokines in the affected brain tis-
vasculature reacts with vasodilation and this multitude of tissue reactions is termed sue11,12. In our opinion, whether the release
extravasation of plasma components and ‘inflammation’. of a pro-inflammatory cytokine alone is
blood cells, establishing three of the four Inflammatory reactions within the indicative of an inflammatory reaction is
classical signs of inflammation: rubor (red- CNS differ substantially from those of debatable. The term ‘immune signalling’
ness), calor (warmth) and tumour (swell- other tissues in several ways. First, the seems to be more appropriate to describe
ing). The fourth sign is dolor (pain). The CNS parenchyma lacks resident dendritic the isolated release of immune-relevant
most violent of these reactions are usually cells; perivascular macrophages3 and molecules without any concomitant expres-
seen during an infection or in an inflam- vascular pericytes4 take over the func- sion of other signs of neuroinflammation.
matory disease and involve presentation tions of mature dendritic cells in the CNS. However, when the respective literature is
NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | JANUARY 2014 | 43
© 2014 Macmillan Publishers Limited. All rights reserved

PERSPECTIVES
reviewed as a whole, it may become clear forms of neuroinflammation18–21 (FIG. 3). are of particular interest as they lead to
that under a given experimental condi- We thus propose the technical term ‘neu- long-term changes in the processing of
tion, the full spectrum of the inflammatory rogenic neuroinflammation’ to describe sensory information in the spinal dorsal
response involving immune cells, vascular those inflammatory reactions within the horn and are identical to those that trigger
cells and neurons takes place; as occurs, for CNS that are triggered by neuronal activity. neurogenic inflammation in the peripheral
example, in the course of epilepsy13. We suggest that neurogenic neuroinflam- tissues (FIG. 2). Effective stimuli in rodent
It is often believed that neuroinflamma- mation may have beneficial effects such as hindpaws include direct electrical nerve
tion is induced only by a pathological state, enabling the nervous system to cope with stimulation at intensities sufficient to acti-
usually in the form of a microbial infection, enhanced metabolic demands, increasing vate C fibres, selective activation of pepti-
exposure to toxins or degeneration (FIG. 1) its computational power and promoting dergic primary afferents that express the
(see REFS 8,10,14 for reviews). However, regeneration. Neurogenic neuroinflam- transient receptor potential V1 (TRPV1)
we feel that neuroinflammation and its mation may become maladaptive when it receptor by capsaicin and chemically
mechanisms do not have to be by definition persists for longer than necessary or when induced inflammation. As in the periphery
pathological and may encompass immune it spreads to remote sites (FIG. 1), and it may (FIG. 2), activation of peptidergic primary
signalling as long as immune cells, vascu- be relevant to conditions as diverse as pain, afferent C fibres also leads to the spinal
lar cells and neurons act in concert. This psychological stress and epileptic seizures. release of various mediators, including
concerted action does not necessarily have glutamate, substance P, calcitonin gene-
to be synergistic at all times: pro- and anti- Neurogenic neuroinflammation related peptide (CGRP), brain-derived neu-
inflammatory processes may occur simul- Classical neurogenic inflammation in rotrophic factor (BDNF), fractalkine and
taneously. FIGURE 1 illustrates the concept peripheral tissues is triggered by action- ATP (FIG. 3). Receptors for these neurotrans-
of parallel and interacting homeostatic and potential-dependent release of substances mitters and neuropeptides are present in
pathological processes and outcomes. from the peripheral terminals of peptider- nearby cells of the immune system, vascular
Many studies demonstrate that, in addi- gic, sensory nerve fibres and involves vaso- cells and higher-order neurons.
tion to the classical instigators of inflam- dilation, plasma extravasation, recruitment
mation described above, enhanced levels of of white blood cells and mast cell degranu- Immune responses to neuronal activity.
neuronal activity can trigger inflammatory lation (BOX 1; FIG. 2). A number of studies Glial cells can be directly activated by
reactions in peripheral tissues, where it has have now shown that similar substances substances that are released from primary
long been known as ‘neurogenic inflam- are released from synapses in the CNS in afferent nerve fibres upon stimulation.
mation’ (REFS 15–17) (BOX 1; FIG. 2). Here, we response to neuronal activity; however, few This includes substance P acting on the
discuss emerging evidence suggesting that studies have considered this response profile neurokinin 1 receptor (NK1; also known as
neuronal activity may also be sufficient to as a whole. substance P receptor), ATP acting on P2X
trigger the concerted actions of immune We focus here on spinal changes in purinoceptor 7 (P2X7) and glutamate act-
cells, vascular cells and neurons within response to stimulation of peptidergic, ing on metabotropic glutamate receptors
the CNS in a manner that resembles other nociceptive nerve fibres. These stimuli (mGluRs) (also see below). Consequently,
Triggers Actions Outcomes
Classical Homeostatic Adaptation

• Infectious microbes • Release of gliotransmitters, neurotrophic • Microbe elimination –
+
• Viral factors, cytokines • Synaptic plasticity
• Bacterial • Vasodilation • Enhanced perfusion
• Fungal and • Phagocytosis • Neuroprotection, repair, regeneration
protozoal
• Autoimmunity Maladaptive Dysfunction
• Toxins +
• Release of pro-inflammatory factors • Hyperexcitability and/or impaired –
• Environmental • Plasma extravasation inhibition
• Disease proteins • Reduced computational power
and danger signals Neuroinflammation
Neurotoxic Degeneration
• Release of pro-inflammatory factors • Progressive CNS loss of function –
Neurogenic
States of enhanced + • Excitotoxicity, apoptosis • Chronic disease
neuronal activity • Blood–CNS-barrier breakdown
• Noxious stimuli
• Psychological stress Anti-inflammatory Resolution
• Epileptic seizure • Release of anti-inflammatory cytokines, • Termination of inflammatory
neuroprotectins, resolvins, neurotrophic response
+
factors, neurotransmitters, neuropeptides,
cell adhesion molecules
• Vasodilation
Figure 1 | Triggers, actions and outcomes of neuroinflammation. homeostatic, leading to adaptation, or dysfunctional and/or
Nature Reviews neurotoxic,
| Neuroscience
Neuroinflammation can be triggered by ‘classical’ factors (infection, autoim- leading to pathology. Anti-inflammatory mechanisms may be triggered in
munity or toxins) but also by factors that lead to enhanced neuronal activity parallel and serve to terminate neuroinflammation and reduce pathological
(including noxious stimuli, psychological stress and epileptic seizures). outcomes (indicated by minus signs). Treatments and interventions may be
Immune cells, vascular cells and neurons promote various independent as targeted at various levels to inhibit the triggers and neuroinflammatory
well as interacting responses (indicated by plus signs). These can be processes, or to promote the resolution of inflammation.
44 | JANUARY 2014 | VOLUME 15 www.nature.com/reviews/neuro

PERSPECTIVES
Box 1 | Neurogenic inflammation

in response to neuronal activity)35,36. In
addition, T cells are activated by serotonin35
Neurogenic inflammation is a local inflammatory state in peripheral tissues induced by neuronal and dopamine37, substances that are also
activity. Upon stimulation, sensory nerve fibres transmit action potentials not only released in spinal dorsal horn upon affer-
orthodromically to the CNS but also antidromically into inactive branches of the afferent fibre ent stimulation38,39 (FIG. 3). Furthermore,
(FIG. 2). Experimentally, various noxious stimuli, such as direct electrical nerve stimulation or
naive, antigen-inexperienced T cells can be
activation of transient receptor potential V1 (TRPV1) channels by capsaicin, lead to the
excitation of C fibres (unmyelinated, nociceptive nerve fibres) and induce neurogenic
recruited to the CNS by chemoattractant
inflammation. At the peripheral endings of peptidergic C fibres, neuropeptides such as signals produced by activated neurons or
substance P, calcitonin gene-related peptide (CGRP) and neuropeptide Y are released and trigger glia and by stressed endothelial cells40.
inflammatory tissue reactions (FIG. 2). Mast cells are particularly implicated, as they rapidly Mast cells are usually activated by
degranulate and release a large number of substances such as cytokines, prostaglandins, immunoglobulin E (IgE) binding to its
serotonin and histamine. Pro-inflammatory mediators, as well as released glutamate, will sensitize receptor FcεRI. However, substances that
nociceptive nerve endings, leading to pain. Further tissue reactions include vasodilation, plasma are released in the spinal cord upon pri-
extravasation and recruitment of leucocytes to the tissue15–17,171. The entire process may be mary afferent stimulation — including sub-
self-amplifying, leading to continuous neuropeptide release. Participation of the CNS is not stance P, CGRP, nerve growth factor and
required for peripheral neurogenic inflammation, although it can clearly amplify it. Neurogenic
vasoactive intestinal polypeptide41 — can
inflammation has initially been described in the skin but has now also been identified in a wide
range of tissues and organs, including peripheral nerves, soft tissue, joints, airway, eye, gums,
also trigger mast-cell degranulation (the
meninges, pancreas and viscera (see REF. 16 for a comprehensive overview). In the skin, neurogenic release of molecules from secretory vesicles
inflammation leads to the classical inflammatory signs of rubor (redness), tumour (swelling), calor known as granules) (FIG. 3). Activation of
(warmth) and dolor (pain). Neurogenic inflammation thus resembles other forms of inflammation in TRPV1‑expressing primary afferent C fibres
many aspects. Neurogenic inflammation may have beneficial effects168,172 or may amplify disease by capsaicin, which leads to the spinal
states such as psoriasis, arthritis, asthma, ocular trauma, periodontitis, migraine, pancreatitis, release of substance P, enhances the number
inflammatory bowel disease, colitis, neuropathic pain, sepsis and cardiovascular disease16. of degranulated spinal dural mast cells42.
Neurons thus seem to be powerful trig-
gers of innate and adaptive immune-cell
markers of activation are upregulated in spi- in the absence of neuronal activity. Glial activation in the CNS. However, it is worth
nal microglia and astrocytes within minutes cells switch to distinct and finely tuned noting that neuronal activity may also trig-
of enhanced neuronal activity. For example, executive phases in response to neuronal ger anti-inflammatory reactions in the CNS,
phosphorylated p38 mitogen-activated activity 6,7. Thus, in addition to the well- as outlined below.
protein kinase is increased in microglia described activation of spinal glial cells
after stimulation of sensory nerve fibres in the course of peripheral neuropathies Vascular responses to neuronal activity.
with formalin in conscious rats22. Microglial or spinal-cord injuries (see REFS 31,32 for Noxious mechanical stimulation, formalin
SRC-family kinases23 are upregulated after reviews), neuronal activity is also sufficient and capsaicin injections into a rat hindpaw
electrical stimulation of C fibres, and con- to activate glial cells in the spinal cord and direct electrical nerve stimulation all
nexin dephosphorylation occurs in astro- (FIG. 3). However, other peripheral triggers increase spinal blood flow 20,43,44. The cou-
cyte gap junctions after capsaicin or C‑fibre of glial-cell activation in the CNS must not pling between neuronal activity and vascular
stimulation in anaesthetized rats24. High- be ignored. For example, cytokines such as responses is mediated by the neurovascular
frequency discharges in primary afferent tumour necrosis factor‑α (TNFα) may be unit, which is comprised primarily of neu-
C fibres induce a rise in intracellular Ca2+ transported in an anterograde direction in rons, astrocytes and endothelial cells (FIG. 3).
concentrations ([Ca2+]i) in spinal astrocytes sensory nerve fibres from the peripheral Vascular cells constitutively express cytokine
within seconds, and enhanced expression of tissues to the spinal cord33, where they receptors such as interleukin‑1 (IL‑1) recep-
immunohistochemical markers of microglia could activate glia. Whether glial-cell acti- tors45, purinergic receptors46, NK1 and CGRP
activation in spinal-cord slices within min- vation and the release of cytokines alone receptors, and soluble guanylyl cyclase (which
utes18. Electrical nerve stimulation induces meet the criteria for being classified as forms part of the signalling pathway activated
morphological changes in microglia and in neuroinflammation is debatable. However, by nitric oxide). Many vasoactive substances
astrocytes in rat spinal-cord and trigeminal as outlined below, neuronal activity also are released from primary afferents, activated
nuclei19,25. Hence, glial activation constitutes recruits additional components of an glial cells and vascular cells in the CNS in
mainly an innate immune response with a inflammatory reaction, and we believe response to primary afferent activity. For
phagocytic macrophage phenotype26 and that together these constitute neurogenic example, spillover beyond the synaptic cleft
probably also involves activation of pat- neuroinflammation. of substance P and other neurokinins that
tern- and danger-recognition receptors Under resting conditions, T cells are cause enhanced capillary permeability 47 and
(such as Toll-like receptor 4)27,28, which are present in the CNS parenchyma in rela- of CGRP, an extremely potent vasodilator 48, is
thought to trigger innate immune responses tively low numbers. CD4+ T cells and, to a known to occur in the spinal cord in response
in the CNS29. greater extent, CD8+ T cells are found in the to afferent nerve stimulation49,50. ATP is
It is becoming increasingly clear intact spinal-cord parenchyma34. Like glial another potent vasodilator 51 in the CNS that
that activation of microglia is not an cells, these T cells express a large number of is released in an activity-dependent manner
‘all‑or‑none’ process and does not take a neurotransmitter receptors and can be acti- in the spinal cord52,53. Other potentially vaso-
linear path with fixed uniform outcomes6. vated in an antigen-independent fashion by active substances, including prostaglandins
Instead, it seems that glial cells are per- glutamate, substance P, CGRP, somatosta- from spinal endothelial cells54 and potassium
manently active but remain in a surveil- tin, BDNF and neuropeptide Y (all of which ions55, are also released in response to neu-
lance mode and are even highly motile30 are released directly from primary afferents ronal activity 56 (FIG. 3).

PERSPECTIVES
CNS
• CB • Complement Macrophage Peripheral tissue

• Complement • Cytokines
• Cytokines • NO DRG
• DAMP • PG
• END Ion channel or ionotropic
• IgE receptor
• NO Antidromic
• PAF Metabotropic receptor
action
• PG Orthodromic
• ATP • GABA potential
• SP action potential Plasmalemmal transporter
• CB • Glu
• Complement • Resolvins
• Cytokines and lipoxins
• DAMP • SP
Peptidergic
C-fibre terminal
Endothelial cell
Noxious stimuli,
inflammation,
Mast cell Ca2+ CD4+ Glu wound, trauma
Cytokines
Dendritic
• Cytokines • ATP • BK cell
• DAMP • BDNF • Complement
• His • CGRP • Cytokines T-cell
• NGF • DAMP • END
• NO • Glu • NO Pericyte
• PAF • NPY • Resolvins • ATP • Glu
• PG • SOM and lipoxins CD8+ • NGF
• ATP • Glu • BDNF
• Serotonin • SP • CB • NA • CB • NPY • CGRP
• TRP • CGRP • NO • CGRP • Resolvins • Complement
• Complement • PG • Complement and lipoxins • Cytokines
• Cytokines • Resolvins • Cytokines • Serotonin • DAMP
• DA and lipoxins • DA • SOM • Resolvins
• DAMP • Serotonin • DAMP • SP and lipoxins
• END • SP Cytokines • END • SP
Figure 2 | Neuronal activity triggers neurogenic inflammation in periph- boxes), creating the ‘inflammatory milieu’. Immune cells, plasma and various
Nature Reviews | Neuroscience
eral tissues. The figure shows a primary afferent, peptidergic nerve fibre mediators can also extravasate into tissue (not shown). Sensory nerve fibres
and elements that contribute to neurogenic inflammation at peripheral can become sensitized and also lower their threshold for further neuro-
nerve terminals. Neurogenic inflammation in the periphery is initiated by transmitter and neuropeptide release. Pro- and anti-inflammatory sub-
neuronal activity generated by a wide range of highly specific (such as tran- stances and signalling molecules that are released (shown in light yellow
sient receptor potential V1 (TRPV1) activation) and less-specific stimuli (such boxes) from various sources bind to receptors on the different cells and
as traumatic injury). This results in the generation of orthodromic action modulate their function. Signalling from higher-order CNS centres (not
potentials that conduct towards the CNS, as well as antidromic action shown) may also dampen or aggravate peripheral neurogenic inflamma-
potentials at branch points that conduct towards the peripheral terminals tion. BDNF, brain-derived neurotrophic factor; BK, bradykinin; CB, cannabi-
to induce neurogenic inflammation. Neurogenic inflammation results from noid; CD, T-cell surface glycoprotein CD; CGRP, calcitonin gene-related
the release of neurotransmitters and neuropeptides from peripheral nerve peptide; DA, dopamine; DAMP, danger-associated molecular patterns;
terminals (blue box). These rapidly affect various cell types, including vascu- DRG, dorsal root ganglia; END, endothelin; Glu, glutamate; His, hista-
lar cells (endothelial cells), mast cells, macrophages and other immune cells mine; IgE, immunoglobulin E; NA, noradrenaline; NGF, nerve growth fac-
(not shown). T cells and dendritic cells may also be recruited. The different tor; NO, nitric oxide; NPY, neuropeptide Y; PAF, platelet activating factor;
cell types themselves also begin to release substances (shown in coloured PG, prostaglandin; SOM, somatostatin; SP, substance P; TRP, tryptase.
Although neuronal activity readily However, a more robust afferent barrage in Contribution of higher-order neurons.
enhances regional blood flow in the CNS, C fibres, which is triggered by direct sciatic Neuronal activity is by definition the pri-
the integrity of the blood–CNS barrier nerve capsaicin application, induces wide- mary trigger for neurogenic neuroinflam-
is substantially more resistant to change. spread disruption of the blood–spinal-cord mation. Peptidergic C fibres are, however,
Formidable neuronal activity is required for barrier 24 hours after stimulation21. not the only logical source for the induction
such changes to occur. For example, the tight Vascular cells in the CNS not only of neurogenic neuroinflammation in the
junction protein occludin is altered in spinal respond to pro-inflammatory sub- spinal cord. In fact, microglia60,61 and CNS
endothelial cells and mild IgG extravasation stances but can also release cytokines and endothelial cells61,62 express receptors for
is detected no earlier than 72 hours after chemokines58,59, possibly contributing to the a wide range of neurotransmitters, some
hindpaw inflammation with carrageenan57. inflammatory process. of which are released from higher-order

PERSPECTIVES
• ATP • Glu
• BDNF • NGF T cell
• CB • NPY
• CGRP • Resolvins • ATP • ATP • Glu
• Complement and lipoxins • BDNF • CB • Gly Descending
• Cytokines • Serotonin CD8+ • Cytokines • CGRP • NA neuron
• DA • SOM • DAMP • Complement • NGF
• DAMP • SP • D-Ser • Cytokines • Resolvins
• END Cytokines • GABA • DA and lipoxins • ATP
• ADO • Glu • Glu • DAMP • PG • Cytokines
• ATP • NA • PG • END • Serotonin • DA
Glu
• CB • NPY CD4+ • GABA • SP • NA
• Complement • Resolvins • Serotonin
• Cytokines and
• DA lipoxins
• DAMP • Serotonin Postsynaptic Astrocyte
• ATP
• END • SOM • CB
• Fractalkine • SP • Cytokines
• GABA • ATP • Fractalkine
Glu
• ATP • CB • GABA
• BDNF K+ • CGRP • Glu
• Complement • Complement • Gly
• Cytokines • Cytokines • NO
H2O • DA
• DAMP
• Glu • ATP • DAMP
Presynaptic
• NO • BDNF • END
• PG Ca2+ • CGRP • Glu
Microglial • Glu • NA
cell • CB Astrocyte • NO
• Cytokines • NPY
• CGRP endfoot • PG
• DAMP • SOM
• Complement Endothelial • Resolvins
• His • SP
• Cytokines cell and lipoxins
• NGF
• DAMP • Cytokines • Serotonin
• NO
• END • DAMP • SP
• PAF
• IgE • Fractalkine
• PG
• NGF • Cytokines
• Serotonin Pericyte
• NO • END
• TRP
Mast cell • NPY Neurovascular unit • NO
• PAF • PG Interneuron
• PG
Blood–CNS barrier • SP CNS
Peripheral tissue
Soma Orthodromic
action potential Plasmalemmal
transporter
Ion channel or
ionotropic receptor
Peptidergic C-fibre terminal Metabotropic
receptor
Nature Reviews | Neuroscience

Figure 3 | Neuronal activity triggers neurogenic neuroinflammation effects against excitotoxicity. However, they may also participate in neu-
in the CNS. This figure illustrates neurogenic neuroinflammation at spi- rogenic neuroinflammation to release pro-inflammatory mediators. As in
nal or trigeminal terminals. In the CNS, enhanced neuronal activity com- the periphery, both pro- and anti-inflammatory mediators, and signalling
ing from peripheral sources will result in neurogenic neuroinflammation molecules and forces can be released from all cell types (key substances
owing to vesicular and non-vesicular release of neurotransmitters and shown in boxes of the respective colour of cell type) in the multipartite
neuropeptides from the primary afferent fibre (blue boxes). This will synaptic region to further affect receptors or channels present on all cell
induce concerted and interacting immune responses, vascular responses types shown (key substances acting on cells shown in light yellow boxes).
and higher-order neuronal network responses in the multipartite synapse. Ongoing neurogenic neuroinflammation may serve to amplify neuronal
This includes, but is not limited to, microglia, astrocytes, the neurovascular network activity and the resulting long-term potentiation may spread far
unit (composed of endothelial cells, other vascular cells such as pericytes, along the neuraxis, enhancing computational power of the neuronal net-
the presynaptic neuron and the astrocyte endfeet) and second-order neu- work. This can serve to elicit appropriate protective responses and behav-
rons within the neuronal network (including interneurons, ascending iours from the organism. In some cases, it may also trigger or aggravate an
neurons and descending neurons), all of which are primary players in the established pathology. Signalling from higher-order CNS centres
response to enhanced C‑fibre activity. Mast cells on the dura, perivascular (descending neurons) can serve to dampen or aggravate neurogenic neu-
macrophages, and CD4+ and CD8+ T cells may also participate and release roinflammation. ADO, adenosine; BDNF, brain-derived neurotrophic fac-
substances. With strong neuronal activity, recruitment of peripheral tor; CB, cannabinoid; CGRP, calcitonin gene-related peptide; DA,
immune cells (including macrophages, T cells and mast cells), and changes dopamine; DAMP, danger-associated molecular patterns; d‑Ser, d‑serine;
at the blood–CNS barrier (for specific substances or involving a regional END, endothelin; Gly, glycine; His, histamine; IgE, immunoglobulin E; NA,
breakdown) can occur, creating further CNS neuroinflammatory noradrenaline; NGF, nerve growth factor; NO, nitric oxide; NPY, neuropep-
responses. Astrocytes found exclusively in the CNS serve to take up exces- tide Y; PAF, platelet activating factor; PG, prostaglandin; SOM, somatostatin;
sive glutamate (Glu) and potassium, thereby providing neuroprotective SP, substance P; TRP, tryptase.

PERSPECTIVES
neurons but not from primary afferents. synapses73–75. The list of relevant synaptic to a rise in InsP3 levels and [Ca2+]i in neu-
Examples are the inhibitory neurotransmit- partners is likely to increase as we broaden rons, microglia and astrocytes, resulting in
ters glycine and GABA, both of which are our knowledge of the mechanisms of neu- glial-cell activation85 and LTP86. However,
released from spinal interneurons, and the rogenic neuroinflammation. Eventually, mGluR5 activation in spinal microglia inhib-
monoamines noradrenaline, serotonin and this growth in knowledge will culminate in its the release of inflammatory mediators
dopamine, which are released from descend- the concept of a ‘multipartite synapse’. The (cytokines or free radicals) both in vitro87
ing-tract neurons. These neurotransmitters actual number of critical cellular and extra- and in vivo88 and a specific group I mGluR
may modulate the functions of both glial and cellular elements modulating the transmis- agonist induces long-term depression at
vascular cells. Monoamines are vasoactive in sion at multipartite synapses will depend spinal Aδ‑fibre synapses89. Evidence from
the CNS63,64 but they also affect the functions upon the context and is likely to differ experiments using in vitro oxidative stress
of immune cells (see REF. 65 for a review). between CNS regions. and excitotoxicity protocols also suggests
In addition, activation of type A GABA anti-inflammatory roles for mGluR1 activa-
receptors leads to the production of oxygen Overlapping signalling pathways. The vari- tion90, and specific stimulation of group I
radicals in rodent microglia66. Noradrenaline ous intracellular signalling pathways that mGluRs in astrocytes leads to increased glu-
causes retraction of microglial processes contribute to neurogenic neuroinflamma- tamate and potassium uptake91. In addition,
through activation of the β2‑adrenergic tion exist in more than one type of cell in vascular cells in the CNS express mGluRs92
receptor under resting conditions and the multipartite synapse. Pharmacologically and it has been suggested that activation
through activation of the adrenergic α2A modulating these signalling pathways of group I mGluRs can increase vascu-
receptor under pro-inflammatory conditions systemically or regionally (but not cell spe- lar permeability 93, although these effects
in tissue culture and brain slices67. Serotonin cifically) may therefore result in complex remain to be investigated further. Hence, the
promotes microglial motility but reduces synergistic and/or antagonist interactions. downstream effects of activity-dependent
phagocytic activity 68. For example, the binding of substance P glutamate release are likely to result in both
In summary, the available evidence sug- to NK1 receptors in spinal neurons after pro- and anti-inflammatory actions.
gests that neuronal activity in primary affer- stimulation of C fibres activates the phos-
ent nerve fibres or higher-order neurons pholipase C and inositol triphosphate Therapeutic resolution
is sufficient to activate innate and adaptive (InsP3) signalling pathway, leading to Most inflammatory conditions are of lim-
immune cells, vascular cells and neurons in increased [Ca2+]i and synaptic long-term ited duration. Resolution of inflammation
the spinal cord. It thus resembles other trig- potentiation (LTP)76. Activation of NK1 is an active process that involves the actions
gers of neuroinflammation in the CNS8,10, receptors on astrocytes can also lead to of anti-inflammatory mediators such as
and we therefore suggest that the term neu- increases in [Ca2+]i levels77 and NK1 recep- IL‑10 (REF. 94), neuroprotectin D1 (REF. 95),
rogenic neuroinflammation should be used tor antagonists can reverse spinal astrocyte resolvins96, neurotrophic factors, and TNFα
to describe this phenomenon. activation78. Activation of microglial NK1 and fractalkine (under some conditions)97,
receptors leads to the activation of the pro- produced by immune, vascular and/or neu-
The multipartite synapse inflammatory nuclear factor-κB pathway 79. ronal cells (FIG. 1). Anti-inflammatory actions
All the elements of neurogenic neuroinflam- NK1 receptor activation in endothelial cells, have been described for dopamine acting
mation described above interact in a com- as in neurons, also leads to phospholipase C on astrocytic dopamine D2 receptors68 (see
plex manner, the details of which have only activation, InsP3 accumulation, and [Ca2+]i REF. 65 for a review), and for somatostatin98,
become better understood in recent years. rises80. Thus, substance P exerts synergistic neuropeptide Y99 and adenosine acting on
For example, neurons and microglia interact pro-inflammatory actions on various cell adenosine A2A receptors on microglia100.
bidirectionally, and the dialogue between types of the multipartite synapse through Other anti-inflammatory responses
these cells involves fractalkine (also known NK1 receptors. to neuronal activity involve major histo-
as CX3CL1), a transmembrane chemokine Release of cytokines may likewise have compatibility complex (MHC) molecules.
that is expressed by neurons and acts synergistic effects. For example, after Upon exposure to interferon‑γ, neurons
through a receptor (CX3CR1) that is exclu- peripheral nerve stimulation56, activation can express MHC molecules at their surface
sively present on microglia69,70. Fractalkine of TNFα and IL‑1 receptors present on to interact with CD8+ cytotoxic T cells101.
is biologically active both as a membrane- superficial dorsal horn neurons, glial cells18 Neuronal activity dampens the neuronal101
bound adhesion molecule and in its soluble and endothelial cells81, can induce prosta- and glial102 expression of MHC molecules
form. For the soluble form of fractalkine to glandin release through cyclooxygenase 1 in part by increasing the release of nerve-
bind CX3CR1, its extracellular domain must (COX1; also known as PTGS1) and COX2 growth factor and BDNF102. Consequently,
be cleaved by cathepsin S, which is released (also known as PTGS2) activation in these silencing neuronal activity by blocking some
from activated microglia. Activity in pri- cell types81–83. This can then drive further voltage-gated sodium channels with tetrodo-
mary afferent nerve fibres can activate spinal primary afferent glutamate, substance P and toxin (TTX) induces upregulation of MHC
microglia, as described above, which in turn CGRP release56. molecules in microglia in vivo103,104 and
releases cathepsin S. This liberates soluble By contrast, the actions of glutamate on activates glial cells30. This finding cannot be
fractalkine from neurons, which boosts cellular signalling are considerably more explained by the expression of TTX-sensitive
microglia activation and is proposed to cause mixed. An example of this is provided sodium channels on glial cells, as blockade of
hyperalgesia (see REFS 71,72 for reviews). by group I mGluRs, which are expressed glial TTX-sensitive sodium channels reduces
In addition, astrocytes, T cells, and the on a wide variety of cell types84, includ- rather than increases cytokine release from
extracellular matrix have profound effects ing neurons, astrocytes, microglia, T cells glia105. The loss of physiologic neuronal and
on synaptic transmission. This has led to and endothelial cells. Activation of group I synaptic activity may also underlie activation
the concepts of tri-, tetra- and pentapartite mGluRs (and particularly mGluR5) leads of microglia after deafferentation106. This is

PERSPECTIVES
consistent with one of the known impor- and antiepileptic drugs that depress C‑fibre synapses in the spinal dorsal horn
tant roles of activated microglia, which is nuclear factor-κB pathways121. Similarly, can- in vitro132 and in vivo133 (see REF. 134 for a
to maintain functional neuronal circuits by nabinoids act on cannabinoid 1 and 2 recep- review). Recent studies have revealed that
eliminating inactive synapses107,108. tors on neuronal, immune and endothelial mediators of neurogenic neuroinflamma-
Neuronal activity may also exert inhibi- cells of the CNS, the effects of which may tion such as BDNF, ATP, TNFα and IL‑1β
tory influences on parenchymal microglia collaborate to reduce neuroinflamma- are also all essential for LTP induction in
through contact-dependent inhibition tion122,123. Conversely, opioids activate innate spinal-cord dorsal horn (see REFS 134,135
involving adhesion molecule–receptor immune cells in the CNS, which contributes for reviews).
pairs (such as CD200–CD200 receptor, to opioid tolerance124, opioid withdrawal Neurogenic neuroinflammation also
CD22–CD45 or HSP60–TREM2 (coupled to LTP125 and paradoxical opioid-induced affects synaptic inhibition in the spinal cord,
DAP12)), soluble adhesion molecules (such hyperalgesia126. which has five essential effects on nocicep-
as intercellular adhesion molecule 5 or extra- Interestingly, anti-inflammatory dietary tion: it prevents hyperalgesia, radiating pain,
cellular fractalkine), neuron-derived IgG109 elements such as omega‑3 polyunsaturated allodynia and spontaneous pain, and reduces
or anti-inflammatory cytokines110,111. fatty acids, neuroprotectin 1 or resolvins can the risk of pain chronicity 134. However, the
Some cytokines exert both pro- and anti- reduce neuroinflammation in the brain127 release of BDNF from central terminals of
inflammatory actions, depending upon the and spinal cord128, block LTP at spinal afferent nerve fibres136, or from spinal glial
context and the CNS region. For example, C‑fibre synapses95 and reduce pain-related cells (in peripheral neuropathy)137 results in
soluble fractalkine has pro-inflammatory behaviour 128 (see REF. 96 for review). impaired inhibition of nociception137,138 (see
and pronociceptive actions in the spinal REF. 139 for a review). Both LTP and reduced
dorsal horn (see REFS 71,72 for reviews). Friend or foe? inhibition can be adaptive if the result-
Conversely, both soluble and membrane- Evidence suggests that neurogenic neuroin- ing hyperalgesia enables better protection
bound forms of fractalkine attenuate flammation has roles in tissue metabolism, of injured tissues. However, they can also
lipopolysaccharide-induced activation of synaptic plasticity, modulation of neuronal become maladaptive when they persist after
microglia in primary cortical glial–neuronal excitability, glutamate excitotoxicity, and healing of the tissue or spread to somatotopi-
co‑cultures112 and reduce microglial neu- degeneration and regeneration. Neurogenic cally inappropriate (uninjured) sites.
rotoxicity in vivo in a murine Parkinson’s neuroinflammation may be beneficial
disease model70. Furthermore, fractalkine- and/or detrimental: the prevailing effect Transitions to pathology. As with other forms
stimulated microglia exert neuroprotective depends on the context and the phase of of inflammation, neurogenic neuroinflam-
effects in vitro through adenosine produc- the responses (FIG. 1). It is therefore possible mation can become pathological (FIG. 1).
tion113 (see REF. 5 for a review). Similarly that broad anti-inflammatory interventions During normal neuronal activity, such as
the cytokine TNFα (usually assumed to be may not only reduce the unwanted effects of that occurring in response to a touch or
pro-inflammatory) may have a physiological neuroinflammation but may also impede its brief pinch, glial cells and the vasculature
and neuroprotective role when present at the beneficial components. perform housekeeping functions. With
low tissue concentrations that are sufficient enhanced levels of activity (such as that fol-
for the activation of TNF receptor 2. Only at Effects in stressed tissue. Enhanced neu- lowing a minor injury), glial and vascular
higher concentrations, which are required ronal activity, such as that occurring during cells become activated in order to cope with
for TNF receptor 1 activation, does TNFα encoding of a noxious stimulus or during enhanced metabolic demands. Synaptic
become a neurotoxic signal (see REF. 114 for psychological stress, increases the metabolic spillover of neurotransmitters and accu-
a review). demands of the neuronal tissue. Neurogenic mulation of toxic metabolites or nitrogen
Thus, the available evidence suggests neuroinflammation, which increases regional and free oxygen radicals140,141 can occur.
that moderate levels of neuronal activity blood flow in the CNS can therefore provide Vasodilation will be engaged without any
exert anti-inflammatory reactions. It may the appropriate oxygen supply and transport detectable extravasation. LTP will be induced
therefore be speculated that the therapeutic capacity for metabolites. Beyond this, neu- at C‑fibre synapses, resulting in hyperalgesia
use of electrical nerve stimulation such as rogenic neuroinflammation has a number of that initially fulfils the homeostatic functions
transcutaneous electrical nerve stimulation, additional effects. described above. With more persistent activ-
electroacupuncture115,116 or transcranial Enhanced activity at glutamatergic syn- ity in peptidergic C fibres (in the case of a
direct-current stimulation117 may exert ben- apses may result in excessive extracellular chronic inflammation or wound and in some
eficial effects in part by modulating neuroin- glutamate concentrations that can become forms of peripheral neuropathy), a transition
flammation and promoting neuroprotective highly toxic to neurons129. Astrocytes express to maladaptive forms of neuroinflamma-
and regenerative mechanisms in the CNS. glutamate transporters that remove gluta- tion starts with changes in the blood–CNS
The emerging roles of neuroinflamma- mate from the extracellular space and that barrier, leading to the presence of novel
tion in CNS functions (and dysfunctions) are upregulated by neuronal activation130 and pro- and/or anti-inflammatory mediators or
likewise call for a fresh look at old drugs. It is group I mGluR signalling 91. Hence, activated cells. Neuroinflammation may reach neigh-
likely that some drugs may exploit their full astrocytes can potentially avoid or reduce bouring areas beyond the termination zones
therapeutic potential by modulating neuro- glutamate excitotoxicity. of activated primary afferents. Finally, a
inflammation rather than by their tradition- Neuroplasticity allows the nervous ‘breakdown’ of the blood–CNS barrier results
ally ascribed modes of action only. Examples system to adapt to changing conditions. in the excessive extravasation of large mol-
include COX inhibitors (which have antino- Usually, this involves direct interactions ecules and recruitment of immune cells into
ciceptive effects in both the periphery and between neurons. A prominent example the CNS parenchyma, which can damage the
the spinal cord118), antipsychotics119, antide- of such an interaction is the induction of neuronal network. Higher-order neurons,
pressants that reduce neuroinflammation120, LTP at glutamatergic synapses131, including including descending-tract neurons, may

PERSPECTIVES
amplify neurogenic neuroinflammation in Repetitive sessions of experimental normal resting mode even if classical mor-
the spinal cord and maintain immune-cell restraint in rats induce chronic stress and phological or immunohistochemical mark-
activation, as well as releasing and promoting lead to an increase in allograft inflammatory ers would suggest so. Instead, it has been
the release of further pro-inflammatory sub- factor 1 IBA1 (also known as AIF1), a micro- proposed that microglia may still bear long-
stances. Glial cells may no longer reduce glu- glia marker, in a number of stress-related lasting (that is, plastic) changes that may
tamate excitotoxicity by uptake mechanisms brain nuclei156. Repeated defeat stress also alter their future responses to similar and/or
but may now release excessive amounts of increases the number of mast cells in the different challenges6, indicating that not only
glutamate (at least when challenged in vitro), brain157. Chronic stress as a result of social neurons and T cells express memory func-
causing excitotoxicity 142 and hyperalgesia in dominance paradigms leads to higher levels tions (see REF. 170 for a review). Furthermore,
non-injured tissues. of inducible nitric oxide synthase and COX2 it is likely that neurogenic neuroinflamma-
gene expression in the rat spinal cord and tion and other forms of inflammation in the
Role in pain, stress and epilepsy to a lowered pain threshold over a similar CNS interact, possibly leading to priming of
Neurogenic neuroinflammation is likely to time course158. Chronic unpredictable stress CNS inflammatory reactions by conditions
have a role in a wide variety of conditions and methamphetamine further disrupt such as pain, psychological stress or epilepsy.
in the normal and diseased CNS, includ- the integrity of the blood–CNS barrier 159. This would be similar to the proposed impact
ing inflammatory and injury-related pain, Interestingly, chronic stress may also increase of systemic infection on the progression of
psychological stress and epilepsy. It may also gastrointestinal permeability with bacterial neurodegenerative disease121.
affect other functions and conditions such lipopolysaccharide translocation leading to An increasing body of literature shows
as neuropathic pain31, migraine143, sleep144, the release of inflammatory mediators in that neuronal activity leads to the activation
learning and memory formation145, mood the CNS160. Thus, neuronal activity patterns of glial cells and to the release of cytokines
disorders146 and autism147. that encode psychological stress responses and chemokines in the CNS. However,
and peripheral immune responses may act whether these responses alone fulfil the cri-
Neurogenic neuroinflammation in pain. synergistically to trigger neuroinflammation teria for ‘inflammation’ has been a matter for
Neurogenic neuroinflammation boosts in the brain. debate. The evidence described here dem-
nociception, as outlined above. Astrocyte onstrates that these reactions are not evoked
signalling through gap junctions and the Neurogenic neuroinflammation in epilepsy. in isolation but that neuronal activity trig-
diffusion of pro-inflammatory mediators Experimental induction of epileptic sei- gers finely orchestrated response patterns in
through spinal cord tissue may lead to zures by kainic-acid injections into rodent CNS areas that involve innate and adaptive
spreading of neurogenic neuroinflamma- cortical areas of the brain11 or electrical immune cells, vascular cells and neurons.
tion beyond the spinal projection zones of stimulation in the CA3 region of the hip- Although it may still be debatable whether
activated C fibres. This may then contribute pocampus161 leads to mRNA upregulation the earliest and mildest responses deserve
to hyperalgesia in uninjured sites (second- of several cytokines (such as TNFα, IL‑1β the label ‘neuroinflammation’, stronger and
ary hyperalgesia), mirror-image pain (pain and IL‑6) and class I MHC162 in brain areas longer-lasting neuronal activity clearly leads
at corresponding contralateral sites) and within hours of stimulation. Similarly, a to classical inflammatory signs, includ-
widespread pain134,148. single epileptic seizure in human patients ing plasma extravasation and activation of
Some forms of neuropathy lead to ectopic raises serum levels of IL‑1 receptor and IL‑6 immune cells. Homeostatic and maladap-
discharges in small afferent nerve fibres, (REF. 163). Surgical removal of the epileptic tive reactions may be active simultaneously
including C fibres149,150. It is therefore possible focus by anterior temporal lobectomy not with anti-inflammatory responses. In some
to speculate that some types of neuropathic only prevents any further epileptic seizures cases, it may not be possible to decide to
pain involve neurogenic neuroinflammation in these patients but also markedly reduces which category a given response should
in the spinal cord. Indeed, most animal mod- circulating levels of TNFα and IL‑1β164. Even be assigned. It therefore seems that no
els of peripheral neuropathy are character- brief epileptic seizures lead to perturbations unequivocal criteria would draw an objec-
ized by the activation of spinal glial cells (see of the blood–CNS barrier, with considerable tive line between homeostatic, physiological
REFS 31,32 for reviews) and by the impair- extravasation of plasma proteins and recruit- para-inflammation on one hand and patho-
ment of the blood–spinal-cord barrier 151, ment of white blood cells into the brain logical neuroinflammation on the other.
including the recruitment of T cells152. parenchyma165. Simultaneously, regenerative Furthermore, evidence suggests that classi-
processes are triggered, and these are also cal neuroinflammation also has a homeo-
Neurogenic neuroinflammation in stress. thought to involve class I MHC162,166. Taken static function. We therefore propose that
It is now becoming increasingly clear that together, the available data suggest that neurogenic neuroinflammation comprises
psychological stress involves not only neu- neuronal activity during epileptic seizures all of the responses outlined in FIG. 1.
rohormonal responses153 but also compo- not only activates glial cells and leads to the In summary, the elaborated inflamma-
nents of neuroinflammation. For example, release of pro-inflammatory cytokines but tory response repertoire of CNS tissue may
in rats, acute stress as a result of immobi- also engages several additional parameters not only be used to deal with infectious,
lization activates mast cells and leads to of neurogenic neuroinflammation. Together, toxic or degenerative processes but also to
plasma extravasation in the diencephalon154. this further enhances the frequency and cope with the demands of increased levels
Inescapable footshock also causes upregu- severity of epileptic seizures27,167,168 (also see of neuronal activity and to enhance the
lation of the microglia activation marker REFS 13,169 for reviews). computational power of neuronal networks
MHCII and downregulation of the neuronal in the CNS. However, neurogenic neuroin-
cell adhesion molecule CD200, which nor- Summary and outlook flammation may become maladaptive and
mally holds microglia in a non-activated It is possible that after a transient glial-cell aggravate clinical conditions such as pain,
state155. response, microglia may not return to their stress and epilepsy.

PERSPECTIVES
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Vol. 51 No. 3/2004
563–585
QUARTERLY
Review
HIF-1: the knowns and unknowns of hypoxia sensing.
Anna Zagórska½ and Józef Dulak½
Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University,

Kraków, Poland
Received: 28 February, 2004; revised: 29 June, 2004; accepted: 09 July, 2004
Key words: hypoxia inducible factor-1, angiogenesis, heme oxygenase, reactive oxygen species, prolyl and
asparaginyl hydroxylases, nitric oxide, carbon monoxide
Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that functions as a

master regulator of cellular and systemic oxygen homeostasis. It consists of two con-
stitutively produced subunits: HIF-1a and HIF-1b. Under normoxic conditions
HIF-1a undergoes hydroxylation at specific prolyl residues which leads to an imme-
diate ubiquitination and subsequent proteasomal degradation of the a subunit. Addi-
.
The work was in part supported by the grants 3 P04 049 22 and P04B 013 21 awarded to J. D. by the
State Committee for Scientific Research (KBN, Poland).
½
Corresponding author: Józef Dulak, Department of Cell Biochemistry, Faculty of Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; phone: (48 12) 664 6375; fax: (48 12)
664 6902; e-mail: jdulak@mol.uj.edu.pl or Anna Zagórska; e-mail: azagorska@op.pl
Abbreviations: AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear
translocator; bHLH, basic helix-loop-helix domain; BMAL, brain muscle ARNT-like protein; CBP,
cAMP-responsive element-binding protein; DMOG, N-dimethyl-oxalylglycine; EPO, erythropoietin;
FIH-1, factor inhibiting HIF-1; Flt-1, fms-like tyrosine kinase-1; FRAP, FKBP-rapamycin associated pro-
tein; HAS, HIF-1 ancillary sequence; HBS, HIF-1 binding site; HIF-1, hypoxia-inducible factor 1; HO-1,
heme oxygenase-1; HRE, hypoxia response element; HUVEC, human umbilical vein endothelial cells;
IPAS, inhibitory PAS protein; MAPK, mitogen-activated protein kinase; MDM2, murine double minute
2; NLS, nuclear localization signal; NOS2, inducible nitric oxide synthase; NPAS2, neuronal PAS do-
main protein 2; ODD, oxygen-dependent degradation domain; PAS, Per-AHR-ARNT-Sim homology do-
main; PHD, PH domain-containing protein; PI3K, posphatidylinositol-3 kinase; PKC, protein kinase C;
pVHL, von Hippel-Lindau tumor suppressor protein; ROS, reactive oxygen species; SNP, sodium
nitroprusside; SOD, superoxide dismutase; TAD, transactivation domain; VEGF, vascular endothelial
growth factor; VSMC, vascular smooth muscle cells.
tionally, hydroxylation of an asparaginyl residue blocks the transcriptional activity

of HIF-1 due to inhibition of its interaction with co-activators. In contrast, under
hypoxic conditions, abolition of prolyl hydroxylation results in HIF-1a stabilization,
whereas the lack of asparaginyl hydroxylation allows the transcriptional activity. Ad-
ditionally, the transcriptional activity may be modulated by phosphorylation or re-
dox modification of HIF-1. Despite its name, HIF-1 is induced not only in response to
reduced oxygen availability but also by other stimulants, such as nitric oxide, various
growth factors, or direct inhibitors of prolyl and asparaginyl hydroxylases. There-
fore, it seems to be a crucial transcription factor elicited by a wide range of stresses
such as impaired oxygenation, inflammation, energy deprivation, or intensive prolif-
eration. However, the mechanisms of normoxic activation, as well as of oxygen sens-
ing, are not yet fully known. Further understanding of the processes that control
HIF-1 activity will be crucial for the development of new diagnostic and therapeutic
strategies.
Oxygen homeostasis is strictly controlled in Although the importance of HIF-1 in the in-
order to maintain intracellular levels of oxy- duction of the complex response to hypoxia is
gen within tight limits dictated by the require- widely appreciated, the molecular mecha-
ment for O2 in many metabolic processes on nisms of HIF-1 activation remain unclear.
the one hand, and its high toxicity on the This review aims to summarize current
other. The control occurs at the level of an en- knowledge on HIF-1 regulation under hypoxic
tire organism as well as at the level of a single and normoxic conditions.
cell. It involves numerous mechanisms of reg-
ulation and adaptation to changes in O2 ten-
sion. At the cellular level, decreased O2 ten- HIF-1 TARGET GENES
sion (hypoxia) leads to the activation of alter-
native metabolic pathways that do not require The consensus DNA sequence for HIF-1 bin-
molecular oxygen. The switch from aerobic ding, 5¢-(A/G)CGTG-3¢, is common for many
metabolism to anaerobic glycolysis is medi- genes upregulated by low oxygen tension
ated by the induction of glycolytic enzymes (Semenza et al., 1996). The list of known
and expression of glucose transporters. Addi- genes activated by HIF-1 grows continuously.
tionally, the expression of various stress pro- It includes genes whose protein products act
teins responsible for cell death or survival is both to maintain O2 homeostasis and to adapt
upregulated. Further adaptations which oc- to changes in the oxygen concentration (Ta-
cur at the tissue and systemic levels lead to ble 1). The first group comprises of genes in-
the increase in O2 delivery. They include involved in the development and functioning of
duction of erythropoiesis (red blood cell pro- the vascular system. They either promote
duction), angiogenesis (new vessel forma- angiogenesis (such as VEGF and its receptor
tion), and hyperventilation. Among the vari- Flt-1) or modulate vascular tone (such as in-
ous upregulated proteins there is erythropoie- ducible nitric oxide synthase, heme oxy-
tin (EPO), the main growth factor inducing genase-1, endothelin 1, adrenomedullin, and
maturation of erythrocytes, and vascular en- a1B-adrenergic receptor). The second group is
dothelial growth factor (VEGF), the major represented by genes whose protein products
mediator of angiogenesis and vascular perme- induce erythropoiesis. Besides erythropoie-
ability. The hypoxia-dependent regulation of tin, HIF-1 upregulates ceruloplasmin, trans-
these and other proteins (Table 1) occurs at ferrin, and transferrin receptor which facili-
the transcriptional level and is mediated by tate the supply of iron to the erythroid tis-
hypoxia-inducible transcription factor sues. The third group consists of genes whose
(HIF-1). products are involved in energy metabolism.
Their cooperation leads to increased glucose HIF-1 STRUCTURE

uptake and a switch to glycolysis as the main
source of energy. The fourth group comprises
HIF-1 is a heterodimer of two bHLH-PAS
genes whose products are responsible for cell
proteins
proliferation and viability. Besides these four
main sets of HIF-1-induced genes, there are Hypoxia-inducible factor-1 is a heterodimer
others also important in response to various composed of the a and b subunits. Both of
stresses (all references in Table 1). them contain two characteristic domains: the
Table 1. Genes upregulated by HIF-1.

basic helix-loop-helix (bHLH) domain and the and PAS-B (aa 228–298) repeats (Wang et al.,
PAS (Per-AHR-ARNT-Sim) domain. The PAS 1995a). Two transactivation domains, N-ter-
domain was termed as an acronym for the minal and C-terminal TADs (also termed
first known members of the family: Dro- NAD and CAD), are localized in the C-termi-
sophila period (Per) and single-minded (Sim) nal half of HIF-1a (aa 531–575 and 786–826,
proteins and mammalian aryl hydrocarbon respectively; Pugh et al., 1997). Moreover, the
receptor (AHR) and aryl hydrocarbon recep- C-terminal part contains a domain responsi-
tor nuclear translocator (ARNT) proteins ble for degradation of HIF-1a under normoxic
(Wang et al., 1995a). It contains two internal conditions (Huang et al., 1998). This oxy-
homology units, the A and B repeats, and is gen-dependent degradation domain (ODD at
involved in interaction between proteins. aa 401–603) contains two PEST-like motifs:
PAS-proteins have been found in many spe- sequences rich in proline (P), glutamic acid
cies, including prokaryotes, which indicates (E), serine (S), and threonine (T) (aa 499–518
their high evolutionary stability (Wang et al., and 581–600) common for many proteins
1995a). with a short half-life (less than 2 h)
The bHLH domain, which is common for a (Rechsteiner & Rogers, 1996). The HIF-1a
large number of transcription factors, is re- half-life under normoxic conditions is less
quired for both protein dimerization and than 10 min and the protein is hardly detect-
DNA binding. In the case of HIF-1, the high- able (Chun et al., 2002). Additionally, HIF-1a
est efficiency of heterodimerization is ob- contains two nuclear localization signals:
tained only if the bHLH and PAS domains are N-NLS (aa 17–74) and C-NLS (aa 718–721).
intact (Jiang et al., 1996a). Moreover, the effi- The C-terminal NLS is crucial in the nuclear
ciency of dimerization and DNA binding of in import of HIF-1a, whereas the N-terminal one
vitro-translated HIF-1a and HIF-1b is weaker seems to be less important (Kallio et al.,
than those isolated from a cell, suggesting 1998). Furthermore, alternative splice vari-
that one or both subunits may undergo ants of HIF-1a and b have been observed
posttranslational modifications (Jiang et al., (Wang et al., 1995a; Gothie et al., 2000) and
1996a). both subunits contain multiple consensus
sites for protein phosphorylation (Wang et
HIF-1a al., 1995b).
HIF-1a is an 826-amino acid (120-kDa) pro- HIF-1b

tein (Fig. 1). In the N-terminal part it contains
the basic domain (aa 17–30), the he- HIF-1b was previously identified as aryl hy-
lix-loop-helix domain (aa 31–71), and the PAS drocarbon nuclear receptor translocator
domain (aa 85–298) with PAS-A (aa 85–158) (ARNT), which heterodimerizes with aryl hy-
Figure 1. Structure of HIF-1a.

Numbers indicate the first and last amino-acid residues of each domain. The main domains are: basic helix-loop-he-
lix domain (bHLH), Per-AHR-ARNT-Sim homology domains (PAS-A and PAS-B), oxygen-dependent degradation
domain (ODD), and transactivation domains (N-TAD and C-TAD). Also position of PEST-like motifs and nuclear lo-
calization signals (NLS) is shown. Further description in the text.
drocarbon receptor (AHR) to form the func- OXYGEN-DEPENDENT HIF-1

tional dioxin receptor. It has two isoforms REGULATION
(774- and 789-aa that constitutes 92 or 94
kDa, respectively) that differ by the presence
of the sequence encoded by a 45-bp alterna-
tive exon (Wang et al., 1995a). Different levels of HIF-1 regulation
Additional HIFs
The regulation of HIF-1 activity concerns
Two other members of the HIF-1a family mostly the a subunit and occurs at multiple
have been identified: HIF-2a, also known as levels such as protein stabilization, post-
endothelial PAS protein (EPAS1), HIF-like translational modifications, nuclear translo-
factor (HLF), HIF-related factor (HRF), or a cation, dimerization, transcriptional activa-
member of PAS domain family 2 (MOP2); and tion, and interaction with other proteins.
HIF-3a. Both of them heterodimerize with Moreover, changes in mRNA expression
one of the members of the ARNT family: (Wang et al., 1995a; Wiener et al., 1996; Yu et
ARNT (HIF-1b), ARNT2, or ARNT3 (BMAL/ al., 1998) and alternative splicing (Gothie et
MOP3) (all references in Semenza, 2000b; al., 2000) of both subunits have been ob-
Wenger, 2002). The structure, regulation and served. Under normoxic conditions, however,
function of all HIFs seem to be similar. How- when HIF-1a and HIF-1b are constitutively
ever, the expression of HIF-2a, HIF-3a, transcribed and translated, the abrogation of
ARNT2, and ARNT3 is tissue specific, which HIF-1 activity results mainly from constitu-
suggests that they may play more specialized tive HIF-1a degradation (Fig. 2).
roles (Semenza, 2000b).
Figure 2. Regulation of HIF-1a protein stability.

Under normoxic conditions HIF-1-prolyl hydroxylases (PHDs) hydroxylate Pro 402 and 564 (P) within the ODD do-
main. After prolyl hydroxylation von Hippel-Lindau tumor suppressor proteins (pVHLs) bind to the ODD domain
and recruit other proteins of E3 ubiquitin ligase complex. HIF-1a is subsequently ubiquitinated and degraded by
the 26S proteasome. Under hypoxic conditions HIF-1-prolyl hydroxylases are inactive which prevents binding of
pVHL. Therefore, HIF-1a escapes ubiquitination and proteasomal degradation, and can be transported to the nu-
cleus where, after dimerization with HIF-1b, it stimulates target genes transcription.
HIF-1a degradation HIF-1 prolyl 4-hydroxylases, members of the

Fe(II)- and 2-oxoglutarate-dependent dioxy-
Ubiquitination genase superfamily. Their activity, however,
is distinct from the activity of procollagen
Under normoxic conditions HIF-1a is sub- prolyl hydroxylases, which belong to the same
jected to ubiquitination and degradation by superfamily (Bruick & McKnight, 2001;
the 26S proteasome proteolysis (Kallio et al., Jaakkola et al., 2001). The prolyl hydroxylase
1999). In HIF-1a ubiquitination the major responsible for HIF-1a hydroxylation was
role is played by the von Hippel-Lindau tumor first identified as a product of egl-9 gene after
suppressor protein (pVHL), whose b-domain searching Caenorhabditis elegans genome da-
interacts directly with the ODD domain of tabase (Epstein et al., 2001). Egl-9 mutant
HIF-1a (Bonicalzi et al., 2001). The inactiva- worms, similarly to vhl-1 mutants, constitu-
tion of pVHL is associated with the von tively expressed HIF-1a. Later three EGL-9
Hippel-Lindau disease, which is a hereditary homologues in mammals were identified and
cancer syndrome characterized by the devel- designated PHD (PH domain containing pro-
opment of highly vascularized tumors with tein) 1, 2, and 3. They are also termed HPHs
constitutive HIF-1 expression (Ivan et al., (HIF-1 prolyl hydroxylases) 3, 2, and 1, re-
2001). pVHL acts as the substrate recognition spectively. They differ in intracellular local-
component of the E3 ubiquitin ligase protein ization: PHD1 was detected exclusively in the
complex and after binding to HIF-1a it re- nucleus, PHD2 — in the cytoplasm, whereas
cruits elongins B and C, cullin 2, and Rbx1 PHD3 in both nucleus and cytoplasm (Metzen
(reviewed in Semenza, 2001). et al., 2003a). Additionally, the existence of a
The interaction between pVHL and HIF-1a fourth PHD has been described (Oehme et al.,
is oxygen-dependent: pVHL associates with 2002). Each of them hydroxylates Pro-564 in
ODD under normoxic but not under hypoxic HIF-1a, whereas only PHD1 and PHD2
conditions, thus the degradation does not oc- hydroxylate Pro-402. Molecular oxygen is a
cur during hypoxia (Maxwell et al., 1999). The substrate of these enzymes, while carbon di-
interaction of pVHL with HIF-1 depends on a oxide and succinate are by-products. During
posttranslational modification of HIF-1a, hypoxia, or in case of a lack of Fe(II) or
which was identified as an oxygen- and 2-oxoglutarate, prolyl hydroxylases cease to
iron-dependent prolyl hydroxylation (Ivan et function, thus HIF-1a does not undergo deg-
al., 2001). The binding of pVHL to the ODD radation (reviewed in Semenza, 2001).
domain, and therefore the ubiquitination of
its N- and C-terminal part (aa 390–417 and
HIF-1 activation
549–582, respectively), must be preceded by
the hydroxylation of proline residues (Pro To obtain full transcriptional activity HIF-1
402 in the N-terminal part and Pro 564 in the must bind to the DNA target sequence and re-
C-terminal part of ODD, Fig. 2). These resi- cruit transcriptional cofactors. This phase
dues are embedded in the conserved amino also undergoes hypoxia-dependent regula-
acid motif, LXXLAP, in which Leu 562 tion. The role of HIF-1a transactivation do-
strongly facilitates the hydroxylation of Pro mains (TADs) is to recruit the transcriptional
564 (Ivan et al., 2001). coactivator complexes to the promoters of
HIF-1 target genes. The central integrating
HIF-1 prolyl hydroxylases coactivator p300/CBP interacts through its
CH1 (cysteine-histidine-rich) domain with
The hydroxylation of the proline residues in HIF-1a TADs (Gu et al., 2001) and recruits
the ODD domain of HIF-1a is catalyzed by the accessory coactivators like histone
acetylotransferases SRC-1, TIF-2, and redox main by preventing its interaction with
factor Ref-1 (Ema et al., 1999). p300/CBP. The asparaginyl hydroxylase that
The molecular mechanism of activation of modifies HIF-1a was recently identified as
the TAD domains was recently discovered. the previously known factor inhibiting HIF-1
Under normoxic conditions the highly con- (FIH-1) (Lando et al., 2002b). The final evi-
served asparagine residue within the C-TAD dence that both residues, Pro within the ODD
domain (Asn 803) is hydroxylated (Lando et and Asn within the C-TAD, are necessary for
al., 2002a) which results in the silencing of the full activation of HIF-1 was provided by
transactivation domains (Fig. 3). The enzyme an experiment in which either critical Pro, or
Figure 3. The role of prolyl and asparaginyl hydroxylation in the stabilization and activation of HIF-1a.
Under normoxic conditions specific prolyl (P) residues within the oxygen-dependent degradation domain (ODD)
and asparaginyl (N) residues within the COOH-terminal transactivation domain (C-TAD) are hydroxylated by re-
spective hydroxylases. The hydroxylation of prolines enables binding of pVHLs and thus constitutes a signal for
proteasomal degradation. The hydroxylation of asparaginyl (N) residues blocks binding of transcriptional
coactivator (p300/CBP) and subsequently inhibits transcriptional activity of HIF-1. In contrast, under hypoxic con-
ditions HIF-1a escapes proteasomal degradation because of abolition of prolyl hydroxylation and may interact with
p300/CBP due to inhibition of asparaginyl hydroxylation.
that hydroxylates the Asn residues is also a Asn, or both were replaced by Ala (Lando et
member of the Fe(II)- and 2-oxoglutarate-deal., 2002a). Replacement of Pro within the
pendant superfamily of dioxygenases, thus it ODD domain resulted in a stable protein even
is blocked by the inhibitors of 2-oxogluta- under normoxia which, however, exhibited
rate-dependent dioxygenases such as dime- low transcriptional activity at normoxia and
thyloxalylglycine (DMOG) and Fe(II) chela- high transcriptional activity at hypoxia. Re-
tors. Treatment of cells with DMOG or iron placement of the critical Asn within the
chelators as well as replacing the critical Asn C-TAD domain does not influence protein ac-
residue with Ala result in activation of C-TAD tivity in comparison to wild-types proteins,
even at normoxia (Lando et al., 2002a). There- because, despite the active C-TAD domain,
fore, p300/CBP interacts with C-TAD only the protein is unstable at normoxia. Finally,
when the Asn is nonhydroxylated which en- the double amino acid substitution resulted in
ables the assembly of transcriptional co- nearly full activity of the protein at normoxia
activator complex, whereas the hydroxylation (Lando et al., 2002a).
of Asn during normoxia silences C-TAD do-
Other levels of HIF-1 regulation HRE may be located within either promoter
or enhancer regions (5¢-flanking, 3¢-flanking,
Nuclear localization of HIF-1a or intervening) of target genes (Fig. 4, refer-
ences in Table 1). Generally, HBS is the mini-
The dimerization of the HIF-1 a and b sub- mal sequence necessary for HIF-1 binding.
units occurs in the nucleus and is necessary However, the structure of HRE, methylation
for the DNA binding and subsequent activa- of the cytosine residue within HBS, or pres-
tion of transcription (Kallio et al., 1997). In ence of additional transcription factors may
contrast to HIF-1b which is present in the nu- influence HIF-1-induced response. Addition-
cleus regardless of oxygen levels, nuclear ally, in the majority of hypoxia-induced genes
translocation of HIF-1a is correlated with HRE contains HIF-1 ancillary sequence
HIF-1 activity. Consequently, it was sug- (HAS), which is located 8–9 nt down- or up-
gested that this translocation could be stream of HBS and is necessary for HIF-1-me-
Figure 4. The localization of hypoxia response element (HRE) in various hypoxia-induced genes.
HRE (black rectangle) may be located within either 5¢-flanking (VEGF and transferrin) or 3¢-flanking (EPO)
enhancer regions, or within the promoter (NOSII) of target genes.
upregulated by hypoxia (Kallio et al., 1998). diated transcription activation (Kimura et al.,
However, when HIF-1a is overexpressed the 2001). HAS is an imperfect inverted repeat of
translocation occurs even under normoxic HBS, thus the secondary structure of HRE
conditions. Therefore, this process seems to seems be crucial for its activatory function
be hypoxia-independent and the nuclear frac- (Fig. 5). It was also shown that HAS recruits
tion of HIF-1a may simply reflect the overall protein complexes distinct from HIF-1
level of this protein in the cell (Hofer et al., (Kimura et al., 2001).
2001). Furthermore, efficient gene activation fre-
quently requires recruiting of more than one
HIF-1 DNA binding HIF-1 or binding of additional transcriptional
factors, which are not hypoxia-dependent.
After the stabilization of the a subunit, nu- Two or three adjacent HBSs were found in
clear translocation, and dimerization, HIF-1 some genes encoding glycolytic enzymes, glu-
binds to its consensus binding site (HBS, cose transporter 1, and transferrin. There is
HIF-1 binding site) within the hypoxia re- also a binding site for the ATF-1/CREB-1 fac-
sponse element (HRE) (Semenza et al., 1996). tor (activating transcription factor-1/cAMP-
The core sequence of HBS is (A/G)CGTG. response element-binding protein-1) in the
HRE of lactate dehydrogenase A gene, for conditions (Frick et al., 2003) or in response
AP-1 (activator protein-1) binding factor in to hypoxia (unpublished data). In contrast, in
VEGF gene, and for the HNF-4 (orphan recep- human microvascular endothelial cells
tor hepatic nuclear factor-4) in the erythropoi- (Józkowicz et al., 2004) and smooth muscle
etin gene (references in Wenger, 2002). All cells (Dulak et al., 2002) hypoxia potently
Figure 5. Human hypoxia response element (HRE) in VEGF, EPO, glucose transporter-1 (GLUT-1), and
lactate dehydrogenase A (LDHA) genes (Kimura et al., 2001).
HRE, besides the HIF-1 binding site (HBS), contains the HIF-1 ancillary sequence (HAS), which is located 8–9 nt
down- or upstream of HBS and constitutes an imperfect inverted repeat of HBS.
these transcription factors modulate HIF-1 modulates VEGF production. These data
response. Such a requirement for additional suggest that the response to hypoxia is cell
transcription factors may (i) amplify hypoxic type specific.
response in particular conditions, (ii) vary re-
sponses of distinct tissues to the hypoxia, and
Molecular mechanism of oxygen sensing
(iii) enable diverse induction of distinct target
genes. Although the mechanisms of HIF-1a stabili-
Another possible level of regulation may be zation and activation are already known, the
the CpG methylation of the cytosine residue signaling pathways that lead to the inhibition
within HBS. Although, in the majority of of prolyl and asparaginyl hydroxylases re-
HIF-1 target genes it remains unmethylated main unclear. The requirement of molecular
as it is located in the methylation-free CpG is- oxygen as a substrate for these enzymes could
lands, the HBS of the erythropoietin gene explain the loss of HIF-1 degradation under
might undergo methylation, which inhibits hypoxic conditions. This assumption, how-
HIF-1 binding (Wenger et al., 1998). To pre- ever, appears to be oversimplified. It has been
vent methylation, HBS is at normoxia occu- shown that collagen prolyl hydroxylases are
pied by other DNA binding factors (reviewed active in hypoxic cells and that maximal
in Wenger, 2002). It might also be hypothe- HIF-1a stabilization occurs under 0.5% O2
sized that methylation of HBS may constitute concentration rather than under anoxia,
a mechanism of cell-specific regulation of which would not be possible if prolyl
hypoxia-induced gene expression. Interest- hydroxylases were direct oxygen sensors
ingly, human macrovascular endothelial cells (Jiang et al., 1996b). Moreover, earlier stud-
do not release VEGF either under normoxic ies provided evidence that other signaling
pathways, such as ROS-, protein phos- b-containing NADPH oxidase that could re-
phorylation-, and nitrosylation-dependent spond to oxygen levels (references in Michiels
pathways are involved in oxygen sensing. et al., 2002), and (iv) induction of HIF-1 target
Therefore, many models of intracellular genes in the presence of exogenous catalase
oxygen sensing have been considered. or antioxidants (Salceda & Caro, 1997). How-
ever, some observations were inconsistent
A hemoprotein as an oxygen sensor with this hypothesis. A nonspecific inhibitor
of NADPH oxidases (diphenylene iodonium,
The initial hypothesis suggested that the DPI) blocked HIF-1 activation in response to
role of oxygen sensor is mediated by an un- hypoxia (Gleadle et al., 1995) and HIF-1 activ-
known protein containing heme as a pros- ity was sustained in cells deficient in a sub-
thetic group. The putative role of heme, unit of NADPH oxidase (Archer et al., 1999).
bound either to HIF-1 PAS domains or to a Moreover, some experiments indicated that
distinct sensor hemoprotein, was suggested the production of ROS was increased rather
because of the fact that HIF-1 activation is in- than decreased during hypoxia (Chandel et
duced not only by the lack of molecular oxy- al., 1998).
gen but also by Fe(II) chelators, such as
desferrioxamine (Wang et al., 1993), and also
by some transient metals that could replace
Fe(II) in heme (Huang et al., 1997). However,
currently these observations seem to confirm
the iron-dependence of prolyl and asparaginyl
hydroxylases rather than hemoproteins
acting as the oxygen sensor.
ROS-dependent signaling pathways Figure 6. Model I of ROS-dependent hypoxia sig-

nal transduction.
Two opposing models postulate signaliza- According to this model hypoxia results in decreased
tion by changes in the cellular levels of reac- reactive oxygen species (ROS) generation, which leads
tive oxygen species (ROS) (reviewed in to HIF-1a stabilization. The same effect is observed in
the presence of antioxidants and catalase (Cat).
Semenza, 2000c; Michiels et al., 2002). The
first of them assumes that ROS are continu-
ously produced by an unknown NADPH The second model concerning ROS-depend-
oxidase that reduces O2 to superoxide anion ent pathway is diametrically opposed to the
(O2·–) which is subsequently converted to hy- previous one as it assumes that hypoxia re-
drogen peroxide (H2O2) by superoxide sults in an increased generation of ROS by
dismutase (SOD). According to this model the mitochondria, which constitutes the signal to
reduction of molecular oxygen concentration HIF-1a stabilization (Fig. 7). According to
would be followed by the reduction of ROS this model under hypoxic conditions the con-
levels, and therefore a decrease in ROS gener- sumption of oxygen at cytochrome c oxidase
ation would be a direct or indirect signal for (mitochondrial complex IV) is lower and elec-
HIF-1 activation (Fig. 6). The experiments trons accumulate at preceding complexes.
confirming this hypothesis showed: (i) de- Such an accumulation leads to increased gen-
creased ROS production under hypoxic condi- eration of ROS at complex III. Compatibly
tions, (ii) suppression of HIF-1 target genes with this theory inhibitors of complexes I and
by exogenous H2O2 in hypoxia, (iii) the pres- III blocked the induction of HIF-1 activity at
ence of a non-mitochondrial cytochrome hypoxia (due to inhibition of ROS produc-
tion), whereas inhibitors of complex IV were chloride or desferrioxamine instead of

able to induce HIF-1-dependent transcription hypoxia (Chandel et al., 1998). The explana-
at normoxia (due to induction of ROS generation of the contradictory results of Chandel et
tion) (Chandel et al., 1998). Another evidence al. (1998), and Vaux et al. (2001) may lie in
that mitochondrial complex III acts as an oxy- different mechanisms of oxygen sensing in
gen sensor was shown in r0 Hep3B cells, lack- the case of anoxia and hypoxia. Prolyl and
ing the functional respiratory chain, in which asparaginyl hydroxylases, which use molecu-
the induction of HIF-1 did not occur under lar oxygen as a substrate, act as direct oxygen
hypoxic conditions (Chandel et al., 1998). sensors in the total absence of oxygen
Moreover, the substrate of complex II (Schroedl et al., 2002). Similarly, des-
(succinate) restored hypoxic response in cells ferrioxamine and cobalt chloride are direct in-
Figure 7. Model II of ROS-dependent hypoxia signal transduction.

According to this model hypoxia results in an increased generation of reactive oxygen species (ROS) due to attenu-
ation of cytochrom c oxidase (mitochondrial complex IV) activity. The generation of ROS is mediated by complex
III at which, in the absence of functional cytochrome c oxidase (due to hypoxia or in the presence of complex IV in-
hibitors), electrons are accumulated. High levels of ROS induce HIF-1a stabilization, whereas low levels lead to
HIF-1a degradation. Low levels of ROS are maintained either when the electron transport chain is fully functional
(normoxia) or when electrons do not reach complex III (in the presence of inhibitors of complex I and II, or III).
with a defect in complex I (Agani et al., 2000). hibitors of the hydroxylases. Thus, at anoxia
Consistent with this theory are also our re- or after treatment with desferrioxamine or
sults showing that overexpression of SOD, cobalt chloride, stabilization of HIF-1a occurs
leading to increased production of H2O2, in- independently of ROS generation and the
duces HIF-1-dependent VEGF expression presence of functional electron transport
(Grzenkowicz-Wydra et al., 2004). chain is not necessary. This hypothesis ex-
However, these findings were not confirmed plains why inhibitors of complex I prevent
in other experiments on r0 cell lines cultured hypoxia-induced but not desferrioxamine- or
under severe hypoxic conditions (0.1% O2) anoxia-induced accumulation of HIF-1a
(Vaux et al., 2001) or after exposure to cobalt (Schroedl et al., 2002). In contrast, in the
hypoxia-mediated HIF-1a stabilization addi- way able to upregulate HIF-1 activity involves
tional intracellular signaling, including the receptor tyrosine kinase®PI3K (pospha-
phosphorylation- and ROS-dependent path- tidylinositol-3 kinase)® prolyl-4-hydroxylases
ways, is required (Schroedl et al., 2002). The (protein kinase B)®FRAP (FKBP-rapamycin
preservation of hypoxia-induced stabilization associated protein) pathway. Some studies
of HIF-1a in cells with a defect in electron showed that inhibition of the PI3K or AKT
transport chain (Vaux et al., 2001) may have kinases impairs HIF-1-dependent gene ex-
resulted either from the presence of marginal pression (references in Wenger, 2002). More-
complex I and III activity, sufficient to gener- over, loss of PTEN (phosphatase and tensin
ate ROS, or from anoxic rather than hypoxic homolog deleted on chromosome ten) activ-
conditions (0.1% O2) used in the experiments. ity, which is a tumor suppressor protein and a
It is possible that signals induced by hypoxia, negative regulator of PI3K, results in in-
as well as signals from some cytokines, creased HIF-1a expression (Zundel et al.,
vasoactive hormones, and nitric oxide (see be- 2000). Finally, FRAP can stimulate HIF-1a
low) lead to the inhibition of proline expression even under normoxic conditions
hydroxylation (Fig. 8). (Zhong et al., 2000). Additionally, the involve-
Figure 8. Prolyl and asparginyl hydroxylases function as direct (anoxia) or indirect (hypoxia) oxygen
sensors.
Phosphorylation cascades ment of ROS-dependent p38 MAP kinase

pathway has been suggested because inhibi-
Initial studies indicated that the activation tion of HIF-1 activity by p38 kinase blockers
of HIF-1 involves protein phosphorylation was observed (Gorlach et al., 2001).
(Wang et al., 1995b). This process might be
mediated by several different protein kinase Other signaling pathways
pathways. First, the p42/p44 (Erk2/Erk1)
mitogen-activated protein kinases (MAPKs) Other suggested signaling pathways con-
are able to phosphorylate HIF-1a (Richard et cern redox-dependent regulation and protein
al., 1999). This modification, however, en- N-nitrosylation. For example, it was shown
hances HIF-1 activity but is not involved in that reduction of cysteine within C-TAD en-
HIF-1a stabilization. Thus, this is rather the hances HIF-1 trans-activation by enabling
way in which growth factors modify HIF-1 the interaction with CBP. This reduction is
function as many of them act via the receptor provided by the system: thioredoxin/the re-
tyrosine kinase®Ras/Raf®MEK (mitogen-ac- dox factor Ref-1 (Ema et al., 1999). Co-ex-
tivated kinase kinase)®MAPK pathway (re- pression of Ref-1 and thioredoxin enhanced
viewed in Wenger, 2002). The second path- the transactivation by C-TAD, but not by
N-TAD, in a hypoxia-dependent manner. Ad- Interaction between HIF-1 and p53

ditionally, upon hypoxic conditions, thio-
redoxin was found to be translocated to the There are many similarities between the two
nucleus where it can interact with Ref-1 transcription factors: HIF-1a and the tumor
(Ema et al., 1999). suppressor p53. Under hypoxic conditions
both proteins accumulate and gain their
transcriptional activity which requires re-
HIF-1 negative regulation
cruitment of p300 as a co-activator. More-
Several negative feedback regulatory path- over, under normoxic conditions p53, simi-
ways that could limit the response to hypoxia larly to HIF-1a, is ubiquitinated by E3 ligase
have been proposed (reviewed in Wenger, (MDM2 for p53) and subsequently degraded
2002). The downregulation of HIF-1a might by 26S proteasome (Giaccia et al., 1998). It
occur on the level of transcription, transla- was observed that the loss of p53 activity re-
tion, protein stabilization, and/or protein ac- sults in an increased accumulation of HIF-1a
tivation. Responsible for the latter could be under hypoxic conditions (Ravi et al., 2000).
Cited2 (also named p35srj), a member of the This relationship was explained by the obser-
CBP/p300-interacting transactivators with a vation that p53 binds to HIF-1a and recruits
glutamic acid and aspartic acid-rich tail. the MDM2 ubiquitin ligase which preferably
Cited2 competes with HIF-1 in binding to the targets HIF-1a for degradation. Thus, in the
cysteine-histidine-rich (CH1) region of p300 case of prolonged anoxic conditions, p53 can
and CBP (Leung et al., 1999). Notably, Cited2 act as a negative regulator of HIF-1 in two
expression is induced by HIF-1 (Bhattacharya ways. First, it inhibits HIF-1 activity by com-
et al., 1999). petitive binding of p300 (Schmid et al., 2003),
Recently it was shown that the down- and second, the interaction between p53 and
regulation occurs also at the level of HIF-1a HIF-1a results in HIF-1a degradation (Ravi et
stabilization as HIF-1 induces synthesis of al., 2000). Such cooperation explains the
prolyl hydroxylases which enable rapid angiogenic switch that occurs during tumori-
HIF-1a hydroxylation and degradation dur- genesis in case of p53 mutation.
ing reoxygenation (D’Angelo et al., 2003).
Also some anti-inflammatory factors could HIF-1 natural inhibitors
act through the inhibition of the HIF-1 path-
way. We recently showed that under hypoxic The natural inhibitors of HIF-1 identified so
conditions prostaglandin-J2 attenuates VEGF far are mainly splice variants of the a subunit
expression by inhibition of HIF-1 activity and thus are able to act as HIF antagonists.
(Józkowicz et al., 2004). Furthermore, heme The first of them, inhibitory PAS protein
oxygenase-1, which is a stress inducible en- (IPAS) is a HIF-a without the transactivation
zyme that degrades heme to carbon monox- domain. High levels of IPAS were indicated in
ide, iron ions, and biliverdin, could enhance the corneal epithelium of the eye where
activity of prolyl and asparaginyl hydroxy- HIF-dependent angiogenesis is significantly
lases by an increased release of iron. impaired (Makino et al., 2001). Other poten-
Biliverdin and its derivate, bilirubin, scaven- tial HIF-1 antagonists are: the zinc-inducible
gers of peroxyl radicals, could also modulate isoform lacking exon 12 (HIF-1aZ) and the
HIF-1a stability due to the attenuation of re- dominant-negative HIF-1a isoform lacking
active oxygen species generation. On the exons 11 and 12. In addition, an antisense
other hand, it was observed that HO-1 over- RNA specific for the 3' untranslated region of
expression enhances VEGF synthesis (re- HIF-1a was identified (references in Wenger,
viewed in Dulak et al., 2004). 2002).
NON-HYPOXIC ACTIVATION OF HIF-1 are responsible for this hypoxia-independent

AND MODULATORS OF HIF-1 induction of HIF-1 in VSCM. The first leads to
ACTIVITY an increase in HIF-1 gene transcription,
whereas the second results in an increase in
Interestingly, an increasing number of stud- translation of HIF-1 mRNA. The central role
ies indicate that HIF-1a stabilization might in both pathways belongs to the diacyl-
occur in a hypoxia-independent manner. The glycerol-sensitive protein kinase C (PKC). It
inducers of HIF-1 activity that act independ- was also shown that the increase in HIF-1
ently of O2 levels may be divided into several translation by angiotensin II is mediated by
groups. The first group consist of direct inhib- ROS-dependent activation of the phospha-
itors of prolyl and asparaginyl hydroxylases tidylinositol-3 kinase (PI3K) pathway (Page et
such as iron chelators, iron replacing mole- al., 2002). Together these two pathways in-
cules, and analogs of 2-oxoglutarate. The sec- crease HIF-1a in VSMC to levels that surpass
ond group is represented by various hor- hypoxic induction.
mones and growth factors. Also hallmarks of Also some growth factors such as insulin, in-
inflammation such as proinflammatory sulin-like growth factor 1 and 2, epidermal
cytokines, nitric oxide, increased tempera- growth factor, fibroblast growth factor 2,
ture, or mechanical stress, may induce HIF-1 platelet-derived growth factor, transforming
activity. growth factor b1, and inflammatory cytokines
such as interleukin-1, or tumor necrosis fac-
tor a, (all references in Wenger, 2002) can
Direct inhibitors of prolyl hydroxylases
evoke HIF-1 activation under normoxic condi-
Transition metal ions, such as cobalt and tions. Most of them act through the tyrosine
nickel, induce HIF-1a stabilization under kinase receptor®PI3K®AKT®FRAP path-
normoxic conditions. Initially this effect was way. This growth factor-dependent HIF-1 acti-
thought to confirm the hypothesis of a heme vation is important in tumorigenesis as the
protein acting as a putative oxygen sensor overexpression of some growth factors may
(Huang et al., 1997). Recently, however, it cause HIF-1-mediated induction of intra-
was suggested that transition metals could in- tumoral angiogenesis.
hibit prolyl hydroxylases by substituting the
ferrous ion coordinated by PHDs (Epstein et
Nitric oxide
al., 2001). Another well-known activator of
HIF-1, desferrioxamine (Wang et al., 1993), HIF-1 induces nitric oxide production
could also inactivate PHDs by removal of iron through the enhancement of inducible nitric
from their catalytic domains. Furthermore, oxide synthase transcription (Table 1). Con-
compounds competing with 2-oxoglutarate versely, NO affects the accumulation and ac-
such as N-dimethyl-oxalylglycine (DMOG), a tivity of HIF-1. Initial studies focusing on the
2-oxoglutarate analog, are also able to inhibit NO influence on HIF-1 activity showed that
PHDs activity (Jaakkola et al., 2001). under hypoxic conditions, or after treatment
with CoCl2, NO inhibits HIF-1a stabilization
and transcriptional activation (Liu et al.,
Vasoactive hormones and cytokines
1998; Sogawa et al., 1998; Huang et al., 1999).
The induction of VEGF expression in vascu- On the other hand, it was indicated that un-
lar smooth muscle cells (VSMC) by vasoactive der normoxic conditions diverse NO donors
hormones like angiotensin II and thrombin is (with the exception of sodium nitroprusside,
mediated through the activation of HIF-1 SNP) or endogenously produced NO (by in-
(Richard et al., 2000). Two separate pathways ducible or endothelial nitric oxide synthase,
NOS2 and NOS3, respectively) causes HIF-1a cently, however, it was reported that NO do-
stabilization and activation of its target genes nor, NOC18, induces HIF-1a synthesis at
(Kimura et al., 2000; Dulak et al., 2000; normoxia, whereas neither hydroxylation nor
Sandau et al., 2001a; 2001b; Józkowicz et al., stabilization of HIF-1a is influenced. Instead,
2001; Dulak & Józkowicz, 2003b). Normoxic it was shown that this effect depends on the
NO-induced upregulation of HIF-1 occurred PI3 and MAP kinases pathways (Kasuno et
through the PI3K/Akt pathway (Sandau et al., 2004).
al., 2001b; Brüne et al., 2001; Natarajan et al.,
2003) and was independent of soluble NO and ROS
guanylyl cyclase (sGC) activity (Brüne et al.,
2001). The type of the NO donor and its con- On the other hand, NO as a free radical re-
centration used in experiments seem to be acts rapidly with ROS, particularly in a diffu-
crucial for the results obtained. The inhibi- sion-controlled fashion with superoxide anion
tory effect of SNP may be evoked by toxic to create peroxynitrate, one of the most reac-
by-products of its decomposition such as tive compounds in nature. If hypoxia acti-
cyanides and iron ions which probably sur- vates HIF-1 through the increase in mitochon-
pass the effect of NO (Dulak et al., 2000; drial ROS generation, NO would inhibit HIF-1
Józkowicz et al., 2001). activation by attenuation of ROS levels. To
confirm this hypothesis it was shown that NO
NO and PHDs blocks HIF-1a stabilization in the presence of
superoxide anion donors or H2O2 (Sandau et
Recently it was reported that the NO-medial., 2001b). Thus ROS and NO might be able
ated activation of HIF-1 at normoxia is caused to induce HIF-1 activity but, when present to-
by attenuation of prolyl hydroxylation gether, they may react to form compounds
(Metzen et al., 2003b) and, therefore, PHDs such as peroxynitrite that lacks this property
might constitute direct or indirect targets of and might cause HIF-1a degradation.
NO. An interaction between NO and iron
could constitute a way of affecting prolyl
Carbon monoxide
hydroxylation as NO directly binds to the fer-
rous ion in heme or non-heme iron-containing Initially it was reported that carbon monox-
proteins. Prolyl hydroxylases contain Fe(II) ide (CO) inhibits hypoxia-induced HIF-1a ex-
in their catalytic domains, thus NO could pression, HIF-1 DNA binding, and HIF-1
compete with molecular oxygen for the cata- transcriptional activity (Huang et al., 1999).
lytic site and subsequently inhibit enzyme Such a correlation would be consistent with
function (Metzen et al., 2003b). This hypothe- the hypothesis of a heme protein acting as an
sis, however, does not explain why NO inhib- oxygen sensor. However the concentrations
its hypoxia-induced HIF-1 accumulation. of CO used in those experiments were very
Helpful in searching for the answer could be high (up to 80%) and therefore non-physiologi-
the fact that NO down-regulates HIF-1a in cal. On the contrary, it is possible that CO, as
hypoxia by activation of PHDs in cytoplasmic an inhibitor of mitochondrial complex IV,
extracts (Wang et al., 2002), although this ef- could lead to an increase in ROS generation
fect has not been confirmed by in vitro pro- and, therefore, induce HIF-1a accumulation.
tein interaction assays (Metzen et al., 2003b). We have shown that low (1% or lower) concen-
Therefore, activation of PHDs by NO might trations of CO strongly induce VEGF expres-
be caused by cytoplasmic components such as sion in vascular smooth muscle cells (Dulak et
protein phosphorylation cascades or/and al., 2002) and in endothelial cells (Józkowicz
ROS-dependent signaling pathways. Re- et al., 2003; reviewed in Dulak & Józkowicz,
2003a). Another interesting hypothesis was in development, physiology, and patho-

proposed after the discovery that mammalian physiology. The central role in the regulation
neuronal PAS domain protein 2 (NPAS2), a of HIF-1 activity belongs to prolyl and aspar-
bHLH-PAS transcription factor involved in aginyl hydroxylases, which function coopera-
regulation of circardian cycle, is regulated not tively as direct or indirect oxygen sensors.
only by the NADPH/NADP ratio but also by The question is why HIF-1 activity is con-
carbon monoxide (Dioum et al., 2002). Incor- trolled by two independent processes. The an-
poration of CO into hemes which are bound to swer might be that multiple levels of the regu-
the PAS domains of NPAS2 inhibits lation enable graded responses to subtle
NPAS2-BMAL1 heterodimerization and, sub- changes in O2 concentration and ensure tight
sequently, its transcriptional activity. The control of the hypoxic response pathway
NPAS2 binding sequence, CACGTG (Rutter (Bruick & McKnight, 2002).
et al., 2001), may be recognized by HIF-1, thus The analysis of HIF-1 expression pattern
reciprocal relationship between these two and the modulation of HIF-1 activity may
transcription factors is possible and requires bring new diagnostic and therapeutic ap-
further studies. proaches in many diseases such as cancer and
vascular disorders (reviewed in Semenza
2000a). It is possible that inhibition of HIF-1
Mechanical and thermal stress
could suppress intratumoral angiogenesis
It has been reported that HIF-1a is present and thus attenuate cancer growth. Addition-
in the nuclei of cardiac myocytes in ally, it was observed that Hif1a+/– mice had
non-hypoxic myocardium (Kim et al., 2002). significantly weaker responses to chronic
This upregulation of HIF-1a occurs as a result hypoxia such as reduced polycythemia, right
of a wall stretch and is mediated by the ventricular hypertrophy, and pulmonary hy-
PI3K®AKT®FRAP pathway. A similar effect pertension (Yu et al., 1999). Thus in case of
was observed in aortic VSMC where hyper- chronic lung disease local downregulation of
tension induced HIF-1a accumulation HIF-1a could be useful in treating or prevent-
(Kuwahara et al., 2002). Therefore, HIF-1 ing these disorders. In contrast, in the case of
seems to be responsible not only for the adap- myocardial ischemia or other ischemic disor-
tation to hypoxia but also for the adaptation ders induction of HIF-1 expression could pre-
to mechanical stress. vent ischemic tissues from permanent dam-
It was also indicated that HIF-1a accumula- ages. HIF-1 upregulation in pro-angiogenic
tion might be evoked by exposure to in- therapies could facilitate the formation of
creased temperature. This effect seems to be fully matured vascularity.
mediated by direct protein stabilization that Therefore, the understanding of the molecu-
involves the heat shock protein HSP90 and is lar mechanisms of HIF-1 activation will be
hypoxia-independent (Katschinski et al., crucial for the development of new drugs act-
2002). ing either as agonists or as antagonists of the
hypoxia response pathway. Possible targets
of future therapies could be not only HIF-1a
CONCLUSIONS itself but also the proteins mediating its stabi-
lization such as prolyl and asparaginyl
Undoubtedly, HIF-1 plays the leading role in hydroxylases.
the induction of complex response to various
stresses and thus contributes to the mainte-
nance of cellular and systemic homeostasis. We are grateful to Dr. Alicja Józkowicz and
This function gives HIF-1 a major importance Prof. Aleksander Koj for useful comments.
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Proceedings of the Nutrition Society (2008), 67, 409–418 doi:10.

Symposium on ‘The challenge of translating nutrition research into public

Session 3: Joint Nutrition Society and Irish Nutrition and Dietetic

Hospital, Tremona Road, Southampton SO16 6YD, UK

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease manifested by

Cytokine: Eicosanoid: Fatty acid: Fish oil: Inflammation

result from loss of tolerance to normally benign environ- inflammation

Free arachidonic acid

COX-1 15-LOX 12-LOX 5-LOX

PGG2 15-HPETE 12-HPETE 5-HPETE

PGH2 15-HETE 12-HETE LTA4 5-HETE

Decreased arachidonic acid

Altered membrane Altered signal

Promotion of a less-inflammatory phenotype

capacity to present antigen (keyhole limpet haemocyanin) Inflammatory mediator production

IL-12 Damage to host tissue

Approx, approximately; NSAID, non-steroidal anti-inflammatory drugs.

Table 2. Summary of the findings of the meta-analysis of Goldberg & Katz(94)

Patient-assessed pain 13 247 254 0.03

NSAID, non-steroidal anti-inflammtory drugs.

functions. Proc Nutr Soc 57, 487–502. Mediators Inflamm 1, 107–115.

Endogenous Receptor Agonists: Resolving

Gerard Bannenberg1,*, Makoto Arita2,*, and Charles N. Serhan3

E-mail: gbannenberg@cnb.uam.es; marita@mol.f.u-tokyo.ac.jp; cnserhan@zeus.bwh.harvard.edu

Controlled resolution or the physiologic resolution of a well-orchestrated inflammatory

KEYWORDS: resolution, inflammation, neutrophil, arachidonic acid (AA), eicosapentaenoic acid

*Corresponding authors. 1440

FIGURE 1. Representation of the temporal cellular events in the evolution of inflammation.

LIPID MEDIATORS AS AGONISTS FOR RESOLUTION

AA-Derived Anti-Inflammatory Lipid Mediators

Lipoxins and Aspirin-Triggered Lipoxins

was subsequently identified to possess potent counter-regulatory actions in inflammation[57]. An

shown to display potent anti-inflammatory actions[49,123,124]. These products are prostaglandins

ω-3 PUFA-Derived Anti-Inflammatory/Proresolution Lipid Mediators

Asprin-Triggered D-Series Resolvins

Protectins and 17S D-Series Resolvins

SUMMARY AND CONCLUSION

resolution of inflammation. Nat. Rev. Immunol. 2, 787–795.

Essent. Fatty Acids 73, 203–210.

This article should be cited as follows:

International Journal of The Scientific Biochemistry

Submit your manuscripts at

BioMed Research International Journal of

Enzyme Research International Journal of

Hindawi Publishing Corporation Hindawi Publishing Corporation Volume 2013

ISRN ISRN ISRN ISRN ISRN

Symposium on ‘The challenge of translating nutrition research into public

Session 3: Joint Nutrition Society and Irish Nutrition and Dietetic

Hospital, Tremona Road, Southampton SO16 6YD, UK

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease manifested by

Cytokine: Eicosanoid: Fatty acid: Fish oil: Inflammation

result from loss of tolerance to normally benign environ- inflammation

Free arachidonic acid

COX-1 15-LOX 12-LOX 5-LOX

PGG2 15-HPETE 12-HPETE 5-HPETE

PGH2 15-HETE 12-HETE LTA4 5-HETE

Decreased arachidonic acid

Altered membrane Altered signal

Promotion of a less-inflammatory phenotype

capacity to present antigen (keyhole limpet haemocyanin) Inflammatory mediator production

IL-12 Damage to host tissue

Approx, approximately; NSAID, non-steroidal anti-inflammatory drugs.

Table 2. Summary of the findings of the meta-analysis of Goldberg & Katz(94)

Gerard Bannenberg1,, Makoto Arita2,, and Charles N. Serhan3