discrete changes in cortical activation during experimentally

Discrete Changes in Cortical Activationduring Experimentally InducedReferred Muscle Pain: A Single-TrialfMRI Study

Vaughan G. Macefield1, S.C. Gandevia1 and Luke A. Henderson2

1Prince of Wales Medical Research Institute and the University

of New South Wales, Sydney, NSW, Australia 2031 and2Department of Anatomy and Histology, University of Sydney,

Sydney, NSW, Australia 2006

Noxious stimulation of skeletal muscle evokes pain that is oftenreferred into distal areas. Despite referred pain being of significantclinical importance, the brain regions responsible for the perceptionof referred pain remain unexplored. The aim of this investigation isto define these regions using functional magnetic resonanceimaging. We induced muscle pain by hypertonic saline injections(0.5 ml) into the tibialis anterior (TA) or flexor carpi radialis (FCR)muscle. TA injections evoked pain that was referred to theankle/foot in 10/17 subjects, whereas FCR injections evoked painthat was projected into the wrist/hand in 6/12 subjects. Regionalbrain responses were statistically tested by convolving thetemporal profile of the subjective pain intensity rating with thehemodynamic response function. For all subjects, signal increasedin the region of primary somatosensory cortex (SI), whichrepresents the leg or arm, that is, the area corresponding to theinjection site. However, for those subjects who reported referredpain, signal intensity increases also occurred in the SI regionrepresenting the foot or hand. Interestingly, differential signalchanges also occurred in anterior cingulate, cerebellar, and insularcortices. This is the first study to provide evidence of corticaldifferentiation in the processing of primary and referred pain.

Keywords: anterior cingulate cortex, cerebellum, insula,somatosensory cortex, somatotopy

Introduction

Pain that originates in muscle has a dull, aching quality that is

diffuse, difficult to localize, and is often referred to sites remote

from the original locus of noxious stimulation, that is, referred

pain. We have recently shown that the deep dull ache induced

by hypertonic saline injection into the tibialis anterior (TA)

muscle is often associated with pain that projects into the ankle

and dorsum of the foot (Graven-Nielsen et al. 1997b; Henderson,

Bandler, et al. 2006). Although the referral of muscle pain was

described over half a century ago (Kellgren 1938; Feinstein et al.

1954), the mechanism(s) responsible for this phenomenon are

poorly understood.

Because referred pain can be induced during a complete

sensory block of the region of the referred pain, it is likely that

the pattern of referral results from a centrally mediated

mechanism (Feinstein et al. 1954). It has been suggested that

pain referred from visceral to cutaneous structures results from

the convergence of nociceptive afferents in the dorsal horn,

that is, viscero-somatic convergence (Foreman et al. 1984).

However, electrophysiological studies reveal only rare conver-

gence at the level of the dorsal horn between nociceptive

afferent neurons innervating different muscles or between

those that innervate muscles and other deep tissues (Hoheisel

and Mense 1990). Given this, it is possible that referral of muscle

pain results from the convergence of nociceptive neurons at

higher brain centers, such as the thalamus and/or somatosen-

sory cortex. In a previous functional magnetic resonance

imaging (fMRI) investigation we found that both muscle and

cutaneous pain evoked in the leg resulted in signal intensity

increases in the region of the primary somatosensory cortex

(SI), which receives sensory input from the region of the

injection, that is, the paracentral lobule. In addition, during

muscle pain, signal intensity increased in the region of SI that

corresponded to the region that receives sensory input from the

site of pain referral (Henderson, Bandler, et al. 2006). These data

suggest that the SI cortex may encode the referral pattern of

muscle pain. However, owing to its location within the sagittal

sulcus, the region of SI cortex, which receives sensory input

from the leg is difficult to distinguish from the immediately

adjacent mid-cingulate cortex, which also displays widespread

signal intensity changes during muscle pain. Furthermore, the

extent of referral of muscle pain varies between subjects, with

some subjects perceiving little or no noticeable referral.

In order to determine whether the referral of muscle pain is

reflected in an expansion of the area of activation of SI cortex,

we used fMRI to investigate the patterns of signal intensity

changes during muscle pain induced by intramuscular injec-

tions of hypertonic saline in the leg and arm. We hypothesized

that subjects with a large region of perceived muscle pain should

have a more extensive activation of SI cortex than subjects with

smaller patterns of pain perception.

Methods

SubjectsTwenty-two healthy subjects (11 females, 11 males) aged 22--49 years

were recruited for the study. Informedwritten consent was obtained for

all procedures and the study was approved by the Human Research

Ethics Committee of the University of New South Wales.

Stimulus and PsychophysicsWith each subject lying in a supine position on the scanner bed, a fine

needle (23 gauge), connected via a 10 cm cannula to a 1-ml syringe filled

with sterile hypertonic (5%) saline, was placed into the right leg: ~1 cm

into the rostral belly of the TA muscle; and/or the right arm: ~1 cm into

the rostral belly of the flexor carpi radialis (FCR) muscle. During

scanning, an intramuscular (0.5 ml) injection was administered. Two

subjects received both the arm and leg muscle injections during a single

scanning session with the injections separated by at least 20 min. In the

remaining 20 subjects, injections were made into 1) TA and FCR in

separate scanning sessions separated by about one month (n = 9), 2) TA

only (n = 9), or 3) FCR only (n = 2). Following a hypertonic saline

injection, subjects pressed a buzzer at the onset of pain, just after the

peak intensity had been reached and when the pain had ceased. After

each pain stimulus, the McGill Pain Questionnaire was read aloud and

the subject selected those words, which described the pain. In addition,

Cerebral Cortex September 2007;17:2050--2059

doi:10.1093/cercor/bhl113

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each subject rated the maximal intensity of the pain on a modified Borg

et al. (1981) scale. The Borg scale was as follows: 0 = infinitely small

amount of pain, 0.5 = just noticeable pain, 1 =minimal pain, 2 =mild pain,

3 = moderate pain, 4 = considerable pain, 5 = large amount of pain, 7 =very large, 10 = extremely large (maximal) pain. Only subjects that rated

the arm or leg muscle pain as 3 (moderate pain) or greater were used for

analysis (TA; n = 17; FCR; n = 12). After scanning, each subject plotted

the profile of the pain intensity over time and using the time points

(onset, maximum, and cessation) indicated by the buzzer, a plot of pain

intensity over time was created for each subject. These pain intensity

time plots were then averaged across subjects to create a mean pain

intensity profile. Each subject also drew the distribution of pain during

each stimulus on a standard diagram.

Imaging Procedures and AnalysisBlood oxygen level--dependent (BOLD) contrast gradient echo, echo-

planar images were collected using a 3-Tesla whole-body scanner

(Philips [Foster City, CA] Intera). One hundred fMRI image volumes

were collected continuously over a 5-min period. Each volume covered

the entire brain and consisted of 32 axial slices (time repetition = 3 s,

time echo = 50 ms, flip angle = 90�, field of view = 220 mm, interslice gap

= 0.4 mm, raw voxel size = 1.72 3 1.95 3 4 mm thick). One minute into

the scanning period an intramuscular injection was administered. A 3D,

T1-weighted anatomical image (voxel size = 0.4 3 0.4 3 0.9 mm) was also

collected.

MRI images were analyzed using statistical parametric mapping 2

(SPM2) (Friston et al. 1995). The first 3 image sets were discarded due to

nonequilibrium effects. The remaining 97 image volumes were motion

corrected, spatially normalized to the Montreal Neurological Institute

(MNI) coordinate system, resampled to 1.5 3 1.5 3 4 mm and then

segmented into gray matter, white matter, and cerebrospinal fluid, and

only thegraymatter imageswereused for further analysis. Thegray image

setswere then spatially smoothed (5-mmfullwidth at halfmaximum)and

their intensity was normalized. The mean pain intensity profile created

from the individual subject’s pain intensity profiles was convolved with

the canonical hemodynamic response function and then used to search

for signal intensity changes. Significant increases and decreases in signal

intensity (random effects; uncorrected P < 0.01) were assessed for the

arm and leg muscle pain groups. To reduce the risk of false positive

activations, a minimum cluster threshold of 10 voxels was used. A subset

of the data collected during leg muscle pain has been used in a previous

investigation (Henderson, Bandler, et al. 2006).

To assess the effects of pain referral, subjects were divided into those

in whom the perceived pain radiated peripherally and those in whom

the pain was limited to a diffuse area around the muscle belly. For the leg

pain, subjects in whom the perceived pain spread into the ankle and

onto the dorsum of the foot were placed into the ‘‘TA-referred’’ group

(n = 10) and those in whom the pain was confined to the lower leg were

placed into the ‘‘TA-nonreferred’’ group (n = 7). Similarly, for the arm

pain, subjects in whom the perceived pain spread into the wrist and into

the hand were placed into the ‘‘FCR-referred’’ group (n = 6) and those

in whom the pain was confined to the forearm were placed into the

‘‘FCR-nonreferred’’ group (n = 6). Regional signal intensity increases and

decreases in each of the TA referred and nonreferred groups and the

FCR-referred and -nonreferred groups were determined using a random

effects analysis (P < 0.01, uncorrected). To determine regions that

responded in a similar fashion, the map of significant signal intensity

changes in the TA referred group was used to mask the significant signal

intensity change map in the TA-nonreferred group. A similar procedure

was performed for the 2 FCR groups. Regions in which signal inten-

sity in the referred groups were significantly greater than the non-

referred groups were also determined (random effects; uncorrected P <

0.01). The percentage changes in signal intensity relative to baseline for

each significant voxel in a cluster were calculated over time to form

individual voxel time trends. These were then averaged across a cluster

to form an average cluster time trend and the cluster time trends from

each subject were then averaged to create a mean (±standard error of

the mean [SEM]) group cluster time trend. The significance of these

trends was verified using a repeated measures analysis of variance

analysis (P < 0.05).

The relationship between S1 signal intensity and area of perceived

spread of pain was then determined by comparing the maximum signal

intensity changes in each significant S1 cluster with the area of

perceived pain. Each subject’s pain distribution drawing was digitized

and the area of perceived pain calculated using ImageJ software

(National Institutes of Health, USA). Each subject’s total pain area was

then expressed as a percentage and plotted against the maximum pain

intensity for each of the significant S1 clusters. Significant relationships

were then determined using a regression analysis. To illustrate further

individual subjects S1 signal intensity changes, a volume of interest

(VOI) analysis of the S1 cortex was performed. Using the S1 template

provided by SPM2, the S1 cortex was divided into multiple VOIs based

on Z-levels. The signal intensity change in each of the S1 VOIs was then

calculated for each individual subject.

Results

Pain Perception

Intramuscular injection of hypertonic saline (5%) evoked pain

after a few seconds, reaching maximal intensity within 50--60 s

and subsiding to preinjection levels after ~5 min. The most

commonly chosen words to describe the muscle pain were

‘‘aching’’ and ‘‘cramping.’’ In all subjects the pain was diffuse and,

despite the small volume of the injectate (0.5 ml), encompassed

the entire region of the muscle belly. In addition to this ‘‘primary

pain,’’ in about half of the subjects the pain was described as

spreading distally to encompass the wrist and hand following

injection into FCR (arm), or into the ankle and dorsum of the

foot following injection into TA (leg). This information was

volunteered by the subjects—they were asked to describe

where they felt the pain but were not specifically asked

whether they felt it projecting distally. The 2 patterns of pain

localization for the leg and arm are illustrated in Figure 1. The

distally projecting pain will be termed ‘‘referred pain.’’ Following

each experiment, subjects were separated into 1 of 2 groups:

those who reported referred pain, and those who did not. There

were no statistically significant differences in the maximal pain

intensity ratings between the ‘‘referred’’ and ‘‘nonreferred’’

groups (P > 0.05; t-test), for pain evoked by intramuscular

injection either into the arm (mean ± SEM 6.3 ± 2 for the

referred group, 7.5 ± 1 for the nonreferred group) or into the leg

(7.0 ± 2 vs. 5.6 ± 2). It should be noted that subjects were asked

only to describe the overall pain, not to differentiate between

the relative intensities at the primary and referred sites.

fMRI Signal Changes

The initial analyses were performed on all subjects, irrespective

of whether or not they reported referred pain, to confirm that

our earlier observations obtained for muscle pain in the leg

(Henderson, Bandler, et al. 2006) held also for the arm. In all

subjects significant changes in fMRI signal intensity were

observed in multiple, yet discrete areas of the brain following

intramuscular injection of hypertonic saline into either the arm

or leg. Increases in signal intensity were found in the cerebel-

lum, the posterior and anterior insula bilaterally, the mid-

cingulate cortex, the somatotopically appropriate area of the

contralateral SI cortex, and the contralateral SII cortex (Fig. 2).

Robust decreases in signal intensity occurred in the perigenual

anterior cingulate cortex (pACC), confirming our earlier findings

(Henderson, Bandler, et al. 2006).

Referred versus Nonreferred

Separation of the subjects into 2 groups, those who reported

referred pain in the foot (for injections into the leg) or hand (for

injections into the arm) and those who did not, revealed

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similarities but also widespread differences in BOLD signal

changes for the groups. Such differences were masked by

pooling the results of the 2 groups in the initial analysis.

Similarities

Irrespective of whether the muscle pain originated in the arm

or leg, robust increases in signal intensity occurred in both the

referred and nonreferred groups in the mid-cingulate, anterior

and posterior insula and secondary somatosensory cortex (SII)

bilaterally. This is illustrated by the red shading in Figure 3.

Differences

For both arm and leg muscle pain, significant signal intensity

differences between the referred and nonreferred pain groups

occurred in the SI, pACC, and cerebellar cortices. During arm

pain only, a region of significant difference also occurred in the

ipsilateral anterior insula.

Within the SI cortex, the signal intensity changes appeared to

correspond to the spread of pain described by each subject.

That is, during leg muscle pain, in both the referred and

nonreferred groups, signal intensity increased in the region of

SI representing the leg and increased in the immediately

adjacent region (representing the foot) in those subjects who

reported a pain referral into the ankle and/or foot. Similarly,

during arm muscle pain both the referred and nonreferred

groups displayed an increase in signal intensity in the region of

SI representing the forearm and, in subjects with pain referred

into the wrist and hand, an increase, which spread laterally into

Figure 1. Line drawings of the leg and arm illustrating the extent of perceived pain in each subject following hypertonic saline injection into either the right FCR or TA muscle.Subjects were divided into 2 groups: those in which the pain was referred into the hand or foot (‘‘referred’’) and those in which the pain was not (‘‘nonreferred’’).

Figure 2. Significant fMRI signal intensity changes correlated to mean pain intensity profile during forearm and leg muscle pain overlaid onto an individual T1-weighted anatomicalimage set. The hot and cold color scales (coded for t-value) indicate regional signal intensity increases and decreases, respectively. Slice positions are indicated by MNI coordinatesat the bottom right of each image. MCC, mid-cingulate cortex; pACC, perigenual anterior cingulate cortex.

2052 Imaging Referred Pain d Macefield et al.Downloaded from https://academic.oup.com/cercor/article-abstract/17/9/2050/270992by gueston 19 February 2018

the region of cortex representing the hand (Fig. 4). In 2 subjects,

the correspondence of the anatomical areas was confirmed by

recording significant increases in signal intensity following a

2-min period of light stroking of the hand or dorsum of the foot

with a brush, relative to a 2-min baseline period.

An examination of maximum signal intensity changes com-

pared with the perceived spread of pain revealed a significant

positive correlation between signal intensity and pain area in

the S1 clusters, which displayed significant differences between

the referred and nonreferred groups (Fig. 5). Furthermore,

there was no significant correlation between signal intensity

and pain area in the S1 clusters, which displayed significant

signal intensity increases in both the referred and nonreferred

groups. Plots of individual subject’s S1 signal intensity changes

further emphasize the greater spread of S1 cortex activation in

subjects with larger pain referral areas (Fig. 6).

A similar pattern also occurred in the ipsilateral insula

following FCR injections. That is, a region of signal intensity

increase in both the referred and nonreferred groups and a

discrete region immediately lateral in which signal intensity

increased in the referred group only (Fig. 3). In contrast to these

signal intensity increases, during both forearm and leg pain,

both the referred and nonreferred groups displayed profound

signal intensity decreases in the pACC. In addition, in the

nonreferred groups only, this region of signal decrease ex-

tended into the immediately adjacent medial prefrontal cortex,

whereas in the referred group signal intensity increased slightly

in this region of prefrontal cortex (Fig. 7). Finally, within the

Figure 3. Significant fMRI signal intensity changes during arm and leg pain in the referred and nonreferred groups. Significant signal intensity increases and decreases in both thereferred and nonreferred groups are indicated by the red and blue shadings, respectively. Significant differences are indicated by the yellow shading. The mean percent (±SEM)change in signal intensity over time for a selection of significant clusters is also shown (light green, referred; dark green, nonreferred). The vertical dashed line indicates the onset ofthe hypertonic saline injection. Slice positions are indicated by MNI coordinates at the bottom left of the lower row of images. MCC, mid-cingulate cortex; pACC, perigenual anteriorcingulate cortex.


cerebellar cortex, discrete clusters occurred in which signal

intensity increased in those subjects reporting referred pain in

the hand or foot but decreased in those subjects who did not

report referred pain.

Discussion

Using whole-brain fMRI we have confirmed that the deep pain

evoked by intramuscular injection of hypertonic saline into the

FCR muscle in the forearm is associated with activation of the

same pain neuromatrix engaged by intramuscular injection into

the TAmuscle in the leg, as demonstrated previously (Henderson,

Bandler, et al. 2006). In addition, we have demonstrated that

somatotopy exists within the SI for deep pain originating in the

leg and forearm. More importantly, we have shown for the first

time that referred pain is reflected in a widespread increase in

neural activity in several discrete cerebral areas, including SI,

perigenual anterior cingulate, ipsilateral anterior insular, and

cerebellar cortices.

Methodological Considerations

Intramuscular injection of hypertonic saline is a well-established

model of deep pain (Graven-Nielsen et al. 1997b) but this is only

the second study to have employed fMRI to investigate central

Figure 4. Significant fMRI signal intensity changes during arm and leg pain in the referred and nonreferred groups. Significant signal intensity increases in both the referred andnonreferred groups are indicated by the red shading and significant differences by the yellow shading. The mean percent (±SEM) change in signal intensity over time for significantclusters in the SI is shown to the right (light green, referred; dark green, nonreferred). The vertical dashed line indicates the onset of the hypertonic saline injection.


processing of pain using this technique. An intramuscular

cannula was inserted into the belly of the FCR or TA muscle

prior to the scanning session and subjects knew that they would

receive an injection that would cause pain. They were also

informed that it would subside after a few minutes. And al-

though the expectancy of painful (Ploghaus et al. 1999) or other

unpleasant stimuli causes brain activations in its own right,

subjects did not anticipate feeling pain that projected into the

hand or foot nor did they receive any suggestion prior to the

experiment that this could occur. We believe we are recording

indicators of cortical neural activity that reflect the perception

of referred pain in an area remote from the site of noxious

stimulation. It has been shown that intramuscular injection of

0.5 ml of saline into the TA muscle forms a stable pool, diffusing

only ~2 cm from the injection site (Graven-Nielsen, McArdle

et al. 1997). Accordingly, we are confident that any referred pain

did not result from the spread of hypertonic saline into distal

structures. Moreover, the connective tissue sleeve around the

muscle would limit spread beyond the muscle into the ankle

and foot. Interestingly, it has been shown that intramuscular

injection of hypertonic saline into the soleus muscle causes

a dull ache that is limited to the small area of the injection

site—subjects do not report any projection of the pain into the

foot nor do they report an ache that is perceived as extending

along the entire muscle belly (Weerakkody et al. 2003). This

would suggest that the large area of primary pain emanating

from TA (and FCR) following injection of hypertonic saline into

this muscle is, of itself, a reflection of the processing of the

primary noxious input to the central nervous system—processing

occurring within the spinal cord and/or within the brain.

Pain Referred from Deep Somatic Tissues

The pain that projects distally from the primary site of noxious

stimulation within muscle has been studied extensively

(Arendt-Nielsen et al. 1997; Graven-Nielsen et al. 1997a;

Arendt-Nielsen and Svensson 2001). Typically, that evoked by

injection of hypertonic saline into the TA muscle refers to the

ankle and dorsum of the foot and, like the area of primary pain in

the muscle belly, shows both temporal and spatial summation

frommultiple injections into the same or separate intramuscular

sites (Arendt-Nielsen et al. 1997; Graven-Nielsen et al. 1997a).

Similar but more expansive sites of projection have been found

following injection into the distal tendon or, especially, the

proximal bone--tendon junction of TA (Gibson et al. 2006).

Referred pain has also been reported following injection of

hypertonic saline into the infrapatellar fat pad, though this

usually projects proximally, towards the groin (Bennell et al.

2004).

Representation of Referred Pain in the Primary andSecondary Somatosensory Cortices

It has been widely suggested that pain is represented in 2 main

systems: a lateral system which includes the lateral thalamus, SI,

and SII cortices and, in some cases, the insula, and a medial

system which includes the medial thalamus, anterior cingulate,

and prefrontal cortices. It is thought that the lateral system

mediates sensory-discriminatory aspects and is somatotopically

organized, whereas the medial system mediates the affective

component of the pain experience and is not somatotopically

organized (Ohara et al. 2005). Although there is no doubt SI

cortex is crucial for the ability to localize somatosensory stimuli

accurately, there is debate as to whether it is also involved in the

localization of noxious stimuli. Electrical stimulation of SI rarely

evokes painful sensations (Mazzola et al. 2005) or alters pain

perception (Head and Holmes 1911). A recent magnetoence-

phalography study has shown that brief laser-induced heating of

the skin evokes a sensation of first pain, mediated by A-delta

fibers and engaging SI and SII cortices, and a C-fiber--mediated

sensation of second pain that primarily recruits the ACC and SII

(Ploner et al. 2002). However, we and others have clearly shown

somatotopic activation of SI during noxious stimulation (Bingel

et al. 2004; Henderson, Bandler, et al. 2006). Furthermore, our

data suggest that the perception of referred pain is represented

Figure 5. Maximum fMRI signal intensity changes in the SI plotted against area ofperceived pain for each individual subject. The overlay on the left indicates thesignificant cluster from which the signal intensity changes were calculated. Note thatin the region of SI in which both the referred and nonreferred groups displayed similarsignal intensity increases (red) there was no significant correlation between signalintensity and area of perceived pain. However, in the SI regions in which the referredand nonreferred groups were significantly different (yellow), there was a significantpositive correlation between the maximum signal intensity change and area ofperceived pain.


Figure 6. Individual subject signal intensity changes plotted over time, in 5 VOI’s in the SI (A--E) during leg and arm muscle pain. The location of the VOIs is indicated to the top rightof each panel. The area of perceived pain is indicated to the left of each set of 5 time trends taken from VOIs (A--E). The vertical dashed line indicates the onset of the hypertonicsaline injection. Note that in the subjects in whom the area of perceived pain is greater, the number of VOIs in which signal intensity increases is greater.


in the SI cortex. That is, we found that signal intensity increased

in the foot area of SI in subjectswho reported referred pain in the

foot and in the hand area for those subjects who per-

ceived referred pain in the hand. We did not however, find

a similar pattern of signal intensity change in the SII cortex. It

may be that SII is not critical for the localization of painful

stimuli or alternatively, that our data are of too low a resolution

to differentiate the fine somatotopic organization of the SII

cortex.

Representation of Referred Pain in the Cingulate andInsular Cortices

Similar to SI, during forearm pain we found a pattern of signal

intensity change in the ipsilateral anterior insula, which

mimicked the pattern of referred pain. We have recently shown

that the right anterior insula displays a somatotopically orga-

nized pattern of fMRI signal increase during noxious muscle and

cutaneous stimulation (Henderson, Gandevia, et al. 2006). It has

been suggested that noxious information is transmitted from

the dorsal horn of the spinal cord to a pain-specific region of the

thalamus—the posterior division of the ventral medial thalamic

nucleus (VMpo). Although the existence of the VMpo in

humans is still a matter for debate, Craig (2003) has proposed

that somatotopically organized noxious information is trans-

mitted from the VMpo to the contralateral posterior insula and

finally to the ipsilateral anterior insular cortices. The results of

direct electrical stimulation, brain imaging, and single-unit

recording investigation have confirmed that the insula is

involved in the processing of noxious information (Robinson

and Burton 1980; Peyron et al. 2000; Ostrowsky et al. 2002;

Mazzola et al. 2005). Magneto- and electroencephalography

studies also indicate that SI and the operculo-insula regions are

the earliest activated structures following noxious stimulation

(Ploner et al. 1999; Frot and Mauguiere 2003). Our data show

that, like SI, the right anterior insula displayed a pattern of signal

intensity increase, which mirrored the perceived extent of

referred muscle pain: subjects who reported significant levels of

referred pain displayed an increase in signal intensity in part

of the right anterior insula in which no changes occurred in

subjects who did not report referred pain. The larger area of

insular activation in those subjects reporting referred pain may,

like SI, reflect the ability to localize noxious stimuli.

In contrast, it has been reported that lesions encompassing

large areas of the insula in humans can result in asymbolia

for pain—a condition in which patients can recognize both

superficial and deep painful stimuli and distinguish their quality

(sharp, dull, etc.) but cannot respond to painful stimuli with

either motor withdrawal, grimacing or appropriate emotional

responses (Berthier et al. 1988). It is well established that the

anterior insula is recruited during emotionally charged chal-

lenges. It is activated primarily by negative emotional states

such as internally generated sadness, anger, fear, frustration, and

disgust, and it has been recently reported to display activity

changes that are significantly correlated with the level of

unpleasantness associated with visceral and muscle pain

(Phillips et al. 1997; Phan, Wager et al. 2004; Abler et al. 2005;

Dunckley et al. 2005; Schreckenberger et al. 2005). Further-

more, Phan, Taylor et al. (2004) report that during the appraisal

of self-relatedness of emotionally charged stimuli, fMRI signal

intensity increases in the same region of the right anterior insula

to that reported here during muscle pain. It has been suggested

that the insula monitors the body’s internal state and acts as an

alarm center, with the anterior insula in particular being en-

gaged during recall of negative emotions (Reiman et al. 1997).

The ‘‘somatic marker’’ hypothesis suggests that the anterior

insula may integrate somatic and external cues of emotional

relevance (Damasio 1996). The right anterior insula may inte-

grate somatotopically organized information from muscle noci-

ceptors with the personal relevance of this emotionally charged

stimulus, helping to ‘‘decide’’ on an appropriate course of ac-

tion. In this context, location of the noxious stimulus would

be likely to be advantageous in deciding on an appropriate

behavioral response.

Figure 7. Significant fMRI signal intensity changes in the cingulate cortex during arm and leg pain in the referred and nonreferred groups. Significant signal intensity increases anddecreases in both the referred and nonreferred groups are indicated by the red and blue shadings, respectively. Significant differences are indicated by the yellow shading. The meanpercent (±SEM) change in signal intensity over time for significant clusters in the perigenual cingulate cortex is shown below (light green, referred; dark green, nonreferred). Thevertical dashed line indicates the onset of the hypertonic saline injection. Slice positions are indicated by MNI coordinates at the top left of each image.


Surprisingly, the pACC, a region, which has also been

implicated in the processing of emotional state changes, also

displayed signal intensity change patterns that appeared to

reflect the perceived spread of pain. Signal intensity decreases

within the pACC during muscle and visceral pain have been

reported previously (Dunckley et al. 2005; Henderson, Bandler,

et al. 2006) and we have speculated that the decreases seen in

this region during muscle pain may reflect the negative affect

associated with pain; the pACC is part of the affective division of

the cingulate cortex (Devinsky et al. 1995; Bush et al. 2000).

Lesions of the pACC result in emotional lability (Hornak et al.

2003), and patients with depression often display decreased

pACC metabolism (Drevets et al. 1997; Mayberg et al. 1997). In

contrast to the S1 and anterior insular cortices, we found that

subjects with a larger referral pattern displayed decreases in

single intensity over a smaller area than those subjects who

reported little referral. It has been shown previously that the

extent of muscle pain referral increases as the perceived

intensity of pain increases an that pain intensity is positively

correlated with pain unpleasantness (Graven-Nielsen et al.

1997b). the data in the present study do not support the

idea that the extent of referral is correlated to perceived

intensity—there were no differences in the magnitude of

pain between the referred and non-referred groups. however,

we did not ask each subject to rate the unpleasantness of

the pain. As the extent of pain referral did not correlate with

a greater signal decline in the pACC region, we suggest that

the right anterior insula, and pACC are involved in different

aspects of the emotional response to painful stimuli. Indeed,

the sensation of unpleasantness has been ascribed largely to

the insula, and the unpleasantness of visceral as well as

muscle stimuli involves signal intensity reductions in the

pACC and increases in the right insula (Dunckley et al. 2005;

Schreckenberger et al. 2005).

Representation of Referred Pain in the Cerebellum

The roles of the cerebellum in the processing of pain have

recently been documented (Bingel et al. 2002; Helmchen et al.

2003; Saab and Willis 2003), though any perceptual role in

nociception has largely been ignored. In our earlier study, in

which we injected hypertonic saline into TA (Henderson,

Bandler, et al. 2006), we saw no consistent changes in signal

intensity within the cerebellum, but given the divergent

responses in the ‘‘referrers’’ and ‘‘nonreferrers’’ we observed in

the present study this is perhaps not surprising. A similar lack of

cerebellar activation was observed during intramuscular nox-

ious electrical stimulation (Niddam et al. 2002). For pain

induced in either the leg or forearmwe saw consistent increases

in signal intensity within cerebellar cortex in those subjects

reporting pain in the ankle/foot or wrist/hand, and decreases in

those subjects who did not. As noted above, there were no

differences in the perceived intensity of pain in the 2 groups

so the disparate responses in cerebellar cortex may reflect

subperceptual sensory processing.

This is the first study to have specifically tested whether re-

ferred pain reflects an increase in neural activity in the cerebral

cortex. We have provided evidence of cortical differentiation in

the processing of primary and referred pain following discrete

noxious stimuli originating in muscle, with widespread yet dis-

crete differences having been observed in the primary sensory,

cingulate, anterior insula, and cerebellar cortices.

Notes

We thank Kathy M. Hughes for help in all imaging procedures and

Dr Paul Macey, University of California, Los Angeles, for help with image

analysis. Scans were performed at the Mayne Clinical Research Imaging

Centre. Thisworkwas supportedbyNationalHealth andMedical Research

Council grant 350889 to V.M. and L.H. Conflict of Interest: None declared.

Address correspondence to Luke A. Henderson, Department of

Anatomy and Histology, University of Sydney, Sydney, NSW, Australia

2006. Email: [email protected].

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