Pain 114 (2005) 239–249

www.elsevier.com/locate/pain

Relationship between pain symptoms and referred sensory and trophic

changes in patients with gallbladder pathology
Maria Adele Giamberardinoa,*, Giannapia Affaitatia, Rosanna Lerzaa, Domenico Lapennab,
Raffaele Costantinic, Leonardo Vecchietb
a
Ce.S.I., ‘G. D’Annunzio’ Foundation; ‘G. D’Annunzio’ University of Chieti, Pescara, Italy
b
Department of Medicine and Science of Aging, ‘G. D’Annunzio’ University of Chieti, Pescara, Italy
c
Department of General Surgery, ‘G. D’Annunzio’ University of Chieti, Pescara, Italy
Received 29 June 2004; received in revised form 11 December 2004; accepted 16 December 2004

Abstract
The relationship was investigated between algogenic potential of gallbladder pathology and occurrence/extent of sensory and trophic
changes in the referred area. Five groups of subjects were studied, with: symptomatic gallbladder calculosis (3–20 colics); asymptomatic
calculosis; symptomatic gallbladder shape abnormality (8–18 colics); asymptomatic shape abnormality; normal gallbladder/no symptoms.
At the cystic point (CP) and contralaterally, all underwent measurement of: pain thresholds to electrical stimulation of skin, subcutis
and muscle; thickness of subcutis and muscle via ultrasounds. Contralaterally to CP, all thresholds were not significantly different in the
five groups. At CP, subcutis and muscle thresholds were significantly lower in symptomatic vs asymptomatic patients and/or normals
(0.0001!P! 0.05). In symptomatic cases, at CP compared to contralaterally, subcutis and muscle thresholds were significantly lower
(0.0001!P!0.02), subcutis thickness was significantly higher and muscle thickness significantly lower (0.006!P!0.02). Subcutis
and muscle thresholds at CP in symptomatic patients were significantly and inversely correlated linearly to the number of colics (P!0.0004;
P!0.0001). Patients with symptomatic calculosis were re-evaluated after 6 months; those not presenting further colics showed a significant
increase in subcutis and muscle thresholds at CP, while those who continued presenting colics showed a further significant threshold decrease
(0.01!P!0.05); tissue thickness did not vary. Referred hyperalgesia and altered trophism from the gallbladder only occur in painful
pathology, their extent being modulated by the amount of perceived pain. The results suggest different mechanisms by which visceral
nociceptive inputs trigger sensory vs trophic changes in the referred area.
q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Keywords: Gallbladder; Calculosis; Shape abnormality; Biliary colic; Pain thresholds; Skin; Subcutis; Muscle; Trophic changes

1. Introduction In clinical studies on different visceral conditions, e.g.

Visceral pain, extremely frequent in medical practice, is
typically referred to somatic structures (skin, subcutis and
muscle) in the same neuromeric field of the affected organ;
here it is most often accompanied by sensory and trophic
changes of tissues (hyperalgesia, increased subcutis thick-
ness, decreased muscle thickness) (Cervero, 1995; Gebhart,
2000; Giamberardino, 2000; Procacci et al., 1986).
* Corresponding author. Address: via Carlo de Tocco n. 3, 66100 Chieti,
Italy. Tel./fax: C39 0871 551086.
E-mail address: mag@unich.it (M.A. Giamberardino).
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2004.12.024
urinary calculosis and dysmenorrhea, referred hyperalgesia
was documented by measuring pain thresholds to different
stimuli; it appeared mostly in deep parietal layers, its extent
being a function of the number of painful episodes
previously experienced (Giamberardino et al., 1994, 1997;
Vecchiet et al., 1989, 1990, 1992). Trophic changes, in
contrast, were mainly detected via clinical means, with
precise quantification—measurement of tissue thickness via
ultrasounds—only in very few cases (Vecchiet et al., 1990).
The nature of referred phenomena has been actively
investigated, especially in recent years. Based on the results
of both clinical and experimental (animal models) studies,
240 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
referred hyperalgesia has been attributed mainly to central
sensitization phenomena, triggered by the massive afferent
visceral barrage (Arendt-Nielsen et al., 2000; Berkley et al.,
1993; Cervero, 1995, 2000; Foreman, 2000; Gebhart, 2000;
Giamberardino et al., 1995, 1996; Roza et al., 1998), with
the referred hyperalgesia at muscle level also probably
being contributed to by a reflex arc mechanism (Aloisi et al.,
2004; Giamberardino et al., 2003; Procacci et al., 1986).
While mechanisms of referred hyperalgesia are being
increasingly explored, those of referred trophic changes
are still poorly investigated. This is due to scarce
documentation of these changes in standardized conditions
in patients and a lack of adequate animal models reprodu-
cing the clinical condition (one exception is the rat model of
uterine inflammation, where neurogenic plasma extravasa-
tion is present in skin of the referred area, first experimental
evidence of referred trophic changes from viscera)
(Wesselmann and Lai, 1997). On the other hand,
investigating these phenomena is of the utmost importance,
as they involve changes—e.g. tendency to muscle atro-
phy—which may impair normal tissue functionality. The
first step in this direction is a quantification of trophic
changes in different visceral pain patterns in clinics, in
parallel with the sensory evaluation.
Based on these premises, this study explored sensory and
trophic referred changes in multiple anatomical/clinical
states of the same internal organ, the gallbladder, with the
aim of correlating the extent of the changes with the
algogenic potential of the visceral trigger. Reasons for
choosing the gallbladder were: possibility to explore the
organ via ultrasounds (innocuous, economic and easily
applicable procedure); high frequency of painful conditions
of this organ in the adult population; existence of various
combinations of anatomical/clinical patterns, i.e. calculosis
with or without painful symptoms, shape abnormality with
or without symptoms, visceral normality and no symptoms
(Ahmed et al., 2000; Calabuig et al., 1996; Canfield et al.,
1998; Caroli-Bosc et al., 1999; Corazziari et al., 1999; Festi
et al., 1999; Goncalves et al., 1998; Velanovich, 1997).
Preliminary results have already been published in abstract
form (Vecchiet et al., 1996).
2. Materials and methods
2.1. Patients and subjects
Patients and normal subjects of both sexes were considered for
the study, the former recruited from the Hospital population and
the latter from the Health Care Personnel of the Department of
Internal Medicine of the ‘G. D’Annunzio’ University of Chieti.
Inclusion criteria for patients were: (a) age range 30–60 years;
(b) ultrasound abdomen evaluation documenting either calculosis
or shape abnormality of the gallbladder; (c) in symptomatic cases
(previous biliary colics), pain perceived strictly on the right side of
the body, first colic not earlier than 5 years prior to examination,
last colic not before 1 month prior to examination; (d) negative
clinical history for any other painful pathology (other than
gallbladder pathology) with area of projection in the upper right
abdominal quadrant, and for any pain in the upper left abdominal
quadrant; (e) informed, written consent for participation.
‘Gallbladder shape abnormality’ was defined as any deviation
from normal conformation, most frequently consisting of abnormal
bend/s of the organ, with three main patterns:
(1) ‘Italic S pattern’, with two bends, one between fundus and
body of the organ, the other between body and infundibulum;
(2) ‘hook-like pattern’, with a bend between fundus and body of
the organ;
(3) ‘infundibulum bend pattern’ with a bend between infundibu-
lum and cystic duct.
These abnormal morphologies may cause colic-type pain
secondary to diskinesia (Cosgrove and McCready, 1986; Ziviello
et al., 1990).
Inclusion criteria for normal subjects were: (a) age range 30–60
years; (b) ultrasound abdomen evaluation documenting absence of
calculosis and normal conformation of the gallbladder; (c) no
clinical and instrumental history of previous calculosis of the
gallbladder (stone-free gallbladder for at least 5 years prior to
examination); (d) negative clinical history for pain in the upper
abdominal quadrants of both sides; (e) informed, written consent
for participation in the study.
A total of 53 patients and 22 normal subjects meeting the
inclusion criteria were selected out of 87 patients and 45
individuals examined. They were classified into five groups, on
the basis of the ultrasound gallbladder examination and clinical
history of painful biliary symptoms [for symptomatic subjects, a
detailed account of the pain previously experienced was
requested, i.e. area of pain distribution, number of colics, date
of first and last colic, mean intensity of the pain via Visual
Analogue Scale (VAS), presence of accompanying neurovege-
tative signs, etc.].
2.1.1. Group 1—patients with symptomatic gallbladder calculosis
Twenty-three patients [14 women and nine men, mean age:
51G8 years (MeanGSD)] were included in this group. A stone
was documented in the gallbladder whose dimension ranged from
1.5 to 4 cm. These patients had experienced 3–20 biliary colics
(9.47G4.89, MeanGSD) in the course of 5 years, with pain strictly
unilateral, i.e. right upper quadrant of abdomen, sometimes
radiating towards the right lateral and posterior aspects of
abdomen, towards the scapula. The mean intensity of the biliary
colics had ranged from 50 to 87 mm of Visual Analogue Scale
(VAS) (65.82G8.37, MeanGSD) and the pain was described as
constrictive or cramplike, often accompanied by nausea, vomiting,
pallor and, sometimes, sweating.
2.1.2. Group 2—patients with asymptomatic gallbladder calculosis
Ten patients [seven women and three men, mean age: 53G8
years] were included in this group. The gallstone had
been discovered 4–5 years before examination (dimensions:
1.6–3.5 cm) and had not changed characteristics in this period.
No painful symptoms had ever been experienced by these patients;
only dyspeptic symptomatology had sometimes been reported.
 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
2.1.3. Group 3—patients with symptomatic gallbladder shape
abnormality
Ten patients [six women and four men, mean age: 50G7 years]
were included in this group. Shape abnormality of the gallbladder
had been ascertained at a previous ultrasound examination 4–5
years before; it was confirmed via ultrasounds at the preliminary
examination before entering the study.
These patients reported 8–18 colic episodes in the course of 5
years (10.5G4.03, MeanGSD), with a mean intensity of
45–72 mm of VAS (60G9.7 SD); the other pain characteristics
were similar to those described for patients of Group 1.
2.1.4. Group 4—patients with asymptomatic gallbladder shape
abnormality
Ten patients [seven women and three men, mean age: 50G7
years] were included in this group. As for group 3, in these patients
shape abnormality of the gallbladder had been ascertained 4–5
years before and confirmed at ultrasound evaluation prior to
commencement of the study. No painful symptoms had ever been
reported by these individuals.
2.1.5. Group 5—subjects with normal gallbladder
and no symptoms
Twenty-two subjects [13 women and nine men, mean age: 53G
8 years] were included in this group. A normal gallbladder was
documented at ultrasounds. No previous calculosis of the biliary
tract had been documented in the clinical history. No pain had ever
been reported in the mentioned areas of the abdomen.
The subjects of the five groups did not differ significantly
regarding mean age (one-way ANOVA). The two groups of
symptomatic patients (symptomatic calculosis and symptomatic
shape abnormality) did not differ significantly regarding the mean
number of colics and mean pain intensity (Student’s t-test for
unpaired samples).
2.2. Study design
At the cystic point (level of junction of 10th rib and outer
margin of right rectus abdominis, typical site of referred tenderness
from painful biliary pathology) (Bonica, 1990) and the correspond-
ing contralateral side, all subjects were submitted to: (a) evaluation
of sensory changes at skin, subcutis and muscle level via clinical
procedures; (b) pain threshold measurement to electrical stimu-
lation of skin, subcutis and muscle; (c) measurement of
subcutaneous and muscle thickness via ultrasounds.
Evaluations were always performed in the morning
(10:00–12:00 h), in the same relative phase of the menstrual
cycle (follicular phase) for women in their fertile phase and in the
pain-free interval for symptomatic subjects (Giamberardino et al.,
1997). The temporal sequence of evaluation involved ultrasound
measurement of tissue thickness first, followed by clinical
procedures for sensory changes and threshold measurement
(to avoid any interference of the manoeuvres for hyperalgesia
detection on tissue trophism).
During the whole period of testing, all patients were lying
comfortably on an adjustable couch, in a quiet room. All of them
had been requested to stay free from any medication for at least
72 h preceding the tests.
Patients of Group 1 (symptomatic gallbladder calculosis)
agreed to be re-evaluated after 6 months for both sensory tests
241
(clinical tests for hyperalgesia detection and pain threshold
measurement) and tissue thickness. Throughout this period they
were asked to keep a diary of their symptoms (number of colics).
The experimental protocol was conducted in agreement with
the Helsinki declaration for human experimental studies and
approved by the local Ethics Committee of the ‘G. D’Annunzio’
University of Chieti.
2.2.1. Sensory evaluation of parietal tissues via clinical procedures
Skin sensitivity was tested via the dermographic procedure and
Head’s technique modified by Galletti (Head, 1920; Teodori and
Galletti, 1962). Subcutis sensitivity was evaluated via pinch
palpation; muscle sensitivity was tested via digital pressure
(Simons et al., 1999).
For the dermographic procedure, vertical parallel lines, about
2 cm apart, are traced on the skin surface using the blunt point of a
calibrated dermograph at a constant pressure (500 g). Red lines
appear as a consequence of the manoeuver (vasodilatation
reaction), which fade away progressively and simultaneously in
normal skin areas. An early interruption of these lines occurs in
hyperalgesic areas, indicating a prevalence of the ischemic phase
of dermographism.
For Head’s procedure, concentric lines are scratched over the
skin surface towards the area of altered dermographic reactivity,
using the tip of a calibrated device, at constant pressure (40 g) and
angle of inclination (258). A painful reaction by the patient
indicates the reaching of the border of the hyperalgesic area.
For the pinch palpation, folds of tissue are grasped between the
thumb and index finger and pressed together. A reaction of
discomfort is shown by the patient if the tissue is hyperalgesic.
For the digital pressure, firm compression is exerted with the tip
of the middle finger at the level of the cystic point (rectus
abdominis) while the subject is lying on a couch and invited to
inspire deeply (a manoeuver meant to put the rectus abdominis into
contraction). A painful reaction by the subject is regarded as
indicative of muscle hyperalgesia (Murphy’s sign) (Bonica, 1990).
Digital pressure was also used to assess if any depression of the
compressed tissues remained for a certain time after the manouever
(‘fovea sign’), which would have been indicative of edema of
superficial somatic tissues (see Galletti et al., 1990).
2.2.2. Pain threshold measurement to electrical stimulation
A computerized constant current electrical stimulator was used
(R.S.D. Stimulator, prototype, Florence 1997) to deliver 18-ms
trains of 0.5-ms monophasic square wave pulses, frequency
310 Hz, repeated automatically every 2 s. The shape of
the stimulating wave was constantly monitored via a double-
trace oscilloscope connected to the stimulating device
(see Giamberardino et al., 2001).
To stimulate the skin, the current was passed through surface
electrodes, consisting of a 10-mm diameter circular plate in
Ag/AgCl (reference electrode) and a cylinder in Ag/AgCl with a
0.3 mm-diameter base (stimulating electrode). They were con-
nected to the skin via conductor paste, 1 cm apart in the
longitudinal sense (with the stimulating electrode being placed
right over the cystic point and the reference electrode placed
distally). An adjustable spring device connected to the stimulating
electrode maintained the pressure exerted on the skin constant
throughout measurement.
242 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
For stimulation of subcutis and muscle, two monopolar needle
electrodes were used (0.3 mm in diameter, 25 mm in length,
isolated with Teflon except for 2 mm at the tip). For subcutis
measurement, the two needles were inserted vertically below the
skin surface, 1.5 cm apart. For muscle measurement these same
needle electrodes were used, their tips made to penetrate deep
under the fascia (the intramuscular position was verified by
observation of the movement of the electrodes under voluntary
contraction and/or low-intensity electrical stimulation of the
muscle). Insertion of these thin electrodes was not reported as
painful by the subjects.
Stimulation began at very low current values (0.01 mA) and the
intensity was automatically increased by the device with each
stimulus repetition in increments of 0.03 mA, until the subject
reported a first, non-painful sensation (of touch in skin, paresthetic
in subcutis, of slight twitch in muscle) and subsequently in
increments of 0.1 mA, until the subjects reported a clear painful
sensation. With the stimulation parameters and electrodes used, the
sensation has distinct characteristics in the three tissues: pricking
pain for skin, linearly radiating prickling pain for subcutis and
cramp-like pain for muscle. Pain thresholds were always measured
by the method of the limits, i.e. the value when pain was first
perceived was stored by the computer device and the stimulus was
then decreased, always at the same rate (0.1 mA), with storing of
the value when pain disappeared. It was increased again until pain
reappeared and the corresponding value was stored. The mean of
the three readings was automatically calculated by the computer
stimulator and displayed as final pain threshold for each tissue.
The subjects were instructed to signal the appearance/disap-
pearance of the sensation by pressing a button connected to the
computer stimulator. They were informed that the assessments
were not intended to be tests of pain endurance, that no
suprathreshold stimuli were supposed to be given, and that they
should therefore not try to bear any pain before reporting it. They
were also informed that they were free to stop the stimulus any
moment for any reason and refuse continued participation at any
time, without penalty of any sort (Giamberardino et al., 1994,
1997, 2001).
Before starting measurement of pain thresholds in the selected
areas, a test measurement was performed in a control area (deltoid
region of one side-skin, subcutis and muscle) to familiarize the
subjects with the procedure. Threshold values recorded on that
occasion were discarded.
2.2.3. Ultrasound evaluation of tissue thickness
The thickness of the whole muscle wall and of the overlying
subcutaneous tissue was measured bilaterally at the level of the
cystic point, in millimeters, by ultrasounds (Galletti et al., 1990;
Obletter et al., 1988; Vecchiet et al., 1990). A SIM 3000 ESA-OTE
Biomedica echograph was employed. Probes of 5 and 7.5 MHz
were used for muscular and subcutaneous measurements, respect-
ively. A 3M kit-echo silicone layer (4 cm in thickness) was
interposed between the end of the probe and the cutaneous surface.
2.3. Statistical analysis
MeansGSEM were calculated of thresholds of all tissues and
subcutis and muscle thickness on both sides of the body for each
group of examined subjects. A two-way analysis of variance
(ANOVA) was applied to evaluate the effects of side [right
(cystic point), left (contralateral symmetrical site)] and clinical
condition (symptomatic calculosis, asymptomatic calculosis,
symptomatic shape abnormality, asymptomatic shape abnormality,
normality) for thresholds in each tissue studied. When appropriate,
the trend between groups for thresholds in each tissue was
evaluated via one-way ANOVA with post hoc multiple compari-
sons (Tukey test) and the difference between the right and left side
of the body in each group was analyzed via Student’s t-test for
paired samples.
For tissue thickness, only comparisons between left and right
side of the body were made within each group (Student’s t-test for
paired samples); no within- group trend was evaluated, given the
high variability of this parameter in different individuals even in
normal conditions.
The linear regression analysis was applied to evaluate a
possible correlation between values of thresholds and tissue
thickness on one hand and number of colic episodes on the
other. The level of significance was established at P!0.05.
3. Results
3.1. Sensory evaluation via clinical procedures
In the site contralateral to the cystic point, all clinical
signs of hyperalgesia detection were negative both in
patients and in normal subjects. At the level of the cystic
point, the dermographic procedure and the Head’s man-
oeuver were negative in all groups, pinch palpation and
Murphy sign were positive in symptomatic patients
(symptomatic calculosis and symptomatic shape abnorm-
ality) and negative in asymptomatic patients and normal
subjects.
Digital compression over both the cystic point and the
contralateral side never produced the classic ‘fovea sign’ in
any of the examined groups.
3.2. Pain thresholds to electrical stimulation
Pain thresholds to electrical stimulation of the three body
wall tissues (skin, subcutis and muscle) at the cystic point
and contralateral side are reported in Figs. 1A–5A for the
five groups of subjects.
Regarding the skin, no significant effect of the side of the
body (right, left) (F: 0.5312) or of the clinical condition
(symptomatic or asymptomatic calculosis, symptomatic or
asymptomatic shape abnormality, normality) (F: 2.101) was
observed on variation of thresholds (two-way ANOVA).
Within each group of subjects, the comparison between the
right and left side of the body never showed any significant
difference.
Regarding the subcutis, a significant effect of the side
of the body (P!0.004; F: 5.403) and of the clinical
condition (P!0.02; F 7.410) was seen for variation of
thresholds (two-way ANOVA). A significant trend among
groups was found for threshold variation on the right side
of the body (one-way ANOVA, P!0.0001), with values
 M.A. Giamberardino et al. / Pain 114 (2005) 239–249 243

Fig. 1. Patients with symptomatic gallbladder calculosis (n. 23). Pain thresholds to electrical stimulation of body wall tissues (skin, subcutis and muscle) and
subcutis and muscle thickness evaluated at ultrasounds at the level of the cystic point and at the contralateral symmetrical site (MeansGSEM). **P!0.01;
***P!0.001: comparison between the two sides of the body (Student’s t-test for paired samples).

Fig. 2. Patients with asymptomatic gallbladder calculosis (n. 10). Legend as for Fig. 1.

in symptomatic calculosis being significantly lower than
in asymptomatic calculosis (P!0.05), asymptomatic
shape abnormality (P!0.01) and normal (P!0.001). No
significant trend was found for the left side of the body.
Within each group of subjects, the comparison between
the two sides of the body showed significantly lower
thresholds on the right side than on the left in
symptomatic calculosis (P!0.0001) and symptomatic
shape abnormality (P!0.002).
Regarding the muscle, a significant effect of the side of
the body (P!0.003; F: 9.771) and of the clinical
condition (P!0.0006; F: 5.396) was seen for variation
of thresholds (two-way ANOVA). A significant trend
among groups was found for threshold variation on the
right side of the body (one-way ANOVA, P!0.0001),
with values in symptomatic calculosis and symptomatic
shape abnormality significantly lower than normal
(P!0.001). No significant trend was found for the left
side of the body. Within each group of subjects, the
comparison between the two sides of the body showed
significantly lower thresholds on the right than on the left
side in symptomatic calculosis (P!0.004) and sympto-
matic shape abnormality (P!0.02).
3.3. Subcutis and muscle thickness
Values of subcutis and muscle thickness are reported in
Figs. 1B–5B for the five groups of subjects.
In symptomatic calculosis and symptomatic shape
abnormality, subcutis thickness was significantly increased

Fig. 3. Patients with symptomatic gallbladder shape abnormality (n. 10). Legend as for Fig. 1. *P!0.05; **P!0.01.
244 M.A. Giamberardino et al. / Pain 114 (2005) 239–249

Fig. 4. Patients with asymptomatic gallbladder shape abnormality (n. 10). Legend as for Fig. 1.

Fig. 5. Normal subjects (n. 22). Legend as for Fig. 1.

(P!0.006 and P!0.02) and muscle thickness significantly
decreased (P!0.008 and P!0.02) on the right side of the
body with respect to the left.
In asymptomatic calculosis and shape abnormality, no
significant difference was found between the two sides of
the body.
In most groups, variations in subcutis thickness were
much more pronounced than those in muscle thickness [see
Fig. 6 for scatter plots of measurements in all groups relative
to the control side (left) of abdomen]. This is likely to be
because of the difference in weight/fat distribution between
the various patients/subjects, which has notable impact on
subcutis thickness, in contrast to the rather homogeneous
degree of muscle training (all patients/subjects were
sedentary), which is the key factor in the determination of
muscle trophism (Galletti et al., 1990; Obletter et al., 1988;
Vecchiet et al., 1990; see also Margonato et al., 1994).
3.4. Correlation between sensory/trophic changes
and painful symptoms
The subcutis threshold at the cystic point in symptomatic
patients was significantly and inversely correlated in a
linear fashion to the number of colics experienced
(P!0.0004; YZK0.1396XC3.048; Fig. 7A).
Similarly, the muscle threshold at the cystic point in
symptomatic patients was significantly and inversely
correlated in a linear fashion to the number of
colics experienced (P!0.0001; YZK0.1969XC4.212;
Fig. 7B).

Fig. 6. Scatter plots of tissue thickness measurements on the side contralateral to cystic point (control side: left). Group 1: symptomatic calculosis; Group 2:
asymptomatic calculosis; Group 3: symptomatic shape abnormality; Group 4: asymptomatic shape abnormality; Group 5: normal gallbladder and no
symptoms.
 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
Fig. 7. Correlation between subcutis threshold at the cystic point and
number of colics (A) and between muscle threshold at the cystic point and
number of colics (B) in symptomatic patients (n. 33, gallbladder
calculosisCgallbladder shape abnormality). See text for details.
3.5. Re-evaluation after 6 months in patients
with symptomatic gallbladder calculosis
During the 6-month period of evaluation, 14 patients of
this group did not report any colic episode (Group 1a—
asymptomatic for 6 months) while the remaining nine
complained of 3–6 colics (Group 1b—symptomatic for 6
months). It was therefore decided to evaluate results of
sensory tests and tissue thickness separately in these
subgroups. In Group 1a: (a) the results of the clinical tests
for hyperalgesia detection did not change with respect to
basal outcome, i.e. in the site contralateral to the cystic
point, all clinical signs of hyperalgesia detection were
Fig. 8. Pain thresholds to electrical stimulation and tissue thickness in a subpopulation of patients with symptomatic gallbladder calculosis who did not present
further colics for a period of 6 months (Group 1a). Evaluation performed in basal conditions and after the asymptomatic 6 months (n. 14, MeansGSEM). Right
side: cystic point; left side: side contralateral to cystic point.
245
negative, while at the level of the cystic point the
dermographic procedure and Head’s manoeuver were
negative, pinch palpation and Murphy sign were positive;
(b) thresholds on the left side remained unchanged;
thresholds on the right side increased significantly with
respect to basal values in subcutis and muscle (Fig. 8, upper
graphs), though remaining still significantly lower than
contralaterally (0.005!P!0.05); (c) tissue thickness did
not vary significantly on either side (Fig. 8, lower graphs).
In Group 1b: (a) thresholds on the left side remained
unchanged, while thresholds on the right side underwent a
further significant decrease in subcutis and muscle (Fig. 9,
upper graphs). Tissue thickness remained unchanged
(Fig. 9, lower graphs).
4. Discussion
Sensory changes in the typical area of pain referral from
the gallbladder (around the cystic point) consist of
hyperalgesia appearing mostly in deep tissues, i.e. subcutis
and muscle, only in symptomatic cases, irrespective of the
organic (calculosis) or dysfunctional (diskinesia due to
shape abnormality) nature of the symptoms. These changes
are related in extent to the number of painful episodes
previously experienced, i.e. the hyperalgesia is more
246 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
Fig. 9. Pain thresholds to electrical stimulation and tissue thickness in a subpopulation of patients with symptomatic gallbladder calculosis who presented
further colics for a period of 6 months (Group 1b)(n. 9, MeansGSEM). Legend as for Fig. 7.
pronounced in the case of a high number of colics. The
evolution in time of the hyperalgesia also depends, at least
in part, on the continuation of painful symptoms, as it tends
to decrease in all cases in whom the spontaneous pain does
not occur for a reasonable length of time (6 months), even
though the visceral focus persists (gallstone still present).
Trophic changes in the same area mainly consist of
increased thickness of the subcutis and decreased thickness
of the muscle. Like the sensory changes, these also occur
only in symptomatic cases. However, their extent and
evolution in time do not appear strictly related to the
algogenic potential of the visceral focus. In fact, they do not
worsen with the repetition of the painful episodes and do not
improve with cessation of spontaneous pain, at least for a
period of several months, as applies in this study.
The present results confirm previous data in different
painful visceral pathologies regarding referred hyperalgesia
as being mostly a deep tissue phenomenon, closely
dependent on the amount of perceived pain (Giamberardino
et al., 1994, 2001; Vecchiet et al., 1989, 1990, 1992). On the
other hand, they document for the first time the profile of the
referred trophic changes in the case of referred pain from
viscera, as there has been only limited documentation of
these phenomena in the past by instrumental means in renal
colics from calculosis (Vecchiet et al., 1990). Though less
pronounced than the sensory changes, and thus needing
further quantification in larger groups of patients in future
studies, trophic changes, too, appear initiated only by
painful visceral inpulses. Unlike the hyperalgesia, however,
they would rather seem to be an ‘on-or-off’ phenomenon.
Both hyperalgesia and trophic changes are thus set off by
afferent impulses from the internal organs that are strong
enough to produce a clear painful sensation, but the different
modulation they apparently receive from the algogenic
activity of the visceral focus suggests they are sustained by
mechanisms at least in part different. As already reported in
the Introduction, referred hyperalgesia is undoubtedly
contributed to by phenomena of central sensitization
triggered by the massive nociceptive visceral barrage
(Cervero, 2000). Central changes after noxious input can
also induce the release of vasoactive peptides from fine
afferents in peripheral tissues not affected by the originating
cause, via dorsal root reflexes conducted antidromically from
the spinal cord (Willis, 1999). The central changes produced
by the visceral input could thus not only be responsible per se
for the secondary hyperalgesia (increased responsiveness of
sensitized viscero-somatic convergent neurons to painful
stimuli in the somatic area of referral) but also contribute to
the phenomenon via dorsal root reflexes which are conducted
centrifugally out to peripheral sensory endings, where they
can release neurotransmitters or alter the excitability of
sensory terminals related to the referred area.
If central changes play a key role in the production of
referred sensory phenomena, it seems logical that they are
accentuated as the painful episodes from the internal organ
are repeated and are reduced as they stop.
 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
The fact that a certain amount of hyperalgesia remains
even quite some time after cessation of the spontaneous pain
could theoretically indicate the persistence of central
changes/sensitization in spite of suppression of the periph-
eral drive (Coderre et al., 1992). At present, however, there
is still a lack of experimental evidence for central
sensitization changes surviving long (days–months) after
the cessation of the initial peripheral triggering insult
(see Cervero, 2000). It is thus plausible that additional
mechanisms contribute to the phenomenon of referred
hyperalgesia, especially at muscle level. One mechanism
could be the reflex arc activation already mentioned in the
Section 1, a hypothesis based on the clinical observation that
the area of pain referral from viscera is often the site
of sustained muscle contraction (Giamberardino, 1999;
Procacci et al., 1986). According to this hypothesis, the
afferent branch of this reflex is represented by sensory fibres
from the internal organ and the efferent branch by somatic
efferences towards the muscle to determine contraction,
which, in turn, could sensitize nociceptors locally. A recent
study in a rat model of referred muscle hyperalgesia from
artificial ureteric calculosis has indeed provided some
experimental support for this hypothesis. Positivity was
found for a number of ultrastructural indices of contraction
in the hyperalgesic muscle ipsilateral to the affected ureter
at lumbar level but not in the contralateral, non-hyperalgesic
muscle, and the extent of these indices was proportional to
the degree of visceral pain behavior and referred hyper-
algesia recorded in the animals. In the same model, c-Fos
activation was found in the spinal cord not only of sensory
neurons but also of motoneurons, significantly more on
the affected side (Aloisi et al., 2004; Giamberardino et al.,
2003). Reflex arc activations have been claimed to be
contributing mechanisms also to the skin/subcutis hyper-
algesia. In this case, the efferent branch of the reflex would
be represented by sympathetic efferences towards the
superficial somatic tissues. This hypothesis is based on the
clinical observation of the reduction of referred superficial
sensory changes in patients after blocking of the sympath-
etic efferences towards the referred area (Galletti
and Procacci, 1966; Jänig and Häbler, 1995; Procacci
et al., 1975).
Regarding interpretation of referred subcutis and muscle
trophic changes from a painful visceral pathology, it seems
difficult to claim purely the intervention of neuronal
sensitization, as central mechanisms alone cannot account
for objective modifications in peripheral tissues. Though the
exact nature of these changes remains to be determined, the
fact that they tend to persist almost unaltered for quite a long
time after cessation of the painful episodes could indicate
some structural tissue alteration, probably triggered via a
reflex mechanism, which is liable to become permanent in
spite of resolution of the visceral condition. With this respect,
it is worth noting that the characteristics of the trophic
changes found in this study are similar to those of changes in
areas of referred pain from deep somatic structures observed
247
in previous studies. In the area of referred pain from
osteoarthritis of the knee, for instance, increased subcutis
thickness and decreased muscle section area – testifying
decreased trophism of the tissue – were documented via
ultrasounds (Galletti et al., 1990). Both the subcutis
thickening found in that study and that recorded in the
present study do not present the characteristics of edema, in
which digital compression leaves a depression (‘fovea sign’)
which lasts for a certain amount of time (no fovea sign was
recorded in our patients). Thus the subcutis trophic change
here recorded is not due to an increase in liquid interstitial
pressure as it is in edema (Guyton et al., 1971). Previous
studies in referred pain areas from somatic tissues evaluated
the subcutis thickness with the methodology of the
‘acceptance’, i.e. standardized measurement of the capacity
of the tissue to accept physiological solution from outside,
using a calibrated apparatus (Galletti et al., 1980, 1990). A
high correlation was found between the increase in thickness
and consistency of the subcutis and the increase in the
resistance to the introduction of the physiological solution.
Thus the increased thickness and consistency of the subcutis
was attributed to an increase in solid tissue pressure that is
exerted on the points of contact of the cells, fibers, collagen,
and intercellular gel, this pressure being an expression of the
resistance that solid structures of the tissue offer to
atmospheric pressure. The hypothesis formulated in these
past studies is that this augmentation in pressure is motivated
by an increase in subcutis interstitial jaluronic acid and not
primarily by an increase in the fat layer (Galletti et al., 1990).
A similar hypothesis could be put forward to account for the
increased thickening of subcutis in the present paper, though
definite conclusions will only be possible after performing
series of biopsies in patients. The same considerations can be
made for precisely clarifying at ultrastructural level the
characteristics of muscle trophic changes in areas of referred
pain from viscera, which will need to be studied with
biopsies, especially in the case of recurrent and/or persisting
painful processes from the internal organs.
Irrespective of possible mechanisms, however, the
results here presented show that algogenic conditions in
internal organs may have long-lasting consequences, which
potentially impair normal tissue structure and possibly
functionality for a long period of time, particularly
regarding the skeletal muscle, which shows a tendency to
atrophy. In our opinion, this outcome points to the necessity
of early intervention to block the visceral pain input, in
order to prevent and/or reduce to a minimum the negative
consequences at the peripheral body wall area of projection.
Acknowledgements
We wish to thank the staff of the Center for Informatics
and Statistics (CIRS) of the ‘G. D’Annunzio’ University of
Chieti for their help with statistical analysis of the results.
248 M.A. Giamberardino et al. / Pain 114 (2005) 239–249
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