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© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
nature publishing group 1205
FUNCTIONAL GI DISORDERS
ORIGINAL CONTRIBUTIONS
INTRODUCTION
In up to 50% of patients seen in gastroenterology outpa-
tient clinics, routine investigations fail to identify underlying
abnormalities that can explain their symptoms and com-
plaints. Because of that reason, these patients are referred to as
having functional gastrointestinal disorders (FGIDs) ( 1,2 ),
comprising a set of diseases that, due to their chronic nature
and high prevalence, constitute a major personal and societal
burden ( 1–3 ).
Recent evidence points the attention to subtle cellular and
molecular alterations in the gastrointestinal tract of patients with
FGIDs ( 4–10 ). Although the presence of so-called ‘low-grade
in ammation’ together with abnormalities at the mucosal level
appear to be key features in patients with FGIDs, they do not fully
justify or explain the presence of (speci c) clinical symptoms
in these patients ( 1 ). Because of its crucial role in the control of
gut sensorimotor functions, the enteric nervous system has been
hypothesized to also have its part in the pathophysiology of FGIDs
Evidence for Neuronal and Structural Changes in
Submucous Ganglia of Patients With Functional
Dyspepsia
Carla Cirillo , MSc, PhD
1
,
2
, Talat Bessissow , MD, PhD
2
,
3
, An-Sofi e Desmet , MSc
1
,
2
, Hanne Vanheel , MSc, PhD
2
,
Jan Tack , MD, PhD
2
and Pieter Vanden Berghe , MSc, PhD
1
,
2
OBJECTIVES:
An intact and well-functioning enteric nervous system is necessary to effi ciently organize gut
function. Functional gastrointestinal disorders are pathological entities in which gut function is
impaired without a clearly established pathophysiology. On the basis of the relative ease with which
intestinal biopsies can be obtained, and taking advantage of a recently developed optical recording
technique, we evaluated whether functional neuronal defects exist in enteric nerves of patients with
functional dyspepsia (FD).
METHODS: The submucous plexus isolated from duodenal biopsies taken from FD patients and control subjects
was used to functionally and morphologically examine nerves and ganglionic architecture (neurons
and glial cells). In light of previous studies reporting eosinophil and mast cell infi ltration in the gut
mucosa of FD patients, we also examined whether these cells infi ltrated the submucous plexus and
whether this correlated with neuronal activity and specifi c clinical symptoms.
RESULTS: We demonstrate that neuronal functioning is impaired in the submucous plexus of FD patients, as
shown by decreased calcium responses to depolarization and electrical stimulation. Glial (S100)
and neuronal (HuCD) markers show signs of gliosis, altered ganglionic architecture, and neuronal
abnormalities in the submucous plexus of FD patients. We found that eosinophils and mast cells
infi ltrated the submucous layer of FD patients to a much larger extent than in controls. A signifi cant
correlation was found between the number of these cells and the calcium transient amplitudes
measured in submucous ganglia.
CONCLUSIONS: We provide the fi rst direct evidence that FD is characterized by functional and structural
abnormalities within the submucous ganglion plexus, which may be of future predictive and
diagnostic value in the treatment of FD patients.
Am J Gastroenterol 2015; 110:1205–1215; doi: 10.1038/ajg.2015.158; published online 16 June 2015
1
Laboratory for Enteric NeuroScience (LENS), University of Leuven , Leuven , Belgium ;
2
Translational Research Center for GastroIntestinal Disorders (TARGID),
University of Leuven , Leuven , Belgium ;
3
Department of Gastroenterology, Royal Victoria Hospital, McGill University Health Center , Montreal , Quebec , Canada .
Correspondence: Pieter Vanden Berghe, MSc, PhD, Laboratory for Enteric Neuroscience (LENS), TARGID, University of Leuven , Herestraat 49, O&N1 mailbox
701 , Leuven 3000 , Belgium . E-mail: pieter.vandenberghe@med.kuleuven.be
Received 29 December 2014 ; accepted 20 April 2015
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Cirillo et al.
( 1,11–13 ). Although highly valuable, the current evidence for
this involvement is at best indirect as it is based on clinical and
basic pharmacological studies ( 14–19 ). e few studies that used
intestinal biopsies to address the possibility of cellular defects
have focused their attention mostly on the mucosal layer ( 8–10 ),
whereas only two ( 7,20 ) also included the nerve plexus and quanti-
ed neuronal numbers. e latter point is of extreme importance,
as neuronal quanti cation is most likely not su cient to identify
‘enteric neuropathies’ ( 1 ). We and others have recently demon-
strated that intestinal biopsies obtained during routine endoscopy
are a source of invaluable biological material, as they contain snip-
pets of submucous plexus, with ganglia and neurons that can be
quanti ed and recorded from ( 21–23 ).
Taking advantage of the recently developed optical recording
techniques ( 21 ) and the ease to obtain biopsies without major risks
( 24 ), we are now able to directly test the hypothesis that nerves in
the intestinal wall of patients with FGIDs are dysfunctional even
when gross morphology seems una ected ( 1 ). We thus used this
advanced optical approach in patients with functional dyspepsia
(FD), which is one of the most prevalent FGIDs. FD is de ned by
Rome III consensus as the presence of symptoms in the absence of
any underlying organic disease and whose pathophysiology is not
clearly established ( 1,25,26 ). Moreover, on the basis of the recent
evidence that low-grade in ammation may have a crucial role in
FD pathophysiology ( 8–10 ) and the fact that immune cells inter-
act with enteric nerves to in uence intestinal function ( 27 ), we
also evaluated how immune cell in ltration in the submucous
plexus of FD patients correlated with both neuronal activity and
the presence of speci c FD symptoms.
MATERIALS AND METHODS
Study population
In this study, we used duodenal biopsies from 18 newly diag-
nosed FD patients ful lling the Rome III criteria ( 2 ) who were
referred to our gastroenterology unit for endoscopic evaluation.
ese patients were invited to complete the Rome III dyspep-
sia module questionnaire ( 2,25,26 ). To facilitate diagnostic and
therapeutic approach to FD patients, the Rome III consensus pro-
posed to subdivide FD into the postprandial distress syndrome
(PDS), characterized by meal-related symptoms, such as early
satiation and postprandial fullness, and the epigastric pain syn-
drome (EPS), characterized by epigastric burning and epigastric
pain ( 2 ). EPS and PDS symptoms were thus assessed. In line with
Rome III criteria, we excluded patients with diabetes or celiac dis-
ease. As controls, we used 20 age- and sex-matched subjects who
were referred to our gastroenterology unit for endoscopic evalu-
ation for other reasons. ese subjects will be termed ‘controls’
throughout the rest of the manuscript. ey had no macroscopic
upper gastrointestinal lesions at endoscopy and did not ful ll the
Rome III criteria for FD. All subjects underwent careful history
taking, clinical examination, and routine biochemistry together
with histological examination of duodenal biopsies. Exclusion
criteria for all participants were intake of non-steroidal anti-
in ammatory drugs, proton-pump inhibitors, corticosteroids, or
other immunosuppressive drugs in the 6 months prior to exami-
nation. All FD patients and controls were asked about food aller-
gies or intolerance. e study protocol (ML7400) was approved
by the Ethics Committee of Leuven University Hospitals. Written
informed consent was obtained from each subject prior to inclu-
sion in the study.
Preparation, loading, and calcium (Ca
2+
)-imaging of submucous
neurons
Duodenal biopsies were taken by experienced endoscopists
(TB, JT) using standard biopsy forceps and put in ice-cold Krebs
solution (in mM: 120.9 NaCl, 5.9 KCl, 1.2 MgCl
2
, 2.5 CaCl
2
, 11.5
glucose, 14.4 NaHCO
3
, and 1.2 NaH
2
PO
4
) ( 21 ). A er removal,
biopsies were quickly transferred to the lab, and the mucosa
was removed by using watchmaker’s forceps to obtain the sub-
mucous plexus. All dissection procedures were performed in
ice-cold Krebs solution to reduce cellular activity and release of
mediators. A er loading (at room temperature) with the uores-
cent Ca
2+
indicator Fluo-4 AM (Molecular Probes, Invitrogen,
Merelbeke, Belgium) and 0.01% Cremophor EL surfactant agent
(Fluka Chemika, Buchs, Switzerland), Ca
2+
-imaging was per-
formed as previously described ( 21 ). Fiber tracts were electrically
stimulated via a platinum electrode (diameter 50 μ m), which
was guided by a mechanical micromanipulator (Narishige,
London, UK). Trains (2 s, 20 Hz) of 300 μ s electrical pulses
(Grass Instruments, Quincy, MA) were used. Neurons were only
included in the Ca
2+
analysis when they displayed a sharply increas-
ing Ca
2+
response to high-K
+
(75 mM) perfusion. Images were
collected using TILL Vision so ware (TILL Photonics, Gräfel ng,
Germany), and analysis was performed using custom-written mac-
ros in IGOR PRO (Wavemetrics, Lake Oswego, OR). To remove
dri and movement artefacts due to perfusion, the image stack was
registered to the rst image. Regions of interest were drawn over
each cell, uorescence intensity was normalized to the basal
uorescence at the onset of the recording for each region of inter-
est (Δ F/F
0
), and peaks were analyzed. A peak was considered
if the signal rose above baseline plus ve times the intrinsic
noise level. e percentage of responsive cells and the maxi-
mum intracellular Ca
2+
concentrations peak amplitude were
determined. All experiments and analyses were performed in a
blinded way.
Immunofl uorescent staining of the submucous plexus
A er xation with paraformaldehyde, submucous plexus prepa-
rations were stained using primary antibodies against neuronal
and glial components (chicken anti-neuro lament 200 kDa
(NF200, 1:500; Abcam, Cambridge, UK), mouse anti-panneu-
ronal HuCD (1:500; Molecular Probes, Invitrogen), rabbit anti-
vasoactive intestinal peptide (VIP, 1:500, Millipore, Overijse,
Belgium), rabbit anti-S100 (1:500; Dako, Glostrup, Denmark)),
eosinophils and mast cells using mouse anti-eosinophilic major
basic protein (1:20; AbD Serotec, Kidlington, UK), and mouse
anti-mast cell tryptase (1:200; Dako). Primary antibody incuba-
tion was followed by a labeling step with appropriate uorescently
labeled secondary antibodies. Immunohistochemical staining was
© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
1207
FUNCTIONAL GI DISORDERS
Submucous Defects in Functional Dyspepsia
visualized under an epi uorescence microscope (BX 41 Olym-
pus, Belgium) with speci c lter cubes (EX/DM/EM in nm) for
blue (325–375/400/435–485), green (460–495/505/510–550),
red (570–590/595/600–660), and far red (620–650/655/660–720)
uorescent probes. Images were recorded using Cell^F so ware
on an XM10 (Olympus) camera. Each preparation was entirely
screened to count ganglia, neurons per ganglion, and also non-
ganglionic (individual) neurons, as described earlier ( 21 ). Confo-
cal images were recorded using Zeiss LSM 510 Meta and LSM 780
confocal microscopes (Cell Imaging Core, KU Leuven, Belgium).
Eosinophils and mast cells were counted in the whole submucous
plexus, and their number was expressed as a number of positive
cells/total surface of submucous plexus.
Analysis of HuCD, S100, and VIP staining in submucous
neurons
As described in an earlier paper ( 28 ), the localization of the HuCD
protein, used to identify neurons, can either be cytosolic or be
predominantly nuclear. In normal conditions, the HuCD protein
is homogeneously spread in the neuronal cell body, whereas in
conditions of experimental neuronal damage HuCD appears to be
present mainly within the nucleus ( 28 ). Each of the samples was
entirely screened, and dual color images were taken of each gan-
glion to evaluate the HuCD staining in a blinded way. Cells that
had a strong nuclear HuCD staining were counted, and the per-
centage over total neurons per ganglion was made for each biopsy.
Similarly, also the mean HuCD intensity signal was measured for
nucleus and cytosol of each neuron (Cell^F so ware, Olympus),
and the mean intensity nucleus/cytosol ratio was calculated for
each biopsy. For volumetric measurements of S100 expressing
cells, we used confocal recordings, which were deconvolved with
Huygens Professional (Scienti c Volume Imaging, Hilversum,
e Netherlands) and visualized in Imaris 7.7 (Bitplane, Zürich,
Switzerland) on an image analysis workstation (Cell Imaging
Core, KU Leuven, Belgium). Cubes of interest were drawn around
individual ganglia, and S100 positive voxels were determined in a
blinded way using the spot detection algorithm available in Imaris
7.7. e total glial volume (in μ m
3
) was calculated and expressed
relative to the number of HuCD-positive neurons present in the
same cube of interest. For VIP analysis, we used a far red (Alexa
647) secondary antibody to avoid spectral overlap with the other
antibody staining. In order to assess VIP staining, all biopsies
were scored in a blinded manner, and ber intensity was quanti-
ed in parallel using IGOR PRO (Wavemetrics).
Data and statistical analysis
All results are presented as mean±s.e.m. e normal distribution
of the data was veri ed by the Kolmogorov–Smirnov test for nor-
mality. Di erences between groups were analyzed using two-tailed
unpaired Student’s t -test or the Mann–Whitney U -test. e num-
ber of neurons/ganglion and ganglia/biopsy was compared using
Fisher’s exact test. e success rate for Ca
2+
-imaging in FD patients
and in controls was evaluated by the
χ
2
test. e di erence in the
number (in %) of neurons responding to high-K
+
or electrical
stimulation (ES) was evaluated using Fisher’s exact test. e cor-
relations between neuronal amplitudes and immune cell numbers
and EPS or PDS symptoms with neuronal amplitudes or immune
cell numbers were performed using Spearman’s rank analysis.
P -values <0.05 were considered signi cant. Statistical analysis
was performed with Microso Excel 2007 (Redmond, WA),
GraphPad Prism 5.0 (GraphPad, San Diego, CA), or SAS enterprise
(Cary, NC).
RESULTS
Study population
Duodenal biopsies taken from 18 FD patients ful lling the Rome
III criteria were included in the study, 10 with EPS and 8 with
PDS ( 2,25,26 ), with 6 of them having overlapping characteristics.
Demographic and clinical characteristics, as well as the indica-
tions for gastro-duodenal endoscopy in FD patients and controls
are summarized in Table 1 . No signi cant di erences in age and
sex distribution were found between controls and FD patients.
None of the participants were taking medication (proton-pump
inhibitors, non-steroidal anti-in ammatory drugs, corticoster-
oids) in the 6 months prior to the examination, none were smok-
ers, and none had food allergies or intolerance. e endoscopic
examination revealed no macroscopic changes in the duodenum
of any of the participants. All subjects were Helicobacter pylori
negative, and clinical examination and routine biochemistry
were not indicative of any organic disease. Routine histology of
Table 1 . Demographic and clinical characteristics of the study
subjects
Controls ( n =20) FD patients
( n =18)
P value
Gender (F:M) 12:8 12:6 —
Age, (range) 43.9±2.7, (22–74) 40.4±3.3,
(26–64)
0.33
a
Indication for
endoscopy ( n )
Anemia (7), follow-up
gastric ulcer (3), weight
loss (6), pre-operative
evaluation for gastro-
esophageal refl ux
disease (4)
FD
b
(18) —
Rome III classifi -
cation
b
(EPS:PDS)
— 10:8
(overlap in 6)
Macroscopic
endoscopy
(duodenum)
Normal Normal —
Routine histology
c
(duodenum)
Normal Normal —
EPS, epigastric pain syndrome; F, female; FD, functional dyspepsia; M, male;
PDS, postprandial distress syndrome.
a
Mann–Whitney U -test.
b
Rome III criteria: postprandial fullness, early satiety, epigastric pain, or epigas-
tric burning ( 2,25,26 ).
c
Routine histology performed by the pathology department at Leuven University
Hospitals.
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Cirillo et al.
duo denal biopsies did not reveal any microscopic change sug-
gestive of duodenopathy. ere were no signi cant di erences
between FD patients and controls in terms of how easily the nerve
layer (submucous plexus) could be dissected and loaded with Ca
2+
indicator.
Quantitative analysis of enteric neurons and ganglia
Immunohistochemical analysis ( Figure 1a ) did not reveal any
di erence in the number of ganglia per biopsy in FD patients
compared with controls (7.9±1 vs. 7.3±1, respectively; P =0.63,
Figure 1b ). In addition, no changes were found in the number
of neurons per ganglion (5.0±0.1 vs. 4.8±0.1, P =0.09, Figure 1c )
nor in the number of non-ganglionic neurons (2.8±0.6 vs.
2.5±0.6, P =0.70, Figure 1d ) between FD patients and controls,
respectively.
Depolarization and electrically induced responses differ
between FD patients and controls
C a
2+
-imaging recordings were performed in 236 neurons from
49 ganglia of controls and 230 neurons from 48 ganglia of FD
patients, as recognized based on morphology and characteris-
tic Fluo-4 loading ( 21 ) ( Figure 2a and c ). Viable neurons were
identi ed with a brief high-K
+
depolarization. In controls, 76%
of the neurons responded to high-K
+
( Figure 2e , right), with an
average Ca
2+
transient amplitude of 8±1% ( Figure 2b and e ). In
FD patients, however, only 56% of neurons responded ( P <0.01,
Figure 2e , right), and their peak amplitude was signi cantly
lower (3±2%, P <0.01, Figure 2d and e ) compared with control
neurons.
Trains of electrical pulses applied to the interconnecting ber
bundles (ES) activated 56% of the high-K
+
responsive neurons
in controls ( Figure 2e , right), with Ca
2+
transient amplitudes
reaching 5±0.2% ( Figure 2b and e ). In FD patients, only 28% of
the identi ed neurons responded to electrical stimuli ( P <0.01,
Figure 2e , right), and lower Ca
2+
transient amplitudes were
measured in responding neurons (2±0.1%, P <0.01, Figure 2d
and e ) compared with controls.
To investigate whether Ca
2+
response characteristics could
be associated with subpopulations of patients, we subdivided
the FD patients (in EPS and PDS) but did not nd any signi -
cant di erence either a er high-K
+
depolarization (3±1 vs. 3±1%,
P =0.42) or a er electrical stimulation (1±0.2 vs. 1±0.2%, P =1.0;
Figure 2f ).
Presence of eosinophils and mast cells in the submucous
plexus of FD patients
Immunostaining of the submucous plexus for eosinophilic major
basic protein revealed a signi cantly higher number of eosino-
phils in FD patients compared with controls (112.8±13.5 vs.
10.2±2.8, P <0.01, total counts per layer, Figure 3a and b ), with
Submucous plexus
NF200
HuCD
NS NS NS
20
bcd
a
15
10
Ganglia/biopsy
Neurons/ganglion
5
0
10
8
6
4
0
2
Isolated neurons/biopsy
10
8
6
4
0
2
Controls Controls FD patients Controls FD patientsFD patients
S100
Merge
Figure 1 . The number of neurons and ganglia does not differ in functional dyspepsia (FD) patients compared with controls. ( a , left) Schematic representa-
tion of the submucous plexus (~5 mm
2
) isolated from duodenal biopsies and ganglia containing neurons (color code: green and red) and glial cells (color
code: blue) with interconnecting fi bers (color code: red and blue). ( a , right) Immunostaining of a submucous ganglion where neurons (HuCD, green), glial
cells (S100, blue), and nerve fi bers (NF200, red) can be identifi ed. Bar = 20 μ m. ( b ) Number of submucous ganglia per biopsy in FD patients compared
with controls. No signifi cant differences were found. ( c ) Number of neurons per ganglion in FD patients compared with controls. No signifi cant differences
were found. ( d ) Number of isolated neurons in the submucous plexus of FD patients compared with controls. No signifi cant differences (NS) were found.
© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
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FUNCTIONAL GI DISORDERS
Submucous Defects in Functional Dyspepsia
of eosinophils and mast cells could be found in close vicinity
(<100 μ m) to neurons ( Figure 3a and c ).
When we compared the total number of in ltrating cells in the
submucous plexus, we found no signi cant di erences in the total
number of eosinophils in PDS compared with EPS (132.9±23.8 vs.
99.6±16.6, respectively, P =0.17; Figure 3e , le ), nor in the total
no di erences between symptom-based subgroups. Similarly,
when stained for the mast cell marker tryptase, the number of
positive cells was signi cantly higher in the submucous plexus
of FD patients compared with controls (223.6±28.2 vs. 69.2±6.0,
P <0.01, total counts per layer, Figure 3c and d ). Double-label
immunostaining using HuCD revealed that a signi cant number
20
15
20
30
10
5
0
15
20
10
5
0
3
4
5
2
1
0
EPS PDS EPS PDS
NS NS
**
**
**
**
8
4
6
10
2
0
4
2
3
5
1
0
4
2
3
5
1
0
Controls FD patients
Controls FD patients
Controls
High K+
High K
+
High K
+
ES
ES
ES
FD patients
Controls
FD patients
15
20
ab
c
e
f
d
High K
+
ES
20 40
Seconds Seconds
60 80 100
10
ΔF/F
0
(%)ΔF/F
0
(%)
ΔF/F
0
(%)
ΔF/F
0
(%)
ΔF/F
0
(%)
ΔF/F
0
(%)
ΔF/F
0
(%)ΔF/F
0
(%)
Controls
1
1
2
2
5
0
0
20 40
Seconds
60 80 100
0
10 20 30 40 60500
10 20 30 40 6050
0
3
4
5
2
1
0
80
40
60
100
20
0
Percentage of
responding neurons
FD patients
2
1
1
2
Figure 2 . Neuronal impairment in the submucous plexus of functional dyspepsia (FD) patients. ( a ) Immunostaining of a typical ganglion (left) from control
and its matched Fluo-4 signal (right). ( b , left) Recordings of the responses of neuron 1 (red arrowhead) and 2 (green arrowhead) to high-K
+
showing
the typical fast upstroke. ( b , right) Recordings of the responses of neuron 1 (red arrowhead) and 2 (green arrowhead) to electrical stimulation (ES).
( c ) Immunostaining of a ganglion (left) from a FD patient and its matched Fluo-4 signal (right). Note that the neurons labeled express a brighter HuCD
staining in the nucleus (green). ( d , left) Recordings of the responses of neuron 1 (red arrowhead) and 2 (green arrowhead) to high-K
+
showing lower
amplitude compared with control neurons. ( d , right) Recordings of the responses of neuron 1 (red arrowhead) and 2 (green arrowhead) to ES: also in this
case, the amplitude is lower compared with control neurons. The arrowheads indicate the neurons of which Ca
2+
responses were plotted in the graphs.
The gray bars indicate the period of exposure to high-K
+
. ( e ) Relative fl uorescence (Δ F/F
0
) recorded from neurons when stimulated with high-K
+
(left)
and ES (middle). Each dot is the average response from neurons in the biopsies from controls (blue dots) and FD patients (red dots). ( e , right) Number
of neurons responding to high-K
+
and ES in FD patients (red bars) compared with controls (blue bars). Each bar shows the percentage of responding
neurons (identifi ed by high-K
+
depolarization) (** P < 0.01). ( f ) Relative fl uorescence (Δ F/F
0
) recorded from neurons when stimulated with high-K
+
(left) and
ES (right) in epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS) subgroups. Each dot is the average response from neurons in the
biopsy. Bars = 20 μ m. NS, not signifi cant.
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Cirillo et al.
number of mast cells per submucous plexus (EPS: 226.2±29.8 vs.
PDS: 242±55.7, P =0.87) ( Figure 3e , right).
Correlation between neuronal impairment and eosinophil/mast
cell number in FD patients and controls
To link the two main ndings of this study, we evaluated whether
impairment of neuronal activation in the submucous plexus
correlated with the increased number of in ltrating eosinophils
and mast cells. To do so, we performed a Spearman correlation
test using the data generated by the Ca
2+
-imaging experiments
and immunohistochemical assessment independently of the clin-
ical classi cation ( Figure 4 ). We found a signi cant non-linear
negative correlation between the response amplitudes and the
amount of in ltrated cells, both for responses elicited by high-
K
+
depolarization (eosinophils: rho=−0.48, P =0.003; mast cells:
rho =−0.34, P =0.042, Figure 4a and b , respectively) or electrical
pulses (eosinophils: rho=−0.56, P =0.001; mast cells: rho =−0.52,
P =0.002, Figure 4c and d , respectively). e Spearman test did
Controls
**
200
300
a
c
e
FD patients
Controls
FD patients
0
100
EPS PDS
200
Eosinophil cell number
300
0
200
EPS PDS
400
Mast cell number
600
Controls
MBP
MBP
HuCD
HuCD
100
0
Eosinophil cell number
FD patients
Controls FD patients
**
NS NS
Tryptase
HuCD
Tryptase HuCD
200
300
100
0
Mast cell number
Figure 3 . The number of eosinophils and mast cells in the submucous plexus is higher in functional dyspepsia (FD) patients than in controls. ( a ) Three
consecutive confocal images (2 μ m apart) of the submucous plexus of control (upper panels) and FD patients (lower panels) where neurons (HuCD, green)
and eosinophils (major basic protein (MBP), color code: magenta) can be identifi ed by immunohistochemistry. ( b ) Number of eosinophils in the
sub mucous plexus of FD patients compared with controls. ( c ) Three consecutive confocal images (2 μ m apart) of the submucous plexus of control
(upper panels) and FD patients (lower panels) where neurons (HuCD, green) and mast cells (tryptase, color code: red) can be identifi ed by immuno-
histochemistry. White arrows indicate immune cells; arrowheads indicate neurons. Bar = 20 μ m. ( d ) Number of mast cells in the submucous plexus of
FD patients compared with controls. ( e ) Number of eosinophils (left) and mast cells (right) in the submucous plexus of epigastric pain syndrome (EPS)
compared with postprandial distress syndrome (PDS) subgroups. ** P < 0.01; NS, not signifi cant.
© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
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FUNCTIONAL GI DISORDERS
Submucous Defects in Functional Dyspepsia
of eosinophils was correlated to the presence of cardinal
PDS symptoms (postprandial fullness: P =0.05, early satiety:
P =0.02; Table 2 ) but not with the presence of cardinal EPS
symptoms (epigastric pain: P =0.18, epigastric burning: P =0.58;
Table 2 ).
not show signi cant correlations between neuronal response
characteristics and the number of eosinophils and mast cells
within the control or FD patient groups.
Correlation between neuronal impairment and clinical symptoms
in FD patients
We evaluated the association between neuronal impairment
in response to high-K
+
or ES in the submucous neurons of FD
patients and the prevalence of EPS or PDS symptoms (assessed
by using standardized questionnaires) ( 2,25,26 ). No correla-
tion was found between neuronal amplitudes and EPS (high-K
+
:
P =0.96 and P =0.51, ES: P =0.53 and P =0.91) or PDS (high-K
+
:
P =0.89 and P =1.00, ES: P =0.81 and P =0.64) symptoms (see
table 2 ).
Correlation between eosinophil/mast cell number and clinical
symptoms in FD patients
e association between the number of eosinophils and mast cells
and the prevalence of EPS or PDS symptoms ( 2,25,26 ) was also
evaluated in the submucous plexus of FD patients. Although no
correlation was found between the amount of mast cells and the
prevalence of EPS or PDS symptoms, intriguingly, the number
Eosinophils Mast cells
15
ΔF/F
0
(%)ΔF/F
0
(%)
ΔF/F
0
(%)ΔF/F
0
(%)
High-K
+
ES
20
25
10
5
0
15
20
25
10
5
0
1
2
3
4
5
1
2
3
4
5
0 50 100 150 200 250
0 50 100
Eosinophil cell number Mast cell number
FD patients Controls
150 200 250
200 400 500300100
200
400
500
300100
Figure 4 . Non-linear relationship between neuronal responses and the number of immune cells infi ltrated in the submucous plexus. ( a , b ) The graphs
show the Ca
2+
response amplitudes (Δ F/F
0
in %) elicited by a high-K
+
stimulus and numbers of eosinophils (left) and mast cells (right). ( c , d ) The graphs
display the data obtained after electrical stimulation (ES) and numbers of eosinophils (left) and mast cells (right). Black lines are non-linear fi ts through the
entire data set; the 95% confi dence interval for the fi tting parameters is indicated as a gray shade. Non-linear correlations (Spearman) were found in each
of the four comparisons. Individual data points were color coded (blue: controls, red: FD) post hoc to indicate the clinically defi ned group they belonged to.
Table 2 . Correlation between neuronal impairment and eosinophil/
mast cell number with prevalence of EPS or PDS symptoms
EPS ( n =10) PDS ( n =8)
Epigastric
pain
Epigastric
burning
Postprandial
fullness
Early
satiety
P value P value P value P value
High-K
+
0.96 0.51 0.89 1.00
ES 0.53 0.91 0.81 0.64
Eosinophils 0.18 0.58 0.05
a
0.02
a
Mast cells 0.79 1.00 0.61 0.87
EPS, epigastric pain syndrome; ES, electrical stimulation; PDS, postprandial
distress syndrome.
a
Spearman’s test.
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Cirillo et al.
The subcellular localization of the neuronal HuCD and glial
S100 is different between FD patients and controls
In this study, we also assessed whether the HuCD staining dis-
played a typical cytosolic pattern or a distinct subcellular localiza-
tion ( 28 ) and found a more explicit nuclear staining in submucous
neurons of FD patients, compared with controls ( Figure 5a
and b ). To quantify this di erence, we used two methods. First,
we counted the neurons per ganglion having a strong nuclear
HuCD staining and found that this percentage was signi cantly
higher in FD patients compared with controls (66.5±5.3 vs.
41.0±4.2, P <0.01, Figure 5c ). To con rm these data, we also used
a second quanti cation method that calculates the mean HuCD
intensity ratio between the nucleus and the cytosol ( 28 ). Again,
this ratio was signi cantly higher in submucous neurons from
FD patients compared with controls (1.7±0.1 vs. 1.3±0.1, P <0.01,
Figure 5d ).
Along with the nuclear HuCD expression pattern, we found
the submucous glial cell labeling (using an antibody against S100)
to be more abundant in FD patients than in controls ( Figure 5a
and b ). We quanti ed this di erence by measuring the total
0
Controls
FD patients
20
Neurons with nuclear HuCD (%)
Mean intensity ratio
nucleus/cytosol (a.u.)
40
60
80
100
0
1
2
3
1000
500
Controls
FD patients
0
2000
Glia volume (mm
3
)/neuron
1500
2500
3000
**
**
**
Controls
HuCD S100
Merge
S100 volume
HuCD
FD patients
S100
Merge
S100 volume
Controls FD patients
Figure 5 . Neuronal and glial abnormalities in the submucous plexus of functional dyspepsia (FD) patients. The images represent neurons (HuCD, green)
and glial cells (S100, color code: magenta), as well as a merged image in controls ( a ) compared with FD patients ( b ). In controls, the staining of panneu-
ronal marker HuCD (green) is homogeneous in cytosol and nucleus (white arrows in a ), whereas in FD patients the intensity of the staining was higher in
the nucleus than in the cytosol (white arrows in b ). In controls, immunostaining shows that glial cells (color code: magenta) entangle neurons (green) and
form a well-defi ned mesh within the ganglion. In FD patients, the glial tangle (color code: magenta) around neurons (green) is more intricate, and single
glial cells are hard to identify. Bars = 20 μ m. ( c – e ): the graphs show the percentage of submucous neurons with HuCD signal more intense in the nucleus
( c ), the mean intensity ratio nucleus/cytosol in submucous neurons, ( d ) and the ratio between the volume of S100 positive glial cells and the number of
neurons per ganglion ( e ) in controls (blue dots) compared with FD patients (red dots). ** P < 0.01.
© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
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Submucous Defects in Functional Dyspepsia
ity. FD and IBS might be considered as a single disorder with
di erent manifestations. However, it is possible that in the two
disorders diverse cellular and molecular pathways are involved,
which may be responsible for di erent clinical manifestations.
Another di erence resides in the experimental approach used.
In our study, we directly assess the function of patient-speci c
neurons, whereas until now only reports were available that
have used neurons from experimental animals or human resec-
tion specimen to investigate the e ect of biopsy supernatants
( 14,15,18,19 ). us, at this moment a similar measurement in
biopsies from patients with IBS would be needed to directly
con rm or refute the fact that submucous neurons from IBS are
indeed hyperexcitable or whether soluble components from IBS
biopsies render neurons more excitable. Whether submucous
neurons themselves, which must have been exposed to these
components for a longer time, are still hyperexcitable is actually
still a matter of debate.
Although it is conceivable that, as a consequence of being
exposed to speci c stressors, maybe already for long time,
neurons might have become damaged, at the moment it is
not known what factors impair neuronal activation in FD
patients. One possible explanation might be found in a neuro–
immune interaction, which has been proposed to underlie
some of the pathophysiological expressions of FGIDs ( 30–34 ).
Indeed, Barbara et al. ( 30 ) demonstrated that mast cells are
in close proximity with nerve endings in the colonic mucosa
of patients with IBS. In line with this hypothesis, some recent
studies have shown immune activation and so-called ‘low-
grade in ammation’ in the duodenum of FD patients ( 8–10 ).
Here we add a new insight to this hypothesis, in that we meas-
ured a signi cant increase in the number of eosinophils and
mast cells, speci cally in the submucous plexus of FD patients.
It is plausible that the decreased neuronal Ca
2+
signaling in
submucous neurons of FD patients is due to the release of
noxious agents from surrounding cells or stroma. At this
moment, no conclusive proof is available, and further studies
will be needed to identify the time course, identity, and
concentration of the releasable factors that would result in a
dampened neuronal Ca
2+
response. An alternative thesis could
be that the neurons themselves are actually not a ected at all but
only become so during dissection, as immune mediators would
be released only from those biopsies containing a signi cant
amount of eosinophils and mast cells. However, the fact that
all dissections were performed at ice-cold temperatures miti-
gates any acute e ect of released biomolecules on neuronal Ca
2+
signaling.
To evaluate the symptomatic relevance of these ndings, we
checked whether eosinophil and mast cell numbers correlated
with cardinal FD symptoms. We found a signi cant association
of submucous eosinophils with early satiety and postprandial
fullness, which are speci c symptoms of PDS. A similar correla-
tion in the duodenal mucosa of PDS patients has recently been
observed by Walker et al. ( 34 ). ey hypothesized that, through
mediator release and recruitment of mast cells, eosinophils
speci cally a ect neuronal and muscle cell functioning. We thus
volume of S100 positive cells and their processes relative to the
number of neurons per ganglion. e total glial volume measured
in the submucous plexus of FD patients was signi cantly higher
than in controls (27224±2214.7 vs. 3659.8±272.3 μ m
3
, P <0.01). As
not all ganglia have the same size or numbers of neurons, we nor-
malized the glial volume to the number of neurons present in the
cubic selection and found this also to be signi cantly higher in FD
patients compared with controls (1993±382.4 vs. 362.1±139.8 μ m
3
,
respectively; P <0.01, Figure 5e ).
Neuronal VIP staining did not differ between FD patients and
controls
In an attempt to identify a certain subpopulation that might have
changed in the submucous plexus of FD patients, we performed
an immunohistochemical staining for VIP neurons, the most
prominent population in the submucous plexus ( 29 ). In all biop-
sies from controls and FD patients, we could detect VIP positivity
in nerve bers and varicosities rather than in individual cell bod-
ies (data not shown). e quanti cation of VIP staining did not
show any di erence between FD patients and controls in our set
of biopsies ( P =0.68).
DISCUSSION
e traditional assumption is that FGIDs are diseases
without organic features, hence the adjective ‘functional’ ( 1 ).
However, a rapidly increasing body of evidence shows that
subtle cellular and molecular alterations can be found in the
gastrointestinal tract of patients with FGIDs ( 4–10 ). In this
paper, we performed live imaging of neuronal activity, using
optical recording techniques in fresh nerve tissue (submucous
plexus) isolated from routine biopsies ( 21 ) to directly assess
whether nerve function is altered in FD patients. We also used
immunohistochemical analyses to assess possible changes in
the number of individual neurons, ganglia per biopsy, and neu-
rons per ganglia, which turned out to be comparable between
FD patients and controls. e latter nding helps explaining
why in classic pathological assessments cellular di erences are
le unnoticed.
e technology to perform live neuron recordings in routine
intestinal biopsies has only recently been established ( 21 ) and
here we demonstrate that it can be applied also in non-healthy
status. We found that there were no obvious di erences between
FD patients and controls in terms of how easily tissues could be
isolated and loaded with Ca
2+
indicator. However, we could show
that the amplitudes of neuronal responses in FD patients were
signi cantly lower compared with controls, both in response
to perfusion with high-K
+
and also to electrical stimulation.
Neuronal activity turned out to be similar when EPS and PDS
patients ( 2,25,26 ) were compared. Our data showing decreased
neuronal activity in FD patients appear in contrast to the nd-
ings of other FGID (including IBS) studies, in which increased
neuronal activation has been suggested ( 14,15,18,19,30 ). ere
are, however, a number of reasons why it is di cult to compare
these discordant ndings, one of which may be disease speci c-
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Cirillo et al.
hypothesized that a correlation might exist between increased
number of in ltrating eosinophils and neuronal impairment.
Although we did not nd any correlation within the control
and the FD patient groups, we describe a signi cant negative
correlation between nerve activity and immune cell numbers
in the overall study population. As this analysis was performed
independently of clinical information and given the charac-
teristic distribution of data as revealed by post hoc labeling of
individual data points, our measured cellular parameters match
surprisingly well with the clinical subdivision between controls
and FD patients. e non-linear correlation also establishes a
link between nerve function and the presence of immune cells
based on information obtained from live human samples. At
the moment it is not clear how acutely the neuronal function
is a ected, and it remains plausible that long-term e ects by
eosinophil-released mediators are needed to in uence neu-
ronal activity in FD patients. We can speculate that eosinophils,
a er migration into the submucous plexus, may a ect speci c
neuronal pathways responsible for early satiety and postpran-
dial fullness in PDS patients. As recently shown in an elegant
series of animal experiments, immune cells also contribute to
organizing motility by interacting with enteric nerves even
in a non-in amed condition ( 27 ). e correlation between
cellular/molecular ndings with clinical symptoms in FD, as
well as in other FGIDs has been evaluated in several studies
( 8–10,30,34 ), but it is still controversial. It may well be that,
although underlying cellular defects are common, symptom
reporting is much more related to individual di erences in
perception and may su er from linguistic limitations in symp-
tom description repertoires, as well as modulation by many
other factors (social, psychological) known to be involved ( 35 ).
Further investigation into these other aspects is thus needed to
narrow the distance between lab bench measurements and clini-
cal evaluations.
Together with neuronal functionality, we analyzed the mor-
phology of submucous neurons in FD patients by using speci c
markers. First of all, immunohistochemical labeling revealed
that the neuronal marker HuCD ( 28,36 ) localized di erently in
neurons of FD patients compared with controls. We and others
( 28,36–38 ) have shown that mouse enteric neurons under path-
ological circumstances, which could be induced by mechanical
damage and hypoxia, display a higher nuclear HuCD appear-
ance. Although at present there is no conclusive explanation
for the altered HuCD expression in submucous neurons of FD
patients, it may be taken along as another measurable cellular
change. In order to identify a speci c neuronal subpopulation
that might be a ected in FD patients, we analyzed VIP stain-
ing, which labels a major class of neurons and nerve bers in
the submucous plexus ( 29 ). In our set of biopsies, we could not
detect di erences in VIP staining, which labeled nerve bers
rather than nerve cell bodies both in FD patients and controls.
However, we cannot exclude that a speci c neuronal subset
might be more a ected compared with other subpopulations
during FD.
As gut function is the result of a combined and fine inter-
play between all enteric nervous system components, we also
analyzed glial morphology in the submucous plexus of FD
patients. Glial cells are tightly packed with neurons to pro-
vide structural and functional support to them. Interestingly,
we measured the abundant presence of the glial marker S100
protein around the submucous neurons of FD patients. It is
widely described that, when triggered, glial cells react and
overexpress glial-derived molecules, a phenomenon called
reactive gliosis and described in many intestinal diseases char-
acterized by inflammation, like celiac disease, ulcerative coli-
tis, and other inflammatory neuropathies ( 39–43 ). Although
it is well established that glial cells are key players in several
gut diseases ( 44,45 ), this is the first study that directly meas-
ured neuronal impairment and gliosis in (FD) patients. On the
basis of our findings, we can speculate that, in the submucous
plexus of FD patients, inflammatory mediators released by
eosinophils and mast cells trigger glial cells to in turn release
factors (cytokines, nitric oxide, nerve growth factor) that affect
neuronal functioning ( 46,47 ).
In summary, we demonstrated impairment of neuronal signal-
ing and altered expression patterns of neuronal and glial markers
in submucous plexus of FD patients. e structural and func-
tional abnormalities identi ed in this study are indicative of a
subtle type of enteric neuro and gliopathy present in FD patients.
Here we show that such abnormalities can be directly monitored
in easy-accessible tissues such as duodenal biopsies by optical
techniques.
ACKNOWLEDGMENTS
We thank the members of LENS for their critical comments and
skilled technical assistance. We also thank Maura Corsetti, Ingrid
Demedts, Florencia Carbone, Natália Pessoa Rocha, and Lukas Van
Oudenhove (TARGID) for the insightful comments and useful
discussions. C.C. and H.V. are post-doctoral fellows and A.-S.D.
a doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek
(FWO, Belgium).
CONFLICT OF INTEREST
Guarantor of the article: Pieter Vanden Berghe, MSc, PhD.
Speci c author contributions : Study concept and design, acquisi-
tion of data, analysis and interpretation of data, dra ing and
editing of the manuscript, and statistical analysis: C.C. and P.V.B;
provision of biopsy material: T.B. and J.T.; enrollment of patients
for the study: T.B. and H.V.; acquisition and analysis of the data:
A.-S.D.; critical revision of the manuscript: T.B., A.-S.D., H.V., and
J.T.; P.V.B. wrote the analysis so ware; and J.T. and P.V.B. obtained
funding.
Financial support: is work was funded by BOF, University of
Leuven (Methusalem Jan Tack; OT/STRT1 Pieter Vanden Berghe)
and FWO (G0889.11; G.0A44.13). Confocal recordings were made
on the equipment of the Cell Imaging Core (University of Leuven)
supported by Hercules foundation grants to P.V.B.
Potential competing interests: None.
© 2015 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
1215
FUNCTIONAL GI DISORDERS
Submucous Defects in Functional Dyspepsia
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Study Highlights
WHAT IS CURRENT KNOWLEDGE
✓ Functional dyspepsia (FD) is a common gastrointestinal
disorder without clear etiology and pathophysiology.
✓ Recent evidence points the attention to cellular and
molecular abnormalities.
✓ The role of enteric nerves in the pathophysiology of FD has
been only indirectly demonstrated.
WHAT IS NEW HERE
✓ Submucous neuronal function is impaired in FD.
✓ Ganglionic architecture, as revealed by glial and neuronal
markers, shows crucial abnormalities in FD.
✓ Altered neuronal function correlates with eosinophil and
mast cell infi ltration.
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